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Drug Metabolism & Pharmacokinetics in Drug Discovery

Drug Metabolism & Pharmacokinetics in Drug Discovery

Drug & in Discovery: A Primer for Bioanalytical Chemists, Part I

Chandrani Gunaratna In the face of advancing technology in combinatorial synthesis and high Bioanalytical Systems Inc. 2701 Kent Avenue throughput screening, the process continues to evolve. West Lafayette, IN Preclinical and pharmacokinetics studies play a key role 47906-1382 in lead identification and optimization. This fast-paced development [email protected] process has imposed an enormous burden on the analytical chemist to design faster and more sensitive assay techniques to aid the drug discovery and development. This article, Part I of a two-part series introduces the analytical chemist to the fundamentals of drug metabolism. Part II of this series will discuss the pharmacokinetics aspects and how drug metabolism data can be used to predict pharmacokinetic parameters.

Technological innovation and the ciency in the optimization of desired liphophilicity and stability are deter- pressures of competition have pharmacological activity in mined by measuring the octanol- caused enormous changes in the while decreasing the reliance on ani- water and pKa. drug discovery process. Progress in mal studies has become a challenge. These measurements are useful in molecular biology and the New chemical entities (NCEs) enter predicting the protein binding, Genome Project has contributed to the drug discovery pipeline through distribution and absorption in the the remarkable advances made in combinatorial synthesis and rational (1). identification of new therapeutic tar- where information The selected leads are further gets. The drug discovery process is about the target of action is used to screened using in vitro tests during rapidly evolving due to the techno- design the . HTS lead optimization. The goal of lead logical developments in target iden- helps the identification of the leads optimization is to select compounds tification along with automation of that provide the required effect at with required biological activity in combinatorial synthesis and high high . In the secon- humans. Relevant pharmacokinetic throughput screening (HTS). In light dary screening stage physicochemi- parameters such as tissue penetra- of these advances, improving effi- cal properties such as , tion, stability, intestinal absorption, F1 metabolism, and elimination are ob- CHEMICAL COMBINATORIAL tained using in vitro systems. These process (IND: in vitro systems include mi- Investigational New Lead Identification Drug, NDA: New Drug DISCOVERY crosomes, or tissue Application) slices for identification HIGH THROUGHPUT SCREENING

Lead Optimiz and evaluation of metabolic path- PHYSIOCHEMICAL PROPERTIES inical ways and rates, and caco-2 cell lines l -c IN VITRO SCREENS re for evaluating transcellular absorp- P udies ation t IN VIVO SCREENS tion. Cytotoxicity data can be ob-

tion S File IND c tained by using organ-specific cell PHASE I lines. Knowledge of the toxic poten- PHASE II nical ug Intera li r C D tial of these early leads and their PHASE III File NDA possible is essential for successful drug discovery. Most REGISTRATION drug candidates fail at this stage and only a few will be judged sufficiently MARKET safe and efficacious to proceed fur- ther into development. Both in vitro

17 Current Separations 19:1 (2000) F2 Oral Intestines drug as schematically shown in F3 is Absorption of a drug after known as ADME studies. administration. Topical Drug Metabolism DRUG IV Interest in drug or (for- IM, SC, IP Membranes eign compounds) metabolism can be dated back to the early 19th century. Inhalation Metabolism then was known as a “detoxication” mechanism in the F3 body. In late 1930s, with the discov- ery of the synthetic azo-dye Pron- Schematic representation sm Metabolites of drug’s path from blood. Drug at metaboli tosil’s metabolism to antibacterial absorption Drug in Absorption agent sulfanilamide in the body, Site Blood elimination studying metabolism has become an important priority. This year BAS distribution and the International Society for the Other Other Study of (ISSX) pro- Distribution Tissues Excretory Fluids Fluids duced a historical calendar celebrat- ing many of the original and in vivo studies are then carried drug metabolism and pharmacoki- contributions to our knowledge of out on the active candidate com- netics aspects in drug discovery. Part the metabolism of organic com- pounds. The objective in these pre- 1 of the article covers the basics of pounds (3). clinical studies is not only to identify drug metabolism. In Part II we will Metabolism is the mechanism of the most active leads with the most discuss the kinetics of drug metabo- elimination of foreign and undesir- appropriate safety profiles but also, lism and the relationship of kinetic able compounds from the body and to select the closest species to data to the pharmacokinetics of a the control of levels of desirable the human for studies (2). drug. compounds such as vitamins in the Understanding of pharmacokinetic body. Since information on the me- and metabolism characteristics of Path of a drug tabolism of a drug plays a significant the selected compounds is needed in role in selection and further charac- designing appropriate human clini- After administration by any route, a terization of the drug, an in-depth cal trials. Various stages of drug dis- drug will reach the blood stream as look at the mechanism of drug meta- covery are illustrated in F1. schematically shown in F2.This bolism is worth the effort. Meeting the objectives of drug process is known as absorption. The The major site of metabolism in metabolism research, whether it be drug in the blood distributes rapidly the body is the . Metabolism in in vitro or in vivo, requires the proc- between the plasma and blood cells liver occurs in two stages: Phase I essing of a very large number of and also between plasma proteins. pathways in liver microsomes where samples for the determination of Most readily cross the capillar- the drug is functionalized and Phase drug candidates and metabolites. ies and reach the II pathways in liver cells where the There is likewise a lot of structural of every organ. soluble drugs parent or the metabolite from Phase to be done to identify me- cross the cell membranes and distrib- I gets conjugated. Liver microsomes tabolites. It is important for analyti- ute into the intracellular fluid of vari- are in the of cal chemists to understand that the ous tissues. This process of liver cells or hepatocytes. Phase I vast majority of compounds (actu- transferring a drug from blood to reactions in microsomes are cata- ally > 99.99%) will never become various tissues is called distribution. lyzed by a group of known drugs. Thus the bioanalytical work A drug is eliminated either di- as the system that must be fast. Often elegance must be rectly through an excretory route plays a significant role in drug meta- traded for speed early in the process. such as urine, bile etc. which is bolism. The common chemical reac- Later on, for example in clinical tri- known as elimination; or indirectly tions involved in Phase I are aromatic als, the number of samples for a par- through enzymatic or biochemical hydroxylation, aliphatic hydroxyla- ticular compound will increase transformation by the liver. The lat- tion, oxidative N-dealkylation, oxi- exponentially and carefully vali- ter path of elimination is called me- dative O-dealkylation, S-oxidation, dated methods are both required and tabolism. The study of this whole reduction and hydrolysis. Most often justified. process of absorption, distribution, this simple functionalization could This article is intended to en- metabolism and elimination of a be sufficient to make a drug more lighten bioanalytical chemists on soluble, facilitating elimination www.currentseparations.com 18 T1 toxic and carcinogenic action of Substrates Known Cytochrome xenobiotics. P450 substrates. CYP1A2 , , , , , There are about 30 human cyto- , , , , chrome P450 enzymes, out of which , , , , , only six, CYP1A2, CYP2C9, , (R)- CYP2C19, CYP2D6, CYP2E1 and CYP2A6 Coumarin, Betadiene, CYP3A4 are the major metabolizing enzymes. CYP3A is the most abun- CYP2C9 Amitriptyline, , Demadex, , , , , , , , dant and most clinically important (S)-Warfarin isozyme in humans. It metabolizes nearly 50% of the clinically available CYP2C19 Amitriptyline, , Clomipramine, , Imipramine, drugs. T1 shows the major CYPs involved in the metabolism of some CYP2D6 Amitriptyline, Betaxolol, Clomipramine, , Clozapine, known drugs. From the table it can , Fluoxetine, Haloperidol, Imipramine, , , , , Olanzapine, be seen that some drugs are metabo- Ondansetron, , Propranolol, , , lized by more than one isozyme. This , multiple- metabolism is the CYP2E1 Acetaminophen, Caffeine, , , cause for metabolism-based drug- Ethanol, Theophylline, Venlafaxine drug interactions (DDIs). Some drugs can be inducers or CYP3A4/5 , Amiodaron, Amitriptyline, , , , Caffeine, , Cerivastatin, , inhibitors of specific isozymes but , Clomipramine, Codeine, Cyclosporine, not necessarily substrates. Enzyme , Dextromethorphan, DHEA, Diazepam, inducers increase specific enzyme , , , , , , Fluoxetine, Imipramine, , , levels by modulating the gene ex- , , , , , pression. Some drugs induce P450 Omeprazole, , Paroxetine, , enzymes that are not involved in their , Rifampin, Sertraline, , , , , , , metabolism. For example, omepra- , Theophylline, Verapami, , (R)-Warfarin zole induces human CYP1A2 but is metabolized by CYP2C19 and through the kidneys. Further conju- Genetic polymorphism CYP3A4 (6). Administration of gation in Phase II occurs by glu- omeprazole can lower the effect of a curonidation, , amino acid Cytochrome P450 enzymes are drug normally metabolized by conjugation, , methyla- grouped into families and sub fami- CYP1A2, e.g., acetaminophen. tion or conjugation to lies based on their structural similar- Enzyme inhibitors function in facilitate elimination. ity (5). Families include CYPs with different ways. The competitive in- >40% amino acid sequence homol- hibitors compete with the substrate Cytochrome P 450 system ogy and are designated by a number for the active site, e.g., fluvoxamine after CYP. Subfamilies are the CYPs and caffeine for CYP1A2 (7). The Cytochrome P450 (CYP) enzyme within a family that have >60% non-competitive inhibitors bind to system, a very large group of en- amino acid sequence homology and the enzyme-substrate complex or to zymes encoded by the P450 gene are designated by a letter following the heme group, e.g., . superfamily, is one of the widely the number. For example CYP3A4 is The third type, irreversible inhibi- studied topics in drug development. a cytochrome P450 enzyme, belong- tors inactivate the enzyme either by CYPs are membrane bound proteins ing to family 3 and subfamily A. The heme binding or protein binding. En- with an approximate molecular last number 4, refers to the sequence zyme inhibition can lead to higher weight of 50 kD, and contain a heme of discovery. systemic levels of a drug causing moiety. CYPs and other mixed func- Several of the drug metabolizing enhanced or toxicity. This tion oxygenases are mainly found in enzymes, for example the CYP2 should be considered when multiple the endoplasmic reticulum of the family, are polymorphic (having drugs are simultaneously prescribed liver. The monooxygenase function more than one variant of the gene). and/or when over-the-counter drugs of CYP450 involves a number of Although the CYP isozymes gener- or neutraceuticals are concomitantly steps but the end reaction is the trans- ally have similar functional proper- administered with prescription fer of one atom to the sub- ties, each one is different and has a drugs. strate (R) that has a site for oxidation distinct role. This polymorphism When several enzymes metabo- as shown below (4). forms a basis for interindividual dif- lize a drug, e.g. propranolol (8), ad- + CYP450 ferences in the efficacy of drug treat- ministration of an NADPH + H +O2 +R-H + ment, side effects of drugs and the will not have a great effect since the NADP +H2O + R-OH

19 Current Separations 19:1 (2000) T2 Ultra extensive metabolism can Known inhibitors Enzyme Inducers Inhibitors cause therapeutic failure due to re- and inducers of duced or lack of acti- CYP isozymes CYP1A2 Cigarette Smoke, , Enoxacin, , , Carbamazepine, Grepafloxacin, vation of the drug whereas poor Charbroiled Foods, Vegetables, Fluvoxamine, Fluoxetine, metabolism can lead to drug toxicity Omeprazole and sometimes death. For optimal CYP2A6 Barbiturates drug , the prescribing physi- cian should have the knowledge of CYP2C9 Rifampin, Carbamazepine, , , the genetic makeup of the CYP en- Ethanol, Phenytoin Fluvoxamine, Fluoxetine, , , zymes in the patient. , Ritonavir, Outcome of drug metabolism CYP2C19 Rifampin Fluvoxamine, Fluoxetine, , Ritonavir Various possibilities of the outcome of drug metabolism are illustrated in F4. CYP2D6 Pregnancy Quinidine, Fluoxetine, Paroxetine, Sertraline, Cytochrome P450 reactions , , make substrates more hydrophilic , for easy elimination through the kid- Haloperidol, Ticlopidine (Ticlid), Ritonavir neys. Although most often this re- sults in inactivation of the drug, CYP2E1 Ethanol, , Ritonavir Cimetidine, Watercress some compounds form active meta- CYP3A4/5 Carbamazepine, Dexamethasone, Ketoconazole, , bolites. These active metabolites can , , Growth Erythromycin, Grapefruit enhance, modify, or inhibit the desir- Hormone, Rifampin, Phenobarbital, Juice, Fluvoxamine, able activity of the drug. Sometimes Phenytoin, Fluoxetine, Diltiazem, Verapamil, Clarithromycin, the active metabolite initiates the Omeprazole), Ritonavir, pharmacological activity. This func- tion is used in designing pro drugs. Pro drugs are defined as therapeutic agents that are inactive but are trans- F4 Toxicity formed into the active form by enzy- End results of drug Toxic Metabolite matic reactions. This is very useful metabolism Altered Activity when the active form is unstable or Active Metabolite poorly water soluble, making the Enhanced DRUG Activity formulation a challenge. Following Inactive Metabolite Loss of oral administration, the hypotensive Activity drug maleate (Vasotec)un- Reversible Metabolite Prolonged dergoes ethyl ester hydrolysis to Activity form enalaprilate, which is the active drug has an alternate pathway. T2 tered to different individuals. Ge- drug. shows some inducers and inhibitors netic polymorphism of CYP450 en- Some drugs have very little of the CYP isozymes. zymes characterize the general therapeutic potential but form a more There is a wide variation in the population into three groups: pharmacologically active metabo- expression, activity and concentra- lite. For example codeine itself has tions of different isozymes among a) Extensive metabolizers (EM): very low analgesic activity. It forms individuals, species and ethnic normal population. , the more active form groups. The expression or the activ- b) Poor metabolizers (PM): Indi- when it is metabolized by CYP2D6. ity of these enzymes is influenced by viduals who inherit two inactive al- Poor metabolizers of CYP2D6 or pa- factors such as species specificity, leles (alternative forms of the gene) tients who are taking CYP2D6 in- genetic polymorphism, gender- hor- showing complete absence of en- hibitors, therefore, do not experience monal control, age, disease and envi- zyme activity. the analgesic property of codeine. ronmental inducers (caffeine, c) Ultra extensive metabolizers In some cases the metabolite ex- cigarette smoke). The variability as- (UEM): Individuals with one com- hibits the same pharmacological ac- sociated with the CYP450 enzymes mon allele and one amplified allele tivity as the parent and is less toxic in each individual results in marked showing enhanced enzyme expres- than the parent. One such example is differences in response when the sion. the antihistamine drug same drug and the is adminis- (Allegra) which is a metabolite of www.currentseparations.com 20 terfenedine (Seldane). Seldane was trials later. Age, hormonal control traceuticals and herbal withdrawn from the market due to its (gender, pregnancy), genetic poly- like St. John’s Wort, Gingko Biloba, fatal interactions with erythromycin morphism, disease state, are all inter- the possibility increases consider- and ketoconazole in some patients nal factors that affect the ably for drug interactions to occur. when concomitantly administered. metabolism. for example, While there are many examples lack Phase II enzymes whereas eld- Influence of Drug Metabolism where both parent and the metabolite erly patients have diminished meta- on Drug Development have the same pharmacological ac- bolism and due to the tivity, some metabolites will show aging process. Although there is no In drug development it is important different pharmacological activity evidence of clinically relevant gen- to have information on the enzymes from the parent. This may lead to the der differences in metabolism of hu- responsible for the metabolism of a discovery of a new drug. is mans, there have been studies drug candidate as early as possible in an antipsychotic drug that undergoes showing the effect of rat sex hor- the design phase. Knowledge of the extensive metabolism. The N-de- mones on bioavailability. Liver dis- metabolic pathways, metabolite sta- methylated metabolite, eases such as hepatitis, liver , bility, toxicity and the specific however has anti depressant activity or cirrhosis impair drug metabolism isozymes involved in the metabolism and is prescribed for that indication. either due to the decreased number are all important information in the Metabolism can also result in of functional hepatocytes or to the drug development process and in toxic metabolites. Formation of re- altered NADPH/NAD ratio in the planning human clinical studies. The active metabolic intermediates is one liver. If the drug is cleared only by rate of metabolism affects the oral of the causes for drug toxicity. Oxi- the liver the impaired metabolism bioavailability and in hu- dation to electrophilic intermediates can result in drug overdose. mans and preclinical species. As dis- or reduction to nucleophilic radicals Genetic or hereditary factors are cussed before, polymorphic that can attack DNA or RNA and the most significant factor in drug enzymes will lead to high interindi- induce carcinogenicity are two ma- metabolism (10). Genetic differ- vidual variability and potential for jor reactions by which toxicity is ex- ences among individuals or ethnic DDIs. Genetic information is used to erted. Although many leads are groups can lead to an excessive or predict the response of individual pa- abandoned early on in drug discov- prolonged therapeutic effect or toxic tients and patient populations to ery stage due to the toxic metabolite overdose. For example, the enzyme drugs and to tailor drug selection and formation, presence of a toxic meta- CYP2D6 which metabolizes a large dosage to fit the individual’s genetic bolite does not always implies toxic- number of drugs has 16 alleles. The constitution. Metabolite profiles are ity in a given drug candidate since activity of this enzyme varies widely important for designing there are other factors that can make among ethnic groups (11). About 1% and pharmacologically active meta- the metabolite toxic or non-toxic. of Arabics, 30% Chinese and 7-10% bolites and for selecting the right Presence of a toxic metabolite how- Caucasians are poor metabolizers of animal species for stud- ever raises a red flag, which must be CYP2D6 drugs. Another example is ies. Structural modification of the extensively examined in animal tox- CYP2C19, which contributes to the drug candidate can alter the metabo- icity studies. metabolism of anxiolytics (e.g. di- lism. Highly hydrophilic or highly Some drugs are metabolized re- azepam). About 14-22% Asians and lipophilic compounds are not suit- versibly. For example, , a 3-6% Caucasians are poor metabo- able because they result in poor nonsteroidal anti-inflammatory drug lizers of CYP2C19. Elevated plasma bioavailability and very slow or very is reversibly metabolized to sulindac drug levels in these populations after fast excretion rates. In these in- sulphide which has anti-inflamma- drug administration can increase the stances replacing an active group tory and analgesic properties and is sedative effect of the drug. with another non-reactive group in irreversibly metabolized to sulindac Environmental factors such as the compound can achieve greater sulphone which has been suggested diet, smoking, consumption metabolic stability. For example, re- to possess antiproliferative effects and concomitant drug therapy also placing a methyl group by a t-butyl against tumors (9). influence the outcome of drug meta- group can prevent demethylation. bolism. Cigarette smoke produces Similarly, oxidation of aromatic Factors Affecting Drug poly aromatic hydrocarbons (PAH) rings can be prevented by substitut- Metabolism which induce CYP1A2 (12). ing them with stronger electron with- CYP1A2 metabolizes the PAHs to drawing groups (e.g. CF3). There are marked differences in drug carcinogens responsible for lung and Information obtained from pre-clini- metabolism across species. Select- colon cancer. Grapefruit juice is a cal drug metabolism studies can be ing a species that closely represents good example of dietary constituent fed back to the design team to intro- the human is very crucial in drug that inhibits CYP3A4 (13). With the duce functional groups which will discovery and in designing clinical new boom in consumption of neu- alter the physical properties to make

21 Current Separations 19:1 (2000) T3 tify the metabolites and the sites at Microsomal incubations are most Information that can be In vitro studies can which metabolism occur. A variety often used to obtain information on obtained from in vitro give information on: studies. of hepatic in vitro systems differing Phase I reactions. One disadvantage • Metabolite stability in biological intricacy are now com- is that the information is not com- • Metabolite profile mercially available for metabolism plete as from the cellular systems. • Metabolite identification studies. Most widely used systems • Interspecies comparisons are discussed below in detail. Isolated Hepatocytes • Toxicology species selection • CYP induction/inhibition Expressed Enzymes Cell cultures or cell suspensions can • Drug/ studies • CYP isoform identification be used to study multiple aspects of • Phase II enzyme studies Advances in molecular biology have drug metabolism, drug transport enabled the identification and char- across cell membranes, cytotoxicity the compounds more metabolically acterization of a large number of in- and enzyme induction in an environ- stable. Thus final selection of a suc- dividual CYP genes. Specific cDNA ment where enzymes and co-factors cessful drug lead depends im- sequences for particular CYP are present in normal physiological mensely on the drug metabolism isozymes have been cloned and ex- concentrations and cellular integrity studies. pressed heterologously. These ex- is maintained. Hepatocytes are used pressed enzymes including human to study both Phase I and Phase II Pre-clinical Drug enzymes are now commercially reactions. Cells can be either primary Metabolism Studies available as pure systems. Since the or permanent cell cultures. Primary conditions of reactions such as con- cell lines are most often used for drug It is important to know how the drug centrations of enzyme, substrate and metabolism studies because perma- is eliminated early in the drug devel- co-factor can be carefully controlled, nent cell lines possess very little or opment process. If elimination is enzyme systems have become a no enzyme activity. Primary cells are mainly by metabolism, then the me- powerful tool to study drug metabo- isolated from fresh liver tissue and tabolic pathways and products need lism. This system is very useful in the can be used immediately after isola- to be understood. Knowing the tox- study of kinetics, specificity and the tion or culture for long-term studies. icity of a drug and its metabolites mechanism of the enzyme reaction. However, cultured or cryopreserved before entering human clinical trials However, folding or the posttransla- cells lose the P450 activity rapidly is essential to avoid failures later on tional modifications and enzyme ac- with time (14). Also, hepatocytes in the process. Both in vitro and ani- tivity of the expressed enzyme may cannot be frozen and thawed or be mal in vivo studies are done in the differ from the native enzyme. prepared from previously frozen pre-clinical stage. liver. Therefore, there is a great need Most promising compounds are Microsomes for research in improving cryopre- selected from in vitro studies and servation technology and stabiliza- their pharmacokinetic parameters Microsomes can be prepared easily tion of P450 activity in primary are obtained in two animal species, from frozen liver tissues. They con- cultures. With the increased avail- commonly in rat and dog in the in tain most of the oxidative drug me- ability of fresh human tissues from vivo animal studies. This article will tabolizing enzymes. Their easy various commercial and non-profit focus on the in vitro studies and in- preparation and good long-term sta- institutions, human hepatocytes formation obtained from them. bility at -80 °C make microsomes the have become the most widely used most frequently used in vitro system and preferred in vitro system. In Vitro Studies in drug metabolism studies. Mi- crosomes are isolated from livercells Tissue Slices The in vitro studies during pre-clini- by disrupting the cellular contents cal screening are low-throughput and centrifugation at 100,000 ✕ g. While tissue slices have been used systems. Primary in vitro metabolic Liver microsomes can be manipu- from other organs like , , systems used in drug metabolism in- lated by induction and inhibition to and , liver is the most com- volve hepatic enzymes or tissue vary the activity or the levels of the monly used tissue type for drug me- preparations. Information (T3)ob- isozymes. The ability to phenotype tabolism experiments. Tissue slices tained by incubating a test drug with microsomes greatly increases the have certain advantages over other these systems can be used as feed- utility of this system in the identifi- systems. With intact cell-cell junc- back to design safer and more meta- cation of specific isozymes responsi- tions, normal hepatic cellular archi- bolically stable drugs. Compounds ble. Metabolic information such as tecture is retained in the tissue. Since can be ranked according to the meta- metabolic profiles, stability, metabo- they contain the complete comple- bolic stabilities. lite identification and kinetics can be ment of drug metabolizing enzymes is used as a qualitative tool to iden- obtained from microsomal systems. with all the cofactors present in rele- www.currentseparations.com 22 T4 6. H. Shih, G. V.Pickwell, D. K. Guenette, B. Bilir, L. C. Quattrochi, Comparison of System Advantages Disadvantages Future Needs Hum. Exp. Toxicol . 18 (1999) in vitro systems. 95-105 Expressed Pure system Single system Integration with other 7. O. V.Olesen, K. Linnet, J Clin Enzymes enzyme systems Psychopharmacol 20 (2000) 35-42. Microsomes Well-used, Long Limited information, 8. Y.Masubuchi, S. Hosokawa, T. term storage at Need cofactors Horie, T.Suzuki, S. Ohmori, M. -80°C,Well- Kitada, and S. Narimatsu, Drug characterized Metab. Disposition, 22 (1994) 909-915. Isolated cells Integrated cellular Short life time, Increased availability 9. N. M. Davies, M. S. Watson Clin. (e.g. Hepatocytes) system Limited enzyme of human cells, Pharmacokinet. 32 (1997) 437-459 stability Better preservation 10. J. van der Weide, L. S. Steijns Ann Slices Easy to prepare, Limited medium Greater availability Clin Biochem 36 (1999) 722-729. Cellular integrity penetration, of human tissues, 11. H. K. Kroemer, M. Eichelbaum Life maintained Short-term viability Cryopreservation Sciences, 26 (1995) 2285-2298. 12. S. Zevin, N. L. Benowitz Clin Pharmacokinet 36 (1999) 425-438. vant concentrations, complete infor- FDA guidelines suggest first using in 13. D. G. Bailey, J. Malcolm, O. Arnold, mation on the metabolism reactions vitro studies to assess the effect of J. D. Spence Br J Clin Pharmacol can be obtained. Liver slices can be drugs on metabolic pathways and if 46 (1998) 101-110. easily and rapidly produced. In addi- the results indicate possible DDIs, to 14. J. G. Hengstler, D. Utesch, P. Steinberg, K. L .Platt, B. Diener, M. tion, liver slices are not exposed to follow up with in vivo assays (15). Ringel, N. Swales, T.Fischer, K. proteolytic enzymes that can destroy The eventual goal of the in vitro Biefang, M. Gerl, T.Bottger, F. important membrane receptors of studies is to predict the in vivo out- Oesch Drug Metab Rev 32 (2000) 81-118. the cell. Although liver slices are in- come in humans. Utility of human in 15. Guidance for Industry, In Vivo creasingly used now in drug metabo- vitro models to predict drug-drug in- Drug Metabolism/Drug Interaction lism studies, they have certain teraction potential and pharmacoki- Studies -Study Design, Data disadvantages. One drawback is the netic variability has been Analysis, and Recommendations for Dosing and Labeling, Food and inadequate penetration of the me- demonstrated successfully for the Drug Administration, November dium. Liver slices cannot be cryopre- anti psychotic drug, olanzapine (16). 1999. served and they have a limited useful Methodology of scaling in vitro data 16. S. A. Wrighton, B. J. Ring Drug experimental period. to predict in vivo outcome, or in vi- Metab. Rev. 31 (1999) 15-28. Some advantages and limitations tro-in vivo correlation, is expanding 17. J. B. Houston Biochemical 47 (1994) of these in vitro systems are summa- due to the increasing availability of 1469-1479. rized in T4. human in vitro systems (17). Part II With greater availability in hu- of this article will discuss the signifi- man tissues and recombinant en- cance of in vitro zymes it is now possible to predict data in the evaluation of in vivo phar- potential DDIs before clinical trials. macokinetic data. Identifying the major metabolic pathways of the drug and its metabo- References lites and exploring the effect of the test drug on the metabolism of other 1. N. Watari, Y.Sugiyama, N. Kaneniwa, M. Hiura. J. drugs and vice versa are two major Pharmacokinetics and goals of the in vitro studies. In vitro Biopharmaceutics, 16 (1988) studies also could help to decide that 279-301. a particular drug is not a substrate for 2. S. A. Wrighton, B. J. Ring, M. VandenBranden, Toxicol. Pathol. 23 certain pathways. This reduces or (1995) 199-208. eliminates the need to study the pos- 3. P.J. Murphy, Ed, Year 2000 sible inhibitory effects of that drug Calendar on History of Drug on other drugs metabolized by that Metabolism, Bioanalytical Systems and ISSX, W. Lafayette, 1999. pathway. 4. M. Spatzenegger, W. Jaeger, Drug Despite the progress in the in Metab. Rev. 27 (1995) 397-417. vitro assays, the general consensus is 5. D. W. Nebert, M. Adenisk, M. J. that in vitro models are too simplistic Coon, R. W. Estabrook, F.J. to substitute for in vivo studies. In Gonzalez, F.P.Guengerich, I. C. Gunsalus, E. F.Johnson, B. vitro studies are best suited to deter- Kemper, W. Levin, I. R. Phillips, R. mine the types of clinical trials Sato, M. R. Waterman, DNA 6 needed to assess potential DDIs. (1987) 1-11

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