US 2001.0034O23A1. (19) United States (12) Patent Application Publication (10) Pub. No.: US 2001/0034023 A1 Stanton, JR. et al. (43) Pub. Date: Oct. 25, 2001

(54) GENE SEQUENCE VARIATIONS WITH Related U.S. Application Data UTILITY IN DETERMINING THE (63) Continuation-in-part of application No. 09/710,467, TREATMENT OF DISEASE, INGENES filed on Nov. 8, 2000, which is a continuation-in-part RELATING TO DRUG PROCESSING of application No. 09/696,482, filed on Oct. 24, 2000. Non-provisional of provisional application No. 60/131,334, filed on Apr. 26, 1999. Non-provisional (76) Inventors: Vincent P. Stanton JR., Belmont, MA of provisional application No. 60/139,440, filed on (US); Martin Zillmann, Shrewsbury, Jun. 15, 1999. MA (US) (30) Foreign Application Priority Data Correspondence Address: Jan. 20, 2000 (WO)...... PCT/USOO/O1392 EDWARD O. KRUESSER BROBECK PHLEGER & HARRISON Publication Classification 12390 EL CAMINO REAL (51) Int. Cl...... C12O 1/68; G06F 19/00 SAN DIEGO, CA 92130 (US) (52) U.S. Cl...... 435/6; 702/20 (57) ABSTRACT (21) Appl. No.: 09/733,000 Methods for identifying and utilizing variances in genes relating to efficacy and Safety of medical therapy and other aspects of medical therapy are described, including methods (22) Filed: Dec. 7, 2000 for Selecting an effective treatment. US 2001/0034023 A1 Oct. 25, 2001

GENE SEQUENCE WARIATIONS WITH UTILITY 0005 For some drugs, over 90% of the measurable IN DETERMINING THE TREATMENT OF interSubject variation in Selected pharmacokinetic param DISEASE, INGENES RELATING TO DRUG eters has been shown to be heritable. For a limited number PROCESSING of drugs, DNA sequence variances have been identified in Specific genes that are involved in drug action or metabo RELATED APPLICATIONS lism, and these variances have been shown to account for the 0001. This application is a continuation-in-part of Stan variable efficacy or safety of the drugs in different individu ton et al., U.S. application Ser. No. 09/710,467, filed Nov. 8, als. AS the Sequence of the human genome is completed, and 2000 entitled GENE SEQUENCE VARIATIONS WITH as additional human gene Sequence variances are identified, UTILITY IN DETERMINING THE TREATMENT OF the power of genetic methods for predicting drug response DISEASE, IN GENES RELATING TO DRUG PROCESS will further increase. This application concerns methods for ING, which is a continuation-in-part of Stanton et al., U.S. identifying and exploiting gene Sequence variances that application Ser. No. 09/696,482, filed Oct. 24, 2000 entitled account for interpatient variation in drug response, particu GENE SEQUENCE VARIATIONS WITH UTILITY IN larly interpatient variation attributable to pharmacokinetic DETERMINING THE TREATMENT OF DISEASE, IN factors and interpatient variation in drug tolerability or GENES RELATING TO DRUG PROCESSING, which is a toxicity. continuation-in-part of U.S. application Ser. No. not yet 0006 The efficacy of a drug is a function of both phar assigned, Attorney Docket No. 030586.0009CIP4, filed Oct. macodynamic effects and pharmacokinetic effects, or bio 6, 2000 entitled GENE SEQUENCE VARIATIONS WITH availability. In the present invention, interpatient variability UTILITY IN DETERMINING THE TREATMENT OF in drug Safety, tolerability and efficacy are discussed in terms DISEASE, IN GENES RELATING TO DRUG PROCESS of the genetic determinants of interpatient variation in ING, which is a continuation-in-part of Stanton et al., U.S. absorption, distribution, metabolism, and excretion, i.e. application Ser. No. 09/639,474, filed Aug. 15, 2000 GENE pharmacokinetic parameters. SEQUENCE VARIATIONS WITH UTILITY IN DETER MINING THE TREATMENT OF DISEASE, IN GENES 0007 Adverse drug reactions are a principal cause of the RELATING TO DRUG PROCESSING, which is a continu low Success rate of drug development programs (less than ation in part of Stanton et al., U.S. application Ser. No. one in four compounds that enters human clinical testing is 09/590,783, filed Jun. 8, 2000 GENE SEQUENCE VARIA ultimately approved for use by the U.S. Food and Drug TIONS WITH UTILITY IN DETERMINING THE TREAT Administration (FDA)). Adverse drug reactions can be cat MENT OF DISEASE, IN GENES RELATING TO DRUG egorized as 1) mechanism based reactions and 2) idiosyn PROCESSING, which is a continuation-in-part of Stanton, cratic, “unpredictable' effects apparently unrelated to the U.S. application Ser. No. 09/501.955, filed Feb. 10, 2000, primary pharmacologic action of the compound. Although which is a continuation-in-part of Stanton, International Some side effects appear Shortly after administration, in Application Ser. No. PCT/US00/01392, filed Jan. 20, 2000, Some instances Side effects appear only after a latent period. Stanton, U.S. application Ser. No. 09/427,835, filed Oct. 26, Adverse drug reactions can also be categorized into revers 1999, and Stanton et al., U.S. application Ser. No. 09/300, ible and irreversible effects. The methods of this invention 747, filed Apr. 26, 1999, and claims the benefit of U.S. are useful for identifying the genetic basis of both mecha Provisional Patent Application, Stanton & Adams, Ser. No. nism based and idiosyncratic toxic effects, whether revers 60/131,334, filed Apr. 26, 1999, and U.S. Provisional Patent ible or not. Methods for identifying the genetic sources of Application, Stanton, Ser. No. 60/139,440, filed Jun. 15, interpatient variation in efficacy and mechanism based tox 1999, which are hereby incorporated by reference in their icity may be initially directed to analysis of genes affecting entireties, including drawings and tables. pharmacokinetic parameters, while the genetic causes of idiosyncratic adverse drug reactions are more likely to be BACKGROUND OF THE INVENTION attributable to genes affecting variation in pharmacody namic responses or immunological responsiveness. 0002 This application concerns the field of mammalian therapeutics and the Selection of therapeutic regimens uti 0008 Absorption is the first pharmacokinetic parameter lizing host genetic information, including gene Sequence to consider when determining the causes of interSubject variances within the human genome in human populations. variation in drug response. The relevant genes depend on the route of administration of the compound being evaluated. 0003. The information provided below is not admitted to For orally administered drugs the major Steps in absorption be prior art to the present invention, but is provided Solely may occur during exposure to Salivary enzymes in the to assist the understanding of the reader. mouth, exposure to the acidic environment of the Stomach, 0004. Many drugs or other treatments are known to have exposure to pancreatic digestive enzymes and bile in the highly variable safety and efficacy in different individuals. A Small intestine, exposure to enteric bacteria and exposure to consequence of Such variability is that a given drug or other cell Surface proteins throughout the gastrointestinal tract. treatment may be effective in one individual, and ineffective For example, uptake of a drug that is absorbed acroSS the or not well-tolerated in another individual. Thus, adminis gastrointestinal tract by facilitated transport may vary on tration of Such a drug to an individual in whom the drug account of allelic variation in the gene encoding the trans would be ineffective would result in wasted cost and time porter protein. Many drugs are lipophilic (a property which during which the patient's condition may significantly promotes passive movement across biological membranes). worsen. Also, administration of a drug to an individual in Variation in levels of Such drugs may depend, for example, whom the drug would not be tolerated could result in a direct on the enterohepatic circulation of the drug, which may be worsening of the patient's condition and could even result in affected by genetic variation in liver canalicular transporters, the patient's death. or intestinal transporters, alternatively renal reabsorbtion US 2001/0034023 A1 Oct. 25, 2001 mechanisms may vary among patients as a consequence of the compound. Experimental means of assessing relevant gene Sequence variances. If a compound is delivered biotransformation Systems are also described. parenterally then absorption is not an issue, however tran 0012 Drug-induced disease or toxicity presents a unique Scutaneous administration of a compound may be Subject to Series of challenges to drug developers, as these reactions genetically determined variation in skin absorptive proper are often not predictable from preclinical Studies and may ties. not be detected in early clinical trials involving Small 0009. Once a drug or candidate therapeutic intervention numbers of subjects. When such effects are detected in later is absorbed, injected or otherwise enters the bloodstream it Stages of clinical development they often result in termina is distributed to various biological compartments via the tion of a drug development program because, until now, blood. The drug may exist free in the blood, or, more there have been no effective tools to seek the determinants commonly, may be bound with varying degrees of affinity to of Such reactions. When a drug is approved despite Some plasma proteins. One classic Source of interpatient variation toxicity, its clinical use is frequently Severely constrained by in drug response is attributable to amino acid polymor the possible occurrence of adverse reactions in even a Small phisms in serum albumin, which affect the binding affinity of group of patients. The likelihood of Such a compound drugs. Such as warfarin. Consequent interpatient variation in becoming first line therapy is Small (unless there are no levels of free warfarin have a significant effect on the degree competing products). Thus, clinical trials that lead to detec of anticoagulation. From the blood a compound diffuses into tion of genetic causes of adverse events and Subsequently to and is retained in interstitial and cellular fluids of different the creation of genetic tests to identify and Screen out organs to different degrees. Interpatient variation in the patients Susceptible to Such events have the potential to (i) levels of a drug in different anatomical compartments may enable approval of compounds for genetically circumscribed be attributable to variation in the genetically encoded chemi populations or (ii) enable repositioning of approved com cal environment of those tissues (cell Surface proteins, pounds for broader clinical use. matrix proteins, cytoplasmic proteins and other factors) 0013 Similarly, many compounds are not approved due 0010. Once absorbed by the gastrointestinal tract, com to unimpressive efficacy. The identification of genetic deter pounds encounter detoxifying and metabolizing enzymes in minants of pharmacokinetic variation may lead to identifi the tissueS of the gastrointestinal System. Many of these cation of a genetically defined population in whom a Sig enzymes are known to be polymorphic in man and account nificant response is occurring. Approval of a compound for for well Studied variation in pharmacokinetic parameters of this population, defined by a genetic diagnostic test, may be many drugs. Subsequently compounds enter the hepatic the only means of getting regulatory approval for a drug. AS portal circulation in a process commonly known as first pass. healthcare becomes increasingly costly, the ability to allo The compounds then encounter a vast array of Xenobiotic cate healthcare resources effectively becomes increasingly detoxifying mechanisms in the liver, including enzymes that urgent. The use of genetic tests to develop and rationally are expressed Solely or at high levels only in liver. These administer medicines represents a powerful tool for accom enzymes include the cytochrome P450s, glucuronly trans plishing more cost effective medical care. ferases, Sulfotransferases, acetyltransferases, methyltrans ferases, the glutathione conjugating System, flavine SUMMARY OF THE INVENTION monooxygenases, and other enzymes known in the art. 0014. The present invention is concerned generally with Polymorphisms have been detected in all of these metabo the field of pharmacology, Specifically pharmacokinetics and lizing Systems, however the genetic factors responsible for toxicology, and more Specifically with identifying and pre interSubject variation have only been partially identified, and dicting inter-patient differences in response to drugs in order in Some cases not yet identified at all. Biotransformation to achieve Superior efficacy and Safety in Selected patient reactions in the liver often have the effect of converting populations. It is further concerned with the genetic basis of lipophilic compounds into hydrophilic molecules that are inter-patient variation in response to therapy, including drug then more readily excreted. Variation in these conjugation therapy, and with methods for determining and exploiting reactions may affect half-life and other pharmacokinetic Such differences to improve medical outcomes. Specifically, parameters. It is important to note that metabolic transfor this invention describes the identification of genes and gene mation of a compound not infrequently gives rise to a Second Sequence variances useful in the field of therapeutics for or additional compounds that have biological activity greater optimizing efficacy and Safety of drug therapy by allowing than, less than, or different from that of the parent com prediction of pharmacokinetic and/or toxicologic behavior pound. Metabolic transformation may also be responsible of Specific drugs in Specific patients. Relevant pharmacoki for producing toxic metabolites. netic processes include absorption, distribution, metabolism 0.011 Biotransformation reactions can be divided into and excretion. Relevant toxicological processes include both two phases. Phase I are oxidation-reduction reactions and dose related and idiosyncratic adverse reactions to drugs, phase II are conjugation reactions. The enzymes involved in including, for example, hepatotoxicity, blood dyscrasias and both of these phases are located predominantly in the liver, immunological reactions. The invention also describes however biotransformation can also occur in the kidney, methods for establishing diagnostic tests useful in (i) the gastrointestinal tract, Skin, lung, and other organs. Phase I development of, (ii) obtaining regulatory approval for and reactions occur predominantly in the endoplasmic reticulum, (iii) Safe and efficacious clinical use of pharmaceutical while phase II reactions occur predominantly in the cytosol. products. These variances may be useful either during the Both types of reactions can occur in the mitochondria, drug development process or in guiding the optimal use of nuclear envelope, or plasma membrane. One skilled in the already approved compounds. DNA sequence variances in art can, for Some compounds, make reasonable predictions candidate genes (i.e. genes that may plausibly affect the concerning likely metabolic Systems given the Structure of action of a drug) are tested in clinical trials, leading to the US 2001/0034023 A1 Oct. 25, 2001 establishment of diagnostic tests useful for improving the Subject to the Signs and Symptoms, or is more likely to development of new pharmaceutical products and/or the develop Such Signs and Symptoms than a normal perSon in more effective use of existing pharmaceutical products. the population. Thus, for example, a perSon Suffering from Methods for identifying genetic variances and determining a condition can include a developing fetus, a perSon Subject their utility in the Selection of optimal therapy for Specific to a treatment or environmental condition which enhances patients are also described. In general, the invention relates the likelihood of developing the Signs or Symptoms of a to methods for identifying and dealing effectively with the condition, or a person who is being given or will be given genetic Sources of interpatient variation in drug response, a treatment which increase the likelihood of the perSon including both variable efficacy as determined by pharma developing a particular condition. For example, tardive cokinetic variability and variable toxicity as determined by dyskinesia is associated with long-term use of anti-psychot pharmacokinetic factors or by other genetic factors (e.g. ics, dyskinesias, paranoid ideation, psychotic episodes and factors responsible for idiosyncratic drug response). depression have been associated with use of L-dopa in 0.015 The inventors have determined that the identifica Parkinson's disease, and dizziness, diplopia, ataxia, Seda tion of gene Sequence variances in genes that may be tion, impaired mentation, weight gain, and other undesired involved in drug action are useful for determining whether effects have been described for various anticonvulsant thera genetic variances account for variable drug efficacy and pies, alopecia and bone marrow Suppression are associated Safety and for determining whether a given drug or other with cancer chemotherapeutic regimens, and immunoSup therapy may be Safe and effective in an individual patient. pression is associated with agents to limit graft rejection Provided in this invention are identifications of genes and following transplantation. Thus, methods of the present Sequence variances which can be useful in connection with invention which relate to treatments of patients (e.g., meth predicting differences in response to treatment and Selection ods for Selecting a treatment, Selecting a patient for a of appropriate treatment of a disease or condition. A target treatment, and methods of treating a disease or condition in gene and variances have utility in pharmacogenetic associa a patient) can include primary treatments directed to a tion Studies and diagnostic tests to improve the use of certain presently active disease or condition, Secondary treatments drugs or other therapies including, but not limited to, the which are intended to cause a biological effect relevant to a drug classes and specific drugs identified in the 1999 Phy primary treatment, and prophylactic treatments intended to sicians’ Desk Reference (53rd edition), Medical Economics delay, reduce, or prevent the development of a disease or Data, 1998, or the 1995 United States Pharmacopeia XXIII condition, as well as treatments intended to cause the National Formulary XVIII, Interpharm Press, 1994, or other development of a condition different from that which would Sources as described below. have been likely to develop in the absence of the treatment. 0018. The term “therapy” refers to a process that is 0016. The terms “disease” or “condition” are commonly intended to produce a beneficial change in the condition of recognized in the art and designate the presence of Signs a mammal, e.g., a human, often referred to as a patient. A and/or Symptoms in an individual or patient that are gener beneficial change can, for example, include one or more of: ally recognized as abnormal. Diseases or conditions may be restoration of function, reduction of Symptoms, limitation or diagnosed and categorized based on pathological changes. retardation of progression of a disease, disorder, or condition Signs may include any objective evidence of a disease Such or prevention, limitation or retardation of deterioration of a as changes that are evident by physical examination of a patient's condition, disease or disorder. Such therapy can patient or the results of diagnostic tests which may include, involve, for example, nutritional modifications, administra among others, laboratory tests to determine the presence of tion of radiation, administration of a drug, behavioral modi DNA sequence variances or variant forms of certain genes in fications, and combinations of these, among others. a patient. Symptoms are Subjective evidence of disease or a 0019. The term “drug” as used herein refers to a chemical patients condition, i.e. the patients perception of an abnor entity or biological product, or combination of chemical mal condition that differs from normal function, Sensation, entities or biological products, administered to a person to or appearance, which may include, without limitations, treat or prevent or control a disease or condition. The physical disabilities, morbidity, pain, and other changes chemical entity or biological product is preferably, but not from the normal condition experienced by an individual. necessarily a low molecular weight compound, but may also Various diseases or conditions include, but are not limited to; be a larger compound, for example, an oligomer of nucleic those categorized in Standard textbooks of medicine includ acids, amino acids, or carbohydrates including without limi ing, without limitation, textbooks of nutrition, allopathic, tation proteins, oligonucleotides, ribozymes, DNAZymes, homeopathic, and Osteopathic medicine. In certain aspects of glycoproteins, lipoproteins, and modifications and combi this invention, the disease or condition is Selected from the nations thereof. A biological product is preferably a mono group consisting of the types of diseases listed in Standard clonal or polyclonal antibody or fragment thereof Such as a texts such as Harrison's Principles of Internal Medicine variable chain fragment; cells, or an agent or product arising (14th Ed) by Anthony S. Fauci, Eugene Braunwald, Kurt J. from recombinant technology, Such as, without limitation, a Isselbacher, et al. (Editors), McGraw Hill, 1997, or Robbins recombinant protein, recombinant vaccine, or DNA con Pathologic Basis of Disease (6th edition) by Ramzi S. Struct developed for therapeutic, e.g., human therapeutic, Cotran, Vinay Kumar, Tucker Collins & Stanley L. Robbins, use. The term “drug may include, without limitation, com WB Saunders Co., 1998, or the Diagnostic and Statistical pounds that are approved for Sale as pharmaceutical prod Manual of Mental Disorders; DSM-IV (4" edition), Ameri ucts by government regulatory agencies (e.g., U.S. Food and can Psychiatric Press, 1994, or other texts described below. Drug Administration (USFDA or FDA), European Medi 0.017. In connection with the methods of this invention, cines Evaluation Agency (EMEA), and a world regulatory unless otherwise indicated, the term "Suffering from a dis body governing the International Conference of Harmoni ease or condition” means that a perSon is either presently zation (ICH) rules and guidelines), compounds that do not US 2001/0034023 A1 Oct. 25, 2001

require approval by government regulatory agencies, food pearance; the remaining patients have only partial tumor additives or Supplements including compounds commonly Shrinkage). Conversely, up to 20-30% of patients receiving characterized as Vitamins, natural products, and completely 5-FU Suffer Serious gastrointestinal or hematopoietic toxic or incompletely characterized mixtures of chemical entities ity, depending on the regimen. including natural compounds or purified or partially purified natural products. The term “drug” as used herein is Synony 0024. Thus, in a first aspect, the invention provides a mous with the terms “medicine”, “pharmaceutical product', method for Selecting a treatment for a patient Suffering from or “product”. Most preferably the drug is approved by a a disease or condition by determining whether or not a gene government agency for treatment of a specific disease or or genes in cells of the patient (in Some cases including both condition. normal and disease cells, Such as cancer cells) contain at least one Sequence variance which is indicative of the 0020. The term “candidate therapeutic intervention” effectiveness of the treatment of the disease or condition. refers to a drug, agent or compound that is under investi The gene or genes are preferably specified herein, in Table gation, either in laboratory or human clinical testing for a 1, 3, or 4. Preferably the at least one variance includes a Specific disease, disorder, or condition. plurality of variances which may provide a haplotype or haplotypes. Preferably the joint presence of the plurality of 0021. A “low molecular weight compound” has a variances is indicative of the potential effectiveness or Safety molecular weight <5,000 Da, more preferably <2500 Da, of the treatment in a patient having Such plurality of Vari still more preferably <1000 Da, and most preferably <700 ances. The plurality of variances may each be indicative of Da. the potential effectiveness of the treatment, and the effects of 0022. Those familiar with drug use in medical practice the individual variances may be independent or additive, or will recognize that regulatory approval for drug use is the plurality of variances may be indicative of the potential commonly limited to approved indications, Such as to those effectiveness if at least 2, 3, 4, or more appear jointly. The patients afflicted with a disease or condition for which the plurality of variances may also be combinations of these drug has been shown to be likely to produce a beneficial relationships. The plurality of variances may include Vari effect in a controlled clinical trial. Unfortunately, it has ances from one, two, three or more gene loci. generally not been possible with current knowledge to 0025. In preferred embodiments of aspects of the inven predict which patients will have a beneficial response, with tion involving genes relating to pharmacokinetic parameters the exception of certain diseases Such as bacterial infections that affect efficacy and Safety, e.g. drug-induced disease or where suitable laboratory methods have been developed. drug-induced, disorder, or dysfunction or other drug-in Likewise, it has generally not been possible to determine in duced pathophysiologic disease, or protection or Sensitivity advance whether a drug will be Safe in a given patient. to toxic compounds, the gene product is involved in a Regulatory approval for the use of most drugs is limited to the treatment of Selected diseases and conditions. The function as described in the Background of the Invention or descriptions of approved drug usage, including the Sug otherwise described herein. gested diagnostic Studies or monitoring Studies, and the 0026. In some cases, the selection of a method of treat allowable parameters of Such Studies, are commonly ment, i.e., a therapeutic regimen, may incorporate Selection described in the “label” or “insert” which is distributed with of one or more from a plurality of medical therapies. Thus, the drug. Such labels or inserts are preferably required by the Selection may be the Selection of a method or methods government agencies as a condition for marketing the drug which is/are more effective or less effective than certain and are listed in common references Such as the Physicians other therapeutic regimens (with either having varying Desk Reference (PDR). These and other limitations or Safety parameters). Likewise or in combination with the considerations on the use of a drug are also found in medical preceding Selection, the Selection may be the Selection of a journals, publications Such as pharmacology, pharmacy or method or methods, which is Safer than certain other meth medical textbooks including, without limitation, textbooks ods of treatment in the patient. of nutrition, allopathic, homeopathic, and Osteopathic medi 0027. The selection may involve either positive selection CC. or negative Selection or both, meaning that the Selection can 0023 Many widely used drugs are effective in a minority involve a choice that a particular method would be an of patients receiving the drug, particularly when one con appropriate method to use and/or a choice that a particular trols for the placebo effect. For example, the PDR shows that method would be an inappropriate method to use. Thus, in about 45% of patients receiving Cognex (tacrine hydrochlo certain embodiments, the presence of the at least one Vari ride) for Alzheimer's disease show no change or minimal ance is indicative that the treatment will be effective or worsening of their disease, as do about 68% of controls otherwise beneficial (or more likely to be beneficial) in the (including about 5% of controls who were much worse). patient. Stating that the treatment will be effective means About 58% of Alzheimer's patients receiving Cognex were that the probability of beneficial therapeutic effect is greater minimally improved, compared to about 33% of controls, than in a perSon not having the appropriate presence or while about 2% of patients receiving CogneX were much absence of particular variances. In other embodiments, the improved compared to about 1% of controls. Thus a tiny presence of the at least one variance is indicative that the fraction of patients had a Significant benefit. Response to treatment will be ineffective or contra-indicated for the many cancer chemotherapy drugs is even worse. For patient. For example, a treatment may be contra-indicated if example, 5-fluorouracil is Standard therapy for advanced the treatment results, or is more likely to result, in undesir colorectal cancer, but only about 20-40% of patients have an able Side effects, or an excessive level of undesirable side objective response to the drug, and, of these, only 1-5% of effects. A determination of what constitutes excessive side patients have a complete response (complete tumor disap effects will vary, for example, depending on the disease or US 2001/0034023 A1 Oct. 25, 2001

condition being treated, the availability of alternatives, the with one or more variances or variant forms of the gene, expected or experienced efficacy of the treatment, and the more preferably in >30%, more preferably in >50%, and tolerance of the patient. AS for an effective treatment, this most preferably in >70% or in more than 90%. means that it is more likely that desired effect will result 0031. Also in other embodiments, the method of select from the treatment administration in a patient with a par ing a treatment includes eliminating a treatment, where the ticular variance or variances than in a patient who has a presence or absence of the at least one variance is indicative different variance or variances. Also in preferred embodi that the treatment will be ineffective or contra-indicated, ments, the presence of the at least one variance is indicative e.g., would result in excessive weight gain. In other pre that the treatment is both effective and unlikely to result in ferred embodiments, in cases in which undesirable Side undesirable effects or outcomes, or vice versa (is likely to effects may occur or are expected to occur from a particular have undesirable side effects but unlikely to produce desired therapeutic treatment, the Selection of a method of treatment therapeutic effects). can include identifying both a first and Second treatment, 0028. In reference to response to a treatment, the term where the first treatment is effective to treat the disease or “tolerance” refers to the ability of a patient to accept a condition, and the Second treatment reduces a deleterious treatment, based, e.g., on deleterious effects and/or effects on effect of the first treatment. lifestyle. Frequently, the term principally concerns the 0032. The phrase “eliminating a treatment” refers to patients perceived magnitude of deleterious effects Such as removing a possible treatment from consideration, e.g., for nausea, weakness, dizziness, and diarrhea, among others. use with a particular patient based on the presence or Such experienced effects can, for example, be due to general absence of a particular variance(s) in one or more genes in or cell-specific toxicity, activity on non-target cells, croSS cells of that patient, or to Stopping the administration of a reactivity on non-target cellular constituents (non-mecha treatment which was in the course of administration. nism based), and/or side effects of activity on the target cellular Substituents (mechanism based), or the cause of 0033). Usually, the treatment will involve the administra toxicity may not be understood. In any of these circum tion of a compound preferentially active or Safe in patients stances one may identify an association between the unde with a form or forms of a gene, where the gene is one Sirable effects and variances in Specific genes. identified herein. The administration may involve a combi nation of compounds. Thus, in preferred embodiments, the 0029 Adverse responses to drugs constitute a major method involves identifying Such an active compound or medical problem, as shown in two recent meta-analyses combination of compounds, where the compound is leSS (Lazarou, J. et al., Incidence of adverse drug reactions in active or is leSS Safe or both when administered to a patient hospitalized patients: a meta-analysis of prospective studies, having a different form of the gene. JAMA279:1200-1205, 1998; Bonn, Adverse drug reactions 0034. Also in preferred embodiments, the method of remain a major cause of death, Lancet 351:1183, 1998). An Selecting a treatment involves Selecting a method of admin estimated 2.2 million hospitalized patients in the United istration of a compound, combination of compounds, or Stated had serious adverse drug reactions in 1994, with an pharmaceutical composition, for example, Selecting a Suit estimated 106,000 deaths (Lazarou et al.). To the extent that able dosage level and/or frequency of administration, and/or Some of these adverse events are due to genetically encoded mode of administration of a compound. The method of biochemical diversity among patients in pathways that effect administration can be selected to provide better, preferably drug action, the identification of variances that are predictive maximum therapeutic benefit. In this context, "maximum of such effects will allow for more effective and safer drug refers to an approximate local maximum based on the Sc. parameters being considered, not an absolute maximum. 0.030. In embodiments of this invention, the variance or 0035 Also in this context, a “suitable dosage level.” variant form or forms of a gene is/are associated with a refers to a dosage level which provides a therapeutically Specific response to a drug. The frequency of a specific reasonable balance between pharmacological effectiveness variance or variant form of the gene may correspond to the and deleterious effects. Often this dosage level is related to frequency of an efficacious response to administration of a the peak or average Serum levels resulting from administra drug. Alternatively, the frequency of a Specific variance or tion of a drug at the particular dosage level. variant form of the gene may correspond to the frequency of an adverse event resulting from administration of a drug. 0036) Similarly, a “frequency of administration” refers to Alternatively the frequency of a Specific variance or variant how often in a Specified time period a treatment is admin form of a gene may not correspond closely with the fre istered, e.g., once, twice, or three times per day, every other quency of a beneficial or adverse response, yet the variance day, once per week, etc. For a drug or drugs, the frequency may still be useful for identifying a patient Subset with high of administration is generally Selected to achieve a pharma response or toxicity incidence because the variance may cologically effective average or peak Serum level without account for only a fraction of the patients with high response excessive deleterious effects (and preferably while still or toxicity. In Such a case the preferred course of action is being able to have reasonable patient compliance for Self identification of a Second or third or additional variances that administered drugs). Thus, it is desirable to maintain the permit identification of the patient groups not usefully serum level of the drug within a therapeutic window of identified by the first variance. Preferably, the drug will be concentrations for the greatest percentage of time possible effective in more than 20% of individuals with one or more without Such deleterious effects as would cause a prudent Specific variances or variant forms of the gene, more pref physician to reduce the frequency of administration for a erably in 40% and most preferably in >60%. In other particular dosage level. embodiments, the drug will be toxic or create clinically 0037. A particular gene or genes can be relevant to the unacceptable side effects in more than 10% of individuals treatment of more than one disease or condition, for US 2001/0034023 A1 Oct. 25, 2001 example, the gene or genes can have a role in the initiation, chloride. (See Current Protocols in Molecular Biology by F. development, course, treatment, treatment outcomes, or M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. D. health-related quality of life outcomes of a number of Seidman, K. Struhl, and V. B. Chanda (editors, John Wiley different diseases, disorders, or conditions. Thus, in pre & Sons.) ferred embodiments, the disease or condition or treatment of the disease or condition is any which involves a gene from 0042. In other preferred embodiments, determining the the gene list described herein as Tables 1, 3, and 4. presence or absence of the at least one variance involves 0.038. Determining the presence of a particular variance Sequencing at least one nucleic acid Sample. The Sequencing or plurality of variances in a particular gene in a patient can involves Sequencing of a portion or portions of a gene and/or be performed in a variety of ways. In preferred embodi portions of a plurality of genes which includes at least one ments, the detection of the presence or absence of at least variance Site, and may include a plurality of Such sites. one variance involves amplifying a Segment of nucleic acid Preferably, the portion is 500 nucleotides or less in length, including at least one of the at least one variances. Prefer more preferably 200 or 100 nucleotides or less, and most ably a segment of nucleic acid to be amplified is 500 preferably 45 nucleotides or less in length. Such Sequencing nucleotides or less in length, more preferably 100 nucle can be carried out by various methods recognized by those otides or less, and most preferably 45 nucleotides or less. skilled in the art, including use of dideoxy termination Also, preferably the amplified Segment or Segments includes methods (e.g., using dye-labeled dideoxy nucleotides) and a plurality of variances, or a plurality of Segments of a gene the use of mass spectrometric methods. In addition, mass or of a plurality of genes. Spectrometric methods may be used to determine the nucle otide present at a variance Site. In preferred embodiments in 0039. In another aspect determining the presence of a set which a plurality of variances is determined, the plurality of of variances in a Specific gene related to treatment of variances can constitute a haplotype or collection of haplo pharmacokinetic parameters associated efficacy or Safety, types. Preferably the methods for determining genotypes or e.g. drug-induced disease, disorder, dysfunction, or other haplotypes are designed to be Sensitive to all the common toxicity-related gene or genes listed in Tables 1,3 and 4 may genotypes or haplotypes present in the population being entail a haplotyping test that requires allele Specific ampli Studied (for example, a clinical trial population). fication of a large DNA segment of no greater than 25,000 nucleotides, preferably no greater than 10,000 nucleotides 0043. The terms “variant form of a gene”, “form of a and most preferably no greater than 5,000 nucleotides. gene’’, or “allele” refer to one specific form of a gene in a Alternatively one allele may be enriched by methods other population, the Specific form differing from other forms of than amplification prior to determining genotypes at Specific the same gene in the Sequence of at least one, and frequently variant positions on the enriched allele as a way of deter more than one, variant Sites within the Sequence of the gene. mining haplotypes. Preferably the determination of the The Sequences at these variant sites that differ between presence or absence of a haplotype involves determining the different alleles of the gene are termed "gene Sequence Sequence of the variant site or Sites by methods Such as chain variances” or "variances” or “variants'. The term “alterna terminating DNA sequencing or miniseduencing, or by tive form” refers to an allele that can be distinguished from oligonucleotide hybridization or by mass spectrometry. other alleles by having distinct variances at least one, and frequently more than one, variant Sites within the gene 0040. The term “genotype' in the context of this inven Sequence. Other terms known in the art to be equivalent tion refers to the alleles present in DNA from a subject or include mutation and polymorphism, although mutation is patient, where an allele can be defined by the particular often used to refer to an allele associated with a deleterious nucleotide(s) present in a nucleic acid Sequence at a par phenotype. In preferred aspects of this invention, the Vari ticular site(s). Often a genotype is the nucleotide(s) present ances are Selected from the group consisting of the variances at a single polymorphic Site known to vary in the human listed in the variance tables herein or in a patent or patent population. application referenced and incorporated by reference in this disclosure. In the methods utilizing variance presence or 0041. In preferred embodiments, the detection of the absence, reference to the presence of a variance or variances presence or absence of the at least one variance involves means particular variances, i.e., particular nucleotides at contacting a nucleic acid Sequence corresponding to one of particular polymorphic Sites, rather than just the presence of the genes identified above or a product of Such a gene with any variance in the gene. a probe. The probe is able to distinguish a particular form of the gene or gene product or the presence or a particular 0044 Variances occur in the human genome at approxi variance or variances, e.g., by differential binding or hybrid mately one in every 500-1,000 bases within the human ization. Thus, exemplary probes include nucleic acid hybrid genome when two alleles are compared. When multiple ization probes, peptide nucleic acid probes, nucleotide alleles from unrelated individuals are compared the density containing probes which also contain at least one nucleotide of variant Sites increases as different individuals, when analog, and antibodies, e.g., monoclonal antibodies, and compared to a reference Sequence, will often have Sequence other probes as discussed herein. Those skilled in the art are variances at different Sites. At most variant Sites there are familiar with the preparation of probes with particular only two alternative nucleotides involving the Substitution Specificities. Those skilled in the art will recognize that a of one base for another or the insertion/deletion of one or variety of variables can be adjusted to optimize the discrimi more nucleotides. Within a gene there may be Several variant nation between two variant forms of a gene, including Sites. Variant forms of the gene or alternative alleles can be changes in Salt concentration, temperature, pH and addition distinguished by the presence of alternative variances at a of various compounds that affect the differential affinity of Single variant Site, or a combination of Several different GC VS. AT base pairs, Such as tetramethyl ammonium variances at different Sites (haplotypes). US 2001/0034023 A1 Oct. 25, 2001

0045. It is estimated that there are 3,300,000,000 bases in Genetics by N. C. Dracoli, J. L. Haines, B. R. Korf, D. T. the Sequence of a single haploid human genome. All human Moir, C. C. Morton, C. E. Seidman, D. R. Smith, and A. cells except germ cells are normally diploid. Each gene in Boyle (editors), John Wiley & Sons.) the genome may span 100-10,000,000 bases of DNA sequence or 100-20,000 bases of mRNA. It is estimated that 0048. In the context of this invention, the term “analyzing there are between 60,000 and 120,000 genes in the human a Sequence” refers to determining at least Some Sequence genome. The “identification' of genetic variances or variant information about the Sequence, e.g., determining the nucle forms of a gene involves the discovery of variances that are otides present at a particular site or Sites in the Sequence, present in a population. The identification of variances is particularly Sites that are known to vary in a population, or required for development of a diagnostic test to determine determining the base Sequence of all of a portion of the whether a patient has a variant form of a gene that is known particular Sequence. to be associated with a disease, condition, or predisposition 0049. In the context of this invention, the term “haplo or with the efficacy or safety of the drug. Identification of type' refers to a cis arrangement of two or more polymor previously undiscovered genetic variances is distinct from phic nucleotides, i.e., variances, on a particular chromo the process of “determining the Status of known variances Some, e.g., in a particular gene. The haplotype preserves by a diagnostic test (often referred to as genotyping). The information about the phase of the polymorphic nucle present invention provides exemplary variances in genes otides-that is, which set of variances were inherited from listed in the gene tables, as well as methods for discovering one parent, and which from the other. A genotyping test does additional variances in those genes and a comprehensive not provide information about phase. For example, an indi written description of Such additional possible variances. vidual heterozygous at nucleotide 25 of a gene (both A and Also described are methods for DNA diagnostic tests to Care present) and also at nucleotide 100 (both G and T are determine the DNA sequence at a particular variant Site or present) could have haplotypes 25A-100G and 25C-100T, or Sites. alternatively 25A-100T and 25C-100G. Only a haplotyping 0046) The process of “identifying” or discovering new test can discriminate these two cases definitively. variances involves comparing the Sequence of at least two alleles of a gene, more preferably at least 10 alleles and most 0050. The terms “variances”, “variants” and “polymor preferably at least 50 alleles (keeping in mind that each phisms”, as used herein, may also refer to a set of variances, Somatic cell has two alleles. The analysis of large numbers haplotypes or a mixture of the two. Further, the term of individuals to discover variances in the gene Sequence variance, variant or polymorphism (singular), as used between individuals in a population will result in detection herein, also encompasses a haplotype. This usage is intended of a greater fraction of all the variances in the population. to minimize the need for cumberSome phrases Such as: ". . Preferably the process of identifying reveals whether there is ... measure correlation between drug response and a variance, a variance within the gene; more preferably identifying variances, haplotype, haplotypes or a combination of Vari reveals the location of the variance within the gene, more ances and haplotypes . . . , throughout the application. preferably identifying provides knowledge of the Sequence Instead, the italicized text in the foregoing Sentence can be of the nucleic acid Sequence of the variance, and most represented by the word “variance”, “variant” or “polymor preferably identifying provides knowledge of the combina phism'. Similarly, the term genotype, as used herein, means tion of different variances that comprise Specific variant a procedure for determining the Status of one or more forms of the gene (referred to as alleles). In identifying new variances in a gene, including a set of variances comprising variances it is often useful to Screen different population a haplotype. Thus phrases Such as ". . . genotype a groups based on racial, ethnic, gender, and/or geographic patient . . . refer to determining the Status of one or more origin because particular variances may differ in frequency variances, including a set of variances for which phase is between Such groups. It may also be useful to Screen DNA known (i.e. a haplotype). from individuals with a particular disease or condition of 0051. In preferred embodiments of this invention, the interest because they may have a higher frequency of certain frequency of the variance or variant form of the gene in a variances than the general population. population is known. Measures of frequency known in the 0047 The process of genotyping involves using diagnos art include “allele frequency', namely the fraction of genes tic tests for Specific variances that have already been iden in a population that have one specific variance or Set of tified. It will be apparent that Such diagnostic tests can only variances. The allele frequencies for any gene should Sum to be performed after variances and variant forms of the gene 1. Another measure of frequency known in the art is the have been identified. Identification of new variances can be "heterozygote frequency' namely, the fraction of individuals accomplished by a variety of methods, alone or in combi in a population who carry two alleles, or two forms of a nation, including, for example, DNA sequencing, SSCP, particular variance or variant form of a gene, one inherited heteroduplex analysis, denaturing gradient gel electrophore from each parent. Alternatively, the number of individuals sis (DGGE), heteroduplex cleavage (either enzymatic as who are homozygous for a particular form of a gene may be with T4 Endonuclease 7, or chemical as with osmium a useful measure. The relationship between allele frequency, tetroxide and hydroxylamine), computational methods heterozygote frequency, and homozygote frequency is (described in “VARIANCE SCANNING METHOD FOR described for many genes by the Hardy-Weinberg equation, IDENTIFYING GENE SEQUENCE VARIANCES filed which provides the relationship between allele frequency, Oct. 14, 1999, Ser. No. 09/419,705, and other methods heterozygote frequency and homozygote frequency in a described herein as well as others known to those skilled in freely breeding population at equilibrium. Most human the art. (See, for example: Cotton, R. G. H., Slowly but variances are substantially in Hardy-Weinberg equilibrium. Surely towards better Scanning for mutations, Trends in In a preferred aspect of this invention, the allele frequency, Genetics 13(2): 43-6, 1997 or Current Protocols in Human heterozygote frequency, and homozygote frequencies are US 2001/0034023 A1 Oct. 25, 2001 determined experimentally. Preferably a variance has an 0055 AS is generally understood, administration of a allele frequency of at least 0.01, more preferably at least particular treatment, e.g., administration of a therapeutic 0.05, still more preferably at least 0.10. However, the allele compound or combination of compounds, is chosen depend may have a frequency as low as 0.001 if the associated ing on the disease or condition which is to be treated. Thus, phenotype is, for example, a rare form of toxic reaction to a in certain preferred embodiments, the disease or condition is treatment or drug. Beneficial responses may also be rare. one for which administration of a treatment is expected to 0.052 In this regard, “population” refers to a defined provide a therapeutic benefit. group of individuals or a group of individuals with a 0056. As used herein, the terms “effective” and “effec particular disease or condition or individuals that may be tiveness” includes both pharmacological effectiveness and treated with a specific drug identified by, but not limited to physiological Safety. Pharmacological effectiveness refers to geographic, ethnic, race, gender, and/or cultural indices. In the ability of the treatment to result in a desired biological most cases a population will preferably encompass at least effect in the patient. Physiological safety refers to the level ten thousand, one hundred thousand, one million, ten mil of toxicity, or other adverse physiological effects at the lion, or more individuals, with the larger numbers being cellular, organ and/or organism level (often referred to as more preferable. In a preferred aspect of this invention, the Side-effects) resulting from administration of the treatment. population refers to individuals with a Specific disease or On the other hand, the term "ineffective' indicates that a condition that may be treated with a specific drug. In an treatment does not provide Sufficient pharmacological effect aspect of this invention, the allele frequency, heterozygote to be therapeutically useful, even in the absence of delete frequency, or homozygote frequency of a Specific variance rious effects, at least in the unstratified population. (Such a or variant form of a gene is known. In preferred embodi treatment may be ineffective in a Subgroup that can be ments of this invention, the frequency of one or more identified by the presence of one or more Sequence variances variances that may predict response to a treatment is deter or alleles.) “Less effective” means that the treatment results mined in one or more populations using a diagnostic test. in a therapeutically significant lower level of pharmacologi 0053. It should be emphasized that it is currently not cal effectiveness and/or a therapeutically greater level of generally practical to study an entire population to establish adverse physiological effects, e.g., greater liver toxicity. the association between a Specific disease or condition or 0057 Thus, in connection with the administration of a response to a treatment and a specific variance or variant drug, a drug which is “effective against a disease or form of a gene. Such studies are preferably performed in condition indicates that administration in a clinically appro controlled clinical trials using a limited number of patients priate manner results in a beneficial effect for at least a that are considered to be representative of the population Statistically significant fraction of patients, Such as a with the disease. Since drug development programs are improvement of Symptoms, a cure, a reduction in disease generally targeted at the largest possible population, the load, reduction in tumor mass or cell numbers, extension of Study population will generally consist of men and women, life, improvement in quality of life, or other effect generally as well as members of various racial and ethnic groups, recognized as positive by medical doctorS familiar with depending on where the clinical trial is being performed. treating the particular type of disease or condition. This is important to establish the efficacy of the treatment in all Segments of the population. 0058 Effectiveness is measured in a particular popula tion. In conventional drug development the population is 0054. In the context of this invention, the term “probe' generally every Subject who meets the enrollment criteria refers to a molecule which detectably distinguishes between (i.e. has the particular form of the disease or condition being target molecules differing in Structure. Detection can be treated). It is an aspect of the present invention that seg accomplished in a variety of different ways depending on the mentation of a study population by genetic criteria can type of probe used and the type of target molecule. Thus, for provide the basis for identifying a Subpopulation in which a example, detection may be based on discrimination of drug is effective against the disease or condition being activity levels of the target molecule, but preferably is based treated. on detection of Specific binding. Examples of Such specific binding include antibody binding and nucleic acid probe 0059) The term “deleterious effects” refers to physical hybridization. Thus, for example, probes can include effects in a patient caused by administration of a treatment enzyme Substrates, antibodies and antibody fragments, and which are regarded as medically undesirable. Thus, for nucleic acid hybridization probes. Thus, in preferred example, deleterious effects can include a wide spectrum of embodiments, the detection of the presence or absence of the toxic effects injurious to health Such as death of normally at least one variance involves contacting a nucleic acid functioning cells when only death of diseased cells is Sequence which includes a variance Site with a probe, desired, nausea, fever, inability to retain food, dehydration, preferably a nucleic acid probe, where the probe preferen damage to critical organs Such as arrythmias, renal tubular tially hybridizes with a form of the nucleic acid Sequence necrosis, fatty liver, or pulmonary fibrosis leading to coro containing a complementary base at the variance site as nary, renal, hepatic, or pulmonary insufficiency among many compared to hybridization to a form of the nucleic acid others. In this regard, the term "adverse reactions' refers to Sequence having a non-complementary base at the variance those manifestations of clinical Symptomology of pathologi Site, where the hybridization is carried out under Selective cal disorder or dysfunction is induced by administration or hybridization conditions. Such a nucleic acid hybridization a drug, agent, or candidate therapeutic intervention. In this probe may span two or more variance Sites. Unless other regard, the term “contraindicated” means that a treatment wise Specified, a nucleic acid probe can include one or more results in deleterious effects Such that a prudent medical nucleic acid analogs, labels or other Substituents or moieties doctor treating Such a patient would regard the treatment as So long as the base-pairing function is retained. unsuitable for administration. Major factors in Such a deter US 2001/0034023 A1 Oct. 25, 2001 mination can include, for example, availability and relative treatment response. Preferably there is a plurality of vari advantages of alternative treatments, consequences of non ances for the gene, preferably a plurality of variances for the treatment, and permanency of deleterious effects of the particular gene. Preferably, the list is recorded in written or treatment. electronic form. For example, identified variances of iden tified genes are recorded for Some of the genes in Tables 3 0060. It is recognized that many treatment methods, e.g., and 4, additional variances for genes in Table 1 are provided administration of certain compounds or combinations of in Table 1 of Stanton & Adams, application Ser. No. 09/300, compounds, may produce Side-effects or other deleterious 747, Supra, and additional gene variance identification tables effects in patients. Such effects can limit or even preclude are provided in a form which allows comparison with other use of the treatment method in particular patients, or may variance information. The possible additional variances in even result in irreversible injury, disorder, dysfunction, or the identified genes are provided in Table 3 in Stanton & death of the patient. Thus, in certain embodiments, the Adams, application Ser. No. 09/300,747, Supra. variance information is used to select both a first method of treatment and a Second method of treatment. Usually the first 0064. In addition to the basic method of treatment, often treatment is a primary treatment which provides a physi the mode of administration of a given compound as a ological effect directed against the disease or condition or its treatment for a disease or condition in a patient is significant Symptoms. The Second method is directed to reducing or in determining the course and/or outcome of the treatment eliminating one or more deleterious effects of the first for the patient. Thus, the invention also provides a method treatment, e.g., to reduce a general toxicity or to reduce a for Selecting a method of administration of a compound to Side effect of the primary treatment. Thus, for example, the a patient Suffering from a disease or condition, by determin Second method can be used to allow use of a greater dose or ing the presence or absence of at least one variance in cells duration of the first treatment, or to allow use of the first of the patient in at least one identified gene from Tables 1, treatment in patients for whom the first treatment would not 3, and 4, where Such presence or absence is indicative of an be tolerated or would be contra-indicated in the absence of appropriate method of administration of the compound. a Second method to reduce deleterious effects or to potentiate Preferably, the selection of a method of treatment (a treat the effectiveness of the first treatment. ment regimen) involves selecting a dosage level or fre quency of administration or route of administration of the 0061. In a related aspect, the invention provides a method compound or combinations of those parameters. In preferred for Selecting a method of treatment for a patient Suffering embodiments, two or more compounds are to be adminis from a disease or condition by comparing at least one tered, and the Selecting involves Selecting a method of variance in at least one gene in the patient, with a list of administration for one, two, or more than two of the com variances in the gene from Tables 1, 3 and 4, or other gene pounds, jointly, concurrently, or Separately. AS understood related to pharmacokinetic parameters, or organ and tissue by those skilled in the art, Such plurality of compounds may damage, or inordinate immune response, which are indica be used in combination therapy, and thus may be formulated tive of the effectiveness or safety of at least one method of in a single drug, or may be separate drugs administered treatment. Preferably the comparison involves a plurality of concurrently, Serially, or Separately. Other embodiments are variances or a haplotype indicative of the effectiveness of at as indicated above for Selection of Second treatment meth least one method of treatment. Also, preferably the list of ods, methods of identifying variances, and methods of variances includes a plurality of variances. treatment as described for aspects above. 0.062 Similar to the above aspect, in preferred embodi 0065. In another aspect, the invention provides a method ments the at least one method of treatment involves the for Selecting a patient for administration of a method of administration of a compound effective in at least Some treatment for a disease or condition, or of Selecting a patient patients with a disease or condition; the presence or absence for a method of administration of a treatment, by comparing of the at least one variance is indicative that the treatment the presence or absence of at least one variance in a gene as will be effective in the patient; and/or the presence or identified above in cells of a patient, with a list of variances absence of the at least one variance is indicative that the in the gene, where the presence or absence of the at least one treatment will be ineffective or contra-indicated in the variance is indicative that the treatment or method of admin patient; and/or the treatment is a first treatment and the istration will be effective in the patient. If the at least one presence or absence of the at least one variance is indicative variance is present in the patient's cells, then the patient is that a Second treatment will be beneficial to reduce a Selected for administration of the treatment. deleterious effect or potentiate the effectiveness of the first treatment, and/or the at least one treatment is a plurality of 0066. In preferred embodiments, the disease or the methods of treatment. For a plurality of treatments, prefer method of treatment is as described in aspects above, ably the Selecting involves determining whether any of the Specifically including, for example, those described for methods of treatment will be more effective than at least one Selecting a method of treatment. other of the plurality of methods of treatment. Yet other embodiments are provided as described for the preceding 0067. In another aspect, the invention provides a method aspect in connection with methods of treatment using for identifying a Subset of patients with enhanced or dimin administration of a compound; treatment of various dis ished response or tolerance to a treatment method or a eases, and Variances in particular genes. method of administration of a treatment where the treatment is for a disease or condition in the patient. The method 0.063. In the context of variance information in the meth involves correlating one or more variances in one or more ods of this invention, the term “list” refers to one or more genes as identified in aspects above in a plurality of patients variances which have been identified for a gene of potential with response to a treatment or a method of administration importance in accounting for inter-individual variation in of a treatment. The correlation may be performed by deter US 2001/0034023 A1 Oct. 25, 2001

mining the one or more variances in the one or more genes the activation or inactivation or elimination of the drug, and in the plurality of patients and correlating the presence or more preferably the genes are involved in the same bio absence of each of the variances (alone or in various chemical process or pathway. combinations) with the patient's response to treatment. The variances may be previously known to exist or may also be 0072. In another aspect, the invention provides a method determined in the present method or combinations of prior for identifying the forms of a gene in an individual, where information and newly determined information may be used. the gene is one Specified as for aspects above, by determin The enhanced or diminished response should be Statistically ing the presence or absence of at least one variance in the Significant, preferably Such that p=0.10 or less, more pref gene. In preferred embodiments, the at least one variance erably 0.05 or less, and most preferably 0.02 or less. A includes at least one variance Selected from the group of positive correlation between the presence of one or more variances identified in variance tables herein. Preferably, the variances and an enhanced response to treatment is indica presence or absence of the at least one variance is indicative tive that the treatment is particularly effective in the group of the effectiveness of a therapeutic treatment in a patient of patients having those variances. A positive correlation of Suffering from a disease or condition and having cells the presence of the one or more variances with a diminished containing the at least one variance. response to the treatment is indicative that the treatment will 0073. The presence or absence of the variances can be be less effective in the group of patients having those determined in any of a variety of ways as recognized by variances. Such information is useful, for example, for those skilled in the art. For example, the nucleotide Sequence Selecting or de-Selecting patients for a particular treatment or of at least one nucleic acid Sequence which includes at least method of administration of a treatment, or for demonstrat one variance site (or a complementary Sequence) can be ing that a group of patients exists for which the treatment or determined, Such as by chain termination methods, hybrid method of treatment would be particularly beneficial or ization methods or by mass spectrometric methods. Like contra-indicated. Such demonstration can be beneficial, for wise, in preferred embodiments, the determining involves example, for obtaining government regulatory approval for contacting a nucleic acid Sequence or a gene product of one a new drug or a new use of a drug of one of the genes with a probe which specifically identifies the presence or absence of a form of the gene. For example, 0068. In preferred embodiments, the variances are in at a probe, e.g., a nucleic acid probe, can be used which least one of the identified genes listed in Tables 1, 3, and 4, Specifically binds, e.g., hybridizes, to a nucleic acid or are particular variances described herein. Also, preferred Sequence corresponding to a portion of the gene and which embodiments include drugs, treatments, variance identifica includes at least one variance Site under Selective binding tion or determination, determination of effectiveneSS, and/or conditions. AS described for other aspects, determining the diseases as described for aspects above or otherwise presence or absence of at least two variances and their described herein. relationship on the two gene copies present in a patient can 0069. In preferred embodiments, the correlation of constitute determining a haplotype or haplotypes. patient responses to therapy according to patient genotype is 0074. Other preferred embodiments involve variances carried out in a clinical trial, e.g., as described herein related to types of treatment, drug responses, diseases, according to any of the variations described. Detailed nucleic acid Sequences, and other items related to variances description of methods for associating variances with clini and variance determination as described for aspects above. cal outcomes using clinical trials are provided below. Fur ther, in preferred embodiments the correlation of pharma 0075. In yet another aspect, the invention provides a cological effect (positive or negative) to treatment response pharmaceutical composition which includes a compound according to genotype or haplotype in Such a clinical trial is which has a differential effect in patients having at least one part of a regulatory Submission to a government agency copy, or alternatively, two copies of a form of a gene as leading to approval of the drug. Most preferably the com identified for aspects above and a pharmaceutically accept pound or compounds would not be approvable in the able carrier, excipient, or diluent. The composition is absence of the genetic information allowing identification of adapted to be preferentially effective to treat a patient with an optimal responder population. cells containing the one, two, or more copies of the form of the gene. 0070 AS indicated above, in aspects of this invention involving Selection of a patient for a treatment, Selection of 0076. In preferred embodiments of aspects involving a method or mode of administration of a treatment, and pharmaceutical compositions, active compounds, or drugs, Selection of a patient for a treatment or a method of the material is Subject to a regulatory limitation, restriction, treatment, the Selection may be positive Selection or nega or recommendation on approved uses or indications, e.g., by the U.S. Food and Drug Administration (FDA), limiting or tive Selection. Thus, the methods can include eliminating a recommending limiting approved use of the composition to treatment for a patient, eliminating a method or mode of patients having at least one copy of the particular form of the administration of a treatment to a patient, or elimination of gene which contains at least one variance. Alternatively, the a patient for a treatment or method of treatment. composition is Subject to a regulatory limitation, restriction, 0071 Also, in methods involving identification and/or or recommendation on approved uses indicating or recom comparison of variances present in a gene of a patient, the mending that the composition is not approved for use or methods can involve Such identification or comparison for a should not be used in patients having at least one copy of a plurality of genes. Preferably, the genes are functionally form of the gene including at least one variance. Also in related to the Same disease or condition, or to the aspect of preferred embodiments, the composition is packaged, and disease pathophysiology that is being Subjected to pharma the packaging includes a label or insert indicating or Sug cological manipulation by the treatment (e.g., a drug), or to gesting beneficial therapeutic approved use of the compo US 2001/0034023 A1 Oct. 25, 2001

Sition in patients having one or two copies of a form of the 0080. The term “differential” or “differentially” generally gene including at least one variance. Alternatively, the label refers to a Statistically Significant different level in the or insert limits or recommends limiting approved use of the specified property or effect. Preferably, the difference is also composition to patients having Zero or one or two copies of functionally significant. Thus, “differential binding or a form of the gene including at least one variance. The latter hybridization” is sufficient difference in binding or hybrid embodiment would be likely where the presence of the at ization to allow discrimination using an appropriate detec least one variance in one or two copies in cells of a patient tion technique. Likewise, “differential effect” or “differen means that the composition would be ineffective or delete tially active' in connection with a therapeutic treatment or rious to the patient. Also in preferred embodiments, the drug refers to a difference in the level of the effect or activity composition is indicated for use in treatment of a disease or which is distinguishable using relevant parameters and tech condition that is one of those identified for aspects above. niques for measuring the effect or activity being considered. Also in preferred embodiments, the at least one variance Preferably the difference in effect or activity is also sufficient includes at least one variance from those identified herein. to be clinically Significant, Such that a corresponding dif ference in the course of treatment or treatment outcome 0077. The term “packaged” means that the drug, com would be expected, at least on a Statistical basis. pound, or composition is prepared in a manner Suitable for distribution or shipping with a box, Vial, pouch, bubble pack, 0081. Also usefully provided in the present invention are or other protective container, which may also be used in probes which Specifically recognize a nucleic acid Sequence combination. The packaging may have printing on it and/or corresponding to a variance or variances in a gene as printed material may be included in the packaging. identified in aspects above or a product expressed from the gene, and are able to distinguish a variant form of the 0078. In preferred embodiments, the drug is selected Sequence or gene or gene product from one or more other from the drug classes or Specific exemplary drugs identified variant forms of that Sequence, gene, or gene product under in an example, in a table herein, and is Subject to a regulatory Selective conditions. Those skilled in the art recognize and limitation or Suggestion or warning as described above that understand the identification or determination of Selective limits or Suggests limiting approved use to patients having conditions for particular probes or types of probes. An Specific variances or variant forms of a gene identified in exemplary type of probe is a nucleic acid hybridization Examples or in the gene list provided below in order to probe, which will selectively bind under selective binding achieve maximal benefit and avoid toxicity or other delete conditions to a nucleic acid Sequence or a gene product rious effect. corresponding to one of the genes identified for aspects above. Another type of probe is a peptide or protein, e.g., an 0079 A pharmaceutical composition can be adapted to be antibody or antibody fragment which specifically or prefer preferentially effective in a variety of ways. In Some cases, entially binds to a polypeptide expressed from a particular an active compound is Selected which was not previously form of a gene as characterized by the presence or absence known to be differentially active, or which was not previ of at least one variance. Thus, in another aspect, the inven ously recognized as a therapeutic compound. Alternatively tion concerns. Such probes. In the context of this invention, the compound was previously known as a therapeutic com a “probe' is a molecule, commonly a nucleic acid, though pound, but the composition is formulated in a manner also potentially a protein, carbohydrate, polymer, or Small appropriate for administration for treatment of a disease or molecule, that is capable of binding to one variance or condition for which a gene of this invention is involved in variant form of the gene to a greater extent than to a form of treatment response, and the active compound had not been the gene having a different base at one or more variance formulated appropriately for Such use before. For example, Sites, Such that the presence of the variance or variant form a compound may previously have been formulated for of the gene can be determined. Preferably the probe distin topical treatment of a skin condition, but is found to be guishes at least one variance identified in Examples, tables effective in IV or other internal treatment of a disease or lists below or in Tables 1 or 3 of Stanton & Adams, identified for this invention. For compounds that are differ application Ser. No. 09/300,747, Supra. entially effective on the gene, Such alternative formulations are adapted to be preferentially effective. In Some cases, the 0082 In preferred embodiments, the probe is a nucleic concentration of an active compound which has differential acid probe 6, 7, 8, 9, 10, 11, 12, 13, 14 or preferably at least activity can be adjusted Such that the composition is appro 17 nucleotides in length, more preferably at least 20 or 22 or priate for administration to a patient with the Specified 25, preferably 500 or fewer nucleotides in length, more variances. For example, the presence of a Specified variance preferably 200 or 100 or fewer, still more preferably 50 or may allow or require the administration of a much larger fewer, and most preferably 30 or fewer. In preferred embodi dose, which would not be practical with a previously utilized ments, the probe has a length in a range from any one of the composition. Conversely, a patient may require a much above lengths to any other of the above lengths (including lower dose, Such that administration of Such a dose with a endpoints). The probe specifically hybridizes under Selective prior composition would be impractical or inaccurate. Thus, hybridization conditions to a nucleic acid Sequence corre the composition may be prepared in a higher or lower unit sponding to a portion of one of the genes identified in dose form, or prepared in a higher or lower concentration of connection with above aspects. The nucleic acid Sequence the active compound or compounds. In yet other cases, the includes at least one and preferably two or more variance composition can include additional compounds useful to Sites. Also in preferred embodiments, the probe has a enable administration of a particular active compound in a detectable label, preferably a fluorescent label. A variety of patient with the Specified variances, which was not in other detectable labels are known to those skilled in the art. previous compositions, e.g., because the majority of patients Such a nucleic acid probe can also include one or more did not require or benefit from the added component. nucleic acid analogs. US 2001/0034023 A1 Oct. 25, 2001

0.083. In preferred embodiments, the probe is an antibody ments, the oligonucleotides are designed and Selected to or antibody fragment which specifically binds to a gene provide variance-specific amplification. product expressed from a form of one of the above genes, where the form of the gene has at least one specific variance 0087. In reference to nucleic acid sequences which “cor with a particular base at the variance Site, and preferably a respond' to a gene, the term “correspond” refers to a plurality of Such variances. nucleotide Sequence relationship, Such that the nucleotide 0084. In connection with nucleic acid probe hybridiza Sequence has a nucleotide Sequence which is the same as the tion, the term “specifically hybridizes” indicates that the reference gene or an indicated portion thereof, or has a probe hybridizes to a Sufficiently greater degree to the target nucleotide Sequence which is exactly complementary in Sequence than to a Sequence having a mismatched base at normal Watson-Crick base pairing, or is an RNA equivalent least one variance Site to allow distinguishing Such hybrid of Such a Sequence, e.g., an mRNA, or is a cDNA derived ization. The term “specifically hybridizes” thus means that from an mRNA of the gene. the probe hybridizes to the target Sequence, and not to non-target Sequences, at a level which allows ready identi 0088. In another aspect, the invention provides a kit fication of probe/target Sequence hybridization under Selec containing at least one probe or at least one primer (or other tive hybridization conditions. Thus, “selective hybridization amplification oligonucleotide) or both (e.g., as described conditions” refer to conditions which allow Such differential above) corresponding to a gene or genes listed in Tables 1, binding. Similarly, the terms “specifically binds” and “selec 3, and 4 or other gene related to a drug-induced disease or tive binding conditions” refer to such differential binding of condition, or other gene involved in absorption, distribution, any type of probe, e.g., antibody probes, and to the condi metabolism, excretion, or in toxicity-related modification of tions which allow such differential binding. Typically a drug. The kit is preferably adapted and configured to be hybridization reactions to determine the Status of variant Suitable for identification of the presence or absence of a Sites in patient Samples are carried out with two different particular variance or variances, which can include or con probes, one specific for each of the (usually two) possible sist of a nucleic acid Sequence corresponding to a portion of variant nucleotides. The complementary information a gene. A plurality of variances may comprise a haplotype of derived from the two separate hybridization reactions is haplotypes. The kit may also contain a plurality of either or useful in corroborating the results. both of Such probes and/or primers, e.g., 2, 3, 4, 5, 6, or more 0085. Likewise, the invention provides an isolated, puri of such probes and/or primers. Preferably the plurality of fied or enriched nucleic acid sequence of 15 to 500 nucle probes and/or primers are adapted to provide detection of a otides in length, preferably 15 to 100 nucleotides in length, plurality of different Sequence Variances in a gene or plu more preferably 15 to 50 nucleotides in length, and most rality of genes, e.g., in 2, 3, 4, 5, or more genes or to amplify preferably 15 to 30 nucleotides in length, which has a and/or Sequence a nucleic acid Sequence including at least Sequence which corresponds to a portion of one of the genes one variance Site in a gene or genes. Preferably one or more identified for aspects above. Preferably the lower limit for of the variance or variances to be detected are correlated the preceding ranges is 17, 20, 22, or 25 nucleotides in with variability in a treatment response or tolerance, and are length. In other embodiments, the nucleic acid Sequence is preferably indicative of an effective response to a treatment. 30 to 300 nucleotides in length, or 45 to 200 nucleotides in In preferred embodiments, the kit contains components (e.g., length, or 45 to 100 nucleotides in length. The nucleic acid probes and/or primers) adapted or useful for detection of a Sequence includes at least one variance site. Such Sequences plurality of variances (which may be in one or more genes) can, for example, be amplification products of a Sequence indicative of the effectiveness of at least one treatment, which spans or includes a variance Site in a gene identified preferably of a plurality of different treatments for a par herein. Likewise, Such a sequence can be a primer, or ticular disease or condition. It may also be desirable to amplification oligonucleotide which is able to bind to or provide a kit containing components adapted or useful to allow detection of a plurality of variances indicative of the extend through a variance Site in Such a gene. Yet another effectiveness of a treatment or treatment against a plurality example is a nucleic acid hybridization probe comprised of of diseases. The kit may also optionally contain other Such a Sequence. In Such probes, primers, and amplification components, preferably other components adapted for iden products, the nucleotide Sequence can contain a Sequence or tifying the presence of a particular variance or variances. Site corresponding to a variance Site or Sites, for example, a Such additional components can, for example, indepen variance site identified herein. Preferably the presence or dently include a buffer or buffers, e.g., amplification buffers absence of a particular variant form in the heterozygous or and hybridization buffers, which may be in liquid or dry homozygous State is indicative of the effectiveness of a form, a DNA polymerase, e.g., a polymerase Suitable for method of treatment in a patient. carrying out PCR (e.g., a thermostable DNA polymerase), 0.086 Likewise, the invention provides a set of primers or and deoxy nucleotide triphosphates (dNTPs). Preferably a amplification oligonucleutides (e.g., 2, 3, 4, 6, 8, 10 or even probe includes a detectable label, e.g., a fluorescent label, more) adapted for binding to or extending through at least enzyme label, light scattering label, or other label. Prefer one gene identified herein. In preferred embodiments the Set ably the kit includes a nucleic acid or polypeptide array on includes primerS or amplification oligonucleotides adapted a Solid phase Substrate. The array may, for example, include to bind to or extend through a plurality of Sequence Vari a plurality of different antibodies, and/or a plurality of ances in a gene(s) identified herein. The plurality of Vari different nucleic acid Sequences. Sites in the array can allow ances preferably provides a haplotype. Those skilled in the capture and/or detection of nucleic acid Sequences or gene art are familiar with the use of amplification oligonucle products corresponding to different variances in one or more otides (e.g., PCR primers) and the appropriate location, different genes. Preferably the array is arranged to provide testing and use of Such oligonucleotides. In certain embodi variance detection for a plurality of variances in one or more US 2001/0034023 A1 Oct. 25, 2001

genes which correlate with the effectiveness of one or more variance provides information about variable patient treatments of one or more diseases, which is preferably a response. In general, the method will involve identifying at variance as described herein. least one variance in at least one gene. An alternative approach is to identify a first patient or Set of patients 0089. The kit may also optionally contain instructions for Suffering from a disease or condition and having a particular use, which can include a listing of the variances correlating genotype, haplotype or combination of genotypes or haplo with a particular treatment or treatments for a disease or types, and a Second patient or Set of patients Suffering from diseases and/or a Statement or listing of the diseases for the same disease or condition that have a genotype or which a particular variance or variances correlates with a haplotype or Sets of genotypes or haplotypes that differ in a treatment efficacy and/or Safety. specific way from those of the first set of patients. Subse 0090 Preferably the kit components are selected to allow quently the extent and magnitude of clinical response can be detection of a variance described herein, and/or detection of compared between the first patient or Set of patients and the a variance indicative of a treatment, e.g., administration of Second patient or Set of patients. A correlation between the a drug, pointed out herein. presence or absence of a variance or variances or haplotypes and the response of the patient or patients to the treatment 0.091 Additional configurations for kits of this invention indicates that the variance provides information about vari will be apparent to those skilled in the art. able patient response and is useful for the present invention. 0092. The invention also includes the use of Such a kit to 0096. The method can utilize a variety of different infor determine the genotype(s) of one or more individuals with mative comparisons to identify correlations. For example a respect to one or more variance Sites in one or more genes plurality of pairwise comparisons of treatment response and identified herein. Such use can include providing a result or the presence or absence of at least one variance can be report indicating the presence and/or absence of one or more performed for a plurality of patients. Likewise, the method variant forms or a gene or genes which are indicative of the can involve comparing the response of at least one patient effectiveness of a treatment or treatments. homozygous for at least one variance with at least one 0093. In another aspect, the invention provides a method patient homozygous for the alternative form of that variance for determining a genotype of an individual in relation to one or variances. The method can also involve comparing the or more variances in one or more of the genes identified in response of at least one patient heterozygous for at least one above aspects by using mass spectrometric determination of variance with the response of at least one patient homozy a nucleic acid Sequence which is a portion of a gene gous for the at least one variance. Preferably the heterozy identified for other aspects of this invention or a comple gous patient response is compared to both alternative mentary Sequence. Such mass spectrometric methods are homozygous forms, or the response of heterozygous patients known to those skilled in the art. In preferred embodiments, is grouped with the response of one class of homozygous the method involves determining the presence or absence of patients and Said group is compared to the response of the a variance in a gene, determining the nucleotide Sequence of alternative homozygous group. the nucleic acid Sequence, the nucleotide Sequence is 100 nucleotides or less in length, preferably 50 or less, more 0097 Such methods can utilize either retrospective or preferably 30 or less, and still more preferably 20 nucle prospective information concerning treatment response vari otides or leSS. In general, Such a nucleotide Sequence ability. Thus, in a preferred embodiment, it is previously includes at least one variance Site, preferably a variance Site known that patient response to the method of treatment is which is informative with respect to the expected response variable. of a patient to a treatment as described for above aspects. 0098. Also in preferred embodiments, the disease or condition is as for other aspects of this invention; for 0094 AS indicated above, many therapeutic compounds example, the treatment involves administration of a com or combinations of compounds or pharmaceutical composi pound or pharmaceutical composition. tions show variable efficacy and/or Safety in various patients in whom the compound or compounds is administered. 0099. In preferred embodiments, the method involves a Thus, it is beneficial to identify variances in relevant genes, clinical trial, e.g., as described herein. Such a trial can be e.g., genes related to the action or toxicity of the compound arranged, for example, in any of the ways described herein, or compounds. Thus, in a further aspect, the invention e.g., in the Detailed Description. provides a method for determining whether a compound has 0100. The present invention also provides methods of a differential effect due to the presence or absence of at least treatment of a disease or condition, preferably a disease or one variance in a gene or a variant form of a gene, where the condition related to pharmacokinetic parameters, e.g. gene is a gene identified for aspects above. absorption, distribution, metabolism, or excretion, that affect 0.095 The method involves identifying a first patient or a drug or candidate therapeutic intervention regarding effi Set of patients Suffering from a disease or condition whose cacy and or Safety, i.e. drug-induced disease, disorder or response to a treatment differs from the response (to the dysfunction or other toxicity effects or clinical Symptoma same treatment) of a Second patient or set of patients tology. Such methods combine identification of the presence Suffering from the same disease or condition, and then or absence of particular variances, preferably in a gene or determining whether the occurrence or frequency of occur genes from Tables 1, 3, and 4, with the administration of a rence of at least one variance in at least one gene differs compound; identification of the presence of particular vari between the first patient or Set of patients and the Second ances with Selection of a method of treatment and admin patient or Set of patients. A correlation between the presence istration of the treatment; and identification of the presence or absence of the variance or variances and the response of or absence of particular variances with elimination of a the patient or patients to the treatment indicates that the method of treatment based on the variance information US 2001/0034023 A1 Oct. 25, 2001

indicating that the treatment is likely to be ineffective or body. The action of a drug also refers to its effects on the contra-indicated, and thus Selecting and administering an Signs or Symptoms of a disease or condition, or effects of the alternative treatment effective against the disease or condi drug that are unrelated to the disease or condition leading to tion. Thus, preferred embodiments of these methods incor unanticipated effects on other processes. Such unanticipated porate preferred embodiments of Such methods as described processes often lead to adverse events or toxic effects. The for Such Sub-aspects. terms “adverse event' or “toxic' event are known in the art 0101 AS used herein, a “gene” is a sequence of DNA and include, without limitation, those listed in the FDA present in a cell that directs the expression of a “biologically reference System for adverse events. active' molecule or “gene product', most commonly by 0104. In accordance with the aspects above and the transcription to produce RNA and translation to produce Detailed Description below, there is also described for this protein. The “gene product is most commonly a RNA invention an approach for developing drugs that are explic molecule or protein or a RNA or protein that is Subsequently itly indicated for, and/or for which approved use is restricted modified by reacting with, or combining with, other con to individuals in the population with Specific variances or Stituents of the cell. Such modifications may include, with combinations of variances, as determined by diagnostic tests out limitation, modification of proteins to form glycopro for variances or variant forms of certain genes involved in teins, lipoproteins, and phosphoproteins, or other the disease or condition or involved in the action or metabo modifications known in the art. RNA may be modified lism or transport of the drug. Such drugs may provide more without limitation by polyadenylation, Splicing, capping or effective treatment for a disease or condition in a population export from the nucleus or by covalent or noncovalent identified or characterized with the use of a diagnostic test interactions with proteins. The term “gene product” refers to for a Specific variance or variant form of the gene if the gene any product directly resulting from transcription of a gene. is involved in the action of the drug or in determining a In particular this includes partial, precursor, and mature characteristic of the disease or condition. Such drugs may be transcription products (i.e., pre-mRNA and mRNA), and developed using the diagnostic tests for Specific variances or translation products with or without further processing variant forms of a gene to determine the inclusion of patients including, without limitation, lipidation, phosphorylation, in a clinical trial. glycosylation, or combinations of Such processing 0105 Thus, the invention also provides a method for 0102) The term “gene involved in the origin or patho producing a pharmaceutical composition by identifying a genesis of a disease or condition” refers to a gene that compound which has differential activity or effectiveness harbors mutations or polymorphisms that contribute to the against a disease or condition in patients having at least one cause of disease, or variances that affect the progression of variance in a gene, preferably in a gene from Tables 1, 3 and the disease or expression of Specific characteristics of the 4, compounding the pharmaceutical composition by com disease. The term also applies to genes involved in the bining the compound with a pharmaceutically acceptable Synthesis, accumulation, or elimination of products that are carrier, excipient, or diluent Such that the composition is involved in the origin or pathogenesis of a disease or preferentially effective in patients who have at least one condition including, without limitation, proteins, lipids, car copy of the variance or variances. In Some cases, the patient bohydrates, hormones, or Small molecules. has two copies of the variance or variances. In preferred embodiments, the disease or condition, gene or genes, 0103) The term “gene involved in the action of a drug” variances, methods of administration, or method of deter refers to any gene whose gene product affects the efficacy or mining the presence or absence of variances is as described Safety of the drug or affects the disease proceSS being treated for other aspects of this invention. by the drug, and includes, without limitation, genes that encode gene products that are targets for drug action, gene 0106 Similarly, the invention provides a method for products that are involved in the metabolism, activation or producing a pharmaceutical agent by identifying a com degradation of the drug, gene products that are involved in pound which has differential activity against a disease or the bioavailability or elimination of the drug to the target, condition in patients having at least one copy of a form of gene products that affect biological pathways that, in turn, a gene, preferably a gene listed in Table 1, having at least affect the action of the drug Such as the Synthesis or one variance and Synthesizing the compound in an amount degradation of competitive Substrates or allosteric effectors Sufficient to provide a pharmaceutical effect in a patient or rate-limiting reaction, or, alternatively, gene products that Suffering from the disease or condition. The compound can affect the pathophysiology of the disease proceSS via path be identified by conventional Screening methods and its ways related or unrelated to those altered by the presence of activity confirmed. For example, compound libraries can be the drug compound. (Particular variances in the latter cat screened to identify compounds which differentially bind to egory of genes may be associated with patient groups in products of variant forms of a particular gene product, or whom disease etiology is more or leSS Susceptible to ame which differentially affect expression of variant forms of the lioration by the drug. For example, there are Several patho particular gene, or which differentially affect the activity of physiological mechanisms in hypertension, and depending a product expressed from Such gene. Alternatively, the on the dominant mechanism in a given patient, that patient design of a compound can exploit knowledge of the vari may be more or less likely than the average hypertensive ances provided herein to avoid significant allele Specific patient to respond to a drug that primarily targets one effects, in order to reduce the likelihood of Significant pathophysiological mechanism. The relative importance of pharmacogenetic effects during the clinical development different pathophysiological mechanisms in individual process. Preferred embodiments are as for the preceding patients is likely to be affected by variances in genes aspect. associated with the disease pathophysiology.) The “action” 0107. In another aspect, the invention provides a method of a drug refers to its effect on biological products within the of treating a disease or condition in a patient by Selecting a US 2001/0034023 A1 Oct. 25, 2001 patient whose cells have an allele of an identified gene, described (in addition to the exemplary methods of preferably a gene Selected from the genes listed in Table 1, examples 13, 14, and 15) which can be utilized. These and determining whether that alteration provides a differen additional methods include chemical cleavage of mis tial effect (with respect to reducing or alleviating a disease matches (see, e.g., Ellis T P, et al., Chemical cleavage of or condition, or with respect to variation in toxicity or mismatch: a new look at an established method. Human tolerance to a treatment) in patients with at least one copy of Mutation 11(5):345-53, 1998), denaturing gradient gel elec at least one allele of the gene as compared to patients with trophoresis (see, e.g., Van OrSouw NJ, et al., Design and at least one copy of one alternative allele. The presence of application of 2-D DGGE-based gene mutational Scanning such a differential effect indicates that altering the level or tests. Genet Anal. 14(5-6):205-13, 1999) and heteroduplex activity of the gene provides at least part of an effective analysis (see, e.g., Ganguly A, et al., Conformation-sensitive treatment for the disease or condition. gel electrophoresis for rapid detection of Single-base differ ences in double-stranded PCR products and DNA fragments: 0108 Preferably the allele contains a variance as shown evidence for solvent-induced bends in DNA heteroduplexes. in Tables 3 and 4 or other variance table herein, or in Table Proc Natl Acad Sci U S A. 90 (21):10325-9, 1993). Table 3 1 or 3 of Stanton & Adams, application Ser. No. 09/300,747, of Stanton & Adams, application Ser. No. 09/300,747, Supra, Supra. Also preferably, the altering involves administering to provides a description of the additional possible variances the patient a compound preferentially active on at least one that could be detected by one skilled in the art by testing an but leSS than all alleles of the gene. identified gene in Tables 1 and 2 using the variance detection 0109 Preferred embodiments include those as described methods described or other methods which are known or are above for other aspects of treating a disease or condition. developed. 0110 AS recognized by those skilled in the art, all the 0115 The present invention provides a method for treat methods of treating described herein include administration ing a patient at risk for drug responsiveness, i.e., efficacy of the treatment to a patient. differences associated with pharmacokinetic parameters, and Safety concerns, i.e. drug-induced disease, disorder, or 0111. In a further aspect, the invention provides a method dysfunction or diagnosed with organ failure or a disease for determining a method of treatment effective to treat a asSociated with drug-induced organ failure. The methods disease or condition by altering the level of activity of a include identifying Such a patient and determining the product of an allele of a gene Selected from the genes listed patient's genotype or haplotype for an identified gene or in Tables 1, 3 or 4, and determining whether that alteration genes. The patient identification can, for example, be based provides a differential effect related to reducing or alleviat on clinical evaluation using conventional clinical metrics ing a disease or condition as compared to at least one and/or evaluation of a genetic variance or variances in one alternative allele or an alteration in toxicity or tolerance of or more genes, preferably a gene or genes from Tables 1, 3 the treatment by a patient or patients. The presence of Such and 4. The invention provides a method for using the a differential effect indicates that altering that level of patient's genotype Status to determine a treatment protocol activity provides at least part of an effective treatment for the which includes a prediction of the efficacy and Safety of a disease or condition. therapy for concurrent treatment in light of drug-induced 0112 Preferably the method for determining a method of disease or an drug-induced or drug associated pathological treatment is carried out in a clinical trial, e.g., as described condition. In a related aspect, the invention features a above and/or in the Detailed Description below. treatment protocol that provides a prediction of patient outcome. Such predictions are based on a demonstrated 0113. In still another aspect, the invention provides a correlation between a particular type of treatment and out method for evaluating differential efficacy of or tolerance to come, efficacy, Safety, likelihood of development of drug a treatment in a Subset of patients who have a particular induced disease, disorder, or dysfunction, or other Such variance or variances in at least one gene, preferably a gene parameter relevant to clinical treatment decisions as evalu in Tables 1, 3, or 4, by utilizing a clinical trial. In preferred ated by a normal prudent physician. embodiments, the clinical trial is a Phase I, II, III, or IV trial. Preferred embodiments include the stratifications and/or 0116. In an another related aspect, the invention provides statistical analyses as described below in the Detailed a method for identifying a patient for participation in a Description. clinical trial of a therapy for the treatment of drug-induced disease, disorder, or dysfunction, or an associated drug 0114. In yet another aspect, the invention provides induced toxicity. The method involves determining the experimental methods for finding additional variances in a genotype or haplotype of a patient with (or at risk for) a gene provided in Tables 3 and 4. A number of experimental drug-induced disease, disorder, or dysfunction. Preferably methods can also beneficially be used to identify variances. the genotype is for a variance in a gene from Table 1. Thus, the invention provides methods for producing cDNA Patients with eligible genotypes are then assigned to a (Example 12) and detecting additional variances in the genes treatment or placebo group, preferably by a blinded ran provided in Tables 1 and 2 using the Single Strand confor domization procedure. In preferred embodiments, the mation polymorphism (SSCP) method (Example 13), the T4 Selected patients have no copies, one copy or two copies of Endonuclease VII method (Example 14) or DNA sequencing a specific allele of a gene or genes identified in Table 1. (Example 15) or other methods pointed out below. The Alternatively, patients Selected for the clinical trial may have application of these methods to the identified genes will Zero, one or two copies of an allele belonging to a set of provide identification of additional variances that can affect alleles, where the Set of alleles comprise a group of related inter-individual variation in drug or other treatment alleles. One procedure for rigorously defining a set of alleles response. One skilled in the art will recognize that many is by applying phylogenetic methods to the analysis of methods for experimental variance detection have been haplotypes. (See, for example: Templeton A. R., Crandall K. US 2001/0034023 A1 Oct. 25, 2001

A. and C. F. Sing A cladistic analysis of phenotypic asso 0119) By “disease management protocol” or “treatment ciations with haplotypes inferred from restriction endonu protocol' is meant a means for devising a therapeutic plan clease mapping and DNA sequence data. III. Cladogram for a patient using laboratory, clinical and genetic data, estimation. Genetics 1992 Oct;132(2):619-33.) Regardless including the patient's diagnosis and genotype. The protocol of the Specific tools used to group alleles, the trial would clarifies therapeutic options and provides information about then test the hypothesis that a Statistically significant differ probable prognoses with different treatments. The treatment ence in response to a treatment can be demonstrated between protocol may provide an estimate of the likelihood that a two groups of patients each defined by the presence of Zero, patient will respond positively or negatively to a therapeutic intervention. The treatment protocol may also provide guid one or two alleles (or allele groups) at a gene or genes. Said ance regarding optimal drug dose and administration and response may be a desired or an undesired response. In a likely timing of recovery or rehabilitation. A "disease man preferred embodiment, the treatment protocol involves a agement protocol’ or “treatment protocol’ may also be comparison of placebo VS. treatment response rates in two or formulated for asymptomatic and healthy Subjects in order more genotype-defined groups. For example a group with no to forecast future disease risks based on laboratory, clinical copies of an allele may be compared to a group with two and genetic variables. In this Setting the protocol Specifies copies, or a group with no copies may be compared to a optimal preventive or prophylactic interventions, including group consisting of those with one or two copies. In this use of compounds, changes in diet or behavior, or other manner different genetic models (dominant, co-dominant, measures. The treatment protocol may include the use of a recessive) for the transmission of a treatment response trait computer program. can be tested. Alternatively, Statistical methods that do not posit a Specific genetic model, Such as contingency tables, 0120 In preferred embodiments, the method further can be used to measure the effects of an allele on treatment involves determining the patient's allele Status and Selecting those patients having at least one wild type allele, preferably response. having two wild type alleles for an identified gene, as 0117. In another preferred embodiment, patients in a candidates likely to develop drug-induced pathological con clinical trial can be grouped (at the end of the trial) accord ditions or drug-associated pathological disease or condi ing to treatment response, and Statistical methods can be tions. In a preferred embodiment, the treatment protocol used to compare allele (or genotype or haplotype) frequen involves a comparison of the allele Status of a patient with cies in two groups. For example responders can be compared a control population and a responder population. This com to nonresponders, or patients Suffering adverse events can be parison allows for a Statistical calculation of a patient's compared to those not experiencing such effects. Alterna likelihood of responding to a therapy, e.g., a calculation of tively response data can be treated as a continuous variable the correlation between a particular allele Status and treat and the ability of genotype to predict response can be ment response. In the context of this aspect, the term measured. In a preferred embodiments patients who exhibit “wild-type allele” refers to an allele of a gene which extreme phenotypes are compared with all other patients or produces a product having a level of activity which is most with a group of patients who exhibit a divergent extreme common in the general population. Two different alleles may phenotype. For example if there is a continuous or Semi both be wild-type alleles for this purpose if both have continuous measure of treatment response (for example the essentially the same level of activity (e.g., specific activity Alzheimer's Disease Assessment Scale, the Mini-Mental and numbers of active molecules). State Examination or the Hamilton Depression Rating Scale) 0121. In preferred embodiments of above aspects involv then the 10% of patients with the most favorable responses ing prediction of drug efficacy, the prediction of drug could be compared to the 10% with the least favorable, or efficacy involves candidate therapeutic interventions that are the patients one Standard deviation above the mean Score known or have been identified to be affected by pharmaco could be compared to the remainder, or to those one Standard kinetic parameters, i.e. absorption, distribution, metabolism, deviation below the mean score. One useful way to select the or excretion. These parameters may be associated with threshold for defining a response is to examine the distri hepatic or extra-hepatic biological mechanisms. Preferably bution of responses in a placebo group. If the upper end of the candidate therapeutic intervention will be effective in the range of placebo responses is used as a lower threshold patients with the genotype of a least one allele, and prefer for an “outlier response then the outlier response group ably two alleles from Tables 1, 3 and 4, but have a risk of should be almost free of placebo responders. This is a useful drug ineffectiveness, i.e. nonresponsive to a drug or candi threshold because the inclusion of placebo responders in a true response group decreases the ability of Statistical date therapeutic intervention. methods to detect a genetic difference between responders 0122) In particular applications of the invention, all of the and nonresponders. above aspects involving a gene variance evaluation or treatment Selection or patient Selection or method of treat 0118. In a related aspect, the invention provides a method ment, the method includes a determination of the genotypic for developing a disease management protocol that entails allele Status of the patient, where a determination of the diagnosing a patient with a disease or a disease Susceptibil patient's allele Status as being heterozygous or homozygous, ity, determining the genotype of the patient at a gene or is predictive of the patient having a poor response to a genes correlated with treatment response and then Selecting candidate therapeutic intervention and development of drug an optimal treatment based on the disease and the genotype (or genotypes or haplotypes). The disease management induced disease, disorder, or dysfunction. protocol may be useful in an education program for physi 0123. In preferred embodiments, the above methods are cians, other caregivers or pharmacists, may constitute part of used for or include identification of a Safety or toxicity a drug label; or may be useful in a marketing campaign. concern involving a drug-induced disease, disorder, or dyS US 2001/0034023 A1 Oct. 25, 2001

function and/or the likelihood of occurrence and/or Severity physiologic dysfunction or Signs and Symptoms of failure or of Said disease, disorder, or dysfunction. dysfunction, or those associated with the pathophysiological 0.124. In preferred embodiments, the invention is suitable manifestations of a disorder. A Suitable therapy can be a for identifying a patient with non-drug-induced disease, pharmacological agent, drug, or therapy that alters a path disorder, or dysfunction but with dysfunction related to ways identified to affect the molecular Structure or function aberrant enzymatic metabolism or excretion of endogenous of the parent candidate therapeutic intervention thereby biologically relevant molecules or compounds. The method affecting drug-induced disease or disorder progression of preferably involves determination of the allele status or any of the described organ System dysfunctions. variance presence or absence determination for at least one 0129. By “drug-induced disease” or “drug-induced syn gene from Tables 1, 3, 4. drome' is meant any physiologic condition that may be 0.125. In another aspect, the invention provides a method correlated with medical therapy by a drug, agent, or candi for treating a patient at risk for a drug-induced disease, date therapeutic intervention. disorder or dysfunction by a) identifying a patient with Such 0.130 By “drug-induced dysfunction” is meant a physi a risk, b) determining the genotypic allele Status of the ologic disorder or Syndrome that may be correlated with patient, and c) converting the data obtained in step b) into a medical therapy by a drug, agent, or candidate therapeutic treatment protocol that includes a comparison of the geno intervention in which Symptomology is similar to drug typicallele Status determination with the allele frequency of induced disease. Specifically included are: a) hemostasis a control population. This comparison allows for a Statistical dysfunction; b) cutaneous disorders; c) cardiovascular dys calculation of the patient's risk for having drug-induced function; d) renal dysfunction; e) pulmonary dysfunction; f) disease, disorder, or dysfunction, e.g., based on correlation hepatic dysfunction, g) systemic reactions; and h) central of the allele frequencies for a population with response or nervous System dysfunction. disease occurrence and/or Severity. In preferred embodi ments, the method provides a treatment protocol that pre 0131 By “drug associated disorder” is meant a physi dicts a patient being heterozygous or homozygous for an ologic dysfunction that may be correlated with medical identified allele to exhibit signs and or Symptoms of drug therapy by a drug, agent, or candidate therapeutic interven induced disease, disorder, or dysfunction and a patient who tion. The drug associated disorder may include disease, is wild-type homozygous for the Said allele, as responding disorder, or dysfunction. favorably to these therapies. 0.132. By “pathway' or “gene pathway' is meant the 0126. In a related aspect, the invention provides a method group of biologically relevant genes involved in a pharma for treating a patient at risk for or diagnosed with drug codynamic or pharmacokinetic mechanism of drug, agent, or induced disease or pathological condition or dysfunction candidate therapeutic intervention. These mechanisms may using the methods of the above aspect and conducting a step further include any physiologic effect the drug or candidate c) which involves determining the gene allele load Status of therapeutic intervention renders. the patient. This method further involves converting the data obtained in steps b) and c) into a treatment protocol that 0.133 AS used herein, a “clinical trial' is the testing of a includes a comparison of the allele Status determinations of therapeutic intervention in a Volunteer human population for these Steps with the allele frequency of a control population. the purpose of determining whether a therapeutic interven This affords a statistical calculation of the patient's risk for tion is Safe and/or efficacious in the human Volunteer or having drug-induced disease, disorder or dysfunction. In a patient population for a given disease, disorder, or condition. preferred embodiment, the method is useful for identifying The analysis of Safety and efficacy in genetically defined drug-induced disease, disorder or dysfunction. In addition, Subgroups differing by at least one variance is of particular in related embodiments, the methods provide a treatment interest. protocol that predicts a patient to be at high risk for 0.134. As used herein “clinical study' is that part of a drug-induced disease, disorder or dysfunction responding by clinical trial that involves determination of the effect a exhibiting Signs and Symptoms of drug-induced toxicity, candidate therapeutic intervention on human Subjects. It disorders, dysfunction if the patient is determined as having includes clinical evaluations of physiologic responses a genotype or allelic difference in the identified gene or including pharmacokinetic (absorption, distribution, bio genes. Such patients are preferably given alternative thera availability, and excretion) as well as pharmacodynamic ple.S. (physiologic response and efficacy) parameters. A pharma 0127. The invention also provides a method for improv cogenetic clinical Study is a clinical Study that involves ing the Safety of candidate therapies for the identification of testing of one or more specific hypotheses regarding the a drug-induced disease, disorder, or dysfunction. The effect of a genetic variance or variances (or set of variances, method includes the Step of comparing the relative Safety of i.e. haplotype or haplotypes) in enrolled Subjects or patients the candidate therapeutic intervention in patients having on response to a therapeutic intervention. These hypotheses different alleles in one or more than one of the genes listed are articulated before the study in the form of primary or in Tables 1, 3, and 4. Preferably, administration of the drug Secondary endpoints. For example the endpoint may be that is preferentially provided to those patients with an allele in a particular genetic Subgroup the rate of objectively type associated with increased efficacy. In a preferred defined responses exceeds Some predefined threshold. embodiment, the alleles of identified gene or genes used are 0.135 AS used herein, “supplemental applications” are wild-type and those associated with altered biological activ those in which a candidate therapeutic intervention is tested ity. in a human clinical trial in order for the product to have an 0128. As used herein, by “therapy associated with drug expanded label to include additional indications for thera induced disease' is meant any therapy resulting in patho peutic use. In these cases, the previous clinical Studies of the US 2001/0034023 A1 Oct. 25, 2001 therapeutic intervention, i.e. those involving the preclinical 0140. By “drug efficacy” is meant the determination of an Safety and Phase I human Safety Studies can be used to appropriate drug, drug dosage, administration Schedule, and Support the testing of the particular candidate therapeutic prediction of therapeutic utility. intervention in a patient population for a different disease, 0.141. By “allele load” is meant the relative ratio of disorder, or condition than that previously approved in the identified gene alleles in the patient's chromosomal DNA. U.S. In these cases, a limited Phase II study is performed in the proposed patient population. With adequate Signs of 0142. By “identified allele” is meant a particular gene efficacy, a Phase III Study is designed. All other parameters isoform that can be distinguished from other identified gene of clinical development for this category of candidate thera isoforms using the methods of the invention. peutic interventions proceeds as described above for inter 0143. By “PCR, PT-PCR, or ligase chain reaction ampli ventions first tested in human candidates. fication' is meant Subjecting a DNA sample to a Polymerase Chain Reaction Step or ligase-mediated chain reaction Step, 0.136 AS used herein, “outcomes” or “therapeutic out or RNA to a RT-PCR step, such that, in the presence of comes” are used to describe the results and value of health appropriately designed primers, a nucleic acid fragment is care intervention. Outcomes can be multi-dimensional, e.g., synthesized or fails to be synthesized and thereby reveals the including one or more of the following: improvement of allele Status of a patient. The nucleic acid may be further Symptoms, regression of the disease, disorder, or condition; analyzed by DNA sequencing using techniques known in the economic outcomes of healthcare decisions. art. 0.137 AS used herein, “pharmacoeconomics” is the 0144. By “gene allele status” is meant a determination of analysis of a therapeutic intervention in a population of the relative ratio of wild type identified alleles compared to patients diagnosed with a disease, disorder, or condition that an allelic variant that may encode a gene product of reduced includes at least one of the following Studies: cost of illness catalytic activity. This may be accomplished by nucleic acid study (COI); cost benefit analysis (CBA), cost minimization sequencing, RT-PCR, PCR, examination of the identified analysis (CMA), or cost utility analysis (CUA), or an gene translated protein, a determination of the identified analysis comparing the relative costs of a therapeutic inter protein activity, or by other methods available to those vention with one or a group of other therapeutic interven skilled in the art. tions. In each of these Studies, the cost of the treatment of a 0145 By “treatment protocol' is meant a therapy plan for disease, disorder, or condition is compared among treatment a patient using genetic and diagnostic data, including the groups. AS used herein, costs are those economic variables patient's diagnosis and genotype. The protocol enhances asSociated with a disease, disorder, or condition fall into two therapeutic options and clarifies prognoses. The treatment broad categories: direct and indirect. Direct costs are asso protocol may include an indication of whether or not the ciated with the medical and non-medical resources used as patient is likely to respond positively to a candidate thera therapeutic interventions, including medical, Surgical, diag peutic intervention that is known to affect physiologic nostic, pharmacologic, devices, rehabilitation, home care, function. The treatment protocol may also include an indi nursing home care, institutional care, and prosthesis. Indi cation of appropriate drug dose, recovery time, age of rect costs are associated with loSS of productivity due to the disease onset, rehabilitation time, Symptomology of attacks, disease, disorder, or condition Suffered by the patient or and risk for future disease. A treatment protocol, including relatives. A third category, the tangible and intangible losses any of the above aspects, may also be formulated for due to pain and Suffering of a patient or relatives often is asymptomatic and healthy Subjects in order to forecast included in indirect cost Studies. future disease risks an determine what preventive therapies 0.138. As used herein, “health-related quality of life” is a should be considered or invoked in order to lessen these measure of the impact of the disease, disorder, or condition disease risks. The treatment protocol may include the use of on an individual’s or group of patient's activities of daily a computer Software program to analyze patient data. living. Preferably, included in pharmacoeconomic Studies is 0146 By “patient at risk for a disease” or “patient with an analysis of the health-related quality of life. Standardized high risk for a disease' is meant a patient identified or Surveys or questionnaires for general health-related quality diagnosed as having drug-induced disease, disorder, dyS of life or disease, disorder, or condition specific determine function or having a genetic predisposition or risk for the impact the disease, disorder, or condition has on an acquiring drug-induced disease, disorder or dysfunction, individuals day to day life activities or Specific activities that where the predisposition or risk is higher than average for are affected by a particular disease, disorder, or condition. the general population or is Sufficiently higher than for other 0.139. As used herein, the term “stratification” refers to individuals as to be clinically relevant. Such risk can be the creation of a distinction between patients on the basis of evaluated, for example, using the methods of the invention a characteristic or characteristics of the patient. Generally, in and techniques available to those skilled in the art. the context of clinical trials, the distinction is used to 0147 By “converting” is meant compiling genotype distinguish responses or effects in different Sets of patients determinations to predict either prognosis, drug efficacy, or distinguished according to the Stratification parameters. For Suitability of the patient for participating in clinical trials of the present invention, Stratification preferably includes dis a candidate therapeutic intervention with known propensity tinction of patient groups based on the presence or absence of drug-induced disease, disorder or dysfunction. For of particular variance or variances in one or more genes. The example, the genotype may be compiled with other patient Stratification may be performed only in the course of analy parameterS Such as age, Sex, disease diagnosis, and known sis or may be used in creation of distinct groups or in other allelic frequency of a representative control population. The ways. converting Step may provide a determination of the Statis US 2001/0034023 A1 Oct. 25, 2001 tical probability of the patient having a particular disease ably in the pharmaceutical and biotechnology industries), by risk, drug response, or patient outcome. enrolling Subjects (e.g., normal and/or patient Subjects) in a clinical drug trial or study unit (preferably a Phase I drug 0.148. By “prediction of patient outcome” is meant a trial or study unit) for the purpose of genotyping the Subjects forecast of the patient’s likely health status. This may in order to assess the contribution of genetic variation to include a prediction of the patient's response to therapy, variation in drug response, genotyping the Subjects to deter rehabilitation time, recovery time, cure rate, rate of disease mine the Status of one or more variances in the Subjects, progression, predisposition for future disease, or risk of administering a compound to the Subjects and measuring a having relapse. Surrogate drug response variable, comparing responses 0149. By “therapy for the treatment of a disease” is meant between two or more genotype-defined groups of Subjects to any pharmacological agent or drug with the property of determine whether there is a genetic component to the healing, curing, or ameliorating any Symptom or disease interperSon variability in response to Said compound; and mechanism associated with drug-induced disease, disorder reporting the results of the Phase I drug trial to a contracting or dysfunction. entity. Clearly, intermediate results, e.g., response data and/ 0150. By “responder population” is meant a patient or or Statistical analysis of response or variation in response patients that respond favorably to a given therapy. can also be reported. 0156. In preferred embodiments, at least some of the 0151. In another aspect, the invention provides a method Subjects have disclosed that they are related to each other for determining whether there is a genetic component to and the genetic analysis includes comparison of groups of interSubject variation in a Surrogate treatment response. The related individuals. To encourage participation of Sufficient method involves administering the treatment to a group of numbers of related individuals, it can be advantageous to related (preferably normal) Subjects and a group of unrelated offer or provide compensation to one or more of the related (preferably normal) Subjects, measuring a Surrogate phar individuals based on the number of subjects related to them macodynamic or pharmacokinetic drug response variable in who participate in the clinical trial, or on whether at least a the Subjects, performing a Statistical test measuring the minimum number of related Subjects participate, e.g., at variation in response in the group of related Subjects and, Separately in the group of unrelated Subjects, comparing the least 3, 5, 10, 20, or more. magnitude or pattern of variation in response or both O157. In a related aspect, the invention provides a method between the groups to determine if the responses of the for recruiting a clinical trial population for Studies of the groups are different, using a predetermined Statistical mea influence of genetic variation on drug response, by Soliciting Sure of difference. A difference in response between the Subjects to participate in the clinical trial, obtaining consent groups is indicative that there is a genetic component to of each of a set of Subjects for participation in the clinical interSubject variation in the Surrogate treatment response. trial, obtaining additional related Subjects for participation in the clinical trial by compensating one or more of the related 0152. In preferred embodiments, the size of the related Subjects for participation of their related Subjects at a level and unrelated groupS is Set in order to achieve a predeter based on the number of related Subjects participating or mined degree of Statistical power. based on participation of at least a minimum specified 0153. In another aspect, the invention provides a method number of related Subjects, e.g., at minimum levels as for evaluating the combined contribution of two or more Specified in the preceding aspect. variances to a Surrogate drug response phenotype in Subjects 0158. In addition to application of the present invention (preferably normal Subjects) by a genotyping a set of to drug-induced diseases and conditions, the present inven unrelated Subjects participating in a clinical trial or Study, tion also provides for the use of variances in genes and gene e.g., a Phase I trial, of a compound. The genotyping is for pathways involved in drug absorption, distribution, metabo two or more variances (which can be a haplotype), thereby lism, or excretion (e.g., as specified in any of Tables 1, 3, and identifying Subjects with Specific genotypes, where the two 4 herein) of a drug. Thus, the above aspects can be utilized or more specific genotypes define two or more genotype in connection with virtually any type of drug. For example, defined groups. A drug is administered to Subjects with two the pharmacogenetic effect, and the determination of Such or more of Said Specific genotypes, and a Surrogate phar effect, of variances in genes in pathways involved in drug macodynamic or pharmacokinetic drug response variable is absorption, distribution, metabolism, or excretion can be measured in the Subjects. A Statistical test or tests is per utilized, for example, for in connection with drugs and drug formed to measure response in the groups separately, where classes as described Stanton, International Application No. the Statistical tests provide a measurement of variation in PCT/USOO/O1392, filed Jan. 20, 2000, entitled GENE response with each group. The magnitude or pattern of SEQUENCE VARIATIONS WITH UTILITY IN DETER variation in response or both is compared between the MINING THE TREATMENT OF DISEASE. Further, the groups to determine if the groups are different using a particular drug and/or pharmacogenetic determination can predetermined Statistical measure of difference. also be applied in the context of any disease, disorder, or 0154) In preferred embodiments, the specific genotypes dysfunction for which a drug treatment is considered or are homozygous genotypes for two variances. In preferred tested, e.g., any of the diseases, disorders, or conditions embodiments, the comparison is between groups of Subjects pointed out in Stanton (Id.). Still further, such analysis and differing in three or more variances, e.g., 3, 4, 5, 6, or even use of pharmacogenetic information for genes involved in more variances. drug adsorption, distribution, metabolism, and excretion can also be combined with any of the different aspects described O155 In another aspect, the invention provides a method for genes involved in treatment response for other diseases, for providing contract research Services to clients (prefer conditions, and dysfunctions as described in Stanton (Id.). US 2001/0034023 A1 Oct. 25, 2001 20

0159. The use of variance information for genes involved operazine hydrochloride and others, all of which share a in drug adsorption, distribution, metabolism, and excretion phenothiazine backbone. Members of the piperidine class for any drug is advantageous, as those processes can affect include, for example, compounds Such as meperidine, diphe the efficacy of any drug. Therefore, variances in Such genes noxylate and loperamide, as well as phenylpiperidines Such that alter one or more of those parameters can be significant as fentanyl, Sufentanil and alfentanil, all of which share the in determining interpatient variation in treatment response. piperidine backbone. Chemical classes and their members Additional aspects and embodiments as described in Stan are recognized by those skilled in the art of medicinal ton, International Application No. PCT/US00/01392, filed chemistry. Jan. 20, 2000, entitled GENE SEQUENCE VARIATIONS WITH UTILITY IN DETERMINING THE TREATMENT 0164. As used herein the term “surrogate marker” refers OF DISEASE, are also included in the scope of this inven to a biological or clinical parameter that is measured in place tion. of the biologically definitive or clinically most meaningful 0160 By “pathway' or “gene pathway' is meant the parameter. In comparison to definitive markers, Surrogate group of biologically relevant genes involved in a pharma markers are generally either more convenient, less expen codynamic or pharmacokinetic mechanism of drug, agent, or Sive, provide earlier information or provide pharmacological candidate therapeutic intervention. These mechanisms may or physiological information not directly obtainable with further include any physiologic effect the drug or candidate definitive markers. Examples of Surrogate biological param therapeutic intervention renders. Included in this are “bio eters: (i) testing erythrocyte membrane acetylcholinesterase chemical pathways' which is used in its usual Sense to refer levels in Subjects treated with an acetylcholinesterase inhibi to a series of related biochemical processes (and the corre tor intended for use in Alzheimer's disease patients (where sponding genes and gene products) involved in carrying out inhibition of brain acetylcholinesterase would be the defini a reaction or Series of reactions. Generally in a cell, a tive biological parameter); (ii) measuring levels of CD4 pathway performs a Significant process in the cell. positive lymphocytes as a Surrogate marker for response to 0.161. By “pharmacological activity” used herein is a treatment for acquired immune deficiency Syndrome meant a biochemical or physiological effect of drugs, com (AIDS). Examples of Surrogate clinical parameters: (i) per pounds, agents, or candidate therapeutic interventions upon forming a psychometric test on normal Subjects treated for administration and the mechanism of action of that effect. a short period of time with a candidate Alzheimer's com pound in order to determine if there is a measurable effect on 0162 The pharmacological activity is then determined by cognitive function. The definitive clinical test would entail interactions of drugs, compounds, agents, or candidate measuring cognitive function in a clinical trial in Alzhe therapeutic interventions, or their mechanism of action, on imer's disease patients. (ii) Measuring blood pressure as a their target proteins or macromolecular components. By Surrogate marker for myocardial infarction. The measure “agonist' or "mimetic' or “activators' is meant a drug, ment of a Surrogate marker or parameter may be an endpoint agent, or compound that activate physiologic components in a clinical Study or clinical trial, hence "Surrogate end and mimic the effects of endogenous regulatory compounds. point”. By “antagonists”, “blockers” or “inhibitors” is meant drugs, agents, or compounds that bind to physiologic components 0.165. As used herein the term “related” when used with and do not mimic endogenous regulatory compounds, or respect to human Subjects indicates that the Subjects are interfere with the action of endogenous regulatory com known to share a common line of descent; that is, the pounds at physiologic components. These inhibitory com Subjects have a known ancestor in common. Examples of pounds do not have intrinsic regulatory activity, but prevent preferred related Subjects include SibS (brothers and sisters), the action of agonists. By "partial agonist' or “partial parents, grandparents, children, grandchildren, aunts, antagonist' is meant an agonist or antagonist, respectively, uncles, cousins, Second cousins and third cousins. Subjects with limited or partial activity. By “negative agonist' or less closely related than third cousins are not Sufficiently “inverse antagonists' is meant that a drug, compound, or related to be useful as “related” Subjects for the methods of agent that can interact with a physiologic target protein or this invention, even if they share a known ancestor, unless macromolecular component and Stabilizes the protein or Some related individuals that lie between the distantly component Such that agonist-dependent conformational related Subjects are also included. Thus, for a group of changes of the component do not occur and agonist medi related individuals, each Subject shares a known ancestor ated mechanism of physiological action is prevented. By within three generations or less with at least one other "modulators' or “factors' is meant a drug, agent, or com Subject in the group, and preferably with all other Subjects pound that interacts with a target protein or macromolecular in the group or has at least that degree of consanguinity due component and modifies the physiological effect of an to multiple known common ancestors. More preferably, agonist. Subjects Share a common ancestor within two generations or less, or otherwise have equivalent level of consanguinity. 0163 AS used herein the term “chemical class” refers to Conversely, as used herein the term “unrelated”, when used a group of compounds that share a common chemical in respect to human Subjects, refers to Subjects who do not scaffold but which differ in respect to the Substituent groups share a known ancestor within 3 generations or less, or linked to the Scaffold. Examples of chemical classes of drugs include, for example, phenothiazines, piperidines, benzodi otherwise have known relatedness at that degree. aZepines and aminoglycosides. Members of the phenothiaz 0166 As used herein the term “pedigree” refers to a ine class include, for example, compounds Such as chlor group of related individuals, usually comprising at least two promazine hydrochloride, mesoridazine beSylate, generations, Such as parents and their children, but often thioridazine hydrochloride, acetophenazine maleate triflu comprising three generations (that is, including grandparents US 2001/0034023 A1 Oct. 25, 2001 or grandchildren as well). The relation between all the variances or haplotypes, from one or more genes, to varia Subjects in the pedigree is known and can be represented in tion in a Surrogate marker in humans. The Surrogate marker a genealogical chart. is generally Selected to provide information on a biological 0167 As used herein the term “hybridization”, when or clinical response, as defined above. used with respect to DNA fragments or polynucleotides 0170 AS used herein, “comparing the magnitude or pat encompasses methods including both natural polynucle tern of variation in response' between two or more groups otides, non-natural polynucleotides or a combination of refers to the use of a Statistical procedure or procedures to both. Natural polynucleotides are those that are polymers of measure the difference between two different distributions. the four natural deoxynucleotides (deoxyadenosine triphos For example, consider two genotype-defined groups, AA and phate dA), deoxycytosine triphosphate dC, deoxyguanine aa, each homozygous for a different variance or haplotype in triphosphate dG) or deoxythymidine triphosphate dT, a gene believed likely to affect response to a drug. The usually designated simply thymidine triphosphate T) or Subjects in each group are Subjected to treatment with the polymers of the four natural ribonucleotides (adenosine drug and a treatment response is measured in each Subject triphosphate A), cytosine triphosphate C), guanine triph (for example a Surrogate treatment response). One can then osphate G) or uridine triphosphate UD). Non-natural poly construct two distributions: the distribution of responses in nucleotides are made up in part or entirely of nucleotides the AA group and the distribution of responses in the aa that are not natural nucleotides; that is, they have one or group. These distributions may be compared in many ways, more modifications. Also included among non-natural poly and the Significance of any difference qualified as to its nucleotides are molecules related to nucleic acids, Such as Significance (often expressed as a p value), using methods peptide nucleic acid (PNA). Non-natural polynucleotides known to those skilled in the art. For example, one can may be polymers of non-natural nucleotides, polymers of compare the means, medians or modes of the two distribu natural and non-natural nucleotides (in which there is at least tions, or one can compare the variance or Standard devia one non-natural nucleotide), or otherwise modified poly tions of the two distributions. Or, if the form of the distri nucleotides. Non-natural polynucleotides may be useful butions is not known, one can use nonparametric Statistical because their hybridization properties differ from those of tests to test whether the distributions are different, and natural polynucleotides. AS used herein the term “comple whether the difference is significant at a specified level (for mentary', when used in respect to DNA fragments, refers to example, the p<0.05 level, meaning that, by chance, the the base pairing rules established by Watson and Crick: A distributions would differ to the degree measured less than pairs with T or U; G pairs with C. Complementary DNA one in 20 similar experiments). The types of comparisons fragments have sequences that, when aligned in antiparallel described are similar to the analysis of heritability in quan orientation, conform to the Watson-Crick base pairing rules at all positions or at all positions except one. AS used herein, titative genetics, and would draw on Standard methods from complementary DNA fragments may be natural polynucle quantitative genetics to measure heritability by comparing otides, non-natural polynucleotides, or a mixture of natural data from related Subjects. and non-natural polynucleotides. 0171 Another type of comparison that can be usefully made is between related and unrelated groups of Subjects. 0168 AS used herein “amplify” when used with respect That is, the comparison of two or more distributions is of to DNA refers to a family of methods for increasing the particular interest when one distribution is drawn from a number of copies of a Starting DNA fragment. Amplification population of related Subjects and the other distribution is of DNA is often performed to simplify Subsequent determi drawn from a group of unrelated Subjects, both Subjected to nation of DNA sequence, including genotyping or haplotyp the same treatment. (The related Subjects may consist of ing. Amplification methods include the polymerase chain Small groups of related Subjects, each compared only to their reaction (PCR), the ligase chain reaction (LCR) and meth relatives.) A comparison of the distribution of a drug ods using Q beta replicase, as well as transcription-based response variable (e.g. a Surrogate marker) between two amplification Systems. Such as the isothermal amplification Such groups may provide information on whether the drug procedure known as Self-sustained Sequence replication (3 response variable is under genetic control. For example, a SR, developed by T. R. Gingeras and colleagues), Strand narrow distribution in the group(S) of related Subjects (com displacement amplification (SDA, developed by G. T. pared to the unrelated Subjects) would tend to indicate that Walker and colleagues) and the rolling circle amplification the measured variable is under genetic control (i.e. the method (developed by P. Lizardi and D. Ward). related Subjects, on account of their genetic homogeneity, 0169. As used herein “contract research services for a are more similar than the unrelated individuals). The degree client” refers to a busineSS arrangement wherein a client to which the distribution was narrower in the related indi entity pays for Services consisting in part or in whole of viduals (compared to the unrelated individuals) would be work performed using the methods described herein. The proportionate to the degree of genetic control. The narrow client entity may include a commercial or non-profit orga neSS of the distribution could be quantified by, for example, nization whose primary business is in the pharmaceutical, computing variance or Standard deviation. In other cases the biotechnology, diagnostics, medical device or contract shape of the distribution may not be known and nonpara research organization (CRO) sector, or any combination of metric tests may be preferable. Nonparametric tests include those Sectors. Services provided to Such a client may include methods for comparing medians Such as the sign test, the any of the methods described herein, particularly including Slippage test, or the rank correlation coefficient (the non clinical trial Services, and especially the Services described parametric equivalent of the ordinary correlation coeffi in the Detailed Description relating to a Pharmacogenetic cient). Pearson's Chi Square test for comparing an observed Phase I Unit. Such services are intended to allow the earliest Set of frequencies with an expected Set of frequencies can possible assessment of the contribution of a variance or also be useful. US 2001/0034023 A1 Oct. 25, 2001 22

0172 The present invention provides a number of advan groups of genes. These pathways contain genes implicated tages. For example, the methods described herein allow for in the etiology or treatment response of the various patient use of a determination of a patient's genotype for the timely outcomes detailed in Table 2. Column 3, headed “Function', administration of the most Suitable therapy for that particular further categorizes the pathways listed in column 2. patient. The methods of this invention provide a basis for Successfully developing and obtaining regulatory approval 0177 Column 4, headed “Name”, lists the genes belong for a compound even though efficacy or Safety of the ing to the class, pathway and function shown to the left (in compound in an unstratified population is not adequate to columns 1-3). The gene names given are generally those justify approval. From the point of View of a pharmaceutical used in the OMIM database or in GenBank, however one or biotechnology company, the information obtained in skilled in the art will recognize that many genes have more pharmacogenetic Studies of the type described herein could than one name, and that it is a Straightforward task to be the basis of a marketing campaign for a drug. For identify Synonymous names. For example, many alternate example, a marketing campaign that emphasized the Supe gene names are provided in the OMIM record for a gene. rior efficacy or Safety of a compound in a genotype or 0178. In column 5, headed “OMIM”, the Online Mende haplotype restricted patient population, compared to a simi lian Inheritance in Man (OMIM) record number is listed for lar or competing compound used in an undifferentiated each gene in column 4. This record number can be entered population of all patients with the disease. In this respect a next to the words: “Enter one or more search keywords:” at marketing campaign could promote the use of a compound the OMIM world wide web site. The url is: http:// in a genetically defined Subpopulation, even though the www3.ncbi.nlm.nih.gov/Omim/searchomim.html. An compound was not intrinsically Superior to competing com OMIM record exists for most characterized human genes. pounds when used in the undifferentiated population with The record often has useful information on the chromosome the target disease. In fact even a compound with an inferior location, function, alleles, and human diseases or disorders profile of action in the undifferentiated disease population asSociated with each gene. could become Superior when coupled with the appropriate 0179 Column 6, headed “GID", provides the GenBank pharmacogenetic test. identification number (hence GID) of a genomic, cDNA, or 0173 By “comprising” is meant including, but not lim partial Sequence of the gene named in column 4. Usually the ited to, whatever follows the word “comprising”. Thus, use GID provides the record of a cDNA sequence. Many genes of the term “comprising indicates that the listed elements have multiple Genbank accession numbers, representing are required or mandatory, but that other elements are different versions of a sequence 5 obtained by different optional and may or may not be present. By “consisting of research groups, or corrected or updated versions of a is meant including, and limited to, whatever follows the Sequence. AS with the gene name, one skilled in the art will phrase “consisting of. Thus, the phrase “consisting of recognize that alternative GenBank records related to the indicates that the listed elements are required or mandatory, named record can be obtained easily. All other GenBank and that no other elements may be present. By “consisting records listing Sequences that are alternate versions of the essentially of is meant including any elements listed after Sequences named in the table are equally Suitable for the the phrase, and limited to other elements that do not interfere inventions described in this application. (One Straightfor with or contribute to the activity or action specified in the ward way to obtain additional GenBank records for a gene disclosure for the listed elements. Thus, the phrase “con is on the internet. General instructions can be found at the Sisting essentially of indicates that the listed elements are NCBI web site at: http://www3.ncbi.nlm.nih.gov. More spe required or mandatory, but that other elements are optional cifically, the GenBank record number in column 6 can be and may or may not be present depending upon whether or entered at the url: http://www3.ncbi.nlm.nih.gov/Entrez/ not they affect the activity or action of the listed elements. nucleotide.html. Once the GenBank record has been retrieved one can click on the “nucleotide neighbors' link 0.174. Other features and advantages of the invention will and additional GenBank records from the same gene will be be apparent from the following description of the preferred listed. embodiments thereof, and from the claims. 0180 Column 7, headed “locus”, provides the chromo DETAILED DESCRIPTION OF THE Some location of the gene listed on the same row. The PREFERRED EMBODIMENTS chromosome location helps confirm the identity of the named gene if there is any ambiguity. 0175 First, the content of tables provided in this descrip 0181 Table 2 is a matrix showing the intersection of tion is briefly described. genes and patient outcomes-that is, which categories of 0176) Table 1, the ADME/Toxicology Gene Table, lists genes are most likely to account for interpatient variation in genes that may be involved in pharmacological responses response to treatments. Column 1 is Similar to the Class involving adsorption, distribution, metabolism, excretion column in Table 1, while column 2 is a combination of the affecting efficacy or Safety of drug response. The table has Pathway and Function columns in Table 1. It is intended seven columns. Column 1, headed “Class” provides broad that the Summary terms listed in columns 1 and 2 be read as groupings of genes relevant to the pharmacology of absorp referring to all the genes in the corresponding Sections of tion, distribution, metabolism, or excretion of drugs. The Table 1. The remaining columns in Table 2 lists potential categories are: adsorption and distribution, Phase I drug effects on efficacy or on eight patient outcomes. The infor metabolism, Phase II drug metabolism, excretion, oxidative mation in the Table lies in the shaded boxes at the intersec StreSS, and immune response. Column 2, headed "Pathway', tion of various 'Pathways” (the rows) and the patient out provides a more detailed categorization of the different comes (the nine columns) An intersection box is shaded classes of genes by indicating the overall purpose of large when a row corresponding to a particular pathway (and by US 2001/0034023 A1 Oct. 25, 2001 extension all the genes listed in that pathway in Table 1) acid residue in the encoded amino acid Sequence which is intersects a column for a specific effect on patient outcome changed); in a sequence 5' to the coding region (5); or in a in response to a candidate therapeutic intervention Such that Sequence 3' to the coding region (3). AS indicated above, the pathway and genes are of possible use in explaining interpreting the location of the variance in the gene depends interpatient differences in response (patient outcomes) to on the correct assignment of the initial ATG of the encoded candidate therapeutic interventions. Thus the Table enables protein (the translation start Site). It should be recognized one skilled in the art to identify therapeutically relevant that assignment of the correct ATG may occasionally be genes in patients with one of the nine patient outcomes for incorrect in GenBank, but that one skilled in the art will the purposes of Stratification of these patients based upon know how to carry out experiments to definitively identify genotype and Subsequent correlation of genotype with drug the correct translation initiation codon (which is not always response. The shaded interSections indicate preferred Sets of an ATG). In the event of any potential question concerning genes for understanding the basis of interpatient variation in the proper identification of a gene or part of a gene, due for response to therapy of the indicated disease indication, and example, to an error in recording an identifier or the absence in that respect are exemplary. Any of the genes in the table of one or more of the identifiers, the priority for use to may account for interpatient variation in response to treat resolve the ambiguity is GenBank accession number, OMIM ments for any of the diseases listed. Thus, the shaded boxes identification number, HUGO identifier, common name indicate the gene pathways that one skilled in the art would identifier. first investigate in trying to understand interpatient variation 0183 If a haplotype for any of the genes listed in this in response to a candidate therapeutic indications with the table has been identified, a series of nucleotides (A, C, G, T) listed patient outcomes. are listed Separated by commas and to the left of each listing 0182 Table 3 lists DNA sequence variances in genes is the associated nucleotide location also separated by com relevant to the methods described in the present invention. mas in brackets. For example, if the haplotype listing is These variances were identified by the inventors in studies TG,C,A12, 245, 385, 612 there is a T at position 12, a G of Selected genes listed in Table 1, and are provided here as at position 246, a C at position 385, and an Aat position 612. useful for the methods of the present invention. The vari Below this list will occur the identified variance start, ances in Table 3 were discovered by one or more of the variance, and CDS context for the identified single nucle methods described below in the Detailed Description or otide polymorphisms as described above. Examples. Table 3 has eight columns. Column 1, the "Name” column, contains the Human Genome Organization 0.184 Table 4 lists additional DNA sequence variances (HUGO) identifier for the gene. Column 2, the “GID” (in addition to those in Table 3) in genes relevant to the column provides the GenBank accession number of a methods of the present invention (i.e. Selected genes from genomic, cDNA, or partial Sequence of a particular gene. Table 1). These variances were identified by various Column 3, the “OMIM ID" column contains the record research groupS and published in the Scientific literature over number corresponding to the Online Mendelian Inheritance the past 20 years. The inventors realized that these variances in Man database for the gene provided in columns 1 and 2. may be useful for understanding interpatient variation in This record number can be entered at the world wide web response to treatment of the diseases listed in Table 2, and site http://www3.ncbi.nlm.nih.gov/Omim/searchomim.html more generally useful for the methods of the present inven to search the OMIM record on the gene. Column 4, the tion. The columns of Table 4 are similar to those of Table 3, VGX Symbol column, provides an internal identifier for the and therefore the descriptions of the rows and columns in gene. Column 5, the “Description' column provides a Table 3 (above) pertain to Table 4, as do the other remarks. descriptive name for the gene, when available. Column 6, 0185 I. Pharmacokinetic Parameters and Effects on Effi the “Variance Start” column provides the nucleotide loca cacy tion of a variance with respect to the first listed nucleotide in the GenBank accession number provided in column 2. 0186 The pharmacokinetic parameters with potential That is, the first nucleotide of the GenBank accession is effects on efficacy are absorption, distribution, metabolism, counted as nucleotide 1 and the variant nucleotide is num and excretion. These parameters affect efficacy broadly by bered accordingly. Column 7, the “variance' column pro modulating the availability of a compound at the site(s) of vides the nucleotide location of a variance with respect to an action. Interpatient variation in the availability of a com ATG codon believed to be the authentic ATG start codon of pound drug, agent, or candidate therapeutic intervention can the gene, where the A of ATG is numbered as one (1) and the result in a reduction of the available compound or more immediately preceding nucleotide is numbered as minus one compound at the Site of action with a corresponding altered (-1). This reading frame is important because it allows the clinical effect. Differences in these parameters, therefore, potential consequence of the variant nucleotide to be inter can be a potential foundation of interpatient variability to preted in the context of the gene anatomy (5' untranslated drug response. region, protein coding sequence, 3' untranslated region). 0187 A. Pharmacokinetic Parameters that Result in a Column 7 also provides the identity of the two variant Reduction of Available Drug nucleotides at the indicated position. For example, in the first entry in Table 3, DG90040, the variance is 191G>A, 0188 1. Absorption-Depending on the solubility indicating the presence of a G or an A at nucleotide 232 of of the drug, and its ability to passively croSS mem GenBank sequence DG90040. Column 8, the “CDS Con branes is fundamental to the ability of the drug, text' column indicates whether the variance is in a coding agent, or candidate therapeutic intervention to effec region but silent (S); in a coding region and results in an tively enter the circulation and gain access to the amino acid change (e.g., R347C, where the letters are one principle Site of action. For enteral delivery or letter amino acid abbreviations and the number is the amino administration, absorption is a critical first Step in the US 2001/0034023 A1 Oct. 25, 2001 24

pharmacologic process. Within the gastrointestinal cannicula or the renal tubule, there is a net result of tract, absorption of a drug, agent, or candidate thera enhanced drug availability. peutic intervention can be affected by the pH of the contents, Speed of gastric emptying, and presence of 0.197 II. Impaired Drug Tolerability and Drug-Induced chelating or binding molecules to the drug, agent or Disease, Disorder, Dysfunction or Toxicity candidate therapeutic intervention. Each of these 0198 In response to chemical Substances, drugs, or xeno parameters can effectively reduce the rate of passive biotics, drug-induced disease, disorder, dysfunction, or tox absorption of the drug acroSS the gastrointestinal icity manifests as cellular damage or organ physiologic mucosal membrane. dysfunction, with one potentially leading to the other. 0189 2. Distribution-Once absorbed, the drug, agent or 0199 Adverse drug reactions can be categorized as 1) candidate therapeutic intervention must be delivered or mechanism based reactions which are exaggerations of distributed to the primary Site of pharmacologic action. pharmacologic effects and 2) idiosyncratic, unpredictable Although distribution is dependent on regional blood flow effects unrelated to the primary pharmacologic action. and cardiac output; distribution may be further affected by Although Some side effects appear Shortly after administra the rate and extent of Sequestration of the drug into biologi tion of a drug, Some side effects appear long after drug cal Spaces that render the product unavailable to the prin administration or after cessation of the drug. Furthermore, ciple or primary Site of pharmacologic Site of action. For these reactions can be categorized by reversible or irrevers example, many drugs are actively transported into biological ible manifestations of the drug-induced toxicity referring to compartments. These processes, if over- or under active may whether the clinical Symptomology Subsides or persists affect the availability and hence reduce the efficacy of the upon withdrawal of the offending agent. product. Further, only unbound drug may be effective to a cell, tissue, or physiological process, and bound product 0200. In the first category, excessive drug effects may may be transported to a Space that is physiologically unre result from alterations of pharmacokinetic parameters by lated to the pharmacologic mechanism of action or may be either drug-drug interactions, pathophysiologic disease of deleterious adverse or toxic consequence. mediated alterations in the organs or processes involved in absorption, distribution, metabolism, or excretion, or genetic 0.190 3. Metabolism-Induction of metabolic enzymes predisposition to heightened pharmacodynamic effect of the to covalently modify the parent drug, agent or candidate drug. The excessive or heightened response may be receptor therapeutic intervention may reduce the ability of the parent or drug target or non-receptor or non-drug target mediated. drug to elicit a pharmacologic action. Metabolism may affect the target active site binding, rate and extent of distribution 0201 There are a large number of adverse events that are and excretion, and overall availability of the active mol Suspected and or known to occur as a result of administration ecule. of a drug, agent, or candidate therapeutic intervention. For example, many antineoplastic agents act by prevention of 0191 4. Excretion-If the excretion of the drug or cell division in dividing cells or promoting cytotoxicity via drug metabolite is rapid, less drug is available to disruption of DNA Synthesis, transcription, and formation of elicit a pharmacologic effect. mitotic Spindles. These agents, unfortunately, do not distin guish between normal and cancerous cells, e.g. normally 0192 B. Pharmacokinetic Parameters that Result in More dividing cells and cancer cells are equally killed. Therefore, Available Drug adverse events of antineoplastic agents include bone marrow 0193 1. Absorption-Enhanced absorption of Suppression leading to anemia, leukopenia, and thrombocy drugs, agents or candidate therapeutic interventions topenia; immunosuppression rendering the patient Suscep may result in increased drug availability. For tible and Vulnerable to infectious agents, and initiation of example, in Some cases of decreased gastric empty mutagenesis and the formation of alternate forms of cancer, ing, there is an enhanced degree of absorption by in many cases, acute myeloid leukemia. prolonging contact with gastrointestinal mucosal 0202) In another example of predictable adverse events membranes. In others, a change in the Solubility of related to drug therapy is immunosuppression as a result of the drug may enhance the passive transport acroSS therapy to reduce or ablate immune response. This therapy the gastrointestinal mucosal membrane. includes but is not exclusive to prevention of graft VS. host 0194 2. Distribution-Since free drug is the form or autoimmune disease. These agents, e.g. corticosteroids, that renderS pharmacologic action and is metabo cyclosporine, and azathioprine, all Suppress humoral or lized and excreted, drug binding may serve to protect cell-mediated immunity. Patients taking these agents are the drug from mechanisms of inactivation. The rate rendered Susceptible to microbial infections, particular and extent of drug binding affects the free drug opportunistic infections Such as cytomegalovirus, pneu concentration relative to the total concentration. mocystis carnii, Candida, and Sperigillus. Furthermore, long-term immunosuppressive therapy is associated with 0.195 3. Metabolism-If drug metabolism induction increased risk of developing lymphoma. Individual drugs is occurring and the inducer is rapidly removed are associated with renal injury (cyclosporine) and intersti without adjustment in the dose of the drug, drug tial pneumonitis (azathioprine). metabolism may be decreased and adverse effects or toxicities may occur. 0203. In the second category of adverse events, idiosyn cratic reactions arise often by unpredictable, unknown 0.196 4. Excretion-If inhibition of active transport mechanisms or reactions that evoke immunologic reactions of the parent drug or metabolite acroSS the bile or unanticipated cytotoxicity. US 2001/0034023 A1 Oct. 25, 2001

0204 Adverse reactions in this category are often found central nervous System which includes tinnitus and dizzi together, because often it is difficult to ascertain the etiology neSS, acute dystonic reactions, parkinsonian Syndrome, of the offending reaction. These toxic events can be specific coma, convulsions, depression and psychosis, and respira for a target organ, e.g. Ototoxicity, nephrotoxicity, hepato tory depression. toxicity, neurotoxicity, etc. or are caused by reactive meta 0209. In the cases whereby severe, fatal reactions occur bolic intermediates and are toxic or create local damage after drug administration, there may be a warning label in usually near the Site of metabolism. the product insert. 0205 Immunologic reactions to drugs are thought or 0210 For example, tricyclic antidepressants can cause result from the combination of the drug or agent with a central nervous System depression, Seizures, respiratory protein to form an antigenic protein-drug complex that arrest, cardiac arrythmias and arrest. The mechanism for the Stimulates the immune System response. Without the forma injury is a result of the increased Synaptic concentrations of tion of a complex, most Small molecular drugs are unable, biogenic amines and inhibition of postsynaptic receptors. alone, to elicit an immunological response. First exposure to the offending drug produces a latent reaction, Subsequent 0211 Acetominophen can cause hepatic necrosis as a exposures usually results in heightened and rapid immuno result of prolonged high dose usage or overdose. In the logical response. These allergic reactions, characterized by hepatocyte, acetominophen is converted to a toxic metabo immunohyperSensitivity, are most dramatic in anaphylaxis. lite that binds to glutathione. AS the concentration of There are other immune responses that result in adverse acetominophen increases the levels of glutathione are reactions or toxicities. They include but are not limited to: 1) depleted and the toxic acetominophen metabolite then binds immune response mediated cytotoxicity which occurs when liver macromolecules. Aggregation of polymorphonuclear the drug-protein complex binds to the Surface of a cell and neutrophils in hepatic microcirculation may cause ischemia this cell-complex is then recognized by circulating antibod and foster necrotic events. ies; 2) serum sickness which occurs when immune com 0212 Halothane can cause hepatic necrosis as well as plexes of drug and antibody are found in the circulation; and prodrome fever and jaundice. Interestingly, the liver effects 3) lupus Syndromes in which the drug or reactive interme of halothane are usually after a first time exposure. The diate interact with nuclear material to Stimulate the forma hepatic reaction is thought to occur via a genetic predispo tion of antinuclear antibodies. Sition to deranged metabolism with the formation of toxic 0206. In addition to the immune phenomena described metabolites. above, there are other drug reactions that are Syndromes 0213 III. Pharmacokinetic Parameters as Potential involving allergic reactions. These reactions include, but are Mechanisms of Drug-Induced Adverse Reactions Leading to not limited to, Skin e rashes, drug induced fever, pulmonary Disease, Disorder, Dysfunction or Toxicities reactions, hepatocellular or cholestatic reactions, interstitial nephritis, and lymphadenopathy. Further, there are Some 0214) A. Absorption drug reactions that mimic allergic reactions but are not 0215. Absorption is the pharmacokinetic parameter that immune related. For example, Such reactions are due to describes the rate and extent of the drug, agent, or candidate direct release of mediators by drugs and are called anaphy therapeutic intervention leaves the Site of administration. lactoid reactions. An example of this type of adverse event Although absorption is critical for the drug, agent, or can is reaction to radiocontrast dye. didate therapeutic intervention to ultimately reach the Site of physiologic action, the term bioavailability is the parameter 0207. These are common adverse drug reactions that may that is clinically relevant. Bioavailability is the term used to prevent a candidate therapeutic intervention from use, con define the extent to which the active component of the drug, tinued development, and marketing rights. Some of these agent, or candidate therapeutic intervention reaches the its reactions are reversible, others are not. Site of physiologic action or a biological fluid to which has 0208 Adverse drug reactions include, but are not limited access to the Site of biological action. Although bioavail to, the following organs Systems: a) hemostasis which ability is related to all pharmacokinetic parameters, e.g. encompass blood dyscrasias (feature of over half of all absorption, distribution, metabolism, and excretion, bio drug-related deaths) which are bone marrow aplasia, granu availability is primarily dependent on the first ability of the locytopenia, aplastic anemia, leukopenia, pancytopenia, drug, agent, or candidate therapeutic intervention to be lymphoid hyperplasia, hemolytic anemia, and thrombocy absorbed from the site of delivery, i.e. cross cellular mem topenia; b) cutaneous which encompass urticaria, macules, branes. papules, angioedema, morbilliform-maculopapular rash, 0216) There are many factors that influence absorption of toxic epidermal necrolysis, erythema multiforme, erythema a drug, agent, or candidate therapeutic intervention. For nodosum, contact dermititis, Vesicles, petechiae, exfoliative example, compound Solubility, conditions of absorption, and dermititis, fixed drug eruptions, and Severe skin rash (Stevens-Johnson Syndrome); c) cardiovascular which route of administration. In the present invention, we concern includes arrythmias, QT prolongation, cardiomyopathy, ourselves with genes that are involved in the active or hypotension, or hypertension; d) renal which includes glom passive process of drug, agent, or candidate therapeutic erulonephritis and tubular necrosis; e) pulmonary which intervention absorption through a biological membrane. includes asthma, acute pneumonitis, eosinophilic pneumoni 0217. The absorption surface is dependent on the route of tis, fibrotic and pleural reactions, and interstitial fibrosis, f) administration. For example, absorption of drugs can occur hepatic which includes Steatosis, hepatocellular damage and via 1) oral (enteral); 2) Sublingual; 3) injections (parenteral, cholestasis, g) systemic which includes anaphylaxis, Vasi i.e., intravenous, intramuscular, intraarterial, intrathecal, culitis, fever, lupus erythematosus Syndrome; and h) the intraperiotoneal, or Subcutaneous); 4) rectal; 5) inhalation US 2001/0034023 A1 Oct. 25, 2001 26

(pulmonary); 6) topical application (skin and eye). In each enzymes there are specific groups that will act as Substrates, of these routes of administration, the adsorption rate and for others there are general classes of chemical compounds extent is dependent on the concentration of the drug at the that will be Suitable Substrates for a given enzyme or Site, the patency of the epithelial cells, local biological enzymes. conditions, and function of the active or passive transport. 0225. In the mammalian liver these mechanisms to 0218 Absorption can affect both the efficacy and safety detoxify and/or enhance the excretion of metabolic by of a drug, agent, or candidate therapeutic intervention. For products, endogenous Substrates, and exogenous molecules. example, for a compound to achieve full pharmacologic The ability to determine whether hepatic function if inad potential, it must be available at the target Site, be active, and equate is based upon clinical observation, e.g., the presence be unbound. In regards to Safety, absorption affects Safety in of jaundice, right upper quadrant abdominal discomfort or one or more of the following: Site of delivery pain, necrosis, pain, pruritis, or by clinical laboratory analyses, e.g., aspar or irritation; rate of administration; and erratic available tate transaminase (AST or SGOT) or alanine transferase concentrations. (ALT or SGPT). The hepatic metabolic and excretory mechanisms are critical for Short- and long-term Survival 0219 B. Distribution and are inheritable characteristics. These hepatic biotrans 0220. The distribution of the drug, agent, or candidate formations mechanisms have broad Substrate specificity that therapeutic intervention is dependent on the rate and extent have been evolutionarily inherited for the host protection the compound enters the bloodstream. Once in the blood from environmental, biological, and chemical Substances. Stream, the compound may be distributed to the interstitial and cellular fluids. The distribution of drugs to target tissues 0226. There are two categories of drug, agent, or candi can be categorized into two phases. The first distribution date therapeutic intervention biotransformation (metabo phase, is dependent on cardiac output and regional blood lism). In the first, phase I, functionalization reactions occur. flow, both of which are dependent on the health and status Phase I reactions introduce or expose a functional group to of the cardiovascular System. In a Second distribution phase, the parent compound. In general, phase I reactions render diffusion into tissues is dependent on the level and extent the parent compound pharmacologically inactive, however that the drug, agent, or candidate therapeutic intervention is there are examples of phase I reaction activation or retention bound. Drug binding by proteins found in the blood can of activity. In phase II reactions, biosynthetic reactions Serve to protect the compound from modifications by occur. Phase II conjugation reactions leads to a covalent enzymes, proteins, or compounds in the circulation and or linkage between a functional group on the parent compound limit the bioavailability of the compound to enter target with glucuronic acid, Sulfate, glutathione, amino acids, or tissueS or individual cells. acetate. The metabolic conversion of drugs is the liver, however, all tissues have enzymatic activity. 0221) Drug entry into tissues requires free drug, and drug binding proteins may limit this active or passive transport. 0227 Factors affecting drug biotransformation are 1) Once distributed into tissues, the drug may be Sequestered induction of metabolizing enzymes, 2) inhibition of enzy within that tissue, to render full pharmacologic activity or to matic reactions, and 3) genetic polymorphisms. It is the prevent that drug from reaching the appropriate target tissue. interplay of these factors and the health and well being of the patient or Subject that determines the fate of parent drug 0222 Distribution can affect both the efficacy and safety molecules in the body. of a drug, agent, or candidate therapeutic intervention. For example, for a compound to achieve full pharmacologic 0228. The first factor affecting drug biotransformation is potential, it must be available at the target Site, be active, and induction of metabolizing enzyme activity. The metabolic be unbound. In regards to Safety, distribution affects Safety processes that modify drugs or chemicals (oxidation, reduc in one or more of the following: distribution to a tissue that tion, or conjugation) can be induced to significant enzymatic is more or leSS affected by the pharmacologic action of the activity. Under physiological conditions, the induction pro compound, erratic available concentrations, and tissue Spe ceSS is in place to coordinately metabolize exceSS Substrates. cific distribution characteristics. The induction process can be both at the level of enzymatic activity and increased protein levels of the pertinent enzyme 0223) C. Metabolism or enzymes. Induction may include one or Several of the 0224 Drugs or xenobiotics, are usually found in the enzymatic pathways or processes in response to the presence circulation bound to plasma proteins, generally but not of drugs, Xenobiotics, endogenous SubStrates, or metabolic exclusive to serum albumin. It is the bound form of the drug by-products. There may or may not be increased toxicity as that is taken up by the hepatocyte. Bile Salts in the circula a result of increased concentrations of metabolites. Further, tion are taken up via organic anion transporters. Once inside induction of phase I reactive processes (oxidation or reduc the hepatocyte, the drug or bile Salt is a Substrate for a Series tion reactions) may or may not induce the phase II reactive of reactions that are either oxidative or reductive or reactions processes (conjugation reactions). that are conjugative Steps in the metabolism of the Substrate. 0229. The second factor affecting drug biotransformation Generally these chemical modifications are a refined proceSS is the inhibition of metabolic enzymes. Enzymatic inhibition to render the Substrate more hydrophilic, or polar, to be more can occur via 1) competition of two or more Substrates for likely excreted in the bile (via the intestinal tract) or urine the enzymatic active site, 2) Suicide inhibitors, or 3) deple (via the kidneys). However, there are exceptions whereby tion of required cofactors for the enzymatic pathways or the redox reactions produce reactive intermediates or prod processes in phase I or phase II reactions. ucts that retard elimination. Except for their role in detoxi fication, there is little in common among the enzymes 0230. In competitive inhibition, two or more drugs, xeno involved in the redox detoxification reactions. For certain biotics, or Substrates present can interact with the active site US 2001/0034023 A1 Oct. 25, 2001 27 of the enzyme. If one drug binds Specifically to the enzy chemotherapy of tuberculosis. Marked interindividual varia matic active site or to an other intracellular regulatory tion in the elimination of this drug was observed and genetic protein molecule, other compounds are blocked from bind Studies of families revealed that this variation was geneti ing and remain unbound. In this case, unmetabolized parent cally controlled. Isoniazid is predominantly metabolized via drug or Xenobiotic remains in the circulation, potentially for N-acetylation. In the analysis of the phenotypically distinct extended periods of time. Competitive inhibition is depen individuals, it was shown that slow acetylators were dent on the relative specificity of the substrates for the homozygous for a recessive gene and fast acetylators were enzymatic active site and the concentration of the drugs or homozygous or heterozygous for the wild type gene. It has Substrates. An example of competitive drug biotransforma been determined that the incidence of the slow acetylator tion inhibition are cimetidine and ketoconazole which phenotype is approximately 50% for U.S. Caucasians and inhibit oxidative drug metabolism by forming a tight com blacks, 60-70% of Northern Europeans, and 5-10% in plex with the heme iron complex of cytochrome P450, and Asians. Other drugs have been shown to be polymorphically macrollide antibiotics Such as erythromycin and troleando acetylated, e.g. Sulfonamides (Sulfadiazine, Sulfamethazine, mycin are metabolized to products bind to heme groups on Sulfapyridine, Sulfameridine, and Sulfadoxine), aminoglute the cytochrome P450 molecules. thimide, amonafide, amrinone, dapsone, dipyrone, endrala 0231. In the second case, the inhibition of enzymes Zine, hydralazine, prizidilol, and procainamide. Other drugs involved in the drug biotransformation process may also that first undergo metabolism and then polymorphically occur by Suicide inactivation. In these cases, the drug or acetylated are clonazepam and caffeine. Xenobiotic may interact and covalently modify or render 0235 Another common genetic polymorphism associ inactive the enzyme involved in the metabolic pathway. In ated with oxidative metabolism is exemplified by the drug this way, the parent drug compound or molecule is not debrisoquine (a sympatholytic antihypertensive). It was dis metabolized, nor is it free to interact with another molecule. covered that variable inter-patient hypotensive response was Examples of Suicide inactivators are Secobarbital and Syn due to differing metabolic rates of debrisoquine 4-hydroxy thetic steroids (norethindrone or ethinyl estradiol) which lase. Further analysis of family Studies revealed that oxida bind to cytochrome P450 and destroy the heme portion of tive metabolic reactions are under monogeneic control. A the enzyme unit. cytochrome P450 enzyme, CYP2D6, was determined to be 0232. In the third case, inhibition of the enzymes the target gene for debrisoquine 4-hydroxylase activity. Poor involved in the drug biotransformation pathway can also metabolizers of desbrisoquine are homozygous for a reces occur by agents or compounds or physiological Status that sive CYP2D6 allele and rapid or fast metabolizers are deplete NADPH or other cofactors required for the enzy homozygous or heterozygous for the wild type CYP2D6 matic reactions to occur. In the cases of phase I oxidation or allele. Urinary metabolic ratio can be determined after reduction, lack of oxygen or NADPH, may reduce the administration of a probe drug and phenotypic assignments efficiency and activity of a particular enzyme. In phase II (poor or extensive metabolizer) can be identified. The extent reactions, cofactors provide specific groups for the enzy of debrisoquine metabolic analysis achieved clinical impor tance as it was determined that other drugs were poorly matic covalent modification of the drug or Xenobiotic. These metabolized in individuals that poorly metabolized debriso phase II cofactors are required for conjugation biotransfor quine. For example, anti-arryhthmicS Such as flecainide, mation reactions to occur and depletion of these cofactors propafenone, and mexiletine, antidepressants Such as amit would be rate limiting. ryptiline, clomipramine, desipramine, fluoetine, imipramine, 0233. The third factor that can affect drug biotransfor maprotiline, mianserin, paroxetine, and nortriptyline; neu mation is genetic polymorphism. Differences among indi roleptics Such as haloperidol, perphenazine, and thior viduals to metabolize drugs have long been known. idazine; antianginals. Such as perhexilene, opioids Such as Observed phenotypic differences, as determined by amount dextromethorphan and codeine; and amphetamines Such as of drug excreted, through polymorphically controlled path methylenedioxymethamphetamine. Further, many B-adren way/S has lead to a generalized classification of Slow (poor) ergic antagonists are metabolized and are Subject to poly metabolizers and fast (rapid or extensive) metabolizers. In morphic influence in elimination patterns. general, poor metabolizers are those with impaired metabo lism of a drug via a polymorphic pathway have been 0236 Another example of a genetic polymorphism asSociated with an increased incidence of adverse effects. In affecting oxidative metabolism was described for mepheny addition, to date all major deficiencies in drug metabolizing toin, a drug prescribed for epilepsy. It was shown that a activity are inherited as autosomal recessive traits. Fast or deficiency in the 4'-hydroxylation of S-mephenytoin is rapid metabolizers are those individuals with processes that inherited as an autosomal recessive trait. The other main extensively metabolize a drug via a polymorphic pathway. metabolic pathway, N-methylation of R-mephenytoin to The fast or rapid metabolizers have been associated with an 5-phenyl-5-ethylhydantoin remains unaffected. Individuals increased incidence of ineffective treatment. In these indi with poor metabolic rate of mephenytoin are Subject to viduals active drug is rapidly metabolized to less active or adverse central effects, i.e. Sedation. Other drugs can be inactive metabolites Such that a reassessment of the phar grouped into the poor mephenytoin metabolizers are macokinetic parameters and dosing regimen may require mephobarbital, hexobarbital, Side-chain oxidization of pro analysis or readjustment, respectively, for appropriate panolol, the demethylation of imipramine, and the metabo therapy to occur. lism of diazepam and desmethyldiazepam. Further analysis of other drugs. Such as the metabolism of antidepressant 0234. The first observed and catalogued genetic polymor drugs (citalopram), the proton pump inhibitor omeprazol, phism associated with drug metabolism was described for the antimalarial drugs pantoprazole and lanSoprazole coSeg isoniazid. Isoniazid is a primary drug prescribed for the regate with mephenytoin metabolites. US 2001/0034023 A1 Oct. 25, 2001 28

0237 Because the majority of metabolic enzymes for the cysteinyl residue. Catalytic cycles of monoamine oxidases. A conduct of drug biotransformation occurs in the liver, or B occur in discrete Steps that take an amine and convert impairment of liver function as a result of hepatic patho it to an aldehyde, while in the process creating hydrogen logical conditions or other disease States can lead to alter peroxide and ammonia. These oxidases have a broad Speci ations of hepatic or other organ metabolic drug biotransfor ficity; they protect mitochondrial proteins from Xenobiotic mation. Liver disease pathologies Such as hepatitis, amines and hydrazines. Further neurotransmitters are alcoholic liver disease, fatty liver disease, biliary cirrhosis, metabolized through this route, e.g. Serotonin, dopamine, and hepatocarcinomas can impair function of normal physi and catecholamines. Primary alkylamines containing unsub ological metabolic pathways. Further, decreases in hepatic Stituted methylene group or groups adjacent to the nitrogen circulation as a result of cardiac insufficiency, hypertension, exhibits activity. Activity increases as the length of a side vascular obstruction, or vascular insult can affect the rate chain, with optimal side length being C6. These enzymes and extent of drug biotransformation. For example drugs also catalyze the oxidation of Secondary and tertiary amines with a high hepatocyte extraction ratio would have different and acyclic amines. Hydrazines can be oxidized by these metabolism rates affected by alterations of hepatic circula oxidases. Substrates for monoamine oxidases include but are tion. Changes in liver blood flow can affect the rate and not eXclusive to the following amines: benzylamine, dopam extent of the metabolism and the clearance of the parent ine, tyramine, epinephrine, N-methylbenzylamine, and N,N- drug. In all cases of hepatic pathological conditions, the dimethlybenzylamine; and the following hydrazines: pro affect on drug biotransformation and clearance of parent carbazine 1,2-dimethylhydrazine. drugs or metabolized products will be dependent on the 0244 Mono-oxygenases are present in liver cell homo Severity and extent of the liver organ and hepatocellular genates and contain two distinct types of Xenobiotic mono damage. oxygenases. They are the cytochrome P450 and the flavin 0238 Although hepatic damage may affect the metabo dependent mono-oxygenases. lism and clearance of a parent drug or metabolic by-product, 0245. The liver microsomal P-450 system consists of a residual concentrations of parent drug or metabolic by flavoprotein, and a family of related, but distinct, hemopro products may be deleterious to the liver and its metabolic teins. The flavoprotein catalyzes the transfer of the electrons functions. Following nonparenteral (enteral) administration from NADPH to the hemoprotein, and is the mono-oxyge of a drug, a significant portion of the drug will be metabo nase. The reaction also requires phosphatidylcholine. The lized by intestinal or hepatic enzymes before it reaches the reductase is a monomeric flavoprotein that contains both general circulation. This first pass effect may generate active FAD and FMN. The reductase is specific for NADPH as a drug (administered drug was a prodrug), inactive drug, or reductant, but other oxidants can be substituted. In addition toxic drug. Prior to circulation of the metabolized product, to cytochrome P-450, the flavoprotein catalyzes reduction of circulation to the kidney, the major organ for excretion of the quinones, nitro, and azo compounds. hydrophilic moiety, and excretion via the urine will occur. Therefore, a metabolic product of hepatic metabolic path 0246 There are many P450 gene families. Subsequent ways can affect the liver, kidney, and other organs of the cloning and Sequence determination has afforded the ability body prior to excretion. to divide this gene family into three main groups, CYP1, CYP2, and CYP3, that are responsible for the majority of 0239) 1. Phase I Drug Biotransformation: Oxidation and drug biotransformation. There are further subdivisions in Reduction Reactions each of these families, examples being CYP2D6, CYP3A4, 0240 Enzymatic Oxidation of Drugs CYP2E1, as well as others. 0241. In oxidative metabolism, oxidases catalyze the 0247 Examples of enzymatic inductive processes that transfer of electrons from Substrate to oxygen, generating affect biotransformation reactions involve the P450 gene either hydrogen peroxide or Superoxide anions. There are family. Specifically, glucocorticoids and anticonvulsants two oxidases present in hepatocytes, they are aldehyde induce CYP3A4, isoniazid, acetone, and chronic ethanol oxidases and monoamine oxidases. Both of these enzymes consumption for CYP2E1. Many inducers of the cyto have broad substrate specificity and contribute broadly to the chrome P450 enzymes also induce conjugation metabolic metabolism of drugs. A third oxidase, Xanthine oxidase, may enzymes, e.g. glucuronosyltransferases. contribute to the oxidation of drugs, due its ability to 0248. In contrast to the monooxygenases, multiple forms catalyze the oxidation of heterocyclic aromatic amines, for of the terminal oxidase (P-450) are present in the hepatocyte. example methotrexate and 6-mercaptopurine. Xanthine oxi There are many distinct isoforms characterized in different dase in intact tissues is present as a NAD-dependent dehy Species including humans. It should be noted that mitochon drogenase, and is converted to an oxidase when there is drial P-450 exhibit little or no activity in the metabolism of disruption of the tissue, for example during hepatic cellular drugs, Xenobiotics, biological compounds, or chemicals. damage. Representative functional groups oxidated by the microSo 0242 Aldehyde oxidase catalyzes the oxidation of fatty mal P-450 System are as follows: alkanes (hexane, decane, hexadecane); alkenes (vinyl chloride, aflatoxin-B1, dield aldehydes to carboxylic acids and the hydroxylation of rin); aromatic hydrocarbons (naphthylene, bromobenzene, Substituted pyridines, pyrimidines, purines, and pteridines. benzo(a)pyrene, biphenyl), alipathic amines (aminopyrine, Generally, Xenobiotic aromatic nitrogen heterocycles are benzphetamine, ethylmorphine); heterocyclic amines metabolized by this enzyme. (3-acetylpyridine, 4,4'-bipyridine, quinoline); amides 0243 Monoamine oxidase is present in two forms, A and (N-acetlyaminofluorene, urethane); ethers (indemethacin, B. They are dimeric proteins consisting of identical Subunits pheancetin, p-nitroanisole); and Sulfides (chloropromazine, and FAD is covalently linked to the protein through a thioanisole). US 2001/0034023 A1 Oct. 25, 2001 29

0249. There are many P450s that have been identified in nases. This class of enzymes is involved in the catalysis of human liver. Substrate specificities vary among these P-450 hydrogen transfer to a hydrogen acceptor, usually a pyridine dependent mono-oxygenases. For example, P4501A1 pre nucleotide. fers polycyclic aromatic hydrocarbons; P-4501A2 prefers arylamines, arylamides, P-450A26 preferS coumarin, 0256 Hydrolysis of Drugs 7-ethoxycoumarin; P-450 2C8, 209, 2C10 prefers tolbuta 0257 Alternative reactions of detoxification and metabo mide, hexobarbital; P-450 2C18 prefers mephenytoin; P-450 lism of drugs and Xenobiotics are initial Steps of hydrolysis. mp-1, mp-2 preferS debrisoquine and related amines, P450 Esters, amides, imides, or other functional groups that are 2E1 prefers ethanol, N-nitroSoalkylamines, Vinyl mono generated as a result of a hydrolysis reaction can alter the mers; P-4503A3, 3A4, 3A5, 3A7 prefers dihydropyridines, hydophilicity of a molecule and enhance urinary excretion. cyclosporin, lovastatin, aflatoxins. Hydrolysis occurs both enzymatically and nonenzymati 0250) The effect of genetic polymorphism of the P450s cally. Hydrolysis of proteins before they are degraded has has been known for Some time. For example, debrisoquine been Suggested as a step in the process of the aging of and related drugs, alfentanil, tolbutamide, (S) mephenytoin. intracellular proteins. Antibodies with an affinity for certain Because the P450s can be induced by xenobiotics, an esters and certain proteases e.g. 3-phosphoglyceraldehyde enhanced metabolic rate or efficiency can lead to one drug dehydrogenase and carbonic anhydrase, have been shown to affecting the potency, efficacy, dosing of another. For have esterase activity. example, women taking rifampicin or barbiturates can lead 0258 Enzymatic hydrolysis of drugs and xenobiotics to metabolic inactivation of Synthetic oral contraceptives. include the following enzymes: esterases, amidases, imi 0251 The flavin-containing mono-oxygenases are the dases, and epoxide hydratases. Examples of drugs undergo principle enzymes catalyzing the N-oxidation of tertiary ing hydrolysis reactions are: procaine, aspirin, clofibrate, amine drugs to N-oxides. The N-oxides are found in abun lidocaine, procainamide, indomethacin. dance in Serum. Although isoforms have been identified and 0259) Other hydrolytic processes include reactions owing the catalytic cycle is similar to the cytochrome P450 system, to both enzymes in tissues, circulation, and those elaborated flavin-containing mono-oxygenases Substrate Specificity dif by microorganisms in the lower bowel; for example, Sulfa fers. Unlike the other flavin-bearing mono-oxygenases, tases, glucoronidases, and phosphatases. these flavin-containing mono-oxygenases are present in the cell as very reactive oxygen-activated form. It is believed 0260 2. Phase II Drug Biotransformation: Conjugation that particular protein Structure Stabilizes the nucleophilic Reactions molecule. Since the molecule is so highly reactive, precise Substrate-to-enzyme fit is unnecessary. The following lists 0261. In addition, to the redox reactions of the hepatocyte Substrate types and examples oxidized by the flavin-con to detoxify or metabolize Xenobiotics, there are a Series of taining mono-oxygenases: tertiary amines (trifluroperazine, conjugation reactions. The Substrates for these reactions are bromopheniramine, morphine, nicotine, pargyline); Second generally the products from the redox reactions described ary amines (desipramine, methamphetamine, propanolol); above. These conjugation reactions involve donation of a hydrazines (1,1-demethlyhydrazine, N-aminopiperidine, Suitable hydrophilic molecular group to an accepting Xeno biotic or its metabolite. The major function of these covalent 1-methyl-1-phenylhydrazine); thiols and disulfides (dithio modifications is to render the parent compound pharmaco threitol, f-mercaptomethanol, thiophenol); thiocarbamides logically inactive. The covalent addition of Such a group to (thiourea, methimazole, propylthiouracil); Sulfides (dimeth a parent drug or compound not only inactivates the Substrate ylsulfide, Sulindac Sulfide). but also renders the recipient molecule more polar and is 0252) Examples of drugs that undergo oxidative reactions more readily excreted via the bile ducts into the intestinal are: N-dealkylation (imipramine, diazepam, codeine, eryth tract or via the urine. romycin, morphine, tamoxifen, theophylline); O-dealkyla tion (codeine, indomethacin, dextromethorphan), alipathic 0262 Lipophilic compounds that have one of the func hydroxylation (tolbutamide, ibuprofen, pentobarbital, mep tional groups that can Serve as an acceptor undergo enzy robamate, cyclosporin, midazolam); aromatic hydroxylation matic catalysis with a Second, donor Substrate. The conju (phenytoin, phenobarbital, propanolol, phenylbutaZone, gation reactions include the following broad categories: ethinyl estradiol); N-oxidation (chlorpheniramine, dapsone); glucuronidation, Sulfation, methylation, N-acetylation, and S-oxidation (cimetidine, chlorpromazine, thioridazine); conjugation with amino acids. The enzymes involved in deamination (diazepam, amphetamine). these reactions are as follows: UDP-glucuronyltransferase, alcohol Sulfotransferase, amine N-Sulfotransferase, phenol 0253 Enzymatic Reduction of Drugs Sulfotransferase, glutathione transferase, catechol O-meth 0254 The reductases are a class of enzymes that are yltransferase, amine N-methyltransferase, histamine N-me involved in the metabolic reduction of Xenobiotics. This thyltransferase, thiol S-methyltransferase, benzoyl-CoA:gly class of enzymes includes the aldehyde and ketone reduc cine acyltransferase, acetyltransacetylase, cysteine tases, the quinone reductases, the nitro and nitroso reduc S-conjugate N-acetyltransferase, cysteine S-conjugate tases, the azoreductases, the N-oxide reductases, and the N-acetyltransferase, cysteine conjugate B-lyase, thioltrans Sulfoxide reductases. These classes of enzymes are involved ferase, and rhodanese. Each of these enzymes has donor and in Sequential one-electron reduction of Some functional acceptor Specificities. The importance of these reactions in groupS and produce radicals that can produce damage cel the detoxification and metabolism of drugs and Xenobiotics lular components directly or indirectly. are discussed in the examples 0255 The dehydrogenases consist of alcohol dehydroge 0263. Examples of drugs that are known to be conjugated nases, aldehyde dehydrogenases, or dihydrodiol dehydroge are: glucuronidation (acetominophen, morphine, diazepam); US 2001/0034023 A1 Oct. 25, 2001 30

Sulfation (acetominophen, Steroids, methyldopa); acetyla branes. For example, the MDR1 protein mediates the can tion (Sulfonamides, isoniazid, dapsone, clonazepan). icular excretion of bulky lipophilic cations, e.g. anticancer drugs, calcium channel blockers, cycloSporine A, and Vari 0264. D. Excretion ous other drugs. In contrast, the MDR3 protein transports 0265 Excretion of parent drugs and metabolites can phosphatidyl choline from the inner leaflet to the outer occur in the excretory organs, namely the kidneys, liver, leaflet of the canicular membrane. Phosphatidylcholine then lungs, skin, and breasts (milk). The kidneys are the most can be Selectively extracted by intracanicular bile Salts and important organs for the excretion of drugs and metabolites. Secreted into bile as vesicles or mixed micelles. MRP2 is Renal excretion involves glomerular filtration, active tubular involved in the transport of amphipathic anionic Substrates absorption, and passive tubule reabsorption. The more e.g. leukotriene C4, glutathione-S conjugates, glucuronides hydrophilic the compound is the more readily excreted via (bilirubin diglucuronide and estradiol-17b-glucuronide), urine. In addition, many drugs and metabolites are excreted Sulfate conjugates, and is responsible for the generation of via the bile into the intestinal tract. These metabolites may bile flow independent of bile salts within the bile cannicula. be excreted in the feces, or may be reabsorbed by the SPGP is the canicular bile salt export pump in the mamma gastrointestinal epithelial cell lining. Organic anions and lian liver. cations, Steroids, fatty acids, and other drugs may be spe 0270. The hepatocyte has the ability to recruit the ATP cifically transported into the bile canniculus. requiring transporters when faced with excessive metabo 0266. In all of the metabolism and excretion routes, the lites. After Synthesis, these transporters are Stored in com physiologic goal is to detoxify and rid the body of drugs, partments that, in response to cAMP, can be actively moved Xenobiotics, endogenous or exogenous chemicals, or com through the cell to the membrane and fused to the cannicula. pounds that may or may not be deleterious to the major The active movement from the intracellular compartment to organs of the body. In principle the detoxification mecha the membrane requires microtubules, cytoplasmic kinesin, nisms function to attain this goal, however there are many cytoplasmic dynesin, and calcium. It has been shown that cases of major organ toxicity upon exposure to drugs or peptides activate phophosinositide 3 kinase, and increased metabolites of drugs. Although drugs and drug metabolites turnover of phosphoinostides drives the formation of 3'pho predominantly affect the liver and kidneys due to the circu phoinositol, which can activate the transporter in the mem latory and physiological processes, other organs can be brane and ultimately increases movement to the cannicular affected. In the present invention, we address Specific genes membrane. Signaling pathways via the activation of rab5 that may have polymorphic Sites affecting metabolic rates to Stimulate the active movement of the transporters to the ultimately affect these major organ functions. internal compartment. 0267 1. Excretion of Drugs and Drug Metabolites via the 0271 2. Excretion of Drugs and Drug Metabolites via the Bile Kidney 0268. After parent drugs or xenobiotics are metabolized 0272 Excretion of drugs or drug metabolites via the by redox and or conjugation reactions, the modified products kidney and into the urine involves three processes: 1) can then be actively transported into the bile cannicula. The glomerular filtration, 2) active tubular Secretion, and 3) transport occurs in an energy dependent fashion requiring passive tubular reabsorption. The amount of drug or metabo ATP. It has been shown that the transporters involved in the lites entering the tubular lumen is dependent on its fractional active transport from the basolateral (Sinusoidal) to the plasma protein binding and glomerular filtration rate. In the apical (canalicular) Surfaces of hepatocytes are members of proximal renal tubule anions and cations are actively trans the ATPbinding cassette (ABC) family. The transmembrane ported by carrier mediated tubular Secretion and bases are electrical potential required to maintain the chemical and transported by a separate System that Secretes choline, electrical potentials required for this active transport is histamine, and other endogenous bases. In the proximal and provided by the Na+/K+ ATPases located on the basolateral distal tubules there is passive reabsorption of these mol membrane. Other ion transporters are the potassium channel, ecules. The concentration gradient for back-diffusion is Sodium-bicarbonate Symporter, chloride-bicarbonate anion created by Sodium and other inorganic ions and water. eXchanger, and the chloride channel. In the cholangiocyte 0273 IV. Identification of Interpatient Variation in there are other ion transporters, for example chloride-bicar Response; Identification of Genes and Variances Relevant to bonate anion exchanger, isoform 2, and other organic-Solute Drug Action; Development of Diagnostic Tests, and Use of transporters. Bile acids, phosphatidyl chorine, organic Variance Status to Determine Treatment anions, organic cations, and cholesterol are actively trans ported. Approximately 5% of the transporters is multi-drug 0274 Development of therapeutics in man follows a resistance protein 1 (MDR1) and the remaining are the course from compound discovery and analysis in a labora phospholipid transporter multi-drug resistance protein 3 tory (preclinical development) to testing the candidate thera (MDR3), alicular multispecific organic-anion transporter peutic intervention in human Subjects (clinical develop (multi-drug resistance associated protein (MRP2 or ment). The preclinical development of candidate therapeutic cMOAT), canalicular bile-salt-export pump (BSEP or interventions for use in the treatment of human diseases, SPGP(sister of p-glycoprotein)), sodium-taurocholate disorders, or conditions begins at the discovery stage cotransporter, organic anion-transporting polypeptide, glu whereby a candidate therapy is tested in Vitro to achieve a tathione transporter, and a chloride-bicarbonate anion desired biochemical alteration of a biochemical or physi eXchanger are also involved in the transport. ological event. If Successful, the candidate is generally tested in animals to determine toxicity, adsorption, distribu 0269. These transporters have been identified to move tion, metabolism and excretion in a living Species. Occa Specific molecules or compounds acroSS biological mem Sionally, there are available animal models that mimic US 2001/0034023 A1 Oct. 25, 2001

human diseases, disorders, and conditions in which testing presence or absence of at least one variance in at least one the candidate therapeutic intervention can provide Support gene; c) an analytical or diagnostic test is provided to ive data to warrant proceeding to test the compound in determine the presence or absence of the at least one humans. It is widely recognized that preclinical data is variance in individual patients; d) the presence or absence of imperfect in predicting response to a compound in man. the variance or variances is used to Select a patient for a Both safety and efficacy have to ultimately be demonstrated treatment or to Select a treatment for a patient, or the in humans. Therefore, given economic constraints, and variance information is used in other methods described considering the complexities of human clinical trials, any herein. technical advance that increases the likelihood of Success fully developing and registering a compound, or getting new 0277 A. Identification of Interpatient Variability in indications for a compound, or marketing a compound Response to a Treatment Successfully against competing compounds or treatment 0278 Interpatient variability is the rule, not the excep regimens, will find immediate use. Indeed, there has been tion, in clinical therapeutics. One of the best Sources of much written about the potential of pharmacogenetics to information on interpatient variability is the nurses and change the practice of medicine. In this application we physicians Supervising the clinical trial who accumulate a provide descriptions of the methods one skilled in the art body of first hand observations of physiological responses to would use to advance compounds through clinical trials the drug in different normal Subjects or patients. Evidence of using genetic Stratification as a tool to circumvent Some of interpatient variation in response can also be measured the difficulties typically encountered in clinical develop Statistically, and may be best assessed by descriptive Statis ment, Such as poor efficacy or toxicity. We also provide tical measures that examine variation in response (beneficial Specific genes, variation in which may account for interpa or adverse) across a large number of Subjects, including in tient variation in treatment response, and further we provide different patient Subgroups (men VS. Women; whites vs. Specific exemplary variances in those genes that may blacks, Northern Europeans vs. Southern Europeans, etc.). account for variation in treatment response. 0279. In accord with the other portions of this descrip 0275. The study of sequence variation in genes that tion, the present invention concerns DNA sequence Vari mediate and modulate the action of drugs may provide ances that can affect one or more of advances at Virtually all Stages of drug development. For example, identification of amino acid variances in a drug 0280) i. The susceptibility of individuals to a dis target during preclinical development would allow develop ease, ment of non-allele Selective agents. During early clinical development, knowledge of variation in a gene related to 0281 ii. The course or natural history of a disease; drug action could be used to design a clinical trial in which 0282) iii. The response of a patient with a disease to the variances are taken account of by, for example, including a medical intervention, Such as, for example, a drug, Secondary endpoints that incorporate an analysis of response a biologic Substance, physical energy Such as radia rates in genetic Subgroups. In later Stages of clinical devel tion therapy, or a specific dietary regimen. (The opment the goal might be to first establish retrospectively terms drug, compound or treatment as used whether a particular problem, Such as liver toxicity, can be herein may refer to any of the foregoing medical understood in terms of genetic Subgroups, and thereby interventions.) The ability to predict either beneficial controlled using a genetic test to Screen patients. Thus or detrimental responses is medically useful. genetic analysis of drug response can aid Successful devel opment of therapeutic products at any Stage of clinical 0283 Thus variation in any of these three parameters development. Even after a compound has achieved regula may constitute the basis for initiating a pharmacogenetic tory approval its commercialization can be aided by the Study directed to the identification of the genetic Sources of methods of this invention, for example by allowing identi interpatient variation. The effect of a DNA sequence vari fication of genetically defined responder Subgroups in new ance or variances on disease Susceptibility or natural history indications (for which approval in the entire disease popu (i and ii, above) are of particular interest as the variances can lation could not be achieved) or by providing the basis for be used to define patient Subsets which behave differently in a marketing campaign that highlights the Superior efficacy response to medical interventions Such as those described in and/or Safety of a compound coupled with a genetic test to (iii). The methods of this invention are also useful in a identify preferential responders. Thus the methods of this clinical development program where there is not yet evi invention will provide medical, economic and marketing dence of interpatient variation (perhaps because the com advantages for products, and over the longer term increase pound is just entering clinical trials) but Such variation in therapeutic alternatives for patients. response can be reliably anticipated. It is more economical 0276 AS indicated in the Summary above, certain aspects to design pharmacogenetic Studies from the beginning of a of the present invention typically involve the following clinical development program than to start at a later Stage process, which need not occur Separately or in the order when the costs of any delay are likely to be high given the Stated. Not all of these described processes must be present resources typically committed to Such a program. in a particular method, or need be performed by a single 0284. In other words, a variance can be useful for cus entity or organization or perSon. Additionally, if certain of tomizing medical therapy at least for either of two reasons. the information is available from other Sources, that infor First, the variance may be associated with a specific disease mation can be utilized in the present invention. The pro Subset that behaves differently with respect to one or more cesses are as follows: a) variability between patients in the therapeutic interventions (i and ii above); Second, the vari response to a particular treatment is observed; b) at least a ance may affect response to a specific therapeutic interven portion of the variable response is correlated with the tion (iii above). Consider for exemplary purposes pharma US 2001/0034023 A1 Oct. 25, 2001 32 cological therapeutic interventions. In the first case, there individuals as an indication that genetic variability in the may be no effect of a particular gene Sequence variance on gene is at least partially responsible for differential treatment the observable pharmacological action of a drug, yet the response. It may be useful to independently analyze vari disease Subsets defined by the variance or variances differ in ances in the different geographic, ethnic and/or racial groups their response to the drug because, for example, the drug acts as the presence of different genetic variances in these groups on a pathway that is more relevant to disease pathophysi (i.e. different genetic background) may influence the effect ology in one variance-defined patient Subset than in another of a Specific variance. That is, there may be a genexgene variance-defined patient Subset. The Second type of useful interaction involving one unstudied gene, however the indi gene Sequence variance affects the pharmacological action cated demographic variables may act as a Surrogate for the of a drug or other treatment. Effects on pharmacological unstudied allele. Statistical measures known to those skilled responses fall generally into two categories, pharmacoki in the art are preferably used to measure the fraction of netic and pharmacodynamic effects. These effects have been interpatient variation attributable to any one variance, or to defined as follows in Goodman and Gilman's Pharmaco measure the response rates in different Subgroups defined logic Basis of Therapeutics (ninth edition, McGraw Hill, genetically or defined by Some combination of genetic, New York, 1986): “Pharmacokinetics” deals with the demographic and clinical criteria. absorption, distribution, biotransformations and excretion of 0289 Useful methods for identifying genes relevant to drugs. The Study of the biochemical and physiological the pharmacological action of a drug or other treatment are effects of drugs and their mechanisms of action is termed known to those skilled in the art, and include review of the “pharmacodynamics.” Scientific literature combined with inferential or deductive 0285) Useful gene sequence variances for this invention reasoning that one skilled in the art of molecular pharma can be described as variances which partition patients into cology and molecular biology would be capable of, large two or more groups that respond differently to a therapy or Scale analysis of gene expression in cells treated with the that correlate with differences in disease Susceptibility or drug compared to control cells, large Scale analysis of the progression, regardless of the reason for the difference, and protein expression pattern in treated VS. untreated cells, or regardless of whether the reason for the difference is known. the use of techniques for identification of interacting pro The latter is true because it is possible, with genetic meth teins or ligand-protein interactions, Such as yeast two-hybrid ods, to establish reliable associations even in the absence of Systems. a pathophysiological hypothesis linking a gene to a pheno 0290 C. Development of a Diagnostic Test to Determine type, Such as a pharmacological response, disease Suscepti Variance Status bility or disease prognosis. 0291. In accordance with the description in the Summary 0286 B. Identification of Specific Genes and Correlation above, the present invention generally concerns the identi of Variances in Those Genes with Response to Treatment of fication of variances in genes which are indicative of the Diseases or Conditions effectiveness of a treatment in a patient. The identification of 0287. It is useful to identify particular genes which do or Specific variances, in effect, can be used as a diagnostic or are likely to mediate the efficacy or Safety of a treatment prognostic test. Correlation of treatment efficacy and/or method for a disease or condition, particularly in View of the toxicity with particular genes and gene families or pathways large number of genes which have been identified and which is provided in Stanton et al., U.S. Provisional Application continue to be identified in humans. AS is further discussed Ser. No. 60/093,484, filed Jul. 20, 1998, entitled GENE in Section C below, this correlation can proceed by different SEQUENCE VARIANCES WITH UTILITY IN DETER paths. One exemplary method utilizes prior information on MINING THE TREATMENT OF DISEASE (concerns the the pharmacology or pharmacokinetics or pharmacodynam Safety and efficacy of compounds active on folate or pyri ics of a treatment method, e.g., the action of a drug, which midine metabolism or action) and Stanton, U.S. Provisional indicates that a particular gene is, or is likely to be, involved Application Ser. No. 60/121,047, filed Feb. 22, 1999, in the action of the treatment method, and further Suggests entitled GENE SEQUENCE VARIANCES WITH UTILITY that variances in the gene may contribute to variable IN DETERMINING THE TREATMENT OF DISEASE response to the treatment method. For example if a com (concerning Alzheimer's disease and other dementias and pound is known to be glucuronidated then a glucuronyl cognitive disorders), which are hereby incorporated by ref transferase is likely involved. If the compound is a phenol, erence in their entireties including drawings. the likely glucuronyltransferase is UGT1 (either the 0292 Genes identified in the examples below and in the UGT1*1 or UGT1*6 transcripts, both of which catalyze the Tables and Figures can be used in the methods of the present conjugation of planar phenols with glucuronic acid). Similar invention. A variety of genes which the inventors realize inferences can be made for many other biotransformation may account for interpatient variation in patient outcome reactions. response to candidate therapeutic interventions are listed in 0288 Alternatively, if such information is not known, Tables 1, 3, and 4. Gene Sequence variances in Said genes are variances in a gene can be correlated empirically with particularly useful for aspects of the present invention. treatment response. In this method, variances in a gene 0293 Methods for diagnostic tests are well known in the which exist in a population can be identified. The presence art. Generally in this invention, the diagnostic test involves of the different variances or haplotypes in individuals of a determining whether an individual has a variance or variant Study group, which is preferably representative of a popu form of a gene that is involved in the disease or condition or lation or populations of known geographic, ethnic and/or the action of the drug or other treatment or effects of Such racial background, is determined. This variance information treatment. Such a variance or variant form of the gene is is then correlated with treatment response of the various preferably one of several different variances or forms of the US 2001/0034023 A1 Oct. 25, 2001

gene that have been identified within the population and are the population as cytosine (C) or thymine (T). Thus a known to be present at a certain frequency. In an exemplary particular copy of gene X may have any of the following method, the diagnostic test involves determining the combinations of nucleotides at positions 150 and 810: Sequence of at least one variance in at least one gene after 150A-810C, 150A-810T, 150G-810C or 150G-810T. Each amplifying a Segment of Said gene using a DNA amplifica of the four possibilities is a unique haplotype. If the two tion method such as the polymerase chain reaction (PCR). In nucleotides interact in either RNA or protein, then knowing this method DNA for analysis is obtained by amplifying a the haplotype can be important. The point of a haplotyping segment of DNA or RNA (generally after converting the test is to determine the haplotypes present in a DNA or RNA to cDNA) spanning one or more variances in the gene cDNA sample (e.g. from a patient). In the example provided sequence. Preferably, the amplified segment is <500 bases in there are only four possible haplotypes, but, depending on length, in an alternative embodiment the amplified Segment the number of variances in the gene and their distribution in is <100 bases in length, most preferably <45 bases in length. human populations there may be three, four, five, Six or more haplotypes at a given gene. The most useful haplotypes for 0294. In some cases it will be desirable to determine a this invention are those which occur commonly in the haplotype instead of a genotype. In Such a case the diag population being treated for a disease or condition. Prefer nostic test is performed by amplifying a Segment of DNA or ably Such haplotypes occur in at least 5% of the population, RNA (cDNA) spanning more than one variance in the gene more preferably in at least 10%, still more preferably in at Sequence and preferably maintaining the phase of the Vari least 20% of the population and most preferably in at least ances on each allele. The term “phase” refers to the rela 30% or more of the population. Conversely, when the goal tionship of variances on a single chromosomal copy of the of a pharmacogenetic program is to identify a relatively rare gene, Such as the copy transmitted from the mother (mater population that has an adverse reaction to a treatment, the nal copy or maternal allele) or the father (paternal copy or most useful haplotypes may be rare haplotypes, which may paternal allele). The haplotyping test may take part in two occur in less than 5%, less than 2%, or even in less than 1% phases, where first genotyping tests at two or more variant of the population. One skilled in the art will recognize that Sites reveal which Sites are heterozygous in each patient or the frequency of the adverse reaction provides a useful guide normal Subject. Subsequently the phase of the two or more to the likely frequency of Salient causative haplotypes. variant Sites can be determined. In performing a haplotyping test preferably the amplified segment is >500 bases in 0298 Based on the identification of variances or variant length, more preferably it is >1,000 bases in length, and forms of a gene, a diagnostic test utilizing methods known most preferably it is >2,500 bases in length. One way of in the art can be used to determine whether a particular form preserving phase is to amplify one Strand in the PCR of the gene, containing specific variances or haplotypes, or reaction. This can be done using one or a pair of oligonucle combinations of variances and haplotypes, is present in at otide primers that terminate (i.e. have a 3' end that stops) least one copy, one copy, or more than one copy in an opposite the variant Site, Such that one primer is perfectly individual. Such tests are commonly performed using DNA complementary to one variant form and the other primer is or RNA collected from blood, cells, tissue Scrapings or other perfectly complementary to the other variant form. Other cellular materials, and can be performed by a variety of than the difference in the 3' most nucleotide the two primers methods including, but not limited to, PCR based methods, are identical (forming an allelic primer pair). Only one of the hybridization with allele-Specific probes, enzymatic muta allelic primerS is used in any PCR reaction, depending on tion detection, chemical cleavage of mismatches, mass Spec which Strand is being amplified. The primer for the opposite trometry or DNA sequencing, including miniseduencing. Strand may also be an allelic primer, or it may prime from Methods for haplotyping are described above. In particular a non-polymorphic region of the template. This method embodiments, hybridization with allele Specific probes can exploits the requirement of most polymerases for perfect be conducted in two formats: (1) allele specific oligonucle complementarity at the 3' terminus of the primer in a otides bound to a Solid phase (glass, Silicon, nylon mem primer-template complex. See, for example: Lo Y M, Patel branes) and the labeled Sample in Solution, as in many DNA P, Newton C R, Markham A F, Fleming KA and J S chip applications, or (2) bound Sample (often cloned DNA or Wainscoat. (1991) Direct haplotype determination by double PCR amplified DNA) and labeled oligonucleotides in solu ARMS: Specificity, Sensitivity and genetic applications. tion (either allele specific or short-e.g. 7mers or 8merS-SO Nucleic Acids Res July 11;19(13):3561-7. as to allow sequencing by hybridization). Preferred methods for diagnostic testing of variances are described in four 0295). It is apparent that such diagnostic tests are per patent applications Stanton et al., entitled A METHOD FOR formed after initial identification of variances within the ANALYZING POLYNUCLEOTIDES, Ser. Nos. 09/394, gene, which allows Selection of appropriate allele Specific 467; 09/394.457; 09/394,774; and 09/394,387; all filed Sep. primerS. 10, 1999. The application of such diagnostic tests is possible 0296 Diagnostic genetic tests useful for practicing this after identification of variances that occur in the population. invention belong to two types: genotyping tests and haplo Diagnostic tests may involve a panel of variances from one typing tests. A genotyping test Simply provides the Status of or more genes, often on a Solid Support, which enables the a variance or variances in a Subject or patient. For example Simultaneous determination of more than one variance in Suppose nucleotide 150 of hypothetical gene X on an One or more genes. autosomal chromosome is an adenine (A) or a guanine (G) 0299) D. Use of Variance Status to Determine Treatment base. The possible genotypes in any individual are AA, AG 0300. The present disclosure describes exemplary gene or GG at nucleotide 150 of gene X. Sequence variances in genes identified in a gene table herein 0297. In a haplotyping test there is at least one additional (e.g., Tables 3 and 4), and variant forms of these gene that variance in gene X, Say at nucleotide 810, which varies in may be determined using diagnostic tests. AS indicated in the US 2001/0034023 A1 Oct. 25, 2001 34

Summary, Such a variance-based diagnostic test can be used or otherwise contra-indicated in an individual who inherits to determine whether or not to administer a specific drug or one or two copies of that form of the gene. other treatment to a patient for treatment of a disease or 0304. In accord with this invention, diagnostic tests for condition. Preferably Such diagnostic tests are incorporated variances and variant forms of genes as described above can in texts. Such as are described in Clinical Diagnosis and be used in clinical trials to demonstrate the Safety and Management by Laboratory Methods (19th Ed) by John B. efficacy of a drug in a Specific population. As a result, in the Henry (Editor) W B Saunders Company, 1996; Clinical case of drugs which show variability in patient response Laboratory Medicine : Clinical Application of Laboratory correlated with the presence or absence of a variance or Data, (6th edition) by R. Ravel, Mosby-Year Book, 1995, or variances, it is preferable that Such drug is approved for Sale other medical textbooks including, without limitation, text or use by regulatory agencies with the recommendation or books of medicine, laboratory medicine, therapeutics, phar requirement that a diagnostic test be performed for a specific macy, pharmacology, nutrition, allopathic, homeopathic, and variance or variant form of a gene which identifies Specific Osteopathic medicine; preferably Such a test is developed as populations in which the drug will be Safe and/or effective. a home brew method by a certified diagnostic laboratory; For example, the drug may be approved for Sale or use by most preferably Such a diagnostic test is approved by regulatory agencies with the Specification that a diagnostic regulatory authorities, e.g., by the U.S. Food and Drug test be performed for a specific variance or variant form of Administration, and is incorporated in the label or insert for a gene which identifies specific populations in which the a therapeutic compound, as well as in the Physicians Desk drug will be toxic. Thus, approved use of the drug, or the Reference. procedure for use of the drug, can be limited by a diagnostic 0301 In such cases, the procedure for using the drug is test for Such variances or variant forms of a gene; or Such a restricted or limited on the basis of a diagnostic test for diagnostic test may be considered good medical practice, but determining the presence of a variance or variant form of a not absolutely required for use of the drug. gene. Alternatively the use of a genetic test may be advised 0305 As indicated, diagnostic tests for variances as as best medical practice, but not absolutely required, or it described in this invention may be used in clinical trials to may be required in a Subset of patients, e.g. those using one establish the safety and efficacy of a drug. Methods for such or more other drugs, or those with impaired liver or kidney clinical trials are described below and/or are known in the art function. The procedure that is dictated or recommended and are described in Standard textbooks. For example, based on genotype may include the route of administration diagnostic tests for a specific variance or variant form of a of the drug, the dosage form, dosage, Schedule of adminis gene may be incorporated in the clinical trial protocol as tration or use with other drugs, any or all of these may inclusion or exclusion criteria for enrollment in the trial, to require Selecting or determination consistent with the results allocate certain patients to treatment or control groups of the diagnostic test or a plurality of such tests. Preferably within the clinical trial or to assign patients to different the use of Such diagnostic tests to determine the procedure treatment cohorts. Alternatively, diagnostic tests for Specific for administration of a drug is incorporated in a text Such as variances may be performed on all patients within a clinical those listed above, or medical textbooks, for example, trial, and Statistical analysis performed comparing and con textbooks of medicine, laboratory medicine, therapeutics, trasting the efficacy or Safety of a drug between individuals pharmacy, pharmacology, nutrition, allopathic, homeo with different variances or variant forms of the gene or pathic, and Osteopathic medicine. AS previously Stated, genes. Preferred embodiments involving clinical trials preferably Such a diagnostic test or tests are required by include the genetic Stratification Strategies, phases, Statistical regulatory authorities and are incorporated in the label or analyses, sizes, and other parameters as described herein. insert as well as the Physicians Desk Reference. 0306 Similarly, diagnostic tests for variances can be 0302 Variances and variant forms of genes useful in performed on groups of patients known to have efficacious conjunction with treatment methods may be associated with responses to the drug to identify differences in the frequency the origin or the pathogenesis of a disease or condition. In of variances between responders and non-responders. Like many useful cases, the variant form of the gene is associated wise, in other cases, diagnostic tests for variance are per with a Specific characteristic of the disease or condition that formed on groups of patients known to have toxic responses is the target of a treatment, most preferably response to to the drug to identify differences in the frequency of the Specific drugs or other treatments. Examples of diseases or variance between those having adverse events and those not conditions ameliorable by the methods of this invention are having adverse events. Such outlier analyses may be par identified in the Examples and tables below; in general ticularly useful if a limited number of patient Samples are treatment of disease with current methods, particularly drug available for analysis. It is apparent that Such clinical trials treatment, always involves Some unknown element (involv can be or are performed after identifying Specific variances ing efficacy or toxicity or both) that can be reduced by or variant forms of the gene in the population. In defining appropriate diagnostic methods. outliers it is useful to examine the distribution of responses in the placebo group; outliers should preferably have 0303 Alternatively, the gene is involved in drug action, responses that exceed in magnitude the extreme responses in and the variant forms of the gene are associated with the placebo group. variability in the action of the drug. For example, in Some cases, one variant form of the gene is associated with the 0307 The identification and confirmation of genetic vari action of the drug Such that the drug will be effective in an ances is described in certain patents and patent applications. individual who inherits one or two copies of that form of the The description therein is useful in the identification of gene. Alternatively, a variant form of the gene is associated variances in the present invention. For example, a Strategy with the action of the drug such that the drug will be toxic for the development of anticancer agents having a high US 2001/0034023 A1 Oct. 25, 2001 therapeutic indeX is described in Housman, International journal articles) and online Sources (databases) to Application PCT/US/94 08473 and Housman, INHIBI identify genes most relevant to the action of a TORS OF ALTERNATIVE ALLELES OF GENES Specific drug or other treatment, particularly with ENCODING PROTEINS VITAL FOR CELL VIABILITY respect to its utility for treating a specific disease, as OR CELL GROWTH AS A BASIS FOR CANCER this beneficially allows the Set of genes to be ana THERAPEUTICAGENTS, U.S. Pat. No. 5,702,890, issued lyzed ultimately in clinical trials to be reduced from Dec. 30, 1997, which are hereby incorporated by reference an initial large Set. Specific Strategies for conducting in their entireties. Also, a number of gene targets and Such Searches are described below. In Some instances asSociated variances are identified in Housman et al., U.S. the literature may provide adequate information to patent application Ser. No. 09/045,053, entitled TARGET ALLELES FOR ALLELE-SPECIFICDRUGS, filed Mar. Select genes to be Studied in a clinical trial, but in 19, 1998, which is hereby incorporated by reference in its other cases additional experimental investigations of entirety, including drawings. the sort described below will be preferable to maxi mize the likelihood that the Salient genes and vari 0308 The described approach and techniques are appli ances are moved forward into clinical Studies. Spe cable to a variety of other diseases, conditions, and/or cific genes relevant to understanding interpatient treatments and to genes associated with the etiology and variation in patient outcome response to candidate pathogenesis of Such other diseases and conditions and the therapeutic interventions are listed in Table 1. In efficacy and Safety of Such other treatments. Table 2 preferred Sets of genes for analysis of vari 0309 Useful variances for this invention can be able therapeutic response in Specific diseases are described generally as variances which partition patients highlighted. These genes are exemplary; they do not into two or more groups that respond differently to a therapy constitute a complete Set of genes that may account (a therapeutic intervention), regardless of the reason for the for variation in clinical response. Experimental data difference, and regardless of whether the reason for the are also useful in establishing a list of candidate difference is known. genes, as described below. 0310 III. From Variance List to Clinical Trial: Identify 0313 (2) Having assembled a list of candidate genes ing Genes and Gene Variances that Account for Variable generally the Second step is to Screen for variances in Responses to Treatment each candidate gene. Experimental and computa tional methods for variance detection are described 0311. There are a variety of useful methods for identify in this invention, and tables of exemplary variances ing a Subset of genes from a large set of candidate genes that are provided (Tables 3, and 4) as well as methods for should be prioritized for further investigation with respect to identifying additional variances and a written their influence on inter-individual variation in disease pre description of Such possible additional variances in disposition or response to a particular drug. These methods the cDNAS of genes that may affect drug action (see include for example, (1) Searching the biomedical literature Stanton & Adams, application Ser. No. 09/300,747, to identify genes relevant to a disease or the action of a drug, filed Apr. 26, 1999, entitled GENE SEQUENCE (2) Screening the genes identified in step 1 for variances. A VARIANCES WITH UTILITY IN DETERMINING large Set of exemplary variances are provided in Tables 3 and THE TREATMENT OF DISEASE, incorporated in 4. Other methods include (3) using computational tools to its entirety. predict the functional effects of variances in Specific genes, (4) using in vitro or in Vivo experiments to identify genes 0314 (3) Having identified variances in candidate which may participate in the response to a drug or treatment, genes the next Step is to assess their likely contri and to determine the variances which affect gene, RNA or bution to clinical variation in patient response to protein function, and may therefore be important genetic therapy, preferably by using informatics-based variables affecting disease manifestations or drug response, approaches Such as DNA and protein Sequence and (5) retrospective or prospective clinical trials. Compu analysis and protein modeling. The literature and tational tools are described in U.S. patent application, Stan informatics-based approaches provide the basis for ton et al., Ser. No. 09/300,747, filed Apr. 26, 1999, entitled prioritization of candidate genes, however it may in GENE SEQUENCE VARIANCES WITH UTILITY IN Some cases be desirable to further narrow the list of DETERMINING THE TREATMENT OF DISEASE, and in candidate genes, or to measure experimentally the Stanton et al., Ser. No. 09/419,705, filed Oct. 14, 1999, phenotype associated with Specific variances or Sets entitled VARIANCE SCANNING METHOD FOR IDEN of variances (e.g. haplotypes). TIFYING GENE SEQUENCE VARIANCES, which are 0315 (4) Thus, as a third step in candidate gene hereby incorporated by reference in their entireties, includ analysis, one skilled in the art may elect to perform ing drawings. Other methods are considered below in Some in Vitro or in Vivo experiments to assess the func detail. tional importance of gene variances, using either 0312 (1) To begin, one preferably identifies, for a biochemical or genetic tests. (Certain kinds of given treatment, a set of candidate genes that are experiments-for example gene expression profiling likely to affect disease phenotype or drug response. and proteome analysis—may not only allow refine This can be accomplished most efficiently by first ment of a candidate gene list but may also lead to assembling the relevant medical, pharmacological identification of additional candidate genes.) Com and biological data from available Sources (e.g., bination of two or all of the three above methods will public databases and publications). One skilled in the provide Sufficient information to narrow and priori art can review the literature (textbooks, monographs, tize the Set of candidate genes and variances to a US 2001/0034023 A1 Oct. 25, 2001 36

number that can be studied in a clinical trial with literature and available online databaseS regarding the patho adequate Statistical power. physiology of the disease and the pharmacology of the drug. Literature or online Sources can provide specific genes 0316 (5) The fourth step is to design retrospective involved in the disease process or drug response, or describe or prospective human clinical trials to test whether biochemical pathways involving multiple genes, each of the identified allelic variance, variances, or haplo which may affect the disease process or drug response. types or combination thereof influence the efficacy or toxicity profiles for a given drug or other therapeutic 0322. Alternatively, biochemical or pathological changes intervention. It should be recognized that this fourth characteristic of the disease may be described; Such infor Step is the crucial Step in producing the type of data mation can be used by one skilled in the art to infer a set of that would justify introducing a diagnostic test for at genes that can account for the biochemical or pathologic least one variance into clinical use. Thus while each changes. For example, to understand variation in response to of the above four Steps are useful in particular a drug that modulates Serotonin levels in a central nervous instances of the invention, this final Step is indis system (CNS) disorder associated with altered levels of pensable. Further guidance and examples of how to Serotonin one would preferably Study, at a minimum, Vari perform these five steps are provided below. ances in genes responsible for Serotonin biosynthesis, release from the cell, receptor binding, presynaptic reuptake, 0317 (6) A fifth (optional) step entails methods for and degradation or metabolism. Genes responsible for each using a genotyping test in the promotion and mar of these functions should be examined for variation that may keting of a treatment method. It is widely appreci account for interpatient differences in drug response or ated that there is a tendency in the pharmaceutical disease manifestations. AS recognized by those skilled in the industry to develop many compounds for well estab art, a comprehensive list of Such genes can be obtained from lished therapeutic targets. Examples include beta textbooks, monographs and the literature. adrenergic blockers, hydroxymethylglutaryl (HMG) CoA reductase inhibitors (Statins), dopamine D2 0323 There are several types of Scientific information, receptor antagonists and Serotonin transporter inhibi described in Some detail below, that are valuable for iden tors. Frequently the pharmacology of these com tifying a set of candidate genes to be investigated with pounds is quite Similar in terms of efficacy and Side respect to a specific disease and therapeutic intervention. effects. Therefore the marketing of one compound First there is the medical literature, which provides basic VS. other members of the class is a challenging information on disease pathophysiology and therapeutic problem for drug companies, and is reflected in the interventions. A Subset of this literature is devoted to specific lesser Success that late products typically achieve description of pathologic conditions. Second there is the compared to the first and Second approved products. pharmacology literature, which will provide additional It occurred to the inventors that genetic Stratification information on the mechanism of action of a drug (pharma can provide the basis for identifying a patient popu codynamics) as well as its principal routes of metabolic lation with a Superior response rate or improved transformation (pharmacokinetics) and the responsible pro Safety to one member of a class of drugs, and that this teins. Third there is the biomedical literature (principally information can be the basis for commercialization genetics, physiology, biochemistry and molecular biology), of that compound. Such a commercialization cam which provides more detailed information on metabolic paign can be directed at caregivers, particularly pathways, protein Structure and function and gene Structure. physicians, or at patients and their families, or both. Fourth, there are a variety of online databases that provide additional information on metabolic pathways, gene fami 0318 1. Identification of Candidate Genes Relevant to lies, protein function and other Subjects relevant to Selecting the Action of a Drug a set of genes that are likely to affect the response to a 0319 Practice of this invention will often begin with treatment. identification of a specific pharmaceutical product, for example a drug, that would benefit from improved efficacy 0324) Medical Literature or reduced toxicity or both, and the recognition that phar 0325 A good starting place for information on molecular macogenetic investigations as described herein provide a pathophysiology of a specific disease is a general medical basis for achieving Such improved characteristics. The ques textbook such as Harrison's Principles of Internal Medi tion then becomes which genes and variances, Such as those cine, 14th edition, (2 Vol Set) by A. S. Fauci, E. Braunwald, provided in this application in Tables 1, 3, and 4, would be K. J. Isselbacher, et al. (editors), McGraw Hill, 1997, or most relevant to interpatient variation in response to the Cecil Textbook of Medicine (20th Ed) by R. L. Cecil, F. Plum drug. AS discussed above, the Set of relevant genes includes and J. C. Bennett (Editors) W B Saunders Co., 1996. For both genes involved in the disease process and genes pediatric diseases texts such as Nelson Textbook of Pediat involved in the interaction of the patient and the treatment rics (15th edition) by R. E. Behrman, R. M. Kliegman, A. M. for example genes involved in pharmacokinetic and phar Arvin and W. E. Nelson (Editors), WB Saunders Co., 1995 macodynamic action of a drug. The biological and biomedi or Oski's Principles and Practice of Pediatrics (3" Edition) cal literature and online databaseS provide useful guidance by J. A. Mamillan & F. A. Oski Lippincott-Raven, 1999 are in Selecting Such genes. Specific guidance in the use of these useful introductions. For obstetrical and gynecological dis resources is provided below. orders texts such as Williams Obstetrics (20th Ed) by F. G. Cunningham, N. F. Gant, P. C. McDonald et al. (Editors), 0320 Review the Literature and Online Sources Appleton & Lange, 1997 provide general information on 0321) One way to find genes that affect response to a drug disease pathophysiology. For psychiatric disorders texts in a particular disease Setting is to review the published such as the Comprehensive Textbook of Psychiatry, VI (2 US 2001/0034023 A1 Oct. 25, 2001 37

Vols) by H. I. Kaplan and B.J. Sadock (Editors), Lippincott, 0340. In addition to these subspecialty texts there are Williams & Wilkins, 1995, or The American Psychiatric many textbooks and monographs that concern more Press Textbook of Psychiatry (3" edition) by R. E. Hales, S. restricted disease areas, or specific diseases. Such books C. Yudofsky and J. A. Talbott (Editors) Amer Psychiatric provide more extensive coverage of pathophysiologic Press, 1999 provide an overview of disease noSology, patho mechanisms and therapeutic options. The number of Such physiological mechanisms and treatment regimens. books is too great to provide examples for all but a few diseases, however one skilled in the art will be able to 0326 In addition to these general texts, there are a variety readily identify relevant texts. One simple way to Search for of more Specialized medical texts that provide greater detail relevant titles is to use the Search engine of an online about Specific disorders which can be utilized in developing bookseller Such as http://www.amazon.com or http://www a list of candidate genes and variances relevant to interpa ..barnesandnoble.com using the disease or drug (or the group tient variation in response to a treatment. For example, of diseases or drugs to which they belong) as Search terms. within the field of medicine there are standard textbooks for For example a Search for asthma would turn up titles Such as each of the Subspecialties. Some specific examples include: Asthma. Basic Mechanisms and Clinical Management (3rd 0327) Heart Disease: A Textbook of Cardiovascular edition) by P. J. Barnes, I. W. Rodger and N. C. Thomson Medicine (2 Volume set) by E. Braunwald (Editor), (Editors), Academic Press, 1998 and Airways and Vascular W B Saunders Co., 1996. Remodelling in Asthma and Cardiovascular Disease. Impli 0328 Hurst's the Heart, Arteries and Veins (9th Ed) cations for Therapeutic Intervention, by C. Page & J. Black (2 Vol Set) by R. W. Alexander, R. C. Schlant, V. (Editors), Academic Press, 1994. Fuster, W. Alexander and E. H. Sonnenblick (Edi 0341 Pathology Literature tors) McGraw Hill, 1998. 0342. In addition to medical texts there are texts that 0329 Principles of Neurology (6th edition) by R. D. Specifically address disease etiology and pathologic changes Adams, M. Victor (editors), and A. H. Ropper (Con asSociated with disease. A good general pathology text is tributor), McGraw Hill, 1996. Robbins Pathologic Basis of Disease (6th edition) by R. S. 0330 Sleisenger & Fordtran's Gastrointestinal and Cotran, V. Kumar, T. Collins and S. L. Robbins, W B Liver Disease. Pathophysiology Diagnosis, Man Saunders Co., 1998. Specialized pathology texts exist for agement (6th edition) by M. Feldman, B. F. Schar each organ System and for Specific diseases, Similar to schmidt and M. Sleisenger (Editors), WB Saunders medical texts. These texts are useful Sources of information Co., 1997. for one skilled in the art for developing lists of genes that 0331 Textbook of Rheumatology (5th edition) by W. may account for Some of the known pathologic changes in N. Kelley, S. Ruddy, E. D. Harris Jr. and C. B. Sledge disease tissue. Exemplary texts are as follows: (Editors) (2 volume set) W B Saunders Co., 1997. 0343 Bone Marrow Pathology 2" edition, by B. J. 0332 Williams Textbook of Endocrinology (9th edi Bain, I. Lampert. & D. Clark, Blackwell Science, tion) by J. D. Wilson, D. W. Foster, H. M. Kronen 1996 berg and Larsen (Editors), WB Saunders Co., 1998. 0344) Atlas of Renal Pathology by F. G. Silva, W. B. 0333 Wintrobe's Clinical Hematology (10th Ed) by Saunders, 1999. G. R. Lee, J. Foerster (Editor) and J. Lukens (Edi 0345) Fundamentals of Toxicologic Pathology by W. tors) (2 Volumes) Lippincott, Williams & Wilkins, M. Haschek and C. G. Rousseaux, Academic Press, 1998. 1997. 0334 Cancer. Principles & Practice of Oncology 0346 Gastrointestinal Pathology by P. Chandra (5th edition) by V. T. Devita, S. A. Rosenberg and S. Soma, Appleton and Lange, 1998. Hellman (editors), Lippincott-Raven Publishers, 1997. 0347 Ophthalmic Pathology with Clinical Correla 0335) Principles of Pulmonary Medicine (3rd edi tions by J. Sassani, Lippincott-Raven, 1997. tion) by S. E. Weinberger & J Fletcher (Editors), W 0348 Pathology of Bone and Joint Disorders by F. B Saunders Co., 1998. McCarthy, F. J. Frassica and A. Ross, W. B. Saun 0336 Diagnosis and Management of Renal Disease ders, 1998. and Hypertension (2nd edition) by A. K. Mandal & 0349 Pulmonary Pathology by M. A. Grippi, Lip J. C. Jennette (Editors), Carolina Academic Press, picott-Raven, 1995. 1994. Massry & Glassock's Textbook of Nephrology 0350 Neuropathology by D. Ellison, L. Chimelli, B. (3rd edition) by S. G. Massry & R. J. Glassock Harding, S. Love & J. Lowe, Mosby Year Book, (editors) Williams & Wilkins, 1995. 1997. 0337 The Management of Pain by J. J. Bonica, Lea and Febiger, 1992 0351) Greenfield's Neuropatholgy 6" edition by J. G. Greenfield, P. L. Lantos & D. I. Graham, Edward 0338 Ophthalmology by M. Yanoff & J. S. Duker, Arnold, 1997. Mosby Year Book, 1998 0339) Clinical Ophthalmology: A Systemic 0352 Pharmacology, Pharmacogenetics and Pharmacy Approach by J. J. Kanski, Butterworth-Heineman, Literature 1994. Essential Otolaryngoloy by J. K. Lee Appleton 0353. There are also both general and specialized texts and Lange 1998. and monographs on pharmacology that provide data on US 2001/0034023 A1 Oct. 25, 2001 38

pharmacokinetics and pharmacodynamics of drugs. The 0363. In addition to the medical, pathology, and pharma discussion of pharmacodynamics (mechanism of action of cology texts listed above there are Several information the drug) in Such texts is often Supported by a review of the Sources that one skilled in the art will turn to for information biochemical pathway or pathways that are affected by the on the genetic, physiologic, biochemical, and molecular drug. Also, proteins related to the target protein are often biological aspects of the disease, disorder or condition or the listed; it is important to account for variation in Such proteins effect of the therapeutic intervention on Specific physiologic as the related proteins may be involved in drug pharmacol processes. The biomedical literature may include informa ogy. For example, there are 14 known Serotonin receptors. tion on nonhuman organisms that is relevant to understand Various pharmacological Serotonin agonists or antagonists ing the likely disease or pharmacological pathways in man. have different affinities for these different receptors. Varia tion in a specific receptor may affect the pharmacology not 0364. Also provided below are illustrative texts which only of drugs targeted to that receptor, but also drugs that are will aid in the identification of a pathway or pathways, and principally agonistS or antagonists of different receptors. a gene or genes that may be relevant to interindividual Such compounds may produce different effects on two variation in response to a therapy. Textbooks of biochemis allelic forms of a non-targeted receptor; for example on try, genetics and physiology are often useful Sources for Such variant form may bind the compound with higher affinity pathway information. In order to ascertain the appropriate than the other, or a compound that is principally an antago methods to analyze the effects of an allelic variance, Vari nist for one allele may be a partial agonist for another allele. ances, or haplotypes in vitro, one skilled in the art will Thus genes encoding proteins Structurally related to the review existing information on molecular biology, cell biol target protein should be Screened for variance in order to ogy, genetics, biochemistry; and physiology. Such texts are Successfully realize the methods of the present invention. A useful Sources for general and Specific information on the good general pharmacology text is Goodman & Gilman's genetic and biochemical processes involved in disease and the Pharmacological Basis of Therapeutics (9th Ed) by J. G. in drug action, as well as experimental procedures that may Hardman, L. E. Limbird, P. B. Molinoff, R. W. Ruddon and be useful in performing in vitro research on an allelic A. G. Gilman (Editors) McGraw Hill, 1996. There are also variance, variances, or haplotype. texts that focus on the pharmacology of drugs for Specific 0365 Texts on gene structure and function and RNA disease areas, or specific classes of drugs (e.g. natural biochemistry will be useful in evaluating the consequences products) or adverse drug interactions, among other Sub of variances that do not change the coding sequence (silent jects. Specific examples include: variances). Such variances may alter the interaction of RNA 0354) The American Psychiatric Press Textbook of with proteins or other regulatory molecules affecting RNA Psychopharmacology (2nd edition) by A. F. processing, polyadenylation, or export. Schatzberg & C. B. Nemeroff (Editors), American 0366 Molecular and Cellular Biology Psychiatric Press, 1998. 0355 Essential Psychopharmacology. Neuroscien 0367 Molecular Cell Biology by H. Lodish, D. tific Basis and Practical Applications by N. Muntner Baltimore, A. Berk, L. Zipurksy & J. Damell, W H and S. M. Stahl, Cambridge Univ Press, 1996. Freeman & Co., 1995. 0356. There are also texts on pharmacogenetics which are 0368 Essentials of Molecular Biology, D. Freifelder particularly useful for identifying genes which may contrib and Malacinski.Jones and Bartlett, 1993. ute to variable pharmacokinetic response. In addition there 0369 Genes and Genomes. A Changing Perspec are texts on Some of the major Xenobiotic metabolizing tive, M. Singer and P. Berg, 1991. University Science proteins, Such as the cytochrome P450 genes. Books 0357 Pharmacogenetics of Drug Metabolism 0370 Gene Structure and Expression, J. D. Hawk (International Encyclopedia of Pharmacology and ins, 1996. Cambridge University Press Therapeutics) by Werner Kalow (Editor) Pergamon Press, 1992. 0371 Molecular Biology of the Cell, 2nd edition, B. 0358 Genetic Factors in Drug Therapy: Clinical Alberts et al., Garland Publishing, 1994. and Molecular Pharmacogenetics by D. A Price 0372 Molecular Genetics Evans, Cambridge Univ Press, 1993. 0373) The Metabolic and Molecular Bases of Inher 0359 Pharmacogenetics (Oxford Monographs on ited Disease by C. R. Scriver, A. L. Beaudet, W. S. Medical Genetics, 32) by W. W. Weber, Oxford Univ Sly (Editors), 7th edition, McGraw Hill, 1995 Press, 1997. 0374 Genetics and Molecular Biology, R. Schleif, 0360 Cytochrome P450:Structure, Mechanism, and 1994. 2nd edition, Johns Hopkins University Press Biochemistry by P. R. Ortiz de Montellano (Editor), Plenum Publishing Corp, 1995. 0375 Genetics, P. J. Russell, 1996. 4th edition, 0361) Appleton & Lange's Review of Pharmacy, 6" Harper Collins edition, (Appleton & Lange's Review Series) by G. 0376 An Introduction to Genetic Analysis, Griffiths D. Hall & B. S. Reiss, Appleton & Lange, 1997. et al. 1993. 5th edition, W. H. Freeman and Company 0362 Genetics, Biochemistry and Molecular Biology 0377 Understanding Genetics. A molecular Literature approach, Rothwell, 1993. Wiley-Liss US 2001/0034023 A1 Oct. 25, 2001 39

0378 General Biochemistry column 3), and constitutes an excellent entry point for identification of references that point to the broader litera 0379 Biochemistry, L. Stryer, 1995. W. H. Freeman ture. Another useful site at NCBI is the Entrez browser, and Company located at http://www3.ncbi.nlm.nih.gov/Entrez/. One can 0380 Biochemistry, D. Voet and J. G. Voet, 1995. Search genomes, polynucleotides, proteins, 3D Structures, John Wiley and Sons taxonomy or the biomedical literature (PubMed) via the EntreZ Site. More generally links to a number of useful Sites 0381 Principles of Biochemistry, A. L. Lehninger, with biomedical or genetic data are maintained at Sites Such D. L. Nelson, and M. M. Cox, 1993. Worth Publish as MedWeb at the Emory University Health Sciences Center CS Library: http://WWW.MedWeb. Emory.Edu/MedWeb/: 0382 Biochemistry, G. Zubay, 1998. Wm. C. Brown Riken, a Japanese web site at: http:/www.rtc.riken.go.jp/ Communications othersite.html with links to DNA sequence, structural, molecular biology, bioinformatics, and other databases, at 0383 Biochemistry, C. K. Mathews and K. E. van the Oak Ridge National Laboratory web site: http://ww Holde, 1990. Benjamin/Cummings w.ornil.gov/hgmis/links.html; or at the Yahoo website of 0384 Transcription Diseases and Conditions: http://dir.yahoo.com/health/dis 0385 Eukaryotic Transcriptiuon Factors, D. S. eases and conditions/index.html. Each of the indicated web Latchman, 1995. Academic Press Sites has additional useful links to other sites. 0400 Another type of database with utility in selecting 0386 Eukaryoticy Gene Transcription,p S. Goodboum the genes on a biochemical pathway that may affect the (ed.), 1996. Oxford University Press. response to a drug are databases that provide information on 0387 Transcription Factors and DNA Replication, biochemical pathways. Examples of Such databases include D. S. Pederson and N. H. Heintz, 1994. CRC Press/ the Kyoto Encyclopedia of Genes and Genomes (KEGG), R. G. Landes Company which can be found at: http://www.genome.ad.jp/kegg/keg g.html. This site has pictures of many biochemical path 0388 Transcriptional Regulation, S. L. McKnight ways, as well as links to other metabolic databaseS Such as and K. Yamamoto (eds.), 1992. 2 volumes, Cold the well known Boehringer Mannheim biochemical path Spring Harbor Laboratory Press ways charts: http://www.expasy.ch/cgi-bin/Search-biochem 0389) RNA index. The metabolic charts at the latter Site are comprehen Sive, and excellent starting points for working out the Salient 0390 Control of Messenger RNA Stability, J. enzymes on any given pathway. Belasco and G. Brawerman (eds.), 1993. Academic PreSS 04.01 Each of the web sites mentioned above has links to 0391) RNA-Protein Interactions, Nagai and Mattai other useful web sites, which in turn can lead to additional (eds.), 1994. Oxford University Press sites with useful information. Research Libraries 0402 Those skilled in the art will often require informa 0392 mRNA Metabolism and Post-transcriptional tion found only at large libraries. The National Library of Gene Regulation, Harford and Morris (eds.), 1997. Medicine (http://www.nlm.nih.gov/) is the largest medical Wiley-Liss library in the World and its catalogs can be Searched online. 0393 Translation Other libraries, such as university or medical school libraries are also useful to conduct Searches. Biomedical bookS Such 0394 Translational Control, J. W. B. Hershey, M. B. as those referred to above can often be obtained from online Mathews, and N. Sonenberg (eds.), 1995. Cold bookstores as described above. Spring Harbor Laboratory Press 0395 General Physiology 0403 Biomedical Literature 04.04 To obtain up to date information on drugs and their 0396 Textbook of Medical Physiology 9" Edition by mechanism of action and biotransformation; disease patho A. C. Guyton and J. E. Hall W. B. Saunders, 1997 physiology, biochemical pathways relevant to drug action 0397 Review of Medical Physiology, 18" Edition by and disease pathophysiology; and genes that encode proteins W. F. Ganong, Appleton and Lange, 1997 relevant to drug action and disease one skilled in the art will consult the biomedical literature. A widely used, publicly 0398. Online Databases accessible web site for Searching published journal articles 0399. Those skilled in the art are familiar with how to is PubMed (http://www.ncbi.nlm.nih.gov/PubMed/). At this Search the biomedical literature, Such as, e.g., libraries, Site, one can Search for the most recent articles (within the online PubMed, abstract listings, and online mutation data last 1-2 months) or older literature (back to 1966). Many bases. One particularly useful resource is maintained at the Journals also have their own sites on the worldwide web and web site of the National Center for Biotechnology Informa can be searched online. For example see the IDEAL web site tion (ncbi): http:/www.ncbi.nlm.nih.gov/. From the ncbisite at: http://www.apnet.com/www/ap/aboutid.html. This site is one can access Online Mendelian Inheritance in Man an online library, featuring full text journals from Academic (OMIM). OMIM can be found at: http:// Press and selected journals from W. B. Saunders and www3.ncbi.nlm.nih.gov/Omim/searchomim.html. OMIM is Churchill Livingstone. The site provides access (for a fee) to a medically oriented database of genetic information with nearly 2000 Scientific, technical, and medical journals. entries for thousands of genes. The OMIM record number is 04.05 Experimental Methods for Identification of Genes provided for many of the genes in Tables 1, 3, and 4 (see Involved in the Action of a Drug US 2001/0034023 A1 Oct. 25, 2001 40

0406. There are a number of experimental methods for Subcellular localization and protein-protein interaction. identifying genes and gene products that mediate or modu Methods for measuring the proteome, or Some part of it, are late the effects of a drug or other treatment. They encompass known in the art. One widely used method is to extract total analyses of RNA and protein expression as well as methods cellular protein and Separate it in two dimensions, for for detecting protein-protein interactions and protein example first by Size and then by isoelectric point. The ligand interactions. Two preferred experimental methods for resulting protein Spots can be stained and quantitated, and identification of genes that may be involved in the action of individual spots can be excised and analyzed by mass a drug are (1) methods for measuring the expression levels spectrometry to provide definitive identification. The results of many mRNA transcripts in cells or organisms treated with can be compared from two or more cell lines or tissues, at the drug (2) methods for measuring the expression levels of least one of which has been treated with a drug. The many proteins in cells or organisms treated with the drug. differential up or down modulation of Specific proteins in response to drug treatment may indicate their role in medi 04.07 RNA transcripts or proteins that are substantially ating the pharmacologic actions of the drug. Another way to increased or decreased in drug treated cells or tissues rela identify the network of proteins that mediate the actions of tive to control cells or tissues are candidates for mediating a drug is to exploit methods for identifying interacting the action of the drug. Preferably the level of an mRNA is proteins. By Starting with a protein known to be involved in at least 30% higher or lower in drug treated cells, more the action of a drug-for example the drug target-one can preferably at least 50% higher or lower, and most preferably use Systems. Such as the yeast two hybrid System and variants two fold higher or lower than levels in non-drug treated thereof (known to those skilled in the art; see Ausubel et al., control cells. The analysis of RNA levels can be performed Current Protocols in Molecular Biology, op.cit.) to identify on total RNA or on polyadenylated RNA selected by oligodT additional proteins in the network of proteins that mediate affinity. Further, RNA from different cell compartments can drug action. The genes encoding Such proteins would be be analyzed independently-for example nuclear VS. cyto useful for Screening for DNA sequence variances, which in plasmic RNA. In addition to RNA levels, RNA kinetics can turn may be useful for analysis of interpatient variation in be examined, or the pool of RNAS currently being translated response to treatments. For example, the protein 5-lipoxy can be analyzed by isolation of RNA from polysomes. Other genase (5LO) is an enzyme which is at the beginning of the useful experimental methods include protein interaction leukotriene biosynthetic pathway and is a target for anti methods Such as the yeast two hybrid System and variants inflammatory drugs used to treat asthma and other diseases. thereof which facilitate the detection of protein-protein In order to detect proteins that interact with 5-lipoxygenase interactions. Preferably one of the interacting proteins is the the two-hybrid System was recently used to isolate three drug target or another protein Strongly implicated in the different proteins, none previously known to interact with action of the compound being assessed. 5LO. (Provost et al., Interaction of 5-lipoxygenase with 0408. The pool of RNAS expressed in a cell is sometimes cellular proteins. Proc. Natl. Acad. Sci. U.S.A. 96: 1881 referred to as the transcriptome. Methods for measuring the 1885, 1999.) A recent collection of articles Summarizing transcriptome, or Some part of it, are known in the art. A Some current methods in proteomics appeared in the August recent collection of articles Summarizing Some current meth 1998 issue of the journal Electrophoresis (volume 19, num ods appeared as a Supplement to the journal Nature Genetics. ber 11). Other useful articles include: Blackstock WP), et al. (The Chipping Forecast. Nature Genetics Supplement, Vol Proteomics: quantitative and physical mapping of cellular ume 21, January 1999.) A preferred method for measuring proteins. Trends Biotechnol. 17 (3): p. 121-7, 1999, and expression levels of mRNAS is to spot PCR products cor Patton W. F., Proteome analysis II. Protein subcellular responding to a large number of Specific genes on a nylon redistribution: linking physiology to genomics via the pro membrane such as Hybond N Plus (Amersham-Pharmacia). teome and Separation technologies involved. J. Chromatogr Total cellular mRNA is then isolated, labeled by random B Biomed Sci App. 722(1-2):203-23. 1999. oligonucleotide priming in the presence of a detectable label 0411 Since many of these methods can also be used to (e.g. alpha 33P labeled radionucleotides or dye labeled assess whether specific polymorphisms are likely to have nucleotides), and hybridized with the filter containing the biological effects, they are also relevant in Section 3, below, PCR products. The resulting Signals can be analyzed by concerning methods for assessing the likely contribution of commercially available Software, Such as can be obtained variances in candidate genes to clinical variation in patient from Clontech/Molecular Dynamics or Research Genetics, responses to therapy. Inc. 0412. 2. Screen for Variances in Genes that may be 04.09 Experiments have been described in model systems Related to Therapeutic Response that demonstrate the utility of measuring changes in the transcriptome before and after changing the growth condi 0413 Having identified a set of genes that may affect tions of cells, for example by changing the nutrient envi response to a drug the next Step is to Screen the genes for ronment. The changes in gene expression help reveal the variances that may account for interindividual variation in network of genes that mediate physiological responses to the response to the drug. There are a variety of levels at which altered growth condition. Similarly, the addition of a drug to a gene can be Screened for variances, and a variety of the cellular or in vivo environment, followed by monitoring methods for variance Screening. The two main levels of the changes in gene expression can aid in identification of variance Screening are genomic DNA Screening and cDNA gene networks that mediate pharmacological responses. Screening. Genomic variance detection may include Screen ing the entire genomic Segment spanning the gene from 2 kb 0410 The pool of proteins expressed in a cell is some to 10 kb upstream of the transcription start site to the times referred to as the proteome. Studies of the proteome polyadenylation site, or 2 to 10 kb beyond the polyadeny may include not only protein abundance but also protein lation Site. Alternatively genomic variance detection may US 2001/0034023 A1 Oct. 25, 2001

(for intron containing genes) include the exons and Some 0416 Methods for variance screening have been region around them containing the Splicing Signals, for described, including DNA sequencing. See for example: example, but not all of the intronic Sequences. In addition to U.S. Pat. No. 5,698,400: Detection of mutation by resolvase Screening introns and eXons for variances it is generally cleavage; U.S. Pat. No. 5,217,863: Detection of mutations in desirable to Screen regulatory DNA sequences for variances. nucleic acids; and U.S. Pat. No. 5,750,335: Screening for Promoter, enhancer, Silencer and other regulatory elements genetic variation, as well as the examples and references have been described in human genes. The promoter is cited therein for examples of useful variance detection generally proximal to the transcription Start Site, although procedures. Detailed variance detection procedures are also there may be Several promoters and Several transcription described in examples 15, 16 and 17. One skilled in the art Start Sites. Enhancer, Silencer and other regulatory elements will recognize that depending on the Specific aims of a may be intragenic or may lie outside the introns and exons, variance detection project (number of genes being screened, possibly at a considerable distance, Such as 100 kb away. number of individuals being screened, total length of DNA Variances in Such Sequences may affect basal gene expres being Screened) one of the above cited methods may be Sion or regulation of gene expression. In either case Such preferable to the others, or yet another procedure may be variation may affect the response of an individual patient to optimal. A preferred method of variance detection is chain a therapeutic intervention, for example a drug, as described terminating DNA sequencing using dye labeled primers, in the examples. Thus in practicing the present invention it cycle Sequencing and Software for assessing the quality of is useful to Screen regulatory Sequences as well as tran the DNA sequence as well as Specialized Software for calling Scribed Sequences, in order to identify variances that may heterozygotes. The use of Such procedures has been affect gene transcription. Frequently the genomic Sequence described by NickerSon and colleagues. See for example: of a gene can be found in the Sources above, particularly by Rieder M. J., et al. Automating the identification of DNA searching GenBank or Medline (PubMed). The name of the variations using quality-based fluorescence re-Sequencing: gene can be entered at a site Such as EntreZ: http://ww analysis of the human mitochondrial genome. Nucleic Acids w.ncbi.nlm.nih.gov/EntreZ/nucleotide.html. Using the Res. 26 (4):967-73, 1998, and: Nickerson D. A., et al. genomic Sequence and information from the biomedical Poly Phred: automating the detection and genotyping of literature one skilled in the art can perform a variance Single nucleotide Substitutions using fluorescence-based detection procedure Such as those described in examples 15, resequencing. Nucleic Acids Res. 25 (14):2745-51, 16 and 17. 1997. Although the variances provided in Tables 3, and 4 consist principally of cDNA variances, it is an aspect of this 0414 Variance detection is often first performed on the invention that detection of genomic variances is also a useful cDNA of a gene for several reasons. First, available data on method for identification of variances that may account for functional Sequence variances Suggests that variances in the interpatient variation in response to a therapy. transcribed portion of a gene may be most likely to have functional consequences as they can affect the interaction of 0417. Another important aspect of variance detection is the transcript with a wide variety of cellular factors during the use of DNA from a panel of human subjects that the complex processes of RNA transcription, processing and represents a known population. For example, if the Subjects translation, with consequent effects on RNA splicing, Sta are being Screened for variances relevant to a specific drug bility, translational efficiency or other processes. Second, as development program it is desirable to include both Subjects a practical matter the cDNA sequence of a gene is often with the target disease and healthy Subjects in the panel, available before the genomic structure is known, although because certain variances may occur at different frequencies the reverse will be true in the future as the sequence of the in the healthy and disease populations and can only be human genome is determined. Third, the cDNA is often reliably detected by Screening both populations. Also, for compact compared to the genomic locus, and can be example, if the drug development program is taking place in Japan, it is important to include Japanese individuals in the Screened for variances with much less effort. If the genomic Screening population. In general, it is always desirable to Structure is not known then only the cDNA sequence can be include Subjects of known geographic, racial or ethnic Scanned for variances. Methods for preparing cDNA are identity in a variance Screening experiment So the results can described in Example 14. Methods for variance detection on be interpreted appropriately for different patient populations, cDNA are described below and in the examples. if necessary. Also, in order to Select optimal Sets of variances 0415. In general it is preferable to catalog genetic varia for genetic analysis of a gene locus it is desirable to know tion at the genomic DNA level because there are an increas which variances have occurred recently-perhaps on mul ing number of well documented instances of functionally tiple different chromosomes-and which are ancient. Inclu important variances that lie outside of transcribed Sequence. Sion of one or more apes or monkeys in the variance Also, to properly use optimal genetic methods to assess the Screening panel is one way of gaining insight into the contribution of a candidate gene to variation in a phenotype evolutionary history of variances. Chimpanzees are pre of interest it is desirable to understand the character of ferred Subjects for inclusion in a variance Screening panel. Sequence variation in the candidate gene: what is the nature 0418 3. Assess the Likely Contribution of Variances in of linkage disequilibrium between different variances in the Candidate Genes to Clinical Variation in Patient Responses gene, are there sites of recombination within the gene; what to Therapy is the extent of homoplasy in the gene (i.e. occurrence of two variant Sites that are identical by State but not identical by 0419. Once a set of genes likely to affect disease patho descent because the same variance arose at least twice in physiology or drug action has been identified, and those human evolutionary history on two different haplotypes); genes have been Screened for variances, said variances (e.g., what are the different haplotypes and how can they be provided in Tables 3, and 4) can be assessed for their grouped to increase the power of genetic analysis? contribution to variation in the pharmacological or toxico US 2001/0034023 A1 Oct. 25, 2001 42 logical phenotypes of interest. Such studies are useful for protein with a treatment method, Such an administered reducing a large number of candidate variances to a Smaller compound, will likely be variable between different patients. number of variances to be tested in clinical trials. There are One skilled in the art will recognize that the application of Several methods which can be used in the present invention computational tools to the identification of functionally for assessing the medical and pharmaceutical implications of consequential variances involves applying the knowledge a DNA sequence variance. They range from computational and tools of medicinal chemistry and physiology to the methods to in vitro and/or in Vivo experimental methods, to analysis. prospective human clinical trials, and also include a variety of other laboratory and clinical measures that can provide 0422 Phylogenetic approaches to understanding evidence of the medical consequences of a variance. In Sequence variation are also useful. Thus if a Sequence general, human clinical trials constitute the highest Standard variance occurs at a nucleotide or encoded amino acid of proof that a variance or Set of variances is useful for residue where there is usually little or no variation in Selecting a method of treatment, however, computational homologs of the protein of interest from non-human species, and in vitro data, or retrospective analysis of human clinical particularly evolutionarily remote species, then the variance data may provide Strong evidence that a particular variance is more likely to affect function of the RNA or protein. will affect response to a given therapy, often at lower cost Computational methods for phylogenetic analysis are and in less time than a prospective clinical trial. Moreover, known in the art, (see below for citations of Some methods). at an early Stage in the analysis when there are many 0423 Computational methods are also useful for analyz possible hypotheses to explain interpatient variation in treat ing DNA polymorphisms in transcriptional regulatory ment response, the use of informatics-based approaches to Sequences, including promoters and enhancers. One useful evaluate the likely functional effects of Specific variances is approach is to compare variances in potential or proven an efficient way to proceed. transcriptional regulatory Sequences to a catalog of all 0420 Informatics-based approaches to the prediction of known transcriptional regulatory Sequences, including con the likely functional effects of variances include DNA and Sensus binding domains for all transcription factor binding protein Sequence analysis (phylogenetic approaches and domains. See, for example, the databaseS cited in: BurkS, C. motifsearching) and protein modeling (based on coordinates Molecular Biology Database List. Nucleic Acids Research in the protein database, or pdb; See http://www.rcsb.org/pdb/ 27: 1-9, 1999, and links to useful databases on the internet ), See, for example: Kawabata et al. The Protein Mutant at: http://www.oup.co.uk/nar/Volume 27/issue 01/sum Database. Nucleic Acids Research 27: 355-357, 1999; also mary/gk.c105 gml.html. In particular see the Transcrip available at: http://pmd.ddb.nig.ac.ip. Such analyses can be tion Factor Database (Heinemeyer, T, et al. (1999) performed quickly and inexpensively, and the results may Expanding the TRANSFAC database towards an expert allow Selection of certain genes for more extensive in vitro System of regulatory molecular mechanisms. Nucleic Acids or in vivo studies or for more variance detection or both. Res. 27: 318-322, or on the internet at: http:// 0421. The three dimensional structure of many medically 193.175.244.40/TRANSFAC/index.html). Any sequence and pharmaceutically important proteins, or homologs of variances in transcriptional regulatory Sequences can be Such proteins in other Species, or examples of domains assessed for their effects on mRNA levels using standard present in Such proteins, is known as a result of X-ray methods, either by making plasmid constructs with the crystallography Studies and, increasingly, nuclear magnetic different allelic forms of the Sequence, transfecting them into resonance Studies. Further, there are increasingly powerful cells and measuring the output of a reporter transcript, or by tools for modeling the Structure of proteins with unsolved assays of cells with different endogenous alleles of vari Structure, particularly if there is a related (homologous) ances. One example of a polymorphism in a transcriptional protein with known Structure. (For reviews See: Rost et al., regulatory element that has a pharmacogenetic effect is Protein fold recognition by prediction-based threading, J. described by Drazen et al. (1999) Pharmacogenetic associa Mol. Biol. 270:471-480, 1997; Firestine et al., Threading tion between ALOX5 promoter genotype and the response to your way to protein function, Chem. Biol. 3:779-783, 1996) anti-asthma treatment. Nature Genetics 22: 168-170. Drazen There are also powerful methods for identifying conserved and co-workers found that a polymorphism in an Sp1 domains and Vital amino acid residues of proteins of transcription factor binding domain, which varied among unknown Structure by analysis of phylogenetic relationships. Subjects from 3-6 tandem copies, accounted for varied (Deleage et al., Protein structure prediction: Implications for expression levels of the 5-lipoxygenase gene when assayed the biologist, Biochimie 79:681-686, 1997; Taylor et al., in vitro in reporter construct assays. This effect would have Multiple protein structure alignment, Protein Sci. 3:1858 been flagged by an informatics analysis that Surveyed the 1870, 1994) These methods can permit the prediction of 5-lipoxygenase candidate promoter region for transcrip functionally important variances, either on the basis of tional regulatory Sequences (resulting in discovery of poly Structure or evolutionary conservation. For example, a crys morphism in the Sp1 motif). tal Structure can reveal which amino acids comprise a Small 0424. 4. Perform in vitro or in vivo Experiments to molecule binding Site. The identification of a polymorphic amino acid variance in the topological neighborhood of Such ASSeSS the Functional Importance of Gene Variances a site, and, in particular, the demonstration that at least one 0425 There are two broad types of studies useful for variant form of the protein has a variant amino acid which assessing the likely importance of variances: analysis of impinges on (or which may otherwise affect the chemical RNA or protein abundance (as described above in the environment around) the Small molecule binding pocket context of methods for identifying candidate genes for differently from another variant form, provides Strong evi explaining interpatient variation in treatment response) or dence that the variance may affect the function of the analysis of functional differences in different variant forms protein. From this it follows that the interaction of the of a gene, mRNA or protein. Studies of functional differ US 2001/0034023 A1 Oct. 25, 2001

ences may involve direct measurements of biochemical genetic background among the Subjects is Substantially activity of different variant forms of an mRNA or protein, or Similar. In particular, in cell lines from a pedigree it is known may involve assaying the influence of a variance or vari that only four parental alleles are Segregating in the children, ances on various cell properties, including both tissue cul and that any two children are on average 50% genetically ture and in Vivo Studies. identical. In a more heterogeneous genetic background (i.e. 0426. The Selection of an appropriate experimental pro cell lines from unrelated subjects) the effect of allelic gram for testing the medical consequences of a variance may variation at multiple genes that modulate the measured drug differ depending on the nature of the variance, the gene, and response phenotypes is more likely to create a nearly con the disease. For example if there is already evidence that a tinuous distribution of responses (except in cases where the protein is involved in the pharmacologic action of a drug, product of one gene accounts for most of the measured drug then the in vitro or in Vivo demonstration that an amino acid response phenotype). variance in the protein affects its biochemical activity is 0429 Many cell lines have been derived from groups of Strong evidence that the variance will have an effect on the related individuals, or pedigrees. A commercial Source of pharmacology of the drug in patients, and therefore that such cell lines is the Human Genetic Cell Respository, patients with different variant forms of the gene may have supported by the National Institute of General Medical different responses to the Same dose of drug. If the variance Sciences (NIGMS) and housed at the Coriell Cell Reposi is Silent with respect to protein coding information, or if it tory, Camden, N.J. A directory of these cell lines is available lies in a noncoding portion of the gene (e.g., a promoter, an on the world wide web: http://locus.umdnj.edu/nigms/. One intron, or a 5'- or 3'-untranslated region) then the appropriate preferred Set of cell lines for pharmacogenetic Studies, biochemical assay may be to assess mRNA abundance, half available from the Coriell Cell Repository, is the set of cell life, or translational efficiency. If, on the other hand, there is lines used by the Centre d’Etudes du Polymorphisme no Substantial evidence that the protein encoded by a par Humain (CEPH) consortium (Paris, France) to establish a ticular gene is relevant to drug pharmacology, but instead is detailed genetic map of man. See, for example: Gyapay, G., a candidate gene on account of its involvement in disease Morissette, J., Vignal, A., et al. (1994) The 1993-94 Gene pathophysiology, then the optimal test may be a clinical thon human genetic linkage map. Nature Genetics 7(2 Spec Study addressing whether two patient groups distinguished No):246-339. More current data on the CEPH genetic link on the basis of the variance respond differently to a thera age map can be found on the World wide web at: http:// peutic intervention. This approach reflects the current reality landru.cephb.fr/cephdb/. Lymphoblastoid cell lines from 57 that biologists do not Sufficiently understand gene regula CEPH families are available from the Coriell Repository. In tion, gene expression and gene function to consistently make most cases the families consist of four grandparents, two accurate inferences about the consequences of DNA parents and between four and twelve children. Sequence variances for pharmacological responses. 0430. The principal attraction of the CEPH cell lines for 0427. In Summary, if there is a plausible hypothesis pharmacogenetic Studies is that a detailed genetic map of regarding the effect of a protein on the action of a drug, then nearly 12,000 polymorphic markers has been established via in vitro and in Vivo approaches, including those described an international effort, and the map data are freely available below, will be useful to predict whether a given variance is on the world wide web. In other words the genotypes of therapeutically consequential. If, on the other hand, there is thousands of polymorphic markers are known in most of the no evidence of Such an effect, then the preferred test is an CEPH cell lines (not all markers were studied in all cell empirical clinical measure of the impact to the variance on lines). As a result, one skilled in the art can determine the efficacy or toxicity in Vivo (which requires no evidence or chromosomal location of any locus that controls a Mende assumptions regarding the mechanism by which the variance lian trait in these cell lines, using Software for linkage may exert an effect on a therapeutic response). However, analysis such as the programs LINKAGE, CRIMAP and given the expense and Statistical constraints of clinical trials, MAPMAKER. (See, for example: Lander, E. S., Green, P., it is preferable to limit clinical testing to variances for which Abrahamson, J., et al. (1987) MAPMAKER: an interactive there is at least Some experimental or computational evi computer package for constructing primary genetic linkage dence of a functional effect. maps of experimental and natural populations. Genomics 0428. In another aspect of the invention a powerful, high 1(2): 174-81. See also: Terwilliger, J. and J. Ott (1994), throughput approach to the genetics of drug response is to Handbook of Human Linkage Analysis. John Hopkins Uni Study variation in drug response phenotypes among cell versity Press, Baltimore for a more exhaustive description of lines derived from related individuals. Consider a cellular linkage analysis methods.) drug response phenotype that is readily measured, and that 0431 One set of interesting Mendelian traits to study varies among cell lines. The demonstration of Mendelian using the CEPH cell lines (or similar cell lines from pedi transmission of the drug response phenotype in cell lines grees) and the genetic approach just described are drug from related individuals would constitute evidence of a response phenotypes. Consider, for example, a G protein genetic component to the drug response phenotype. The coupled receptor that exists in two allelic forms that behave expected pattern of Segregation depends on making an differently in the presence of a compound being developed assumption about the genetic model: recessive, dominant or for human clinical use (e.g. one receptor binds the com co-dominant alleles will produce different proportions in the pound with higher affinity than the other). Methods for progeny of a croSS. The value of Studying cell lines as assaying G protein mediated Signal transduction are well Surrogates for people is that experiments can be performed known in the art. By adding the compound (either at a fixed for a small fraction of the cost. The value of studying cell concentration or at a series of different concentrations) to a lines from related individuals is that genetic effects on drug family-derived set of lymphoblastoid cell lines (which of response are likely to be much easier to identify when course must express the G protein coupled receptor) and US 2001/0034023 A1 Oct. 25, 2001 44 measuring the Signal produced it should be possible to detect cell lines, the effort already expended to produce a human the Segregation of the grandparental alleles in the parents linkage map in the CEPH cell lines can be exploited. and the Segregation of parental alleles in the children. For 0434 Cell responses that could be usefully characterized example, consider two alleles of the receptor: if allele A by the above methods include for example the level of produces a greater Signal than allele B at a given concen Signaling in a pathway that mediates the response to a tration of the compound, and if one parent is an AB compound (as in the G protein coupled receptor assays heterozygote while the other parent is a BB heterozygote where levels of a second messenger are measured), com then the levels of signal in the children should be medium (in pound uptake, compound metabolism, levels of metabolites AB heterozygotes) or low (in BB homozygotes). The detec affected by a compound, levels of proteins (including tion of such a pattern in cell lines of the family would enzymes in biochemical pathways related to the action of the constitute evidence that the G protein coupled receptor compound), levels of an inhibitory complex formed by a polymorphism was responsible for interSubject differences compound, and other assays known to those skilled in the art in response to the compound. (More generally, the detection of pharmacology and assay development. For example, a of any discrete partitioning of responses in the data-high Study of the genetic basis of variation in response to the and low, or high medium and low-is Suggestive of genetic antineoplastic drug 5-fluorouracil might include measure control, with the genetic model to be inferred from the ment of cell uptake of 5-FU, conversion of 5-FU to inactive pattern of inheritance, and Support for the hypothesis to metabolites such as 5,6-dihydrofluorouridine or fluoro-beta come from the analysis of multiple families.) It is not alanine, conversion of 5-FU to active metabolites Such as necessary to know the identify of the variant gene in advance 5-fluorodeoxyuridine, levels of thymidylate synthetase (an (as in the G protein coupled receptor example just provided). enzyme inhibited by 5-FU), levels of 5, 10 methylenetet The pattern of Segregation of the drug response phenotype in rahydrofolate (a folate co-factor essential for 5-FU mediated the cell lines of the various members of the CEPH families inhibition of thymidylate synthetase) and the enzymes that can be compared to the pattern of Segregation of the thou produce it, or levels of nucleotide pools or the enzymes that Sands of polymorphic markers already typed in the same cell produce them. All of the relevant transporters and enzymes lines. are expressed in lymphoblastoid cells, even though 5-FU is 0432 Those polymorphic markers that co-segregate with not routinely used in the therapy of lymphoid malignancies. the drug response phenotype are candidates for marking the 0435 However, a limitation of lymphoblastoid cell lines location of the locus responsible for the drug response for the methods described above is that they are not suitable phenotype. By performing the same experiment in cell lines for all of the different types of assays one might wish to from multiple (e.g. from two up to 57 CEPH) families the perform. One alternative is to use fibroblast cell lines, which list of candidate polymorphic markers generally narrows to have already been derived from multiple different families. a few, all of which (or nearly all of which) are from the same Fibroblasts are not available from the CEPH pedigrees, chromosomal region-viz. the region harboring the gene however a set of fibroblasts from known pedigrees could be responsible for the drug response phenotype. Knowing (i) genotyped at a Set of highly polymorphic markers to produce the chromosomal location of the gene (or genes) implicated a genetic map. Another approach is to treat lymphoblastoid by the linkage analysis, together with (ii) information about cells with a procedure or agent that induces differentiation to the location and function of genes in that chromosomal a different cell type, Such as an adipocye or a myocyte. For region (available from online databases, for example, those example, there are genes which effectively control differen at the U.S. National Center for Biotechnology Information; tiation programs (e.g. peroxisome proliferator activated See http://www.ncbi.nlm.nih.gov/LocusLink?), and further receptor PPARgamma mediates adipocyte differentiation, (iii) knowing Something of the pharmacology of the com myoD mediates myocyte differentiation); introduction of pound and consequently the metabolic and regulatory path Such a gene into a cell line of one type can alter its ways likely to influence its action, should constrain the list differentiated State to another cell type. Alternatively, Stimu of candidate genes likely to be responsible for the observed lation of the gene product of Such a regulatory gene (e.g. variation to a small number of genes. These genes (if there treatment of cells with the PPARgamma agonist troglita is more than one) can be Systematically evaluated for Zone) can be used to induce differentiation to a different cell pharmacogenetic impact by identifying polymorphisms and type. Such procedures are known in the art, and may be testing whether they coSegregate with drug response phe effectively applied to human lymphoblasts. notypes in the pedigrees, in new pedigrees, in cells from unrelated individuals, or in Vivo in a population of nonre 0436. In preferred embodiments of the above methods the lated individuals, for example in a clinical trial. cells used are from the CEPH pedigrees. Preferably at least one pedigree is Studied, more preferably two pedigrees, Still 0433 Some drug response phenotypes may not behave as more preferably five pedigrees and most preferably eight Mendelian traits, but may rather be continuous (quantitative) pedigrees or more. It is useful to perform a Statistical traits under the control of Several genes. Variation at any of calculation to determine how many pedigrees and cell lines the relevant gene loci could affect drug response, often to should be Studied to achieve a given power to detect an different extents. Robust methods for mapping quantitative effect, making assumptions about the magnitude of the trait loci (QTL) are known in the art. For example, see: Shugart, Y.Y. and Goldgar, D. E. (1999) Multipoint genomic effect. Scanning for quantitative loci: effects of map density, Sibship 0437. In another aspect, described below, the methods Size and computational approach. Eur J Hum Genet described above can be used to identify mRNAS that vary in 7(2):103-9. It is worth emphasizing that in the approach levels between cell lines as a result of genetically controlled described (using the CEPH cell lines) there is no need for regulatory factors, Such as, for example, polymorphisms in genotyping in order to map the drug response traits in the promoters that affect the binding or action of transcriptional US 2001/0034023 A1 Oct. 25, 2001

regulatory factors. Such variation in mRNA levels may be individuals as a result of genetically controlled factors. responsible for interSubject variation in drug response. Genetic factors include both cis-acting polymorphisms, Such as might be present in promoters (e.g. polymorphisms that 0438 Experimental Methods: Genomic DNA Analysis affect the binding or action of transcription factors) as well 0439 Variances in DNA may affect the basal transcrip as trans-acting factorS Such as might be present in transcrip tion or regulated transcription of a gene locus. Such vari tion factors (e.g. an amino acid polymorphism that affects ances may be located in any part of the gene but are most the interaction of a transcription factor with a promoter likely to be located in the promoter region, the first intron, element, or that might affect levels of the transcription factor or in 5" or 3' flanking DNA, where enhancer or silencer itself). Variation in mRNA levels may contribute to inter elements may be located. Methods for analyzing transcrip Subject variation in drug response, disease Susceptibility or tion are well known to those skilled in the art and exemplary disease manifestations. (See above for example of promoter methods are briefly described above and in some of the texts polymorphism in 5-lipoxygenase and its effect on response cited elsewhere in this application. Transcriptional run off assay is one useful method. Detailed protocols can be found to anti-asthma medications.) in texts such as: Current Protocols in Molecular Biology 0444 The methods for identifying mRNAS which vary in edited by: F. M. Ausubel, et al. John Wiley & Sons, Inc, abundance as a consequence of genetic mechanisms are 1999, or: Molecular Cloning. A Laboratory Manual by J. Similar to those described above for drug response pheno Sambrook, E. F. Fritsch and T Maniatis. 1989. 3 vols, 2nd types. First, by examining whether levels of an mRNA edition, Cold Spring Harbor Laboratory Press Segregate in one or more pedigrees it is possible to infer 0440 Experimental Methods: RNA Analysis whether there is a genetic component to the variation. Second, by inspecting the CEPH genotype data it is possible 0441) RNA variances may affect a wide range of pro to identify genetic markers that coSegregate with the mRNA ceSSes including RNA Splicing, polyadenylation, capping, expression levels (either increased or decreased) and thereby export from the nucleus, interaction with translation initia map the chromosomal location of the locus or loci that tion, elongation or termination factors, or the ribosome, or control mRNA levels. Third, by inspection of the genes at interaction with cellular factors including regulatory pro the chromosomal locus controlling mRNA levels it should teins, or factors that may affect mRNA half life. However, be possible to identify one or a few genes that are likely the effect of most RNA sequence variances on RNA func responsible for the effect. These genes can then be defini tion, if any, Should ultimately be measurable as an effect on tively evaluated by discovering variances and testing if they RNA or protein levels--either basal levels or regulated predict mRNA levels (or other phenotypes) in the pedigree levels or levels in Some abnormal cell State, Such as cells cell lines, in cell lines from unrelated individuals, or in Vivo. from patients with a disease. Therefore, one preferred Fourth, the above analysis can be performed on cell lines method for assessing the effect of RNA variances on RNA Subjected to various pharmacological or nutritional manipu function is to measure the levels of RNA produced by lations. For example cell lines from one or more pedigrees different alleles in one or more conditions of cell or tissue can be treated with a drug, or deprived of an amino acid and growth. Said measuring can be done by conventional meth mRNA levels measured at various times after treatment. Any ods such as Northern blots or RNAase protection assays variable differences in mRNA levels in response to the (kits available from Ambion, Inc.), or by methods such as treatment, if they Segregate in pedigrees, can be Subjected to the Taqman assay (developed by the Applied BioSystems StepS 1-3. Fifth, this analysis can be performed at very large Division of the Perkin Elmer Corporation), or by using scale using arrays of gridded cDNAS, PCR products or arrays of oligonucleotides or arrays of cDNAS attached to oligonucleotides corresponding to an unlimited number of solid surfaces. Systems for arraying cDNAS are available genes. In each experiment the RNA from the pedigree cell commercially from companies Such as Nanogen and General lines (treated or not) is isolated, labeled using standard Scanning. Complete Systems for gene expression analysis methods and hybridized to the grids containing the nucleic are available from companies Such as Molecular Dynamics. acids corresponding to the genes being investigated. Current For recent reviews of systems for high throughput RNA commercial methods permit up to 400,000 oligonucleotides expression analysis see the Supplement to Volume 21 of (more than the total number of human genes) to be queried Nature Genetics entitled “The Chipping Forecast', espe in one experiment, although lower density formats are also cially articles beginning on pages 9, 15, 20 and 25. well Suited to the methods described. Thus, in a compara 0442. Additional methods for analyzing the effect of tively modest number of experiments the entire transcript variances on RNA include Secondary Structure probing, and population of lymphoblasts (probably <25,000 unique tran direct measurement of half life or turnover. Secondary Scripts) can be queried for genetically controlled variation in Structure can be determined by techniques Such as enzymatic mRNA abundance. Other types of cell lines can be subjected probing (using enzymes Such as T1, T2 and S1 nuclease), to Similar analysis. chemical probing or RNAase H probing using oligonucle 0445. The variation in mRNA levels due to gene poly otides. Most RNA structural assays are performed in vitro, morphisms is likely to be of Small magnitude (generally however Some techniques can be performed on cell extracts two-fold differences or less are expected). Therefore a key or even in living cells, using fluorescence resonance energy aspect of experimental Systems used to measure mRNA transfer to monitor the state of RNA probe molecules. levels is their accuracy. Preferably a System capable of 0443) In another aspect the methods described above resolving mRNAs that differ in abundance (measured in (relating to the use of cell lines from pedigrees to genetically molecules per cell, or relative to a Standard Such as total map phenotypes that can be studied in tissue culture cells) mRNA or one or more specific RNAS Such as actin or can be used to identify mRNAS that vary in levels between clathrin or glucose-6-phosphare dehydrogenase) is Suffi US 2001/0034023 A1 Oct. 25, 2001 46 ciently sensitive to detect differences as small as 50%, more transgenic methods, or a combination of approaches may be preferably as Small as 30%, and most preferably as small as used. To create animal Strains with targeted gene disruptions 20%. a DNA construct is created (using DNA sequence informa tion from the host animal) that will undergo homologous 0446. There are 757 individuals in the 57 CEPH cell recombination when inserted into the nucleus of an embry lines. Thus all the CEPH cell lines could fit in eight 96 well onic Stem cell. The targeted gene is effectively inactivated microtiter plates. Microtiter plates provide a convenient due to the insertion of non-natural Sequence-for example a format for growing cells and for performing cell manipula translation Stop codon or a marker gene Sequence that tions, Such as those described above, using multichannel interrupts the reading frame. Well known PCR based meth pipettes or automated pipetting robots. By growing cells in ods are then used to Screen for those cells in which the large Volume flasks, counting them (by hemocytometer or desired homologous recombination event has occurred. Coulter counter or other means) and then aliquoting them Gene knockouts can be accomplished in Worms, droSophila, robotically to 96 well plates it is possible to assure that each mice or other organisms. Once the knockout cells are created well has nearly the Same number of cells. A large number of (in whatever species) the candidate therapeutic intervention plates can be prepared in this way and then Stored frozen in can be administered to the animal and pharmacological or appropriate medium until needed for experiments. biological responses measured, including gene expression 04.47 Experimental Methods: Protein Analysis levels. If variant forms of the gene are useful in explaining interpatient variation in response to the compound in man, 0448. There are a variety of experimental methods for then complete absence of the gene in an experimental investigating the effect of an amino acid variance on organism should have a major effect on drug response. As a response of a patient to a treatment. The preferred method next step various human forms of the gene can be introduced will depend on the availability of cells expressing a particu into the knockout organism (a technique Sometimes referred lar protein, and the feasibility of a cell-based assay VS. assays to as a knock-in). Again, pharmacological Studies can be on cell extracts, on proteins produced in a foreign host, or on performed to assess the impact of different human variances proteins prepared by in Vitro translation. on drug response. Methods relevant to the experimental 0449 For example, the methods and systems listed below approaches described above can be found in the following can be utilized to demonstrate differential expression, Sta exemplary texts: bility and/or activity of different variant forms of a protein, 0454 General Molecular Biology Methods or in phenotype/genotype correlations in a model System. 04.55 Molecular Biology. A project approach, S.J. 0450 For the determination of protein levels or protein Karcher, Fall 1995. Academic Press activity a variety of techniques are available. The in vitro protein activity can be determined by transcription or trans 0456 DNA Cloning: A Practical Approach, D. M. lation in bacteria, yeast, baculovirus, COS cells (transient), Glover and B. D. Hayes (eds). 1995. IRL/Oxford Chinese Hamster Ovary (CHO) cells, or studied directly in University Press. Vol. 1-Core Techniques; Vol human cells, or other cell Systems can be used. Further, one 2-Expression Systems; Vol. 3-Complex can perform pulse chase experiments to determine if there Genomes; Vol. 4-Mammalian Systems. are changes in protein Stability (half-life). 0457 Short Protocols in Molecular Biology, 0451 One skilled in the art can construct cell based Ausubel et al. October 1995. 3rd edition, John Wiley assays of protein function, and then perform the assays in and Sons cells with different genotypes or haplotypes. For example, identification of cells with different genotypes, e.g., cell 0458 Current Protocols in Molecular Biology lines established from families and Subsequent determina Edited by: F. M. Ausubel, R. Brent, R. E. Kingston, tion of relevant protein phenotypes (e.g., expression levels, D. D. Moore, J. G. Seidman, K. Struhl, (Series post translational modifications, activity assays) may be Editor: V. B. Chanda), 1988 performed using Standard methods. 0459 Molecular Cloning. A laboratory manual, J. 0452 Assays of protein levels or function can also be Sambrook, E. F. Fritsch. 1989. 3 vols, 2nd edition, performed on cell lines (or extracts from cell lines) derived Cold Spring Harbor Laboratory Press from pedigrees in order to determine whether there is a genetic component to variation in protein levels or function. 0460 Polymerase Chain Reaction (PCR) The experimental analysis is as above for RNAS, except the 0461) PCR Primer. A laboratory manual, C. W. assays are different. Experiments can be performed on naive Diffenbach and G. S. Dveksler (eds.). 1995. Cold cells or on cells Subjected to various treatments, including Spring Harbor Laboratory Press. pharmacological treatments. 0462) The Polymerase Chain Reaction, K. B. Mullis 0453. In another approach to the study of amino acid et al. (eds.), 1994. Birkhauser variances one can express genes corresponding to different alleles in experimental organisms and examine effects on 0463 PCR Strategies, M. A. Innis, D. H. Gelf, and disease phenotype (if relevant in the animal model), or on J. J. Sninsky (eds.), 1995. Academic Press response to the presence of a compound. Such experiments may be performed in animals that have disrupted copies of 0464 General Procedures for Discipline Specific Studies the homologous gene (e.g. gene knockout animals engi 0465 Current Protocols in Neuroscience Edited by: neered to be deficient in a target gene), or variant forms of J. Crawley, C. Gerfen, R. McKay, M. Rogawski, D. the human gene may be introduced into germ cells by Sibley, P. Skolnick, (Series Editor: G. Taylor), 1997. US 2001/0034023 A1 Oct. 25, 2001 47

0466 Current Protocols in Pharmacology Edited many patients will need to be enrolled to have adequate by: S. J. Enna/M. Williams, J. W. Ferkany, T. Statistical power to measure an effect of a specified magni Kenakin, R. E. Porsolt, J. P. Sullivan, (Series Editor: tude (power analysis), (iii) definition of any primary or G. Taylor),1998. Secondary genetic endpoints, and (iv) definition of methods of Statistical genetic analysis, as well as other aspects. In the 0467 Current Protocols in Protein Science Edited outline below Some of the major types of genetic hypothesis by: J. E. Coligan, B. M. Dunn, H. L. Ploegh, D. W. testing, power analysis and Statistical testing and their appli Speicher, P. T. Wingfield, (Series Editor: Virginia cation in different Stages of the drug development process Benson Chanda), 1995. are reviewed. One skilled in the art will recognize that 0468. Current Protocols in Cell Biology Edited by: certain of the methods will be best Suited to specific clinical J. S. Bonifacino, M. Dasso, J. Lippincott-Schwartz, Situations, and that additional methods are known and can be J. B. Harford, K. M. Yamada, (Series Editor: K. used in particular instances. Morgan) 1999. 0475 A. Performing a Clinical Trial: Overview 0469 Current Protocols in Cytometry Managing 0476 AS used herein, a “clinical trial' is the testing of a Editor: J. P. Robinson, Z. Darzynkiewicz (ed)/P. therapeutic intervention in a Volunteer human population for Dean (ed), A. Orfao (ed), P. Rabinovitch (ed), C. the purpose of determining whether it is Safe and/or effica Stewart (ed), H. Tanke (ed), L. Wheeless (ed), cious in the treatment of a disease, disorder, or condition. (Series Editor: J. Paul Robinson), 1997. The present invention describes methods for achieving Supe 0470 Current Protocols in Human Genetics Edited rior efficacy and/or Safety in a genetically defined Subgroup by: N. C. Dracopoli, J. L. Haines, B. R. Korf, et al., defined by the presence or absence of at least one gene (Series Editor: A. Boyle), 1994. Sequence variance, compared to the effect that could be 0471 Current Protocols in Immunology Edited by: obtained in a conventional trial (without genetic Stratifica J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. tion). M. Shevach, W. Strober, (Series Editor: R. Coico), 0477. A “clinical study' is that part of a clinical trial that 1991. involves determination of the effect of a candidate thera peutic intervention on human Subjects. It includes clinical 0472. IV. Clinical Trials evaluation of physiologic responses including pharmacoki 0473 A clinical trial is the definitive test of the utility of netic (bioavailability as affected by drug absorption, distri a variance or variances for the Selection of optimal therapy. bution, metabolism and excretion) and pharmacodynamic A clinical trial in which an interaction of gene variances and (physiologic response and efficacy) parameters. A pharma clinical outcomes (desired or undesired) is explored will be cogenetic clinical Study (or clinical trial) is a clinical study referred to herein as a “pharmacogenetic clinical trial'. that involves testing of one or more specific hypotheses Pharmacogenetic clinical trials require no knowledge of the regarding the interaction of a genetic variance or variances biological function of the gene containing the variance or (or set of variances, i.e. haplotype or haplotypes) on variances to be assessed, nor any knowledge of how the response to a therapeutic intervention. Pharmacogenetic therapeutic intervention to be assessed works at a biochemi hypotheses are formulated before the Study, and may be cal level. The pharmacogenetics effects of a variance can be articulated in the Study protocol in the form of primary or addressed at a purely Statistical level: either a particular Secondary endpoints. For example an endpoint may be that variance or Set of variances is consistently associated with a in a particular genetic Subgroup the rate of objectively Significant difference in a Salient drug response parameter defined responses exceeds the response rate in a control (e.g. response rate, effective dose, Side effect rate, etc.) or group (either the entire control group or the Subgroup of not. On the other hand, if there is information about either controls with the same genetic Signature as the treatment the biochemical basis of a therapeutic intervention or the Subgroup they are being compared to) or exceeds that in the biochemical effects of a variance, then a pharmacogenetic whole treatment group (i.e. without genetic stratification) by clinical trial can be designed to test a specific hypothesis. In Some predefined relative or absolute amount. preferred embodiments of the methods of this application 0478 For a clinical study to commence enrollment and the mechanism of action of the compound to be genetically proceed to treat Subjects at an institution that receives any analyzed is at least partially understood. federal Support (most medical institutions in the U.S.), an 0474 Methods for performing clinical trials are well application that describes in detail the Scientific premise for known in the art. (see e.g. Guide to Clinical Trials by Bert the therapeutic intervention and the procedures involved in Spilker, Raven Press, 1991; The Randomized Clinical Trial the Study, including the endpoints and analytical methods to and Therapeutic Decisions by Niels Tygstrup (Editor), Mar be used in evaluating the data, must be reviewed and cel Dekker; Recent Advances in Clinical Trial Design and accepted by a review panel, often termed an Institutional Analysis (Cancer Treatment and Research, Ctar 75) by Peter Review Board (IRB). Similarly any clinical study that will F. Thall (Editor) Kluwer Academic Pub, 1995. Clinical ultimately be evaluated by the FDA as part of a new drug or Trials. A Methodologic Perspective by Steven Piantadosi, product application (or other application as described Wiley Series in Probability and Statistics, 1997). However, below), must be reviewed and approved by an IRB. The IRB performing a clinical trial to test the genetic contribution to is responsible for determining that the trial protocol is Safe, interpatient variation in drug response entails additional conforms to established ethical principles and guidelines, design considerations, including (i) defining the genetic has risks proportional to any expected benefits, assures hypothesis or hypotheses, (ii) devising an analytical Strategy equitable Selection of patients, provides Sufficient informa for testing the hypothesis, including determination of how tion to patients (via a consent form) to insure that they can US 2001/0034023 A1 Oct. 25, 2001 48 make an informed decision about participation, and insures in the different responder or nonrespondergroups measured. the privacy of participants and the confidentiality of any data One or more gene Sequence variances or a combination of collected. (See the report of the National Commission for variances and/or haplotypes may be Studied. Protection of Human Subjects of Biomedical and Behavioral 0482. The informed consent document is a description of Research (1978). The Belmont Report: Ethical Principles the therapeutic intervention and the clinical protocol in and Guidelines for the Protection of Human Subjects of Simple language (e.g. third grade level) for the patient to Research. Washington, D.C.: DHEW Publication Number read, understand, and, if willing, agree to participate in the (OS) 78-0012. For a recent review see: Coughlin, S. S. (ed.) Study by Signing the document. In a pharmacogenetic clini (1995) Ethics in Epidemiology and Clinical Research. Epi cal Study the informed consent document will describe, in demiology Resources, Newton, Mass.) The European coun Simple language, the use of a genetic test or a limited Set of terpart of the U.S. FDA is the European Medicines Evalu genetic tests to determine the Subject or patient's genotype ation Agency (EMEA). Similar agencies exist in other at a particular gene variance or variances, and to further countries and are responsible for insuring, via the regulatory ascertain whether, in the Study population, particular vari process, that clinical trials conform to Similar Standards as ances are associated with particular clinical or physiological are required in the U.S. The documents reviewed by an IRB responses. The consent form should also describe proce include a clinical protocol containing the information dures for assuring privacy and confidentiality of genetic described above, and a consent form. information. 0479. It is also customary, but not required, to prepare an investigator's brochure which describes the scientific 0483 The U.S. FDA reviews proposed clinical trials hypothesis for the proposed therapeutic intervention, the through the process of an Investigational New Drug Appli preclinical data, and the clinical protocol. The brochure is cation (IND). The IND is composed of the investigator's made available to any physician participating in the pro brochure, the Supporting in vitro and in Vivo animal or posed or ongoing trial. previous human data, the clinical protocol, and the informed consent forms. In each of the sections of the IND, a specific 0480. The supporting preclinical data is a report of all the description of a Single allelic variance or a number of in vitro, in Vivo animal or previous human trial or other data variances to be tested in the clinical study will be included. that Supports the Safety and/or efficacy of a given therapeutic For example, in the investigator's brochure a description of intervention. In a pharmacogenetic clinical trial the preclini the gene or genes hypothesized to account, at least in part, cal data may also include a description of the effect of a for differential responses will be included as well as a Specific genetic variance or variances on biochemical or description of a genetic variance or variances in one or more physiologic experimental variables in vitro or in Vivo, or on candidate genes. Further, the preclinical data may include a treatment outcomes, as determined by in Vivo Studies in description of in vivo, in vitro or in silico studies of the animals or humans (for example in an earlier trial), or by biochemical or physiologic effects of a variance or variances retrospective genetic analysis of clinical trial or other medi (e.g., haplotype) in a candidate gene or genes, as well as the cal data (see below) used to formulate or strengthen a predicted effects of the variance or variances on efficacy or pharmacogenetic hypothesis. For example, case reports of toxicology of the candidate therapeutic intervention. The unusual pharmacological responses in individuals with rare results of retrospective genetic analysis of response data in alleles (e.g. mutant alleles), or the observation of clustering patients treated with the candidate therapy may be the basis of pharmacological responses in family members may pro for formulating the genetic hypotheses to be tested in the vide the rationale for a pharmacogenetic clinical trial. prospective trial. The U.S. FDA reviews applications with particular attention to Safety and toxicological data to ascer 0481. The clinical protocol provides the relevant scien tain whether candidate compounds should be tested in tific and therapeutic introductory information, describes the humans. inclusion and exclusion criteria for human Subject enroll ment, including genetic criteria if relevant (e.g. if genotype 0484. The established phases of clinical development are is to be among the enrollment criteria), describes in detail Phase I, II, III, and IV. The fundamental objectives for each the exact procedure or procedures for treatment using the phase become increasingly complex as the Stages of clinical candidate therapeutic intervention, describes laboratory development progreSS. In Phase I, Safety in humans is the analyses to be performed during the Study period, and primary focus. In these Studies, dose-ranging designs estab further describes the risks (both known and possible) involv lish whether the candidate therapeutic intervention is Safe in ing the use of the experimental candidate therapeutic inter the Suspected therapeutic concentration range. However, it is vention. In a clinical protocol for a pharmacogenetic clinical common practice to obtain information about Surrogate trial, the clinical protocol will further describe the genetic markers of efficacy even in phase I clinical trials. In a variance and/or variances hypothesized to account for dif pharmacogenetic clinical trial there may be an analysis of ferential responses in the normal human Subjects or patients the effect of a variance or variances on Phase I safety or and Supporting preclinical data, if any, a description of the Surrogate efficacy parameters. At the same time, evaluation methods for genotyping, genetic data collection and data of pharmacokinetic parameters (e.g., adsorption, distribu handling as well as a description of the genetic Statistical tion, metabolism, and excretion) may be a secondary objec analysis to be performed to measure the interaction of the tive; again, in a pharmacogenetic clinical Study there may be variance or variances with treatment response. Further, the an analysis of the effect of Sequence variation in genes that clinical protocol for a pharmacogenetic clinical trial will affect absorption, distribution, metabolism and excretion of include a description of the genetic Study design. For the candidate compound on pharmacokinetic parameters example patients may be stratified by genotype and the such as peak blood levels, half life or tissue distribution of response rates in the different groups compared, or patients the compound. AS clinical development Stages progress, trial may be Segregated by response and the genotype frequencies objectives focus on the appropriate dose and method of US 2001/0034023 A1 Oct. 25, 2001 49 administration required to elicit a clinically relevant thera ment and control groups are balanced for race, SeX and age peutic response. In a pharmacogenetic clinical trial, there composition and other non-genetic factors relevant to dis may be a comparison of the effectiveness of Several doses of ease will be Sufficient to assure that genetic background is a compound in patients with different genotypes, in order to controlled, however a preferred practice is to explicitly test identify interactions between genotype and optimal dose. for genetic Stratification between test and control groups. For this purpose the doses Selected for late Stage clinical Methods for minimizing the possibility of spurious results testing may be greater, equal or less than those chosen based attributable to genetic Stratification between two comparison upon preclinical Safety and efficacy determinations. Data on groups include the use of Surrogate markers of geographic, the function of different alleles of genes affecting pharma racial and/or ethnic background, Such as have been cokinetic parameters could provide the basis for Selecting an described by Rannala and coworkers. (See, for example: optimal dose or range or doses of a compound or biological. Rannala B, and J L Mountain. 1997 Detecting immigration Genes involved in drug metabolism may be particularly by using multilocus genotypes. Proc Natl AcadSci USA Aug useful to study in relation to understanding interpatient 19;94(17):91.97-201.) One procedure would be to assure that variation in optimal dose. Genes involved in drug metabo Surrogate markers of genetic background (Such as those lism include the cytochrome P450s, especially 2D6, 3A4, described by Rannala and Mountain) occur at comparable 2C9, 2E1, 2A6 and 1A1; the glucuronyltransferases; the frequency in two comparison groups. acetyltransferases, the methyltransferases, the Sulfotrans 0488 Open label trials are unblinded; in single blind ferases, the glutathione System; the flavine monooxygenases trials patients are kept unaware of treatment assignments, in and other enzymes known in the art. double blind trials both patients and investigators are 0485 An additional objective in the latter stages of unaware of the treatment groups, a combination of these clinical development is demonstration of the effect of the procedures may be instituted during the trial period. Phar therapeutic intervention on a broad population. In phase III macogenetic clinical trial design may include one or a trials, the number of individuals enrolled is dictated by a combination of open label, single blind, or double blind power analysis. The number of patients required for a given clinical trial designs. Reduction of biases attributable to the pharmacogenetic clinical trial will be determined by prior knowledge of either the type of treatment or the genotype of knowledge of variance or haplotype frequency in the Study the normal Subjects or patients is an important aspect of population, likely response rate in the treated population, Study design. So, for example, even in a Study that is Single expected magnitude of pharmacogenetic effect (for example, blind with respect to treatment, it should be possible to keep the ratio of response rates between a genetic Subgroup and both patients and caregivers blinded to genotype during the the unfractionated population, or between two different Study. genetic Subgroups); nature of the genetic effect, if known 0489. In designing a clinical trial it is important to (e.g. dominant effect, codominant effect, recessive effect); include termination endpoints Such as adverse clinical and number of genetic hypotheses to be evaluated (including events, inadequate Study participation either in the form of number of genes and/or variances to be Studied, number of lack of adherence to the clinical protocol or loSS to follow gene or variance interactions to be studied). Other consid up, (e.g. Such that adequate power is no longer assured), lack erations will likely arise in the design of Specific trials. of adherence on the part of trial investigators to the trial 0486 Clinical trials should be designed to blind both protocol, or lack of efficacy or positive response within the human Subjects and Study coordinators from biasing that test group. In a pharmacogenetic clinical trial these consid may otherwise occur during the testing of a candidate erations obtain not only in the entire treatment group, but therapeutic invention. Often the candidate therapeutic inter also in the genetically defined Subgroups. That is, if a vention is compared to best medical treatment, or a placebo dangerous toxic effect manifests itself predominantly or (a compound, agent, device, or procedure that appears exclusively in a genetically defined Subpopulation of the identical to the candidate therapeutic intervention but is total treatment population it may be deemed inappropriate to therapeutically inert). The combination of a placebo group continue treating that genetically defined Subgroup. Such and blind controls for potentially confounding factorS Such decisions are typically made by a data Safety monitoring as prejudice on the part of Study participants or investigators, committee, a group of experts not including the investiga insures that real, rather than perceived or expected, effects of tors, and generally not blinded to the analysis, who review the candidate therapeutic intervention are measured in the the data from an ongoing trial on a regular basis. trials. Ideally blinding extends not only to trial Subjects and 0490. It is important to note that medicine is a conser investigators but also to data review committees, ancillary Vative field, and clinicians are unlikely to change their perSonnel, Statisticians, and clinical trial monitors. behavior on the basis of a single clinical trial. Thus it is 0487. In pharmacogenetic clinical studies, a placebo arm likely that, in most instances, two or more clinical trials will or best medical control group may be required in order to be required to convince physicians that they should change ascertain the effect of the allelic variance or variances on the their prescribing habits in View of genetic information. efficacy or toxicology of the candidate therapeutic interven Large Scale trials represent one approach to providing tion as well as placebo or best medical therapy. It will be increased data Supporting the utility of a genetic Stratifica important to assure that the composition of the control and tion. In Such trials the Stringent clinical and laboratory data test populations are matched, to the degree possible, with collection characteristic of traditional trials is often relaxed respect to genetic background and allele frequencies. This is in exchange for a larger patient population. Important goals particularly true if the variances being investigated may in large Scale pharmacogenetic trials will include establish have an effect on disease manifestations (in addition to a ing whether a pharmacogenetic effect is detectable in all hypothesized effect on response to treatment). It is likely that Segments of a population. For example, in the North Ameri Standard clinical trial procedures Such as insuring that treat can population one might Seek to demonstrate a pharmaco US 2001/0034023 A1 Oct. 25, 2001 50 genetic effect in people of African, Asian, European and once human testing has begun and costs therefore mount Hispanic (i.e. Mexican and Puerto Rican) origin, as well as quickly. Timely information may allow a drug to be killed in native American people. (It generally will not be practical early, or may result in an accelerated program of clinical to Segment patients by geographical origin in a Standard trials. In addition to information about efficacy and Safety, it clinical trial, due to loss of power.) Another goal of a large is useful to have information about the existence and mag Scale clinical trial may be to measure more precisely, and nitude of genetic effects on efficacy and toxicity at the with greater confidence, the magnitude of a pharmacoge earliest possible Stage. If properly managed, genetically netic effect first identified in a smaller trial. Yet another determined heterogeneity in drug response may not be an undertaking in a large Scale clinical trial may be to examine obstacle to development. On the contrary, it may provide the the interaction of an established pharmacogenetic variable basis for identification of a patient population in whom both (e.g. a variance in gene A, shown to affect treatment high efficacy and Safety can be achieved. Clear delineation response in a smaller trial) with other genetic variances of Such a population may facilitate Smaller, more targeted (either in gene A or in other candidate genes). A large Scale trials and more rapid clinical development. Consequently, trial provides the Statistical power necessary to test Such the early identification of genetic determinants of drug interactions. response will, in the future, increasingly become a priority 0491 In designing all of the above stages of clinical of clinical development. testing investigators must be attentive to the Statistical 0497 Phase I trials are not necessarily confined to the problems raised by testing multiple different hypotheses, initial Stages of human clinical development. It is not including multiple genetic hypotheses, in Subsets of patients. unusual for Phase I trials to be initiated at a later Stage of Bonferroni’s correction or other Suitable statistical methods clinical development in order to, for example, clarify basic for taking account of multiple hypothesis testing will need to questions about clinical pharmacology that have arisen as a be judiciously applied. However, in the early Stages of result of Phase II study data. It may be that the most efficient clinical testing, when the main goal is to reduce the large way to advance the genetic understanding of pharmacologi number of potential hypotheses that could be tested to a few cal responses to a compound in Phase II is to perform a that will be tested, based on limited data, it may be imprac Phase I trial using a specific genetic design, as described tical to rigidly apply the multiple testing correction. below. 0492 B. Phase I Clinical Trials 0498 2. Phase I Trials Designed for Genetic Analysis 0499. In this invention we describe two exemplary novel 0493 1. Introduction methods for organization of Phase I trials that will facilitate 0494 Phase I clinical trials are generally designed pri identification and measurement of the genetic component of marily to establish a Safe dose and Schedule of administra variation in treatment response using modest numbers of tion for a new compound. At the same time, Phase I is the subjects. We describe how these methods can be practiced first opportunity to Study the clinical pharmacology of a new by Selectively enrolling Subjects who share genetic charac compound in man. Relevant Studies may include aspects of teristics, either as a result of a familial relationship or as a pharmacokinetic behavior, Side effects and toxicity. In addi result of genetic homogeneity at candidate loci believed to tion to these well established purposes, Phase I trials are affect response to the candidate treatment. We show how the increasingly being used to gather information relevant to analysis of Such individuals Substantially increases the early assessment of efficacy. Such information can be useful power of genetic analysis compared to analysis of unrelated in making an early yes/no decision about the further devel individuals. We also describe methods for operating a Phase opment of a compound, or a family of related compounds, I unit capable of carrying out the novel genetic analyses all being tested simultaneously in Phase I trials. Since Phase 0500. The two types of Pharmacogenetic Phase I Units I trials are typically conducted in normal volunteers (com described in this application will be referred to as the pounds for cancer and Some other terminal diseases are an Pharmacogenetic Phase I Relatives Unit and the Pharmaco exception), Surrogate markers of drug effect are measured, genetic Phase I Outliers Unit, or the Relatives Unit and the rather than disease response. The development of Sophisti Outliers Unit for short. The term Pharmacogenetic Phase I cated Surrogate markers of pharmacodynamic effects has Unit will be used to refer to both types of Phase I Unit. The allowed more information on efficacy to be gathered in Relatives Unit requires a population comprised of groups of Phase I, and this trend will almost certainly continue as basic related individuals. The related individuals may be parents understanding of disease pathophysiology increases, and as and offspring, groups of SibS, or of cousins, or any mixture more products are developed for disease prophylaxis. of these or other groups of related individuals. The Outliers Unit requires the initial enrollment of a large number of 0495 Phase I studies are typically performed on a small unrelated Volunteers (at least Several hundreds of Subjects, number (<60) of healthy volunteers. Consequently, Phase I preferably at least one thousand, more preferably at least five Studies as currently designed are not amenable to genetic thousand, and most preferably ten thousand or more indi analysis: the number of Subjects is simply too small to viduals) willing to provide DNA for genotyping on an detect, with adequate Statistical certainty, any genetic effects as-needed basis (many of these volunteers will never par on drug response that are short of all or none in magnitude. ticipate in a trial). Subsequently, Small numbers of individu In fact, no genetic analyses of Phase I Studies have been als are drawn from this large population for Specific clinical published or described in public meetings. trials, based on their genetic homogeneity at candidate loci 0496 AS described in detail elsewhere in this application, believed likely to account for interSubject variation in it is highly desirable to gather the information necessary to response to the candidate compound. make informed decisions about clinical development as 0501) The concept underlying these two types of Phar early as possible in the development process, particularly macogenetic Phase I Units is similar: the idea is to recruit US 2001/0034023 A1 Oct. 25, 2001 multiple Small groups of Subjects who are genetically more candidate loci is required at all (albeit knowledge of the homogeneous than would be possible with Standard nonge relevant genes is required to develop a useful genetic netic recruitment criteria. If there is a genetic component to diagnostic test at a later Stage). Thus, the Relatives Unit treatment response then there should be more intragroup provides a clear picture of the importance of heredity factors homogeneity and more intergroup heterogeneity in drug in determining drug response, regardless of our understand response measures (e.g. Surrogate measures of drug ing of the mechanism of action of the drug, or any other response) than would be expected by chance, and there aspect of drug pharmacology. should be Statistically significant differences in drug response measures between the different groups. The mag 0506 The rationale is as follows: if a surrogate drug nitude of Such differences can provide an estimate of the response trait (i.e., a Surrogate marker of pharmacodynamic magnitude of the genetic component of interSubject varia effect that can be measured in normal Subjects) is under tion in drug response. genetic control, then related individuals, Such as SibS (who share 50% of their alleles at autosomal loci on average), 0502. 3. Pharmacogenetic Phase I Relatives Unit should have more similar responses than unrelated individu als, who share a much Smaller fraction of alleles. In other 0503. In the Pharmacogenetic Phase I Relatives Unit, one words, individuals who share more alleles at the loci that is comparing groups of related individuals to each other and affect drug response should be more Similar to each other to other groups of related individuals. The underlying than individuals who, on average, share fewer alleles. By assumption is that one can assess the magnitude of the using Statistical methods known in the art the distribution of genetic component of variation in drug response (if any) by traits of related individuals can be compared to the degree of comparing drug response traits in related individuals with variation in a set of unrelated individuals. The potential for those of unrelated individuals. Two types of effect would insight from this kind of analysis is reflected in the fact that Suggest the presence of a genetic component to variation in twin studies (in which traits of identical twins are compared drug response measures. First, the distribution of drug to those of fraternal twins) indicate that differences among responses in related individuals may be different from that individuals in pharmacokinetic variables (e.g. compound observed in the entire group, or in a group comprised of half life, peak concentration) can be strongly genetically unrelated individuals. For example, a Statistically significant determined. (For a Summary of Such pharmacokinetic Stud narrowing of the distribution (e.g. Smaller Standard deviation ies, see Propping, P. 1978 Pharmacogenetics. Rey: Physiol. in groups of related individuals compared to unrelated Biochem. Pharmacol. 83: 123-173.) Such studies are impor individuals) would indicate that individuals who share alle tant because they clearly reveal genetic determination of les are more Similar to each other than individuals who do pharmacogenetic traits (although they may overestimate its not share (as many) alleles, implying that the drug response degree; see Falconer, D. S. and Mackay, T. 1996 Introduc trait is partially affected by a heritable factor or factors. tion to Quantitative Genetics, Addison Wesley Longman Second, the mean value of the drug response measure (whether blood pressure or a cognitive test) may vary Ltd.). between groups of related individuals, indicating that dif 0507 The type of study proposed here, whether it ferent alleles at loci relevant to drug response are present in involves comparison of parents and offspring, groups of the different families. (Note that the relevant trait is not Sibs, or other groups of relatives, will also reveal the extent blood pressure or cognition, but the response of blood of genetic determination, and without requiring twins. This pressure or cognition to a pharmacological intervention.) is a two-fold advantage; pairs of twins are more difficult to obtain than parent-child or Sib-Sib pairs, and one avoids the 0504 Individuals can be related in any of several ways, uncertainty about the genetic inferences gained from twin most preferably as parent and child or as Siblings. Parent analysis. child pairs, in particular, enable one to use simple Statistical techniques (e.g., regression) in order to assess the degree to 0508 Drug responses among related and unrelated indi which response to Surrogate markers is influenced by genetic viduals may be continuously or discretely distributed. In the differences among individuals. However, parent-child pairs former case, it is likely that many loci have Some effect on may be less Suitable for Some Surrogate markers, especially the trait, while in the latter case, variation could be attrib those related to candidate drugs used to treat age-related utable to Mendelian Segregation of alleles in a family (or disorders. In Such a context, one can readily use clusters of families) with, for example, AA homozygotes giving one Siblings and/or cousins, uncle/nephew pairs or other groups phenotype and Aa heterozygotes and aa homozygotes giving of related individuals to assess the degree of genetic deter a Second phenotype, all in the context of a relatively mination of response to a Surrogate marker. homogeneous genetic background. 0505) An attractive aspect of the Pharmacogenetic Phase 0509. There is a wealth of analytical techniques known in I Relatives Unit (unlike the Outliers Unit) is that it does not the art that can be used to assess the mode of inheritance for require any laboratory tests to implement. One infers the a particular trait and to determine the degree to which degree of gene sharing between individuals from their differences among individuals are genetically determined. relationship to each other. A parent is 50% genetically These techniques include cluster analysis and discriminant identical to each of his or her children; sibs are 50% analysis used to define traits with variable expression and genetically identical to each other on average; uncles/aunts the fitting of a variety of genetic models to the data, are 25% identical to nieces/nephews on average, and So including generalized Single-locus models, mixed models in forth. Thus the degree to which two related individuals are which a trait is determined by a major locus and by many expected to be Similar as a result of genetic factorS is known. minor loci, and a So-called polygenic model in which many Therefore no tests to determine genetic Status are required loci contribute variation to the trait, the result being a (i.e. no genotyping); in fact, no knowledge of the relevant continuously-distributed phenotype (For further details, see US 2001/0034023 A1 Oct. 25, 2001 52

Eaves, L.J. 1977 Inferring the causes of human variation, early assessment of the extent of genetically determined Journal of the Royal Statistical Society A 140: 324-355 and variation in drug response for a given therapeutic com Cloninger, C. R. 1988 Complex Human Traits. Pp. 312 pound. Such information provides an informed basis for 317 in: Proceedings of the Second International Conference either stopping development at the earliest possible Stage or, on Quantitative Genetics, eds., B. S. Weir, E. J. Eisen, M. M. preferably, continuing development, but with a plan to Goodman, and G. Namkoong, Sinauer ASSociates, Inc). identify and control for genetic variation So as to allow rapid Specific Statistical techniques involved in the fitting and progression through the regulatory approval process. analysis of these genetic models are also well known in the 0514 For example, it is well known that clinical trials to art; they include parametric and nonparametric correlation, assess the efficacy of candidate drugs for Alzheimer's dis regression, and one-way and two-way analysis of variance ease are long and expensive, and most Such drugs are only (For further details, see Mather, K. and Jinks, J. L. 1977 effective in a fraction of patients. Using Surrogate measures Introduction to Biometrical Genetics, Cornell University of response in normals drawn from a population of related Press and Falconer, D. S. and Mackay, T. 1996 Introduc individuals might help to assess the contribution of genetic tion to Quantitative Genetics, Addison Wesley Longman variation to variation in treatment response. For an acetyl Ltd.) Many, perhaps most, traits of pharmacogenetic interest cholinesterase inhibitor, relevant Surrogate pharmacody will be continuously-distributed. In this context, the central namic measures might include testing erythrocyte mem Statistical comparison is one between the differences among brane acetylcholinesterase levels in drug treated normal average traits of different families (say, groups of Sibs), or Subjects, or testing performance on a psychometric test of among all the members of Several Such families, as com pared to the differences among traits within families (among Short term memory, or other measures that are affected by Sibs). If Such differences in So-called mean Squares are large treatment (and ideally that correlate with clinical efficacy). enough (as compared to the differences expected under the 0515 Similarly, antidepressant drugs can produce a vari null hypothesis of no family differences), one can infer that ety of effects on mood in normal Subjects. Careful measure there is a genetic component to differences among families. ment and Statistical analysis of Such responses in related and unrelated normal Subjects could provide an early indication 0510 Standard theory known in the art indicates that of whether there is a genetic component to drug response there is an inverse relationship between Study size and the (and hence clinical efficacy). The observation of significant ability to detect a given genetic effect. So, for example, variation among families would provide evidence of a assume that the 50% of the variation among individuals is pharmacogenetic effect and justify the Substantial expendi due to genetic differences. A Phase 1 trial composed of Sixty ture necessary for a full pharmacogenetic drug development individuals consisting of thirty parent-child pairs may or program. Conversely, the absence of any significant familial may not allow one to detect Such a genetic effect, given the influence on drug response in a Pharmacogenetics Relatives standard criterion for statistical significance (P<0.05), Unit could provide an early termination point for pharma depending on assumptions one makes about the number of cogenetic Studies. loci that have major effects. However, a trial composed of 120 individuals consisting of sixty parent-child pairs would 0516 Again, the proposed studies do not require any likely be Sufficient to provide Statistically Significant evi knowledge of candidate loci, nor is DNA collection or dence for a 50% heritable drug response effect. Once one genotyping required. One needs only a reliable Surrogate parent-child pair is recruited, it is generally advantageous pharmacodynamic assay and groups of related normal indi Statistically to add additional parent-child combinations as viduals. Standard Statistical methods should permit the mag opposed to adding additional children for a given parent. nitude of the pharmacogenetic effect to be estimated. It should be a criteria for deciding whether to proceed with 0511) If 75% or more of the variation in drug response more intensive, gene-focused pharmacogenetic analysis dur among individuals is due to genetic differences, a Phase 1 ing later Stages of development. trial composed of Sixty individuals consisting of thirty parent-child pairs would allow one to detect Such a genetic 0517. 4. Pharmacogenetic Phase I Outliers Unit effect, given the Standard criterion for Statistical Significance 0518. The prerequisites for a Pharmacogenetic Phase I (P<0.05). Outliers Unit, as well as the type of information that can be 0512 Similar calculations can be made if one analyzes obtained, differ in Several respects from a Pharmacogenetic Siblings in a Phase I trial, instead of using parent-child pairs. Phase I Relatives Unit. First, the Outliers Unit requires some These calculations indicate that the more powerful approach knowledge of the molecular pharmacology of the candidate for a Relatives Unit is generally to focus on parent-child compound-enough knowledge to Select at least one can pairs as opposed to the use of groups of Siblings, especially didate gene. Second, the Outliers Unit provides information if minimizing the number of Subjects is an objective of the on the effect, if any, of known genetic variation in the Study. However, the use of groups of Siblings may be candidate gene or genes on variation in the drug response necessary or preferable, especially if the trait in question is measures. This is advantageous in that it Sets the Stage for manifested only at a specific age. In Such a case, one can pharmacogenetic analysis in later Stages of clinical devel readily use Standard theory to compare alternative designs opment. Third, the Outliers Unit does not require recruit for the study. The overall point is that the statistical frame ment of relatives. Instead, one initially recruits a large work associated with the Relatives Unit will allow one to population of individuals from which Small Subsets are choose the approach that is best-Suited for a given trait. drawn as necessary for Specific trials based on their geno types. All of the individuals in the large population are 0513. In general, techniques for measuring whether phar initially asked to provide DNA samples (from blood or other macodynamic traits are under genetic control using Surro readily available tissue Such as buccal mucosa) which can gate markers of drug efficacy will be useful in obtaining an Subsequently be genotyped at candidate loci of potential US 2001/0034023 A1 Oct. 25, 2001

relevance to a particular candidate drug of interest. Over would perform a Phase I Study consisting of measuring a time a database of genotypes can be assembled, potentially Surrogate drug response in individuals with genotypes reducing the need for genotyping later. From this large AA/BB, aa/BB, AA/bb and aa/bb and then statistically collection of Subjects one then Selects a group of individuals comparing the distribution of a trait in each of these groups with genotypes expected to homogeneous for the drug with the distribution of the same trait in the other groups response trait of interest (assuming that the candidate and/or in the unfractionated (total) population. The statisti gene(s) play a significant role in drug response). The indi cal techniques for Such comparisons are known in the art and viduals with identical (and preferably homozygous) geno include parametric and nonparametric analyses to detect types at the candidate gene(s) might comprise a collection of differences in population averages, Such as the t-test and the the common genotypes or haplotypes, or they may include Mann-Whitney U test. If individuals of a given rare geno Some rare genotypes/haplotypes as well. The main point is type do have significantly different Surrogate drug responses that one can recruit groups consisting of any mixture of when compared to each other, or when compared to the rest genotypes or haplotypes in order to assess the role that of the population, one can infer that the locus likely affects variation in the candidate gene(s) may play in trait deter the trait. mination. In this method, then, one recruits a population for clinical genetic investigation utilizing methods in Statistical 0521. The size requirements of the source population of genetics to optimize the size and genetic composition of the individuals will depend on the range of allele frequencies to population. be analyzed. For example, if the allele frequencies for A and a are, say, 0.15 and 0.85, and for B and b are 0.2 and 0.8 then 0519. The mechanics of an Outlier Unit are as follows. the frequency of AA homozygotes is expected to be 2.25% Several thousand subjects are enrolled in the Outlier Unit and BB homozygotes 4%. In the absence of any linkage with the understanding that they provide a blood Sample between the loci, the frequency of AA/BB double homozy from which DNA is extracted and stored. Each time a new gotes is expected to be 0.0225x0.04=0.0009 or about one outlier Study is performed their Sample may be genotyped. subject in 1000. At least five subjects of each genotype (It will not be necessary to genotype all Subjects for all should be recruited for the Outlier Unit, and preferably at trials-just enough to identify Subjects with the desired least ten subjects. Thus, for studies of two loci in which the genotypes or haplotypes. Subjects may be paid a fee for each minor allele frequency for both loci is in the 0.15-0.20 range, genotyping analysis done on their Sample, regardless of the recruitment of individuals that are potential outliers for whether the sample is used.) Only rarely will a particular the trait under investigation (i.e., homozygotes at the can Subject have a genotype that meets the criteria for a specific didate loci) will require at least 1,000 individuals and outlier study (see below). When a match occurs, that subject preferably 5,000 or more. will be invited to participate in that Study. The genotyping done to identify subjects for a study will be determined by 0522 One of the most useful aspects of the Outlier Unit the candidate genes deemed relevant to pharmacology of the is that individuals with rare genotypes can be pharmaco candidate drug, and by the polymorphisms or haplotypes in logically assessed in a Small Study. This addresses a Serious those candidate genes. Ideally DNA samples from Several limitation of conventional clinical trials with respect to the thousand subjects will be arrayed in 96 or 384 well plates so investigation of polygenic traits or the effect of rare alleles. that the genotyping or haplotyping of large numbers of Even conventional Phase III studies, which typically have Subjects can be performed using automated methods. Any the largest number of patients, are usually of insufficient size highly accurate and inexpensive genotyping procedure will to address simple one-locus hypotheses about efficacy or Suffice, Such as the methods described elsewhere in this toxicity with adequate statistical power (e.g. 80% or 90% application. Clearly it is desirable to have a Stable population power). The problem is that for each new allele that must be for genotyping, given the investment required to recruit considered (e.g. five common haplotypes at a candidate Subjects, isolate and array DNA, and accumulate a database locus) the comparison groups are reduced and Statistical of genotype data. Since most Subjects will only rarely be power is diminished. It is therefore an especially challenging invited to participate in clinical trials, the ongoing partici problem to test the effect of multiple alleles at a single locus, pation of subjects in the Outliers Unit must be assured by let alone interaction of alleles at Several loci in determining other means-for example, by a modest annual payment for drug response. The Outlier Unit provides a way to efficiently remaining in the Outliers Unit, plus a fee for each occasion test for the effects of multiple alleles at a candidate locus on which their Sample is genotyped. (e.g. haplotypes), or to test for interactions between two or 0520. The power of the Outliers Unit lies in the ability to more candidate loci by allowing ready identification of rapidly enroll individuals with Virtually any desired geno groups of individuals who, on account of being homozygous type in a Phase I clinical trial. Suppose, for example, that one at one or several loci of interest, should be outliers for the wants to determine the drug response phenotype of indi drug response traits of interest. viduals homozygous for rare alleles at candidate loci. Con 0523 The information that can be gained from an Out sider a compound for which there are two loci believed liers Unit is of great value in designing Subsequent efficacy likely to influence response to treatment. The first locus has trials, as it provides a basis for constraining the number of alleles A and a, while the second has alleles B and b. If these hypotheses to be tested. In lieu of Such information, one is loci do in fact contribute significantly to treatment response compelled to Statistically test a variety of genetic models for then homozygotes would be expected to exhibit the most a number of candidate loci. The correction for multiple extreme responses (assuming a dominant or codominant testing necessitated by Such uncertainty about the genetic model). One could also measure epistatic (gene X gene) model is frequently large enough to put Statistically signifi interactions on the presumption that drug response measures cant results beyond reach. On the other hand, if the pheno might be extreme in individuals homozygous for Specific typic effect of each allele at a locus (or the effect of at least alleles of the two candidate genes. So, for example, one some alleles) is known from the Outliers Unit study, one is US 2001/0034023 A1 Oct. 25, 2001 54 then able to design a Phase II or Phase III study that tests a Smaller variance of one distribution reduces the overlap relatively Small number of genetic hypotheses, thereby con between it and the other trait distributions to which it is siderably improving the Statistical power of the genetic being compared). In fact, the assumption of identical vari analysis in efficacy trials. ances in the homozygote and total groups is not necessarily 0524 Consider a locus with two alleles, one with fre the biologically most likely case: it is reasonable to expect quency 0.95 and the other 0.05, as revealed by genotyping that the variance of the trait in the genetically more homo the individuals in the large Source population for the Outliers geneous group may be less (if the locus in question in fact Unit. The two alleles combine to make three genotypes contributes to variation in the drug response trait). This which are observed to differ in their response to a candidate effect would result in a Smaller population being adequate to compound of interest. There are Several Statistical compari show a genetically determined component to the difference Sons that one can undertake in order to determine whether in treatment effect between the two groups. different alleles at this locus are associated with differences 0526 Serious adverse effects occuring at low frequency in response. One is to compare the average response of, Say, are often detected in the later Stages of drug development. In individuals who are homozygous for the rare allele with the Some cases Such effects have a Significant genetic compo average response of individuals chosen at random from the nent. To address this issue preemptively, an Outlier Unit can Source population. In this instance, the Outlier Unit is perform trials in which Subjects are Selected to represent composed of a group of individuals with the rare genotype only the rare alleles at one or more loci that are candidates and an equal-sized group composed of random genotypes for influencing the response to treatment. For example, (including the rare genotype). (In general, equal group sizes variances occurring at 5% allele frequency are expected to are Statistically more efficient; they are not necessary, how occur in homozygous form in 0.25% of the population ever, which is fortunate Since Some alleles of interest might (0.05x0.05), and therefore may rarely, if ever, be encoun be So rare that finding, Say, even ten individuals who are tered in early clinical development. Yet Such Subjects could homozygous would be difficult.) A Second kind of Statistical readily be identified by genotyping the hundreds to thou comparison would be to compare equal-sized groups of the sands of patients enrolled in a Phase I Outliers Unit. three genotypes (AA, Aa, aa), in order to determine whether the presence or absence of a particular allele has a significant 0527. Alternatively, by insuring that all common geno effect on the drug response trait. In this instance, the Outlier types are represented in an Outlier Unit Study the contribu Unit is preferably composed of equal-sized groups of the tion of a major candidate locus can be tested with a powerful three genotypes. Statistical design. Consider a locus with five haplotypes, A, B, C, D and E, with frequencies 0.3, 0.25, 0.2, 0.15, and 0.05 0525 ASSume that being a homozygote for the rare allele (plus several additional alleles with frequency lower than of the locus described in the preceding paragraph causes a 0.05). A comparison of groups of homozygous for each of 15% average difference in a pharmacokinetic parameter the haplotypes-that is AA, BB, CC, DD and EE homozy (e.g., the area under curve of drug concentration in blood) as gotes—each group of equal size, provides a powerful design compared to random individuals. ASSume further that the to measure the contribution of variation at the candidate Outliers Unit has a total of sixty individuals, including thirty locus to variation in drug response In this case, determina individuals of the rare genotype and thirty individuals cho tion of Sample sizes rests upon assumptions about the Sen at random. Finally, assume that the variance of indi differences in average trait values for each haplotype. All vidual responses is identical within the two groupS and that other things being equal, detecting a difference is easiest it is equal to 0.1. Standard Statistical theory indicates that when a Subset of the haplotypes appears to be appreciably thirty individuals per group is not adequate to Statistically distinct from the rest. Such a situation allows one to make prove that there is a significant difference in average uptake a reasonably principled decision to lump haplotypes So that rate between the groups (P<0.05). Instead, with an increase one compares, Say, one haplotype with all of the others. In to 108 individuals in each group, one would be able to Such a circumstance, Sample size calculations for testing a provide statistical evidence for this effect. However, if we difference in average responses would be roughly similar to assume that homozygosity for an allele at the candidate those described above. More generally, one can assess the locus causes a 30% difference in area under curve then the overall heterogeneity of the traits associated with each number of individuals required to provide Statistical evi haplotype (say, with a parametric or nonparametric analysis dence for a difference between the two groups (for P-0.05 of variance) and one can also make individual comparisons and holding all other assumptions constant) is only twenty between haplotypes (by using a multiple comparison pro seven. The number of individuals required to detect a 60% difference in area under curve (all other assumptions con cedure if the initial analysis of variance reveals significant Stant) is only Seven. This calculation assumes that the loci in heterogeneity) The identification of genetically determined question affect only the average trait in each of the two phenotypic variation at Such a locus the can reduce the groupS and that the shapes of the trait distribution are likelihood of discrepant results due to genetic Stratification identical in the two groups. While conclusions based upon in later trials. Such an assumption are biologically meaningful and Statis 0528. In another embodiment of the invention, it would tically robust, in Some circumstances there may be differ be useful to prospectively determine the Status of polymor ences in the shape of the trait distributions associated with phisms at genes that are involved in the pharmacokinetic or different genotypes. In particular, one or more classes of pharmacodynamic action of many drugs. This would Save homozygous genotypes may have a narrower trait distribu genotyping the large Outliers Unit population each time a tion (Smaller variance) than another, or than the population new project is initiated. Demand for genotyped groups of as a whole. Such a difference can be accounted for in the patients can be anticipated from pharmaceutical and bio analysis, in fact, it would be expected to reduce the number technology companies and contract research organizations of subjects needed for the Outliers Unit trial (since the (CROS). Genotyping might initially focus on common phar US 2001/0034023 A1 Oct. 25, 2001 macological targets Such as estrogen receptorS or other justifying the costs of the Pharmacogenetic Phase I Unit. nuclear receptors, or on adrenergic receptors, Serotonin Nonetheless, it is clearly desirable to operate a Pharmaco receptors, dopamine receptors and other G protein coupled genetic Phase I Unit as efficiently as possible. In order to receptors. The pre-genotyped Outlier Unit population could make a Phase I unit an efficient busineSS operation it is be part of a package of Services (along with genotyping useful to (i) use Statistical genetic methods to design studies assay development capability, high-throughput genotyping that require the minimal number of Subjects to achieve capacity and Software and expertise in Statistical genetics) adequate Statistical power (e.g. power of 80% to detect an designed to accelerate pharmacogenetic Phase I Studies. effect at the P-0.05 level), in order to keep subject costs at Eventually, as the databank of genotypes is expanded, a minimum, (ii) take measures to reduce the turnover of individuals with Virtually any genotype or combination of participating Subjects, in View of the long term investment genotypes can be called in for precisely designed physi made in patient recruitment and (in the case of the Outliers ological or toxicological Studies designed to test for phar Unit) genotyping. This may be accomplished by offering macogenetic effects. Subjects financial or other incentives to encourage Sustained 0529. As noted earlier, the Pharmacogenetic Phase I participation in the Pharmacogenetic Phase I Unit. The types Relatives Unit and the Pharmacogenetic Phase I Outlier Unit of incentives that would be useful differ between the two can provide useful information at almost any Stage of types of Phase I Units (see below). (iii)Secure rights to reuse clinical development. It is not unusual, for example, for a genotype data and, ideally, phenotypic data collected during product in Phase II or even Phase III testing to be remanded each Pharmacogenetic Phase I Unit trial, in order to create to Phase I in order to clarify Some aspect of toxicology or a database that over time will Save costs by eliminating the physiology. In this context, either or both of the Pharmaco need to repetitively genotype the same loci, and may even genetic Phase I Units would be extremely useful to a drug tually produce information of broad utility in clinical phar development company, as Studies in groups of related indi macology research: namely a database on the heritability of viduals (Relatives Unit) or in defined genetic Subgroups phenotypic responses to various broad classes of compounds drawn from a large genotyped population (Outliers Unit) (benzodiazepines, statins, taxanes, etc.) and the major would be an economical and efficient way to clarify the classes of genes involved. Such a database could become a nature and extent of pharmacogenetic effects, if any, thereby product. paving the way for future rational development of the 0535) In order to efficiently set up a Phase I Pharmaco compound. genetic Relatives Unit family participation can be encour 0530) 5. Surrogate Endpoints aged by appropriate incentive compensation. For example, subjects with no participating family members might be paid 0531 AS explained above, some of the most attractive S200 for participation in a study; two Sibs participating in the applications of Pharmacogenetic Phase I Units depend on same study might each be paid S300; if they could encourage the availability of Surrogate markers for pharmacodynamic another Sib (or cousin) to participate the three related drug action. The most useful Surrogate markers are those individuals might each be paid S350 for each study; parent which can be used in normal subjects in Phase I; which can sib pairs might be paid S400 for each study, and so forth. be measured easily, inexpensively and accurately, and for This type of compensation would encourage Subjects to which there is compelling data linking the Surrogate marker recruit their relatives to participate in Phase I studies. To the with Some clinically important aspect of disease biology, extent that certain types of blood relationship are more Such as disease manifestations in various organ Systems, useful for efficient genetical analysis, those types of related disease progression, disease morbidity or mortality, or dis individuals could be compensated most highly. This type of parate other clinical indices known in the art. The utility of compensation would increase the cost of Studies, however Surrogate markers increases in proportion to the difficulty the increased speed of Setting up the Relatives Unit, and the and cost of clincal development. Thus for a disease like increased retention of Subjects, would compensate over Alzheimer's, where long trials involving many patients are time. The optimal location to establish a Pharmacogenetic Standard, the use of Surrogate measures of, for example, Relatives Unit is in a city with a stable population, many cognitive ability, are highly desirable. large families, and a open attitudes toward modern technol 0532. The standard endpoints of Phase I trials are also ogy. The size of a Relatives Unit need be little more than 150 useful measures for analysis in a Pharmacogenetic Phase I subjects, though 250 would allow greater flexibility in Unit. For example, Studies of compound adsorption, distri drawing related Subjects from different racial or ethnic bution, metabolism, excretion and bioavailability may be groups (see below), and allow for more trials to be per analyzed for their genetic component. Similarly, toxic formed simultaneously. 400-500 subjects would be most responses and dose-related Side effects may be analyzed by preferable. Greater than 500 subjects would provide little the pharmacogenetic methods of this invention. benefit while increasing costs Substantially. 0536 Ideally subjects in the pharmacogenetic Phase I 0533 6. Establishing and Operating a Phase I Pharma unit are of known ethnic/racial/geographic background and cogenetic Relatives Unit Willing to participate in Phase I Studies, for pay, over a 0534 First, it should be noted that the information that period of years. For specific studies in a Relatives Unit can be gained from a Pharmacogenetic Phase I Unit provides Subjects from one or more racial, ethnic or geographically for Substantial cost Savings in later Stages of clinical devel defined group may be analyzed in order to (i) mirror the opment. Therefore it is to be expected that even if the cost population in which Phase II or Phase III trials are to be of operating a Pharmacogenetic Phase I Unit exceeds the conducted; (ii) determine if there are measurable differences cost of operating a conventional Phase I Unit, the overall in pharmacogenetic effects in different racial, ethnic or costs of clinical development are likely to be lower, thereby geographically defined groups; (iii) Study the most homo US 2001/0034023 A1 Oct. 25, 2001 56

geneous group possible in order to increase the chances of normals, the Status of a variance or variances Suspected to detecting a particular type of genetic effect. affect the efficacy of the candidate therapeutic intervention may be used as part of the inclusion criteria. Alternatively, 0537 Ideally consent for genotyping should be obtained analysis of variances or haplotypes in patients with different at the same time that Subjects are enrolled. Appropriate treatment responses may be among the endpoints. It is not consent forms will be drafted and approved by an indepen unusual for Such a Phase I Study design to include a dent review board. It would be most efficient if blanket double-blind, balanced, random-order, croSSOver Sequence consent for genotyping any polymorphic Site or Sites deemed (separated by washout periods), with multiple doses on relevant to the pharmacology of any candidate drug could be Separate occasions and both pharmacokinetic and pharma obtained. However, if this somewhat broad type of consent codynamic endpoints. is deemed inappropriate by the review board then consent could be Somewhat narrowed by adding the qualification 0542 2. Phase I Trials with Subjects Drawn from Large that any loci that are genotyped be relevant to a customer Populations and/or from Related Volunteer Subjects: The project. A third, more onerous arrangement would be obtain Pharmacogenetic Phase I Unit Concept consent to genotype polymorphic Sites in loci relevant to 0543. In general it is useful to be able to assess the Specific families of compounds, or to obtain consent for contribution of genetic variation to treatment response at the genotyping a specific list of genes. Another, Still leSS desir earliest possible Stage of clinical development. Such an able Solution would be to obtain consent for genotyping on assessment, if accurate, will allow efficient prioritization of a project-by-project basis (for example by mailing out reply candidate compounds for Subsequent detailed pharmacoge cards to all Subjects for each study), after the specific netic Studies, only those treatments where there is early polymorphic Sites to be genotyped have been Selected. evidence of a significant interaction of genetic variation with 0538 Another essential element of operating a Relatives treatment response would be advanced to pharmacogenetic Unit is having adequate quality control measures. One Studies in later Stages of development. In this invention we crucial aspect of quality control is an independent testing describe methods for achieving early insight-in Phase method to confirm the relatedness of the recruited Subjects I-into the contribution of genetic variation to variation in This can be accomplished by genotyping multiple (10-50) Surrogate treatment response variables. It occurred to the highly polymorphic loci, Such as Short tandem repeat inventors that this can be accomplished by bringing the Sequences, in individuals believed to be related. By com power of genetic linkage analysis and outlier analysis to paring the degree of genetic identity observed with that Phase I testing via the recruitment of a very large Phase I expected from the purported relation (e.g. 50% in the case of population including a large number of individuals who Sibs) it is possible to ensure with considerable certainty that have consented in advance to genetic studies (occasionally all related individuals are in fact related as they believe referred to hereinafter as a Pharmacogenetic Phase I Unit). themselves to be. (Inconsistency between genotyping and In one embodiment of a Pharmacogenetic Phase I Unit many reported relationship would be dealt with simply by not of the subjects are related to each other by blood. (Currently enrolling the unrelated individuals in any trials.) Phase I trials are performed in unrelated individuals, and there is no consideration of genetic recruitment criteria, or of 0539 AS indicated above, methods for retention of Sub genetic analysis of Surrogate markers.) There are several jects in a Phase I Outliers Unit preferably consist of making novel ways in which a large population, or a population modest payments for continuing participation (i.e. continued comprised at least in part of related individuals, could be permission to genotype under the limits of the consent); useful in early clinical trials. Some of the most attractive additional payments for genotyping analysis, whether or not applications depend on the availability of Surrogate markers it results in a request to participate in a clinical Study; and, for pharmacodynamic drug action which can be used early of course, generous compensation for participation in each in clinical development, preferably in normal Subjects in Outliers Unit clinical study. Phase I. Such Surrogate markers are increasingly used in 0540 Phase I of clinical development is generally Phase I, as drug development companies Seek to make early focused on Safety, although drug companies are increasingly yes/no decisions about compounds. obtaining information on pharmacokinetics and Surrogate 0544 Recruitment of a population optimized for clinical pharmacodynamic markers in early trials. Phase I Studies are genetic investigation may entail utilization of methods in typically performed with a small number (<60) of normal, Statistical genetics to Select the Size and composition of the healthy Volunteers usually at Single institutions. The primary population. For example powerful methods for detecting and endpoints in these Studies usually relate to pharmacokinetic mapping quantitative trait loci in Sibpairs have been devel parameters (i.e. adsorption, distribution, metabolism and oped. These methods can provide Some estimate of the bioavailability), and dose-related side effects. In a Phase I Statistical power derived from a given number of groups of pharmacogenetic clinical trial, Stratification based upon closely related individuals. Ideally Subjects in the pharma allelic variance or variances of a candidate gene or genes cogenetic Phase I unit are of known ethnic/racial/geographic related to pharmacokinetic parameters may allow early background and Willing to participate in Phase I Studies, for assessment of potential genetic interactions with treatment. pay, Over a period of years. The population is preferably 0541) Phase I studies of some diseases (e.g. cancer or Selected to achieve a specified degree of Statistical power for other medically intractable diseases for which no effective genetic association Studies, or is Selected in order to be able medical alternative exists) may include patients who satisfy to reliably identify a certain number of individuals with rare Specified inclusion criteria. These Safety/limited-efficacy genotypes, as discussed below. Family participation could Studies can be conducted at multiple institutions to ensure be encouraged by appropriate incentive compensation. For rapid enrollment of patients. In a pharmacogenetic Phase I example, individual subjects might be paid S200 for partici Study that includes patients, or a mixture of patients and pation in a study; two SibS participating in the same Study US 2001/0034023 A1 Oct. 25, 2001 57 might each be paid S300; if they could encourage another sib unrelated individuals (where, for example, variation might (or cousin) to participate the three related individuals might be more nearly continuous). Such a pattern could be attrib each be paid S350, and so forth. This type of compensation utable to Mendelian inheritance operating on a restricted Set would encourage Subjects to recruit their relatives to par of alleles in a family (or families) with, for example, AA ticipate in Phase I Studies. (It would also increase the cost of homozygotes giving one phenotype and AB heterozygotes Studies, however the type of data that can be obtained can and BB homozygotes giving a Second phenotype, all in the not be duplicated with conventional approaches.) The opti context of a relatively homogeneous genetic background. In mal location to establish such a Phase I unit is a city with a contrast, variation among non-related Subjects would be leSS Stable population, many large families, and a positive atti discrete due to a greater degree of variation in genetic tude about gene technology. The Pharmacogenetic Phase I background and the presence of additional alleles C, D and Unit population can then be used to test for the existence of E at the candidate locus. Statistical measures of the Signifi genetic variation in response to any drug as a first Step in cance of Such differences in distribution, including nonpara deciding whether to proceed with extensive pharmacoge metric methods Such as chi Square and contingency tables, netic Studies in later Stages of clinical development. Specific are known in the art. uses of a large Phase I unit in which Some or all Subjects are related include: 0547 The methods described herein for measuring 0545 a. It should be possible, for virtually any com whether pharmacodynamic traits are under genetic control, pound, to assess the magnitude of the genetic contribution to using Surrogate markers of drug efficacy in phase I Studies variation in drug response (if any) by comparing variation in which include groups of related individuals, will be useful in drug response traits among related VS. non-related individu obtaining an early assessment of the extent of genetically als. The rationale is as follows: if a Surrogate drug response determined variation in drug response for a given therapeu trait (i.e., a Surrogate marker of pharmacodynamic effect that tic compound. Such information provides an informed basis can be measured in normal Subjects) is under strong genetic for either Stopping development at the earliest possible Stage control then related individuals, who share 25% (cousins) or or, preferably, continuing with development but with a plan 50% (sibs) of their alleles, should have less divergent for identifying and controlling for genetic variation So as to responses (less intragroup variance) than unrelated individu allow rapid progression through the regulatory approval als, who share a much Smaller fraction of alleles. That is, proceSS. individuals who share alleles at the genes that affect drug 0548. For example, it is well known that Alzheimer's response should be more similar to each other (i.e. have a trials are long and expensive, and most drugs are only narrower distribution of responses, whether measured by effective in a fraction of patients. Using Surrogate measures variance, Standard deviation or other means) than individu of response in normals drawn from a population of related als who, on average, share very few alleles. By using individuals would help to assess the contribution of genetic Statistical methods known in the art the degree of variation variation to variation in treatment response. For an acetyl in a set of data from related individuals (each individual cholinesterase inhibitor, relevant Surrogate pharmacody would only be compared with his/her relatives, but such namic measures could include testing erythrocyte membrane comparisons would be performed within each group of acetylcholinesterase levels in drug treated normal Subjects, relatives and a Summary statistic developed) could be com or performing psychometric tests that are affected by treat pared to the degree of variation in a Set of unrelated ment (and ideally that correlate with clinical efficacy) and individuals (the same Subjects could be used, but the Second measuring the effect of treatment. AS another example, comparison would be across related groups). Account would antidepressant drugs can produce a variety of effects on be taken of the degree of Similarity expected between related mood in normal Subjects-or no effect at all. Careful moni individuals, based on the fraction of the genome they shared toring and measurement of Such responses in related VS. by descent. Thus the extent of variation in the Surrogate unrelated normal Subjects, and Statistical comparison of the response marker between identical twins should be less than degree of variation in each group, could provide an early between Sibs, which should be less than between first readout on whether there is a genetic component to drug cousins, which should be less than that between Second response (and hence clinical efficacy). The observation of cousins, and So forth, if there is a genetic component to the Similar effects in family members, and comparatively dis variation. It is well known from twin studies (in which, for Similar effects in unrelated Subjects would provide compel example, variation between identical twins is compared to ling evidence of a pharmacogenetic effect and justify the variation between fraternal twins) that pharmacokinetic Substantial expenditure necessary for a full pharmacogenetic variables (e.g. compound half life, peak concentration) are drug development program. Conversely, the absence of any frequently over 90% heritable; the type of study proposed Significant family influence on drug response would provide here (comparison of variation within groups of SibS and an early termination point for pharmacogenetic Studies. Note cousins to variation between unrelated Subjects) would also that the proposed Studies do not require any knowledge of show this genetic effect, without requiring the recruitment of candidate genes, nor is DNA collection or genotyping monozygotic twins. For a Summary of pharmacokinetic required-simply a reliable Surrogate pharmacodynamic studies in twins see: Propping, Paul (1978) Pharmacogenet assay and Small groups of related normal individuals. ics. Rev. Physiol. Biochem. Pharmacol. 83: 123-173. Refined Statistical methods should permit the magnitude of 0546. It may be that the pattern of drug responses that the pharmacogenetic effect to be measured, which could be distinguishes related individuals from non-related individu a further criteria for deciding whether to proceed with als is more complex than, for example, variance or Standard pharmacogenetic analysis. The greater the differential in deviation. For example, there may be two discrete pheno magnitude or pattern of variance between the related and the types characteristic of intrafamilial variation (a bimodal unrelated Subjects, the greater the extent of genetic control distribution) that are not a feature of variation between of the trait. US 2001/0034023 A1 Oct. 25, 2001 58

0549. Not all drug response traits are under the predomi 0.0225x0.04=0.0009 or 0.09%, or about 1 subject in 1000. nant control of one locus. Many Such traits are under the Ideally at least 5 Subjects of each genotype are recruited for control of multiple genes, and may be referred to as quan the Phase I study, and preferably at least 10 subject. Thus, titative trait loci. It is then desirable to identify the major loci even for variances of moderately low allele frequency (15%, contributing to variation in the drug response trait. This can 20%), the identification of potential outliers (i.e. homozy be done for example, to map quantitative trait loci in a gotes) for the candidate genes of interest will require a large population of drug treated related normals. Either a candi population. Preferably the Phase I unit has enrolled at least date gene approach or a genome wide Scanning approach 1,000 normal individuals, more preferably 2,000, still more can be used. (For review of some relevant methods see: Hsu preferably 5,000 and most preferably 10,000 or more. In L., Aragaki C, Quiaoit F. (1999) A genome-wide scan for a another application of the large, genotyped Phase I popula Simulated data set using two newly developed methods. tion it may be useful to identify individuals with rare Genet Epidemiol 17 Suppl 1:S621-6; Zhao LP, Aragaki C, variances in candidates genes (either homozygous or het Hsu L, Quiaoit F. (1998) Mapping of complex traits by erozygous), in order to determine whether those variances Single-nucleotide polymorphisms. Am J Hum Genet are predisposing to extreme pharmacological responses to 63(1):225-40; Stoesz M R, Cohen J C, Mooser V, et al. the compound. For example, variances occurring at 5% (1997) Extension of the Haseman-Elston method to multiple allele frequency are expected to occur in homozygous form alleles and multiple loci: theory and practice for candidate in 0.25% of the population (0.05x0.05), and therefore may genes. Ann Hum Genet 61 (Pt 3):263-74)) However, this rarely, if ever, be encountered in early clinical development. method would require at least 100 patients (preferably 200, Yet it may be serious adverse effects occurring in just Such and still more preferably >300) to have adequate statistical a Small group that create problems in later Stages of drug power, and each patient would have to be genotyped at a few development. In yet another application of the large geno polymorphic loci (candidate gene approach) or hundreds of typed Phase I population, Subjects may be Selected to polymorphic loci (genome Scanning approach). represent the known common variances in one or more 0550 b. With a large Phase I population of normal genes that are candidates for influencing the response to Subjects that need not be related (a second type of Pharma treatment. By insuring that all common genotypes are rep cogenetic Phase I Unit) it is possible to efficiently identify resented in a Phase I trial the likelihood of misleading results and recruit for any Phase I trial a set of individuals com due to genetic Stratification (resulting in discrepancy with prising virtually any combination of genotypes present in a results of later, larger trials can be reduced. population (for example, all common genotypes, or a group 0552. It would be useful to prospectively genotype the of genotypes expected to represent outliers for a drug large Phase I population for variances that are commonly the response trait of interest). This method preferably entails Source of interpatient variation in drug response, Since obtaining blood or other tissue (e.g. buccal Smear) in demand for genotyped groups of Such patients can be advance from a large number of the subjects in the Phase I anticipated from pharmaceutical companies and contract unit. Ideally consent for genotyping would be obtained at the research organizations (CROS). For example, genotyping same time. It would be most efficient if blanket consent for might initially focus on common pharmacological targets genotyping any polymorphic Site or Sites could be obtained. Such as estrogen receptors, adrenergic receptors, or Seroto Second best would be consent for testing any Site relevant to nin receptors. The pre-genotyped Phase I population could any customer project (not specific at the time of initial be part of a package of Services (along with genotyping consent). Third best would be consent to genotype polymor assay development capability, high throughput genotyping phic Sites relevant to Specific disease areas. Another, leSS capacity and Software and expertise in Statistical genetics) desirable, Solution would be to obtain consent for genotyp designed to accelerate pharmacogenetic Phase I Studies. ing on a project by project basis (for example by mailing out Eventually, as the databank of genotypes built up, individu reply cards), after the specific polymorphic Sites to be als with Virtually any genotype or combination of genotypes genotyped are known. could be called in for precisely designed physiological or 0551 One useful way to screen for pharmacogenetic toxicological Studies designed to test for pharmacogenetic effects in Phase I is to recruit homozygotes for a variance or effects. variances of interest in one or more candidate genes. For 0553) One of the most useful aspects of the Pharmaco example, consider a compound for which there are two genetic Phase I Unit is that Subjects with rare genotypes can genes that are Strong candidates for influencing response to be pharmacologically assessed in a Small Study. This treatment. Gene X has alleles A and A', while gene Y has addresses a Serious limitation of conventional clinical trials alleles B and B'. If these genes do in fact contribute with respect to the investigation of polygenic traits or the Significantly to response then one would expect that, regard effect of rare alleles. Unfortunately even Phase III studies, as less of the mode of inheritance (recessive, codominant, currently performed, are often barely powered to address dominant, polygenic) homozygotes would exhibit the most Simple one variance hypotheses about efficacy or toxicity. extreme responses. One would also expect epistatic inter The problem, of course, is that each time a new genetic actions, if any, to be most extreme in double homozygotes. variable is introduced the comparison groups are cut in Thus one would ideally perform a Surrogate drug response halves or thirds (or even Smaller groups if there are multiple test in Phase I volunteers doubly homozygous at both X and haplotypes at each gene). It is therefore a challenging Y. That is, test AA/BB, AA/BB, AA/B'B' and A'A'/'B' problem to test the interaction of Several genes in determin subjects. If the allele frequencies for A and A are 0.15 and ing drug response. Yet the character of drug response data in 0.85, and for B and B' 0.2 and 0.8 then the frequency of AA populations-there is often a continuous distribution of homozygotes is expected to be 2.25% and BB homozygotes responses among different individuals-Suggests that drug 4%. In the absence of any linkage between the genes, the responses may often be mediated by Several genes. (On the frequency of AA/BB double homozygotes is expected to be other hand, there are an increasing number of well docu US 2001/0034023 A1 Oct. 25, 2001 59 mented Single gene, or even Single Variance, pharmacoge other objective measures known to those skilled in the art. netic effects in the literature, showing that it is possible to In a pharmacogenetic Phase II trial the inclusion/exclusion detect the effect of a single variance.) One approach to criteria may include one or more genotypes or haplotypes. identifying pharmacogenetic effects is to focus on finding Alternatively, genetic analysis may be performed at the end the Single gene variances that have the largest effects. This of the trial. The primary goals in Phase II testing may approach can be undertaken within the Scale of current include (i) identification of the optimal medical indication clinical trials. However, in order to develop a test which for the compound, (ii) definition of an optimal dose or range predicts a large fraction of the quantitative variation in a or doses, balancing Safety and efficacy considerations (dose drug response trait it may be desirable to test the effect of finding Studies), (iii) extended Safety studies (complement multiple genes, including the interaction of variances at ing Phase I safety studies), (iv) evaluation of efficacy in different genes, which may be non-additive (referred to as patients with the targeted disease or condition, either in epistasis). The Pharmacogenetic Phase I Unit provides a way comparison to placebo or to current best therapy. To Some to efficiently test for gene interactions or multigene effects extent these goals may be achieved by performing multiple by, for example, allowing easy identification of individuals trials with different goals. Likewise, Phase II trials may be who, on account of being homozygous at Several loci of designed specifically to evaluate pharmacogenetic aspects of interest, should be outliers for the drug response phenotypes the drug candidate. Primary efficacy endpoints typically of interest if there is a genexgene interaction. Testing drug focus on clinical benefit, while Surrogate endpoints may response in a Small number of Such individuals will provide measure treatment response variables Such as clinical or a quick read on gene interaction. Obtaining genetic data on laboratory parameters that track the progreSS or extent of the pharmacodynamic action of a compound in Phase I disease, often at lesser time, cost or difficulty than the should also provide a crude measure of allele affects—which definitive endpoints. A good Surrogate marker must be variances or haplotypes increase pharmacological responses convincingly associated with the definitive outcome. and which decrease them. This information is of great value Examples of Surrogate endpoints include tumor Size as a in designing Subsequent trials, as it constrains the number of Surrogate for Survival in cancer trials, and cholesterol levels hypotheses to be tested, thereby enabling powerful Statistical as a Surrogate for heart disease (e.g. myocardial infarction) designs. This is because when the effect of variances on drug in trials of lipid lowering cardiovascular drugs. Secondary response measures is unknown one is forced to Statistically endpoints Supplement the primary endpoint and may be test all the possible effects of each allele (e.g. two tailed Selected to help guide further clinical Studies. tests). As the number of genetically defined groups increases 0557. In a pharmacogenetic Phase II clinical trial, retro (e.g. as a result of multiple variances or haplotypes) there is spective or prospective design will include the stratification a loss of Statistical power due to multiple testing correction. of patients based upon a variance or variances in a gene or On the other hand, if the relative phenotypic effect of each genes Suspected of affecting treatment response. The gene or allele at a locus is known (or can be hypothesized) from genes may be involved in mediating pharmacodynamic or Phase I data then each individual in a Subsequent clinical pharmacokinetic response to the candidate therapeutic inter trial contributes useful information-there is a specific pre vention. The parameters evaluated in the genetically Strati diction of response based on that individuals combination of fied trial population may include primary, Secondary or genotypes or haplotypes, and testing the fit of the actual data Surrogate endpoints. Pharmacokinetic parameters-for to those predictions provides for powerful Statistical designs. example, dosage, absorption, toxicity, metabolism, or excre (It is also possible to measure allele effects biochemically, of tion-may also be evaluated in genetically Stratified groups. course, to establish which alleles have positive and which Other parameters that may be assessed in parallel with negative effects, but at considerable cost.) genetic Stratification include gender, race, ethnic or geo 0554. It is important to note that Phase I trials can provide graphic origin (population history) or other demographic useful information at almost any stage of clinical develop factors. ment. It is not unusual, for example, for a product in Phase 0558 While it is optimal to initiate pharmacogenetic II or even Phase III testing to be remanded to Phase I in order Studies in phase I, as described above, it may be the case that to clarify Some aspect of toxicology or physiology. In this pharmacogenetic Studies are not considered until phase II, context a Pharmacogenetic Phase I Unit would be extremely when problems relating either to efficacy or toxicity are first useful to a drug development company. Phase I Studies in encountered. It is highly desirable to initiate pharmacoge defined genetic Subgroups drawn from a large genotyped netic studies no later than Phase II of a clinical development population, or in groups of related individuals, would be the plan because (1) phase III Studies tend to be large and most economical and efficient way to clarify the existence of expensive-not an optimal Setting in which to explore pharmacogenetic effects, if any, paving the way for future untested pharmacogenetic hypotheses; (2) phase III Studies rational development of the product. are typically designed to test one fairly narrow hypothesis 0555 C. Phase II Clinical Trials regarding efficacy of one or a few dose levels in a specific disease or condition. Phase II Studies are often numerous, 0556 Phase II studies generally include a limited number and are intended to provide a broad picture of the pharma of patients (<100) who satisfy a set of predefined inclusion cology of the candidate compound. This is a good Setting for criteria and do not Satisfy any predefined exclusion criteria initial pharmacogenetic Studies. Several pharmacogenetic of the trial protocol. Phase II studies can be conducted at hypotheses may be tested in phase II, with the goal of Single or multiple institutions. Inclusion/exclusion criteria eliminating all but one or two. may include historical, clinical and laboratory parameters for a disease, disorder, or condition, age; gender, reproduc 0559) D. Phase III Clinical Trials tive status (i.e. pre- or postmenopausal), coexisting medical 0560 Phase III studies are generally designed to measure conditions, psychological, emotional or cognitive State, or efficacy of a new treatment in comparison to placebo or to US 2001/0034023 A1 Oct. 25, 2001 60 an established treatment method. Phase II studies are often higher response rate, greater magnitude of response or fewer performed at multiple Sites. The design of this type of trial Side effects-in a genetically defined Subgroup. The genetic includes power analysis to ensure the Sufficient data will be Subgroup with Superior response may constitute a larger gathered to demonstrate the anticipated effect, making fraction of the total patient population than the new drug assumptions about response rate based on earlier trials. AS a would likely achieve otherwise. In this instance, there is a result Phase III trials frequently include large numbers of clear rationale for performing a Phase IV pharmacogenetic patients (up to 5,000). Primary endpoints in Phase III studies trial to identify a variance or variances that mark a patient may include reduction or arrest of disease progression, population with Superior clinical response. Subsequently a improvement of Symptoms, increased longevity or increased marketing campaign can be designed to alert patients, phy disease-free longevity, or other clinical measures known in Sicians, pharmacy managers, managed care organizations the art. In a pharmacogenetic Phase III clinical Study, the and other parties that, with the use of a rapid and inexpensive endpoints may include determination of efficacy or toxicity genetic test to identify eligible patients, the new drug is in genetically defined Subgroups. Preferably the genetic Superior to other members of the class (including the market analysis of outcomes will be confined to an assessment of leading first, Second and third drugs introduced). The high the impact of a Small number of variances or haplotypes at responder Subgroup defined by a variance or variances may a Small number of genes, said variances having already been also exhibit a Superior response to other drugs in the class (a Statistically associated with outcomes in earlier trials. Most class pharmacogenetic effect), or the Superior efficacy in the preferably variances at only one or two genes will be genetic Subgroup may be specific to the drug tested (a assessed. compound-specific pharmacogenetic effect). 0561. After successful completion of one or more Phase 0564) In a Phase IV pharmacogenetic clinical trial, both III studies, the data and information from all trials conducted retrospective and prospective analysis can be performed. In to test a new treatment method are compiled into a New both cases, the key element is genetic Stratification based on Drug Application (NDA) and Submitted for review by the a variance or variances or haplotype. Phase IV trials will U.S. FDA, which has authority to grant marketing approval often have adequate Sample size to test more than one in the U.S. and its territories. The NDA includes the raw pharmacogenetic hypothesis in a Statistically Sound way. (unanalyzed) clinical data, i.e. the patient by patient mea Surements of primary and Secondary endpoints, a Statistical 0565) F. Unconventional Clinical Development analysis of all of the included data, a document describing 0566 Although the above listed phases of clinical devel in detail any observed Side effects, tabulation of all patients opment are well-established, there are cases where strict who dropped-out of trials and detailed reasons for their Phase I, II, III development does not occur, for example, in termination, and any other available data pertaining to the clinical development of candidate therapeutic interven ongoing in vitro or in Vivo Studies Since the Submission of tions for debilitating or life threatening diseases, or for the investigational new drug (IND) application. If pharma diseases where there is presently no available treatment. coeconomic objectives are a part of the clinical trial design Some of the mechanisms established by the FDA for such then data Supporting cost or economic analyses are included studies include Treatment INDs, Fast-Track or Accelerated in the NDA. In a pharmacogenetic clinical Study, the phar reviews, and Orphan Drug Status. In a clinical development macoeconomic analyses may include genetically Stratified program for a candidate therapeutic of this type there is a assessment of the candidate therapeutic intervention in a useful role for pharmacogenetic analysis, in that the candi cost benefit analysis, cost of illness Study, cost minimization date therapeutic may not produce a Sufficient benefit in all Study, or cost utility analysis. The analysis may also be patients to justify FDA approval, however analysis of out Simultaneously Stratified by Standard criteria Such as race/ come in genetic Subgroups may lead to identification of a ethnicity/geographic origin, Sex, age or other criteria. Data variance or variances that predict a response rate Sufficient from a genetically Stratified analysis may be used to Support for FDA approval. an application for approval for marketing of the candidate 0567 As used herein, “supplemental applications” are therapeutic intervention. those in which a candidate therapeutic intervention is tested 0562 E. Phase IV Clinical Trials in a human clinical trial in order to gain an expanded label indication, expanding recommended use to new medical 0563 Phase IV studies occur after a therapeutic interven indications. In these applications, previous clinical Studies of tion has been approved for marketing, and are typically the therapeutic intervention, i.e. preclinical Safety and Phase conducted for Surveillance of Safety, particularly occurrence I human Safety Studies can be used to Support the testing of of rare side effects. The other principal reason for Phase IV the therapeutic intervention in a new indication. Pharmaco Studies is to produce information and relationships useful for genetic analysis is also useful in the context of clinical trials marketing a drug. In this regard pharmacogenetic analysis to Support Supplemental applications. Since these are, by may be very useful in Phase IV trials. Consider, for example, definition, focused on diseases not Selected for initial devel a drug that is the fourth or fifth member of a drug class (say opment the Overall efficacy may not be as great as for the Statins, or thiazidinediones or fluoropyrimidines) to obtain leading indication(s). The identification of genetic Sub marketing approval, and which does not differ significantly groups with high response rates may enable the rapid in clinical effects--efficacy or safety-from other members approval of Supplemental applications for expanded label of the drug class. The first, Second and third drugs in the indications. In Such instances part of the label indication class will likely have a dominant market position (based on may be a description of the variance or variances that define their earlier introduction into the marketplace) that is diffi the group with Superior response. cult to overcome, particularly in the absence of differenti ating clinical effects. However, it is possible that the new 0568. As used herein, “outcomes” or “therapeutic out drug produces a Superior clinical effect-for example, comes' describe the results and value of healthcare inter US 2001/0034023 A1 Oct. 25, 2001

vention. Outcomes can be multi-dimensional, and may 0575. The degree of precision may be expressed in terms include improvement of Symptoms, regression of a disease, of the maximal acceptable Standard error of a measurement, disorder, or condition; prevention of a disease or Symptom; the magnitude of variation in which the 95% confidence cost Savings or other measures. interval must be confined or the minimal magnitude of 0569 Pharmacoeconomics is the analysis of a therapeutic difference in a clinical or laboratory value that must be intervention in a population of patients diagnosed with a detectable (at a statistically significant level, and with a disease, disorder, or condition that includes at least one of Specified power for detection) in a comparison to be per the following studies: cost of illness study (COI); cost formed at the end of the trial (hypothesis test). The minimal benefit analysis (CBA), cost minimization analysis (CMA), magnitude is generally Set at the level that represents the or cost utility analysis (CUA), or an analysis comparing the minimal difference that would be considered of clinical relative costs of a therapeutic intervention with one or a importance. group of other therapeutic interventions. In each of these 0576. In pharmacogenetic clinical trials there are two Studies, the cost of the treatment of a disease, disorder, or countervailing effects with respect to power. First, the com condition is compared among treatment groups. Costs have parison groups are reduced in size (compared to a conven both direct (therapeutic interventions, hospitalization) and tional trial) due to genetic partitioning of both the treatment indirect (loss of productivity) components. Pharmacoeco and control groups into two or more Subgroups. However, it nomic factors may provide the motivation for pharmacoge is reasonable to expect that variability for a trait is Smaller netic analysis, particularly for expensive therapies that ben within groups that are genetically homogeneous with respect efit only a fraction of patients. For example, interferon alpha to gene variances affecting the trait. If this is the case then is the only treatment that can cure hepatitis C virus infection, power is increased as a function of the reduction in vari however viral infection is completely and permanently ability within (genetically defined) groups. eliminated in less than a quarter of patients. Nearly half of patients receive virtually no benefit from alfa interferon, but 0577. In general it is preferable to power a pharmacoge may Suffer Significant Side effects. Treatment costs are netic clinical trial to See an effect in the largest genetically ~$10,000 per course. A pharmacogenetic test that could defined Subgroups. For example, for a variance with allele predict responders would save much of the cost of treating frequencies of 0.7 and 0.3 the common homozygote group patients not able to benefit from interferon alpha therapy, will comprise 49% of all patients (0.7x0.7x100). It is most and could provide the rationale for treating a population in desirable to power the trial to observe an effect (either a cost efficient manner, where treatment would otherwise be positive or a negative) in this group. If it is desirable to unaffordable. measure an effect of therapy in a Small genetic group (for example, the 9% of patients homozygous for the rare allele) 0570 AS used herein, “health-related quality of life” is a then genotyping should be considered as an enrollment measure of the impact of a disease, disorder, or condition on criterion to insure a Sufficient number of patients are enrolled a patient's activities of daily living. An analysis of the to perform an adequately powered Study. health-related quality of life is often included in pharmaco economic Studies. 0578 Statistical methods for powering clinical trials are known in the art. See, for example: Shuster, J. J. (1990) 0571 AS used herein, the term “stratification” refers to Handbook of Sample Size Guidelines for Clinical Trials. the partitioning of patients into groups on the basis of CRC Press, Boca Raton, Fla.; Machin, D. and M. J. Camp clinical or laboratory characteristics of the patient. “Genetic bell (1987) Statistical Tables for the Design of Clinical Stratification” refers to the partitioning of patients or normal Trials. Blackwell, Oxford, UK; Donner, A. (1984) Subjects into groups based on the presence or absence of a Approaches to Sample Size Estimation in the Design of variance or variances in one or more genes. The Stratification may be performed at the end of the trial, as part of the data Clinical Trials- A Review. Statistics in Medicine3: 199-214. analysis, or may come at the beginning of a trial, resulting 0579. H. Statistical Analysis of Clinical Trial Data in creation of distinct groups for Statistical or other purposes. 0580. There are a variety of statistical methods for mea 0572 G. Power Analysis in Pharmacogenetic Clinical Suring the difference between two or more groups in a Trials clinical trial. One skilled in the art will recognize that 0573 The basic goal of power calculations in clinical different methods are Suited to different data Sets. In general, trial design is to insure that trials have adequate patients and there is a family of methods customarily used in clinical controls to fairly assess, with Statistical Significance, trials, and another family of methods customarily used in whether the candidate therapeutic intervention produces a genetic epidemiological Studies. Methods in quantitative and clinically significant benefit. population genetics designed to measure the association between genotypes and phenotypes, and to map and measure 0574 Power calculations in clinical trials are related to the effect of quantitative trait loci are also relevant to the task the degree of variability of the drug response phenotypes of measuring the impact of a variance on response to a measured and the treatment difference expected between treatment. Methods from any of these disciplines may be comparison groups (e.g. between a treatment group and a Suitable for performing Statistical analysis of pharmacoge control group). The Smaller the variance within each group being compared, and the greater the difference in response netic clinical trial data, as is known to those skilled in the art. between the two groups, the fewer patients are required to 0581 Conventional clinical trial statistics include produce convincing evidence of an effect of treatment. hypothesis testing and descriptive methods, as elaborated These two factors (variance and treatment difference) deter below. Guidance in the Selection of appropriate Statistical mine the degree of precision required to answer a specific tests for a particular data Set is provided in texts. Such as: clinical question. BioStatistics. A Foundation for Analysis in the Health Sci US 2001/0034023 A1 Oct. 25, 2001 62 ences, 7th edition (Wiley Series in Probability and Math mal and logistic distributions, and, by using a log ematical Statistics, Applied Probability and statistics) by transformation, exponential, Weibull, lognormal, Wayne W. Daniel, John Wiley & Sons, 1998; Bayesian loglogistic and gamma distributions. Methods and Ethics in a Clinical Trial Design (Wiley Series in Probability and Mathematical Statistics. Applied Prob 0595 (13) Compute non-parametric estimates of ability Section) by J. B. Kadane (Editor), John Wiley & Survival distribution with right-censored data and Sons, 1996. Examples of Specific hypothesis testing and compute rank tests for association of the response descriptive Statistical procedures that may be useful in variable with other variables. analyzing clinical trial data are listed below. 0596 B. Descriptivep Statistical Methods 0582 A. Hypothesis Testing Statistical Procedures 0583 (1) One-sample procedures (binomial confi 0597 Factor analysis with rotations dence interval, Wilcoxon signed rank test, permuta 0598 Canonical correlation tion test with general Scores, generation of exact permutational distributions) 0599 Principal component analysis for quantitative 0584) (2) Two-sample procedures (t-test, Wilcoxon variables. Mann-Whitney test, Normal score test, Median test, 0600 Principal component analysis for qualitative Van der Waerden test, Savage test, Logrank test for data. censored Survival data, Wilcoxon-Gehan test for censored Survival data, Cochran-Armitage trend test, 0601 Hierarchical and dynamic clustering methods permutation test with general Scores, generation of to create tree Structure, dendrogram or phenogram. exact permutational distributions) 0602 Simple and multiple correspondence analysis 0585 (3) RXC contingency tables (Fisher's exact using a contingency table as input or raw categorical test, Pearson's chi-squared test, Likelihood ratio test, Kruskal-Wallis test, Jonckheere-Terpstra test, Lin data. ear-by linear association test, McNemar's test, mar 0603 Specific instructions and computer programs for ginal homogeneity test for matched pairs) performing the above calculations can be obtained from 0586 (4) Stratified 2x2 contingency tables (test of companies such as: SAS/STAT Software, SAS Institute Inc., homogeneity for odds ratio, test of unity for the Cary, N.C., U.S.A.; BMDP Statistical Software, BMDP common odds ratio, confidence interval for the com Statistical Software Inc., Los Angeles, Calif., USA; SYS mon odds ratio) TAT software, SPSS Inc., Chicago, Ill., USA; StatXact & 0587 (5) Stratified 2xC contingency tables (all two LogXact, CYTEL Software Corporation, Cambridge, Mass., Sample procedures listed above with Stratification, USA. confidence intervals for the odds ratioS and trend, 0604 C. Statistical Genetic Methods Useful for Analysis generation of exact permutational distributions) of Pharmacogenetic Data 0588 (6) General linear models (simple regression, multiple regression, analysis of variance 0605. A wide spectrum of mathematical and statistical -ANOVA-, analysis of covariance, response-Sur tools may be useful in the analysis of data produced in face models, weighted regression, polynomial pharmacogenetic clinical trials, including methods regression, partial correlation, multiple analysis of employed in molecular, population, and quantitative genet variance -MANOVA-, repeated measures analy ics, as well as genetic epidemiology. Methods developed for plant and animal breeding may be useful as well, particularly sis of variance). methods relating to the genetic analysis of quantitative traits. 0589 (7) Analysis of variance and covariance with a nested (hierarchical) structure. 0606 Analytical methods useful in the analysis of genetic variation among individuals, populations and Species of 0590 (8) Designs and randomized plans for nested various organisms are described in the following texts: and crossed experiments (completely randomized Molecular Evolution, by W- H. Li, Sinauer Associates, Inc., design for two treatment, Split-Splot design, hierar 1997; Principles of Population Genetics, by D. L. Hartland chical design, incomplete block design, latin Square A. G. Clark, 1996; Genetics and Analysis of Quantitative design) Traits, By M. Lynch and B. Walsh, Sinauer Associates, Inc., 0591 (9) Nonlinear regression models Principles of Quantitative Genetics, by D. S. Falconer and T. F. C. Mackay, Longman, 1996; Genetic Variation and 0592 (10) Logistic regression for unstratified or Human Disease, by K. M. Weiss, Cambridge University Stratified data, for binary or ordinal response data, Press, 1993; Fundamentals of Genetic Epidemiology, by M. using the logit link function, the normit function or J. Khoury, T. H. Beaty, and B. H. Cohen, Oxford University the complementary log-log function. Press, 1993; Handbook of Genetic Linkage, by J. Terwilliger 0593 (11) Probit, logit, ordinal logistic and gompit J. Ott, Johns Hopkins University Press, 1994. regression models. 0607. The types of statistical analysis performed in dif 0594) (12) Fitting parametric models to failure time ferent branches of genetics are outlined below as a guide to data that may be right-, left-, or interval-censored. the relevant literature and publicly available Software, Some Tested distributions can include extreme value, nor of which is cited. US 2001/0034023 A1 Oct. 25, 2001

0608 Molecular Evolutionary Genetics 0629 Determination of quantitative trait nucleotide (QTN) using a combination linkage disequilibrium 0609 Patterns of nucleotide variation among indi viduals, families/populations and acroSS Species and methods and cladistic approaches genera, 0630 Determination of individual causal nucleotide in the diploid or haploid State on the phenotype using 0610 Alignment of sequences and description of the method of measured genotype approaches, and variation/polymorphisms among the aligned combined effects or Synergistic interaction of the Sequences, amounts of Similarities and dissimilari causal mutations on the phenotype ties, 0631 Determination of relative importance of each 0611 Measurement of molecular variation among of the mutations on a given phenotype using multi various regions of a gene, testing of neutrality mod variate methods, Such as discriminant function, prin els, cipal component and Step-wise regression methods 0.612) Rates of nucleotide changes among coding 0632) Determination of direct and indirect effect of and the non-coding regions within and among popu polymorphisms on a complex phenotype using path lations, analysis (partial regression) methods 0613 Construction of phylogenetic trees using 0633 Determination of the effects of specific envi methods Such as neighborhood joining and maxi ronment on a given genotype-genotypexenviron mum parSimony; estimation of ages of variances ment interactions using joint regression and additive using coalescent models, and multiplicative parameter methods. 0614 Population Genetics 0634 Genetic Epidemiology 0.615 Patterns of distribution of genes among geno 0635 Determination of sample size based on the types and populations. Hardy-Weinberg equilibrium, disease and the marker frequency in the “case' and departures form the equilibrium in the “control” populations 0616 Genotype and haplotype frequencies, levels of 0636 Stratification of studvy poppopulation based on heterozygosities, polymorphism information con gender, ethnic, Socio-economic variation tents of genes, estimation of haplotypes from geno 0637 Establishing a “causal relationship” between types; the E-M algorithm, and parsimony methods genotype and disease, using, using Various associa 0.617 Estimation of linkage disequilibrium and tion and linkage approaches—viz., case-control recombination designs, family Studies (if available), transmission disequilibrium tests etc., 0618. Hierarchical structure of populations, the F-Statistics, estimation of inbreeding, Selection and 0638 Linkage analysis between markers and a can drift didate locus using two-point and multipoint approaches. Computer programs used for genetic 0619 Genetic admixture/migration and mutation analysis are: Dna SP version 3.0, by Juilo Rozas, frequencies University of Barcelona, Spain. Http://www.bi 0620 Spatial distribution of genotypes using spatial o.ub.es/-Julio; Arlequin 1.1 by S. Schnieder, J-M Kueffer, D. Roessli and L. Excoffier. University of autocorrelation methods Geneva, Switzerland, http://anthropologie.unige.ch/ 0621) Kin-structured maintenance of variation and arlequin. PAUP*4, by D. L. Swofford, Sinauer Asso migration ciates, Inc., 1999. SYSTAT Software, SPSS Inc., Chicago, Ill., 1998; . Linkage User's Guide, by J. 0622 Quantitative Genetics Ott, Rockefeller University, Http://Linkage.rock 0623 Phenotype as the product of the interaction efeller.edu/Soft/linkage between genotype and environment 0639 Guidance in the selection of appropriate genetic Statistical tests for analysis of data can be obtained from 0624. Additive, dominance and epistatic variance on texts. Such as: Fundamentals of Genetic Epidemiology the phenotype (Monographs in Epidemiology and Biostatistics, Vol 22) by 0625 Effects of homozygosity, heterozygosity and M. J. Khoury, B. H. Cohen & T. H. Beaty, Oxford Univ developmental homeostasis Press, 1993; Methods in Genetic Epidemiology by Newton E. Morton, S. Karger Publishing, 1983; Methods in Obser 0626 Estimation of heritability: broad sense and vational Epidemiology, 2nd edition (Monographs in Epide OW SCSC miology and Biostatistics, V. 26) by J. L. Kelsey (Editor), A. 0627 Determination of number of genes governing S. Whittemore & A. S. Evans, 1996; Clinical Trials. Design, a character Conduct, and Analysis (Monographs in Epidemiology and Biostatistics, Vol 8) by C. L. Meinert & S. Tonascia, 1986) 0628 Determination of quantitative trait loci (QTLS) using family information or population 0640 I. Retrospective Clinical Trials information, and using linkage and/or association 0641. In general the goal of retrospective clinical trials is Studies to test and refine hypotheses regarding genetic factors that US 2001/0034023 A1 Oct. 25, 2001 are associated with drug responses. The best Supported as the mini-mental status exam (MMSE) or the hypotheses can Subsequently be tested in prospective clini Alzheimer's Disease ASSessment Scale cal trials, and data from the prospective trials will likely (ADAS), which includes a cognitive compo comprise the main basis for an application to register the nent (ADAS-COG). There are also clinical drug and predictive genetic test with the appropriate regu assessment Scales for many other diseases, latory body. In Some cases, however, it may become accept including cancer. able to use data from retrospective trials to Support regula tory filings. Exemplary Strategies and criteria for Stratifying 0657 ii. Disease manifestations (clinical pre patients in a retrospective clinical trial are provided below. Sentation). 0642 Clinical Trials to Study the Effect of One Gene 0658) iii. Radiological staging criteria. Locus on Drug Response 0659 c. Pathological criteria: 0643 A. Stratify Patients by Genotype at One Candidate 0660 i. Histopathologic features of disease tis Variance in the Candidate Gene Locus Sue, or pathological diagnosis. (For example 0644) 1. Genetic stratification of patients can be there are many varieties of lung cancer: Squa accomplished in Several ways, including the follow mous cell carcinoma, adenocarcinoma, Small ing (where A is the more frequent form of the cell carcinoma, bronchoalveolar carcinoma, variance being assessed and 'a' is the leSS frequent etc., each of which may-which, in combina form): tion with genetic variation, may correlate with 0661 ii. Pathological stage. A variety of dis 0645 (a) AA vs. aa eases, particularly cancer, have pathological 0646) (b) AA vs. Aa vs. aa Staging Schemes 0647 (c) AA vs. (Aa+aa) 0662) iii. Loss of heterozygosity (LOH) 0648 (d) (AA+Aa) vs. aa. 0663 iv. Pathology studies such as measuring 0649 2. The effect of genotype on drug response levels of a marker protein phenotype may be affected by a variety of nonge 0664) v. Laboratory studies such as hormone netic factors. Therefore it may be beneficial to mea levels, protein levels, Small molecule levels Sure the effect of genetic Stratification in a Subgroup of the overall clinical trial population. Subgroups can 0665 3. Measure frequencvC y of respondersp in each be defined in a number of ways including, for genetic Subgroup. Subgroups may be defined in example, biological, clinical, pathological or envi Several ways. ronmental criteria. For example, the predictive value 0666) i. more than two age groups of genetic Stratification can be assessed in a Subgroup 0667 ii. reproductive status such as pre or post or Subgroups defined by: menopausal 0650 a. Biological Criteria 0668 4. Stratify by haplotype at one candidate locus 0651) i. gender (males vs. females) where the haplotype is made up of two variances, 0652) ii. age (for example above 60 years of three variances or greater than three variances. age). Two, three or more age groups may be 0669 Data from already completed clinical trials can be useful for defining Subgroups for the genetic retrospectively reanalyzed. Since the questions are new, the analysis. data can be treated as if it were a prospective trial, with 0653) iii. hormonal status and reproductive his identified variances or haplotypes as Stratification criteria or tory, including pre- VS. post-menopausal Status endpoints in clinically Stratified data (e.g. what is the fre quency of a particular variance in a response group com of women, or multiparous VS. nulliparous pared to nonresponsders). Care should be taken to in Study WOC ing a population in which there may be a link between 0654) iv. ethnic, racial or geographic origin, or drug-related genes and disease-related genes. Surrogate markers of ethnic, racial or geo graphic origin. (For a description of genetic 0670 Retrospective pharmacogenetic trials can be con markers that Serve as Surrogates of racial/ethnic ducted at each of the phases of clinical development, if origin See, for example: Rannala, B. and J. L. Sufficient data is available to correlate the physiologic effect Mountain, Detecting immigration by using of the candidate therapeutic intervention and the allelic variance or variances within the treatment population. In the multilocus genotypes. Proc Natl AcadSci USA case of a retrospective trial, the data collected from the trial , 94 (17): 9197-9201, 1997. Other surrogate can be re-analyzed by imposing the additional Stratification markers could be used, including biochemical on groups of patients by Specific allelic variances that may markers.) exist in the treatment groupS. Retrospective trials can be 0655 b. Clinical Criteria useful to ascertain whether a hypothesis that a specific 0656 i. Disease status. There are clinical grad variance has a Significant effect on the efficacy or toxicity ing Scales for many diseases. For example, the profile for a candidate therapeutic intervention. Status of Alzheimer's Disease patients is often 0671 A prospective clinical trial has the advantage that measured by cognitive assessment Scales Such the trial can be designed to ensure the trial objectives can be US 2001/0034023 A1 Oct. 25, 2001 met with Statistical certainty. In these cases, power analysis, 0674) The design of a pharmacogenetics clinical trial will which includes the parameters of allelic variance frequency, include a description of the allelic variance impact on the number of treatment groups, and ability to detect positive observed efficacy between the treatment groups. Using this outcomes can ensure that the trial objectives are met. type of design, the type of genetic and phenotypic relation 0672. In designing a pharmacogenetic trial, retrospective ship display of the efficacy response to a candidate thera analysis of Phase II or Phase III clinical data can indicate peutic intervention will be analyzed. For example, a geno trial variables for which further analysis is beneficial. For typically dominant allelic variance or variances will be those example, Surrogate endpoints, pharmacokinetic parameters, in which both heterozygotes and homozygotes will demon dosage, efficacy endpoints, ethnic and gender differences, Strate a specific phenotypic efficacy response different from and toxicological parameters may result in data that would the homozygous recessive genotypic group. A pharmacoge require further analysis and re-examination through the netic approach is useful for clinicians and public health design of an additional trial. In these cases, analysis involv professionals to include or eliminate Small groups of ing Statistics, genetics, clinical outcomes, and economic responderS or non-responders from treatment in order to parameters may be considered prior to proceeding to the avoid unjustified Side-effects. Further, adjustment of dosages Stage of designing any additional trials. Factors involved in when clear clinical difference between heterozygous and the consideration of Statistical significance may include homozygous individuals may be beneficial for therapy with Bonferroni analysis, permutation testing, with multiple test the candidate therapeutic intervention ing correction resulting in a difference among the treatment 0675. In another example, a recessive allelic variance or groups that has occurred as a result of a chance of no greater variances will be those in which only the homozygote than 20%, i.e. p-0.20. Factors included in determining recessive for that or those variances will demonstrate a clinical outcomes to be relevant for additional testing may Specific phenotypic efficacy response different from the include, for example, consideration of the target indication, heterozygotes or homozygous dominants. An extension of the trial endpoints, progression of the disease, disorder, or these examples may include allelic variance or variances condition during the trial Study period, biochemical or organized by haplotypes from additional gene or genes. pathophysiologic relevance of the candidate therapeutic intervention, and other variables that were not included or 0676 V. Variance Identification and Use anticipated in the initial Study design or clinical protocol. Factors to be included in the economic Significance in 0677 A. Initial Identification of Variances in Genes determining additional testing parameters include Sample 0678 Selection of Population Size and Composition size, accrual rate, number of clinical Sites or institutions required, additional or other available medical or therapeutic 0679 Prior to testing to identify the presence of sequence interventions approved for human use, and additional or variances in a particular gene or genes, it is useful to other available medical or therapeutic interventions concur understand how many individuals should be Screened to rently or anticipated to enter human clinical testing. Further, provide confidence that most or nearly all pharmacogeneti there may be patients within the treatment categories that cally relevant variances will be found. The answer depends present data that fall outside of the average or mean values, on the frequencies of the phenotypes of interest and what or there may be an indication of multiple allelic loci that are assumptions we make about heterogeneity and magnitude of involved in the responses to the candidate therapeutic inter genetic effects. Prior to testing to identify the presence of vention. In these cases, one could propose a prospective Sequence Variances in a particular gene or genes, it is useful clinical trial having an objective to determine the Signifi to understand how many individuals should be Screened to cance of the variable or parameter and its effect on the provide confidence that most or nearly all pharmacogeneti outcome of the parent Phase II trial. In the case of a cally relevant variances will be found. The answer depends pharmacogenetic difference, i.e. a single or multiple allelic on the frequencies of the phenotypes of interest and what difference, a population could be Selected based upon the assumptions we make about heterogeneity and magnitude of distribution of genotypes. The candidate therapeutic inter genetic effects. At the beginning we only know phenotype vention could then be tested in this group of Volunteers to frequencies (e.g. responders vs. nonresponders, frequency of test for efficacy or toxicity. The repeat prospective Study various side effects, etc.). could be a Phase I limited study in which the subjects would 0680 The most conservative assumption (resulting in the be healthy human volunteers, or a Phase II limited efficacy lowest estimate of allele frequency, and consequently the Study in which patients which Satisfy the inclusion criteria largest Suggested Screening population) is (i) that the phe could be enrolled. In either case, the Second, confirmatory notype (e.g. toxicity or efficacy) is multifactorial (i.e. can be trial could then be used to Systematically ensure an adequate caused by two or more variances or combinations of Vari number of patients with appropriate phenotype is enrolled in ances), (ii) that the variance of interest has a high degree of a Phase III trial. penetrance (i.e. is consistently associated with the pheno 0673 A placebo controlled pharmacogenetics clinical type), and (iii) that the mode of transmission is Mendelian trial design will be one in which target allelic variance or dominant. Consider a pharmacogenetic Study designed to variances will be identified and a diagnostic test will be identify predictors of efficacy for a compound that produces performed to Stratify the patients based upon presence, a 15% response rate in a nonstratified population. If half the absence, or combination thereof of these variances. In the response is Substantially attributable to a given variance, and Phase II or Phase III stage of clinical development, deter the variance is consistently associated with a positive mination of a Specific Sample size of a prospective trial will response (in 80% of cases) and the variance need only be be described to include factorS Such as expected differences present in one copy to produce a positive result then ~10% between a placebo and treatment on the primary or Second of the Subjects are likely heterozygotes for the variance that ary endpoints and a consideration of the allelic frequencies. produces the response. The Hardy-Weinberg equation can be US 2001/0034023 A1 Oct. 25, 2001 used to infer an allele frequency in the range of 5% from 0683) The table above shows the probability (expressed these assumptions (given allele frequencies of 5%/95% then: as percent) of detecting both alleles (i.e. detecting heterozy 2x0.05x0.95=0.095, or 9.5% heterozygotes are expected, gotes) at a biallelic locus as a function of (i) the allele and 0.05x0.05=0.0025, or 0.25% homozygotes are frequencies and (ii) the number of individuals genotyped. expected. They sum to 9.5%+0.25%=9.75% likely respond The chances of detecting heterozygotes increases as the ers, 80% of whom, or 7.6%, are likely real responders due frequencies of the two alleles approach 0.5 (down a col to presence of the positive response allele. Thus about half umn), and as the number of individuals genotyped increases of the 15% responders are accounted for.). From the Table (to the right along a row). The numbers in the table are given it can be seen that, in order to have a 99% chance of by the formula: 1-(p)"-(q)". Allele frequencies are des detecting an allele present at a frequency of 5% nearly 50 ignated p and q and the number of individuals tested is Subjects should be Screened for variances, assuming that the designated n. (Since humans are diploid, the number of variances occur in the Screening population at the same alleles tested is twice the number of individuals, or 2n.) frequency as they occur in the patient population. Similar 0684. While it is preferable that numbers of individuals, analyses can be performed for other assumptions regarding or independent Sequence Samples, are Screened to identify likely magnitude of effect, penetrance and mode of genetic variances in a gene, it is also very beneficial to identify transmission. variances using Smaller numbers of individuals or Sequence 0681 At the beginning we only know phenotype frequen Samples. For example, even a comparison between the cies (e.g. responders vs. nonresponders, frequency of vari Sequences of two Samples or individuals can reveal Sequence ous side effects, etc.). As an example, the occurrence of variances between them. Preferably, 5, 10, or more samples Serious 5-FU/FA toxicity-e.g. toxicity requiring hospital or individuals are Screened. ization is often >10%. The occurrence of life threatening toxicity is in the 1-3% range (Buroker et al. 1994). The 0685 Source of Nucleic Acid Samples occurrence of complete remissions is on the order of 2-8%. 0686 Nucleic acid samples, for example for use in vari The lowest frequency phenotypes are thus on the order of ance identification, can be obtained from a variety of Sources ~2%. If we assume that (i) homogeneous genetic effects are as known to those skilled in the art, or can be obtained from responsible for half the phenotypes of interest and (ii) for the genomic or cDNA Sources by known methods. For example, most part the extreme phenotypes represent recessive geno the Coriell Cell Repository (Camden, N.J.) maintains over types, then we need to detect alleles that will be present at 6,000 human cell cultures, mostly fibroblast and lympho ~10% frequency (0.1x0.1=0.01, or 1% frequency of blast cell lines comprising the NIGMS Human Genetic homozygotes) if the population is at Hardy-Weinberg equi Mutant Cell Repository. A catalog (http://locus.umdnj.edu/ librium. To have a ~99% chance of identifying such alleles nigms) provides racial or ethnic identifiers for many of the would require Searching a population of 22 individuals (see cell lines. It is preferable to perform polymorphism discov Table below). If the major phenotypes are associated with ery on a population that mimics the population to be heterozygous genotypes then we need to detect alleles evaluated in a clinical trial, both in terms of racial/ethnic/ present at -0.5% frequency (2x0.005x0.995=0.00995, or geographic background and in terms of disease Status. ~1% frequency of heterozygotes). A 99% chance of detect Otherwise, it is generally preferable to include a broad ing such alleles would require ~40 individuals (Table population sample including, for example, (for trials in the below). Given the heterogeneity of the North American United States): Caucasians of Northern, Central and South population we cannot assume that all genotypes are present ern European origin, Africans or African-Americans, His in Hardy-Weinberg proportions, therefore a substantial over panics or Mexicans, Chinese, Japanese, American Indian, Sampling may be done to increase the chances of detecting East Indian, Arabs and Koreans. relevant variances: For our initial Screening, usually, 62 individuals of known race/ethnicity are Screened for Vari 0687 Source of Human DNA, RNA and cDNA Samples ance. Variance detection Studies can be extended to outliers 0688 PCR based screening for DNA polymorphism can for the phenotypes of interest to cover the possibility that be carried out using either genomic DNA or cDNA produced important variances were missed in the normal population from mRNA. For many genes, only cDNA sequences have Screening. been published, therefore the analysis of those genes is, at

Allele frequencies n = 5 n = 10 n = 15 n = 20 m = 25 n = 30 n = 35 n = 50 p = .99, 9.56 18.21 26.03 33.10 39.5O 45.28 50.52 63.40 p = .97, 26.26 45.62 59.90 70.43 78.19 83.92 88.14 95.24 p = .95, 40.13 64.15 78.53 87.15 92.3O 95.39 97.24 99.65 p = .93, 51.60 76.58 88.66 94.51 97.34 98.71 99.38 99.93 p = .9, q = 65.13 87.84 95.76 98.52 99.48 99.82 99.94 >99.9 p = .8, q = 89.26 98.84 99.88 99.99 99.9 99.9 99.9 >99.9 p = .7, q = 97.17 99.92 99.99 99.9 99.9 99.9 99.9 >99.9

0682 Likelihood of Detecting Polymorphism in a Popu least initially, at the cDNA level since the determination of lation as a Function of Allele Frequency & Number of intron-exon boundaries and the isolation of flanking Individuals Genotyped Sequences is a laborious proceSS. However, Screening US 2001/0034023 A1 Oct. 25, 2001 67 genomic DNA has the advantage that variances can be 0695) The T4E7 Variance Imaging procedure has been identified in promoter, intron and flanking regions. Such used to screen particular genes. The efficiency of the T4E7 variances may be biologically relevant. Therefore prefer enzyme to recognize and cleave at all mismatches has been ably, when variance analysis of patients with outlier tested and reported in the literature. One group reported responses is performed, analysis of Selected loci at the detection of 81 of 81 known mutations (Youil et al. 1995) genomic level is also performed. Such analysis would be while another group reported detection of 16 of 17 known contingent on the availability of a genomic Sequence or mutations (Mashal and Sklar, 1995). Thus, the T4E7 method intron-exon boundary Sequences, and would also depend on provides highly efficient variance detection. the anticipated biological importance of the gene in connec tion with the particular response. 0696 DNA Sequencing 0697 A Subset of the samples containing each unique 0689. When cDNA is to be analyzed it is very beneficial T4E7 cleavage Site is Selected for Sequencing. DNA to establish a tissue Source in which the genes of interest are Sequencing can, for example, be performed on ABI 377 expressed at Sufficient levels that cDNA can be readily automated DNA sequencers using BigDye chemistry and produced by RT-PCR. Preliminary PCR optimization efforts cycle Sequencing. Analysis of the Sequencing runs will be for 19 of the 29 genes in Table 2 reveal that all 19 can be limited to the 30-40 bases pinpointed by the T4E7 procedure amplified from lymphoblastoid cell mRNA. The 7 untested as containing the variance. This provides the rapid identifi genes belong on the same pathways and are expected to also cation of the altered base or bases. be PCR amplifiable. 0698. In some cases, the presence of variances can be 0690 PCR Optimization inferred from published articles which describe Restriction 0691 Primers for amplifying a particular sequence can be Fragment Length Polymorphisms (RFLP). The sequence designed by methods known to those skilled in the art, variances or polymorphisms creating those RFLPS can be including by the use of computer programs Such as the readily determined using convention techniques, for PRIMER Software available from Whitehead Institute/MIT example in the following manner. If the RFLP was initially Genome Center. In Some cases it is preferable to optimize discovered by the hybridization of a cDNA, then the the amplification proceSS according to parameters and meth molecular sequence of the RFLP can be determined by ods known to those skilled in the art; optimization of PCR restricting the cDNA probe into fragments and Separately reactions based on a limited array of temperature, buffer and hybridizing to a Southern blot consisting of the restriction primer concentration conditions is utilized. New primers are digestion with the enzyme which reveals the polymorphic obtained if optimization fails with a particular primer Set. Site, identifying the Sub-fragment which hybridizes to the polymorphic restriction fragment, obtaining a genomic 0692 Variance Detection Using T4 Endonuclease VII clone of the gene (e.g., from commercial Services Such as Mismatch Cleavage Method Genome Systems (Saint Louis, Mo.) or Research Genetics 0693. Any of a variety of different methods for detecting (Ala. ) which will provide appropriate genomic clones on variances in a particular gene can be utilized, Such as those receipt of appropriate primer pairs). Using the genomic described in the patents and applications cited in Section A clone, restrict the genomic clone with the restriction enzyme above. An exemplary method is a T4 Endo VII method. The which revealed the polymorphism and isolate the fragment enzyme T4 endonuclease VII (T4E7) is derived from the which contains the polymorphism, e.g., identifying by bacteriophage T4. T4E7 specifically cleaves heteroduplex hybridization to the cDNA which detected the polymor DNA containing Single base mismatches, deletions or inser phism. The fragment is then Sequenced acroSS the polymor tions. The site of cleavage is 1 to 6 nucleotides 3' of the phic site. A copy of the other allele can be obtained by PCT mismatch. This activity has been exploited to develop a from addition Samples. general method for detecting DNA sequence variances 0699 Variance Detection Using Sequence Scanning (Youil et al. 1995; Mashal and Sklar, 1995). A quality controlled T4E7 variance detection procedure based on the 0700. In addition to the physical methods, e.g., those T4E7 patent of R. G. H. Cotton and co-workers. (Del Tito et described above and others known to those skilled in the art al., in press) is preferably utilized. T4E7 has the advantages (see, e.g., Housman, U.S. Pat. No. 5,702,890; of being rapid, inexpensive, Sensitive and Selective. Further, 0701 Housman et al., U.S. patent application Ser. No. Since the enzyme pinpoints the Site of Sequence variation, 09/045,053), variances can be detected using computational Sequencing effort can be confined to a 25-30 nucleotide methods, involving computer comparison of Sequences from Segment. two or more different biological Sources, which can be 0694. The major steps in identifying sequence variations obtained in various ways, for example from public Sequence in candidate genes using T4E7 are: (1) PCR amplify 400 databases. The term “variance Scanning refers to a process 600 bp segments from a panel of DNA samples; (2) mix a of identifying Sequence variances using computer-based fluorescently-labeled probe DNA with the sample DNA; (3) comparison and analysis of multiple representations of at heat and cool the samples to allow the formation of hetero least a portion of one or more genes. Computational variance duplexes; (4) add T4E7 enzyme to the samples and incubate detection involves a process to distinguish true variances for 30 minutes at 37 C., during which cleavage occurs at from Sequencing errors or other artifacts, and thus does not Sequence variance mismatches; (5) run the samples on an require perfectly accurate Sequences. Such Scanning can be ABI 377 Sequencing apparatus to identify cleavage bands, performed in a variety of ways, preferably, for example, as which indicate the presence and location of variances in the described in Stanton et al., filed Oct. 14, 1999, Ser. No. Sequence; (6) a Subset of PCR fragments showing cleavage 09/419,705. are Sequenced to identify the exact location and identity of 0702 While the utilization of complete cDNA sequences each variance. is highly preferred, it is also possible to utilize genomic US 2001/0034023 A1 Oct. 25, 2001

Sequences. Such analysis may be desired where the detec lation of Such a response to a particular gene is performed tion of variances in or near splice Sites is Sought. Such first.) Such a differential response can be determined using Sequences may represent full or partial genomic DNA prospective and/or retrospective data. Thus, in Some cases, Sequences for a gene or genes. Also, as previously indicated, published reports will indicate that the course of treatment partial cDNA sequences can also be utilized although this is will vary depending on the presence or absence of particular leSS preferred. AS described below, the variance Scanning variances. That information can be utilized to create a analysis can Simply utilize Sequence overlap regions, even diagnostic test and/or incorporated in a treatment method as from partial Sequences. Also, while the present description is an efficacy or Safety determination Step. provided by reference to DNA, e.g., cDNA, Some Sequences may be provided as RNA sequences, e.g., mRNA sequences. 0709. Usually, however, the effect of one or more vari Such RNA sequences may be converted to the correspond ances is separately determined. The determination can be ing DNA sequences, or the analysis may use the RNA performed by analyzing the presence or absence of particu Sequences directly. lar variances in patients who have previously been treated with a particular treatment method, and correlating the 0703 B. Determination of Presence or Absence of variance presence or absence with the observed course, Known Variances outcome, and/or development of adverse events in those patients. This approach is useful in cases in which observa 0704. The identification of the presence of previously tion of treatment effects was clearly recorded and cell identified variances in cells of an individual, usually a Samples are available or can be obtained. Alternatively, the particular patient, can be performed by a number of different analysis can be performed prospectively, where the presence techniques as indicated in the Summary above. Such meth or absence of the variance or variances in an individual is ods include methods utilizing a probe which specifically determined and the course, outcome, and/or development of recognizes the presence of a particular nucleic acid or amino adverse events in those patients is Subsequently or concur acid Sequence in a Sample. Common types of probes include rently observed and then correlated with the variance deter nucleic acid hybridization probes and antibodies, for mination. example, monoclonal antibodies, which can differentially bind to nucleic acid Sequences differing in one or more 0710 Analysis of Haplotypes Increases Power of Genetic variance Sites or to polypeptides which differ in one or more Analysis amino acid residues as a result of the nucleic acid Sequence 0711. In some cases, variation in activity due to a single variance or variances. Generation and use of Such probes is gene or a single genetic Variance in a single gene may not be well-known in the art and So is not described in detail herein. Sufficient to account for a clinically significant fraction of the 0705 Preferably, however, the presence or absence of a observed variation in patient response to a treatment, e.g., a variance is determined using nucleotide Sequencing of a drug, there may be other factors that account for Some of the Short Sequence Spanning a previously identified variance variation in patient response. Drug response phenotypes Site. This will utilize validated genotyping assays for the may vary continuously, and Such (quantitative) traits may be polymorphisms previously identified. Since both normal and influenced by a number of genes (Falconer and Mackay, tumor cell genotypes can be measured, and Since tumor Quantitative Genetics, 1997). Although it is impossible to material will frequently only be available as paraffin embed determine a priori the number of genes influencing a quan ded sections (from which RNA cannot be isolated), it will be titative trait, potentially only one or a few loci have large necessary to utilize genotyping assays that will work on effects, where a large effect is 5-20% of total variation in the genomic DNA. Thus PCR reactions will be designed, opti phenotype (Mackay, 1995). mized, and validated to accommodate the intron-exon Struc 0712 Having identified genetic variation in enzymes that ture of each of the genes. If the gene Structure has been may affect action of a Specific drug, it is useful to efficiently published (as it has for some of the listed genes), PCR address its relation to phenotypic variation. The Sequential primers can be designed directly. However, if the gene testing for correlation between phenotypes of interest and structure is unknown, the PCR primers may need to be Single nucleotide polymorphisms may be adequate to detect moved around in order to both span the variance and avoid asSociations if there are major effects associated with Single exon-intron boundaries. In some cases one-sided PCR meth nucleotide changes, certainly it is useful to this type of ods such as bubble PCR (Ausubel et al. 1997) may be useful analysis. However there is no way to know in advance to obtain flanking intronic DNA for Sequence analysis. whether there are major phenotypic effects associated with 0706. Using such amplification procedures, the standard Single nucleotide changes and, even if there are, there is no method used to genotype normal and tumor tissues will be way to be Sure that the Salient variance has been identified DNA sequencing. PCR fragments encompassing the Vari by Screening cDNAS. A more powerful way to address the ances will be cycle sequenced on ABI 377 automated question of genotype-phenotype correlation is to assort Sequencers using Big Dye chemistry genotypes into haplotypes. (A haplotype is the cis arrange ment of polymorphic nucleotides on a particular chromo 0707) C. Correlation of the Presence or Absence of Spe Some.) Haplotype analysis has several advantages compared cific Variances with Differential Treatment Response to the Serial analysis of individual polymorphisms at a locus 0708 Prior to establishment of a diagnostic test for use in with multiple polymorphic Sites. the Selection of a treatment method or elimination of a 0713 (1) Of all the possible haplotypes at a locus (2" treatment method, the presence or absence of one or more haplotypes are theoretically possible at a locus with n binary Specific variances in a gene or in multiple genes is correlated polymorphic Sites) only a Small fraction will generally occur with a differential treatment response. (AS discussed above, at a significant frequency in human populations. Thus, usually the existence of a variable response and the corre asSociation Studies of haplotypes and phenotypes will US 2001/0034023 A1 Oct. 25, 2001 involve testing fewer hypotheses. As a result there is a Samples with known haplotypes. The Set of haplotypes smaller probability of Type I errors, that is, false inferences determined by pedigree analysis can be useful in computa that a particular variant is associated with a given phenotype. tional methods, including those utilizing the E-M algorithm. 0714 (2) The biological effect of each variance at a locus 0719 Haplotypes can also be determined directly from may be different both in magnitude and direction. For cDNA using the T4E7 procedure. T4E7 cleaves mismatched example, a polymorphism in the 5' UTR may affect trans heteroduplex DNA at the site of the mismatch. If a hetero lational efficiency, a coding Sequence polymorphism may duplex contains only one mismatch, cleavage will result in affect protein activity, a polymorphism in the 3' UTR may the generation of two fragments. However, if a Single affect mRNA folding and half life, and so on. Further, there heteroduplex (allele) contains two mismatches, cleavage may be interactions between variances: two neighboring will occur at two different Sites resulting in the generation of polymorphic amino acids in the same domain-Say cyS/arg three fragments. The appearance of a fragment whose size at residue 29 and met/val at residue 166-may, when corresponds to the distance between the two cleavage Sites combined in one Sequence, for example, 29 cyS-166 Val, is diagnostic of the two mismatches being present on the have a deleterious effect, whereas 29 cys-166 met, 29 same strand (allele). Thus, T4E7 can be used to determine arg-166 met and 29 arg-166 Val proteins may be nearly equal haplotypes in diploid cells. in activity. Haplotype analysis is the best method for assess 0720. An alternative method, allele specific PCR, may be ing the interaction of variances at a locus. used for haplotyping. The utility of allele specific PCR for 0715 (3) Templeton and colleagues have developed pow haplotyping has already been established (Michalatos-Be erful methods for assorting haplotypes and analyzing hap loin et al., 1996; Chang et al. 1997). Opposing PCR primers lotype/phenotype associations (Templeton et al., 1987). are designed to cover two sites of variance (either adjacent Alleles which share common ancestry are arranged into a Sites or sites Spanning one or more internal variances). Two tree structure (cladogram) according to their (inferred) time versions of each primer are Synthesized, identical to each of origin in a population (that is, according to the principle other except for the 3' terminal nucleotide. The 3' terminal of parSimony). Haplotypes that are evolutionarily ancient nucleotide is designed so that it will hybridize to one but not will be at the center of the branching Structure and new ones the other variant base. PCR amplification is then attempted (reflecting recent mutations) will be represented at the with all four possible primer combinations in Separate wells. periphery, with the linkS representing intermediate Steps in Because Taq polymerase is very inefficient at extending 3 evolution. The cladogram defines which haplotype-pheno mismatches, the only samples which will be amplified will type association tests should be performed to most effi be the ones in which the two primers are perfectly matched ciently exploit the available degrees of freedom, focusing for Sequences on the same strand (allele). The presence or attention on those comparisons most likely to define func absence of PCR product allows haplotyping of diploid cell tionally different haplotypes (Haviland et al., 1995). This lines. At most two of four possible reactions should yield type of analysis has been used to define interactions between products. This procedure has been Successfully applied, for heart disease and the apolipoprotein gene cluster (Haviland example, to haplotype the DPD amino acid polymorphisms. et al 1995) and Alzheimer's Disease and the Apo-E locus 0721 Parsimony methods are also useful for classifying (Templeton 1995) among other Studies, using populations as DNA sequences, haplotypes or phenotypic characters. Par small as 50 to 100 individuals. The methods of Templeton Simony principle maintains that the best explanation for the have also been applied to measure the genetic determinants observed differences among sequences, phenotypes (indi of variation in the angiotensin-I converting enzyme gene. viduals, species) etc., is provided by the Smallest number of (Keavney, B., McKenzie, C. A., Connoll, J. M. C., et al. evolutionary changes. Alternatively, Simpler hypotheses are Measured haplotype analysis of the angiotensin-I converting preferable to explain a Set of data or patterns, than more enzyme gene. Human Molecular Genetics 7: 1745-1751.) complicated ones, and ad hoc hypotheses should be avoided 0716 Methods for Determining Haplotypes whenever possible (Molecular Systematics, Hillis et al., 0717 The goal of haplotyping is to identify the common 1996). Parsimony methods thus operate by minimizing the haplotypes at Selected loci that have multiple sites of Vari number of evolutionary steps or mutations (changes from ance. Haplotypes are usually determined at the cDNA level. one sequence/character) required to account for a given set Several general approaches to identification of haplotyes can of data. be employed. Haplotypes may also be estimated using 0722 For example, supposing we want to obtain rela computational methods or determined definitively using tionships among a set of Sequences and construct a structure experimental approaches. Computational approaches gener (tree/topology), we first count the minimum number of ally include an expectation maximization (E-M) algorithm mutations that are required for explaining the observed (see, for example: Excoffier and Slatkin, Mol. Biol. Evol. evolutionary changes among a set of Sequences. A structure 1995) or a combination of Parsimony (see below) and E-M (topology) is constructed based on this number. When once methods. this number is obtained, another structure is tried. This 0718 Haplotypes can be determined experimentally process is continued for all reasonable number of Structures. without requirement of a haplotyping method by genotyping Finally, the structure that required the smallest number of Samples from a set of pedigrees and observing the Segrega mutational StepS is chosen as the likely structure/evolution tion of haplotypes. For example families collected by the ary tree for the Sequences Studied. Centre d’Etude du Polymorphisme Humaine (CEPH) can be 0723 For haplotypes identified herein, haplotypes were used. Cell lines from these families are available from the identified by examining genotypes from each cell line. This Coriell Repository. This approach will be useful for cata list of genotypes was optimized to remove variance siteS/ loging common haplotypes and for validating methods on individuals with incomplete information, and the genotype US 2001/0034023 A1 Oct. 25, 2001 70 from each remaining cell line was examined in turn. The variances provides useful information for Selecting or number of heterozygotes in the genotype were counted, and designing or preparing methods or materials for medical use those genotypes containing more than one heterozygote in the aspects of this invention. AS previously Stated, Such were discarded, and the rest were gathered in a list for variance determination or diagnostic methods can be per Storage and display. For haplotypes identified herein, hap formed in various ways as understood by those skilled in the lotypes were identified by examining genotypes from each art. cell line. This list of genotypes was optimized to remove 0730. In certain variance determination methods, it is variance Sites/individuals with incomplete information, and necessary or advantageous to amplify one or more nucle the genotype from each remaining cell line was examined in otide Sequences in one or more of the genes identified turn. The number of heterozygotes in the genotype were herein. Such amplification can be performed by conven counted, and those genotypes containing more than one tional methods, e.g., using polymerase chain reaction (PCR) heterozygote were discarded, and the rest were gathered in amplification. Such amplification methods are well-known a list for Storage and display. to those skilled in the art and will not be specifically 0724 D. Selection of Treatment Method Using Variance described herein. For most applications relevant to the Information present invention, a Sequence to be amplified includes at least one variance Site, which is preferably a site or Sites 0725 1. General which provide variance information indicative of the effec 0726 Once the presence or absence of a variance or tiveness of a method of treatment or method of administra variances in a gene or genes is shown to correlate with the tion of a treatment, or effectiveness of a Second method of efficacy or Safety of a treatment method, that information treatment which reduces a deleterious effect of a first treat can be used to Select an appropriate treatment method for a ment method, or which enhances the effectiveness of a first particular patient. In the case of a treatment which is more method of treatment. Thus, for PCR, such amplification likely to be effective when administered to a patient who has generally utilizes primer oligonucleotides which bind to or at least one copy of a gene with a particular variance or extent through at least one Such variance Site under ampli variances (in Some cases the correlation with effective fication conditions. treatment is for patients who are homozygous for a variance 0731. For convenient use of the amplified sequence, e.g., or set of variances in a gene) than in patients with a different for Sequencing, it is beneficial that the amplified Sequence be variance or Set of variances, a method of treatment is of limited length, but Still long enough to allow convenient Selected (and/or a method of administration) which corre and Specific amplification. Thus, preferably the amplified lates positively with the particular variance presence or Sequence has a length as described in the Summary. absence which provides the indication of effectiveness. AS indicated in the Summary, Such Selection can involve a 0732 Also, in certain variance determination, it is useful variety of different choices, and the correlation can involve to Sequence one or more portions of a gene or genes, in a variety of different types of treatments, or choices of particular, portions of the genes identified in this disclosure. methods of treatment. In Some cases, the Selection may AS understood by perSons familiar with nucleic acid include choices between treatments or methods of adminis Sequencing, there are a variety of effective methods. In tration where more than one method is likely to be effective, particular, Sequencing can utilize dye termination methods or where there is a range of expected effectiveneSS or and mass spectrometric methods. The Sequencing generally different expected levels of contra-indication or deleterious involves a nucleic acid Sequence which includes a variance effects. In Such cases the Selection is preferably performed to Site as indicated above in connection with amplification. Select a treatment which will be as effective or more effec Such Sequencing can directly provide determination of the tive than other methods, while having a comparatively low presence or absence of a particular variance or Set of level of deleterious effects. Similarly, where the selection is Variances, e.g., a haplotype, by inspection of the Sequence between method with differing levels of deleterious effects, (visually or by computer). Such sequencing is generally preferably a method is selected which has low such effects conducted on PCR amplified Sequences in order to provide but which is expected to be effective in the patient. Sufficient Signal for practical or reliable Sequence determi nation. 0727. Alternatively, in cases where the presence or absence of the particular variance or variances is indicative 0733. Likewise, in certain variance determinations, it is that a treatment or method of administration is more likely useful to utilize a probe or probes. AS previously described, to be ineffective or contra-indicated in a patient with that Such probes can be of a variety of different types. variance or variances, then Such treatment or method of 0734 VI. Pharmaceutical Compositions, Including Phar administration is generally eliminated for use in that patient. maceutical Compositions Adapted to be Preferentially Effective in Patients Having Particular Genetic Character 0728 2. Diagnostic Methods istics 0729. Once a correlation between the presence and absence of at least one variance in a gene or genes and an 0735. A. General indication of the effectiveness of a treatment, the determi 0736. The methods of the present invention, in many nation of the presence or absence of that at least one variance cases will utilize conventional pharmaceutical composi provides diagnostic methods, which can be used as indicated tions, but will allow more advantageous and beneficial use in the Summary above to Select methods of treatment, of those compositions due to the ability to identify patients methods of administration of a treatment, methods of Select who are likely to benefit from a particular treatment or to ing a patient or patients for a treatment and others aspects in identify patients for whom a particular treatment is leSS which the determination of the presence or absence of those likely to be effective or for whom a particular treatment is US 2001/0034023 A1 Oct. 25, 2001

likely to produce undesirable or intolerable effects. How remaining in cancer cells following loSS of heterozygosity ever, in Some cases, it is advantageous to utilize composi affecting that gene. Thus, even though normal cells of the tions which are adapted to be preferentially effective in patient may contain a form of the gene which correlates with patients who possess particular genetic characteristics, i.e., effective treatment response, the absence of that form in in whom a particular variance or variances in one or more cancer cells will mean that the treatment would be less likely genes is present or absent (depending on whether the pres to be effective in that patient than in another patient who ence or the absence of the variance or variances in a patient retained in cancer cells the form of the gene which correlated is correlated with an increased expectation of beneficial with effective treatment response. Those skilled in the art response). Thus, for example, the presence of a particular will understand whether the variances or gene forms in variance or variances may indicate that a patient can ben normal or disease cells are most indicative of the expected eficially receive a significantly higher dosage of a drug than treatment response, and will generally utilize a diagnostic a patient having a different test with respect to the appropriate cells. Such a cell type indication or Suggestion may also be contained in a regula 0737 B. Regulatory Indications and Restrictions tory Statement, e.g., on a label or in a product insert. 0738. The sale and use of drugs and the use of other 0741. C. Preparation and Administration of Drugs and treatment methods usually are Subject to certain restrictions Pharmaceutical Compositions Including Pharmaceutical by a government regulatory agency charged with ensuring Compositions Adapted to be Preferentially Effective in the Safety and efficacy of drugs and treatment methods for Patients Having Particular Genetic Characteristics medical use, and approval is based on particular indications. In the present invention it is found that variability in patient 0742 A particular compound useful in this invention can response or patient tolerance of a drug or other treatment be administered to a patient either by itself, or in pharma often correlates with the presence or absence of particular ceutical compositions where it is mixed with Suitable car variances in particular genes. Thus, it is expected that Such riers or excipient(s). In treating a patient exhibiting a dis a regulatory agency may indicate that the approved indica order of interest, a therapeutically effective amount of a tions for use of a drug with a variance-related variable agent or agents Such as these is administered. A therapeu response or toleration include use only in patients in whom tically effective dose refers to that amount of the compound the drug will be effective, and/or for whom the administra that results in amelioration of one or more Symptoms or a tion of the drug will not have intolerable deleterious effects, prolongation of Survival in a patient. Such as excessive toxicity or unacceptable Side-effects. Con 0743 Toxicity and therapeutic efficacy of such com versely, the drug may be given for an indication that it may pounds can be determined by Standard pharmaceutical pro be used in the treatment of a particular disease or condition cedures in cell cultures or experimental animals, e.g., for where the patient has at least one copy of a particular determining the LDs (the dose lethal to 50% of the popu variance, variances, or variant form of a gene. Even if the lation) and the EDso (the dose therapeutically effective in approved indications are not narrowed to Such groups, the 50% of the population). The dose ratio between toxic and regulatory agency may Suggest use limited to particular therapeutic effects is the therapeutic indeX and it can be groups or excluding particular groups or may state advan expressed as the ratio LDs/EDs. Compounds which tages of use or exclusion of Such groups or may state a exhibit large therapeutic indices are preferred. The data warning on the use of the drug in certain groups. Consistent obtained from these cell culture assays and animal Studies with Such Suggestions and indications, Such an agency may can be used in formulating a range of dosage for use in Suggest or recommend the use of a diagnostic test to identify human. The dosage of Such compounds lies preferably the presence or absence of the relevant variances in the within a range of circulating concentrations that include the prospective patient. Such diagnostic methods are described EDso with little or no toxicity. The dosage may vary within in this description. Generally, Such regulatory Suggestion or this range depending upon the dosage form employed and indication is provided in a product insert or label, and is the route of administration utilized. generally reproduced in references Such as the Physician's Desk Reference (PDR). Thus, this invention also includes 0744) For any compound used in the method of the drugs or pharmaceutical compositions which carry Such a invention, the therapeutically effective dose can be esti Suggestion or Statement of indication or warning or Sugges mated initially from cell culture assayS. For example, a dose tion for a diagnostic test, and which may also be packaged can be formulated in animal models to achieve a circulating with an insert or label Stating the Suggestion or indication or plasma concentration range that includes the ICso as deter warning or Suggestion for a diagnostic test. mined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in 0739. In accord with the possible variable treatment plasma may be measured, for example, by HPLC. responses, an indication or Suggestion can Specify that a patient be heterozygous, or alternatively, homozygous for a 0745. The exact formulation, route of administration and particular variance or variances or variant form of a gene. dosage can be chosen by the individual physician in View of Alternatively, an indication or Suggestion may specify that a the patient's condition. (See e.g. Finglet. al., in The Phar patient have no more than one copy, or Zero copies, of a macological Basis of Therapeutics, 1975, Ch. 1 p.1). It particular variance, variances, or variant form of a gene. should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust adminis 0740. A regulatory indication or Suggestion may concern tration due to toxicity, or to organ dysfunctions. Conversely, the variances or variant forms of a gene in normal cells of the attending physician would also know to adjust treatment a patient and/or in cells involved in the disease or condition. to higher levels if the clinical response were not adequate For example, in the case of a cancer treatment, the response (precluding toxicity). The magnitude of an administrated of the cancer cells can depend on the form of a gene dose in the management of disorder of interest will vary with US 2001/0034023 A1 Oct. 25, 2001 72 the severity of the condition to be treated and the route of facilitate processing of the active compounds into prepara administration. The Severity of the condition may, for tions which can be used pharmaceutically. The preparations example, be evaluated, in part, by Standard prognostic formulated for oral administration may be in the form of evaluation methods. Further, the dose and perhaps dose tablets, dragees, capsules, or Solutions. The pharmaceutical frequency, will also vary according to the age, body weight, compositions of the present invention may be manufactured and response of the individual patient. A program compa in a manner that is itself known, e.g., by means of conven rable to that discussed above may be used in Veterinary tional mixing, dissolving, granulating, dragee-making, levi medicine. tating, emulsifying, encapsulating, entrapping or lyophiliz 0746) Depending on the specific conditions being treated, ing processes. Such agents may be formulated and administered Systemi 0751 Pharmaceutical formulations for parenteral admin cally or locally. Techniques for formulation and administra istration include acqueous Solutions of the active compounds tion may be found in Remington's Pharmaceutical Sciences, in water-Soluble form. Additionally, Suspensions of the 18th ed., Mack Publishing Co., Easton, Pa. (1990). Suitable active compounds may be prepared as appropriate oily routes may include oral, rectal, transdermal, vaginal, trans injection Suspensions. Suitable lipophilic Solvents or mucosal, or intestinal administration; parenteral delivery, vehicles include fatty oils Such as Sesame oil, or Synthetic including intramuscular, Subcutaneous, intramedullary fatty acid esters, Such as ethyl oleate or triglycerides, or injections, as well as intrathecal, direct intraventricular, liposomes. Aqueous injection Suspensions may contain Sub intravenous, intraperitoneal, intranasal, or intraocular injec stances which increase the Viscosity of the Suspension, Such tions, just to name a few. as Sodium carboxymethyl cellulose, Sorbitol, or dextran. 0747 For injection, the agents of the invention may be Optionally, the Suspension may also contain Suitable Stabi formulated in aqueous Solutions, preferably in physiologi lizers or agents which increase the Solubility of the com cally compatible bufferS Such as Hanks's Solution, Ringer's pounds to allow for the preparation of highly concentrated Solution, or physiological Saline buffer. For Such transmu Solutions. cosal administration, penetrants appropriate to the barrier to 0752 Pharmaceutical preparations for oral use can be be permeated are used in the formulation. Such penetrants obtained by combining the active compounds with Solid are generally known in the art. excipient, optionally grinding a resulting mixture, and pro 0748 Use of pharmaceutically acceptable carriers to for cessing the mixture of granules, after adding Suitable aux mulate the compounds herein disclosed for the practice of iliaries, if desired, to obtain tablets or dragee cores. Suitable the invention into dosages Suitable for Systemic administra excipients are, in particular, fillerS Such as Sugars, including tion is within the scope of the invention. With proper choice lactose, Sucrose, mannitol, or Sorbitol, cellulose preparations of carrier and Suitable manufacturing practice, the compo Such as, for example, maize Starch, wheat Starch, rice Starch, Sitions of the present invention, in particular, those formu potato Starch, gelatin, gum tragacanth, methyl cellulose, lated as Solutions, may be administered parenterally, Such as hydroxypropylmethyl-cellulose, Sodium carboxymethylcel by intravenous injection. The compounds can be formulated lulose, and/or polyvinylpyrrolidone (PVP). If desired, dis readily using pharmaceutically acceptable carriers well integrating agents may be added, Such as the croSS-linked known in the art into dosages Suitable for oral administra polyvinyl pyrrollidone, agar, or alginic acid or a Salt thereof tion. Such carriers enable the compounds of the invention to Such as Sodium alginate. Dragee cores are provided with be formulated as tablets, pills, capsules, liquids, gels, Syrups, Suitable coatings. For this purpose, concentrated Sugar Solu Slurries, Suspensions and the like, for oral ingestion by a tions may be used, which may optionally contain gum patient to be treated. arabic, talc, polyvinyl pyrrollidone, carbopol gel, polyethyl ene glycol, and/or titanium dioxide, lacquer Solutions, and 0749 Agents intended to be administered intracellularly Suitable organic Solvents or Solvent mixtures. Dyestuffs or may be administered using techniques well known to those pigments may be added to the tablets or dragee coatings for of ordinary skill in the art. For example, Such agents may be identification or to characterize different combinations of encapsulated into liposomes, then administered as described active compound doses. above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous Solution at the 0753 Pharmaceutical preparations which can be used time of lipoSome formation are incorporated into the aque orally include push-fit capsules made of gelatin, as well as ous interior. The liposomal contents are both protected from Soft, Sealed capsules made of gelatin and a plasticizer, Such the external microenvironment and, because liposomes fuse as glycerol or Sorbitol. The push-fit capsules can contain the with cell membranes, are efficiently delivered into the cell active ingredients in admixture with filler Such as lactose, cytoplasm. Additionally, due to their hydrophobicity, Small binderS Such as Starches, and/or lubricants Such as talc or organic molecules may be directly administered intracellu magnesium Stearate and, optionally, Stabilizers. In Soft cap larly. Sules, the active compounds may be dissolved or Suspended in Suitable liquids, Such as fatty oils, liquid paraffin, or liquid 0750 Pharmaceutical compositions suitable for use in the polyethylene glycols. In addition, Stabilizers may be added. present invention include compositions wherein the active ingredients are contained in an effective amount to achieve 0754) The invention described herein features methods its intended purpose. Determination of the effective amounts for determining the appropriate identification of a patient is well within the capability of those skilled in the art, diagnosed with a neurological disease or neurological dyS especially in light of the detailed disclosure provided herein. function based on an analysis of the patient's allele Status for In addition to the active ingredients, these pharmaceutical a gene listed in Tables 1, 3, and 4. Specifically, the presence compositions may contain Suitable pharmaceutically accept of at least one allele indicates that a patient will respond to able carriers comprising excipients and auxiliaries which a candidate therapeutic intervention aimed at treating a US 2001/0034023 A1 Oct. 25, 2001 neurological clinical Symptoms. In a preferred approach, the and diagnostic genotyping can lead to improved medical patient's allele Status is rapidly diagnosed using a Sensitive management of a disease and/or approval of a drug, or PCR assay and a treatment protocol is rendered. The inven broader use of an already approved drug. Gene pathways tion also provides a method for forecasting patient outcome including, but not limited to, those that are outlined in the and the Suitability of the patient for entering a clinical drug gene pathway, Table 1, are useful in identifying the Sources trial for the testing of a candidate therapeutic intervention of interpatient variation in efficacy as well as in the adverse for a neurological disease, condition, or dysfunction. events Summarized in the column headings of Table 2, 0755. The findings described herein indicate the predic Discussed in detail below are exemplary candidate genes for tive value of the target allele in identifying patients at risk for the analysis of pharmacokinetic variability in clinical devel neurologic disease or neurologic dysfunction. In addition, opment, using the methods described above. because the underlying mechanism influenced by the allele 0762. Advantages of Inclusion of Pharmacogenetic Status is not disease-specific, the allele Status is Suitable for Stratification in Clinical Development of Agents: Impact on making patient predictions for diseases not affected by the Efficacy pathway as well. 0763 The advantages of a clinical research and drug 0756. The following examples, which describe exem development program that includes the use of polymorphic plary techniques and experimental results, are provided for genotyping for the Stratification of patients for the appro the purpose of illustrating the invention, and should not be priate Selection of candidate therapeutic intervention construed as limiting. includes 1) identification of patients that may respond earlier and show signs and Symptoms of efficacious therapy, 2) EXAMPLE 1. identification of the primary gene and relevant polymorphic variance that directly affects efficacy endpoints, 3) identifi Effect of Pharmacokinetic parameters on Efficacy cation of pathophysiologic relevant variance or variances of Drugs and Candidate Therapeutic Interventions and potential therapies affecting those allelic genotypes or haplotypes, and 4) identification of allelic variances or 0757. The efficacy of a compound is determined by a haplotypes in genes that indirectly affects efficacy, Safety or combination of pharmacodynamic and pharmacokinetic both. effects. Both types of effect are under genetic control. In the present invention, the genetic determinants of efficacy are 0764. By identifying Subsets of patients, based upon discussed in terms of variation in the genes that encode genotype, that experience efficacious therapeutic benefit in proteins responsible for absorption, distribution, metabo response to the administration of a drug, agent or candidate lism, and excretion of compounds, i.e. pharmacokinetic therapeutic intervention, optimal Selection may reduce level parameterS. and extent of the appearance or manifestation of a side effect or toxicity. Appropriate genotyping and correlation to dosing 0758. The pharmacokinetic parameters with potential regimen, or Selection of optimal therapy would be beneficial effects on efficacy include absorption, distribution, metabo to the patient, caregivers, medical perSonnel, and the lism, and excretion. These parameters affect efficacy broadly by controlling the availability of a compound at the site(s) of patient's loved ones. action. Interpatient variability in the availability of a com 0765. As an example of identification of the primary gene pound can result in undertreatment or overtreatment, or in and relevant polymorphic variance that directly affects effi adverse reactions due to levels of a compound or its metabo cacy, Safety, or both one could Select an gene pathway as lite(s). Differences in the genes responsible for pharmaco described in the Detailed Description, and determine the kinetic variation, therefore, can be a potential Source of effect of genetic polymorphism and therapy efficacy, Safety, interpatient variability in drug response. or both within that given pathway. For example, referring to Table 2, genes involved in absorption and distribution, phase 0759) Impact of Stratification Based Upon Genotype in I and phase II metabolism, and excretion the optimization of Drug Development for Drugs, Compounds, or Candidate therapy of by an agent known to have an efficacious effect Therapeutic Interventions that may Efficacy by determining whether the patient has a predisposing 0760 Clozapine induced agranulocytosis has been asso genotype in which the Selected agents are more effective and ciated in Some reports with Specific HLA haplotypes or with or are more Safe. In considering an optimization protocol, HSP70 variants. These reports suggest that a gene within the one could potentially predetermine the genotypic profile of HLA region is associated with agranulocytosis in response these genes involved in the manifestation of the adverse to clozapine therapy. In a recent Study, two ethnic groups effect, or those genes preeminently responsible for drug were analyzed for genetic markers for agranulocytosis. response. By embarking on the previously described gene Tumor necrosis factor microsatellites d3 and b4 were found pathway approach, it is technical feasibility to determine the in higher frequencies in patients that experience clozapine relevant genes within Such a targeted drug development induced agranulocytosis. These data, while they need to be program. confirmed by additional Studies, are Suggestive that tumor 0766 Identification of pathophysiologic relevant vari necrosis factor polymorphisms may also be associated with ance or variances and potential therapies affecting those clozapine-induced agranulocytosis. allelic genotypes or haplotypes may speed drug develop 0761. In this invention we provide additional genes and ment for therapeutic alternatives. There is a need for thera gene Sequence variances that may account for variability in pies that are targeted to a disease and Symptom management toxic responses. The Detailed Description above demon with limited or no undesirable side effects. Identification of Strates how identification of a candidate gene or genes (e.g. a specific variance or variances within genes involved in the gene pathways), genetic Stratification, clinical trial design, manifestation of clinical efficacious endpoints or therapeutic US 2001/0034023 A1 Oct. 25, 2001 74 benefit and Specific genetic polymorphisms of these critical dramatic depletion of one or more of the following cell genes may assist the development of novel agents and the types: neutrophils, platelets, or reticulocytes. Drugs associ identification of those patients that may best benefit from ated with producing aplastic anemia are: 1) agents or com therapy of these candidate therapeutic alternatives. pounds that produce bone marrow depression, for example 0767 By identifying allelic variances or haplotypes in cytotoxic drugs used in cancer chemotherapy; 2) agents or genes that indirectly affects efficacy, Safety of any class of compounds that frequently, but inevitably, produce marrow drugs, one could target Specific Secondary drug or agent aplasia, for example benzene; 3) agents or compounds that therapeutic actions that affect the overall therapeutic action are associated with aplastic anemia, for example chloram of these agents. phenicol, antiprotozoals, and Sulfonamides. 0774 Aplastic anemia is almost always a result of dam 0768 Pharmacogenomics studies for these drugs, or age to the hematopoietic Stem cells. There are two possible other agent, compound, drug, or candidate therapeutic inter routes for the destruction of these cells: 1) direct damage to vention, could be performed by identifying genes that are the stem cell DNA, and 2) cell cycle dependant depletion of involved in the function of a drug including, but not limited later Stage progenitor cells. In the first case, drugs or agents to absorption, distribution, metabolism, or elimination, the bind to and randomly damage the genetic material. This type interaction of the drug with its target as well as potential of aplasia is associated with both early aplasia (immediate or alternative targets, the response of the cell to the binding of direct cytotoxicity) or later myelodysplasia and leukemia. In a drug to a target, the metabolism (including Synthesis, the latter case, mitotically and metabolically active progeni biodistribution or elimination) of natural compounds which tor cells are preferentially affected and progenitor cell deple may alter the activity of the drug by complementary, com tion may lead to unregulated proliferation of Spared Stem petitive or allosteric mechanisms that potentiate or limit the effect of the drug, and genes involved in the etiology of the cells. disease that alter its response to a particular class of thera 0775 Leukopenia is defined when the circulating peutic agents. It will be recognized to those skilled in the art peripheral white cell count falls below 5-10x10 cells per that this broadly includes proteins involved in pharmacoki liter. Circulating leukocytes consist of neutrophils, mono netics as well as genes involved in pharmacodynamics. This cytes, basophils, eosinophils, and lymphocytes. also includes genes that encode proteins homologous to the 0776 Neutropenia is defined when the peripheral neutro proteins believed to carry out the above functions are also phil count falls below 2x10 cells per liter. There are a Worth evaluation as they may carry out Similar functions. number of drugs families that can cause neutropenia includ Together the foregoing proteins constitute the candidate ing, but not exclusive to, antiarrythmics (procainamide, genes for affecting response of a patient to the therapeutic propanolol, quinidine), antibiotics (chloramphenicol, peni intervention. Using the methods described above, variances cillins, Sulfonamides, trimethorpimmethoxazole, para-ami in these genes can be identified, and research and clinical noSalicyclic acid, rifampin, Vancomycin, isoniazid, nitro Studies can be performed to establish an association between furantoin), antimalarials (dapsone, quinine, pyrimethamine), a drug response or toxicity and Specific variances. anticonvulsants (phenytoin, mephenytoin, trimethadione, EXAMPLE 2 ethoSuximide, carbamazepine), hypoglycemic agents (tolb utamide, chlorpropamide), antihistamines (cimetadine, bro Drug-Induced Toxicity: Blood Dyscrasias mpheniramine, tripelennamine), antihypertensives (methy 0769) I. Description of Blood Dyscrasias dopa, captopril), antiinflammatory agents (aminopyrine, phenylbutaZone, gold salts, ibuprofen, indomethacin), 0770 Blood dyscrasias are a feature of over half of all diuretics (acetazolamide, hydrochlorothiazide, chlorthali drug-related deaths and include, but are not limited to, bone done), phenothiazines (chlorpormazine, promazine, marrow aplasia, granulocytopenia, aplastic anemia, leuko prochlorperazine), antimetabolite immunosuppresive penia, lymphoid hyperplasia, hemolytic anemia, and throm agents, cytotoxic agents (alkylating agents, antimetabolites, bocytopenia. All of these Syndromes include pancytopenia anthracyclines, Vinca alkyloids, cis-platinum, hydroxy area, to Some degree. actinomycin D), and other agents (alpha and gamma inter 0771 Bone marrow aplasia is defined as a profound feron, allopurinol, ethanol, levamisole, penicillamine). loSS of bone marrow resulting in pancytopenia. Drugs 0777 Lymphoid hyperplasia- is characterized by reac known to cause bone marrow aplasia include, but are not tive changes within the T-cell regions of the lymph node that limited to, chloramphenicol, gold Salts, mephenytoin, peni encroach on, and at times appear to efface, the germinal cillamine, phenylbutaZone, and trimethadione. In general follicles. In these regions, the T-cells undergo progressive these drugs are not first line therapy due to the rare occur transformation to immunoblasts. These reactions are rence of marrow aplasia. Specific forms of aplasia include: encountered particularly in response to drug-induced immu 0772 Granulocytopenia-is defined as a loss of poly noreactivity. Drugs known to cause lymphoid hyperplasia morphonuclear neutrophils to a count lower than 500. are phenytoin, and mephenytoin. Granulocytopenia primarily predisposes the patient to bac 0778 Hemolytic anemia- is characterized by the prema terial and fungal infections. Drugs known to cause granu ture destruction of red cells, accumulation of hemoglobin locytopenia include, but are not limited to, captopril, cepha metabolic by-products, and a marked increase in erythro losporins, choral hydrate, chlorpropamide, penicillins, poresis within the bone marrow. Drugs know to cause phenothiazines, phenylbutaZone, phenytoin, procainamide, hemolytic anemia include, but are not excluded to, methyl propranolol, and tolbutamide. dopa, penicillin, Sulfonamides, and Vitamin E deficiency. 0773 Aplastic anemia-is a disorder involving an inabil 0779 Thrombocytopenia is characterized by a marked ity of the hematologic cells to regenerate and thus there is a reduction in the number of circulating platelets to a level US 2001/0034023 A1 Oct. 25, 2001 below 100,000/mm. Drug-induced thrombocytopenia may 0785. By identifying Subsets of patients, based upon result from decreased production of platelets or decreased genotype, that experience blood dyscrasias in response to platelet Survival or both. Drugs known to cause thrombocy the administration of a drug, agent or candidate therapeutic topenia include, but are not excluded to, ethanol, acetomi intervention, optimal Selection may reduce level and extent nophen, acetazolamide, acetylsalicyclic acid, 5-aminoSali of the hemostatic damage. Appropriate genotyping and cylic acid, carbamazepine, chlorpheniramine, cimetadine, correlation to dosing regimen, or Selection of optimal digitoxin, diltiazem, ethychlorynol, gold Salts, heparin, therapy would be beneficial to the patient, caregivers, medi hydantoins, isoniazid, levodopa, meprobamate, methyldopa, cal perSonnel, and the patient's loved ones. penicillamine, phenylbutaZone, procainamide, quinidine, quinine, ranitidine, Rauwolfa alkaloids, rifampin, Sulfona 0786. As an example of identification of the primary gene mides, Sulfonylureas, cytotoxic drugs, and thiazide diuretics. and relevant polymorphic variance that directly affects effi cacy, Safety, or both one could Select an gene pathway as 0780 II. Impact of Stratification Based Upon Genotype described in the Detailed Description, and determine the in Drug Development for Drugs, Compounds, or Candidate effect of genetic polymorphism and therapy efficacy, Safety, Therapeutic Interventions that may Induce Blood Dyscrasias or both within that given pathway. For example, referring to 0781 Clozapine induced agranulocytosis is associated Table 2, genes involved in drug transport, phase I and phase with differing HLA types and HSP70 variants in patients for II metabolism, protection from reactive intermediate dam whom responded to clozapine therapy but developed age, and immune responsiveness the optimization of therapy agranulocytosis. This is Suggestive that a gene within the of by an agent known to have a blood dyscrasia Side effect MHC region is associated with the manifestation of agranu by determining whether the patient has a predisposing locytosis in response to clozapine therapy. In a recent Study, genotype in which the Selected agents are more effective and two ethnic groups were analyzed for genetic markers for the or are more Safe. In considering an optimization protocol, agranulocytosis. Tumor necrosis factor microSatellites d3 one could potentially predetermine the genotypic profile of and b4 were found in higher frequencies in patients that these genes involved in the manifestation of the adverse experience clozapine-induced agranulocytosis. These data effect, or those genes preeminently responsible for drug are Suggestive that there is an involvement of tumor necrosis response. By embarking on the previously described gene factor constellation polymorphism and clozapine-induced pathway approach, it is technical feasibility to determine the agranulocytosis. relevant genes within Such a targeted drug development program. 0782. There is evidence to suggest that there are safety response differences to drug therapy in reference to devel 0787. Identification of pathophysiologic relevant vari opment of blood dyscrasias which may be attributable to ance or variances and potential therapies affecting those genotypic differences between individuals. There is pro allelic genotypes or haplotypes may speed drug develop Vided in this invention examples of gene pathways that are ment for therapeutic alternatives. There is a need for thera implicated in the disease process or its therapy and those that pies that are targeted to a disease and Symptom management potentially cause this variability. The Detailed Description with limited or no undesirable side effects. Identification of above demonstrates how identification of a candidate gene a specific variance or variances within genes involved in the or genes and gene pathways, Stratification, clinical trial pathophysiologic manifestation of blood dyscrasisas and design, and implementation of genotyping for appropriate Specific genetic polymorphisms of these critical genes may medical management of a given disease can be used to assist the development of novel agents and the identification identify the genetic cause of variations in clinical response of those patients that may best benefit from therapy of these to therapy, new diagnostic tests, new therapeutic approaches candidate therapeutic alternatives. for treating this disorder, and new pharmaceutical products 0788. By identifying allelic variances or haplotypes in or formulations for therapy. Gene pathways including, but genes that indirectly affects efficacy, Safety of any class of not limited to, those that are outlined in the gene pathway drugs that has an effect on the prevention, progression, or Table 1, and pathway matrix Table 2 and discussed below Symptoms of blood dyscrasias, one could target specific are candidates for the genetic analysis and product devel Secondary drug or agent therapeutic actions that affect the opment using the methods described above. overall therapeutic action of hemoprotective agents. 0783 Advantages of Inclusion of Pharmacogenetic 0789 Pharmacogenomics studies for these drugs, or Stratification in Clinical Development of Agents that May other agent, compound, drug, or candidate therapeutic inter Cause Blood Dyscrasias vention, could be performed by identifying genes that are 0784 The advantages of a clinical research and drug involved in the function of a drug including, but not limited development program that includes the use of polymorphic to absorption, distribution, metabolism, or elimination, the genotyping for the Stratification of patients for the appro interaction of the drug with its target as well as potential priate Selection of candidate therapeutic intervention alternative targets, the response of the cell to the binding of includes 1) identification of patients that may respond earlier a drug to a target, the metabolism (including Synthesis, and show signs and Symptoms of blood dyscrasias, 2) biodistribution or elimination) of natural compounds which identification of the primary gene and relevant polymorphic may alter the activity of the drug by complementary, com variance that directly affects manifestation of a blood dis petitive or allosteric mechanisms that potentiate or limit the order, 3) identification of pathophysiologic relevant variance effect of the drug, and genes involved in the etiology of the or variances and potential therapies affecting those allelic disease that alter its response to a particular class of thera genotypes or haplotypes, and 4) identification of allelic peutic agents. It will be recognized to those skilled in the art variances or haplotypes in genes that indirectly affects that this broadly includes proteins involved in pharmacoki efficacy, Safety or both. netics as well as genes involved in pharmacodynamics. This US 2001/0034023 A1 Oct. 25, 2001 76 also includes genes that encode proteins homologous to the 0796 Toxic epidermal necrolysis and erythroderma and proteins believed to carry out the above functions are also exfoliative dermititis Worth evaluation as they may carry out Similar functions. Together the foregoing proteins constitute the candidate 0797 Cutaneous erythroderma, edema, scaling, and fis genes for affecting response of a patient to the therapeutic Suring may occur in response to certain drugs. Drugs asso ciated with these types of cutaneous reactions include, but intervention. Using the methods described above, variances are limited to, allopurinol, amikacin, captopril, carbam in these genes can be identified, and research and clinical aZepine, chloral hydrate, chlorambucil, chloroquine, chlor Studies can be performed to establish an association between promazine, cycloSporine, diltiazem, ethambutol, ethylene a drug response or toxicity and Specific variances. diamine, glutethimide, gold Salts, griseofulvin, hydantoins, hydroxychloroquine, minoxidil, nifedipine, nonsteroid anti EXAMPLE 3 inflammatory agents, penicillin, phenobarbital, rifampin, Spironolactone, Sulfonamides, trimethadione, trimethoprim, Drug-Induced Toxicity: Cutaneous Toxicity tocainamide, tocainide, Vancomycin, Verpamil. 0790 Drug-induced cutaneous toxicity includes, but is 0798 Erythema mutliforme- is characterized by a not excluded to, eczematous: photodermititis (phototoxic hypersensitivity reaction in blood vessels of the dermis. The and photoallergic), exfoliative dermititis; maculopapular hyperSensitivity is the result of immune complexes formed eruption; papuloSquamous reactions: psoriaform, lichus pla by Small molecules interacting with proteinaceous compo nus, or pityriasis rosea-like; Vesiculobullous reactions, toxic nents of the blood vessels. In cases whereby the mucosal epidermal necrolysis, pustular-acneiform reactions, urticaria membranes of the mouth and eye are involved, is referred to and erythemas: urticaria, erythema multiforme; nodular as Stevens-Johnson Syndrome. Typically the cutaneous lesions: erythema nodosum, Vasiculitis reaction; telangiec lesions, blisters and painful erosions occur in the mout and tatic and LE reactions, pigmentary reaction; other cutaneous eye. reactions: fixed drug reactions, alopecia, hypertrichosis, macules, papules, angioedema, morbilliform-maculopapular 0799 Drugs associated with erythema mulitforme rash, toxic epidermal necrolysis, erythema multiforme, include, but are not limited to, allopurinol, acetominophen, erythema nodosum, contact dermititis, Vesicles, petechiae, amikacin, barbiturates, carbamazepine, chloroquine, chlor exfoliative dermititis, fixed drug eruptions, and Severe skin poramide, clindamycin, ethambutol, ethoSuximide, gold rash (Stevens-Johnson Syndrome). Salts, glucocorticoids, hydantoins, hydralazine, hydrox yurea, mechlorethamine, meclofenamate, penicillins, phe 0791 Drugs known to be associated with cutaneous nothiazides, phenophthalein, phenylbutaZone, rifampin, toxicities include, but are not eXclusive of, antineoplastic Streptomycin, Sulfonamides, Sulfonylureas, Sulindac, Vac agents, Sulfonamides, hydantoins and others listed for each type of toxicity. CCS. 0800 Fixed drug eruptions 0792 Uticaria and angioedema-is defined as the tran Sient appearance of elevated, erythematous pruitic wheals 0801 Drug associated with fixed drug eruptions include, (hives) or Serpiginous exanthem. The appearance of uticaria but are not excluded to, acetominophen, 5-aminoSalicyclic is perceived as ongoing immediate hyperSensitivity reaction. acid, aspirin, barbiturates, benzodiazepines, barbiturates, Angioedema is defined as uticaria, but involving deeper chloroquine, dapsone, dimethylhydrinate, gold Salts, dermal and Subdermal Sites. Uticaria and angioedema appear hydralazine, hyoscine, ibuprofen, iodides, meprobamate, to result from dilation of local postcapillary Venules. methanamine, metronidazole, penicillins, phenobarbital, Degranulation of cutaneous mast cells may be involved. phenolphthalein, phenothiazides, phenylbutaZone, procarba Zine, pseudoephedrine, quinine, Saccharin, Streptomycin, 0793 Drugs associated with uticaria and angioedema Sulfonamides, and tetracyclines. include, but are not excluded to, antimicrobials include, but not exclusive of, 5-aminoSalicylic acid, aminoglycosides, 0802 Erythema nodosum is an innflammatory reaction cephalosporins, ethambutol, isoniazid, metronidazole, in Subcutaneous fat which represents a hyperSentivity reac miconazole, nalidixic acid, penicillins, quinine, rifampin, tion to a number of antigenic Stimuli. Multiple red, painful Spectinomycin, Sulfonamides, and other drugs: asparagi nodules do not ulcerate but involute and leave a yellow nase, aspirin and other non-Steroidal antiinflammatory agen purple bruises. Small molecules intreracting with proteina ets, calcitonin, chloral hydrate, chlorambucil, cimetidine, ceous components forma asensitizing antigen. cyclophosphamide, daunorubicin, ergotamine, ethchlor Vynol, doxorubicin, ethoSuximide, ethylenediamine, gluco 0803 Drugs associated with producing erythema nodo corticoids, melphalan, penicillamine, phenothiazines, dum include, but are not excluded to, bromides, oral con procainamide, procarbazine, quinidine, tartazine, thiazide traceptives, penicillins, and Sulfonamides. diuretics, thiotepa. 0804 Contact dermititis-is characterized by eruptions 0794. Morbilliform-maculopapular rash-are rashes that on histological analysis to epidermal intercellular edema result in eruptions or are morbilliform in nature. (Spongiosis). Contact dermititis can be caused by allergic or irritant mechanisms. Allergic contact dermititis is a delayed 0795 Drugs associated with rashes include, but are not hyperSensitivity reaction that can occur in response to a limited to, 5-aminosalicyclic acid, cephalosporins, erythro variety of Small molecules that when bound to proteinaceous mycin, gentamicin, penicillins, Streptomycin, Sulfonamides, components of the skin form a Sensitizing antigen. The allopurinol, barbiturates, captopril, coumarin, gold Salts, antigen is processed by Langerhans cells in the epidermis, hydantoins, thiazide diuretics. presenting the antigen to the circulating T lymphocytes. US 2001/0034023 A1 Oct. 25, 2001 77

Irritant dermititis is produced by Substances that irritate or 0811. By identifying subsets of patients, based upon have a direct toxic effect on the skin. genotype, that experience cutaneous reactions in response to the administration of a drug, agent or candidate therapeutic 0805 Drugs associated with contact dermititis side intervention, optimal Selection may reduce level and extent effects include, but are not limited to, ambroXol, amikacin, of the skin damage. Appropriate genotyping and correlation antihistamines, bacitracin, benzalkonium chloride, ben to dosing regimen, or Selection of optimal therapy would be Zocaine, benzyl chloride, cetl alcohol, chloramphenicol, chlorpormazine, clioquinol, colophony, ethylenediamine, beneficial to the patient, caregivers, medical perSonnel, and fluorouracil, formaldehyde, gentamycin, glucocorticoids, the patient's loved ones. glutaraldehyde, heparin, hexachlorophene, iodochlorhy 0812. As an example of identification of the primary gene droxyquin, lanolin, local anesthestics, minoxidil, naftin, and relevant polymorphic variance that directly affects effi neimycin, nitrofuraZone, opiates, para-aminobenzoic acid, cacy, Safety, or both one could Select an gene pathway as parabens, penicillins, phenothiazines, proliflavine, propylene described in the Detailed Description, and determine the glycol, Streptomycin, Sulfonamides, thimerosal, timolol. effect of genetic polymorphism and therapy efficacy, Safety, or both within that given pathway. For example, referring to 0806 Impact of Stratification Based Upon Genotype in Table 2, genes involved in drug transport, phase I and phase Drug Development for Drugs, Compounds, or Candidate II metabolism, protection from reactive intermediate dam Therapeutic Interventions that May Induce Cutaneous Reac age, and immune responsiveness, the optimization of tions therapy of by an agent known to have a cutaneous Side effect 0807 Recently, it has been described that there is a by determining whether the patient has a predisposing deletion polymorphism in the B2 bradykinin receptor gene genotype in which the Selected agents are more effective and (B2BKR). It was revealed that there is a 9 base pair deletion or are more Safe. In considering an optimization protocol, in exon 1 of the B2BKR gene and upon inspection of one could potentially predetermine the genotypic profile of patients experienceing angioedema, patients with immu these genes involved in the manifestation of the adverse nochemical evidence of angioedema were homozygous for effect, or those genes preeminently responsible for drug no deletion at that Site. These results were Suggestive of response. By embarking on the previously described gene B2BKR genotype influence on the clinical Status and mani pathway approach, it is technical feasibility to determine the festation angioedema. relevant genes within Such a targeted drug development program. 0808 There is evidence to suggest that there are safety response differences to drug therapy in reference to devel 0813 Identification of pathophysiologic relevant vari opment of cutaneous reactions which may be attributable to ance or variances and potential therapies affecting those genotypic differences between individuals. There is pro allelic genotypes or haplotypes may speed drug develop Vided in this invention examples of gene pathways that are ment for therapeutic alternatives. There is a need for thera implicated in the disease process or its therapy and those that pies that are targeted to a disease and Symptom management potentially cause this variability. The Detailed Description with limited or no undesirable side effects. Identification of above demonstrates how identification of a candidate gene a specific variance or variances within genes involved in the or genes and gene pathways, Stratification, clinical trial pathophysiologic manifestation of cutaneous reactions and design, and implementation of genotyping for appropriate Specific genetic polymorphisms of these critical genes may medical management of a given disease can be used to assist the development of novel agents and the identification identify the genetic cause of variations in clinical response of those patients that may best benefit from therapy of these to therapy, new diagnostic tests, new therapeutic approaches candidate therapeutic alternatives. for treating this disorder, and new pharmacuetical products 0814. By identifying allelic variances or haplotypes in or formulations for therapy. Gene pathways including, but genes that indirectly affects efficacy, Safety of any class of not limited to, those that are outlined in the gene pathway drugs that has an effect on the prevention, progression, or Table 1, and pathway matrix Table 2 and discussed below Symptoms of cutaneous reactions, one could target specific are candidates for the genetic analysis and product devel Secondary drug or agent therapeutic actions that affect the opment using the methods described above. overall therapeutic action. 0809 Advantages of Inclusion of Pharmacogenetic 0815 Pharmacogenomics studies for these drugs, or Stratification in Clinical Development of Agents that May other agents, compounds, or candidate therapeutic interven Cause Cutaneous Reactions tions, could be performed by identifying genes that are 0810) The advantages of a clinical research and drug involved in the the function of a drug including, but not development program that includes the use of polymorphic limited to absorption, distribution, metabolism, or elimina genotyping for the Stratification of patients for the appro tion, the interaction of the drug with its target as well as priate Selection of candidate therapeutic intervention potential alternative targets, the response of the cell to the includes 1) identification of patients that may respond earlier binding of a drug to a target, the metabolism (including and show signs and Symptoms of cutaneous reactions, 2) Synthesis, biodistribution or elimination) of natural com identification of the primary gene and relevant polymorphic pounds which may alter the activity of the drug by comple variance that directly affects manifestation of a cutaneous mentary, competitive or allosteric mechanisms that potenti disorder, 3) identification of pathophysiologic relevant vari ate or limit the effect of the drug, and genes involved in the ance or variances and potential therapies affecting those etiology of the disease that alter its response to a particular allelic genotypes or haplotypes, and 4) identification of class of therapeutic agents. It will be recognized to those allelic variances or haplotypes in genes that indirectly affects skilled in the art that this broadly includes proteins involved efficacy, Safety or both. in pharmacokinetics as well as genes involved in pharma US 2001/0034023 A1 Oct. 25, 2001 78 codynamics. This also includes genes that encode proteins 0821. By identifying Subsets of patients, based upon homologous to the proteins believed to carry out the above genotype, that experience CNS toxicity in response to the functions are also worth evaluation as they may carry out administration of a drug, agent or candidate therapeutic Similar functions. Together, the foregoing proteins constitute intervention, optimal Selection may reduce level and extent the candidate genes for affecting response of a patient to the of the neurologic damage. Appropriate genotyping and therapeutic intervention. Using the methods described correlation to dosing regimen, or Selection of optimal above, variances in these genes can be identified, and therapy would be beneficial to the patient, caregivers, medi research and clinical Studies can be performed to establish cal perSonnel, and the patient's loved ones. an association between a drug response or toxicity and Specific variances. 0822. As an example of identification of the primary gene and relevant polymorphic variance that directly affects effi EXAMPLE 5 cacy, Safety, or both one could Select an gene pathway as described in the Detailed Description, and determine the Drug-Induced CNS Toxicity effect of genetic polymorphism and therapy efficacy, Safety, 0816 Drug-induced central nervous system toxicity or both within that given pathway. For example, referring to includes CNS stimulation or CNS depression. Characteris Table 2, genes involved in drug transport, phase I and phase tics of CNS toxicity include, but are not limited to, tinnitus II metabolism, protection from reactive intermediate dam and dizziness, acute dystonic reactions, parkinsonian Syn age, the optimization of therapy of by an agent known to drome, coma, convulsions, depression and psychosis, Sweat inpart CNS toxic or undesirable side effect or effects by ing, mydriasis, hyperpyrexia, centrally mediated cardiovas determining whether the patient has a predisposing genotype cular involvement (hypertension, tachycardia, extrasystoles, in which the Selected agents are more effective and or are arrythmias, circulatory collapse) and respiratory depression more Safe. In considering an optimization protocol, one or tachypnea. Drugs known to be associated with CNS could potentially predetermine the genotypic profile of these toxicity include, but are not exclusive of, Salicylates, antip genes involved in the manifestation of the adverse effect, or Sychotics, Sedatives, cholinergics, those genes preeminently responsible for drug response. By 0817) Impact of Stratification Based Upon Genotype in embarking on the previously described gene pathway Drug Development for Drugs, Compounds, or Candidate approach, it is technical feasibility to determine the relevant Therapeutic Interventions that May Induce CNS Toxicity genes within Such a targeted drug development program. 0818. There is evidence to suggest that there are safety 0823. Identification of pathophysiologic relevant vari response differences to drug therapy in reference to devel ance or variances and potential therapies affecting those opment of CNS toxicities which may be attributable to allelic genotypes or haplotypes may speed drug develop genotypic differences between individuals. There is pro ment for therapeutic alternatives. There is a need for thera Vided in this invention examples of gene pathways that are pies that are targeted to a disease and Symptom management implicated in the disease process or its therapy and those that with limited or no undesirable side effects. Identification of potentially cause this variability. The Detailed Description a specific variance or variances within genes involved in the above demonstrates how identification of a candidate gene pathophysiologic manifestation of CNS toxicities and Spe or genes and gene pathways, Stratification, clinical trial cific genetic polymorphisms of these critical genes may design, and implementation of genotyping for appropriate assist the development of novel agents and the identification medical management of a given disease can be used to of those patients that may best benefit from therapy of these identify the genetic cause of variations in clinical response candidate therapeutic alternatives. to therapy, new diagnostic tests, new therapeutic approaches 0824. By identifying allelic variances or haplotypes in for treating this undesirable adverse effect, and new phar genes that indirectly affects efficacy, Safety of any class of macuetical products or formulations for therapy. Gene path drugs that has an effect on the prevention, progression, or ways including, but not limited to, those that are outlined in Symptoms of CNS toxicities, one could target Specific Sec the gene pathway Table 1, and pathway matrix Table 2 and ondary drug or agent therapeutic actions that affect the discussed below are candidates for the genetic analysis and overall therapeutic action of neuroprotective agents. product development using the methods described above. 0825 Pharmacogenomics studies for these drugs, or 0819 Advantages of Inclusion of Pharmacogenetic other agent, compound, drug, or candidate therapeutic inter Stratification in Clinical Development of Agents that May vention, could be performed by identifying genes that are Cause CNS Toxicities involved in the the function of a drug including, but not 0820) The advantages of a clinical research and drug limited to absorption, distribution, metabolism, or elimina development program that includes the use of polymorphic tion, the interaction of the drug with its target as well as genotyping for the Stratification of patients for the appro potential alternative targets, the response of the cell to the priate Selection of candidate therapeutic intervention binding of a drug to a target, the metabolism (including includes 1) identification of patients that may respond earlier Synthesis, biodistribution or elimination) of natural com and show signs and Symptoms of CNS toxicities, 2) iden pounds which may alter the activity of the drug by comple tification of the primary gene and relevant polymorphic mentary, competitive or allosteric mechanisms that potenti variance that directly affects manifestation of a CNS toxic ate or limit the effect of the drug, and genes involved in the ity, 3) identification of pathophysiologic relevant variance or etiology of the disease that alter its response to a particular variances and potential therapies affecting those allelic class of therapeutic agents. It will be recognized to those genotypes or haplotypes, and 4) identification of allelic skilled in the art that this broadly includes proteins involved variances or haplotypes in genes that indirectly affects in pharmacokinetics as well as genes involved in pharma efficacy, Safety or both. codynamics. This also includes genes that encode proteins US 2001/0034023 A1 Oct. 25, 2001 79 homologous to the proteins believed to carry out the above 0833 Hepatocellular damage may be the result of direct functions are also worth evaluation as they may carry out chemical mediated effects, may be severe, and usually is Similar functions. Together the foregoing proteins constitute asSociated with damage within organelles, DNA and mem the candidate genes for affecting response of a patient to the branes. Clinically there is a marked elevation of SGOT and therapeutic intervention. Using the methods described SGPT as well as other enzymes. In cases of cholestasis there above, variances in these genes can be identified, and is jaundice, pruritis, a marked elevation of bile Salts and research and clinical Studies can be performed to establish alkaline phosphatase activity, but not an elevation of SGOT an association between a drug response or toxicity and or SGPT. In cases of toxic liver disease there is difficulty, at Specific variances. least initially to determine the underlying etiology. Clini cally, Symptoms may not appear as clear as described above. EXAMPLE 6 Further, depending on the rate and extent of the damage, hepatocellular damage may be masked or asymptomatic Drug-Induced Liver Toxicity until liver impairment has induced cholestasis. 0826 Drug-induced liver disease or drug-induced liver 0834 Potentially hepatotoxic agents can be divided toxicity can manifest as Zonal necrosis, nonspecific focal broadly into two groups: intrinsic hepatotoxins and idiosyn hepatitis, Viral hepatitis-like reactions, inflammatory or non cratic hepatotoxins. Intrinsic hepatotoxins produce acute inflammatory cholestasis, Small or large droplet fatty liver, liver damage in a predictable, dose-dependent fashion granulomas, chronic hepatitis, fibrosis, tumors, or vascular Shortly after ingestion or exposure. Generally, all Subjects lesions. exposed will uniformly exhibit signs and Symptoms. In this category, the effects Seen in humans can be mimicked in 0827. In the majority of the cases of known drug-induced animal models. Examples of intrinsic hepatotoxins are car liver toxicity, the drug is metabolized to a form that is bon tetrachloride, 2-nitropropane, trichloroethane, the deleterious to hepatic, or extrahepatic function. There are octapeptide toxins of the Amanita mushroom species, and many endogenous or exogenous compounds that may be the antipyretic, acetominophen. In Some of these cases, toxic considered to attenuate or ablate toxic hepatocyte-produced metabolites result in covalent modification of hepatocyte metabolite mechanisms or effects of hepatic or extrahepatic macromolecules or reactive oxygen intermediates leads to damage. peroxidation of cell membrane lipids or other intracellular 0828. In hepatocellular damage, free oxygen radicals molecules. may be generated in the hepatic metabolic processes that are deleterious to intracellular organelles, DNA, or metabolic 0835. In contrast, idiosyncratic hepatotoxins produce pathways. There are endogenous cytoprotective agents that liver damage in an unpredictable, dose-independent manner may prevent free radical-mediated damage Such as retinoids, after a latent period of ingestion or exposure. Animal models flavins, reduced glutathione, Vitamin E.S-adenylylmethion or experimental data is generally incapable of predicting the ine, and the enzyme Superoxide dismutase (SOD). In animal effect in humans. Further, idiosyncratic hepatotoxins do not models in which SOD activity is diminished or absent, the uniformly affect a population; a Subset of the group exposed liver function was normal, but the sensitivity to toxin may or may not exhibit signs or Symptoms. Range of challenge was heightened. Symptoms are from mild to Severe and is thought to coincide with differences in the pathways of drug or Xenobiotic 0829. In cholestatic damage, the bile salt uptake, metabo biotransformation or immune-mediated drug Sensitivity lism, Secretion, or transport is compromised and the residual (drug allergy). In idiosyncratic drug-induced liver disease, increased bile Salt concentrations are deleterious to hepato fever, arthralgias, rash, eosinophilia, are often prominent cyte function. The increase in bile Salts is the main metabolic and indicate a hyperSensitivity reaction. disturbance that initially leads to jaundice and pruritis and can progreSS to pancreatitis, hyperbilirubinemia, biliary cir 0836 Impact of Stratification Based Upon Genotype in rhosis, and hepatic encephalopathy. Drug Development for Drugs, Compounds, or Candidate Therapeutic Interventions that may Induce Hepatotoxicity 0830. In both cases of drug-induced liver toxicity, the drug must first be absorbed and enter in the hepatic circu 0837 Genes encoding proteins with catalytic function lation. Further, clinically it is often difficult to determine that are involved in the metabolism of drugs or xenobiotics whether cholestatic damage leads to hepatocellular damage are listed in Tables 1 and 2 below. Further listed are those or whether hepatocellular damage leads to cholestatic dam proteins that are involved in the uptake, transport, or Secre age. In many cases, until the patient is Symptomatic, the tion into the bile cannicula. Below are further specific underlying damage mechanisms may be clinically over example of drug-specific effects on the liver. looked. By the time the drug-induced liver disease is Symp 0838 Acetaminophen-Induced Liver Disease tomatic, the damage, be it hepatocellular or cholestatic or both, may be irreversible. 0839 Acetominophen is a readily available, easy to administer analgesic that is an example of a intrinsic hepa 0831) Identification of Genes involved in Drug-Induced totoxin. This hepatotoxin causes Zonal necrosis and acute Liver Toxicity liver failure and is associated with renal failure. Although a 0832. Thus, in the process of identifying drug- or xeno high dose (10-15 grams) is required for significant liver biotic-induced liver toxicity, one skilled in the art would injury to occur, the onset of initial Symptoms does not occur identify key metabolic enzymes or bile cannicula transport until hours after ingestion. The progression of Symptoms processes that would be linked with either hepatocellular occurs including progressive liver failure with hepatic damage or cholestasis or combination of hepatocellular encephalopathy, prolongation of prothrombin time, damage or cholestasis. hypoglycemia, and lactic acidosis. The liver injury is caused US 2001/0034023 A1 Oct. 25, 2001 80 by a toxic metabolite of acetominophen via the P450 0849) Isoniazid (INH)-Induced Liver Disease metabolizing System. This toxic intermediate at low con 0850. Isoniazid is used as a single drug in the prophylaxis centrations is conjugated with glutathione. However, in of tuberculosis. In 10-20% of of the persons taking INH, toxic doses, the conjugating enzymes Stores are exhausted subclinical liver injury occurs. The conversion of INH to and the reactive intermediate reacts with intracellular pro acetylhydrazine is via acetylation. In slow acetylators, INH teins and results in cellular dysfunction and ultimately death. is more hepatotoxic. The conversion of INH to acetylhydra The rate of metabolism is dependent on the concentrations Zine to diacetylhydrazine is impaired. In Slow acetylators, of both P450 and glutathione. Speeding this toxic pathway the acetylhydrazine is not well metabolized and is further may include increasing the available P450 or reducing the oxidized by one of the P450 enzymes to a toxic, reactive availablility of glutathione, e.g. using known inducers of molecule that is responsible for the liver disease. Discon P450 such as ethanol and and phenobarbital; and known tinuation of the drug returns the enzymatic levels to normal inhibitors of glutathione concentrations, e.g., ethanol and fasting. Acetominophen toxicity is completely reversed if and the liver is able to restore activity. the drug is removed. Chronic ingestion may produce Sub 0851 Sodium Valproate-Induced Liver Disease clinical liver injury, centrilobular necrosis, or chronic hepa 0852. Sodium valproate is an anti-epileptic agent that is titis; however all reversible if the drug is removed. routinely prescribed for petit mal epilepsy and in Some cases produces severe hepatotoxicity. Similar to INH, sodium 0840 Amiodarone-Induced Liver Disease Valproate is accompanied by a high incidence of transient, 0841 Amiodarone is used in treatment of refractory Slight and asymptomatic increases in Serum transaminases. arrythmias. In Some patients amiodarone produces mild to Usually the increased enzyme activity appears after weeks moderate increases of Serum transaminases which are gen of treatment. In rare cases of Severe liver toxicity, the erally accompanied by engorgement of lySOSomes with nonspecific Systemic and digestive Symptoms are followed phospholipid. In a fraction of the patients, a more Severe by jaundice, evidence of liver failure, as well as encephal liver injury develops which histologically resembles alco opathy and coagulopathy. The mechanism of hepatotoxicity holic hepatitis: fat infiltration of hepatocytes, focal necrosis, is unknown, however there are theories that there is impair fibrosis, polymorphonuclear leukocyte infiltrates, and Mal ment of mitochondiral oxidation of long-chain fatty acids by lory bodies. The lesion may progreSS to micronodular cir a metabolite of the parent drug. Symptoms subside with little rhosis, with portal hypertension and liver failure. Hepatome to no residual liver dysfunction after discontinuing the drug. galy is Seen, but jaundice is rare. 0853) Oral Contraceptive Induced Liver Disease 0842) Amiodarone accumulates in lysosomes and inhibits 0854 Estrogen, progesterone, and combination oral con lySOSomal phopholipases, however the connection between traceptives can produce Several adverse effects on the hep this mechanism and alcoholic hepatitis histopathology is tobiliary System. They are 1) hepatocellular cholestasis, 2) unknown. Unfortunately, rapid discontinuation of amio liver cell neoplasias, 3) increased predisposition to choles darone increases the risk of cardiac arrythmias. terol and gall Stone formation, 4) hepatic vein thrombosis. 0843. Chlopromazine-Induced Liver Disease These cholestatic hepatotoxic effects are attributed to estro gen's direct effect on bile formation. The mechanism of 0844 Chlorpromazine is an anti-psychotic agent which, action is unknown. in a Small portion of the patient population can produce a 0855. There is evidence to suggest that there are safety cholestatic reaction. Symptoms include fever, anorexia, response differences to drug therapy in reference to devel arthalgias, pruritis, jaundice, and eosinophilia is common. opment of drug-induced liver toxicity which may be attrib This idiosyncratic type of liver toxicity Suggests a hyper utable to genotypic differences between individuals. There is Sensitivity type reaction. The Symptoms Subside over a provided in this invention examples of gene pathways that period of weeks following discontinuation, Rarely, residual are implicated in the disease proceSS or its therapy and those cholestatic disease occurs, treatment for pruritis and fat that potentially cause this variability. The Detailed Descrip Soluble Vitamin Supplementation may be required, but even tion above demonstrates how identification of a candidate tual recovery almost always occurs. gene or genes and gene pathways, Stratification, clinical trial 0845 Erythromycin-Induced Liver Disease design, and implementation of genotyping for appropriate medical management of a given disease can be used to 0846 Erythromycin, a broad spectrum antibiotic, can be identify the genetic cause of variations in clinical response accompanied by a cholestatic reaction. Inflammatory cell to therapy, new diagnostic tests, new therapeutic approaches infiltration and liver cell necrosis may occur. The hepatop for treating this disorder, and new pharmacuetical products toxicity presents as right upper quadrant pain, fever, and or formulations for therapy. Gene pathways including, but variable cholestatic Symptoms. The prognosis is uniform not limited to, those that are outlined in the gene pathway and will occur after readminstration of the drug, The mecha Table 1, and pathway matrix Table 2 and discussed below nism of action is unknown. are candidates for the genetic analysis and product devel 0847 Halothane-Induced Liver Disease opment using the methods described above. 0848 Halothane is a gaseous anthesthetic and can, in rare 0856 Advantages of Inclusion of Pharmacogenetic instances, cause a viral-like hepatitis Syndrome. In Severe Stratification in Clinical Development of Agents that May cases, this hepatotoxicity, may cause fatal massive heaptic Cause Liver Toxicity necrosis. Severe reactions seem to appear after previous or 0857. The advantages of a clinical research and drug multiple exposure to halothane. It is known that the P450 development program that includes the use of polymorphic metabolites of this xenobiotic are responsible for the mecha genotyping for the Stratification of patients for the appro nism of hepatic injury. priate Selection of candidate therapeutic intervention US 2001/0034023 A1 Oct. 25, 2001 includes 1) identification of patients that may respond earlier potential alternative targets, the response of the cell to the and show signs and Symptoms of liver toxicity, 2) identifi binding of a drug to a target, the metabolism (including cation of the primary gene and relevant polymorphic Vari Synthesis, biodistribution or elimination) of natural com ance that directly affects manifestation of a liver disorder, 3) pounds which may alter the activity of the drug by comple identification of pathophysiologic relevant variance or vari mentary, competitive or allosteric mechanisms that potenti ances and potential therapies affecting those allelic geno ate or limit the effect of the drug, and genes involved in the types or haplotypes, and 4) identification of allelic variances etiology of the disease that alter its response to a particular or haplotypes in genes that indirectly affects efficacy, Safety class of therapeutic agents. It will be recognized to those or both. skilled in the art that this broadly includes proteins involved in pharmacokinetics as well as genes involved in pharma 0858. By identifying Subsets of patients, based upon codynamics. This also includes genes that encode proteins genotype, that experience drug-induced liver toxicity in homologous to the proteins believed to carry out the above response to the administration of a drug, agent or candidate functions are also worth evaluation as they may carry out therapeutic intervention, optimal Selection may reduce level Similar functions. Together the foregoing proteins constitute and extent of the hepatic damage. Appropriate genotyping the candidate genes for affecting response of a patient to the and correlation to dosing regimen, or Selection of optimal therapeutic intervention. Using the methods described therapy would be beneficial to the patient, caregivers, medi above, variances in these genes can be identified, and cal perSonnel, and the patient's loved ones. research and clinical Studies can be performed to establish 0859. As an example of identification of the primary gene an association between a drug response or toxicity and and relevant polymorphic variance that directly affects effi Specific variances. cacy, Safety, or both one could Select an gene pathway as described in the Detailed Description, and determine the EXAMPLE 7 effect of genetic polymorphism and therapy efficacy, Safety, or both within that given pathway. For example, referring to Drug-Induced Cardiovascular Toxicity Table 2, genes involved in drug transport, phase I and phase 0863 Drug induced cardiovascular toxicities include but II metabolism, excretion, hepatic cannicular uptake and are not excluded to arrythmias, tachycardia, extrasyStoles, concentration, and protection from reactive intermediate circulatory collapse, QT prolongation, cardiomyopathy, damage the optimization of therapy by an agent known to hypotension, or hypertension. Drugs known to elicit these have a hepatic Side effect by determining whether the patient type of responses include but are not excluded to theophyl has a predisposing genotype in which the Selected agents are line, hydantoins, doxorubicin, daunorubicin. more effective and or are more Safe. In considering an optimization protocol, one could potentially predetermine 0864 Arrythmias-If the normal sequence of electrical the genotypic profile of these genes involved in the mani impulse and propagation through myocardial tissue is per festation of the adverse effect, or those genes preeminently turbed, an arrythmia occurs. Broadly, arrythmias fall into responsible for drug response. By embarking on the previ one of three categories: bradyarrythmias (slowing or failure ously described gene pathway approach, it is technical of the initiating impulse), heart block (an impaired propa feasibility to determine the relevant genes within Such a gation through node tissue or atrial or ventricular muscle), targeted drug development program. and tachyarrythmias (abnormal rapid heart rhythms). Sub categories include: Sinus bradycardia, atrioventricular block 0860) Identification of pathophysiologic relevant vari (AV block), sinus tachycardia, Ventricular tachycardia, atrial ance or variances and potential therapies affecting those flutter, multifocal atrial tachycardia, polymorphic ventricu allelic genotypes or haplotypes may speed drug develop lar tachycardia with or without QT prolongation, frequent or ment for therapeutic alternatives. There is a need for thera difficult to terminate Ventricular tachycardia, atrial tachycar pies that are targeted to a disease and Symptom management dia with or without AV block, ventricular bigeminy, and with limited or no undesirable side effects. Identification of Ventricular fibrillation. Drugs known to induce these types a specific variance or variances within genes involved in the of arrythmias include, but are not excluded to, digitalis, pathophysiologic manifestation of drug-induced liver toxic Verapamil, diltiazem, b-adrenergic blockers, clonidine, ity and Specific genetic polymorphisms of these critical methyldopa, quinidine, flecainide, propafenone, theophyl genes may assist the development of novel agents and the line, Sotalol, procainamide, disopyramide, certain non-car identification of those patients that may best benefit from dioactive drugs (), and amiodarone. therapy of these candidate therapeutic alternatives. 0865 Heart Rate, Tachycardia-Heart rate is under both 0861. By identifying allelic variances or haplotypes in Sympathetic and parasympathic control. The influence of genes that indirectly affects efficacy, Safety of any class of heart rate on cardiac output is paramount. Drugs affecting drugs that has an effect on the prevention, progression, or heart rate include, but are not limited to, Sympathomimetics, Symptoms of drug induced liver toxicity, one could target parasympathomimetics, and agents or compounds affecting Specific Secondary drug or agent therapeutic actions that these two central inputs. affect the overall therapeutic action of hepatoprotective agents. 0866 Extasystoles is defined as premature myocardial excitation. Extrasystoles can include atrial, nodal, or ven 0862 Pharmacogenomics studies for these drugs, or tricular. Other asynchronous pathologies may result from other agent, compound, drug, or candidate therapeutic inter these Systoles. Drugs known to be associated with extra vention, could be performed by identifying genes that are Systoles include, but are not excluded to, agents that prolong involved in the the function of a drug including, but not the depolarization time, agents that leave a residual available limited to absorption, distribution, metabolism, or elimina intracellular calcium, or agents that alter the function of the tion, the interaction of the drug with its target as well as K+ or Na+ channel activity. US 2001/0034023 A1 Oct. 25, 2001 82

0867 QT Prolongation is the interval on an electrocar 0875 Impact of Stratification Based Upon Genotype in diogram that indicates Ventricular action potential duration. Drug Development for Drugs, Compounds, or Candidate QT prolongation can lead to uncoordinated atrial and ven Therapeutic Interventions that may Induce Cardiovascular tricular action potentials. In these circumstances of delayed Toxicity or prolonged polymorphic ventricular after depolarizations, 0876 There is evidence to suggest that there are safety resultant abnormal triggering of Secondary, uncoordinated response differences to drug therapy in reference to devel depolarizations can occur. Two of these conditions are opment of cardiovascular toxicity which may be attributable explained as follows and may be associated with underlying to genotypic differences between individuals. There is pro rapid or slow heart rate: 1) under conditions of residual Vided in this invention examples of gene pathways that are excess intracellular calcium (myocardial ischemia, adrener implicated in the disease process or its therapy and those that gic stress, digitalis intoxication), and 2) under conditions of potentially cause this variability. The Detailed Description marked prolongation of cardiac action potential (agents above demonstrates how identification of a candidate gene (antiarrythmics or others) that prolong action potential dura or genes and gene pathways, Stratification, clinical trial tion). design, and implementation of genotyping for appropriate 0868 Cardiomyopathy- There are broadly three catego medical management of a given disease can be used to ries of cardiomyopathies: dilated, hypertrophic, and restric identify the genetic cause of variations in clinical response tive. These cardiac muscular diseases can be of mechanical to therapy, new diagnostic tests, new therapeutic approaches or acquired origin. for treating this disorder, and new pharmacuetical products 0869 Dilated cardiomyopathies are generally caused by or formulations for therapy. Gene pathways including, but myocardial injury that results in depressed Systolic function not limited to, those that are outlined in the gene pathway and progressive ventricular dilatation. Drug induced dilated Table 1, and pathway matrix Table 2 and discussed below cardiomyopathy can occur in the presence of, but are not are candidates for the genetic analysis and product devel excluded to, ethanol, chenotherapeutic agents, elemental opment using the methods described above. compounds, and catecholamimetics. 0877 Advantages of Inclusion of Pharmacogenetic 0870 Hypertrophic cardiomyopathy is the presentation Stratification in Clinical Development of Agents that May of grossly assymetric (eccentric) or symmetric (concentric) Cause Cardiovascular Toxicity hyoertrophy of the left ventricle in the absence of another 0878 The advantages of a clinical research and drug cardiac or Systemic disease capable of producing the dis development program that includes the use of polymorphic proportionate increase in Ventricle mass. In drug induced genotyping for the Stratification of patients for the appro hypertrophic cardiomyopathy, there may be compensatory priate Selection of candidate therapeutic intervention hypertrophy of the left ventricle in response to inordinate includes 1) identification of patients that may respond earlier and or Sustained hypertension or prolonged reduced or and show signs and Symptoms of cardiovascular toxicity, 2) insufficient cardiac output as a result of myocardial injury or identification of the primary gene and relevant polymorphic noncardiac mediated physiological events. variance that directly affects manifestation of a cardiovas 0871 Restrictive cardiomyopathies are the result of a cular disorder, 3) identification of pathophysiologic relevant primary abnormality of diastolic function (impaired filling). variance or variances and potential therapies affecting those Impaired diastolic function can occur as a result of morpho allelic genotypes or haplotypes, and 4) identification of logically detectable myocardial or endomyocardial disease, allelic variances or haplotypes in genes that indirectly affects interstitial deposition of deposition of abnormal Substances efficacy, Safety or both. (infiltrative), intracellular accumulation of abnormal Sub 0879. By identifying Subsets of patients, based upon stances (strage diseases), or as a result of endomyocardial genotype, that experience cardiovascular toxicities in disease. In the last category, anthracyclines have been asso response to the administration of a drug, agent or candidate ciated with both dilated and restrictive cardiomyopathies. therapeutic intervention, optimal Selection may reduce level 0872 Blood Pressure-Blood pressure is regulated in a and extent of the cardiovascular damage. Appropriate geno complex interplay of neural and endocrine mechanisms. typing and correlation to dosing regimen, or Selection of These mechanisms are aimed at the physiologic contorl of optimal therapy would be beneficial to the patient, caregiv cardiac output, delivery of blood components to the tissues, ers, medical perSonnel, and the patient's loved ones. and removal of metabolic by-products from the tissues. 0880. As an example of identification of the primary gene 0873. Hypertension is defined as the elevated arterial and relevant polymorphic variance that directly affects effi blood pressure either an increase of Systolic or diastolic cacy, Safety, or both one could Select an gene pathway as preSSure or both. Secondary hypertension can be associated described in the Detailed Description, and determine the with drugs and chemicals including, but not limited to, effect of genetic polymorphism and therapy efficacy, Safety, cyclosporine, oral contraceptives, glucocorticoids, miner or both within that given pathway. For example, referring to alocorticoids, Sympathomimetics, tyramine, and MAO Table 2, genes involved in drug transport, phase I and phase inhibitors. II metabolism, and protection from reactive intermediate 0874) Hypotension is defined as the reduction in blood damage the optimization of therapy of by an agent known to preSSure that is associated with Orthostatic hypotension, have a cardiovascular Side effect by determining whether the Syncope, head injury, hepatic failure, antidiuresis, myocar patient has a predisposing genotype in which the Selected dial infarction and cardiogenic Shock. Drug-induced agents are more effective and or are more Safe. In consid hypotension is associated drugs including, but not exclusive ering an optimization protocol, one could potentially prede of, parasympathomimetics, diuretics, and direct acting car termine the genotypic profile of these genes involved in the diac agents. manifestation of the adverse effect, or those genes preemi US 2001/0034023 A1 Oct. 25, 2001 nently responsible for drug response. By embarking on the 0886. There is evidence to suggest that there are safety previously described gene pathway approach, it is technical response differences to drug therapy in reference to devel feasibility to determine the relevant genes within Such a opment of pulmonary toxicities which may be attributable to targeted drug development program. genotypic differences between individuals. There is pro 0881 Identification of pathophysiologic relevant vari Vided in this invention examples of gene pathways that are ance or variances and potential therapies affecting those implicated in the disease process or its therapy and those that allelic genotypes or haplotypes may speed drug develop potentially cause this variability. The Detailed Description above demonstrates how identification of a candidate gene ment for therapeutic alternatives. There is a need for thera or genes and gene pathways, Stratification, clinical trial pies that are targeted to a disease and Symptom management design, and implementation of genotyping for appropriate with limited or no undesirable side effects. Identification of medical management of a given disease can be used to a specific variance or variances within genes involved in the identify the genetic cause of variations in clinical response pathophysiologic manifestation of cardiovascular toxicities to therapy, new diagnostic tests, new therapeutic approaches and Specific genetic polymorphisms of these critical genes for treating this disorder, and new pharmacuetical products may assist the development of novel agents and the identi or formulations for therapy. Gene pathways including, but fication of those patients that may best benefit from therapy not limited to, those that are outlined in the gene pathway of these candidate therapeutic alternatives. Table 1, and pathway matrix Table 2 and discussed below 0882. By identifying allelic variances or haplotypes in are candidates for the genetic analysis and product devel genes that indirectly affects efficacy, Safety of any class of opment using the methods described above. drugs that has an effect on the prevention, progression, or 0887 Advantages of Inclusion of Pharmacogenetic Symptoms of cardiovascular toxicities, one could target Stratification in Clinical Development of Agents that May Specific Secondary drug or agent therapeutic actions that Cause Pulmonary Toxicities affect the overall therapeutic action of cardiovascular pro tective agents. 0888. The advantages of a clinical research and drug development program that includes the use of polymorphic 0883 Pharmacogenomics studies for these drugs, or genotyping for the Stratification of patients for the appro other agent, compound, drug, or candidate therapeutic inter priate Selection of candidate therapeutic intervention vention, could be performed by identifying genes that are includes 1) identification of patients that may respond earlier involved in the the function of a drug including, but not and show signs and Symptoms of pulmonary toxicity, 2) limited to absorption, distribution, metabolism, or elimina identification of the primary gene and relevant polymorphic tion, the interaction of the drug with its target as well as variance that directly affects manifestation of a pulmonary potential alternative targets, the response of the cell to the disorder, 3) identification of pathophysiologic relevant vari binding of a drug to a target, the metabolism (including ance or variances and potential therapies affecting those Synthesis, biodistribution or elimination) of natural com allelic genotypes or haplotypes, and 4) identification of pounds which may alter the activity of the drug by comple allelic variances or haplotypes in genes that indirectly affects mentary, competitive or allosteric mechanisms that potenti efficacy, Safety or both. ate or limit the effect of the drug, and genes involved in the etiology of the disease that alter its response to a particular 0889. By identifying Subsets of patients, based upon class of therapeutic agents. It will be recognized to those genotype, that experience pulmonary toxicities in response skilled in the art that this broadly includes proteins involved to the administration of a drug, agent or candidate thera in pharmacokinetics as well as genes involved in pharma peutic intervention, optimal Selection may reduce level and codynamics. This also includes genes that encode proteins extent of the pulmonary damage. Appropriate genotyping homologous to the proteins believed to carry out the above and correlation to dosing regimen, or Selection of optimal functions are also worth evaluation as they may carry out therapy would be beneficial to the patient, caregivers, medi Similar functions. Together the foregoing proteins constitute cal perSonnel, and the patient's loved ones. the candidate genes for affecting response of a patient to the 0890. As an example of identification of the primary gene therapeutic intervention. Using the methods described and relevant polymorphic variance that directly affects effi above, variances in these genes can be identified, and cacy, Safety, or both one could Select an gene pathway as research and clinical Studies can be performed to establish described in the Detailed Description, and determine the an association between a drug response or toxicity and effect of genetic polymorphism and therapy efficacy, Safety, Specific variances. or both within that given pathway. For example, referring to Table 2, genes involved in drug transport, phase I and phase EXAMPLE 8 II metabolism, excretion, protection from reactive interme Drug-Induced Pulmonary Toxicity diate damage, and immune responsiveness, the optimization of therapy of by an agent known to have a pulmonary Side 0884 Drug induced pulmonary toxicity includes, but is effect by determining whether the patient has a predisposing not excluded to, asthma, acute pneumonitis, eosinophilic genotype in which the Selected agents are more effective and pneumonitis, fibrotic and pleural reactions, and interstitial or are more Safe. In considering an optimization protocol, fibrosis. Drug know to elicit pulmonary toxicity include, but one could potentially predetermine the genotypic profile of are not excluded to, Salicylates, nitrofuratoin, buSulfan, these genes involved in the manifestation of the adverse nitrofurantoin, and bleomycin. effect, or those genes preeminently responsible for drug 0885 Impact of Stratification Based Upon Genotype in response. By embarking on the previously described gene Drug Development for Drugs, Compounds, or Candidate pathway approach, it is technical feasibility to determine the Therapeutic Interventions that may Induce Pulmonary Tox relevant genes within Such a targeted drug development icities program. US 2001/0034023 A1 Oct. 25, 2001

0891) Identification of pathophysiologic relevant vari potentially cause this variability. The Detailed Description ance or variances and potential therapies affecting those above demonstrates how identification of a candidate gene allelic genotypes or haplotypes may speed drug develop or genes and gene pathways, Stratification, clinical trial ment for therapeutic alternatives. There is a need for thera design, and implementation of genotyping for appropriate pies that are targeted to a disease and Symptom management medical management of a given disease can be used to with limited or no undesirable side effects. Identification of identify the genetic cause of variations in clinical response a specific variance or variances within genes involved in the to therapy, new diagnostic tests, new therapeutic approaches pathophysiologic manifestation of pulmonary toxicity and for treating this disorder, and new pharmacuetical products Specific genetic polymorphisms of these critical genes may or formulations for therapy. Gene pathways including, but assist the development of novel agents and the identification not limited to, those that are outlined in the gene pathway of those patients that may best benefit from therapy of these Table 1, and pathway matrix Table 2 and discussed below candidate therapeutic alternatives. are candidates for the genetic analysis and product devel 0892. By identifying allelic variances or haplotypes in opment using the methods described above. genes that indirectly affects efficacy, Safety of any class of 0897 Advantages of Inclusion of Pharmacogenetic drugs that has an effect on the prevention, progression, or Stratification in Clinical Development of Agents that May Symptoms of pulmonary toxicity, one could target Specific Cause or are Associated with Renal Toxicity Secondary drug or agent therapeutic actions that affect the 0898 The advantages of a clinical research and drug overall therapeutic action of pulmonary protective agents. development program that includes the use of polymorphic 0893 Pharmacogenomics studies for these drugs, or genotyping for the Stratification of patients for the appro other agent, compound, drug, or candidate therapeutic inter priate Selection of candidate therapeutic intervention vention, could be performed by identifying genes that are includes 1) identification of patients that may respond earlier involved in the the function of a drug including, but not and show signs and Symptoms of renal toxicity, 2) identifi limited to absorption, distribution, metabolism, or elimina cation of the primary gene and relevant polymorphic vari tion, the interaction of the drug with its target as well as ance that directly affects manifestation of a renal disorder, 3) potential alternative targets, the response of the cell to the identification of pathophysiologic relevant variance or vari binding of a drug to a target, the metabolism (including ances and potential therapies affecting those allelic geno Synthesis, biodistribution or elimination) of natural com types or haplotypes, and 4) identification of allelic variances pounds which may alter the activity of the drug by comple or haplotypes in genes that indirectly affects efficacy, Safety mentary, competitive or allosteric mechanisms that potenti or both. ate or limit the effect of the drug, and genes involved in the etiology of the disease that alter its response to a particular 0899. By identifying Subsets of patients, based upon class of therapeutic agents. It will be recognized to those genotype, that experience renal toxicities in response to the skilled in the art that this broadly includes proteins involved administration of a drug, agent or candidate therapeutic in pharmacokinetics as well as genes involved in pharma intervention, optimal Selection may reduce level and extent codynamics. This also includes genes that encode proteins of the renal damage. Appropriate genotyping and correlation homologous to the proteins believed to carry out the above to dosing regimen, or Selection of optimal therapy would be functions are also worth evaluation as they may carry out beneficial to the patient, caregivers, medical perSonnel, and Similar functions. Together the foregoing proteins constitute the patient's loved ones. the candidate genes for affecting response of a patient to the 0900. As an example of identification of the primary gene therapeutic intervention. Using the methods described and relevant polymorphic variance that directly affects effi above, variances in these genes can be identified, and cacy, Safety, or both one could Select an gene pathway as research and clinical Studies can be performed to establish described in the Detailed Description, and determine the an association between a drug response or toxicity and effect of genetic polymorphism and therapy efficacy, Safety, Specific variances. or both within that given pathway. For example, referring to Table 2, genes involved in drug transport, phase I and phase EXAMPLE 9 II metabolism, and renal tubular uptake and concentration the optimization of therapy of by an agent known to have a Drug-Induced Renal Toxicity renal Side effect by determining whether the patient has a predisposing genotype in which the Selected agents are more 0894) Drug-induced renal toxicity includes, but is not effective and or are more Safe. In considering an optimiza exclusived to, glomerulonephritis and tubular necrosis. tion protocol, one could potentially predetermine the geno Drugs associated with eliciting renal toxicity include, but are typic profile of these genes involved in the manifestation of not excluded to, penicillamine, aminoglycoside antibiotics, the adverse effect, or those genes preeminently responsible cyclosporine, amphotericin B, phenacetin, and Salicylates. for drug response. By embarking on the previously 0895 Impact of Stratification Based Upon Genotype in described gene pathway approach, it is technical feasibility Drug Development for Drugs, Compounds, or Candidate to determine the relevant genes within Such a targeted drug Therapeutic Interventions that may InduceRenal Toxicity development program. 0896. There is evidence to suggest that there are safety 0901) Identification of pathophysiologic relevant vari response differences to drug therapy in reference to devel ance or variances and potential therapies affecting those opment of renal toxicity which may be attributable to allelic genotypes or haplotypes may speed drug develop genotypic differences between individuals. There is pro ment for therapeutic alternatives. There is a need for thera Vided in this invention examples of gene pathways that are pies that are targeted to a disease and Symptom management implicated in the disease process or its therapy and those that with limited or no undesirable side effects. Identification of US 2001/0034023 A1 Oct. 25, 2001 a specific variance or variances within genes involved in the and a (AA, 2Aa, aa) are conventionally described in terms pathophysiologic manifestation of renal toxicity and Specific of p-2pq+q in which p and q are the allele frequencies of genetic polymorphisms of these critical genes may assist the A and a. In this equation (p +2pq+q=1), p is defined as the development of novel agents and the identification of those frequency of one allele and q as the frequency of another patients that may best benefit from therapy of these candi allele for a trait controlled by a pair of alleles (A and a). In date therapeutic alternatives. other words, p equals all of the alleles in individuals who are 0902. By identifying allelic variances or haplotypes in homozygous dominant (AA) and half of the alleles in genes that indirectly affects efficacy, Safety of any class of individuals who are heterozygous (Aa) for this trait. In drugs that has an effect on the prevention, progression, or mathematical terms, this is Symptoms of renal toxicity, one could target Specific Sec ondary drug or agent therapeutic actions that affect the 0905 Likewise, q equals the other half of the alleles for overall therapeutic action of renal protective agents. the trait in the population, or 0903 Pharmacogenomics studies for these drugs, or other agent, compound, drug, or candidate therapeutic inter vention, could be performed by identifying genes that are 0906. Because there are only two alleles in this case, the involved in the the function of a drug including, but not frequency of one plus the frequency of the other must equal limited to absorption, distribution, metabolism, or elimina 100%, which is to say tion, the interaction of the drug with its target as well as p+q=1 potential alternative targets, the response of the cell to the binding of a drug to a target, the metabolism (including 0907 Alternatively, Synthesis, biodistribution or elimination) of natural com pounds which may alter the activity of the drug by comple 0908 All possible combinations of two alleles can be mentary, competitive or allosteric mechanisms that potenti expressed as: ate or limit the effect of the drug, and genes involved in the etiology of the disease that alter its response to a particular (p+q)°=1 class of therapeutic agents. It will be recognized to those 0909 or more simply, skilled in the art that this broadly includes proteins involved in pharmacokinetics as well as genes involved in pharma codynamics. This also includes genes that encode proteins 0910. In this equation, if p is assumed to be dominant, homologous to the proteins believed to carry out the above then p' is the frequency of homozygous dominant (AA) functions are also worth evaluation as they may carry out individuals in a population, 2pd is the frequency of het Similar functions. Together the foregoing proteins constitute erozygous (Aa) individuals, and q is the frequency of the candidate genes for affecting response of a patient to the homozygous recessive (aa) individuals. therapeutic intervention. Using the methods described 0911. From observations of phenotypes, it is usually only above, variances in these genes can be identified, and possible to know the frequency of homozygous dominant or research and clinical Studies can be performed to establish recessive individuals, because both dominant and recessives an association between a drug response or toxicity and will express the distinguishable traits. However, the Hardy Specific variances. Weinberg equation allows us to determine the expected frequencies of all the genotypes, if only p or q is known. EXAMPLE 10 Knowing p and q, it is a simple matter to plug these values into the Hardy-Weinberg equation (p+2pq+q=1). This then Hardy-Weinberg Equilibrium provides the frequencies of all three genotypes for the 0904 Evolution is the process of change and diversifi selected trait within the population. This illustration shows cation of organisms through time, and evolutionary change Hardy-Weinberg frequency distributions for the genotypes affects morphology, physiology and reproduction of organ AA, Aa, and aa at all values for frequencies of the alleles, p isms, including humans. These evolutionary changes are the and q. It should be noted that the proportion of heterozygotes result of changes in the underlying genetic or hereditary increases as the values of p and q approach 0.5. material. Evolutionary changes in a group of interbreeding 0912 Linkage Disequilibirum individuals or Mendelian population, or simply populations, are described in terms of changes in the frequency of 0913 Linkage is the tendency of genes or DNA genotypes and their constituent alleles. Genotype frequen Sequences (e.g. SNPs) to be inherited together as a conse cies for any given generation is the result of the mating quence of their physical proximity on a single chromosome. among members (genotypes) of their previous generation. The closer together the markers are, the lower the probabil Thus, the expected proportion of genotypes from a random ity that they will be separated during DNA crossing over, union of individuals in a given population is essential for and hence the greater the probability that they will be describing the total genetic variation for a population of any inherited together. Suppose a mutational event introduces a Species. For example, the expected number of genotypes that “new” allele in the close proximity of a gene or an allele. could form from the random union of two alleles, A and a, The new allele will tend to be inherited together with the of a gene are AA, Aa and aa. The expected frequency of alleles present on the “ancestral, chromosome or haplotype. genotypes in a large, random mating population was dis However, the resulting association, called linkage disequi covered to remain constant from generation to generation; or librium, will decline over time due to recombination. Link achieve Hardy-Weinberg equilibrium, named after its dis age disequilibrium has been used to map disease genes. In coverers. The expected genotypic frequencies of alleles. A general, both allele and haplotype frequencies differ among US 2001/0034023 A1 Oct. 25, 2001 populations. Linkage disequilibrium is varied among the populations, being absent in Some and highly significant in others. 0914) Quantification of the Relative Risk of Observable Outcomes of a Pharmacogenetics Trial 0924) For example, if q=0.4, PlaR=40% and ObsRR=1.5 0915) Let PlaR be the placebo response rate (0% (PlaR (i.e. TntR=60%), then 1.6 (ExpRR (2.4. This means that the (100%) and TntR be the treatment response rate (0% (TntR best treatment response rate we can expect in a genotypic (100%) of a classical clinical trial. ObsRR is defined as the subgroup of patients in these conditions would be 95.6% relative risk between TntR and PlaR: instead of 60%. 0925. This can also be expressed in terms of maximum ObSRR=Tntri PiaR. potential gain between the observed difference in response 0916 Suppose that in the treatment group there is a rates (TntR-PlaR) without any pharmacogenetic hypothesis polymorphism in relation to drug metabolism Such as the and the maximum expected difference in response rates treatment response rate is different for each genotypic Sub (max(ExpRR)*PlaR-TntR) with a strong pharmacogenetic group of patients. Let q be the allele a frequency of a hypothesis: recessive biallelic locus (e.g. SNP) and p=1-q the allele A frequency. Following Hardy-Weinberg equilibrium, the rela tive frequency of homozygous and heterozygous patients are (max(ExpRR): PlaR-TntR) = (ObsRR-2pg - q2)f p2: PlaR as follow: TntR e (max(ExpRR): PlaR-TntR) = TntR - PlaR: (2pq + q2) - Tntr; p2f p2 = (max(ExpRR): PlaR TntR) = TintR: (1 - p2) - PlaR: (2pq + q2)f p2 ce. (max(ExpRR): PlaR-TntR) = 0917 with (1 - p2)f p2: (TntR - PlaR)

0918) Let's define AAR, AaR, aaR as respectively the 0926) that is for the previous example, response rates of the AA, Aa and aa patients. We have the (95.6%–60%)=(1-0.62)/0.62 (60%-40%)=35.6% following relationship: 0927 Suppose that, instead of one SNP, we have ploci of SNPs for one gene. This means that we have 2p possible haplotypes for this gene and (2p)(2p-1)/2 possible geno 0919 Suppose that the aa genotypic group of patients has types. And with 2 genes with p1 and p2 SNP loci, we have the lowest response rate, i.e. a response rate equal to the (2p1)(2p1-1)/2 (2p2)(2p2-1)/2) possibilities; and so on. placebo response rate (which means that the polymorphism Examining haplotypes instead of combinations of SNPs is has no impact on natural disease evolution but only on drug especially useful when there is linkage disequilibrium action) and let's define ExpRR as the relative risk between enough to reduce the number of combinations to test, but not AAR and aaR, as complete since in this latest case one SNP would be suffi cient. Yet the problem of frequency above still remains with ExpRR=AAR/aaR. haplotypes instead of SNPs since the frequency of a haplo 0920. From the previous equations, we have the follow type cannot be higher than the highest SNP frequency ing relationships: involved. Hence cladograms. 0928 Statistical Methods to be used in Objective Analy SCS 0921. The maximum of the expected relative risk, max 0929. The statistical significance of the differences (ExpRR), corresponding to the case of heterozygous patients between variance frequencies can be assessed by a Pearson having the same response rate as the placebo rate, is Such chi-squared test of homogeneity of proportions with n-1 that: degrees of freedom. Then, in order to determine which variance(s) is(are) responsible for an eventual significance, 0922) we can consider each variance individually against the rest, up to n comparisons, each based on a 2x2 table. This should result in chi-squared tests that are individually valid, but ObsRR = ExpRRp2+2pq + q2 => ExpRR = (ObsRR-2pg - q2)f p2 taking the most Significant of these tests is a form of multiple testing. A Bonferroni’s adjustment for multiple testing will thus be made to the P-values, such as p=1-(1-p)". Chi Square on 3 genotypes, on haplotypes. 0923. The minimum of the expected relative risk, min (ExpRR), corresponding to the case of heterozygous patients 0930 The statistical significance of the difference having the same response rate as the homozygous non between genotype frequencies associated to every variance affected patients, is Such that: can be assessed by a Pearson chi-squared test of homoge US 2001/0034023 A1 Oct. 25, 2001 87 neity of proportions with 2 degrees of freedom, using the 0941) 8) If there is a disagreement between the Same Bonferroni’s adjustment as above. experimentally determined haplotypes and the com 0931 Testing for unequal haplotype frequencies between putationally derived haplotypes, drop the computa cases and controls can be considered in the same framework tionally derived rare haplotypes, construct cla as testing for unequal variance frequencies Since a single dograms from these haplotypes using the Templeton variance can be considered as a haplotype of a single locus. (1987) algorithm. The relevant likelihood ratio test compares a model where 0942 9) Note regions of high recombination. Divide two separate Sets of haplotype frequencies apply to the cases regions of high recombination further to See patterns and controls, to one where the entire Sample is characterized of linkage disequilibria. by a single common Set of haplotype frequencies. This can be performed by repeated use of a computer program 0943) 10) Establish association between cladograms (Terwilliger and Ott, 1994, Handbook of Human Linkage and clinical variables using the nested analysis of Analysis, Baltimore, John Hopkins University Press) to variance as presented by Templeton (1995), and Successively obtain the log-likelihood corresponding to the assign causal variance to a specific haplotype. Set of haplotpe frequency estimates on the cases (1 nL), on the controls (1nleontrol), and on the Overall (1n Leontined). 0944) 11) For variances in the regions of high The test statistic 201n Lease)+(1nleonto)-(1n Lentined)) is recombination, use permutation tests for establishing then chi-squared with r-1 degrees of freedom (where r is the asSociations between variances and the phenotypic number of haplotypes). variables. 0932. To test for potentially confounding effects or 0945) 12) If two or more genes are found to affect a effect-modifiers, Such as Sex, age, etc., logistic regression clinical variable determine the relative contribution can be used with case-control Status as the outcome variable, of each of the genes or variances in relation to the and genotypes and covariates (plus possible interactions) as clinical variable, using Step-wise regression or dis predictor variables. criminant function or principal component analysis. 0946 13) Determine the relative magnitudes of the EXAMPLE 11 effects of any of the two variances on the clinical variable due to their genetic (additive, dominant or Exemplary Pharmacogenetic Analysis Steps epistasis) interaction. 0933. In accordance with the discussion of distribution frequencies for variances, alleles, and haplotypes, variance 0947 14) Using the frequency of an allele or hap detection, and correlation of variances or haplotypes with lotypes, as well as biochemical/clinical variables treatment response variability, the points below list major determined in the in vitro or in vivo studies, deter items which will typically be performed in an analysis of the mine the effect of that gene or allele on the expres pharmacogenetic determination of the effects of variances in Sion of the clinical variable, according to the mea the treatment of a disease and the Selection/optimization of Sured genotype approach of Boerwinkle et al (Ann. treatment. Hum. Genet 1986). 0934) 1) List candidate gene/genes for a known 0948) 15) Stratify ethnic/clinical populations based genetic disease, and assign them to the respective on the presence or absence of a given allele or a metabolic pathways. haplotype. 0935 2) Determine their alleles, observed and 0949) 16) Optimize drug dosages based on the fre expected frequencies, and their relative distributions quency of alleles and haplotypes as well as their among various ethnic groups, gender, both in the effects using the measured genotype approach as a control and in the study (case) groups. guide. 0936 3) Measure the relevant clinical/phenotypic EXAMPLE 12 (biochemical/physiological) variables of the disease. Method for Producing cDNA 0937 4) If the causal variance/allele in the candidate gene is unknown, then determine linkage disequilib 0950 In order to identify sequence variances in a gene by ria among variances of the candidate gene(s). laboratory methods it is in Some instances useful to produce cDNA(s) from multiple human subjects. (hi other instances 0938 5) Divide the regions of the candidate genes it may be preferable to study genomic DNA.). Methods for into regions of high linkage disequilibrium and low producing cDNA are known to those skilled in the art, as are disequilibrium. methods for amplifying and Sequencing the cDNA or por 0939 6) Developp haplotvipesnaplotyp among9. variances that tions thereof. An example of a useful cDNA production show Strong linkage disequilibrium using the com protocol is provided below. AS recognized by those skilled putation methods. in the art, other Specific protocols can also be used. 0940) 7) Determine the presence of rare haplotypes 0951) cDNA Production experimentally. Confirm if the computationally 0952 Make sure that all tubes and pipette tips are RNase determined rare haplotypes agree with the experi free. (Bake them overnight at 100° C. in a vaccum oven to mentally determined haplotypes. make them RNase-free.) US 2001/0034023 A1 Oct. 25, 2001 88

0953 1. Add the following to a RNase-free 0.2 ml et al. (Detection of polymorphisms of human DNA by gel micro-amp tube and mix gently: electrophoresis as Single-Strand conformation polymor phisms. Proc Natl AcadSci USA. 86(8):2766-70, 1989), the 0954 24 ul water (DEPC treated) technique was used on genomic DNA, however the same 0955) 12 ul RNA (lug/ul) group showed that the technique works very well on PCR amplified DNA as well. In the last 10 years the technique has 0956] 12 ul random hexamers (50 ng/ul) been used in hundreds of published papers, and modifica 0957 2. Heat the mixture to 70° C. for ten minutes. tions of the technique have been described in dozens of papers. The enduring popularity of the technique is due to 0958) 3. Incubate on ice for 1 minute. (1) a high degree of Sensitivity to single base differences 0959 4. Add the following: (>90%) (2) a high degree of selectivity, measured as a low frequency of false positives, and (3) technical ease. SSCP is 0960) 16 ul 5xSynthesis Buffer almost always used together with DNA sequencing because 0961) 8ul 0.1M DTT SSCP does not directly provide the sequence basis of differential fragment mobility. The basic steps of the SSCP 0962) 4 u110 mM dNTP mix (10 mM each dNTP) procedure are described below. 0963) 4 ul SuperScript RT II enzyme 0972. When the intent of SSCP screening is to identify a 0964 Pipettepelle genuygentlv to mix. large number of gene variances it is useful to Screen a relatively large number of individuals of different racial, 0965 5. Incubate at 42° C. for 50 minutes. ethnic and/or geographic origins. For example, 32 or 48 or 0966 6. Heat to 70° C. for ten minutes to kill the 96 individuals is a convenient number to Screen because gel enzyme, then place it on ice. electrophoresis apparatus are available with 96 Wells (Applied Biosystems Division of Perkin Elmer Corpora 0967 7. Add 160 ul of water to the reaction so that tion), allowing 3x32, 2x48 or 96 samples to be loaded per the final volume is 240 ul. gel. 0968 8. Use PCR to check the quality of the cDNA. 0973) The 32 (or more) individuals screened should be Use primer pairs that will give a ~800 base pair long representative of most of the Worlds major populations. For piece. See “PCR Optimization” for the PCR proto example, an equal distribution of Africans, Europeans and col. Asians constitutes a reasonable Screening Set. One useful 0969. The following chart shows the reagent amounts for Source of cell lines from different populations is the Coriell a 20 ul reaction, a 80 ul reaction, and a batch of 39 (which Cell Repository (Camden, N.J.), which sells EBV immor makes enough mix for 36) reactions: talized lyphoblastoid cells obtained from several thousand Subjects, and includes the racial/ethnic/geographic back ground of cell line donors in its catalog. Alternatively, a panel of cDNAS can be isolated from any Specific target 20 ul X1 tube 80 ul X1 tube 80 ul X39 tubes population. water 6 ul 24 ul 936 RNA 3 ul 12 ul 0974 SSCP can be used to analyze cDNAS or genomic random hexamers 3 ul 12 ul 468 DNAS. For many genes cINA analysis is preferable because synthesis buffer 4 ul 16 ul 624 for many genes the full genomic Sequence of the target gene O.1 M DTT 2 ul 8 ul 312 is not available, however, this circumstance will change over 10 mM dNTP 1 ul 4 ul 156 the next few years. To produce cDNA requires RNA. There SSRT 1 ul 4 ul 156 fore each cell lines is grown to mass culture and RNA is isolated using an acid/phenol protocol, Sold in kit form as Trizol by Life Technologies (Gaithersberg, Md.). The EXAMPLE 13 unfractionated RNA is used to produce cDNA by the action of a modified Maloney Murine Leukemia Virus Reverse Method for Detecting Variances by Single Strand Transcriptase, purchased in kit form from Life Technologies Conformation Polymorphism (SSCP) Analysis (SuperScript II kit). The reverse transcriptase is primed with 0970) This example describes the SSCP technique for random hexamer primers to initiate cDNA Synthesis along identification of Sequence variances of genes. SSCP is the whole length of the RNAS. This proved useful later in usually paired with a DNA sequencing method, Since the obtaining good PCR products from the 5' ends of some SSCP method does not provide the nucleotide identity of genes. Alternatively, oligodT can be used to prime cDNA variances. One useful Sequencing method, for example, is Synthesis. DNA cycle sequencing of P labeled PCR products using 0975) Material for SSCP analysis can be prepared by the Femtomole DNA cycle sequencing kit from Promega PCR amplification of the cDNA in the presence of one op (WI) and the instructions provided with the kit. Fragments labeled dNTP (usually Cup dCTP). Usually the concentra are selected for DNA sequencing based on their behavior in tion of nonradioactive dCTP is dropped from 200 uM (the the SSCP assay. standard concentration for each of the four dNTPs) to about 0971 Single strand conformation polymorphism screen 100 uM, and p dCTP is added to a concentration of about ing is a widely used technique for identifying an discrimi 0.1-0.3 uM. This involves adding a 0.3-1 ul (3-10 uCi) of P nating DNA fragments which differ from each other by as cCTP to a 10 ul PCR reaction. Radioactive nucleotides can little as a Single nucleotide. AS originally developed by Orita be purchased from DuPont/New England Nuclear. US 2001/0034023 A1 Oct. 25, 2001

0976 The customary practice is to amplify about 200 0981 1. Amplification by the polymerase chain base pair PCR products for SSCP, however, an alternative reaction (PCR) of 400-600 bp regions of the candi approach is to amplify about 0.8-1.4 kb fragments and then date gene from a panel of DNA samples The DNA use Several cocktails of restriction endonucleases to digest samples can either be cDNA or genomic DNA and those into Smaller fragments of about 0.1-0.4 kb, aiming to will represent Some croSS Section of the World popu have as many fragments as possible between 0.15 and 0.3 lation. kb. The digestion strategy has the advantage that less PCR is required, reducing both time and costs. Also, Several 0982) 2. Mixing of a fluorescently labeled probe different restriction enzyme digests can be performed on DNA with the sample DNA. each set of samples (for example 96 cDNAs), and then each 0983 Heating and cooling the mixtures causing hetero of the digests can be run separately on SSCP gels. This duplex formation between the probe DNA and the sample redundant method (where each nucleotide is Surveyed in DNA three different fragments) reduces both the false negative and false positive rates. For example: a Site of variance 0984) 3. Addition of T4 endonuclease VII to the might lie within 2 bases of the end of a fragment in one heteroduplex DNA samples. T4 endonuclease will digest, and as a result not affect the conformation of that recognize and cleave at Sequence variance mis Strand; the same variance, in a Second or third digest, would matches formed in the heteroduplex DNA. likely lie in a location more prone to affect Strand folding, 0985 4. Electrophoresis of the cleaved fragments on and therefore be detected by SSCP. an ABI sequencer to determine the Site of cleavage. 0977. After digestion, the radiolabelled PCR products are diluted 1:5 by adding formamide load buffer (80% forma 0986 5. Sequencing of a subset of PCR fragments mide, 1xSSCP gel buffer) and then denatured by heating to identified by T4 endonuclease VI to contain vari 90% C for 10 minutes, and then allowed to renature by ances to establish the Specific base variation at that quickly chilling on ice. This procedure (both the dilution and location. the quick chilling) promotes intra- (rather than inter-) Strand 0987. A more detailed description of the procedure is as asSociation and Secondary Structure formation. The Second follows: ary Structure of the Single Strands influences their mobility on nondenaturing gels, presumably by influencing the num 0988 A candidate gene sequence is downloaded from an ber of collisions between the molecule and the gel matrix appropriate database. Primers for PCR amplification are (i.e., gel sieving). Even single base differences consistently designed which will result in the target Sequence being produce changes in intrastrand folding Sufficient to register divided into amplification products of between 400 and 600 bp. There will be a minimum of a 50 bp of overlap not as mobility differences on SSCP. including the primer Sequences between the 5' and 3' ends of 0978 The single strands were then resolved on two gels, adjacent fragments to ensure the detection of variances one a 5.5% acrylamide, 0.5xTBE gel, the other an 8% which are located close to one of the primerS. acrylamide, 10% glycerol, 1XTTE gel. (Other gel recipes are known to those skilled in the art.) The use of two gels 0989 Optimal PCR conditions for each of the primer provides a greater opportunity to recognize mobility differ pairs is determined experimentally. Parameters including but ences. Both glycerol and acrylamide concentration have not limited to annealing temperature, pH, MgCl2 concentra been shown to influence SSCP performance. By routinely tion, and KCl concentration will be varied until conditions analyzing three different digests under two gel conditions for optimal PCR amplification are established. The PCR (effectively 6 conditions), and by looking at both Strands conditions derived for each primer pair is then used to under all 6 conditions, one can achieve a 12-fold Sampling amplify a panel of DNA samples (cDNA or genomic DNA) of each base pair of cDNA. However, if the goal is to rapidly which is chosen to best represent the various ethnic back Survey many genes or cDNAS then a leSS redundant proce grounds of the World population or Some designated Subset dure would be optimal. of that population. 0990 One of the DNA samples is chosen to be used as a EXAMPLE 1.4 probe. The same PCR conditions used to amplify the panel are used to amplify the probe DNA. However, a floures Method for Detecting Variances by T4 cently labeled nucleotide is included in the deoxy-nucleotide Endonuclease VII (T4E7) Mismatch Cleavage mix So that a percentage of the incorporated nucleotides will Method be fluorescently labeled. 0979 The enzyme T4 endonuclease VII is derived from 0991. The labeled probe is mixed with the corresponding the bacteriophage T4. T4 endonuclease VII is used by the PCR products from each of the DNA samples and then bacteriophage to cleave branched DNA intermediates which heated and cooled rapidly. This allows the formation of form during replication So the DNA can be processed and heteroduplexes between the probe and the PCR fragments packaged. T4 endonuclease can also recognize and cleave from each of the DNA samples. T4 endonuclease VII is heteroduplex DNA containing Single base mismatches as added directly to these reactions and allowed to incubate for well as deletions and insertions. This activity of the T4 30 min. at 37 C. 10 ul of the Formamide loading buffer is endonuclease VII enzyme can be exploited to detect added directly to each of the Samples and then denatured by Sequence variances present in the general population. heating and cooling. A portion of each of these Samples is 0980 The following are the major steps involved in electrophoresed on an ABI 377 sequencer. If there is a identifying Sequence variations in a candidate gene by T4 Sequence variance between the probe DNA and the Sample endonuclease VII mismatch cleavage: DNA a mismatch will be present in the heteroduplex frag US 2001/0034023 A1 Oct. 25, 2001 90 ment formed. The enzyme T4 endonuclease VII will recog conditions derived for each primer pair is then used to nize the mismatch and cleave at the Site of the mismatch. amplify a panel of DNA samples (cDNA or genomic DNA) This will result in the appearance of two peaks correspond which is chosen to best represent the various ethnic back ing to the two cleavage products when run on the ABI 377 grounds of the World population or Some designated Subset Sequencer. of that population. 0992 Fragments identified as containing sequencing 1003 PCR reactions are purified using the QIAquick 8 variances are Subsequently Sequenced using conventional PCR purification kit (Qiagen cath 28142) to remove nucle methods to establish the exact location and Sequence Vari otides, proteins and buffers. The PCR reactions are mixed CC. with 5 volumes of Buffer PB and applied to the wells of the QlAquick Strips. The liquid is pulled through the Strips by EXAMPLE 1.5 applying a vacuum. The Wells are then washed two times with 1 ml of buffer PE and allowed to dry for 5 minutes Method for Detecting Variances by DNA under vacuum. The PCR products are eluted from the strips Sequencing using 60 ul of elution buffer. 0993 Sequencing by the Sanger dideoxy method or the 1004 The purified PCR fragments are sequenced in both Maxim Gilbert chemical cleavage method is widely used to directions using the Perkin Elmer ABI PrismTM Big DyeTM determine the nucleotide Sequence of genes. terminator Cycle Sequencing Ready Reaction Kit (Cath 0994 Presently, a worldwide effort is being put forward 4303150). The following sequencing reaction is set up: 8.0 to Sequence the entire human genome. The Human Genome ul Terminator Ready Reaction Mix, 6.0 ul of purified PCR Project as it is called has already resulted in the identification fragment, 20 picomoles of primer, deionized water to 20 ul. and Sequencing of many new human genes. Sequencing can The reactions are run through the following cycles 25 times: not only be used to identify new genes, but can also be used 96 C. for 10 second, annealing temperature for that par to identify variations between individuals in the Sequence of ticular PCR product for 5 seconds, 60° C. for 4 minutes. those genes. 1005 The above sequencing reactions are ethanol pre 0995 The following are the major steps involved in cipitated directly in the PCR plate, washed with 70% etha identifying Sequence variations in a candidate gene by nol, and brought up in a Volume of 6 ul of formamide dye. Sequencing: The reactions are heated to 90° C. for 2 minutes and then quickly cooled to 4 C. 1 ul of each Sequencing reaction is 0996 1. Amplification by the polymerase chain then loaded and run on an ABI 377 sequencer. reaction (PCR) of 400-700 bp regions of the candi date gene from a panel of DNA samples The DNA 1006. The output for the ABI sequencer appears as a samples can either be cDNA or genomic DNA and Series of peaks where each of the different nucleotides, A, C, will represent Some croSS Section of the World popu G, and Tappear as a different color. The nucleotide at each lation. position in the Sequence is determined by the most promi nent peak at each location. Comparison of each of the 0997 2. Sequencing of the resulting PCR fragments Sequencing outputs for each Sample can be examined using using the Sanger dideoxy method. Sequencing reac Software programs to determine the presence of a variance tions are performed using floureScently labeled in the Sequence. One example of heterozygote detection dideoxy terminators and fragments are separated by using Sequencing with dye labeled terminators is described electrophoresis on an ABI 377 sequencer or its by Kwok et. al (Kwok, P -Y.; Carlson, C.; Yager, T. D., equivalent. Ankener, W., and D. A. Nickerson, Genomics 23, 138-144, 0998. 3. Analysis of the resulting data from the ABI 1994). The software compares each of the normalized peaks 377 Sequencer using Software programs designed to between all the samples base by base and looks for a 40% identify Sequence variations between the different decrease in peak height and the concomitant appearance of Samples analyzed. a new peak underneath. PoSSible variances flagged by the Software are further analyzed visually to confirm their 0999 A more detailed description of the procedure is as validity. follows: 1000. A candidate gene sequence is downloaded from an EXAMPLE 16 appropriate database. Exemplary Pharmacogenetic Analysis 1001 Primers for PCR amplification are designed which StepS-Biological Function Analysis will result in the target Sequence being divided into ampli fication products of between 400 and 700 bp. There will be 1007. In many cases when a gene which may affect drug a minimum of a 50 bp of overlap not including the primer action is found to exhibit variances in the gene, RNA, or Sequences between the 5' and 3' ends of adjacent fragments protein Sequence, it is preferable to perform biological to ensure the detection of variances which are located close experiments to determine the biological impact of the Vari to one of the primers. ances on the Structure and function of the gene or its 1002 Optimal PCR conditions for each of the primer expressed product and on drug action. Such experiments pairs is determined experimentally. Parameters including but may be performed in Vitro or in Vivo using methods known not limited to annealing temperature, pH, MgCl2 concentra in the art. tion, and KCl concentration will be varied until conditions 1008 The points below list major items which may for optimal PCR amplification are established. The PCR typically be performed in an analysis of the effects of US 2001/0034023 A1 Oct. 25, 2001 91 variances in the treatment of a disease and the Selection/ alters the transcription of a gene and translation into optimization of treatment using biological Studies to deter a gene product. This involves producing amounts of mine the Structure and function of variant forms of a gene or the gene by molecular cloning Sufficient for in vitro its expressed product. or in Vivo Studies. Methods for producing genes and gene products are known in the art and include 1009) 1) List candidate gene/genes for a known cloning of Segments of genetic material in prokary genetic disease, and assign them to the respective otes or eukarotic hosts, run off transcription and metabolic pathways. cell-free translation assays that can be performed in 1010 2) Identify variances in the gene sequence, the cell free extracts, transfection of DNA into cultured expressed mRNA sequence or expressed protein cells, introduction of genes into live animals or Sequence. embryos by direct injection or using vehicles for gene delivery including transfection mixtures or 1011 3) Match the position of variances to regions viral vectors. of the gene, mRNA, or protein with known biologi cal functions. For example, Specific Sequences in the 1015 7) Experimental methods known in the art can promotor of a gene are known to be responsible for be used to determine whether a specific variance determining the level of expression of the gene, alters the ability of a gene to be transcribed into Specific Sequences in the mRNA are known to be RNA. For example, run off transcription assays can involved in the processing of nuclear mRNA into be performed in Vitro or expression can be charac cytoplasmic mRNA including Splicing and polyade terized in transfected cells or transgenic animals. nylation; and certain Sequences in proteins are 1016 8) Experimental methods known in the art can known to direct the trafficking of proteins to specific be used to determine whether a specific variance locations within a cell and to constitute active Sites of alters the processing, Stability, or translation of RNA biological functions including the binding of pro into protein. For example, reticulocyte lysate assays teins to other biological consituents or catalytic can be used to Study the production of protein in cell functions. Variances in Sites Such as these, and others free Systems, transfection assays can be designed to known in the art, are candidates for biological effects Study the production of protein in cultured cells, and on drug action. the production of gene products can be measured in 1012 4) Model the effect of the variance on mRNA transgenic animals. or protein structure. Computational methods for pre 1017 9) Experimental methods known in the art can dicting the structure of mRNA are known and can be be used to determine whether a specific variant alters used to assess whether a specific variance is likely to the activity of an expressed protein product. For cause a Substantial change in the Structure of mRNA. example, protein can be producted by reticulocyte Computational methods can also be used to predict lyStae Systems or by introducing the gene into the Structure of peptide Sequences enabling predic prokaryotic organisms. Such as bacteria or lowre tions to be made concerning the potential impact of eukaryotic organisms such as yeast or fungus), or by the variance on protein function. Most useful are introducing the gene into cultured cells or transgenic structures of proteins determined by X-ray diffrac animals. Protein produced in Such Systems can be tion, NMR or other methods known in the art which extracted or purified and Subjected to bioassays provide the atomic structure of the protein. Compu known to those in the art as measures of the nction tational methods can be used to consider the effect of of that particular protein. Bioassays may involve, but changing an amino acid within Such a structure to are not limited to, binding, inhibiton, or catalytic determine whether Such a change would disrupt the functions. Structure and/or function of the protein. Those skilled in the art will recognize that this analysis can 1018) 10) Those skilled in the art will recognize that be performed on crystal Structures of the protein it is Sometimes preferred to perform the above known to have a variance as well as homologous experiments in the presence of a specific drug to proteins expressed from different loci in the human determine whether the drug has differential effects on genome, or homologous proteins from other Species, the activity being measured. Alternatively, Studies or non-homologous but analogous proteins with may be performed in the presence of an analogue or Similar functions from humans or other Species. metabolite of the drug. 1013 5) Produce the gene, mRNA or protein in 1019 11) Using methods described above, specific amounts Sufficient to experimentally characterize the variances which alter the biological function of a Structure and function of the gene, mRNA or protein. gene or its gene product that could have an impact on It will be apparent to those skilled in the art that by drug action can be identified. Such variances are then comparing the activity of two genes or their products Studied in clinical trial populations to determine which differ by a single variance, the effect of the whether the presence or absence of a Specific vari variance can be determined. Methods for producing ance correlates with observed clinical outcomes Such genes or gene products which differ by one or more as efficacy or toxicity. bases for the purpose of experimental analysis are known in the art. 1020 12) It will be further recognized that there may be more than one variance within a gene that is 1014 6) Experimental methods known in the art can capable of altering the biological function of the be used to determine whether a specific variance gene or gene product. These variances may exhibit US 2001/0034023 A1 Oct. 25, 2001 92

Similar, Synergistic effects, or may have opposite multiple testing correction should be required. If consider effects on gene function. In Such cases, it is necessary ation of more than one Subgroup constitutes multiple testing, to consider the haplotype of the gene, namely the or if individual patients are included in multiple Subgroups, combination of variances that are present within a then statistical corrections may required in the values of Z. Single allele, to assess the composite function of the or Zee which would increase the number of patients required. gene or gene product. 1031. It should be emphasized that a clinical trial of this 1021 13) Perform clinical trials with stratification nature may not provide Statistically significant data concern of patients based on presence or absence of a given ing associations with any genotype other than “i'. The total variance, allele or haplotype of a gene. Establish number of patients that would be required in a clinical trial asSociations between observed drug responses Such to test more than one genetically defined Subgroup would be as toxicity, efficacy, drug response, or dose toleration determined by the maximum value of N for any single and the presence or absense of a Specific variance, Subgroup. allele, or haplotype. 1032 The power of pharmacogenomics to improve the 1022) 14) Optimize drug dosage or drug usage based efficiency of clinical trials arises from the fact it is possible on the presence of the variant. to have N-N. The goal of pharmacogenomic analysis is to identify a genetically define Subgroup in which the magni EXAMPLE 1.7 tude of the clinical response is greater and the variability in response is reduced. These observations correspond to an Stratification of Patients by Genotype in increase in the magnitude of the (mean) observed response Prospective Clinical Trials 8 or a decrease the degree of variability O. Since the value 1023. In a prospective clinical trial, patients will be of N calculated in equation 1.2 decreases non-linearly as the stratified by genotype to determine whether the observed Square of these changes, the total number of patients N can outcomes are different in patients having different geno also decrease non-linearly, resulting in a clinical trial that types. A critical issue is the design of Such trials to assure requires fewer patients to achieve Statistical significance. If that a Sufficient number of patients are Studied to observe 8, and O, are not different than 6 and O, then N is greater genetic effects. than N as given by N=N/P, Values of 8, and O, that give 1024. The number of patients required to achieve statis N

1038. It will be apparent that such an analysis remains observed clinical outcome, for example efficacy or toxicity Sensitive to increases in Ö, but is leSS Sensitive to changes in and performing a maximum likelihood analysis for the O which are not also reflected in the control group. asSociation between a given gene and the observed pheno 1039 Certain analysis may be performed by comparing type based on the allele frequencies and a range of values for individuals with one haplotype against the entire normal A (the association constant between a specific allele and the population. Such an analysis may be used to establish the observed outcome used to stratify patients). Selectivity of the response associated with a specific haplo 1044. This analysis is performed by comparing the type. For example, it may be desirable to establish that the observed gene frequencies in a patient population with an response or toxicity observed in a specific Subgroup is observed outcome to gene frequencies in a table in which the greater than that associated observed with the entire popu predicted frequencies of different alleles of the gene assum lation. It may also be of interest to compare the response to ing different values of the association constant A for that treatment between two different subgroups. If O differs allele. This table of predicted gene frequencies can be between the groups, then the estimate of the number of constructed by those skilled in the art based on the frequency patients that need to be enrolled in the trial must be calcu of any Specific allele in the normal population, the predicted lated using equations 2.1 with N being the maximum of inheritance of the effect (e.g. dominant or recessive) and the N/P, for the different subgroups. fraction of a Subgroup with a specific outcome who would 1040 Another issue in controls is the relative size of the have that allele based on the association constant A. treatment and control groups. In a prospectively designed 1045. For example, if a specific outcome was only clinical trial which Selectively incorporates patients with observed in the presence of a Specific allele of a gene, the haplotype “i” the number of patients in the control and expected frequency would be 1. If a specific outcome was treatment group will be essentially equivalent. If the control never observed in the presence of a Specific allele of a gene, group is different, or if haplotypes are used for Stratification the expected fecuency would be 0. If there was no associa but not inclusion, Statistical corrections may need to be made tion between the allele and the observed outcome, the for having populations of different size. frequency of that allele among individuals with an observed outcome would be the same as in the general population. A EXAMPLE 1.8 Statistical analysis can be performed to compare the observed allele frequencies with the predicted allele fre Stratification of Patients by Phenotype quencies and determine the best fit or maximum likelihood of 1041. The identification of genetic associations in Phase the association. For example, a chi Square analysis will II or retrospective Studies can be performed by Stratifying determine whether the observed outcome is statistically patients by phenotype and analyzing the distribution of Similar to predicted outcomes calculated for different modes genotypes/haplotypes in the Separate populations. A particu of inheritance and different potential values of A. P values larly important aspect of this analysis is that any gene may can then be calculated to determine the likelihood that any have only a partial effect on the observed outcome, meaning Specific association is Statistically Significant. A curve can be that there will be an association value (A) corresponding to calculated based on different values of A, and the maximal the fraction of patients in a phenotypically-defined Subgroup likelihood of an association determined from the peak of who exhibit that phenotype due to a specific genotype/ Such a curve. Methods for chi Square analysis are known to phenotype. those in the art. 1042. It will be recognized to those skilled in the art that 1046. A multidimensional analysis can also be performed the fraction of individuals who exhibit a phenotype due to to determine whether an observed outcome is associated any specific allele will be less than 1 (i.e. A-1). This is true with more than one allele at a specific genetic locus. An for Several reasons. The observed phenotype may occur by example of this analysis considering the potential effects of random chance. The observed phenotype may be associated two different alleles of a single gene is shown. It will be with environmental influences, or the observed phenotype apparent to those skilled in the art that this analysis can be may be due to different genetic effects in different individu extended to n dimensions using computer programs. als. Furthermore, the onstruction of haplotypes and analysis 1047 This analysis can be used to determine the maxi of recombination may not group all alleles with pheontypi mum likelihood that one or more alleles at a given locus are cally-significant variances within a Single haplotype or hap asSociated with a specific clinical outcome. lotype cluster. In this case, causative variances at a single locus may be associated with more than one haplotype or 1048. It will be apparent to those skilled in the art that haplotype cluster and the association constant A for the locus critical issues in this analysis include the fidelity of the would be A=A+A+. . . +A-1. It is likely that many phenotypic association and identification of a control group. phenotypes will be associated with multiple alleles at a In particular, it may be useful to perform an identical given locus, and it is particularly important that Statistical analysis in patients receiving a placebo to eliminate other methods be sufficiently robust to identify association with a forms of bias which may contribute to statistical errors. locus even if A is reduced by the presence of Several causative alleles. Other Embodiments 1043 Statistical methods can be used to identify genetic 1049. The invention described herein provides a method effects on an observed outcome in patient groups Stratified for identifying patients with a risk of developing drug by phenotype, eg the presence or absence of the observed induced liver disease or hepatic dysfunction by determining response. One Such method entails determining the allele the patients allele Status for a gene listed in Tables 1 and 2 frequencies in two populations of patients Stratified by an and providing a forecast of the patients ability to respond to US 2001/0034023 A1 Oct. 25, 2001 94 a given drug treatment. In particular, the invention provides ferred embodiments are exemplary and are not intended as a method for determining, based on the presence or absence limitations on the Scope of the invention. Changes therein of a polymorphism, a patient's likely response to drug and other uses will occur to those skilled in the art, which are therapies as drug-induced liver disease or hepatic dysfunc encompassed within the Spirit of the invention, are defined tion. Given the predictive value of the described polymor by the Scope of the claims. phisms acroSS two different classes of drug, having different 1054 It will be readily apparent to one skilled in the art mechanisms of action, the candidate polymorphism is likely that varying Substitutions and modifications may be made to to have a similar predictive value for other drugs acting the invention disclosed herein without departing from the through other pharmacological mechanisms. Thus, the Scope and Spirit of the invention. For example, using other methods of the invention may be used to determine a compounds, and/or methods of administration are all within patient's response to other drugs including, without limita the Scope of the present invention. Thus, Such additional tion, antihypertensives, anti-obesity, anti-hyperlipidemic, or embodiments are within the Scope of the present invention anti-proliferative, antioxidants, or enhancers of terminal and the following claims. differentiation. 1055. The invention illustratively described herein suit 1050. In addition, while determining the presence or ably may be practiced in the absence of any element or absence of the candidate allele is a clear predictor determin elements, limitation or limitations which is not specifically ing the efficacy of a drug on a given patient, other allelic disclosed herein. Thus, for example, in each instance herein variants of reduced catalytic activity are envisioned as any of the terms “comprising”, “consisting essentially of predicting drug efficacy using the methods described herein. and “consisting of may be replaced with either of the other In particular, the methods of the invention may be used to two terms. The terms and expressions which have been treat patients with any of the possible variances, e.g., as employed are used as terms of description and not of described in Table 3 of Stanton & Adams, application Ser. limitation, and there is no intention that in the use of Such No. 09/300,747, Supra. terms and expressions of eXcluding any equivalents of the 1051. In addition, while the methods described herein are features shown and described or portions thereof, but it is preferably used for the treatment of human patient, non recognized that various modifications are possible within the human animals (e.g., pets and livestock) may also be treated Scope of the invention claimed. Thus, it should be under using the methods of the invention. stood that although the present invention has been Specifi cally disclosed by preferred embodiments and optional 1052 All patents and publications mentioned in the features, modification and variation of the concepts herein specification are indicative of the levels of skill of those disclosed may be resorted to by those skilled in the art, and skilled in the art to which the invention pertains. All refer that Such modifications and variations are considered to be ences cited in this disclosure are incorporated by reference within the Scope of this invention as defined by the appended to the same extent as if each reference had been incorporated claims. by reference in its entirety individually. 1056. In addition, where features or aspects of the inven 1053) One skilled in the art would readily appreciate that tion are described in terms of Markush groups or other the present invention is well adapted to carry out the objects grouping of alternatives, those skilled in the art will recog and obtain the ends and advantages mentioned, as well as nize that the invention is also thereby described in terms of those inherent therein. The methods, variances, and compo any individual member or Subgroup of members of the Sitions described herein as presently representative of pre Markush group or other group.

TABLE 1.

Class Pathway Function Name OMIM GID Locus Absorption Gastrointestinal Glycosidases Sucrase-isomaltase/S1 222900 NM 001041 3q25-q26 and Drug Metabolism maltase-glucoamylase/alpha-glucosidase/MGAM 54360 NM 004668 Chr.7 Distribution actase-phlorizin hydrolase/LPH/lactase/LCT 603202 NM 002299 2q21 salivary amylase A/AMY1A 04700 NM 004038 1.p21 salivary amylase B/AMY1B O4701 * * * * * * 1p21 salivary amylase C/AMY1C O4702 * * * * * * 1p22 pancreatic amylase A/AMY2A O465O XO7057 1p21 pancreatic amylase B/AMY2B O4660 * * * * * * 1p21 Proteases and dipeptidylpeptidase IV/CD26/ADA complexing 02720 NM 001935 2q23 Peptidases protein 2/DPP4 pepsinogen A/PGA/PG 69700 AHOO1519 11q13 pepsinogen, group 3/PGA3 69710 * * * * * * 11q13 6974O JO4443 6p21.3- pepsinogen C/PGC q21.1 47910 AHO02853 19q13.2- q13.4 chymotrypsin-like protease 18888 X71875 16q22.1 rypsinogen 1/TRY1/protease, serine 1/PRSS1 276000 NM 002769 7q35 rypsinogen 1/TRY2/protease, serine 2/PRSS2 601564 NM 002770 7q35 rypsinogen 1/TRY3/protease, serine 3/PRSS3 * * * * * * NM 002771 * * * * * * enterokinase 1/TRY3/protease, serine.7/PRSS7 226200 NM 002772 21q21 chymotrypsinogen 1/CTRB1 118890 NM 001906 16923.2– q23.3 US 2001/0034023 A1 Oct. 25, 2001 95

TABLE 1-continued

Class Pathway Function Name GID Locus carboxypeptidase A1/CPA1 NM OO1868 carboxypeptidase A2/CPA2 NM OO1869 :::::::::::::::::: carboxypeptidase Z/CPZ NM OO3652 elastase 1/ELA1 DOO158 12q13 renal microsomal dipeptidase/DPEP1 (b-lactam ring NM 004413 16q24.3 hydrolysis) tripeptidyl peptidase II/TPP2 90470 NM OO3291 13.q.32 q33 protease inhibitor 1/alpha-1-antitrypsin/AAT/PI NM OOO295 14q32.1 protease inhibitor/alpha-1-antichymotrypsin/AACT NM OO1085 14q32.1 protease inhibitor 1 (alpha-1-antitrypsin)-like/PIL NM OO6220 14q32.1 Lipases Carboxyl ester lipase (bile salt-stimulated M852O1 9q34.3 lipase)/CEL Carboxyl ester lipase-like (bile salt-stimulated lipase NM OO1808 like)/CELL Pancreatic colipase/CLPS M95529 6pter p21.1 Pancreatic triglyceride lipase/PNLTP AHOO3527 Oq26.1 Lipoprotein lipase/LPL NM OOO237 8p22 Hepatic triglyceride lipase/LIPC AHOO5429 Oxidases salivary peroxidase/SAPX U39573 :::::::::::::::::: alcohol dehydrogenases 6/ADH6 NM OOO6721 Esterases paraoxonase 2/PON2 L4851.3 Phosphatases intestinal alkaline phosphatase/ALPI NM OO163 tissue non-specific alkaline phosphatase/liver 71760 alkaline phosphatase/ALPL NM 000478 Drug Binding Blood Transport serum albumin/ALB NM 000477 alpha fetoprotein/AFP NM OO1134 4 alpha albumin/afamin/AFM/ALB2 NM OO1133 4 vitamin D-binding protein?group-specific AHOO4448 4 component?GC orosomucoid 1/alpha 1 acid glycoprotein/ORM1 M13692 orosomucoid 2/alpha 1 acid glycoprotein, type NM OOO608 2fORM2 transthyretin (prealbumin, amyloidosis type I)/TTR NM OOO371 thyroxin-binding globulin/TBG NM OOO354 corticosteroid binding globulin precursor/CBG NM OO1756 sex hormone-binding globulin/SHBG X16349 mannose-binding lectin, soluble/MBL2 NM OOO242

Bile Acid Hepatic fatty acid binding protein/FABP1 346SO :::::::::::::::::: Binders Intestinal fatty acid binding protein/FABP2 34.640 NM OOO134 Muscle fatty acid binding protein/mammary-derived 34651 NM 004102 growth inhibitor/MDGI/FABP3 Adipocyte fatty acid binding protein/FABP4 NM OO1442 Ileal fatty acid binding protein/FABP6 U19869 Brain fatty acid binding protein/FABP7 D88648 Adipocyte long chain fatty acid transport :::::::::::::::::: :::::::::::::::::: protein/FATP Drug Uptake ABC Retina-specific ATP binding cassette NM OOO350 Transporters transporter/ABCR ATP binding cassette 1/ABC1 AO12376 ATP binding cassette 2/ABC2 U18235 ATP binding cassette 3/ABC3 NM OO1089 ATP binding cassette 7/ABC7 ABOO5289 ATP binding cassette 8/ABC8 6O3O76 AFO381752 ATP-binding cassette 50/ABC50 603429 AFO273O26 Placenta-specific ATP-binding cassette 603756 NM OO4827 transporter/ABCP cystic fibrosis transmembrane conductance 6O2421 NM 000492 regulator/CFTR adrenoleukodystrophy fadrenomyeloneuropathy/ALD NM OOOO33 adrenoleukodystrophy related protein/ALDR U2815O sulfonylurca receptor (hyperinsulinemia)/SUR 600509 NM OOO352 peroxisomal membrane protein 1/PXMP1 170995 NM OO2858 peroxisomal membrane protein 1-like/PXMP1L 603214 NM O05050 antigen peptide transporter 1/MHC 1/TAP1 170260 NM 000593 US 2001/0034023 A1 Oct. 25, 2001 96

TABLE 1-continued

Class Pathway Function Name OMIM GID Locus antigen peptide transporter 2/MHC 2/TAP2 170261 NM 000544 6p21.3 multidrug resistance associated protein MRP1 158343 LO5628 16p13.1 multidrug resistance associated protein 601.107 NM 000392 10q24 MRP2/CMOAT ATP-binding cassette, sub-family C (CFTR/MRP), :::::::::::::::::: :::::::::::::::::: member 3/CMOAT2 NM OO3786 ATP-binding cassette, sub-family C (CFTR/MRP), * * * * * * NM 005845 * * * * * * member 4/MOATB ATP-binding cassette, sub-family C (CFTR/MRP), * * * * * * NM 005688 * * * * * * member 5/SMRP ATP-binding cassette, sub-family C (CFTR/MRP), 6O1439 NM OO5691 ****** member 9/SUR2 multidrug resistance protein MDR1 171050 X96395 7q21.1 multidrug resistance protein MDR3/P-glycoprotein 6O2347 XO6181 7q21.1 3/PGY3 anthracyleline resistance-related protein/ARA 603234 NM 001171 16p13.1 bile salt export pump/BSEP 603201, NM 003742 2d24 familial intrahepatic cholestasis 1/FIC1 602397 NM 005603 18q21 Human sorcin/SRI 18252O L12387 7q21.1 Solute Solute carrier family 1, member 1/SLC1A1 1335SO UO8989 9p24 Antiporters (glutamate) Solute carrier family 1, member 2/SLC1A2 6OO3OO UO3SOS 11p13 (glutamate) p12 Solute carrier family 1, member 3/SLC1A3 600111 UO3SO4 5p13 (glutamate) Solute carrier family 1, member 4/SLC1A4 600229 NM 003038 2p15-p13 (glutamate) Solute carrier family 1, member 5/SLC1A5 (neutral O9190 AF105230 19q13.3 AA) Solute carrier family 1, member 6/SLC1A6 600637 NM 005071 * * * * * * (glutamate) Solute carrier family 2, member 1/SLC2A1/SGLT1 82380 NM 006516 22d 13.1 (glucose) Solute carrier family 2, member 2/SLC2A2/GLUT2 38160 NM 006516 3q26.1- (glucose) q26.3 Solute carrier family 2, member 3/SLC2A3/GLUT3 3817O M2O681 12p13.3 (glucose) Solute carrier family 2, member 4/SLC2A4/GLUT4 38.190 * * * * * * 17p13 (glucose) Solute carrier family 2, member 5/SLC2A5/GLUT5 38230 NM_003039 1p36.2 (glucose) Solute carrier family 3 member 1/SLC3A1 (aa O4614 * * * * * * 2p16.3 transporter) Solute carrier family 5 member 1/SLC5A2 (glucose) 82.381 * * * * * * 16p11.2 Solute carrier family 5 member 3/SLC5A3 6OO444 L385OO 21q22 Solute carrier family 5 member 6/SLC5A6 (folate, 604024 * * * * * * 2p23 biotin, lipoate) Solute carrier family 6 member 1/SLC6A1 (GABA) 37165 X54673 3p25-p24 Solute carrier family 6 member 2/SLC6A2 63970 NM 001043 16q12.2 (noradrenalin) Solute carrier family 6 member 3/SLC6A3 26455 L241.78 5p15.3 (dopamine) Solute carrier family 6 member 4/SLC6A4 82138 XTO697 17q11.1- (serotonin) q12 Solute carrier family 6, member 5/SLC6A5 (glycine) 604159 NM 004211 ****** Solute carrier family 6, member 6/SLC6A6 (taurine) 86854 U1612O 3p25-q24 Solute carrier family 6, member 8/SLC6A8 (creatine) 3OOO36 NM OO5629 300036 Solute carrier family 6, member 9/SLC6A9 (glycine) 6O1019 S7O612 1p313 Solute carrier family 6, member 10/SLC6A10 6O1294 * * * * * * 16p11.2 (creatine-testis) Solute carrier family 6, member 12/SLC6A12 603080 NM 003044 12p13 (GABA-betaine) Solute carrier family 7, member 1/SLC7A1 (cationic 104615 * * * * * * 13q12.3 AA Solute carrier family 7, member 2/SLC7A2 (cationic 6O1872 D29990 8p22 AA Solute carrier family 7, member 4/SLC7A4 (cationic 603752 * * * * * * 22q11.2 AA Solute carrier family 7, member 5/SLC7A5 (neutral 600182 M8O244 16q24.3 AA Solute carrier family 7, member 7/SLC7A7 (dibasic 603593 Y18474 14q11.2 AA Solute carrier family 7, member 9/SLC7A9 (neutral 604144 * * * * * * 19q13.1 AA US 2001/0034023 A1 Oct. 25, 2001 97

TABLE 1-continued

Class Pathway Function Name OMIM GID Locus Solute carrier family 10, member 1/SLC10A 182396 NM 003049 chir. 14 (taurocholate) Solute carrier family 10, member 2/SLC10A2 601295 NM 000452 13.q.33 (taurocholate) Solute carrier family 11, member 1/SLC11A1 (?) 600266 AHO02806 2.d35 Solute carrier family 11, member 2/SLC11A2 (iron) 6OO523 L37347 12q13 Solute carrier family 13, member 2/SLC13A2 604148 NM 003984 17p11.1– (dicarboxylic acids) q11.1 Solute carrier family 14, member 1/SLC14A1 (urea) 111000 * * * * * * 18q11 q12 Solute carrier family 14, member 2/SLC14A2 (urea) 6O1611 X96969 18q12.1- q21.1 Solute carrier family 15, member 1/SLC15A 6OO544 U13173 13.q.33 (peptides) q34 Solute carrier family 15, member 2/SLC15A2 6O2339 SA82O3 :::::::::::::::::: (peptides) Solute carrier family 16, member 1/SLC16A 600682 NM 003051 1p13.2- (monocarboxylic acids) p12 Solute carrier family 16, member 2/SLC16A2 300095 NM 006517 Xq13.2 (monocarboxylic acids) Solute carrier family 16, member 3/SLC16A3 603877 NM OO4207 * * * * * * (monocarboxylic acids) Solute carrier family 16, member 4/SLC16A4 603878 * * * * * * :::::::::::::::::: (monocarboxylic acids) Solute carrier family 16, member 5/SLC16A5 603879 NM 004695 * * * * * * (monocarboxylic acids) Solute carrier family 16, member 6/SLC16A6 60388O NM 004694 ****** (monocarboxylic acids) Solute carrier family 16, member 7/SLC16A7 603654 AFO49608 2d 13 (monocarboxylic acids) Solute carrier family 18, member 1/VAT1/SLC18A1 193OO1 LO9118 Oq25 (monoamines) Solute carrier family 18, member 2/VAT2/SLC18A2 193002 : * * * * * 8p21.3 (monoamines) Solute carrier family 18, member 3/VAT3/SLC18A3 600336 NM 003055 10q11.2 (monoamines) Solute carrier family 19, member 1/SLC19A1 6OO424 U1972O 21q22.3 (reduced folate) Solute carrier family 19, member 2/SLC19A2 6O3941 AF16O186 d23.2- (thiamine) q23.3 Solute carrier family 21, member 2/SLC21A2 6O1460 NM 005630 3g21 (prostaglandin) Solute carrier family 21, member 3/SLC21A3 602883 NM 005075 12p12 (organic anion) Solute carrier family 22, member 1/SLC22A1 602607 NM OO3058 6d26 (organic cation) Solute carrier family 22, member 1-like/SLC22A1 L 6O2631 AFO37O64 1p15.5 (organic cation) Solute carrier family 22, member 2/SLC22A2 602608 NM OO3058 6d26 (organic cation) Solute carrier family 22, member 4/SLC22A4 604190 NM OO3059 Chr. 5 (organic cation) Solute carrier family 22, member 5/SLC22A5 603377 NM OO3060 5.d33.1 (camitine) Solute carrier family 25, member 1/SLC25A1 190315 X96924 22q11 (tricarboxylic acids) (mitochondrial) Solute carrier family 25, member 11/SLC25A11 604165 NM 003562 17p13.3 (oxoglutarate/malate) (mitochondrial) Solute carrier family 25, member 12/SLC25A12 (?) 603.667 NM 003705 ****** (mitochondrial) Solute carrier family 25, member 13/SLC25A13 (?) 603859 * * * * * * 7q21.3 (mitochondrial) Solute carrier family 25, member 15/SLC25A15 603861 * * * * * * 13q14 (ornithine) (mitochondrial) Solute carrier family 25, member 16/SLC25A16 13908O M31659 10q21.3- (ADP/ATP) (mitochondrial) q22.1 Solute carrier family 29, member 1/SLC29A1/ENT1 602193 NM 004955 6p21.2- (nucleoside) (mitochondrial) p21.1 Solute carrier family 29, member 2/SLC29A2/ENT2 6O2110 X86681 11q13 (nucleoside) (mitochondrial) Phase I Drug Monooxigenases Flavin- Flavin-containing monooxygenase 1/FMO1 136130 NM 002021 1923-q25 Metabolism (mixed function containing Flavin-containing monooxygenase 3/FMO3 136132 AHOO6707 1923-q25 (Oxidation and Oxidases) Mono- Flavin-containing monooxygenase 4/FMO4 136131 NM 001460 1923-q25 reduction) OXygenases Flavin-containing monooxygenase 5/FMO5 603957 NM 001461 1921.1 US 2001/0034023 A1 Oct. 25, 2001 98

TABLE 1-continued

Class Pathway Function Name OMIM GID Locus P450 Aryl hydrocarbon receptor nuclear 126110 NM 001668 1921 Cytochromes translocator? ARNT Aryl hydrocarbon receptor nuclear translocator- 602550 NM 001178 11p15 like? ARNTL Aryl hydrocarbon receptor/AHR 600253 NM 001621 7p15 Nuclear receptor subfamily 1, group I, member 603065 NM 003889 * * * * * * 2/NR12 Constitutive androstane receptor, beta/orphan nuclear 603881, NM OO5122 ****** hormone receptor/CAR Nuclear receptor subfamily 1, group H, member 600380 UO7132 9q13.3 2/NR1H2 Retinoic acid receptor, alpha/RARA 180240 NM 000964 17q12 Retinoic acid receptor, beta/RARB 180220 NM 000965 3p24 Retinoic acid receptor, gamma/RARG 18O190 M57707 2d 13 Retinoid X receptor alpha/RXRA 180245 NM 005693 9q34.3 Retinoid X receptor beta/RXRB 18O246 X66424 6p21.3 Retinoid X receptor gamma/RXRG 18O247 U3848O q22-q23 RAR-related orphan receptor A/RORA 600825 NM 002943 15q21– d22 RAR-related orphan receptor B/RORB 60O825 * * * * * * 5q21 d22 RAR-related orphan receptor C/RORC 602943 NM 005060 1921 cellular retinoic acid-binding protein, type 1802.31 * * * * * * q21.3 2/CRABP2 glucocorticoid receptor/GRL 138040 NM 000176 5q31 Peroxisome proliferative activated receptor, 170998 NM 005036 22g 12 alpha/PPARA d13.1 Peroxisome proliferative activated receptor, 601487 NM 005037 3p25 gamma/PPARG Peroxisome proliferative activated receptor, 600409 NM 006238 1921.3 delta/PPARD cytochrome P450, subfamily I, polypeptide 1 (aryl 108330 NM 000499 15q22 hydrocarbon oxidase)/CYP1A1 q24 cytochrome P450, subfamily I, polypeptide 2 124060 AHOO2667 5q22 (phenacetin metabolism)/CYP1A2 Cter cytochrome P450, subfamily IB, polypeptide 1 601771 NM 0001.04 2p22-p21 (dioxin inducible)/CYP1B cytochrome P450, subfamily II, polypeptide 1 12396O X13897 9q13.2 (phenobarbital inducible)/CYP2A cytochrome P450, subfamily IIA, polypeptide 6 122720 NM OOO762 19q13.2 (coumarin-7-hydroxylase)/CYP2A6 cytochrome P450, subfamily IIB (phenobarbital 12393O M2987.4 9q13.2 inducible)/CYP2B cytochrome P450, subfamily IIC, polypeptide 6O1129 * * * * * * Oq24 8/CYP2C8 cytochrome P450, subfamily IIC, polypeptide 9 6O1130 * * * * * * Oq24 (hydroxylation of tolbutamide)/CYP2C9 Cytochromescytochrome P450, subfamily IIC, polypeptide601131 ****** Oq25 18/CYP2C18 cytochrome P450, subfamily IIC, polypeptide 19 24020 NM 000769 10q24.1- (mephenytoin 4-hydroxylase)/CYP2C19 d24.3 cytochrome P450, subfamily IID, polypeptide 6 24030 NM OOO106 22d 13.1 (debrisoquine hydroxylation)/CYP2D6 cytochrome P450, subfamily IIE (ethanol 24O4O JO2843 Oq24.3- inducible)/CYP2E Cter cytochrome P450, subfamily IIF (ethoxycoumarin 24O7O NM OOO774 19q13.2 monooxygenase), polypeptide 1/CYP2F1 cytochrome P450, subfamily IIJ (arachidonate 6O1258 NM 000775 1p31.3- epoxygenase), polypeptide 2/CYP2J2 p31.2 cytochrome P450, subfamily IIIA (niphedipine 24010 NM 000776 7q22.1 Oxidase), polypeptide 3/CYP3A3 cytochrome P450, subfamily IVA (fatty acid W- 6O1310 NM OOO778 Chr.1 hydroxylase), polypeptide 11/CYP4A11 cytochrome P450, subfamily IVB, polypeptide 24075 NM 000779 1p34-p12 1/CYP4B1 cytochrome P450, subfamily IVF (leukotriene B4-W- 601270 NM 000896 19p13.2 hydroxylase), polypeptide 3/CYP4F3 cytochrome P450, subfamily VIIA (cholesterol 7-a- 18455 M898O3 8q11-q12 hydroxylase), polypeptide 1/CYP7A1 cytochrome P450, subfamily VIIB (oxysterol 7-a- 603711 AF029403 8q21.3 hydroxylase), polypeptide 1/CYP7B1 cytochrome P450, subfamily VIIIB (sterol 12-a- 602172 * * * * * * 3p21.3- hydroxylase), polypeptide 1/CYP8B1 p22 cytochrome P450, subfamily XIA (cholesterol side- 118485 NM 000781 15q23 US 2001/0034023 A1 Oct. 25, 2001 99

TABLE 1-continued

Class Pathway Function Name OMIM GID chain cleavage)/CYP11A cytochrome P450, subfamily XIB, polypeptide 2 12408O NM OOO498 (steroid 11-b-hydroxylase)/CYP11B2 cytochrome P450, subfamily XDX (androgen 107910 NM OOO103 aromatase)/CYP19 cytochrome P450, subfamily XXI (sterol 21-a- 2O1910 M13936 hydroxylase)/CYP21 cytochrome P450, subfamily XXIV (25 S67623 hydroxyvitamin D24-hydroxylase)/CYP24 cytochrome P450, subfamily XXVIA, polypeptide 1 NM OOO783 (retinoic acid hydroxylase)/CYP26A1 cytochrome P450, subfamily XXVIIA, polypeptide 1 2137OO NM OOO105 (25-hydroxyvitamin D-1-a-hydroxylase)/CYP27A1 adrenodoxin?ferredoxin 1FFDX1/ADX 10326O NM OO4109 adrenodoxin reductase/ferredoxin:NADP(+) 103270 NM 004110 reductase/FDXRADXR cytochrome P450, subfamily XXVIIB, polypeptide 1 2647OO NM OOO785 (25-hydroxyvitamin D-1-a-hydroxylase)/CYP27B1 cytochrome P450, subfamily XLVI (cholesterol 24 604087 NM OO6668 hydroxylase)/CYP46 cytochrome P450, subfamily LI (lanosterol 14-a- 6O1637 U51692 demethylase)/CYP51 General General Monoamine Oxidase A. MAOA 30985O M69226 Oxidases Oxidases Monoamine Oxidase B; MAOB 309860 M69.177 Copper-containing amine Oxidase/AOC3 603735 NM OO3734 Xanthine dehydrogenase/XDH 2783OO NM OOO379 tryptophan 2,3-dioxygenase/TDO2 NM OO5651 sulfite oxidase/SUOX NM 000456 Dehydrogenases Cofactor molybdenum factor synthesis 1/MOCS1 AJ224328 Synthesis molybdenum factor synthesis 2/MOCS2 :::::::::::::::::: Alcohol alcohol dehydrogenases 1, alpha subunit/ADH1 NM OOO667 Dehydrogenases cohol dehydrogenases 2, beta subunit/ADH2 NM OOO668 cohol dehydrogenases 3, gamma subunit/ADH3 M12272 cohol dehydrogenases 4/pi isozyme? ADH4 M15943 hol dehydrogenases 5/chi isozyme/ADH5 NM OOO671 hol dehydrogenases 6/ADH6 NM OOO672 hol dehydrogenases 7/ADH7 AHOO6682 Aldehyde ehyde dehydrogenase 1/ALDH1 (liver cytosol) AHOO2598 Dehydrogenases ehyde dehydrogenase 2/ALDH2 (liver KO3OO1 ochondria) ehyde dehydrogenase 3/acetaldehyde M74542 hydrogenase/ALDH3 (stomach) ehyde dehydrogenase 5/acetaldehyde NM OOO692 hydrogenase/ALDH5 ehyde dehydrogenase 5, member Al/succinic 27198O NM OO1080 emialdehyde dehydrogenase/ALDH5A1 ehyde dehydrogenase 6/acetaldehyde NM OOO693 hydrogenase/ALDH6 ehyde dehydrogenase 7/acetaldehyde NM OOO694 hydrogenase/ALDH7 ehyde dehydrogenase 8/ALDH8 6O1917 NM OOO695 ehyde dehydrogenase 9/g-aminobutyraldehyde 6O2733 NM OOO696 hydrogenase/ALDH9 ehydedehydrogenase 10/ALDH10 2702OO NM OOO382 7p11.2 Dihydro ihydropyrimidine dehydrogenase (5-fluoroacil 274270 UO9178 p22 pyrimidine d e Oxification) Dehydrogenase Fatty Acid f Peroxisome Peroxisome proliferative activated receptor, 170998 NM OO5036 Oxidation Proliferation alpha/PPARA Peroxisome proliferative activated receptor, NM OO5037 gamma/PPARG Peroxisome proliferative activated receptor, 18O231 NM OO6238 q21.3 delta/PPARD Peroxisome peroxisome biogenesis factor 1/PEX1 6O2136 ABOO8112 Synthesis peroxisomal membrane pro ein 3 (35kD, Zeliweger 170993 NM OOO318 syndrome)/PXMP3/PEX2 peroxisomal biogenesis fac or 3/PEX3 6O3164 NM OO3630 :::::::::::::::::: peroxisomal biogenesis fac or 6/PEX6 601498 NM OOO287 peroxisomal biogenesis fac or 7/PEX7 6O1757 NM OOO288 peroxisomal biogenesis fac or 10/PEX10 6O2859 :::::::::::::::::: :::::::::::::::::: peroxisomal biogenesis fac or 11A/PEX11A 603866 NM OO3847 :::::::::::::::::: peroxisomal biogenesis fac or 11B/PEX11B 603867 NM OO3846 :::::::::::::::::: peroxisomal biogenesis fac or 12/PEX12 6O1758 NM OOO286 peroxisomal biogenesis fac or 13/PEX13 6O1789 U71374 2p15