USOO8158362B2

(12) United States Patent (10) Patent No.: US 8,158,362 B2 Helgadottir et al. (45) Date of Patent: Apr. 17, 2012

(54) METHODS OF DIAGNOSING 4,448,784. A 5/1984 Glamkowski et al. SUSCEPTIBILITY TO MYOCARDAL 4.450,171 A 5/1984 Hoffman et al. 4,499,289 A 2f1985 Baran et al. NFARCTION AND SCREENING FORAN 4,613,610 A 9/1986 Wareing LTA4H HAPLOTYPE 4,647,576 A 3, 1987 Hoefleet al. 4,681,893 A 7, 1987 Roth (75) Inventors: Anna Helgadottir, Reykjavík (IS); 4,683.202 A 7, 1987 Mullis Hákon Hákonarson, Reykjavík (IS); 4,686,237 A 8, 1987 Anderson Jeffrey R. Gulcher, Lake Barrington, IL 4,736,866 A 4, 1988 Leder et al. (US); Mark E. Gurney, Grand Rapids, 4,870,009 A 9, 1989 Evans et al. MI (US) (Continued) (73) Assignee: deCODE Genetics ehf. Reykjavik (IS) FOREIGN PATENT DOCUMENTS CA 2337571 8, 2002 (*) Notice: Subject to any disclaimer, the term of this (Continued) patent is extended or adjusted under 35 U.S.C. 154(b) by 1395 days. OTHER PUBLICATIONS (21) Appl. No.: 11/270,804 Ioannidis, J. P.A. PLoS Medicine 2(8):696-701 (Aug. 2005).* (Continued) (22) Filed: Nov. 9, 2005 Primary Examiner — Diana B Johannsen (65) Prior Publication Data (74) Attorney, Agent, or Firm — Marshall, Gerstein & Borun US 2007/O280917 A1 Dec. 6, 2007 LLP Related U.S. Application Data (57) ABSTRACT (63) Continuation-in-part of application No. 1 1/096, 191, Polymorphisms in the FLAP and LTA4H gene are shown by filed on Mar. 30, 2005. genetic association analysis to be susceptibility markers for myocardial infarction (MI) and ACS, as well as stroke and (51) Int. Cl. PAOD. Pathway targeting for treatment and diagnostic appli CI2O I/68 (2006.01) cations in identifying those who are at risk of developing MI, CO7H21/04 (2006.01) ACS, stroke or PAOD, in particular are described. The inven (52) U.S. Cl...... 435/6.18:536/23.2:536/23.5 tion also provides methods of prophylaxis therapy for MI in (58) Field of Classification Search ...... None human Subjects having a race including black African ances See application file for complete search history. try by administering to the Subject a composition comprising a therapeutically effective amount of MI therapeutic agent (56) References Cited that inhibits synthesis in vivo. The invention also provides for compositions comprising a leukotriene synthesis U.S. PATENT DOCUMENTS inhibitor and a statin and methods of using these composi 3,983,140 A 9, 1976 Endo et al. tions to reduce C-reactive protein in a human Subject at risk of 4,231,938 A 11/1980 Monaghan et al. MI, ACS, stroke and/or PAOD. 4,346,227 A 8, 1982 Terahara et al. 4,376, 110 A 3, 1983 David et al. 1 Claim, 11 Drawing Sheets

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Crosslin et al., Genetic effects in the leukotriene biosynthesis path- Genbank Accession No. Z52271, Homo sapiens (D13S1238) DNA way and association with atherosclerosis. Hum. Genet. 125:217-229 segment containing (CA) repeat; clone AFMal42Zb5; single read, (2009). sequence tagged site, Sep. 9, 2004. Genbank Accession No. Z24370, Homo sapiens (D13S289) DNA segment containing (CA) repeat; clone AFM321xbl; single read, Nov. 28, 1994. * cited by examiner U.S. Patent US 8,158,362 B2

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US 8,158,362 B2 1. 2 METHODS OF DAGNOSING medical risks and Susceptibilities. This may especially be true SUSCEPTIBILITY TO MYOCARDAL in the United States and other countries that are apt to have INEARCTION AND SCREENING FOR AN people of mixed ancestry. LTA4H HAPLOTYPE Atherosclerosis can affect vascular beds in many large and medium arteries. Myocardial infarction and unstable angina This application is a continuation-in-part of U.S. patent (acute coronary syndrome (ACS)) stem from coronary artery application Ser. No. 11/096,191, filed Mar. 30, 2005 (now atherosclerosis, while ischemic stroke most frequently is a abandoned), which is incorporated herein by reference in its consequence of carotid or cerebral artery atherosclerosis. entirety. Limb ischemia caused by peripheral arterial occlusive disease The file copy of the sequence listing is Submitted on a 10 (PAOD) may occur as a consequence of iliac, femoral and Compact-Disc Read Only Memory (CD-ROM). Three copies popliteal artery atherosclerosis. The atherosclerotic diseases of the CD-ROM are submitted (denoted as Copy 1, Copy 2 remain common despite the wide-spread use of medications and CRF) and the content of the 3 submitted CD-ROMs are that inhibit thrombosis (aspirin) or treat medical risk factors identical. The sequence listing is saved on each CD-ROM as such as elevated cholesterol levels in blood (statins), diabetes, an ASCII text file named 40807.txt (571 kB), which was 15 or hypertension (diuretics and anti-hypertensives). created on Nov. 8, 2005. The contents of each CD-ROM are Atherosclerotic disease is initiated by the accumulation of incorporated herein by reference in its entirety. lipids within the artery wall, and in particular, the accumula tion of low-density lipoprotein (LDL) cholesterol. The BACKGROUND OF THE INVENTION trapped LDL becomes oxidized and internalized by macroph ages. This causes the formation of atherosclerotic lesions Myocardial infarction (MI) and Acute Coronary Syndrome containing accumulations of cholesterol-engorged macroph (ACS), e.g., unstable angina, non-ST-elevation myocardial ages, referred to as “foam cells'. As disease progresses, infarction (NSTEMI) or ST-elevation myocardial infarction smooth muscle cells proliferate and grow into the artery wall (STEMI), are the leading causes of hospital admissions in forming a “fibrous cap' of extracellular matrix enclosing a industrialized countries. Cardiovascular disease continues to 25 lipid-rich, necrotic core. Present in the arterial walls of most be the principle cause of death in the United States, Europe people throughout their lifetimes, fibrous atherosclerotic and Japan. The costs of the disease are high both in terms of plaques are relatively stable. Such fibrous lesions cause morbidity and mortality, as well as in terms of the financial extensive remodeling of the arterial wall, outwardly displac burden on health care systems. ing the external, elastic membrane, without reduction in lumi Myocardial infarction generally occurs when there is an 30 nal diameter or serious impact on delivery of oxygen to the abrupt decrease in coronary blood flow following a throm heart. Accordingly, patients can develop large, fibrous athero botic occlusion of a coronary artery previously damaged by sclerotic lesions without luminal narrowing until late in the atherosclerosis. In most cases, infarction occurs when an disease process. However, the coronary arterial lumen can atherosclerotic plaque fissures, ruptures or ulcerates and become gradually narrowed over time and in some cases when conditions favor thrombogenesis. In rare cases, infarc 35 compromise blood flow to the heart, especially under high tion may be due to coronary artery occlusion caused by coro demand States Such as exercise. This can result in reversible nary emboli, congenital abnormalities, coronary spasm, and a ischemia causing chest pain relieved by rest called stable wide variety of systemic, particularly inflammatory diseases. angina. Medical risk factors for MI include cigarette smoking, diabe In contrast to the relative stability offibrous atherosclerotic tes, hypertension and serum total cholesterol levels >200 40 lesions, the culprit lesions associated with myocardial infarc mg/dL, elevated serum LDL cholesterol, and low serum HDL tion and unstable angina (each of which are part of the acute cholesterol. Event rates in individuals without a prior history coronary syndrome) are characterized by a thin fibrous cap, a of cardiovascular disease are about 1%. In individuals who large lipid core, and infiltration of inflammatory cells Such as have had a first MI or ACS, the risk of a repeat MI within the T-lymphocytes and monocyte/. Non-invasive next year is 10-14%, despite maximal medical management 45 imaging techniques have shown that most MI's occurat sites including angioplasty and stent placement. with low- or intermediate-grade Stenoses, indicating that Cardiovascular disease is one of the primary causes of coronary artery occlusion is due most frequently to rupture of death among African Americans and over 4 in every 10 non culprit lesions with consequent formation of a thrombus or Hispanic black adults have CVD, including myocardial inf blood clot and not solely due to luminal narrowing by Steno arction, stroke and congestive heart failure (American Heart 50 sis. Plaque rupture may be due to erosion or uneven thinning Association, 2005 statistics). Race-based therapeutics are of the fibrous cap, usually at the margins of the lesion where emerging to treat populations of human patients that are at a macrophages enter, accumulate, and become activated by a high risk based on their race. For example, BiDil (isosorbide local inflammatory process. Thinning of the fibrous cap may dinitrate hydralazine hydrochloride) is currently approved for result from degradation of the extracellular matrix by pro treatment of heart failure in self-identified black patients to 55 teases released from activated macrophages. These changes improve Survival, to prolong time to hospitalization for heart producing plaque instability and risk of MI may be aug failure, and to improve patient-reported functional status. In mented by production of tissue-factor procoagulant and other addition, U.S. Pat. No. 6,465,463 purports to describe meth factors increasing the likelihood of thrombosis. ods of reducing mortality associated with heart failure in In acute coronary syndrome, the culprit lesion showing black patients comprising administering to these patients 60 rupture or erosion with local thrombosis typically is treated hydralazine compounds in combination with isosorbide dini by angioplasty or by balloon dilation and placement of a stent trate or isosorbide mononitrate. to maintain luminal patency. Patients experiencing ACS are at Although self-reporting is an effective way to identify race, high risk for a second coronary event due to the multi-vessel ethnic ancestry and other family and medical history infor nature of coronary artery disease with event rates approach mation that may be relevant to medical diagnosis and treat 65 ing 10-14% within 12 months after the first incident. ment, it is imperfect. A need exists for improved tools, espe The emerging view of MI is as an inflammatory disease of cially genetic tools, to more reliably correlate patients with the arterial vessel wall on preexisting chronic atherosclerotic US 8,158,362 B2 3 4 lesions, sometimes triggering rupture of culprit lesions and patients with such a genetic alteration, inhibition of the Bcr leading to local thrombosis and Subsequent myocardial inf Abl kinase leads to rapid elimination of the tumor cells and arction. The process that triggers and Sustains arterial wall remission from leukemia. inflammation leading to plaque instability is unknown, how Many general inflammatory markers predict risk of coro ever, it results in the release into the circulation of tumor 5 nary heart disease, although these markers are not specific to necrosis factor alpha and interleukin-6. These and other atherosclerosis. For example, Stein (Stein, S. Am J Cardiol, cytokines or biological mediators released from the damaged 87 (suppl):21A-26A (2001)) discusses the use of any one of vessel wall stimulate an inflammatory response in the liver the following serum inflammatory markers as Surrogates for causing elevation in several non-specific general inflamma predicting risk of coronary heart disease including C-reactive tory markers including C-reactive protein. Although not spe 10 protein (CRP), serum amyloid A, fibrinogen, interleukin-6, cific to atherosclerosis, elevated C-reactive protein (CRP) and tissue necrosis factor-alpha, Soluble vascular cell adhesion serum amyloid A appear to predict risk for MI, perhaps as molecules (sVCAM), soluble intervascular adhesion mol Surrogates for vessel wall inflammation. ecules (sICAM), E-selectin, matrix metalloprotease type-1, Although classical risk factors such as Smoking, hyperlipi 15 matrix metalloprotease type-2, matrix metalloprotease type demia, hypertension, and diabetes are associated with many 3, and matrix metalloprotease type-9. Elevation in one more cases of coronary heart disease (CHD) and MI, many patients of these serum inflammatory markers is not specific to coro do not have involvement of these risk factors. In fact, many nary heart disease but also occurs with age or in association patients who exhibit one or more of these risk factors do not with cerebrovascular disease, peripheral vascular disease, develop MI. Family history has long been recognized as one non-insulin dependent diabetes, osteoarthritis, bacterial of the major risk factors. Although some of the familial clus infection, and sepsis. tering of MI reflects the genetic contribution to the other Serum C-reactive protein (CRP) is viewed as a convenient conventional risk factors, a large number of studies have and sensitive marker of systemic inflammation. Generally Suggested that there are significant genetic susceptibility fac CRP is measured in serum samples using commercially avail tors, beyond those of the known risk factors (FriedlanderY, et 25 able enzyme-linked immunosorbent assays (EIA). Consistent al., Br: Heart J. 1985; 53:382-7, Shea S. et al., J. Am. Coll. across multiple published studies is the finding of a correla Cardiol. 1984; 4:793-801, and Hopkins P. N., et al., Am. J. tion between increased risk for coronary artery disease with Cardiol. 1988: 62:703-7). Major genetic susceptibility fac increased serum CRP. For example, in the Women's Health tors have only been identified for the rare Mendelian forms of Study, CRP was measured in 27.939 apparently healthy hyperlipidemia Such as a familial hypercholesterolemia. 30 American women. The cut-off points for quintiles of serum Genetic risk is conferred by subtle differences in genes CRP in women were: less than or equal to 0.49, more than among individuals in a population. Genes differ between 0.49 to 1.08, more than 1.08 to 2.09, more than 2.09 to 4.19, individuals most frequently due to single nucleotide polymor and more than 4.19 mg CRP per liter, see Ridker, P. M. et al., phisms (SNP), although other variations are also important. New England. J. Med., 347: 1557-1565 (2001). In compari SNP are located on average every 1000 base pairs in the 35 son to the lowest quintile, and even when adjusting for age, human genome. Accordingly, a typical human gene contain every quintile more than 0.49 mg CRP per liter was associated ing 250,000 base pairs may contain 250 different SNP. Only with increased risk for coronary heart disease with the highest a minor number of SNP are located in exons and alter the relative risk of 4.5 seen for those women in the highest quin amino acid sequence of the protein encoded by the gene. Most tile of serum CRP (more than 4.19 mg CRP per liter). A SNP have no effect on gene function, while others may alter 40 similar correlation between increased serum CRP and transcription, splicing, translation, or stability of the mRNA increased risk for coronary heart disease in women has been encoded by the gene. Additional genetic polymorphism in the reported (Ridker, P. M. et al., New Engld. J. Med., 342:836 human genome is caused by insertion, deletion, translocation, 843 (2000) and Bermudez, E. A. et. al., Arterioscler: Thromb. or inversion of either short or long stretches of DNA. Genetic Vasc. Biol., 22: 1668-1673 (2002)). Men also show a corre polymorphisms conferring disease risk may therefore 45 lation between increased serum inflammatory markers such directly alter the amino acid sequence of proteins, may as CR and increased risk for coronary heart disease has been increase the amount of protein produced from the gene, or reported (Doggen, C. J. M. et al., J. Internal Med., 248:406 may decrease the amount of protein produced by the gene. 414 (2000) and Ridker, P. M. et al., New England. J. Med., As genetic polymorphisms conferring risk of disease are 336:973-979 (1997)). Quintiles for serum CRP as reported uncovered, genetic testing for Such risk factors is becoming 50 by Doggen et al., were less than 0.65, more than 0.65 to 1.18, important for clinical medicine. Examples are apolipoprotein more than 1.18 to 2.07, more than 2.07 to 4.23, and more than E testing to identify genetic carriers of the apoE4 polymor 4.23 mg CRP per liter. Unlike women, elevated serum CRP phism in dementia patients for the differential diagnosis of correlates with increased relative risk for coronary heart dis Alzheimer's disease, and of Factor V Leiden testing for pre ease only in the 4' and 5' quintiles of CRP (relative risk of disposition to deep venous thrombosis. More importantly, in 55 1.7x and 1.9x, respectively). the treatment of cancer, diagnosis of genetic variants in tumor Serum CRP in women also has been measured in conjunc cells is used for the selection of the most appropriate treat tion with lipid markers such as levels of serum low density ment regime for the individual patient. In breast cancer, lipoprotein-cholesterol (LDL-C). In the study by Ridker, P. genetic variation in estrogen receptor expression or heregulin M. et al. (2002), serum CRP and LDL-C are minimally cor type 2 (Her2) receptor tyrosine kinase expression determine 60 related, screening for both serum markers provided better if anti-estrogenic drugs (tamoxifen) or anti-Her2 antibody prognostic indication than either alone. Thus, women with (Herceptin) will be incorporated into the treatment plan. In serum CRP above median values (more than 1.52 mg CRP per chronic myeloid leukemia (CML) diagnosis of the Philadel liter) and also serum LDL-C above median values (more than phia chromosome genetic translocation fusing the genes 123.7 mg LDL-C per deciliter) were at highest risk for coro encoding the Bcrand Abl receptor tyrosine kinases indicates 65 nary heart disease. that Gleevec (STI571), a specific inhibitor of the Bcr-Abl Elevated CRP or other serum inflammatory markers is also kinase should be used for treatment of the cancer. For CML prognostic for increased risk of a second myocardial infarctin US 8,158,362 B2 5 6 patients with a previous myocardial infarct (Retterstol, L. et types, etc. that are assayed. Numerous FLAP and LTA4H al., Atheroscler, 160: 433-440 (2002)). embodiments are described below in greater detail. Since CRP is produced in the liver, there is no a priori The invention also pertains to methods of treatment (pro mechanistic explanation for why elevation in CRP and other phylactic and/or therapeutic) for certain diseases and condi serum inflammatory markers should be prognostic for coro tions (e.g., MI, ACS, atherosclerosis, stroke, PAOD) associ nary artery disease. As discussed by Doggen, C.J. M., et al., ated with FLAP or with other members of the leukotriene one or more of the following factors were speculated to pathway (e.g., biosynthetic enzymes or proteins such as account for the correlation observed: (1) intrinsic inflamma FLAP arachidonate 4-lipoxygenase (5-LO), leukotriene C4 tion and tissue damage within arterial lesions, (2) prior infec synthase (LTC4S), leukotriene A4 hydrolase (LTA4H), leu 10 kotriene B4 12-hydroxydehydrogenase (LTB4DH)); recep tion by Helicobacter pylori or by Chlamydia pneumoniae, (3) tors and/or binding agents of the enzymes; and receptors for release of peptide cytokines including interleukin-6, or (4) the leukotrienes LTA4, LTB4, LTC4, LTD4, LTE4, Cys LT1, activation of the complement system. Cys LT2, including leukotriene B4 receptor 1 (BLT1), leu The end products of the leukotriene pathway are potent kotriene B4 receptor 2 (BLT2), cysteinyl leukotriene receptor inflammatory lipid mediators derived from arachidonic acid. 15 1 (CysLTR1), cysteinyl leukotriene receptor 2 (CysLTR2). They can potentially contribute to development ofatheroscle The methods include the following: methods of treatment for rosis and destabilization of atherosclerotic plaques through myocardial infarction or Susceptibility to myocardial infarc lipid oxidation and/or proinflammatory effects. LTC4, LTD4. tion; methods of phophylaxis therapy for myocardial infarc and LTE4, are known to induce vasoconstriction. Allen et al., tion; methods of treatment for transientischemic attack, tran Circulation, 97:2406-2413 (1998) described a novel mecha sient monocular blindness or stroke, or Susceptibility to nism in which atherosclerosis is associated with the appear stroke; methods of treatment for claudication, PAOD or sus ance of a leukotriene receptor(s) capable of inducing hyper ceptibility to PAOD; methods of treatment for acute coronary activity of human epicardial coronary arteries in response to syndrome (e.g., unstable angina, non-ST-elevation myocar LTC4 and LTD4. LTB4, on the other hand, is a strong proin dial infarction (NSTEMI) or ST-elevation myocardial infarc flammatory agent. Increased production of these end prod 25 tion (STEMI)); methods for reducing risk of MI, stroke or ucts, of the leukotriene pathway, could therefore serve as a PAOD in persons with asymptomatic ankle/brachial index risk factor for MI and atherosclerosis, whereas both inflam less than 0.9; methods for decreasing risk of a second myo mation and vasoconstriction/vasospasm have a well estab cardial infarction or stroke; methods of treatment for athero lished role in the pathogenesis of MI and atherosclerosis. It Sclerosis, such as for patients requiring treatment (e.g., angio has also been shown that a heterozygous deficiency of the 30 plasty, Stents, revascularization procedure) to restore blood 5-LO enzyme in a knockout mouse model decreases athero flow in arteries (e.g., coronary, carotid, and/or femoral arter scleroticlesion size in LDLR-/- mice by about 95%. (Mehra ies); methods of treatment for asymptomatic ankle?brachial bian et al., Circulation Research. 91: 120 (2002)). However, index of less than 0.9; and/or methods for decreasing leukot such genetic evidence for leukotriene involvement in MI or riene synthesis (e.g., for treatment of myocardial infarction, atherosclerosis in humans has not been reported. Mehrabian 35 stroke or PAOD). et al. did report a very Small genetic association study looking The invention provides for methods of prophylaxis therapy for correlation between promoter polymorphisms of 5-LO for myocardial infarction (MI). These methods comprise and carotid intimal thickening in normal individuals (Dwyer selecting a human Subject Susceptible to MI, administering to et al. New England Journal of Medicine, 350: 29-37, 2004). the Subject a composition comprising a therapeutically effec However, their data paradoxically suggest that a lower 40 tive amount of an MI therapeutic agent that inhibits leukot amount of leukotriene production correlates with carotid ath riene synthesis in vivo, wherein the MI therapeutic agent erosclerosis. inhibits leukotriene synthesis by inhibiting the activity of at least one protein selected from 5-Lipoxygenase activating SUMMARY OF THE INVENTION protein (FLAP) and 5-lipoxygenase (5-LO). The methods 45 also (optionally) comprise monitoring myeloperoxidase level As described herein, a gene on chromosome 13q12-13 has before and during the prophylaxis treatment, wherein the MI been identified as playing a major role in myocardial infarc therapeutic agent is administered in an amount effective to tion (MI). This gene, herein after referred to as the MI gene, reduce MPO levels in a subject. These methods may further comprises nucleic acid that encodes 5-lipoxygenase activat comprise monitoring at least one additional or alternative ing protein (ALOX5AP or FLAP) herein after referred to as 50 inflammatory marker, Such as C-reactive protein, in the FLAP. The gene has also been shown to play a role in stroke human Subject before and during the prophylaxis therapy. and PAOD. In addition, a gene on chromosome 12q23 has In some methods of the invention, a leukotriene synthesis been identified as playing a major role in myocardial infarc inhibitor is administered to an individual in a therapeutically tion (MI). The gene comprises nucleic acid that encodes effective amount. The leukotriene synthesis inhibitor can be leukotriene A4 hydrolase, herein after referred to as LTA4H. 55 an agent that inhibits or antagonizes a member of the leukot One aspect of the invention includes materials and methods riene synthesis pathway (e.g., FLAP, 5-LO, LTC4S, LTA4H, for identifying or diagnosing a person or screening for and LTB4DH). For example, the leukotriene synthesis inhibi increased risk (susceptibility) to MI or other conditions based tor can be an agent that inhibits or antagonizes FLAP on FLAP and/or LTA4H markers that can be assayed from polypeptide activity (e.g., a FLAP inhibitor) and/or FLAP their DNA or RNA. For example, in some embodiments, the 60 nucleic acid expression, as described herein (e.g., a FLAP invention is a method of screening a human Subject for Sus nucleic acid antagonist). In another embodiment, the leukot ceptibility or elevated risk for MI or other adverse cardiovas riene synthesis inhibitor is an agent that inhibits or antago cular events by assaying nucleic acid from the Subject for one nizes polypeptide activity and/or nucleic acid expression of or more polymorphisms, markers, haplotypes, alleles or another member of the leukotriene biosynthetic pathway genotypes, and assaying or predicting the presence or absence 65 (e.g., LTC4S, LTA4H) or that increases breakdown of leukot of the susceptibility or elevated risk from the presence or rienes (e.g., LTB4DH). In preferred embodiments, the agent absence or identity of the polymorphisms, markers, haplo alters activity and/or nucleic acid expression of FLAP or of US 8,158,362 B2 7 5-LO. Preferred agents include those set forth in the Agent human administration, and preferably further comprise a (at Table I herein. In another embodiment, preferred agents can least one) pharmaceutically acceptable diluent, adjuvant, be: 1-((4-chlorophenyl)methyl)-3-((1,1-dimethylethyl)thio)- excipient, or carrier. Materials and methods for formulation alpha,alpha-dimethyl-5-(2-quinolinylmethoxy)-1H-Indole and co-formulation are well known, and many are described herein in greater detail. In one variation, formulation of the 2-propanoic acid otherwise known as MK-0591, (R)-(+)- composition into convenient unit dose formulations, such as alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic pills or capsules for oral administration, including Sustained acid otherwise known as BAY-X-1005, 3-(3-(1,1-dimethyl release formulations, is specifically contemplated. In another ethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chlorometh variation, co-administration transdermally, e.g., through a ylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde oXime skin patch, is contemplated. In still another variation, admin 0-2-acetic acid otherwise known as A-81834, optically pure 10 istration of one or both agents through a drug eluting stent is enantiomers, salts, chemical derivatives, and analogues; or specifically contemplated. In particular, the compositions can be , atreleuton, 6-((3-fluoro-5-(tetrahydro-4- may comprises a leukotriene synthesis inhibitor that inhibits methoxy-2H-pyran-4-yl)phenoxy)methyl)-1-methyl-2(1H)- the activity of a member of the leukotriene synthesis pathway quinlollinone otherwise known as ZD-2138, 1-((4-chlorophe Such as 5-lipoxygenase, 5-lipoxygenase activating protein nyl)methyl)-3-((1,1dimethylethyl)thio)-alpha,alpha 15 (FLAP), leutokriene C4 synthase, leukriene A4 hydolase, dimethyl-5-(2-quinolinylmethoxy)-1H-Indole-2-propanoic arachidonate 4-lipoxygenase, leukotriene B4 12-hydroxyde acid otherwise known as MK-886, 4-(3-(4-(2-Methyl-imida hydrogenase, leukotriene A4 receptor, leukotriene B4 recep Zol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-car tor, leukotriene C4 receptor, leukotriene D4 receptor, leukot boxylic acid amide otherwise known as CJ-13610, their opti riene E4 receptor, leukotriene B4 receptor 1, leukotriene B4 cally pure enantiomers, salts, chemical derivatives, and receptor 2, cysteinyl leukotriene receptor 1 and cysteinyl analogues. In another embodiment, the agent alters metabo leukotriene receptor 2. Any LT inhibitor is suitable for prac lism or activity of a leukotriene (e.g., LTA4. LTB4, LTC4. tice of the invention, and several LT inhibitors are described LTD4, LTE4, Cys LT1, Cys LT2), such as leukotriene antago herein. To help minimize side effects, an LT inhibitor that is nists or antibodies to leukotrienes, as well as agents which specific for a member of the LT synthesis pathway is pre alter activity of a leukotriene receptor (e.g., BLT1, BLT2, 25 ferred. Exemplary inhibitors include both small molecules, CysLTR1, and CysLTR2). biological inhibitors of proteins, (e.g., antibody Substances, In other preferred embodiments, the agent alters activity peptides), and biological inhibitors that operate at the nucleic and/or nucleic acid expression of LTA4H. Preferred agents acid level (e.g., antisense nucleic acids and interfering RNA include those set forth in the Agent Table II and in the Addi nucleic acids and Zinc finger proteins). tional LTA4H Agent List herein. In another embodiment, 30 Preferred agents that inhibit the activity of a member of the preferred agents can be: ethyl-1-(2-4-(phenylmethyl)phe leukotriene pathway are listed in the Agent Table I herein, noxyethyl-4-piperidine-carboxylate, otherwise known as including the following agents: 1-((4-chlorophenyl)methyl)- SC-56938: 4-5-(3-Phenyl-propyl)thiophen-2-ylbutoxy 3-((1,1-dimethylethyl)thio)-alpha,alpha-dimethyl-5-( acetic acid, otherwise known as RP64966; (R)-S-4-(dim 2-quinolinylmethoxy)-1H-Indole-2-propanoic acid, (R)-(+)- ethylamino)phenyl)methyl-N-(3-mercapto-2methyl-1-oxo 35 alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic propyl-L-cycteine, otherwise known as SA6541; optically acid, 3-(3-(1,1-dimethylethylthio-5-(quinoline-2-yl pure enantiomers, salts, chemical derivatives, and analogues. methoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dim The results in Example 10 demonstrate that inpatients with ethylpropionaldehyde oXime-0-2-acetic acid, Zileuton, the at-risk FLAP and LTA haplotypes, a FLAP inhibitor atreleuton, 6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran (DG-031 also known as Bay-X-1005) had a highly significant 40 4-yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone, 1-((4- and dose-dependent effect at the cellular, whole bood and chlorophenyl)methyl)-3-((1.1 dimethylethyl)thio)-alpha,al urinary metabolite level including a 26% reduction in leukot pha-dimethyl-5-(2quinolinylmethoxy)-1H-Indole-2- riene B production by activated , a 13% reduction propanoic acid and 4-(3-(4-(2-Methyl-imidazol-1-yl)- of myeloperoxidase in whole blood, and a 27% increase in phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylic acid urinary leukotriene E. Furthermore, there was evidence of a 45 amide. In one variation, the LT inhibitor is an inhibitor of persistent effect, following discontinuation of the FLAP FLAP. One preferred group of compounds are described inhibitor, on high senstivity C-reactive protein and serum herein as BAY X 1005 (also known as DG-031) as well as amyloid A. This reduction in CRP and serum amyloid A was related compounds described in Mohrs et al., U.S. Pat. No. observed on top of the beneficial effects that may have been 4,970.215, incorporated herein by reference in its entirety. In acheived by statins taken by 85% of the study subjects. 50 another variation, the LT inhibitoris a LTA4H inhibitor. Other The invention includes compositions comprising a leukot preferred agents include those set forth in the Agent Table II riene synthesis inhbitor and a statin. The invention also and the LTA4H Agent list set out herein. Additional preferred includes the use of a leukotriene synthesis inhibitor and a agents include those described in Penning et al., Med Chem. statin for the manufacture of a medicament for reducing CRP 200245(16):3482-90, Penning, Curr Pharm Des. 2001, 7(3): levels in a human Subject. Such compositions are intended for 163-79 and Penning et al., J Med Chem. 2000 43(4):721-35. AGENT TABLE II

Target Compound ID Chemical Name Patent Reference

LTA4H SC-57461A 3-methyl 3-4- Penning, T. D. et al. Bioorg Med. Inhibitor (phenylmethyl)phenoxy Chem. Letters (2003), 13, 1137-1139. propylaminopropionic Penning, T. D. et al. Bioorg Med. acid Chem. Letters, (2002), 12,3383-3386 LTA4H SC-56938 Ethyl-1-(2-4- Penning, T. D. et al. Bioorg Med. Inhibitor (phenylmethyl)phenoxyethyl Chem. Letters (2003), 13, 1137-1139. 4-piperidine Penning, T. D. et al. Bioorg Med. US 8,158,362 B2 9 10 AGENT TABLE II-continued

Target Compound ID Chemical Name Patent Reference carboxylate Chem. Letters, (2002), 12,3383-3386. U.S. Pat. No. 6,506,876A1 LTA4H RP 64966 4-5-(3-Phenyl WO9627585 Inhibitor propyl)thiophen-2- ylbutoxyacetic acid LTA4H SA 6541 (R)-S-4- WO9809943 Inhibitor (dimethylamino)phenylmethyl N-(3-mercapto 2methyl-1-oxopropyl-L- cycteine LTA4H SA-94.99 (R)-3-(4- Inhibitor SA-6541 Dimethylamino benzylsulfanyl)-2-((R)- 3-mercapto-2-methyl propionylamino)- propionic acid LTB4 Amelubant Carbamic acid, (4-((3- U.S. Pat. No. 6,576,669 Receptor BIIL-284 ((4-(1-(4- Antagonist hydroxyphenyl)-1- methylethyl)phenoxy)methyl) phenyl) methoxy)phenyl)iminomethyl)- ethyl ester LTB4 BIRZ-227 5-Chloro-2-3-(4- ournal of Organic Chemistry Receptor methoxy-phenyl)-2- 998, 63: 2(326-330). Antagonist pyridin-2-yl-pyrrollidin 1-yl)-benzooxazole LTB4 CP 1955.43 2-((3S,4R)-3,4-dihydro Process: WO 98/11085 1998, Receptor 4-hydroxy-3- priority US 60/26372 1996; J. Antagonist (phenylmethyl)-2H-1- Pharamacology and Expert. benzopyran-7-yl)-4- Therapy, 1998, 285:946-54 (trifluoromethyl)benzoic acid LTB4 Ebselen 2-Phenyl ournal of Cerebral Blood Flow and Receptor benzodisoselenazol-3- Metabolism 1995, July 2-6 (S162): Antagonist Ole Drugs of the Future 1995, 20:10 (1057) LTB4 4-5-(4-Carbamimidoyl ACS Meeting 1994, 207th: San Receptor phenoxy)-pentyloxy Diego (MEDI 003); International Antagonist N,N-diisopropyl-3- Congress of the Inflammation methoxy-benzamide Research Association 1994, maleate 7th: White Haven (Abs W23) LTB4 LY 210073 5-(2-Carboxy-ethyl)-6- Med Chem 1993 36 (12) 1726-1734 Receptor 6-(4-methoxy-phenyl)- Antagonist hex-5-enyloxy)-9-oxo 9H-xanthene-2- carboxylic acid LTB4 LY 213024 5-(3-carboxybenzoyl)-2- J Med Chem 1993 36 (12) 1726-1734 Receptor (decyloxy)benzenepropanoic Antagonist acid LTB4 LY 255.283 -5-ethyl-2-hydroxy-4- EP 276.064 B 1990, priority US Receptor 6-methyl-6-(1H 2479 1987 Antagonist etrazol-5- yl)heptyloxy phenylethanone LTB4 LY 264086 7-carboxy-3-(decyloxy)- U.S. Pat. No. 4996230 1991, priority US Receptor 9-oxo-9H-xanthene-4- 481413 1990 Antagonist propanoic acid LTB4 LY 292728 7-carboxy-3-3-(5- EP 743064. A 1996, priority US Receptor ethyl-4'-fluoro-2- 44.3179 1995 Antagonist hydroxy(1,1'-biphenyl 4-yl)oxypropoxy-9- oxo-9H-xanthene-4- propanoic acid disodium sal LTB4 LY-293111 Benzoic acid.2-(3-(3-((5- Proceedings of the American Receptor (VML-295) ethyl-4'-fluoro-2- Society for Clinical Oncology Antagonist hydroxy(1,1'-biphenyl)- 2002, 21: 1 (Abs 343) LY-293111 4-yl)oxy)propoxy)-2- for Cancer propylphenoxy)- SCRIP World Pharmaceutical News 1997, 2272 (13) for VML-295) LTB4 ONO 4057; (E)-2-(4- EP4OS116 A 1991 Receptor LB 457 carboxybutoxy)-6-6- Antagonist (4-methoxyphenyl)-5- hexenyloxybenzenepropanoic acid LTB4 PF 10042 -(5-hydroxy-5-8-(1- EP 422329 B 1995, priority US Receptor hydroxy-2-phenylethyl)- 40963O 1989 US 8,158,362 B2 11 12 AGENT TABLE II-continued Target Compound ID Chemical Name Patent Reference Antagonist 2-dibenzofuranyl)-1- oXopentylpyrrollidine LTB4 RG-14893 8-Benzyloxy-4-(methyl SCRIP World Pharmaceutical News Receptor phenethyl-carbamoyl)- 1996, 2168 (20) Antagonist methyl-naphthalene-2- carboxylic acid LTB4 SB-201993 3-6-(2-Carboxy-vinyl)- WO-09SOO487 Receptor 5-8-(4-methoxy Antagonist phenyl)-octyloxy pyridin-2- ylmethylsulfanylmethyl benzoic acid LTB4 SC-52798 7-3-(2- Bioorganic and Medicinal Receptor Cyclopropylmethyl-3- Chemistry Letters 1994, 4: 6 (811-816); Antagonist methoxy-4-thiazol-4-yl- Journal of Medicinal phenoxy)-propoxy-8- Chemistry 1995, 38: 6 (858–868) propyl-chroman-2- carboxylic acid LTB4 SC-53228 3-7-3-(2- International Congress of the Receptor Cyclopropylmethyl-3- Inflammation Research Association Antagonist methoxy-4- 1994, 7th: White Haven (Abs W5) methylcarbamoyl phenoxy)-propoxy-8- propyl-chroman-2-yl)- propionic acid LTB4 WAY 3-fluoro-4'-(2- Drugs under Experimental and Receptor 121006 quinolinylmethoxy)- Clinical research 1991, 17: 8 (381-387) Antagonist 1,1'-biphenyl-4- acetic acid LTB4 ZD-2138 3-Amino-3-(4-methoxy International Symposium on Receptor tetrahydro-pyran-4-yl)- Medicinal Chemistry Antagonist acrylic acid 1-methyl-2- 1994, 13th: Paris (P 197) oxo-1,2-dihydro quinolin-6- ylmethyl ester

In addition the following LTA4H inhibitors are described 35 the enzyme that converts HMG-CoA to the cholesterol pre in USP2003/0004101A1, the teachings of which are incorpo cursor mevalonic acid. Numerous compounds with high rated herein by reference in their entirety: specificity for this enzyme have been developed and approved for human therapy. Compositions of the invention may com Additional LTA4H Agent List prise a statin that is listed in Agent Table III herein, such as 40 roVuvastatin (also known visastatin), fluvastatin, atorvastatin, 1. 1-2-4-(phenylmethyl)phenoxyethyl-2-methyl-4-tet lovastatin (also known as mevolin), simvastatin, pravastatin, razolylpieridine pitavastatin, mevastatin, crevastatin, ML-236A, ML-236B, 2. 1-2-4-(4-oxazolyl)phenoxy)phenoxyethylpyrroli MBV-530A and MB-53OB. dine References to agents should be understood to include phar 3. 3-methyl 3-4-(2-thienylmethyl)phenoxypropyl 45 maceutically acceptable salts, acids, bases, esters, pro-drugs, aminopropionic acid metabolites, and other common formulation variants of the 4. methyl 3-methyl 3-4-(2-thienylmethyl)phenoxypro agents. pylaminopropionate An increasing body of emerging evidence identifies serum 5. 3-methyl 3-4-(3-thienylmethyl)phenoxypropyl CRP as a marker for cardiovascular morbidity/mortality, and aminopropionic acid 50 correlates reductions in serum CRP to better clinical out 6. methyl-3-methyl 3-4-(3-theinylmethyl)phenoxypro comes. (See, e.g., U.S. Pat. No. 6,040,147, Ridker et al., N. pylaminopropionate Engl. J. Med. 352(1): 20-28 (2005); Nissen et al., N. Engl. J. 7. 3-methyl 3-4-(4-fluorophenoxy)phenoxypropyl Med. 352(1): 29-38 (2005); and Pearson et al., Circulation aminopropionic acid 107: 499-511 (2003).) Serum CRP in excess of 3.0 mg/L is 8. 3-methyl 3-4-(4-biphenyloxy)phenoxypropyl 55 considered high risk; from 1.0 to 3.0 average risk; and below aminopropionic acid 1 mg/L low risk. (Pearson et al.) Compositions and methods 9. N-3-3-4-(phenylmethyl)phenoxypropyl)methy of the invention provide tools for reducing serum CRP. laminopropionylbenzenesulfonamide Reductions in CRP can be measured on a concentration basis, 10. 1-2-4-(phenylmethyl)phenoxyethyl-2-methyl-4- where compositions and methods that achieve CRP below 3.0 (1H-tetrazol-5-yl)piperidine 60 mg/L are preferred; with still more preferred targets of 2.75 11. 1-2-[4-(phenylmethyl)phenoxyethyl-4-(1H-tetra mg/L, 2.5 mg/L, 2.25 mg/L, 2.0 mg/L, 1.75 mg/L, 1.5 mg/L. Zol-5-yl)piperidine 1.25 mg/L, 1.0 mg/L, 0.75 mg/L, and 0.5 mg/L. Reductions in In some embodiments, compositions of the invention com CRP also can be measured on a percentage basis, where prise a statin, and methods of the invention comprise admin clinical effectiveness is evaluated as a percentage reduction in istration of a statin. In this context, the term "statin' should be 65 CRP in a patient compared to no drug therapy or compared to understood to refer to any of the class of inhibitors of 3-hy single drug therapy. Depending on the initial CRP measure droxy-3-methylglutarlcoenzyme A (HMG-CoA) reductase, ment, compositions and methods that reduce CRP anywhere US 8,158,362 B2 13 14 from 10%-90% or more are contemplated, e.g., reductions of therapy of a longer duration may result in further CRP reduc 10%, 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, tion than the 20-30% effect observed in the short term study. 80%, or any target in between these values. Reductions in In an embodiment of the invention, the compositions com CRP can also be measured relative to quartile or quintile prise a leukotriene synthesis inhibitor in an amount effective distribution of CRP in the normal population (i.e., from 4" to reduce serum CRP in a human subject and a statin. In quartile to 3" quartile or from 3" quartile to 2" quartile). For another embodiment, the compositions comprise a statin in an example, upper quartiles, having serum CRP greater than amount effective to reduce serum LDL-C in a human subject about 2.0 mg/L, defines a population to target for treatment and a leukotriene synthesis inhibitor. The invention also according to the invention to reduce CRP an/or redule risk of encompasses compositions comprising a leukotriene synthe MI or other adverse events. 10 sis inhbitor and a statin in amounts effective to synergistically The invention also includes methods of reducing MPO and reduce CRP in a human subject. method of monitoring MPO levels. Reductions in MPO can In one variation, the leukotriene inhibitor and the statin are be measured on a concentration basis, where compositions included in the composition of the invention in amounts effec and methods that reduce MPO level relative to the quartile tive to synergistically reduce serum C-reactive protein in a distribution of MPO in the normal population (i.e., from ath 15 human Subject. quartile to 3' or from 3' to 2') are preferred. Reductions in For practice of the invention with BAY-X1005, doses of MPO also can be measured on a percentage basis, where 50-750 mg per day for adult human patients are contem clinical effectiveness is evaluated as a percentage reduction in plated. Doses of 100-500 mg, from one to five times per day, MPO in a patient compared to no drug therapy or compared to is contemplated. Doses of 250-375 mg, from one to three single drug therapy. Depending on the initial MPO measure times per day, is preferred. ment, compositions and methods that reduce MPO anywhere Dosing for clinically approved statins have been developed from 10%-90% or more are contemplated, e.g., reductions of and published by the manufacturers. In a preferred embodi 10%, 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, ment, the statin is co-formulated with the LT inhibitor in a pill 80%, or any target in between these values. or capsule for administrations 1-4 times per day. Soluble CD40 ligand (SCD40L) is a marker of inflamma 25 The invention provides for methods of using these compo tion as well as a marker for pro-thrombotic and pro-athero sitions to reduce risk factors for cardiovascular diseases Such genic processes. Elevated levels of SCD40L are associated as for MI, ACS, stroke, or PAOD. In one method, a compo with increased cardiovascular risk, including a heightened sition comprising a leukotriene synthesis inhibitor and a sta risk of death in patients with acute coronary syndromes (Varo tin is administered to a human Subject exhibiting one or more et al. Circulation 108:1049-1052, 2003 and Schonbeck et al. 30 risk factors for MI, ACS, stroke or PAOD, wherein the com Circ Res 89:1092-1103, 2001). Patients having an increased position is administered in an amount effective to reduce at risk of ACS, such as MI, have levels of SCD40L that are least one risk factor for MI, ACS, stroke or PAOD. Preferably, greater than the 75th percentile, particularly patients in the the risk factor is elevated serum LDL-C oran elevated inflam 90-99th percentile are at a great risk. In addition, patients matory marker Such as CRP or serum amyloid A. In a highly having an increased risk of ACS, including MI, have levels of 35 preferred embodiment, LDL-C and CRP are both reduced sCD40L that are in the third or forth quartile. clinically significant amounts, where a clinically significant The invention also contemplates methods of reducing amount is an amount that correlates with a statistically sig serum sGD40L levels and method of monitoring serum nificant measurable reduction in risk for an adverse cardio sCD40L levels. Reductions in SCD40L can be measured on a vascular event, when analyzed in a population, e.g., in a concentration basis, where compositions and methods that 40 clinical study. reduce SCD40L levels relative to the quartile distribution of The invention also provides for method of using these sCD40L in the normal population (i.e., from 4" quartile to 3" compounds to reduce CRP in human Subject. In one variation, or from 3" to 2") are preferred. Reductions in scD40L also the invention is a method of reducing C reactive protein can be measured on a percentage basis, where clinical effec (CRP) in a human Subject, comprising administering to a tiveness is evaluated as a percentage reduction in SCD40L in 45 human in need of treatment to reduce CRP a composition of a patient compared to no drug therapy or compared to single the invention containing the LT inhibitor and the statin as drug therapy. Depending on the initial SCD40L measurement, described above, in an amount effective to reduce serum C compositions and methods that reduce SCD40L anywhere reactive protein in the human subject. The identification of a from 10%-90% or more are contemplated, e.g., reductions of human in need of treatment for CRP reduction can be based 10%, 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 50 on a variety of factors described herein, including genetic 80%, or any target in between these values. Adminsitration of factors, CRP measurements, measurements of other inflam leukotriene synthesis inhibitors, such as the molecules matory markers, and measurements of non-genetic and non described herein, is specificially contemplated in amounts inflammatory markers for risk of MI. In one variation, the effective to achieve the desired reduction in SCD40 lignad. method includes selecting for the administering step a human Repeated measurements to monitor and maintain the SCD40L 55 subject at risk for a disease or condition selected from the reduction and adjust dosing is specifically contemplated. group consisting of myocardial infarction, acute coronary In some variations of the invention, the composition of the syndrome, stroke, or peripheral arterial occlusive disease. invention includes the leukotriene synthesis inhibitor in an Thus, the invention provides a method that comprises select amount effective to reduce serum C-reactive protein (CRP) in ing a human subject at risk for MI, ACS, stroke or PAOD and a human Subject. In some variations, the composition of the 60 administering to the Subject a composition comprising a leu invention includes the statin in an amount effective to reduce kotriene synthesis inhibitor and a statin wherein the compo serum low density lipoprotein cholesterol (LDL) and reduce sition is in an amount effective to reduce serum CRP in a serum CRP in a human Subject. In at least one preliminary and human subject. The method may further comprise the step of short term study described herein, human subjects that measuring serum CRP in the human Subject to monitor thera already enjoyed the CRP-lowering benefits of statin therapy 65 peutic efficacy of the composition, wherein a decrease in were administered the LT inhibitor BAY-X1005, and signifi serum CRP following the administering of the composition cant further reductions in CRP were detected. Combination indicates therapeutic efficacy. US 8,158,362 B2 15 16 In still another variation, the monitoring of risk factors DG00AAHII) (SEQ ID NO: 1, position 1765.79), allele G, and/or toxicity is used to adjust dose or dosing. For example, SG13S30 (SEQ ID NO: 1, position 193840), allele G; and dose or dosing of a statin or a leukotriene synthesis inhibitor SG13S42 (SEQID NO: 1, position 203877), allele A, iden is increased if serum CRP and/or LDL and/or serum or uri tifies the subject as having a susceptibility to MI, the presence nary leukotriene measurements do not decrease to a target in said Subject of a haplotype comprised of markers: level, such as a level equivalent to the bottom 50 percentile, 40 SG13S377 (SEQ ID NO: 1, position 169965), allele A: percentile, 30 percentile, 20 percentile, 10 percentile, 1 per SG13S114 (SEQ ID NO: 1, position 178096), allele A: centile of a population, or other target percentile in between SG13S41 (SEQ ID NO: 1, position 202045), allele A; and these exemplary targets. As described above, monitoring also SG13S35 (SEQID NO: 1, position 206117), allele G, iden can be used to adjust dosing to achieve a target level of serum 10 CRP, or to achieve a target percentage reduction in CRP for a tifies the subject as having a susceptibility to MI. particular human Subject. In another embodiment, the invention provides for a The monitoring may involve parameters in addition to method of selecting a human Subject Susceptible to MI com CRP. A benefit of the statin for many human subjects will be prising analyzing nucleic acid of a human Subject for the the reduction in serum LDL, and methods of the invention 15 presenece or absence of at least one FLAP polymorphism that include administering the composition of the invention in an correlates with a susceptibility to MI. FLAP polymorphisms amount effective to reduce serum LDL and serum leukot that that correlate to susceptibility to MI include SG13S377 rienes in the human subject. In this embodiment, serum LDL (SEQID NO: 1, position 169965), allele A: SG13S114 (SEQ may be monitored. Other markers described herein, including ID NO: 1, position 178096), allele A; SG13S41 (SEQID NO: serum amyloid A nad myeloperoxidase, may be monitored. 1, position 202045), allele A; and SG13S35 (SEQID NO: 1, In certain embodiments of the invention, the individual or position 206117), allele G. Additional FLAP polymorphisms human subject selected for treatment is an individual who has that that correlate to a susceptibility to MI include SG13S375 at least one risk factor. Such as an at-risk haplotype for myo (SEQ ID NO: 1, position 164874), allele T, SG13S25 (SEQ cardial infarction, stroke or PAOD; an at-risk haplotype in the ID NO: 1, position 165553), allele G: SG13S32 (SEQID NO: FLAP gene; a polymorphism in a FLAP nucleic acid; an 25 1, position 176579), allele A; and SG13S106 (SEQID NO: 1, at-risk polymorphism in the 5-LO gene promoter. The inven position 198547), allele G or A. The methods may further tion provides for methods of selecting a human Subject Sus comprise selecting a subject with the presence of at least one ceptible to MI comprising determining a FLAP genotype or FLAP polymorphism and with the presence of elevated CRP, haplotype of a human Subject, and selecting for treatment a SCD4OL or MPO. human subject with a FLAP genotype or haplotype that cor 30 relates with an increased risk of MI. The methods of the An another embodiment, the invention provides for meth invention include selecting a human subject with the presence ods of prophylaxis therapy for myocardial infarction (MI) of at least one at-risk haplotype within or near the FLAP gene comprising analyzing nucleic acid of a human Subject for the Such as a haplotype shown in Table 14, a haplotype shown in presence and absence of a FLAP haplotype, wherein the Table 15; a haplotype shown in Table 21; haplotype B4: 35 haplotype is comprised of markers: SG13S377 (SEQID NO: haplotype B5; haplotype B6; haplotype A4; haplotype A5; 1, position 169965), allele A; SG13S114 (SEQ ID NO: 1, haplotype HapB, haplotype HapC1, haplotype HapC2, hap position 178096), allele A: SG13S41 (SEQ ID NO: 1, posi lotype HapC3, haplotype HapC4-A and haplotype HapC4-B. tion202045), allele A; and SG13S35 (SEQID NO: 1, position The methods of the invention also include selecting a 206117), allele G, and selecting for treatment a human sub human Subject for treatment, wherein the presence in said 40 ject having nucleic acid with the presence of the FLAP hap subject of a haplotype comprising marker SG13S106 (SNP lotype. This method further comprises administering to the DG00AAHII) (SEQ ID NO: 1, position 176579), allele G, Subject a composition comprising a therapeutically effective identifies the subject as having a susceptibility to MI; the amount of an MI therapeutic agent that inhibits leukotriene presence of a haplotype comprised of markers SG13S99 synthesis in vivo, wherein the MI therapeutic agent inhibits (DG00AAFIU), allele T (SEQ ID NO: 1, position 138551); 45 leukotriene synthesis by inhibiting the activity of at least one SG13S377 (DG00AAJFF) (SEQID NO: 1, position 169965), protein selected from 5-Lipoxygenase activating protein allele G: SG13S106 SNP DG00AAHII (SEQ ID NO: 1, (FLAP) and 5-lipoxygenase (5-LO). position 1765.79), allele G: SG13S32 (SEQ ID NO: 1, posi In one embodiment, the invention provides for methods of tion 1985.47), allele A; and SG13S35 (SEQID NO: 1, position decreasing risk of a Subsequent myocardial infarction in an 206117), allele G identifies the subject as having a suscepti 50 individual who has had at least one myocardial infarction, bility to MI; the presence in said subject of a haplotype comprising administering a therapeutically effective amount comprised of markers: SG13S375 (SEQ ID NO: 1, position of an MI therapeutic agent to the individual, wherein the MI 164874), allele T: SG13S25 (SEQ ID NO: 1, position therapeutic agent inhibits leukotriene synthesis by inhibiting 165553), allele G: SG13S32 (SEQ ID NO: 1, position the activity of at least one protein selected from 5-Lipoxyge 176579), allele A; and SG13S106 (SEQ ID NO: 1, position 55 nase activating protein (FLAP) and 5-lipoxygenase (5-LO) 198547), allele G or A identifies the subject as having a and monitoring myeloperoxidase (MPO) in the individual susceptibility to MI, the presence in said subject of a haplo before and during administration of the therapeutic agent, type comprised of marker SG13S375(SNP DG00AAJFC) wherein the therapeutic agent is administered in an amount (SEQ ID NO: 1, position 164874), allele T: and SG13S25 effective to reduce the leukotriene level in a subject. (SEQ ID NO: 1, position 165553), allele G, identified the 60 In another embodiment, the invention provides for meth Subject as having a susceptibility to MI; the presence in said ods of screening a human subject for susceptibility for MI subject of a haplotype comprised of marker SG13S375(SNP comprising analyzing nucleic acid of a human Subject for the DG00AAJFC) (SEQ ID NO: 1, position 164874), allele T: presence and absence of the FLAP haplotype comprised of and SG13S25 (SEQIDNO: 1, position 165553), allele G, and markers: SG13S377 (SEQID NO: 1, position 169965), allele SG13S32 (SEQ ID NO: 1, position 1985.47) identified the 65 A: SG13S114 (SEQ ID NO: 1, position 178096), allele A: Subject as having a susceptibility to MI, the presence in said SG13S41 (SEQ ID NO: 1, position 202045), allele A; and subject of a haplotype comprised of marker SG13S106 (SNP SG13S35 (SEQ ID NO: 1, position 206117), allele G, and US 8,158,362 B2 17 18 identifying the Subject as having a susceptibility to MI. For example, the invention provides for methods of assess wherein the presence of the FLAP haplotype correlates with ing a susceptibility to myocardial infarction (MI) or acute an increased risk of MI. coronary syndrome (ACS) in a human individual, comprising In another embodiment, the invention provides methods of screening nucleic acid of the individual for at least one poly screening a human Subject for Susceptibility to MI compris 5 morphism in a LTA4H nucleic acid that correlates with ing analyzing nucleic acid of a human Subject for the presence increased occurrence of myocardial infarction or ACS in a or absence of the LTA4H haplotype comprised of markers: human population, wherein the presence of the at least one DG12S1664, SG12S16, SG12S17, SG12S18, SG12S21, polymorphism identifies the individual as having elevated SG12S22, SG12S23, SG12S24, SG12S25, SG12S26, susceptibility to MI or ACS, and wherein the absence of the at DG12S1666, SG12S100, SG12S28, and SG12S144, with 10 least one polymorphism in the nucleic acid identifies the alleles 0, C, A, T, G, G, T, T, A, T, 0, and T, T, and A, individual as not having the elevated susceptibility. Such respectively. The allelic frequency of a shorter version of this polymorphisms include allele Tat marker SG12S551 or allele haplotype including markers DG12S 1664, SG12S26, A at marker SG12S540 or allele C at marker rs250477 and DG12S1666, and SG12S144, with alleles 0, T, 0, and A, allele T at marker rs2660896 or allele A at marker SG12S540 respectively, is 51% in male MI patients and 43% in controls 15 (re2540500) and allele A at marker rs107353540 (carried by 76% of male patients and 67% of controls). Allelic The study described in Example 21 demonstrates that the frequency of this haplotype is higher, or 56%, in a subgroup of frequency of the LTA4H haplotype, HapK, is rare in black patients that have had more than one MI. African American Subjects but confers a Surprisingly great Table 42 shows the results of the haplotype association relative risk for MI. The invention also provides for methods analysis using 1560 unrelated MI patients and 953 unrelated of prophylaxis therapy for myocardial infarction (MI) in a population controls. A haplotype comprised of the consecu human comprising selecting a human Subject having a race tive markers was highly significant in MI patients that had that includes black African ancestry, and administering to the also had either stroke or peripheral arterial occlusive disease Subject a composition comprising a therapeutically effective (PAOD) (P-value adjusted for multiple comparisons=0.007). amount of an MI therapeutic agent that inhibits leukotriene The fact that the haplotypes shown in Table 42 are more 25 synthesis in vivo. significant in MI patients that have more than one clinically Human Subjects having a race that includes black African evident cardiovascular complication might indicate that the ancestry may be persons of African descent or lineage. Black gene played a role in clinical activity or severity of the ath African ancestry may be determined by self reporting as erosclerotic disease. The significantly associated haplotype is African-Americans, Afro-Americans, Black Americans, comprised of the following consecutive markers; SG12S438, 30 being a member of the black race or being a member of the DG12S1664, SG12S16, SG12S21, SG12S23, SG12S25, negro race. For example, African Americans or Black Ameri SG12S26, DG12S1666, SG12S100, SG12S28, SG12S143, cans are those persons living in North America and having SG12S144, SG12S221, SG12S222, SG12S223, SG12S225, origins in any of the black racial groups of Africa. For SG12S226, SG12S233, SG12S237, and DG12S1668 with example, self-reported persons of black African ancestry may alleles C, 0, C, G, T, A, T, 0, T, TT, A, G, C, C, G, G, C, T, and 35 have at least one parent of black African ancestry or at least O. one grandparent of black African ancestry. Particular LTA4H haplotypes, HapK, Hapl and HapQ, are Human Subjects having a race that includes black African set out in Table 47. The invention includes methods of screen ancestry may also be determined by genetic analysis. Genetic ing a human Subject for Susceptibility to MI comprising ana analysis of ancestry may be carried out using unlinked mic lyzing nucleic acid of a human Subject for the presence or 40 rosatellite markers such as those set out in Smith et al. Am J absence of the HapK haplotype comprised of the following Hum Genet 74, 1001-13 (2004). Particularly, African ances markers: SG12S16, SG12S21, SG12S23, SG12S25, try may be determined using the unlinked microsatellite SG12S26, SG12S100, SG12S28, SG12S143, SG12S144, markers set out in Table 54 herein and described in Example SG12S221 with alleles C, G, T, A, T, T, T, C, G and G, 21. A person’s alleles for each tested marker are identified and respectively. The invention also provides methods of screen 45 using standard computational techniques, compared to the ing a human Subject for Susceptibility to MI comprising ana allelic frequencies of each marker in different races. The use lyzing nucleic acid of a human Subject for the presence or of increasing numbers of these markers generally provides an absence of the Hap haplotype comprised of the following increasing arsenal of information to assess probable racial markers: SG12S16, SG12S21, SG12S23, SG12S25, origin/mixture. For example, the selecting of human Subjects SG12S26, SG12S100, SG12S28, SG12S143, SG12S144, 50 of black African ancestry comprises analyzing nucleic acid SG12S221 with alleles C, G, T, A, T, T, T, T, A and G, from the human Subject to determine a racial genotype com respectively. The invention also provides methods of screen prised of at least 8 microsatellite markers set forth in Table 54, ing a human Subject for Susceptibility to MI comprising ana and selecting for therapy a Subject with a genotype that cor lyzing nucleic acid of a human Subject for the presence or relates with the race. Preferably, the racial genotype is com absence of the HapQ haplotype comprised of the following 55 prised of alleles of the markers D1S466, D3S1583, D3S4011, markers SG12S16, SG12S21, SG12S23, SG12S25, D4S3014, D6S1037, D9S1777, D20S113 and D22S1172, SG12S26, SG12S100, SG12S28, SG12S143, SG12S144, that correlate with African and European ancestry. SG12S221 with alleles C, G, T, G, T, C, T, T, A and G, In some variations of the invention, the methods comprise respectively. selecting a human Subject having both a race that includes As described herein, smaller subsets of markers, or even 60 black African ancestry and at least one additional genetic, single individual markers, may be used as a Surrogate for family or medical history risk factor of cardiovascular dis screening for a predictive haplotype because the Subsets/ ease. In some variations, the additional family or medical markers with predictive values include marker SG12S551 history risk factor may be a race that includes European having allele T or SG12S540 having allele A or marker ancestry, whereby the subject has mixed African and Euro rs250477 having allele C and marker rs2660896 having allele 65 pean ancestry. An exemplary Subject having a race including T or marker SG12S540 (re2540500) having allele A and a mixed African and European ancestry may have at least one marker rs107353540 having allele A. parent or grandparent of African ancestry and at least one US 8,158,362 B2 19 20 parent or grandparent of European ancestry. European ances In another variation of the invention, the selected human try may be determined by self reporting as European Subject may be determined to have the HapK Surrogate hap ancestor(s) (from a country in Europe), European American, lotype comprising marker rs250477 having allele C and white or Caucasian. European ancestry may also be deter marker rs2660896 having allele T. The correlation of this 2 mined by genetic analysis using unlinked microsatellite SNP surrogate haplotype to HapK is R=0.95 for both African markers as described in Example 21 and set out in Table 54. American patients and Caucasian patients. In addition, the In some variations, the additional risk factor comprises a selected human subject may be determined to have the HapK genetic variation that correlates with susceptibility to MI, in surrogate haplotype comprising marker SG12S540 at least one gene selected from 5-lipoxygenase activating (re2540500) having allele A and marker rs107353540 having protein (FLAP) and leukotriene A4 hydrolase (LTA4H). In 10 allele A. The correlation of this 2 SNP haplotype to HapK is Some variations of the method, the selecting step comprises R=0.95 for both African American patients and Caucasian determining a FLAP genotype or haplotype of a human Sub patients. ject, and selecting for therapy a human Subject with the race In some variations of the method, the selecting of human and with a FLAP genotype that correlates with susceptibility Subjects comprises analyzing nucleic acid of a human Subject to MI. In particular, the FLAP genotype or haplotype that 15 for the presence or absence of at least one FLAP polymor correlates with susceptibity of MI is Hap A or HapB or the phism or LTA4H polymorphism that correlates with a sus haplotypes provided in Table 5, Table 7 and Table 34. ceptibility to myocardial infarction, and selecting for therapy In other variations of the method, the selecting step com a human subject with the race and with the at least one prises determining a LTA4H genotype or haplotype of a polymorphism that correlates with susceptibility to MI. The human Subject, and selecting for therapy a human Subject FLAP polymorphic markers that correlate with MI include with the race and with a LTA4H genotype that correlates with those SNPs set out in Table 5, Table 7 and Table 34 and those susceptibility to MI. In particular, the LTA4H genotype or SNPs within the Hap A or HapB FLAP haplotype. The haplotype that correlates with susceptibility to MI is HapK, LTA4H polymorphic markers that correlate with MI include Hapl or HapQ or the haplotypes provided in Table 47 or 51. those SNPs set out in Table 47 or 51 and those SNPs within The selected human subject may be determined to have a 25 the HapK, Hapl and HapQLTA4H haplotype. LTA4H haplotype comprising markers SG12S25 having The individuals or human subjects selected for treatment in allele A, SG12S26 having allele T, SG12S143 having allele any of the methods of the invention may have at least one C, SG12S144 having allele G and SG12S221 having allele G. family or medical history risk factor Such as diabetes; hyper The selected human subject may also be determined to have a tension; hypercholesterolemia; elevated triglycerides; LTA4H haplotype comprising markers SG12S25 having 30 elevated lp(a); obesity; ankle/brachial index (ABI) less than allele A, SG12S26 having allele T, SG12S100 having allele T. 0.9; a past or current Smoker, transient ischemic attack; tran SG12S28 having allele T, SG12S143 having allele C, sient monocular blindness; carotid endarterectomy; asymp SG12S144 having allele G and SG12S221 having allele G. In tomatic carotid stenosis; claudicatioin; limb ischemia leading addition, selected human Subject may also be determined to to gangrene, ulceration or amputation; a vascular or periph have a LTA4H haplotype comprising markers SG12S16 hav 35 eral artery revascularization graft, increased serum LDL cho ing allele C, SG12S21 having allele G, SG12S23 having lesterol and/or decreased HDL cholesterol; serum total cho allele T, SG12S25 having allele A, SG12S26 having allele T. lesterol D-200 mg/dl., increased leukotriene synthesis; and/or SG12S100 having allele T, SG12S28 having allele T. at least one previous myocardial infarction, ACS, stable SG12S143 having allele C, SG12S144 having allele G and angina, previous transientischemic attack, transient monocu SG12S221 having allele G. 40 lar blindness, or stroke, asymptomatic carotid Stenosis or A Surrogate haplotype is a haplotype that correlates with carotid endarterectomy, atherosclerosis, requires treatment the original haplotype, but may be defined by different mark for restoration of coronary artery blood flow (e.g., angio ers. A perfect Surrogate haplotype is one that will identify plasty, stent, revascularization procedure). exactly the same individuals as the original haplotype. For In addition, the individuals or human subjects selected for example, all subjects that carry the original haplotype will 45 treatment in any of the methods of the invention may have an also carry the the Surrogate haplotype and all that carry the elevated inflammatory marker, e.g., a marker Such as an end Surrogate also carry the original haplotype. A perfect Surro product of the leukotriene pathway, such as LTB4, C-reactive gate will have a correlation coefficient (R) of 1. Surrogate protein (CRP), serum sGD40L, serum amyloid A, fibrinogen, haplotypes may not be perfect, for example if the frequency of a leukotriene, a leukotriene metabolite, interleukin-6, tissue the Surrogate is different than that of original haplotype then 50 necrosis factor-alpha, a soluble vascular cell adhesion mol it is likely that all Subjects that carry the original haplotype ecule (sVCAM), a soluble intervascular adhesion molecule will also carry the Surrogate haplotype but not all that carry (sICAM), E-selectin, matrix metalloprotease type-1, matrix the Surrogate will carry the original (if the Surrogate is more metalloprotease type-2, matrix metalloprotease type-3. frequent than the originalhaplotype). In this case the R is less matrix metalloprotease type-9, myeloperoxidase (MPO), and than 1. 55 N-tyrosine). The invention provides for methods of prophy In some variations of the invention, the selected human laxis therapy for MI comprising administering a MI therapeu Subject may be determined to have a HapK Surrogate haplo tic agent in an amount effective to reduce the elevated serum type. For example, the selected human Subject may be deter level of at least one elevated inflammatory markers. mined to have a LTA4H genotype comprising marker In a particular embodiment, individuals or human Subjects SG12S551 having allele T, or SG12S540 having allele A. The 60 selected for treatment in any of the methods of the invention SG12S551 may serve as a single SNP surrogate for HapK, have an elevated CRP level in excess of 3.0 mg/L. In addition, since its Tallele correlates to HapKin HapMap CEU samples the invention contemplates selecting human Subjects for (The International HapMap Project. Nature 426, 789-96 treatment that have greater than 2.75 mg/L, greater than 2.5 (2003)) with R=0.74, D-1. The SG12S540 may also serve mg/L or greater than 2.25 mg/L, greater than 2.0 mg/L. The as a single SNP surrogate for HapK, since its A allele corre 65 invention also contemplates selecting human Subjects for leates to HapK in HapMap CEU samples with R=0.81 and treatment that have a median CRP level, e.g. CRP level D'=1. greater than 1.75mg/L or greater than 1.5 mg/L. US 8,158,362 B2 21 22 In some variations of the invention, individuals or human In certain embodiments of the invention, the individual or subjects selected for treatment in any of the methods of the human Subject selected for treatment using any of the preced invention have an elevated serum SOD40L level in excess of ing methods of the invention is an individual who has at least 1.4 ng/ml. In addition, the invention contemplates selecting one risk factor, such as an at-risk haplotype for myocardial human Subjects for treatment that have greater than 1.5 ng/ml. 5 infarction, stroke or PAOD; an at-risk haplotype in the FLAP greater than 2.0 ng/ml, greater than 2.5 ng/ml, greater than 3.0 gene; a polymorphism in a FLAP nucleic acid; an at-risk ng/ml, greater than 3.5 ng/ml, greater than 4.0 ng/ml, greater polymorphism in the 5-LO gene promoter, diabetes; hyper than 4.5 ng/ml or greater than 5.0 ng/ml. tension; hypercholesterolemia; elevated triglycerides; In a particular embodiment, the invention provides for elevated lp(a); obesity; ankle/brachial index (ABI) less than methods of prophylaxis for myocardial infarction (MI) com 10 0.9; a past or current Smoker, transient ischemic attack; tran prising administering to a Subject in need of prophylaxis for sient monocular blindness; carotid endarterectomy; asymp tomatic carotid stenosis; claudicatioin; limb ischemia leading myocardial infraction a composition comprising a therapeu to gangrene, ulceration or amputation; a vascular or periph tically effective amount of an MI therapeutic agent that inhib eral artery revascularization graft; an elevated inflammatory its leukotriene synthesis in Vivo, and monitoring myeloper 15 marker (e.g., a marker Such as C-reactive protein (CRP), oxidase (MPO) level in the human subject before and during serum SCD40, serum amyloid A, fibrinogen, a leukotriene, a the prophylaxis treatment, wherein the MI therapeutic agent leukotriene metabolite, interleukin-6, tissue necrosis factor is administered in an amount effective to reduce the MPO alpha, a soluble vascular cell adhesion molecule (sVCAM), a level in a subject. soluble intervascular adhesion molecule (sICAM), E-selec In another embodiment, the invention provides for meth tin, matrix metalloprotease type-1, matrix metalloprotease ods of prophylaxis for myocardial infarction (MI) comprising type-2, matrix metalloprotease type-3 matrix metallopro administering to a Subject in need of prophylaxis for myocar tease type-9, myeloperoxidase (MPO), and N-tyrosine); dial infraction a composition comprising a therapeutically increased LDL cholesterol and/or decreased HDL choles effective amount of an MI therapeutic agent that inhibits terol, increased leukotriene synthesis; and/or at least one leukotriene synthesis in vivo, and monitoring soluble CD40 25 previous myocardial infarction, ACS. stable angina, previous ligand (CD40L) level in the serum of the human subject transient ischemic attack, transient monocular blindness, or before and during the prophylaxis treatment, wherein the MI stroke, asymptomatic carotid stenosis or carotid endarterec therapeutic agent is administered in an amount effective to tomy, atherosclerosis, requires treatment for restoration of reduce the SCD40L level in a subject. coronary artery blood flow (e.g., angioplasty, Stent, revascu The invention also provides for methods of Screening a 30 larization procedure). human Subject for risk of developing myocardial infarction, The invention additionally pertains to methods of assessing comprising contacting a blood sample from the human sub an individual for an increased risk of MI, ACS, atherosclero ject with a calcium ionophore to stimulate production of a sis, stroke, or PAOD, by assessing or monitoring a level of a leukotriene; and measuring production of a leukotriene in the leukotriene metabolite (e.g., LTE4, LTD4, LTB4) in the indi blood sample after the contacting step, wherein elevated leu 35 vidual (e.g., in a sample of blood, serum, plasma or urine). An kotriene production compared to a control correlates with individual or human subject selected for treatment may have increased risk of developing myocardial infarction (MI). The an elevated measurement of a leukotriene or leukotriene control in these methods may be a human of the same sex as metabolite, such as LTC4, LTD4, LTB4 and LTE4. The level the Subject selected for treatment or may be a human age of leukotrienes and leukotriene metabolites may be measured matched to the subject selected for treatment. 40 in serum, plasma, blood or urine in the individual. An Human Subjects that already are treated with statins can increased level of leukotriene metabolite is indicative of an enjoy the benefit of the present invention if the subjects increased risk. The invention also encompasses methods of therapy is modified to include an LTantagonist. Thus, in still assessing an individual for an increased risk of MI, ACS, another embodiment, the invention is a method of reducing C atherosclerosis, stroke, transient ischemic attack, transient reactive protein (CRP) in a human Subject, comprising: 45 monocular blindness, asymptomatic carotid Stenosis, PAOD, selecting a human Subject that receives statin therapy to claudication, or limb ischemia, by stimulating production of reduce serum LDL, wherein the statin therapy optionally a leukotriene or a leukotriene metabolite in a test sample from reduces serum CRP in the human Subject; and administering the individual (e.g., a sample comprising neutrophils), using to the human Subject a leukotriene synthesis antagonist, in an a calcium ionophore, and comparing the level of the leukot amount effective to further reduce CRP in the human subject. 50 riene or leukotriene metabolite with a control level. A level of In still another embodiment, the invention is a method of production of the leukotriene or leukotriene metabolite that is reducing C reactive protein (CRP) in a human Subject, com significantly greater than the control level, is indicative of prising: identifying a human Subject in need of treatment to increased risk. reduce serum CRP; administering to the human subject a The invention further pertains to methods of assessing composition comprising a statin; and administering to the 55 response to treatment with a leukotriene synthesis inhibitor, human Subject a composition comprising a leukotriene Syn by assessing or monitoring a level of a leukotriene or leukot thesis inhibitor, wherein the statin and the leukotrience syn riene metabolite in the individual before treatment, and com thesis inhibitor are administered in amounts effective to paring the level to a level of the leukotriene or leukotriene reduce serum CRP in the human subject. The statin and the LT metabolite assessed during or after treatment. A level that is inhibitor can be simultaneously administered as a single com 60 significantly lower during or after treatment, than before position, as described above; can be simultaneously admin treatment, is indicative of efficacy of the treatment with the istered as separate compositions; or can be sequentially leukotriene synthesis inhibitor. The level of leukotriene may administered. Depending on the dosing schedule, the daily be monitored in serum, plasma, blood or urine collected from administration regimen may include simultaneous adminis the subject before, during and after treatment. The invention tration at Some times and separate administration at other 65 additionally pertains to methods of assessing response to times, e.g., if one agent is administered twice daily and treatment with a leukotriene synthesis inhibitor, by stimulat another three times daily. ing production of a leukotriene or a leukotriene metabolite in US 8,158,362 B2 23 24 a first test sample from the individual (e.g., a sample com In an embodiment of the invention, the method of diagnos prising neutrophils) before treatment, using a calcium iono ing comprises screening for a LTA4H haplotype comprising phore, and comparing the level of the leukotriene or leukot markers SG12S25 having allele A, SG12S26 having allele T. riene metabolite with a level of production of the leukotriene SG12S143 having allele C, SG12S144 having allele G and or leukotriene in a second test sample from the individual, SG12S221 having allele G. The LTA4H haplotype screened during or after treatment. A level of production of the leukot for may also comprise markers SG12S25 having allele A, riene or leukotriene metabolite in the second test sample that SG12S26 having allele T, SG12S100 having allele T. is significantly lower than the level in the first test sample, is SG12S28 having allele T, SG12S143 having allele C, indicative of efficacy of the treatment, for example, the treat SG12S144 having allele G and SG12S221 having allele G. ment or therapeutic agent reduces the leukotriene level in the 10 Further, the LTA4H haplotype screened for may comprise subject to the medial level of leukotrienes in human subjects markers SG12S16 having allele C, SG12S21 having allele G, in the general population or lower than that medial level. SG12S23 having allele T, SG12S25 having allele A, Similarly, the invention encompasses methods of assessing SG12S26 having allele T, SG12S100 having allele T. response to treatment with a leukotriene synthesis inhibitor, 15 SG12S28 having allele T, SG12S143 having allele C, by assessing or monitoring a level of an inflammatory marker SG12S144 having allele G and SG12S221 having allele G. in the individual before treatment, and during or after treat A method of assessing a Susceptibility to myocardial inf ment. A level of the inflammatory marker during or after arction (MI) or acute coronary syndrome (ACS) in a human treatment, that is significantly lower than the level of inflam individual, comprising screening nucleic acid of the indi matory marker before treatment, is indicative of efficacy of vidual for at least one polymorphism in a LTA4H nucleic acid the treatment. that correlates with increased occurrence of myocardial inf To determine the effectiveness of compositions of the arction or ACS in a human population, present invention comprising a statin, total cholesterol, wherein the presence of the at least one polymorphism LDL-C and/or triglycerides may be assessed from measure identifies the individual as having elevated susceptibility to ments of risk factor markers in the serum of a human Subject 25 MI or ACS, and administered the composition. A level of serum total choles wherein the absence of the at least one polymorphism in the terol, LDL-C and/or triglycerides during or after treatment, nucleic acid identifies the individual as not having the that is significantly lower than the level of total cholesterol, elevated susceptibility. LDL-C and/or triglycerides before treatment is indicative of It is well established that different racial and ethnic groups the efficacy of the treatment. 30 show greater or lesser Susceptibility to certain diseases and The invention also pertains to use of leukotriene synthesis conditions, and there is at least one drug (BiLDilTM) that is inhibitors for the manufacture of a medicament for the treat FDA-approved for race-specific administration. A need exists ment of MI, ACS, stroke, PAOD, and/or atherosclerosis, as for improved materials and methods for diagnosing racial or described herein, as well as for the manufacture of a medica ethnic ancestry using genetic techniques. Genetic techniques ment for the reduction of leukotriene synthesis. 35 offer a potential improvement overself-reporting, especially Injurisdictions that forbid the patenting of methods that are for individuals having unknown parents, such as adoptees, or practiced on the human body, the following restrictions are for providing information about prior generations that are intended: (1) the selecting of a human Subject shall be con beyond an individuals known family tree. Another aspect of Strued to be restricted to selecting based on testing of a bio the invention is novel materials and methods for genetic logical sample that has previously been removed from a 40 assessment of race. For example, the invention includes a human body and/or based on information obtained from a method of determining racial ancestry of a human individual, medical history, patient interview, or other activity that is not comprising assaying or analyzing nucleic acid of a human practiced on the human body; and (2) the administering of a individual to determine a racial genotype comprised of one or composition to a human Subject shall be restricted to prescrib more microsatellite marker(s) selected from the group con ing a controlled Substance that a human Subject will self 45 sisting of D1S493, D1S2866, D1S2630, D1S466, D1S2847, administer by any technique (e.g., orally, inhalation, topical D2S166, D6S405 D6S446, DG8S156, D8S1719, D8S1831, application, injection, insertion, etc.). The broadest reason D8S1746, D9S1839, D9S1777, D10S1698, D11S4130, able interpretation that is consistent with laws or regulations D11S1321 D11S4206, D11S1320, D12S1723, D13S152, defining patentable Subject matteris intended. Injurisdictions D14S741, D16S404, D17S745, D17S1799, D18S464, that do not forbid the patenting of methods that are practiced 50 D19S534, D19S113, D2OS878, and D21S1884; wherein on the human body, the selecting of Subjects and the admin racial ancestry is determined from alleles of the one or more istering of compositions includes both methods practiced on microsatellite markers, and determining racialancestry based the human body and also the foregoing activities. on the racial genotype. In some variations, in addition to these The invention further provides for methods of diagnosising one or more markers, one assays one or more of the additional Susceptibility to myocardial infarction in a human Subject 55 Table 54 markers to generate the racial genotype. comprising: Screening for black African ancestry in the The invention also includes methods of assessing risk for a human Subject, and screening for an LTA4H genotype or disease with race-dependent risks in a human individual com haplotype in a nucleic acid of the human Subject, wherein the prising analyzing nucleic acid of a human individual to deter presence of the a black African ancestry and the genotype or mine a racial genotype comprised of one or more microsat haplotype in the nucleic acid is indicative of a Susceptibility to 60 ellite marker(s) selected from the group consisting of cardiovascular disease. The screening for black African D1 S493, D1S2866, D1S2630, D1 S466, D1S2847, D2S166, ancestry may be determined by any of the techniques D6S405 D6S446, DG8S156, D8S1719, D8S1831, D8S1746, described herein, such as patient self-reporting African D9S1839, D9S1777, D10S1698, D11S4130, D11S1321 ancestory or identification of African ancestory by genetic D11S4206, D11S1320, D12S1723, D13S152, D14S741, testing. Likewise, the at-risk haplotype is determined by any 65 D16S404, D17S745, D17S1799, D18S464, D19S534, technique described herein, and is any at-risk haplotype D19S113, D20S878, and D21S1884; wherein racial ancestry described herein Such as a HapK genotype or haplotype. is determined from alleles of the one or more microsatellite US 8,158,362 B2 25 26 markers, determining racial ancestry based on the racial of the genus and each value within the range is intended as an genotype and evaluating risk for the disease based on the aspect of the invention. Likewise, various aspects and fea racial genotype. tures of the invention can be combined, creating additional Studies with the ethnicity panel of markers described aspects which are intended to be within the scope of the herein have shown that ethnicity is a quantifiable trait. The invention. Although the applicant(s) invented the full scope of applications of Such an ethnicity panel extends beyond its the claims appended hereto, the claims appended hereto are utility for determining degree of ancestry in the context of not intended to encompass within their scope the prior art HapK and MI risk. Although a strong correspondence work of others. Therefore, in the event that statutory prior art between self-reported ethnicity and genetically estimated within the scope of a claim is brought to the attention of the ancestry has been found, self-report is not entirely reliable. 10 Therefore, there are several scenarios where the ethnicity applicants by a Patent Office or other entity or individual, the panel of markers described herein (using some or all of our applicant(s) reserve the right to exercise amendment rights markers beyond those previously reported) can be useful. under applicable patent laws to redefine the subject matter of In one embodiment, the determination of racial or ethnic Such a claim to specifically exclude Such statutory prior art or ancestry using the panel of genetic markers may assess Sus 15 obvious variations of statutory prior art from the scope of ceptibility or risk for certain diseases. Many diseases have such a claim. Variations of the invention defined by such race-dependent incidences or risks. For example, hyperten amended claims also are intended as aspects of the invention. sion is more common in African-Americans than in Cauca sians. Traditionally, ethnicity or race is taken into account BRIEF DESCRIPTION OF THE DRAWINGS when defining a given individual’s risk for a disease. How ever, that depends on self-reporting by the individual of his/ The foregoing and other objects, features and advantages her ethnicity. Using an ethnicity panel of markers may of the invention will be apparent from the following more improve reliability of risk assessments for any given disease particular description of preferred embodiments of the inven that shows race-dependent risks. tion. In another embodiment, the determination of racial or eth 25 FIG. 1 shows the results from a haplotype association nic ancestry using the panel of genetic markers will allow for case-control analysis of 437 female MI patients versus 721 the matching of the genetic backgrounds of case and control controls using combinations 4 and 5 microsatellite markers to groups in case-controlled clinical studies. In a traditional define the test haplotypes. The p-value of the association is case-control association study with any protein, RNA, DNA plotted on the y-axis and position of markers on the X-axis. or analyte marker it is valuable if the genetic backgrounds of 30 Only haplotypes that show association with a p-value <10 the case and control groups be matched. Using an racial panel are shown in the figure. The most significant microsatellite may improve reliability of matching case and control group marker haplotype association is found using markers beyond what can be achieved through self-reporting of race. DG13S1103, DG 13S166, DG13S1287, DG13S1061 and This would have the benefit of decreasing chance of finding DG13S301, with alleles 4, 0, 2, 14 and 3, respectively false-positive associations and would lead to more accurate 35 (p-value of 1.02x107). Carrier frequency of the haplotype is determination of the magnitude of risk of markers that 7.3% in female MI patients and 0.3% in controls. The seg showed true association. ment that is common to all the haplotypes shown in the figure Difference in ethnic or racial ancestry may also be consid includes only one gene, FLAP. ered as a risk marker (genetic, RNA, protein, other analyte). FIG. 2 shows the alleles of the markers defining the most Therefore, the determination of race or ancestry using the 40 significant microsatellite marker haplotypes. The segment panel of genetic markers may be useful to condition that risk defined with a black Square is common to all the of most in any given subject based on his/her relative ethnic makeup. significantly associated haplotypes. The FLAP nucleic acid is In addition, analysis of case-control studies based on an eth located between makers DG 13S166 and D13S1238. Two nicity panel may lead to discoveries that certain markers marker haplotype involving alleles 0 and -2 for markers ((genetic, RNA, protein, other analyte) may show race-de 45 DG13S166 and D13S1238, respectively, is found in excess in pendent risks. Use of an ethnicity panel may improve the patients. Carrier frequency of this haploype is 27% in patients sensitivity of finding ethnic differences in risk, especially and 15.4% in controls (p-value 1x10). Therefore, associa when compared to self-reporting. tion analysis confirms that the most tightly MI-associated Additional features and variations of the invention will be gene within the linkage peak is FLAP. apparent to those skilled in the art from the entirety of this 50 FIG.3 shows the relative location of key SNPs and exons of application, including the drawing and detailed description, the ALOX5AP/FLAP gene (exons shown in vertical rect and all Such features are intended as aspects of the invention. angles). Haplotype length varies between 33 to 68 kb. Likewise, features of the invention described herein can be FIG. 4 shows a significant positive correlation between re-combined into additional embodiments that also are serum LTE4 levels and serum CRP levels. intended as aspects of the invention, irrespective of whether 55 FIG. 5 shows a linkage scan using framework microsatel the combination of features is specifically mentioned above lite markers on chromosome 13 for male patients with as an aspect or embodiment of the invention. Also, only Such ischemic stroke or TIA (n=342 in 164 families at 6 meiosis). limitations which are described herein as critical to the inven The LOD score is expressed on the y axis and the distance tion should be viewed as such; variations of the invention from the pter in Kosambi cM on the x axis. lacking limitations which have not been described herein as 60 FIG. 6 shows a pairwise linkage disequilibrium (LD) critical are intended as aspects of the invention. between SNPs in a 60 kb region encompassing FLAP. The In addition to the foregoing, the invention includes, as an markers are plotted equidistantly. Two measures of LD are additional aspect, all embodiments of the invention narrower shown: D' in the upper left triangle and P values in the lower in scope in any way than the variations specifically mentioned right triangle. Shaded lines indicate the positions of the exons above. For example, although aspects of the invention may 65 of FLAP and the stars indicate the location of the markers of have been described by reference to a genus or a range of the at-risk haplotype A4. Scales for the LD strength are pro values for brevity, it should be understood that each member vided for both measures to the right. US 8,158,362 B2 27 28 FIG. 7 provides a schematic of the clinical trial schedule. gene, the FLAP nucleic acid sequence. The two marker hap This figure shows that at Visit 2 (on Day 1 of study) subjects lotype involving alleles 0 and -2 for markers DG 13S166 and were randomised into each of the three arms and to either D13S1238, respectively, was found in excess in patients. placebo or active drug within each arm. A 2-week washout Specific variants of the gene were then sought that were period separated the 4-week treatment periods. Cross-over associated with MI. was performed at week 6. In order to screen for SNPs in the FLAP gene, the whole FIG. 8 shows the analysis of carry-over effect for CRP and gene was sequenced, both exons and introns. Initially, 9 SNPs SAA (on log-scale). identified within the gene were genotyped in patients and FIG. 9 shows the results of the first step of the linkage controls. Additional microsatellite markers close to or within analysis: multipoint non-parametric LOD scores for a frame 10 the FLAP gene were also genotyped in all patients and con work marker map on chromosome 12. A LOD score Sugges trols. Five publicly known SNPs that are located within a 200 tive of linkage of 1.95 was found at marker D12S2081. kb distance 5' to the FLAP gene were also genotyped in FIG. 10 shows the results of the second step of the linkage patients and controls. Haplotype association analysis in this analysis: multipoint non-parametric LOD scores for the fami case-control study including these additional markers lies after adding 20 fine mapping markers to the candidate 15 showed several different variants of the same haplotype that region. The inclusion of additional microsatellite markers were all significantly associated with female MI (see, e.g., increased the information on sharing by decent from 0.8 to Table 8). Table 9 shows two haplotypes that are representative 0.9, around the markers that gave the highest LOD scores. of these female MI risk haplotypes which are referred to FIG. 11 shows LD and haplotypes in the LTA4H region. herein as the female MI"at risk” haplotypes. The relative risk FIG. 11, left panel, shows a pairwise LD between the 10 for male MI patients that had the female MI-'at risk” haplo genotyped SNPs in a 48-kb region encompassing LTA4H. type was increased (see, e.g., Table 9), indicating that the The markers are plotted equidistantly. Two measures of LD female MI-"at risk” haplotype also increased the risk of hav are shown: D' in the upper left triangle and R-values in the ing an MI in males. These results further strengthened the lower right triangle. Scales for both measures of LD are hypothesis that the FLAP gene was an MI susceptibility gene. shown on the right. FIG. 11, right panel, depicts all haplo 25 types found in Icelandic population controls that have allelic SNP Haplotype Association to MI, and Subsequently frequency greater than 1%. The haplotype showing strongest to Stroke and PAOD association to MI in Iceland (Hapk) is bold. In an effort to identify haplotypes involving only SNP DETAILED DESCRIPTION OF THE INVENTION 30 markers that associate with MI, additional SNPs were iden tified by sequencing the FLAP gene and the region flanking Extensive genealogical information has been combined the gene. Currently, a total of 45 SNPs in 1343 patients and with powerful gene sharing methods to map a gene on chro 624 unrelated controls have been genotyped. Two correlated mosome 13q12-13 that is associated with myocardial infarc series of SNP haplotypes have been observed in excess in tion. A genome wide search for Susceptibility genes for MI. 35 patients, denoted as A and B in Table 7. The length of the using a framework map of 1000 microsatellite markers, haplotypes varies between 33 and 69 kb, and the haplotypes revealed a locus Suggestive of linkage on 13q12-13. Sixty cover one or two blocks of linkage disequilibrium. Both series families with 159 femaleMI patients that clustered within and of haplotypes (Hap A and HapB) contain the common allele G including 6 meiotic events were used in linkage analysis. At of the SNPSG13S25. HapC2, identified in the analysis of the first, only female MI patients were used in the linkage analy 40 North American cohort (see Example 13), also contains the sis in an effort to enrich for patients with stronger genetic allele G of the SNPSG13S25. All haplotypes in the A series factors contributing to their risk for MI. The epidemiological contain the SNP SG13S114, while all haplotypes in the B study of a population-based sample of Icelandic MI patients series contain the SNPSG13S106. In the B series, the hap had previously suggested that the genetic factors for MI might lotypes B4, B5, and B6 have a relative risk (RR) greater than be stronger for females than males, as the relative risk for 45 2 and with allelic frequencies above 10%. The haplotypes in siblings of female MI patients was significantly higher than the A series have slightly lower RR and lower p-values, but the relative risk for siblings of male probands (1.59 (CI 1.47 higher frequency (15-16%). The haplotypes in series B and A 1.73) vs. 1.35 (CI 1.28-1.42)) (unpublished data). The highest are strongly correlated, i.e., the haplotypes in B define a LOD score (2.5) was found at marker D13S289. The LOD subset of the haplotypes in A. Hence, haplotypes in series B score results for the families remained the same after adding 50 are more specific than A. However, haplotypes in series A are 14 microsatellite markers to the candidate region. The inclu more sensitive, i.e., they capture more individuals with the sion of the additional markers increased the information on putative mutation, as is observed in the population attribut sharing by descent from 0.7 to 0.8, around the markers that able risk which is less for B than for A. Furthermore, these gave the highest LOD scores. This linkage analysis mapped a haplotypes show similar risk ratios and allelic frequencies for gene contributing to MI to chromosome 13q12-13. 55 early-onset patients (defined as onset of first MI before the The candidate MI locus on chromosome 13q12-13 was age of 55) and for both genders. In addition, analyzing various then finely mapped with microsatellite markers. Patients with groups of patients with known risk factors, such as hyperten myocardial infarction and controls were initially genotyped Sion, high cholesterol, Smoking and diabetes, does not reveal with microsatellite markers with an average spacing between any significant correlation with these haplotypes, suggesting markers of less than 100 kb over the 12 Mb candidate region. 60 that the haplotypes in the FLAP gene represent an indepen Initial haplotype association analysis that included all geno dent genetic susceptibility factor for MI. typed microsatellite markers across the MI candidate locus, Analysis of the North American cohort (described in resulted in several extended haplotypes composed of 4 and 5 Example 12) identified another haplotype C which is associ microsatellite markers that were significantly associated with ated with MI as demonstrated in Table 35 (Example 13). female MI (see, e.g., Tables 14 and 15 below). A region 65 HapC is defined by the Tallele of marker SG13S375. There common to all these extended haplotypes, is defined by mark are 4 additional variations of the HapC haplotype which ers DG13S166 and D13S1238. This region includes only one comprise SNPs in addition to the Tallele of SG13S375. US 8,158,362 B2 29 30 HapC2 is defined by allele T of the SNPs SG13S375 and leukotrienes from arachidonic acid. It catalyzes the conver allele G of the SNPSG13S25. HapC3 is defined by allele T of sion of arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11, the SNPS SG13S375 and allele G of the SNP SG13S25 and 14-cis-eicosatetraenoic acid (5-HPETE), and further to the allele A of SNPSG13S32. HapC4-A is defined by allele G of allylic epoxide 5 (S)-trans7.9 trans 11,14-cis-eicosatet the SNP SG13S106 in addition to allele T of the SNPs 5 raenoic acid (leukotriene A4, LTA4). SG13S375, allele G of the SNPSG13S25 and allele A of SNP The leukotrienes are a family of highly potent biological SG13S32. HapC4-B is defined by allele A of the SNP mediators of inflammatory processes produced primarily by SG13S106 in addition to allele T of the SNPs SG13S375, bone marrow derived leukocytes such as monocytes, mac allele G of the SNP SG13S25 and allele A of SNPSG13S32. rophages, and neurophils. Both FLAP and 5-LO are detected HapC4-A correlates with Hap A and HapB. 10 within atherosclerosis lesions (Proc Natl Acad Sci U S A. In addition, genetic analysis in Icelandic and North Ameri 2003 Feb. 4:100(3):1238-43.), indicating that the vessel itself can cohorts (Examples 17) identified LTA4H haplotypes can be a source of leukotrienes. It was found at first that the associated MI as demonstrated in Tables 42 and 44. HapK is MI-risk FLAP haplotype was associated with higher serum defined by allele C of the SNPs SG12S16, allele G of the leukotriene levels. Increased production of leukotriene in SNPs SG12S21, allele T of the SNPs SG12S23, allele A of the 15 individuals with pre-existing atherosclerosis lesions may lead SNPs SG12S25, allele T of the SNPs SG12S26, allele Tof the to plaque instability or friability of the fibrous cap leading to SNPs SG12S100, allele T of the SNPs SG12S28, allele C of local thrombotic events. If this occurs in coronary artery the SNPs SG12S143, allele G of the SNPs SG12S144, and arteries it leads to MI or unstable angina. If it occurs in the allele G of the SNPS SG12S221. cerebrovasculature it leads to stroke or transient ischemic For Icelandic and European American cohorts, essentially 20 attack. If it occurs in large arteries to the limbs, it causes or the HapKassociation to MI can be obtained by analyzing the exacerbates limb ischemia in persons with peripheral arterial following 5 SNPs: allele A of the SNPs SG12S25, allele T of occlusive disease (PAOD). Therefore, those with genetically the SNPs SG12S26, allele C of the SNPs SG12S143, allele G influenced predisposition to produce higher leukotriene lev of the SNPs SG12S144, and allele G of the SNPs SG12S221. els have higher risk for events due to pre-existing atheroscle However, for African American cohorts, the HapK associa- 25 rosis such as MI. tion to MI can be obtained analyzing the following 7 SNPs: Inhibitors of FLAP function impede translocation of 5-LO allele A of the SNPs SG12S25, allele Tof the SNPs SG12S26, from the cytoplasm to the cell membrane and inhibit activa allele T of the SNPs SG12S100, allele T of the SNPs tion of 5-LO and thereby decrease leukotriene synthesis. SG12S28, allele C of the SNPs SG12S143, allele G of the As a result of these discoveries, methods are now available SNPs SG12S144, and allele G of the SNPs SG12S221. The 30 for the treatment of myocardial infarction (MI) and acute addition of these two SNPs to the 5 SNPs that define HapK coronary syndrome (ACS), as well as stroke and PAOD, adds additional information to improve accuracy of determi through the use of leukotriene inhibitors, such as agents that nation of the carrier status of a tested individual for this inhibit leukotriene biosynthesis or antagonize signaling haplotype. This extra information is more helpful in African through leukotriene receptors. The term, “treatment as used American cohorts than Caucasian since there is a greater 35 herein, refers not only to ameliorating symptoms associated diversity of haplotypes in African-Americans, although the with the disease or condition, but also preventing or delaying 5-SNP HapK has good accuracy for determining carrier sta the onset of the disease or condition; preventing or delaying tus in African-Americans. Single SNPs, such as SG12S551 the occurrence of a second episode of the disease or condi with allele T and SG12S540 with allele A, have been defined tion; and/or also lessening the severity or frequency of symp that are highly correlated to HapK and can in large measure 40 toms of the disease or condition. In the case of atherosclero substitute for HapK in terms of defining MI risk. sis, “treatment” also refers to a minimization or reversal of the HapD is defined by allele C of the SNPs SG12S16, allele G development of plaques. Methods are additionally available of the SNPs SG12S21, allele T of the SNPs SG12S23, allele for assessing an individual’s risk for MI, ACS, stroke or A of the SNPs SG12S25, allele T of the SNPs SG12S26, PAOD. In a preferred embodiment, the individual to be allele T of the SNPs SG12S100, allele T of the SNPs 45 treated is an individual who is susceptible (at increased risk) SG12S28, allele T of the SNPs SG12S143, allele A of the for MI, ACS, stroke or PAOD, such as an individual who is in SNPs SG12S144, and allele G of the SNPs SG12S221. HapO one of the representative target populations described herein. is defined by allele C of the SNPs SG12S16, allele G of the SNPs SG12S21, allele T of the SNPs SG12S23, allele G of Representative Target Populations the SNPs SG12S25, allele T of the SNPs SG12S26, allele C of 50 the SNPs SG12S100, allele T of the SNPs SG12S28, allele T In one embodiment of the invention, an individual who is at of the SNPs SG12S143, allele A of the SNPs SG12S144, and risk for MI, ACS, stroke or PAOD is an individual who has an allele G of the SNPS SG12S221. at-risk haplotype in FLAP, as described herein. In one Because stroke and PAOD are diseases that are closely embodiment, a haplotype associated with a susceptibility to related to MI (all occur on the basis of atherosclerosis), the 55 myocardial infarction, ACS, stroke or PAOD comprises SNP haplotype in the FLAP gene that confers risk to MI was markers SG13S99, SG13S25, SG13S377, SG13S106, assessed to determine whether it also conferred risk of stroke SG13S32 and SG13S35 at the 13q12-13 locus. In another and/or PAOD. Table 21 shows that haplotype A4 increases the embodiment, a haplotype associated with a susceptibility to risk of having a stroke to a similar extent as it increases the myocardial infarction, ACS, stroke or PAOD comprises risk of having an MI. Table 37 demonstrates that Hap A is 60 markers SG13S99, SG13S25, SG13S106, SG13S30 and associated with risk of stroke in a Scottish chort (Example SG13S42 at the 13q12 locus. In a third embodiment, a hap 14). Although not as significantly, haplotype A4 also confers lotype associated with a susceptibility to myocardial infarc risk of developing PAOD. tion, ACS, stroke or PAOD comprises markers SG13S25. The FLAP nucleic acid encodes a 5-lipoxygenase activat SG13S106, SG13S30 and SG13S42 at the 13q12-13 locus. In ing protein, which, in combination with 5-lipoxygenase 65 a fourth embodiment, a haplotype associated with a suscep (5-LO), is required for leukotriene synthesis. FLAP acts coor tibility to myocardial infarction, ACS, stroke or PAOD com dinately with 5-LO to catalyze the first step in the synthesis of prises markers SG13S99, SG13S25, SG13S114, SG13S89 US 8,158,362 B2 31 32 and SG13S32 at the 13q12-13 locus. In a fifth embodiment, a and/or arterial vessel wall. In another embodiment of the haplotype associated with a susceptibility to myocardial inf invention, an individual who is at risk for MI or ACS is an arction, ACS, stroke or PAOD comprises markers SG13S25, individual who has a polymorphism in an LTA4H gene, in SG13S114, SG13S89 and SG13S32 at the 13q12-13 locus. In which the presence of the polymorphism is indicative of a another embodiment, a haplotype associated with a suscepti 5 susceptibility to MI or ACS. The term “gene.” as used herein, bility to myocardial infarction, ACS, stroke or PAOD com refers to not only the sequence of nucleic acids encoding a prises marker SG13S375 at the 13q12-13 locus. In another polypeptide, but also the promoter regions, transcription embodiment, a haplotype associated with a susceptibility to enhancement elements, splice donor/acceptor sites, and other myocardial infarction, ACS, stroke or PAOD comprises non-transcribed nucleic acid elements. Representative poly markers SG13S25 and SG13S375 at the 13q12-13 locus. In 10 morphisms include those presented in Table 40. Along the another embodiment, a haplotype associated with a suscepti same lines, certain variants in the FLAP gene and other mem bility to myocardial infarction, ACS, stroke or PAOD com bers of the leukotriene biosynthetic and response pathway prises markers SG13S25, SG13S375 and SG13S32 at the (see, U.S. Provisional Application No. 60/419,432, filed on 13q12-13 locus. In an additional embodiment, a haplotype Oct. 17, 2002; U.S. patent application Ser. No. 10/829,674, associated with a Susceptibility to myocardial infarction, 15 filed on Apr. 22, 2004 (now U.S. Pat. No. 7,507,531) may ACS, stroke or PAOD comprises markers SG13S25, indicate one’s increased risk for MI and ACS. Other repre SG13S375, SG13S32 and SG13S106 at the 13q12-13 locus. sentatibe at-risk haplotypes are shown in Table 41 and Table Additional haplotypes associated with a susceptibility to 42. Additional "at-risk” haplotypes can be determined using myocardial infarction, ACS, stroke or PAOD include the hap linkage disequilibrium and/or haplotype blocks. lotypes shown in Tables 5, 7, 8, 9, 1014, 15, 17 and 19, as well In an embodiment, an individual who is at risk for MI, ACS, as haplotypes comprising markers shown in Table 13. stroke or PAOD is an individual who has an elevated inflam Increased risk for MI, ACS, stroke or PAOD in individuals matory marker. An "elevated inflammatory marker, as used with a FLAP at-risk haplotype is logically conferred by herein, is the presence of an amount of an inflammatory increased production of leukotrienes in the arterial vessel wall marker that is greater, by an amount that is statistically sig or in bone-marrow derived inflammatory cells within the 25 nificant, than the amount that is typically found in control blood and/or arterial vessel wall. It is shown herein that FLAP individual(s) or by comparison of disease risk in a population at-risk haplotypes are associated with higher production of associated with the lowest band of measurement (e.g., below LTB4 ex vivo. It is further shown herein that serum leukot the mean or median, the lowest quartile or the lowest quintile) riene levels (specifically, leukotriene E4) correlate with compared to higher bands of measurement (e.g., above the serum CRP levels in myocardial infarction patients. FLAP 30 mean or median, the second, third or fourth quartile; the genetic variation may drive high leukotriene levels (within second, third, fourth or fifth quintile). An “inflammatory the blood vessel and/or systemically), which in turn may drive marker” refers to a molecule that is indicative of the presence higher CRP levels which has been shown as a risk factor for of inflammation in an individual, for example, C-reactive MI. Accordingly, individuals with a FLAP at-risk haplotype protein (CRP), serum amyloid A, fibrinogen, leukotriene lev are likely to have elevated serum CRP as well as other serum 35 els (e.g., leukotriene B4, leukotriene C4), leukotriene inflammatory markers. The level of serum CRP or other metabolites (e.g., leukotriene E4), interleukin-6, tissue necro serum inflammatory markers can be used as a Surrogate for sis factor-alpha, Soluble vasculare cell adhesion molecules the level of arterial wall inflammation initiated by lipid depo (sVCAM), soluble intervascular adhesion molecules (sI sition and atherogenesis conferred by the presence of the CAM), E-selectin, matrix metalloprotease type-1, matrix at-risk FLAP haplotype. 40 metalloprotease type-2, matrix metalloprotease type-3. In another embodiment of the invention, an individual who matrix metalloprotease type-9, myeloperoxidase (MPO), is at risk for MI, ACS, stroke or PAOD is an individual who N-tyrosine) or other markers (see, e.g., Doggen, C.J. M. et al., has a polymorphism in a FLAP gene, in which the presence of J. Internal Med., 248:406-414 (2000); Ridker, P. M. et al., the polymorphism is indicative of a susceptibility to MI, ACS, New Englind. J. Med 1997:336:973-979, Rettersol, L. et al., stroke or PAOD. The term “gene, as used herein, refers to not 45 2002: 160:433-440; Ridker, P. M. et al., New England. J. only the sequence of nucleic acids encoding a polypeptide, Med., 2002: 347: 1557-1565; Bermudez, E. A. et al., Arte but also the promoter regions, transcription enhancement ele rioscler. Thromb. Vasc. Biol., 2002: 22:1668-167). In certain ments, splice donor/acceptor sites, and other non-transcribed embodiments, the presence of Such inflammatory markers nucleic acid elements. Representative polymorphisms can be measured in serum or urine. include those presented in Table 13, below. 50 In an embodiment, an individual who is at risk for MI, ACS, In a further embodiment of the invention, an individual stroke or PAOD is an individual who has increased LDL who is at risk for MI, ACS, stroke or PAOD is an individual cholesterol and/or decreased HDL cholesterol levels. For who has an at-risk polymorphism in the 5-LO gene in the example, the American Heart Association indicates that an promoter region, as described herein. LDL cholesterol level of less than 100 mg/dL is optimal; from In one embodiment of the invention, an individual who is at 55 100-129 mg/dL is near/above optimal; from 130-159 mg/dL risk for MI or ACS is an individual who has an at-risk haplo is borderline high; from 160-189 is high; and from 190 and up type in LTA4H, as described herein. In one embodiment, the is very high. Therefore, an individual who is at risk for MI, haplotype can comprise alleles 0, T, 0, and A, of markers ACS, stroke or PAOD because of an increased LDL choles DG12S1664, SG12S26, DG12S1666, and SG12S144, terol level is, for example, an individual who has more than respectively, at the 12q23 locus. This LTA4H "at-risk” hap 60 100 mg/dL cholesterol, such as an individual who has a near/ lotype is detected in over 76% of male patients who have above optimal level, a borderline high level, a high level or a previously had an MI, conferring an increased relative risk of very high level. Similarly, the American Heart Association 1.4 fold and in 72% of female MI patients with a relative risk indicates that an HDL cholesterol level of less than 40 mg/dL of 1.2. Increased risk for MI or ACS in individuals with an is a major risk factor for heart disease; and an HDL choles LTA4H at-risk haplotype is logically conferred by increased 65 terol level of 60 mg/dL or more is protective against heart production of leukotrienes in the arterial vessel wall or in disease. Thus, an individual who is at risk for MI, ACS, stroke bone-marrow derived inflammatory cells within the blood or PAOD because of a decreased HDL cholesterol level is, for US 8,158,362 B2 33 34 example, an individual who has less than 60 mg/dL HDL 13. In a certain embodiment, the haplotype can comprise one cholesterol. Such as an individual who has less than 40 mg/dL or more alleles (e.g., a haplotype containing a single SNP), HDL cholesterol. two or more alleles, three or more alleles, four or more alleles, In another embodiment, an individual who is at risk for MI, or five or more alleles. The genetic markers are particular ACS, stroke or PAOD is an individual who has increased 5 “alleles' at “polymorphic sites' associated with FLAP. A leukotriene synthesis. “Increased leukotriene synthesis,” as nucleotide position at which more than one sequence is pos used herein, indicates an amount of production of leukot sible in a population (eithera natural population or a synthetic rienes that is greater, by an amount that is statistically signifi population, e.g., a library of synthetic molecules), is referred cant, than the amount of production of leukotrienes that is typically found in control individual(s) or by comparison of to herein as a “polymorphic site'. Where a polymorphic site leukotriene production in a population associated with the 10 is a single nucleotide in length, the site is referred to as a lowest band of measurement (e.g., below the mean or median, single nucleotide polymorphism (“SNP). For example, if at the lowest quartile or the lowest quintile) compared to higher a particular chromosomal location, one member of a popula bands of measurement (e.g., above the mean or median, the tion has an adenine and another member of the population has second, third or fourth quartile; the second, third, fourth or a thymine at the same position, then this position is a poly fifth quintile). For example, the FLAP at-risk haplotypes 15 morphic site, and, more specifically, the polymorphic site is a correlate with increased serum leukotriene synthesis levels, SNP Polymorphic sites can allow for differences in and with increased production of leukotrienes ex vivo. An sequences based on Substitutions, insertions or deletions. individual can be assessed for the presence of increased leu Each version of the sequence with respect to the polymorphic kotriene synthesis by a variety of methods. For example, an site is referred to herein as an “allele' of the polymorphic site. individual can be assessed for an increased risk of MI, ACS, Thus, in the previous example, the SNP allows for both an stroke, PAOD or atherosclerosis, by assessing the level of a adenine allele and a thymine allele. leukotriene metabolite (e.g., LTE4) in a sample (e.g., serum, Typically, a reference sequence is referred to for a particu plasma or urine) from the individual. Samples containing lar sequence. Alleles that differ from the reference are blood, cells, or tissue can also be obtained from an individual referred to as “variant alleles. For example, the reference and used to assess leukotriene or leukotriene metabolite pro 25 FLAP sequence is described herein by SEQ ID NO: 1. The duction ex vivo under appropriate assay conditions. An term, “variant FLAP', as used herein, refers to a sequence that increased level of leukotriene metabolites, and/or an increased level of leukotriene production ex vivo, is indica differs from SEQ ID NO: 1, but is otherwise substantially tive of increased production of leukotrienes in the individual, similar. The genetic markers that make up the haplotypes and of an increased risk of MI, ACS, stroke, PAOD or athero described herein are FLAP variants. Sclerosis. 30 Additional variants can include changes that affect a In a further embodiment, an individual who is at risk for polypeptide, e.g., the FLAP polypeptide. These sequence MI, ACS, or stroke is an individual who has already experi differences, when compared to a reference nucleotide enced at least one MI, ACS event or stroke, or who has stable sequence, can include the insertion or deletion of a single angina, and is therefore at risk for a second MI, ACS event or nucleotide, or of more than one nucleotide, resulting in a stroke. In another embodiment, an individual who is at risk 35 frame shift; the change of at least one nucleotide, resulting in for MI, ACS, stroke or PAOD is an individual who has ath a change in the encoded amino acid; the change of at least one erosclerosis or who requires treatment (e.g., angioplasty, nucleotide, resulting in the generation of a premature stop stents, revascularization procedure) to restore blood flow in codon; the deletion of several nucleotides, resulting in a dele arteries. tion of one or more amino acids encoded by the nucleotides; Infurther embodiments, an individual who is at risk for MI, 40 the insertion of one or several nucleotides. Such as by unequal stroke or PAOD is an individual having asymptomatic ankle/ recombination or gene conversion, resulting in an interrup brachial index of less than 0.9; an individual who is at risk for tion of the coding sequence of a reading frame; duplication of stroke, is an individual who has had one or more transient all or a part of a sequence; transposition; or a rearrangement ischemic attacks; who has had transient monocular blindness; of a nucleotide sequence, as described in detail above. Such has had a carotid endarterectomy; or has asymptomatic sequence changes alter the polypeptide encoded by a FLAP carotid stenosis; an individual who is at risk for PAOD, is an nucleic acid. For example, if the change in the nucleic acid individual who has (or had) claudication, limb ischemia lead ing to gangrene, ulceration or amputation, or has had a revas sequence causes a frame shift, the frame shift can result in a cularization procedure. change in the encoded amino acids, and/or can result in the In additional embodiments, an individual who is at risk for generation of a premature stop codon, causing generation of MI, ACS, stroke or PAOD is an individual who has diabetes: a truncated polypeptide. Alternatively, a polymorphism asso hypertension; hypercholesterolemia; elevated triglycerides ciated with a susceptibility to MI, ACS, stroke or PAOD can (e.g., >200 mg/dl); elevated lp(a); obesity; ankle/brachial be a synonymous change in one or more nucleotides (i.e., a index (ABI) less than 0.9; and/or is a past or current smoker. change that does not result in a change in the amino acid Individuals at risk for MI, ACS, stroke or PAOD may fall sequence). Such a polymorphism can, for example, alter into more than one of these representative target populations. 55 splice sites, affect the stability or transport of mRNA, or For example, an individual may have experienced at least one otherwise affect the transcription or translation of the MI, ACS event, transient ischemic attack, transient monocu polypeptide. The polypeptide encoded by the reference lar blindness, or stroke, and may also have an increased level nucleotide sequence is the “reference' polypeptide with a of an inflammatory marker. As used therein, the term “indi particular reference amino acid sequence, and polypeptides vidual in a target population” refers to an individual who is at 60 encoded by variant alleles are referred to as “variant” risk for MI, ACS, stroke or PAOD who falls into at least one polypeptides with variant amino acid sequences. of the representative target populations described above. Haplotypes are a combination of genetic markers, e.g., particular alleles at polymorphic sites. The haplotypes Assessment for At-Risk Haplotypes described herein, e.g., having markers such as those shown in 65 Table 13, are found more frequently in individuals with MI, A“haplotype.” as described herein, refers to a combination ACS, stroke or PAOD than in individuals without MI, ACS, of genetic markers (“alleles'), such as those set forth in Table stroke or PAOD. Therefore, these haplotypes have predictive US 8,158,362 B2 35 36 value for detecting a susceptibility to MI, ACS, stroke or ACS, stroke or PAOD comprises marker SG13S375 at the PAOD in an individual. The haplotypes described herein are 13q12-13 locus. In another embodiment, a haplotype associ in Some cases a combination of various genetic markers, e.g., ated with a susceptibility to myocardial infarction, ACS, SNPs and microsatellites. Therefore, detecting haplotypes stroke or PAOD comprises markers SG13S25 and SG13S375 can be accomplished by methods known in the art for detect at the 13q12-13 locus. In another embodiment, a haplotype ing sequences at polymorphic sites, such as the methods associated with a Susceptibility to myocardial infarction, described above. ACS, stroke or PAOD comprises markers SG13S25, In certain methods described herein, an individual who is at SG13S375 and SG13S32 at the 13q12-13 locus. In an addi risk for MI, ACS, stroke or PAOD is an individual in whom an tional embodiment, a haplotype associated with a suscepti at-risk haplotype is identified. In one embodiment, the at-risk 10 bility to myocardial infarction, ACS, stroke or PAOD com haplotype is one that confers a significant risk of MI, ACS, prises markers SG13S25, SG13S375, SG13S32 and stroke or PAOD. In one embodiment, significance associated SG13S106. In other embodiments, the at-risk haplotype is with a haplotype is measured by an odds ratio. In a further selected from the group consisting of haplotype B4, B5, B6, embodiment, the significance is measured by a percentage. In A4, A5, C1, C2, C3, C4-A and C4-B. The at-risk haplotype one embodiment, a significant risk is measured as an odds 15 can also comprise a combination of the markers in the hap ratio of at least about 1.2, including by not limited to: 1.2, 1.3, lotypes B4, B5, B6, A4, A5, C1, C2, C3, C4-A and/or C4-B. 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. In a further embodiment, an In further embodiments, the at-risk haplotype can be haplo odds ratio of at least 1.2 is significant. In a further embodi type HapB. In other embodiments, the at-risk haplotype com ment, an odds ratio of at least about 1.5 is significant. In a prises a polymorphism shown in Table 13. further embodiment, a significant increase in risk is at least Standard techniques for genotyping for the presence of about 1.7 is significant. In a further embodiment, a significant SNPs and/or microsatellite markers can be used, such as increase in risk is at least about 20%, including but not limited fluorescent based techniques (Chen, et al., Genome Res. 9. to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 492 (1999)), PCR, LCR, Nested PCR and other techniques 70%, 75%, 80%, 85%, 90%, 95%, and 98%. In a further for nucleic acid amplification. In a preferred embodiment, the embodiment, a significant increase in risk is at least about 25 method comprises assessing in an individual the presence or 50%. In yet another embodiment, an at-risk haplotype has ap frequency of SNPs and/or microsatellites in, comprising por value <0.05. It is understood however, that identifying tions of the FLAP gene, wherein an excess or higher fre whether a risk is medically significant may also depend on a quency of the SNPs and/or microsatellites compared to a variety of factors, including the specific disease, the haplo healthy control individual is indicative that the individual is type, and often, environmental factors. 30 susceptible to MI, ACS, stroke or PAOD. See, for example, An at-risk haplotype in, or comprising portions of the Table 13 (below) for SNPs and markers that can form haplo FLAP gene, in one where the haplotype is more frequently types that can be used as screening tools. These markers and present in an individual at risk for MI, ACS, stroke or PAOD SNPs can be identified in at-risk haploptypes. For example, (affected), compared to the frequency of its presence in a an at-risk haplotype can include microsatellite markers and/ healthy individual (control), and wherein the presence of the 35 or SNPs such as those set forth in Table 13. The presence of haplotype is indicative of susceptibility to MI, ACS, stroke or the haplotype is indicative of a susceptibility to MI, ACS, PAOD. As an example of a simple test for correlation would stroke or PAOD, and therefore is indicative of an individual be a Fisher-exact test on a two by two table. Given a cohort of who falls within a target population for the treatment methods chromosomes the two by two table is constructed out of the described herein. number of chromosomes that include both of the haplotypes, 40 Haplotype analysis involves defining a candidate Suscep one of the haplotype but not the other and neither of the tibility locus using LOD scores. The defined regions are then haplotypes. ultra-fine mapped with microsatellite markers with an aver In certain embodiments, an at-risk haplotype is an at-risk age spacing between markers of less than 100 kb. All usable haplotype within or near FLAP that significantly correlates microsatellite markers that are found in public databases and with a haplotype Such as a halotype shown in Table 14, a 45 mapped within that region can be used. In addition, micro haplotype shown in Table 15; a haplotype shown in Table 19; satellite markers identified within the deCODE genetics haplotype B4; haplotype B5; haplotype B6; haplotype A4; sequence assembly of the human genome can be used. The haplotype A5; or haplotype HapB. In other embodiments, an frequencies of haplotypes in the patient and the control at-risk haplotype comprises an at-risk haplotype within or groups can be estimated using an expectation-maximization near FLAP that significantly correlates with susceptibility to 50 algorithm (Dempster A. et al., 1977. J. R. Stat. Soc. B, 39:1- myocardial infarction or stroke. In a particular embodiment, a 389). An implementation of this algorithm that can handle haplotype associated with a susceptibility to myocardial inf missing genotypes and uncertainty with the phase can be arction, ACS, stroke or PAOD comprises markers SG13S99, used. Under the null hypothesis, the patients and the controls SG13S25, SG13S377, SG13S106, SG13S32 and SG13S35 at are assumed to have identical frequencies. Using a likelihood the 13q12-13 locus. In another embodiment, a haplotype 55 approach, an alternative hypothesis is tested, where a candi associated with a Susceptibility to myocardial infarction, date at-risk-haplotype, which can include the markers ACS, stroke or PAOD comprises markers SG13S99, described herein, is allowed to have a higher frequency in SG13S25, SG13S106, SG13S30 and SG13S42 at the 13d 12 patients than controls, while the ratios of the frequencies of 13 locus. In a third embodiment, a haplotype associated with other haplotypes are assumed to be the same in both groups. a susceptibility to myocardial infarction, ACS, stroke or 60 Likelihoods are maximized separately under both hypotheses PAOD comprises markers SG13S25, SG13S106, SG13S30 and a corresponding 1-df likelihood ratio statistic is used to and SG13S42 at the 13q12-13 locus. In a fourth embodiment, evaluate the statistic significance. a haplotype associated with a Susceptibility to myocardial To look for at-risk-haplotypes in the 1-1 od drop, for infarction, ACS, stroke or PAOD comprises markers example, association of all possible combinations of geno SG13S99, SG13S25, SG13S114, SG13S89 and SG13S32 at 65 typed markers is studied, provided those markers span a prac the 13q12-13 locus. In another embodiment, a haplotype tical region. The combined patient and control groups can be associated with a Susceptibility to myocardial infarction, randomly divided into two sets, equal in size to the original US 8,158,362 B2 37 38 group of patients and controls. The haplotype analysis is then polypeptide activity and/or nucleic acid expression of another repeated and the most significant p-value registered is deter member of the leukotriene biosynthetic pathway (e.g., 5-LO; mined. This randomization scheme can be repeated, for LTC4S: LTA4H; LTE4DH). In still another embodiment, a example, over 100 times to construct an empirical distribution leukotriene synthesis inhibitor is an agent that alters activity of p-values. In a preferred embodiment, a p-value of <0.05 is 5 or metabolism of a leukotriene (e.g., an antagonist of a leu indicative of an at-risk haplotype. kotriene; an antagonist of a leukotriene receptor). In preferred A detailed discussion of haplotype analysis is described in embodiments, the leukotriene synthesis inhibitor alters activ International Application No. PCT/US03/32556, filed on ity and/or nucleic acid expression of FLAP or of 5-LO, or Oct. 16, 2003, which is incorporated by reference herein in its alters interaction between FLAP and 5-LO. entirety. 10 Leukotriene synthesis inhibitors can alter polypeptide activity or nucleic acid expression of a member of the leukot Methods of Treatment riene pathway by a variety of means, such as, for example, by catalytically degrading, downregulating or interfering with The present invention encompasses methods of treatment the expression, transcription or translation of a nucleic acid (prophylactic and/or therapeutic, as described above) for MI, 15 encoding the member of the leukotriene pathway; by altering ACS, stroke or PAOD in individuals, such as individuals in posttranslational processing of the polypeptide; by altering the target populations described above, as well as for other transcription of splicing variants; or by interfering with diseases and conditions associated with FLAP or with other polypeptide activity (e.g., by binding to the polypeptide, or by members of the leukotriene pathway (e.g., for atherosclero binding to another polypeptide that interacts with that mem sis). Members of the “leukotriene pathway,” as used herein, ber of the leukotriene pathway, such as a FLAP binding agent include polypeptides (e.g., enzymes, receptors) and other as described herein or some other binding agent of a member molecules that are associated with production of leukot of the leukotriene pathway; by altering interaction among two rienes: for example, proteins or enzymes Such as FLAP. or more members of the leukotriene pathway (e.g., interaction 5-LO, other leukotriene biosynthetic enzymes (e.g., leukot between FLAP and 5-LO); or by antagonizing activity of a riene C4 synthase, leukotriene A4 hydrolase); receptors or 25 member of the leukotriene pathway. binding agents of the enzymes; leukotrienes such as LTA4. Representative leukotriene synthesis inhibitors include the LTB4, LTC4, LTD4, LTE4; and receptors of leukotrienes following: agents that inhibit activity of a member of the (e.g., leukotriene B4 receptor 1 (BLT1), leukotriene B4 leukotriene biosynthetic pathway (e.g., FLAP, 5-LO), receptor 2 (BLT2), cysteinyl leukotriene receptor 1 (Cys LTC4S, LTA4H, such as the agents presented in the Agent LTR1), cysteinyl leukotriene receptor 2 (CysLTR2)). 30 Tables I and II herein; agents that inhibit activity of receptors In particular, the invention relates to methods of treatment of members of the leukotriene pathway, such as FLAP recep for myocardial infarction or susceptibility to myocardial inf tors, LTA4 receptors, LTB4 receptors (e.g. BLT 1, BLT2), arction (for example, for individuals in an at-risk population LTC4 receptors, LTD4 receptors, LTE4 receptors, Cys LT1 such as those described above); as well as methods of treat receptors, Cys LT2 receptors, 5-LO receptors; BLT1: BLT2: ment for acute coronary syndrome (e.g., unstable angina, 35 CysLTR1. CysLTR2; agents that bind to the members of the non-ST-elevation myocardial infarction (NSTEMI) or ST leukotriene pathway, such as FLAP binding agents (e.g., elevation myocardial infarction (STEMI)); methods for 5-LO) or agents that bind to receptors of members of the reducing risk of MI, stroke or PAOD in persons with asymp leukotriene pathway (e.g., leukotriene receptor antagonists); tomatic ankle/brachial index less than 0.9; for decreasing risk agents that bind to a leukotriene (e.g., to LTA4, LTB4, LTC4. of a second myocardial infarction; for stroke or Susceptibility 40 LTD4, LTE4, Cys LT1, Cys LT2); agents that increase break to stroke; for transientischemic attack; for transient monocu down of leukotrienes (e.g., LTB4DH); or other agents that lar blindness; for decreasing risk of a second stroke; for otherwise affect (e.g., increase or decrease) activity of the PAOD or susceptibility to PAOD; for ABI less than 0.9; for leukotriene; claudication or limb ischemia; for atherosclerosis, such as for antibodies to leukotrienes; patients requiring treatment (e.g., angioplasty, stents, revas 45 antisense nucleic acids or Small double-stranded interfer cularization procedure) to restore blood flow in arteries (e.g., ing RNA, to nucleic acids encoding FLAP, 5-LO, LTA4H, or coronary, carotid, and/or femoral arteries); for treatment of a leukotriene synthetase or other member of the leukotriene asymptomatic ankle?brachial index of less than 0.9; and/or for pathway, or fragments or derivatives thereof, including anti decreasing leukotriene synthesis (e.g., for treatment of MI. sense nucleic acids to nucleic acids encoding the FLAP, 5-LO ACS, stroke or PAOD). The invention additionally pertains to 50 or leukotriene synthetase polypeptides, and vectors compris use of one or more leukotriene synthesis inhibitors, as ing Such antisense nucleic acids (e.g., nucleic acid, cDNA, described herein, for the manufacture of a medicament for the and/or mRNA, double-stranded interfering RNA, or a nucleic treatment of MI, ACS, stroke, PAOD and/or atherosclerosis, acid encoding an active fragment or derivative thereof, or an e.g., using the methods described herein. The invention also oligonucleotide; for example, the complement of one of SEQ provides for the use of one or more leukotriene synthesis 55 ID Nos. 1, 3, 718 or 719, or a nucleic acid complementary to inhibitors, as described herein, for the manufacture of a medi the nucleic acid encoding SEQID NO: 2 or 718, or fragments cament for reducing the risk for MI, ACS, PAOD, stroke or derivatives thereof); and/or artherosclerosis using the methods described herein. peptidomimetics; fusion proteins or prodrugs thereof; These medicaments may comprise a leukotriene synthesis ribozymes; other Small molecules; and inhibitor alone or in combination with a statin, as described 60 other agents that alter (e.g., inhibit or antagonize) expres herein. sion of a member of the leukotriene pathway, such as FLAP, In the methods of the invention, a “leukotriene synthesis 5-LO or LTA4H nucleic acid expression or polypeptide activ inhibitor is used. In one embodiment, a “leukotriene synthe ity, or that regulate transcription of FLAP splicing variants, sis inhibitor is an agent that inhibits FLAP polypeptide activ 5-LO splicing variants or LTA4H splicing variants(e.g., ity and/or FLAP nucleic acid expression, as described herein 65 agents that affect which splicing variants are expressed, or (e.g., a nucleic acid antagonist). In another embodiment, a that affect the amount of each splicing variant that is leukotriene synthesis inhibitor is an agent that inhibits expressed). US 8,158,362 B2 39 40 More than one leukotriene synthesis inhibitor can be used ficient to treat the disease or condition, such as by ameliorat concurrently, if desired. ing symptoms associated with the disease or condition, pre The therapy is designed to alter activity of a FLAP venting or delaying the onset of the disease or condition, polypeptide, a 5-LO polypeptide, or another member of the and/or also lessening the severity or frequency of symptoms leukotriene pathway in an individual. Such as by inhibiting or 5 antagonizing activity. For example, a leukotriene synthesis of the disease or condition). The amount which will be thera inhibitor can be administered in order to decrease synthesis of peutically effective in the treatment of a particular individu leukotrienes within the individual, or to downregulate or als disease or condition will depend on the symptoms and decrease the expression or availability of the FLAP nucleic severity of the disease, and can be determined by standard acid or specific splicing variants of the FLAP nucleic acid. clinical techniques. In addition, in vitro or in vivo assays may Downregulation or decreasing expression or availability of a 10 optionally be employed to help identify optimal dosage native FLAP nucleic acid or of a particular splicing variant ranges. The precise dose to be employed in the formulation could minimize the expression or activity of a defective will also depend on the route of administration, and the seri nucleic acid or the particular splicing variant and thereby ousness of the disease or disorder, and should be decided minimize the impact of the defective nucleic acid or the 15 according to the judgment of a practitioner and each patients particular splicing variant. Similarly, for example, a leukot circumstances. Effective doses may be extrapolated from riene synthesis inhibitor can be administered in order to dose-response curves derived from in vitro or animal model downregulate or decrease the expression or availability of the test systems. nucleic acid encoding 5-LO or specific splicing variants of the In preferred embodiments of the invention, the leukotriene nucleic acid encoding 5-LO. synthesis inhibitor agent is an agent that inhibits activity of The leukotriene synthesis inhibitor(s) are administered in a FLAP and/or of 5-LO. Preferred agents include the following, therapeutically effective amount (i.e., an amount that is Suf as set forth in Agent Table I below:

US 8,158,362 B2 43 44

VOWN

QUITENIboIULIOLIO

US 8,158,362 B2 49 50 In preferred methods of the invention, the agents set forth bonatoms; Z is thiazolyl, optionally substituted with alkyl of in the Agent Table III can be used for prophylactic and/or from one to six carbon atoms or haloalkyl of from one to six therapeutic treatment for diseases and conditions associated carbon atoms; L is selected from the group consisting of (a) with FLAP or with other members of the leukotriene path alkylene of from 1-6 carbonatoms, (b) alkenylene of from 2-6 way, or with increased leukotriene synthesis. In particular, carbon atoms, (c) alkynylene of from 2-6 carbon atoms, (d) they can be used for treatment for myocardial infarction or hydroxyalkyl of 1-6 carbon atoms, (e) >C=O, (f) >C=N Susceptibility to myocardial infarction, such as for individu OR, where R is hydrogen or C-C alkyl, (g) -(CHR1), als in an at-risk population as described above, (e.g., based on (CO)(CHR), where n and m are independently selected identified risk factors such as elevated cholesterol, elevated from an integer from one to six and R and R2 are indepen C-reactive protein, and/or genotype); for individuals suffer 10 dently selected from hydrogen and C-C-alkyl, (h) ing from acute coronary syndrome, Such as unstable angina, —(CHR),C=NOR, where R, R and n are as defined non-ST-elevation myocardial infarction (NSTEMI) or ST above: (i) —(CHR), ON=CR, where R, R and n are as: elevation myocardial infarction (STEMI); methods for reduc defined above; () —(CHR), O—(CHR), , where R. ing risk of MI, stroke or PAOD in persons with asymptomatic 15 R., n and m are as defined above, (k) —(CHR), NR ankle/brachial index less than 0.9; for decreasing risk of a (CHR), , where R, R2, n and mare as defined above and Subsequent myocardial infarction, Such as in individuals who R is selected from hydrogen and C-C-alkyl: (1)—(CHR), have already had one or more myocardial infarctions; for —S-CHR), , where R. R., n and m are as defined stroke or Susceptibility to stroke; for decreasing risk of a above; and (m) —(CHR), (SO)—(CHR), , where R. second stroke; for PAOD or susceptibility to PAOD; for treat R., n and m are as defined above: A is carbocyclic aryl ment of atherosclerosis, such as in patients requiring treat optionally substituted with alkyl of from one to six carbon ment (e.g., angioplasty, Stents, revascularization procedure) atoms, haloalkyl of from one to six carbon atoms, hydroxy to restore blood flow in arteries (e.g., coronary, carotid, and/or alkyl of from one to six carbonatoms, alkoxy of from one to femoral arteries); for treatment of asymptomatic ankle/bra twelve carbonatoms, alkoxyalkoxyl in which the two alkoxy chial index of less than 0.9; and/or for decreasing leukotriene 25 portions may each independently contain from one to six synthesis (e.g., for treatment of myocardial infarction, ACS, carbon atoms, alkylthio of from one to six carbon atoms, Stroke or PAOD hydroxy, halogen, cyano, amino, alkylamino of from one to In one preferred embodiment of the invention, the leukot six carbon atoms, dialkylamino in which the two alkyl groups riene synthesis inhibitor is an inhibitor of FLAP such as may independently contain from one to six carbon atoms, 1-((4-chlorophenyl)methyl)-3-((1,1-dimethylethyl)thio)-al 30 alkanoylamino of from two to eight carbon atoms, N-al pha,alpha-dimethyl-5-(2-quinolinylmethoxy)-1H-Indole-2- kanoyl-N-alkylamino in which the alkanoyl is of from two to propanoic acid otherwise known as MK-0591, (R)-(+)-alpha eight carbon atoms and the alkyl group is of from one to six cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid carbon atoms, alkylaminocarbonyl of from two to eight car otherwise known as BAY-X-1005, 3-(3-(1,1-dimethyleth bon atoms, dialkylaminocarbonyl in which the two alkyl ylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphe 35 groups are independently of from one to six carbon atoms, nyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-0-2- carboxyl, alkoxycarbonyl or from two to eight carbon atoms, acetic acid otherwise known as A-81834, their optically pure phenyl, optionally substituted with alkyl of from one to six enantiomers, salts, chemical derivatives, analogues, or other carbon atoms, haloalkyl of from one to six carbon atoms, compounds inhibiting FLAP that effectively decrease leukot alkoxy of from one to six carbon atoms, hydroxy or halogen, riene biosynthesis when administered to humans. 40 phenoxy, optionally substituted with alkyl of from one to six In another preferred embodiment of the invention, the leu carbon atoms, haloalkyl of from one to six carbon atoms, kotriene synthesis inhibitor is an inhibitor of 5LO such as alkoxy of from one to six carbon atoms, hydroxy or halogen, zileuton, atreleuton, 6-((3-fluoro-5-(tetrahydro-4-methoxy and phenylthio, optionally substituted with alkyl of from one 2H-pyran-4-yl)phenoxy)methyl)-1-methyl-2(1H)- to six carbon atoms, haloalkyl of from one to six carbon quinlollinone otherwise known as ZD-2138, 1-((4-chlorophe 45 atoms, alkoxy of from one to six carbon atoms, hydroxy or nyl)methyl)-3-((1,1dimethylethyl)thio)-alpha,alpha halogen. Preferably, the compound is a compound or phar dimethyl-5-(2-quinolinylmethoxy)-1H-Indole-2-propanoic maceutically acceptable Salt thereof having the name (R)— acid otherwise known as MK-886, 4-(3-(4-(2-Methyl-imida N-3--5-(4-fluorophenylmethyl)thiazo-2-yl)-1 methyl-2- Zol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-car propynyl)-N-hydroxyurea. See U.S. Pat. No. 4,615,596, boxylic acid amide otherwise known as CJ-13610, their opti 50 incorporated herein by reference. cally pureenantiomers, salts, chemical derivatives, analogues The compound is represented by the following formula: or other compounds inhibiting 5-LO that effectively decrease leukotriene biosynthesis when administered to humans. The compound can be represented by the following for mula: 55

A Z OM n1L

Y B -N NH2 60 or a pharmaceutically acceptable Salt thereof, wherein A is O selected from the group consisting of straight or branched divalent alkylene of from one to twelve carbon atoms and in M is selected from the group consisting of hydrogen, a divalent cycloalkylene of from three to eight carbon atoms: pharmaceutically acceptable cation, and a pharmaceutically 65 R is selected from the group consisting of hydrogen, alky acceptable metabolically cleavable group; B is a straight or lthio of from one to six carbon atoms, phenylthio, optionally branched divalent alkylene group of from one to twelve car substituted with alkyl of from one to six carbonatoms, alkoxy US 8,158,362 B2 51 52 of from one to six carbon atoms, or halogen, phenylalkylthio atoms, haloalkyl of from one to six carbon atoms, alkoxy of in which the alkyl portion contains from one to six carbon from one to six carbonatoms, hydroxy or halogen, phenoxy, atoms, and the phenyl group is optionally substituted with optionally substituted with alkyl of from one to six carbon alkyl of from one to six carbon atoms, alkoxy of from one to atoms, haloalkyl of from one to six carbon atoms, alkoxy of six carbon atoms, or halogen, R is selected from the group from one to six carbon atoms, hydroxy or halogen, or phe consisting of COOB wherein B is selected from hydrogen, a nylthio, optionally substituted with alkyl of from one to six pharmaceutically acceptable cation, or a metabolically cleav carbon atoms, haloalkyl of from one to six carbon atoms, able group, —COOalkyl where the alkyl portion contains alkoxy of from one to six carbon atoms, hydroxy or halogen. from one to six carbon atoms, —COOalkylcarbocyclicaryl Preferably, the compound is a compound or a pharmaceuti 10 cally acceptable salt thereof selected from the group consist where the alkylportion contains from one to six carbonatoms ing of N-3-(5-(4-fluorophenylmethyl)fur-2-yl)-3-butyn-2- and the aryl portion is optionally substituted with alkyl of yl)-N-hydroxyurea; N-3-(5-(4-fluorophenylmethyl)-2- from one to six carbon atoms, alkoxy of from one to six thienyl)-1-methyl-2-propynyl)-N-hydroxyurea; (R)-N-3- carbon atoms, or halogen, —CONRSR wherein Rs is (5-(4-fluorophenylmethyl)-2-thienyl)-1-methyl-2- selected from the group consisting of hydrogen, hydroxyl, 15 propynyl)-N-hydroxyurea; and (R) N-3-(5-(4- alkyl of from one to six carbonatoms, and alkoxy of from one chlorophenylmethyl)-2-thienyl)-1-methyl-2-propynyl)-N- to six carbon atoms, and R is selected from the group con hydroxyurea; (S) N-3-5-(4-fluorophenylmethyl)-2- sisting of hydrogen and alkyl of from one to six carbonatoms, thienyl-1-methyl-2-propynyl)-N-hydroxyurea. See U.S. —COR, and —OH: R is selected from the group consisting Pat. No. 5.288,751, incorporated by reference herein. of phenylalkyl in which the alkyl portion contains from one to The compound can be represented by the formula: six carbon atoms, and the phenyl group is optionally substi tuted with alkyl of from one to six carbon atoms, alkoxy of from one to six carbonatoms, or halogen, R is selected from RI O the group consisting of thiazolylalkyloxy in which the alkyl 21 N1N7 portion contains from one to six carbon atoms, and the het 25 eroaryl portion is optionally substituted with alkyl of from R" N N A1 NR6 one to six carbon atoms, alkoxy of from one to six carbon atoms, or halogen, and thiazolyloxy optionally Substituted with alkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, or halogen. See U.S. Pat. No. 5.288, 30 or a pharmaceutically acceptable Salt thereof, wherein A is 743, incorporated herein by reference. selected from the group consisting of straight or branched The compound can be represented by the formula: divalent alkylene of one to twelve carbon atoms, straight or branched divalent alkenylene of two to twelve carbon atoms, and divalent cycloalkylene of three to eight carbon atoms; R' An-7 S 'M 35 is alkylthio of one to six carbonatoms; R is selected from the N -N NH2 group consisting of hydrogen and alkyl of one to six carbon B atoms; R is selected from the group consisting of (carboxyl) alkyl in which the alkyl portion is of one to six carbon atoms, O (alkoxycarbonyl)alkyl in which the alkoxycarbonyl portion is 40 of two to six carbonatoms and the alkylportion is of one to six or a pharmaceutically acceptable salt thereof, wherein Mis carbon atoms, (aminocarbonyl)alkyl in which the alkyl por selected from the group consisting of hydrogen, and a phar tion is of one to six carbon atoms, ((alkylamino)carbonyl) maceutically acceptable cation; B is a straight or branched alkyl in which each alkyl portion independently is of one to divalent alkylene group of from one to twelve carbon atoms; six carbon atoms, and ((dialkylamino)carbonyl)alkyl in Z is selected from the group consisting of: (a) furyl, option 45 which eachalkylportion independently is of one to six carbon ally substituted with alkyl of from one to six carbonatoms, or atoms; R is phenylalkyl in which the alkyl portion is of one haloalkyl of from one to six carbon atoms, and (b) thienyl, to six carbon atoms; R is 2-, 3- or 6-quinolylmethoxy, optionally substituted with alkyl of from one to six carbon optionally substituted with alkyl of one to six carbon atoms, atoms, or haloalkyl of from one to six carbon atoms; and L is haloalkyl of one to six carbon atoms, alkoxy of one to twelve alkylene of from 1-6 carbon atoms; A is phenyl optionally 50 carbonatoms, halogen, or hydroxy. Preferably, the compound substituted with alkyl of from one to six carbon atoms, is selected from the group consisting of 3-(3-1,1-dimethyl haloalkyl of from one to six carbon atoms, hydroxyalkyl of ethylthio)-5-(quinolin-2-ylmethoxy-1-(4-chlorophenylm from one to six carbon atoms, alkoxy of from one to twelve ethyl)-indol-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2 carbon atoms, alkoxyalkoxyl in which the two alkoxy por acetic acid; 3-(3-(1,1-dimethylethylthio)-5-(quinolin-2-yl tions may each independently contain from one to six carbon 55 methoxy)-1-(4-chloro-phenylmethyl) indol-2-yl)-2,2-dim atoms, alkylthio of from one to six carbon atoms, hydroxy, ethylpropionaldehyde oxime-O-2-(3-methyl)butyric acid; halogen, cyano, amino, alkylamino of from one to six carbon 3-(3-(1,1-dimethylethylthio)-5-(6,7-dichloroquinolin-2-yl atoms, dialkylamino in which the two alkyl groups may inde methoxy)-1-(4-chlorophenylmethyl)indol-2-yl)-2,2-dimeth pendently contain from one to six carbon atoms, alkanoy ylpropionaldehyde oxime-O-2-acetic acid; and 3-(3-(1,1- lamino of from two to eight carbon atoms, N-alkanoyl-N- 60 dimethylethylthio)-5-(6-fluoroquinolin-2-ylmethoxy)-1- alkylamino in which the alkanoyl is of from two to eight (4chlorophenylmethyl) indol-2-yl)-2.2- carbonatoms and the alkyl group is of from one to six carbon dimethylpropionaldehyde oXime-O-2-propionic acid; or a atoms, alkylaminocarbonyl of from two to eight carbon pharmaceutically acceptable salt or ester thereof. See U.S. atoms, dialkylaminocarbonyl in which the two alkyl groups Pat. No. 5,459,150, incorporated by reference herein. are independently of from one to six carbon atoms, carboxyl, 65 The compound can be represented by the formula: alkoxycarbonyl of from two to eight carbon atoms, phenyl, optionally substituted with alkyl of from one to six carbon

US 8,158,362 B2 55 56 3-( 1-methyl-2-thioXo-1,2,3,4-tetrahydroquinolin-6- lmethoxy)phenyl-4-methoxytetrahydropyran, 4-(5-fluoro 3-(1-methyl-2-thioxo-1,2,3,4-tetrahydroquinolin-6-ylthio) phenyl-4-methoxytetrahydropyran and pharmaceutically (-CH) acceptable salt thereof. See EP 466452 B1, incorporated herein by reference. The compound can be a Substituted 4-(quinolin-2-61 wherein R represents hydrogen, lower alkyl orphenyland methoxy)phenylacetic acid derivative represented by the fol in denotes a number of 0 to 5, Z represents norbornyl, or lowing formula: 10 represents a group of the formula:

15 O

wherein R and R'' are identical or different and denote hydrogen, lower alkyl or phenyl, or R and R' can together form a saturated carbocyclic ring having up to 6 carbonatoms and m denotes a number from 1 to 6, and A and Bareidentical or different and denote hydrogen, lower alkyl or halogen, or a or pharmaceutically acceptable salt thereof, wherein R' pharmaceutically acceptable salt thereof. Preferably the com represents a group of the formula: 25 pounds are selected from the group consisting of 2-4- (quinolin-2-yl-methoxy)phenyl-2-cyclopentylacetic acid, 2-4-(duinolin-2-yl-methoxy)phenyl-2-cyclohexylacetic acid, and 2-4-(duinolin-2-yl-methoxy)phenyl-2-cyclohep -OR2 O tylacetic acid, (+)-enantiomer of 2-4-(duinolin-2-yl-meth 30 oxy)phenyl-2-cyclopentylacetic acid, (-)-enantiomer of 2-4-(duinolin-2-yl-methoxy)phenyl-2-cyclopentylacetic acid and pharmaceutically acceptable salts thereof. See U.S. RandR are identical ordifferent and represent hydrogen, Pat. No. 4,970.215, incorporated herein by reference. lower alkyl, phenyl, benzyl or a group of the formula: The compound can be represented by the formula:

R R5 N R4 X^n y 21 4. cio- N (CRRI), Y-(CRR)-Q R2 42NN V (O), R8

wherein R. R. R. R and R are independently hydrogen, R4 R4 halogen, lower alkyl, lower alkenyl, lower alkynyl, -CF3, CN, NO2, N3, C(OH)RR, CO2R, SR, -CH-COR5 -CH-CH-OR5 50 R4 -CH3, -CF3, C(O)H, X-R or X-R; R and R are -CH R7 independently: alkyl, —(CH2)uPh(R)2 or —(CH2)uTh(R)2: R4 r R is —CF3 or R; R is hydrogen or X R; each R is indepen 55 dently hydrogen or lower alkyl, or two Rs on same carbon - CH-O-F O O atom are joined to form a cycloalkyl ring of 3 to 6 carbon atoms; R is hydrogen, lower alkyl or —CH2R, R is lower alkyl or —(CH2)rR; R is —CF3 or R; R is hydrogen, —C(O) R. R. or two Rs on the same nitrogen may be joined to form 60 a monocyclic heterocyclic ring of 4 to 6 atoms containing up R" represents hydrogen, lower alkyl, phenyl or benzyl, to 2 heteroatoms chosen from O, S or N; R is hydrogen, which can optionally be substituted by hydroxyl, carboxyl, —CF3, lower alkyl, lower alkenyl, lower alkynyl or—(CH2) lower alkoxycarbonyl, lower alkylthio, heteroaryl or carbam rR; R is -(CH2)s-C(RR)-(CH2)s-R or -CH2C(O)NRR: oyl, R represents hydrogen, lower alkyl, phenylor benzyl, R' R is hydrogen or lower alkyl, R is a) a monocyclic or bicyclic represents a group of the formula—COR or CO R. R. 65 heterocyclic ring containing from 3 to 9 nuclear carbonatoms represents hydrogen, lower alkyl or phenyl, Y represents a and 1 or 2 nuclear hetero-atoms selected from N, S or O and group of the formula: with each ring in the heterocyclic radical being formed of 5 or

US 8,158,362 B2 59 60 3-N-(4-chlorobenzyl)-3-phenylacetyl-5-(quinolin-2-yl methylamino-carbonyl, ethylaminocarbonyl, iso-propylami methoxy)indol-2-yl)-2,2-dimethylpropanoic acid, nocarbonyl and the like. The term “alkylthio’ refers to an 3-N-(4-fluorobenzyl)-3-(3.3-dimethyl-1-oxo-1-butyl)-5- alkyl group, as defined above, attached to the parent molecu (quinolin-2-ylmethoxy)indol-2-yl)-2,2-dimethylpro lar moiety through a Sulfur atom and includes Such examples panoic acid, as methylthio, ethylthio, propylthio, n-, sec- and tert-bu 3-N-(4-bromobenzyl)-3-(3.3-dimethyl-1-oxo-1-butyl)-5- tylthio and the like. The term “alkanoyl represents an alkyl (quinolin-2-ylmethoxy)indol-2-yl)-2,2-dimethylpro group, as defined above, attached to the parent molecular panoic acid, moiety through a carbonyl group. Alkanoyl groups are exem 3-N-(4-iodobenzyl)-3-(3,3-dimethyl-1-oxo-l -butyl)-5- plified by formyl, acetyl, propionyl, butanoyl and the like. (quinolin-2-ylmethoxy)indol-2-yl)-2,2-dimethylpro 10 The term "alkanoylamino” refers to an alkanoyl group, as panoic acid, previously defined, attached to the parent molecular moiety 3-N-(4-chlorobenzyl)-3-(1,1-dimethylbutyl)-5-(quinolin-2- through a nitrogenatom. Examples of alkanoylamino include ylmethoxy)indol-2-yl)-2,2-dimethylpropanoic acid, formamido, acetamido, and the like. The term "N-alkanoyl 3-N-(4-chlorobenzyl)-3-(1,1-dimethylpropyl)-5-(quinolin N-alkylamino” refers to an alkanoyl group, as previously 2-ylmethoxy)indol-2-yl)-2,2-dimethylpropanoic acid, 15 defined, attached to the parent molecular moiety through an 3-N-(3-fluorobenzyl)-3-(1,1-dimethylethyl)-5-(quinolin-2- aminoalkyl group. Examples of N-alkanoyl-N-alkylamino ylmethoxy)indol-2-yl)-2,2-dimethylpropanoic acid, include N-methylformamido, N-methyl-acetamido, and the 3-N-(4-chlorobenzyl)-3-(3-methylethyl)-5-(quinolin-2-yl like. The terms “alkoxy” or “alkoxyl denote an alkyl group, methoxy)indol-2-yl)-2,2-dimethylpropanoic acid, as defined above, attached to the parent molecular moiety 3-N-(4-chlorobenzyl)-3-cyclopropyl-5-(quinolin-2-yl through an oxygen atom. Representative alkoxy groups methoxy)indol-2-yl)-2,2-dimethylpropanoic acid, include methoxyl, ethoxyl, propoxyl, butoxyl, and the like. 3-N-(4-chlorobenzyl)-3-(1-methyl-1-cyclopropyl)-5- The term “alkoxyalkoxyl refers to an alkyl group, as defined (quinolin-2-ylmethoxy)indol-2-yl)-2,2-dimethylpro above, attached through an oxygen to an alkyl group, as panoic acid, defined above, attached in turn through an oxygen to the 3-N-(4-chlorobenzyl)-3-cyclopentyl-5-(quinolin-2-yl 25 parent molecular moiety. Examples of alkoxyalkoxyl include methoxy)indol-2-yl)-2,2-dimethylpropanoic acid, methoxymethoxyl, methoxyethyoxyl, ethoxyethoxyl and the 3-N-(4-chlorobenzyl)-3-cyclohexyl-5-(quinolin-2-yl like. The term “alkoxyalkyl refers to an alkoxy group, as methoxy)indol-2-yl)-2,2-dimethylpropanoic acid, defined above, attached through an alkylene group to the 3-N-(4-chlorobenzyl)-3-(alpha-, alpha-dimethylbenzyl)-5- parent molecular moiety. The term “alkoxycarbonyl repre (quinolin-2-ylmethoxy)indol-2-yl)-2,2-dimethylpro 30 sents an ester group; i.e., an alkoxy group, attached to the panoic acid, parent molecular moiety through a carbonyl group Such as 3-N-(4-chlorobenzyl)-3-(2-4-chloro-alpha, alpha-dimeth methoxycarbonyl, ethoxycarbonyl, and the like. The term ylbenzyl-5-(quinolin-2-ylmethoxy)indol-2-yl)-2,2-dim1 “alkenyl denotes a monovalent group derived from a hydro ethylpropanoic acid, carbon containing at least one carbon-carbon double bond by 3-N-(4-chlorobenzyl)-3-(1-adamantyl)-5-(quinolin-2-yl 35 the removal of a single hydrogen atom. Alkenyl groups methoxy)indol-2-yl)-2,2-dimethylpropanoic acid, include, for example, ethenyl, propenyl, butenyl, 1-methyl 3-N-(4-chlorobenzyl)-3-((1-adamantyl)methyl)-5-(quino 2-buten-1-yl and the like. The term “alkylene' denotes a lin-2-ylmethoxy)indol-2-yl)-2,2-dimethylpropanoic acid, divalent group derived from a straight or branched chain 3-N-(1,1-dimethylethyl)-3-(4-chlorobenzyl)-6-(quinolin-2- saturated hydrocarbon by the removal of two hydrogen ylmethoxy)indol-2-yl)-2,2-dimethylpropanoic acid, 40 atoms, for example methylene, 1.2-ethylene, 1,1-ethylene, 3-N-(1,1-dimethylpropyl)-3-(4-chlorobenzyl)-6-(quino 1,3-propylene, 2,2-dimethylpropylene, and the like. The term line-2-ylmethoxy)indol-2-yl)-2,2-dimethylpropanoic “alkenylene' denotes a divalent group derived from a straight acid, or branched chain hydrocarbon containing at least one car 3-N-(4-chlorobenzyl)-3-(3.3-dimethyl-1-oxo-1-butyl)-5- bon-carbon double bond. Examples of alkenylene include (quinolin-2-ylmethoxy)indol-2-yl)-2,2-diethylpropanoic 45 -CH=CH-, -CH-CH=CH-, - C(CH)—CH-, acid, —CH-CH=CHCH , and the like. The term "cycloalky methyl 3-N-(4-chlorobenzyl)-3,6-bis(acetyl)-5-(quinolin-2- lene' refers to a divalent group derived from a saturated ylmethoxy)indol-2-yl)-2.2 dimethyl propanoate or carbocyclic hydrocarbon by the removal of two hydrogen methyl 3-N-(4-chlorobenzyl)-3,6-bis(cyclopropanecarbo atoms, for example cyclopentylene, cyclohexylene, and the nyl)-5-(quinolin-2-ylmethoxy)indol-2-yl)-2,2-dimethyl 50 like. The term “cycloalkyl denotes a monovalent group propanoate. See EP 419049 B1, incorporated herein by derived from a monocyclic or bicyclic Saturated carbocyclic reference. ring compound by the removal of a single hydrogen atom. The term “alkyl refers to a monovalent group derived Examples include cyclopropyl, cyclobutyl, cyclopentyl, from a straight or branched chain Saturated hydrocarbon by cyclohexyl, bicyclo2.2.1]heptanyl, and bicyclo2.2.2]octa the removal of a single hydrogen atom. Alkyl groups are 55 nyl. The term “alkynylene' refers to a divalent group derived exemplified by methyl, ethyl, n- and iso-propyl. n-, sec-, iso by the removal of two hydrogen atoms from a straight or and tert-butyl, and the like. The term “hydroxyalkyl repre branched chain acyclic hydrocarbon group containing a car sents an alkyl group, as defined above, Substituted by one to bon-carbon triple bond. Examples of alkynylene include three hydroxyl groups with the proviso that no more than one CH=CH , CH=CH-CH , CH=CH CH hydroxy group may be attached to a single carbonatom of the 60 (CH)—, and the like. The term “carbocyclic aryl' denotes a alkyl group. The term “alkylamino” refers to a group having monovalent carbocyclic ring group derived by the removal of the structure —NHR' wherein R' is alkyl, as previously a single hydrogen atom from a monocyclic or bicyclic fused defined, examples of alkylamino include methylamino, ethy or non-fused ring system obeying the “4n+2 p electron’ or lamino, iso-propylamino and the like. The term “alkylami Huckel aromaticity rule. Examples of carbocyclic aryl groups nocarbonyl refers to an alkylamino group, as previously 65 include phenyl, 1- and 2-maphthyl, biphenylyl, fluorenyl, and defined, attached to the parent molecular moiety through a the like. The term “(carbocyclic aryl)alkyl refers to a car carbonyl group. Examples of alkylaminocarbonyl include bocyclic aryl ring group as defined above, attached to the US 8,158,362 B2 61 62 parent molecular moiety through an alkylene group. Repre group. The term "heteroaryloxy' denotes a heteroaryl group, sentative (carbocyclic aryl)alkyl groups include phenylm as defined above, attached to the parent molecular moiety ethyl, phenylethyl, phenylpropyl, 1-naphthylmethyl, and the through an oxygen atom. The term "heteroarylalkoxy” like. The term “carbocyclicarylalkoxy” refers to a carbocy denotes a heteroarylalkyl group, as defined above, attached to clicarylalkyl group, as defined above, attached to the parent the parent molecular moiety through an oxygen atom. molecular moiety through an oxygen atom. The term "car bocyclic aryloxyalkyl refers to a carbocyclic aryl group, as Method of Reducing Risk Factors for Cardiovascular defined above, attached to the parent molecular moiety Disease through an oxygen atom and thence through an alkylene group. Such groups are exemplified by phenoxymethyl, 1 10 The present invention encompasses compositions and and 2-naphthyloxymethyl, phenoxyethyl and the like. The methods for reducing risk factors for MI, ACS, stroke, and/or term “(carbocyclic aryl)alkoxyalkyl denotes a carbocyclic PAOD. The method of reducing risk factors comprise admin aryl group as defined above, attached to the parent molecular istering a composition comprising a leukotriene synthesis moiety through an alkoxyalkyl group. Representative (car inhibitor, described in detail herein alone, or in combination bocyclic aryl)alkoxyalkyl groups include phenylmethoxym 15 with a statin, to an individuals at risk for any of these condi ethyl, phenylethoxymethyl, 1- and 2-maphthylmethoxyethyl, tions. Individuals at risk include the target population and the like. “Carbocyclic arylthioalkyl represents a car described herein, especially individuals with elevated CRP, bocyclic aryl group as defined above, attached to the parent and those at risk for other diseases and conditions associated molecular moiety through a sulfur atom and thence through with FLAP and/or other members of the leukotriene pathway. an alklyene group and are typified by phenylthiomethyl, 1 In particular, the invention encompasses methods of reducing and 2-maphthylthioethyl and the like. The term “dialky plasma CRP levels or plasma serum amyloid A levels com lamino” refers to a group having the structure —NR'R'" prising administering an effective amount of leukotriene wherein R' and R" are independently selected from alkyl, as inhibitor alone or in combination with a statin. previously defined. Additionally, R' and R" taken together Statins are competitive inhibitors of 3-hydroxy-3-methyl may optionally be —(CH), wherekk is an integer of from 25 glutarlcoenzyme A (HMG-CoA) reductase, the enzyme that 2 to 6. Examples of dialkylamino include, dimethylamino, converts HMG-CoA to the cholesterol precursor mevalonic diethylaminocarbonyl, methylethylamino, piperidino, and acid. Upon binding to the active site of HMG-CoA redu the like. The term “halo or halogen denotes fluorine, chlo catase, statins alter the conformation of the enzyme, thereby rine, bromine or iodine. The term “haloalkyl denotes an preventing it from attaining a functional structure. The con alkyl group, as defined above, having one, two, or three halo 30 formational change of the HMG-CoA reducatase active site gen atoms attached thereto and is exemplified by Such groups makes statin drugs very effective and specific. Inhibition of as chloromethyl, bromoethyl, trifluoromethyl, and the like. HMG-CoA reducatase reduces intracellular cholesterol syn The term "hydroxyalkyl represents an alkyl group, as thesis in hepatocytes. The reduction of intracellular choles defined above, substituted by one to three hydroxyl groups terol results in an increase in hepatic LDL receptors on the with the proviso that no more than one hydroxy group may be 35 cell surface, which in turn reduces the level of circulating attached to a single carbon atom of the alkyl group. The term LDL and its precursors, intermediate density lipoproteins “phenoxy' refers to a phenyl group attached to the parent (IDL) and very low density lipoproteins (VLDL). In addition, molecular moiety through an oxygen atom. The term "phe statins inhibit hepatic synthesis of apolipoprotein B-100, nylthio’ refers to a phenyl group attached to the parent which results in a decrease in the synthesis and Secretion of molecular moiety through a sulfur atom. The term "pyridy 40 triglyercide rich lipoproteins. Additional beneficial effects of loxy' refers to a pyridyl group attached to the parent molecu statins on lipid biosynthesis include inhibition of LDL oxi lar moiety throughan oxygenatom. The terms "heteroaryl' or dation, and inhibition of the expression of Scavenger recep "heterocyclic aryl as used herein refers to substituted or tors. Statins also reduce the accumulation of esterified cho unsubstituted 5- or 6-membered ring aromatic groups con lersterol into macrophages, increase endothelial cell nitric taining one oxygen atom, one, two, three, or four nitrogen 45 oxide synthesis, reduce inflammatory processes, increase the atoms, one nitrogen and one Sulfur atom, or one nitrogen and stability of artherosclerotic plaques, and restore platelet activ one oxygen atom. The term heteroaryl also includes bi-or ity and the coagulation process. tricyclic groups in which the aromatic heterocyclic ring is Because of their beneficial effects and high specificity, fused to one or two benzene rings. Representative heteroaryl statins have become some of the most prescribed medicines in groups are pyridyl, thienyl, indolyl pyrazinyl, isoquinolyl, 50 the Western industrialized world. In preferred embodiments pyrrolyl pyrimidyl, benzothienyl, furyl, benzobfuryl, imi of the invention, the statin is one of the following agents: dazolyl, thiazolyl, carbazolyl, and the like. The term “het roVuvastatin, fluvastatin, atorvastatin, lovastatin, simvastatin, eroarylalkyl denotes a heteroaryl group, as defined above, pravastatin or pitavastatin. These agents are described in attached to the parent molecular moiety through an alkylene detail in the Statin Agent Table III below.

US 8,158,362 B2 69 70 Mevastatin and related compounds are disclosed in U.S. agent therapy, and also reduces at least one other cardiovas Pat. No. 3,983,140. Lovastatin (mevinolin) and related com cular risk factor more effectively than single agent therapy pounds are disclosed in U.S. Pat. No. 4,231.938. Keto analogs alone. of mevinolin (lovastatin) are disclosed in European Patent In one variation, the invention provides a composition Application No. 0,142,146 A2, and quinoline and pyridine comprising a leukotriene synthesis inhibitor and a statin for derivatives are disclosed in U.S. Pat. Nos. 5,506,219 and simultaneous administration, e.g., in one dose. A composition 5,691322. in tablet, pill, or capsule form, including Sustained release Pravastatin and related compounds are disclosed in U.S. formulations, are specifically contemplated. In another varia Pat. No. 4.346,227. Simvastatin and related compounds are tion, a unit dose comprising a single dose of the leukotriene disclosed in U.S. Pat. Nos. 4,448,784 and 4,450,171. 10 synthesis inhibitor and a single dose of the statin, packaged Fluvastatin and related compounds are disclosed in U.S. together but not in admixture, is contemplated. In another Pat. No. 5,354,772. Cerivastatin and related compounds are variation, methods of the invention involve administering a disclosed in U.S. Pat. Nos. 5,006,530 and 5,177,080. Altorv composition comprising a leukotriene inhibitor and a com astatin and related compounds are disclosed in U.S. Pat. Nos. 15 position comprising a statin at the same or different times, 4,681,893: 5,273,995; 5,385,929 and 5,686,104. e.g., administering the leukotriene synthesis inhibitor before Pitavastatin (nisvastatin (NK-104) or itavastatin) and or after administration of a composition comprising a statin. related compounds are disclosed in U.S. Pat. No. 5,011,930. Compositions for and methods of administering the agents to Rosuvastatin (visastatin (ZD-4522)) and related compounds an individual continuously (e.g., through a patch or i.v.), one are disclosed in U.S. Pat. No. 5,260,440. to twelve times a day, once a day, every other day, twice a Other possible HMG CoA reductase molecules are week, weekly, or monthly for one or more weeks, months, or described in U.S. Pat. Nos. 5,753,675; 4,613,610; 4,686,237; years, or for the entire life of a patient, depending on the level 4,647,576; and 4,499,289; and British patent no. GB of risk for the individual, is specifically contemplated, to 2205837. manage serum CRP and other cardiovascular risk factor lev The patents cited in relation to statins or other agents iden 25 els. It is contemplated that these compositions will be used for tified herein describe how to make and use the statins/agents, treatment and lifestyle management plans for primary or sec as well as biochemically active homologs thereof, salts, pro ondary MI, ACS, stroke, or PAOD prevention. drugs, metabolites, and the like. Such patents are incorpo rated herein by reference in their entirety. Dosings for the Nucleic Acid Therapeutic Agents 30 statins also have been described in patent and trade literature In another embodiment, a nucleic acid of the invention; a (e.g., Physician's Desk Reference 2004, incorporated herein nucleic acid complementary to a nucleic acid of the invention; by reference) and by the manufacturers and clinical practitio or a portion of such a nucleic acid (e.g., an oligonucleotide as ners that prescribe them. Combination therapy using statin described below); or a nucleic acid encoding a member of the dosings similar to what is used when prescribing statins 35 leukotriene pathway (e.g., 5-LO), can be used in “antisense' alone, or less, is specifically contemplated. therapy, in which a nucleic acid (e.g., an oligonucleotide) Compositions comprising a leukotriene synthesis inhibitor which specifically hybridizes to the mRNA and/or genomic alone or in combination with a statin may comprises a leu DNA of a nucleic acid is administered or generated in situ. kotriene synthesis inhibitor in an amount effective to reduce a The antisense nucleic acid that specifically hybridizes to the risk factor such as CRP or serum amyloid A. Effective daily 40 mRNA and/or DNA inhibits expression of the polypeptide doses of the leukotriene synthesis inhibitors are between 0.01 encoded by that mRNA and/or DNA, e.g., by inhibiting trans mg and 100 g, more preferably 0.1 mg to 1 g, and all indi lation and/or transcription. Binding of the antisense nucleic vidual doses within these ranges are specifically contem acid can be by conventional base pair complementarity, or, for plated. Exemplary single adult doses include 10 mg, 25 mg. example, in the case of binding to DNA duplexes, through 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg,300 mg, 350 45 specific interaction in the major groove of the double helix. mg, 400 mg, 500 mg and 750 mg, from one to four times daily. An antisense construct can be delivered, for example, as an The compositions may comprise a statin in an amount effec expression plasmid as described above. When the plasmid is tive to reduce total serum cholesterol, serum LDL, and/or transcribed in the cell, it produces RNA that is complemen serum CRP. Effective daily doses are between 0.01 mg and tary to a portion of the mRNA and/or DNA that encodes the 100 g, more preferably 0.1 mg to 1 g, and all individual doses 50 polypeptide for the member of the leukotriene pathway (e.g., within these ranges are specifically contemplated. Exemplary FLAP or 5-LO). Alternatively, the antisense construct can be individual doses include 5 mg, 10 mg, 15mg, 20 mg, 30 mg. an oligonucleotide probe that is generated ex vivo and intro 40 mg 50 mg, 60 mg, and 80 mg, 100 mg, 150 mg, 200 mg. duced into cells; it then inhibits expression by hybridizing 250 mg, and 500 mg, from one to four times daily. with the mRNA and/or genomic DNA of the polypeptide. In Emerging evidence Suggests that elevated CRP is an inde 55 one embodiment, the oligonucleotide probes are modified pendent risk factor for adverse clinical outcomes. See, e.g., oligonucleotides that are resistant to endogenous nucleases, Ridkeret al., N. Engl.J.Med. 352: 1 (Jan. 6, 2005). In another e.g., exonucleases and/or endonucleases, thereby rendering variation, the invention provides compositions, unit doses, them stable in vivo. Exemplary nucleic acid molecules for use and methods of treatment where a leukotriene synthesis as antisense oligonucleotides are phosphoramidate, phospho inhibitor and a statin are included or administered in amounts 60 thioate and methylphosphonate analogs of DNA (see also that synergistically act to reduce serum CRP levels. Syner U.S. Pat. Nos. 5,176,996, 5,264,564 and 5,256,775). Addi gistically effective amounts are amounts that either (a) tionally, general approaches to constructing oligomers useful achieve a greater percentage reduction in CRP than is in antisense therapy are also described, for example, by Van achieved in an average patient using either type of agent der Kroletal. (Biotechniques 6:958-976 (1988)); and Steinet alone, at a safe and effective amount, or (b) reduces CRP a 65 al. (Cancer Res. 48:2659-2668 (1988)). With respect to anti comparable amount to single agent therapy, with fewer side sense DNA, oligodeoxyribonucleotides derived from the effects; or (c) reduces CRP a comparable amount to single translation initiation site are preferred. US 8,158,362 B2 71 72 To perform antisense therapy, oligonucleotides (mRNA, In another embodiment of the invention, small double cDNA or DNA) are designed that are complementary to stranded interfering RNA (RNA interference (RNAi)) can be mRNA encoding the polypeptide. The antisense oligonucle used. RNAi is a post-transcription process, in which double otides bind to mRNA transcripts and prevent translation. Stranded RNA is introduced, and sequence-specific gene Absolute complementarity, although preferred, is not 5 silencing results, though catalytic degradation of the targeted required. A sequence "complementary’ to a portion of an mRNA. See, e.g., Elbashir, S. M. et al., Nature 411:494-498 RNA, as referred to herein, indicates that a sequence has (2001); Lee, N. S., Nature Biotech. 19:500-505 (2002); Lee, sufficient complementarity to be able to hybridize with the S-K. et al., Nature Medicine 8(7):681-686 (2002); the entire RNA, forming a stable duplex; in the case of double-stranded teachings of these references are incorporated herein by ref 10 erence. RNAi is used routinely to investigate gene function in antisense nucleic acids, a single strand of the duplex DNA a high throughput fashion or to modulate gene expression in may thus be tested, or triplex formation may be assayed. The human diseases (Chi et al., PNAS, 100 (11):6343-6346 ability to hybridize will depend on both the degree of comple (2003)). Introduction of long double standed RNA leads to mentarity and the length of the antisense nucleic acid, as sequence-specific degradation of homologous gene tran described in detail above. Generally, the longer the hybridiz 15 scripts. The long double stranded RNA is metabolized to ing nucleic acid, the more base mismatches with an RNA it small 21-23 nucleotide siRNA (small interfering RNA). The may contain and still form a stable duplex (or triplex, as the siRNA then binds to protein complex RISC (RNA-induced case may be). One skilled in the art can ascertain a tolerable silencing complex) with dual function helicase. The helicase degree of mismatch by use of standard procedures. has RNAas activity and is able to unwind the RNA. The The oligonucleotides used in antisense therapy can be 20 unwound siRNA allows an antisense strand to bind to a DNA, RNA, or chimeric mixtures or derivatives or modified target. This results in sequence dependent degradation of versions thereof, single-stranded or double-stranded. The oli cognate mRNA. Aside from endogenous RNAi, exogenous gonucleotides can be modified at the base moiety, Sugar moi RNAi, chemically synthesized or recombinantly produced ety, orphosphate backbone, for example, to improve stability can also be used. Using non-intronic portions of the FLAP of the molecule, hybridization, etc. The oligonucleotides can 25 gene, such as corresponding mRNA portions of SEQID NO. include other appended groups such as peptides (e.g. for 1, or portions of SEQID NO: 3, target regions of the FLAP targeting host cell receptors in vivo), or agents facilitating gene that are accessible for RNAi are targeted and silenced. transport across the cell membrane (see, e.g., Letsinger et al., With this technique it is possible to conduct a RNAi gene Proc. Natl. Acad. Sci. USA 86:6553-6556 (1989); Lemaitreet walk of the nucleic acids of the FLAP gene and determine the al., Proc. Natl. Acad. Sci. USA 84:648-652 (1987); PCT Inter- 30 amount of inhibition of the protein product. Thus it is possible national Publication No. WO 88/09810) or the blood-brain to design gene-specific therapeutics by directly targeting the barrier (see, e.g., PCT International Publication No. WO mRNAs of the gene. 89/10134), or hybridization-triggered cleavage agents (see, Endogenous expression of a member of the leukotriene e.g., Krolet al., BioTechniques 6:958-976 (1988)) or interca pathway (e.g., FLAP, 5-LO) can also be reduced by inacti lating agents. (See, e.g., Zon, Pharm. Res. 5: 539-549 (1988)). 35 Vating or "knocking out the gene or its promoter using tar To this end, the oligonucleotide may be conjugated to another geted homologous recombination (e.g., see Smithies et al., molecule (e.g., a peptide, hybridization triggered cross-link Nature 317:230-234 (1985); Thomas & Capecchi, Cell ing agent, transport agent, hybridization-triggered cleavage 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989)). agent). For example, an altered, non-functional gene of a member of The antisense molecules are delivered to cells that express 40 the leukotriene pathway (or a completely unrelated DNA the member of the leukotriene pathway in vivo. A number of sequence) flanked by DNA homologous to the endogenous methods can be used for delivering antisense DNA or RNA to gene (either the coding regions or regulatory regions of the cells; e.g., antisense molecules can be injected directly into gene) can be used, with or without a selectable marker and/or the tissue site, or modified antisense molecules, designed to a negative selectable marker, to transfect cells that express the target the desired cells (e.g., antisense linked to peptides or 45 gene in vivo. Insertion of the DNA construct, via targeted antibodies that specifically bind receptors or antigens homologous recombination, results in inactivation of the expressed on the target cell Surface) can be administered gene. The recombinant DNA constructs can be directly systematically. Alternatively, in a preferred embodiment, a administered or targeted to the required site in vivo using recombinant DNA construct is utilized in which the antisense appropriate vectors, as described above. Alternatively, oligonucleotide is placed under the control of a strong pro- 50 expression of non-altered genes can be increased using a moter (e.g., pol III or pol II). The use of Such a construct to similar method: targeted homologous recombination can be transfect target cells in the patient results in the transcription used to insert a DNA construct comprising a non-altered of sufficient amounts of single stranded RNAs that will form functional gene, or the complement thereof, or a portion complementary base pairs with the endogenous transcripts thereof, in place of a gene in the cell, as described above. In and thereby prevent translation of the mRNA. For example, a 55 another embodiment, targeted homologous recombination vector can be introduced in vivo such that it is taken up by a can be used to insert a DNA construct comprising a nucleic cell and directs the transcription of an antisense RNA. Such a acid that encodes a polypeptide variant that differs from that vector can remain episomal or become chromosomally inte present in the cell. grated, as long as it can be transcribed to produce the desired Alternatively, endogenous expression of a member of the antisense RNA. Such vectors can be constructed by recom- 60 leukotriene pathway can be reduced by targeting deoxyribo binant DNA technology methods standard in the art and nucleotide sequences complementary to the regulatory region described above. For example, a plasmid, cosmid, YAC or of the member of the leukotriene pathway (i.e., the promoter viral vector can be used to prepare the recombinant DNA and/or enhancers) to form triple helical structures that prevent construct that can be introduced directly into the tissue site. transcription of the gene in target cells in the body. (See Alternatively, viral vectors can be used which selectively 65 generally, Helene, C., Anticancer Drug Des., 6(6):569-84 infect the desired tissue, in which case administration may be (1991); Helene, C. et al., Ann. N.Y Acad. Sci. 660:27-36 accomplished by another route (e.g., systemically). (1992); and Maher, L. J., Bioassays 14(12):807-15 (1992)). US 8,158,362 B2 73 74 Likewise, the antisense constructs described herein, by that is lower during or after treatment can be shown, for antagonizing the normal biological activity of one of the example, by decreased serum or urinary leukotrienes, or members of the leukotriene pathway, can be used in the decreased ex vivo production of leukotrienes, or decreased manipulation of tissue, e.g., tissue differentiation, both in leukotriene, metabolites. A level that is “significantly lower. vivo and for ex vivo tissue cultures. Furthermore, the anti- 5 as used herein, is a level that is less than the amount that is sense techniques (e.g., microinjection of antisense molecules, typically found in control individual(s), or is less in a com or transfection with plasmids whose transcripts are anti-sense parison of disease risk in a population associated with the with regard to a nucleic acid RNA or nucleic acid sequence) other bands of measurement (e.g., the mean or median, the can be used to investigate the role of one or more members of highest quartile or the highest quintile) compared to lower the leukotriene pathway in the development of disease-re- 10 bands of measurement (e.g., the mean or median, the other lated conditions. Such techniques can be utilized in cell cul quartiles; the other quintiles). ture, but can also be used in the creation of transgenic ani For example, in one embodiment of the invention, the level mals. of a leukotriene or leukotriene metabolite is assessed in an The therapeutic agents as described hereincan be delivered individual before treatment with a leukotriene synthesis in a composition, as described above, or by themselves. They 15 inhibitor; and during or after treatment with the leukotriene can be administered systemically, or can be targeted to a synthesis inhibitor, and the levels are compared. A level of the particular tissue. The therapeutic agents can be produced by a leukotriene or leukotriene metabolite during or after treat variety of means, including chemical synthesis; recombinant ment that is significantly lower than the level of the leukot production; in Vivo production (e.g., a transgenic animal, riene or leukotriene metabolite before treatment, is indicative such as U.S. Pat. No. 4,873.316 to Meade et al.), for example, 20 of efficacy of treatment with the leukotriene synthesis inhibi and can be isolated using standard means such as those tor. In another embodiment, production of a leukotriene or a described herein. In addition, a combination of any of the leukotriene metabolite is stimulated in a first test sample from above methods of treatment (e.g., administration of non-al the individual, using a calcium ionophore, before treatment tered polypeptide in conjunction with antisense therapy tar with a leukotriene synthesis inhibitor, and is also stimulated geting altered mRNA for a member of the leukotriene path- 25 in a second test sample from the individual, using a calcium way; administration of a first splicing variant in conjunction ionophore, during or after treatment with the leukotriene Syn with antisense therapy targeting a second splicing variant) thesis inhibitor, and the level of production in the first test can also be used. sample is compared with with the level of production of the The invention additionally pertains to use of such thera leukotriene or leukotriene metabolite in the second test peutic agents, as described herein, for the manufacture of a 30 sample. A level of the leukotriene or leukotriene metabolite in medicament for the treatment of MI, ACS, stroke, PAOD the second test sample that is significantly lower than the level and/or atherosclerosis, e.g., using the methods described of the leukotriene or leukotriene metabolite in the first test herein. sample, is indicative of efficacy of treatment with the leukot riene synthesis inhibitor. Monitoring Progress of Treatment 35 In another embodiment of the invention, an individual who is a member of a target population of individuals at risk for The current invention also pertains to methods of monitor MI, ACS, stroke or PAOD (e.g., an individual in a target ing the response of an individual. Such as an individual in one population described above. Such as an individual at-risk due of the target populations described above, to treatment with a to elevated C-reactive protein) can be assessed for response to leukotriene synthesis inhibitor. 40 treatment with a leukotriene synthesis inhibitor, by examin Because the level of inflammatory markers can be elevated ing levels of inflammatory markers in the individual. For in individuals who are in the target populations described example, levels of an inflammatory marker in an appropriate above, an assessment of the level of inflammatory markers of test sample (e.g., serum, plasma or urine) can be measured the individual both before, and during, treatment with the before, and during or after treatment with the leukotriene leukotriene synthesis inhibitor will indicate whether the treat- 45 synthesis inhibitor. The level of the inflammatory marker ment has successfully decreased production of leukotrienes before treatment is compared with the level of the inflamma in the arterial vessel wall or in bone-marrow derived inflam tory marker during or after treatment. The efficacy of treat matory cells. For example, in one embodiment of the inven ment is indicated by a decrease in the level of the inflamma tion, an individual who is a member of a target population as tory marker, that is, a level of the inflammatory marker during described above (e.g., an individual at risk for MI, ACS, 50 or after treatment that is significantly lower (e.g., significantly stroke or PAOD, such as an individual who is at-risk due to a lower), than the level of inflammatory marker before treat FLAP haplotype) can be assessed for response to treatment ment, is indicative of efficacy. Representative inflammatory with a leukotriene synthesis inhibitor, by examining leukot markers include: C-reactive protein (CRP), serum amyloid A. riene levels or leukotriene. metabolite levels in the individual. fibrinogen, serum SOD40L, a leukotriene (e.g., LTB4, LTC4. Blood, serum, plasma or urinary leukotrienes (e.g., leukot- 55 LTD4, LTE4), a leukotriene metabolite, interleukin-6, tissue riene E4, cysteinyl leukotriene 1), or ex vivo production of necrosis factor-alpha, Soluble vascular cell adhesion mol leukotrienes (e.g., in blood samples stimulated with a calcium ecules (sVCAM), soluble intervascular adhesion molecules ionophore to produce leukotrienes), or leukotriene metabo (sICAM), E-selectin, matrix metalloprotease type-1, matrix lites, can be measured before, and during or after treatment metalloprotease type-2, matrix metalloprotease type-3. with the leukotriene synthesis inhibitor. The leukotriene or 60 matrix metalloprotease type-9, myeloperoxidase (MPO), and leukotriene metabolite level before treatment is compared N-tyrosine. In a preferred embodiment, the marker is CRP, with the leukotriene or leukotriene metabolite level during or SCD4OL or MPO. after treatment. The efficacy of treatment is indicated by a The efficacy of treatment of a leukotriene synthesis inhibi decrease in leukotriene production: a level of leukotriene or tor may be monitored by measuring at-risk biomarkers in leukotriene metabolite during or after treatment that is sig- 65 plasma, serum or urine. Clinical assays are available for the nificantly lower than the level of leukotriene or leukotriene following biomarkers: CRP, serum amyloid A., IL-1B, IL-6, metabolite before treatment, is indicative of efficacy. A level IL-8, IL-10, TNF-C, SCD40L, E-selectin, P-selectin and US 8,158,362 B2 75 76 intracellular adhesion molecule-1, Vascular cell ashesion an agent that is a leukotriene synthesis inhibitor as described molecule-1. The relative risk of a cardiovascular event pre herein. For instance, a leukotriene synthesis inhibitor can be dicted by CRP levels is low risk has less thatn 1 mg/L, average formulated with a physiologically acceptable carrier or is 1.0-3.0 mg/L and high risk patients have greater than 3.0 excipient to prepare a pharmaceutical composition. The car mg/L. Thus, optimal therapeutic effect of a leukotriene Syn rier and composition can be sterile. The formulation should thesis inhibitor alone or in combination with a statin is reduc Suit the mode of administration. ing CRP level to 2.0 mg/L or lower. The invention also provides for compositions comprising a The efficacy of treatment of a statin is monitored by mea leukotriene synthesis inhibit, as set out in Agent Table I, and suring the level of total serum cholesterol, serum LDL and/or serum triglycerides. A level of serum total cholesterol, a statin, as set out in the Agent Table III. The leukotriene LDL-C and/or triglycerides during or after treatment, which O synthesis inhibitor and the statin may be coformulated with a is significantly lower than the level of total cholesterol, physiological acceptable carrier or expedient to prepare a LDL-C and/or triglycerides before treatment is indicative of pharmaceutical composition. This composition may be for the efficacy of the treatment. For cholesterol management mulation to deliver the leukotriene synthesis inhibitor and purposes, “high risk patients' have an LDL level of 130 statin in a single dose. The processes for the isolation and mg/Dl or higher and optimally the statin treatment will reduce 15 purification of statins and other HMG-CoA reductase inhibi the LDL level to less than 100 mg/dL. “Moderately-high risk tors include different combinations of extraction, chromatog patients' are those individuals with two or more risk factors raphy, lactonization and crystallization methods. Examples for coronary heart disease with a 10-20% risk of heart attack of formulations for statins, statin derivatives and statin Salts within ten years. Optimally, the statin treatment will keep the are found in the following, all incorporated by reference in LDL level under 129 mg/dL. More recent studies show an their entirety, U.S. Pat. Nos. 6,316,460, 6,589.959, RE37, additional benefit on morbidity and mortality when statin 314, 5,354,772, 5,356,896, 5,686,104, 5,969,156, 6,126,971, therapy decreased serum LDL-C to less than 70 mg/dL. (Rid 5,030,447, 5, 180,589, 5,622,985, 6,825,015, 6,838,566, ker et al., N. Engl. J. Med. 352(1):20-28, 2005; Nissenet al., 5,403,860, 5,763,653, and 5,763,646, International Patent N. Engl. J. Med. 352(1): 29-38, 2005). Thus optimal thera Publications WO 86/03488, WO 86/07054, French Patent peutic effect of a statin would be to lower LDL-C levels to 25 No. 2596393, European Patent Application No. 0221025, under 70 mg/dL. as described by Ridker et al., N. Engl. J. British Patent Nos. 2055100A and 2073199A and European Med. 352(1): 20-28, 2005 and Nissen et al., N. Engl. J. Med. 352(1): 29-38, 2005, statin therapy may reduce CRP, CRP is Patent No. 65,835. an additional parameter that may be monitored in connection Suitable pharmaceutically acceptable carriers include but with statin therapy. are not limited to water, Salt solutions (e.g., NaCl), Saline, 30 buffered saline, alcohols, glycerol, ethanol, gum arabic, Veg Assessment of Increased Risk etable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, The present invention additionally pertains to methods for magnesium Stearate, talc, silicic acid, viscous paraffin, per assessing an individual (e.g., an individual who is in a target fume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl population as described herein, Such as an individual who is at 35 pyrolidone, etc., as well as combinations thereof. The phar risk for MI, ACS, stroke or PAOD), for an increased risk of maceutical preparations can, if desired, be mixed with auxil MI, ACS, atherosclerosis, stroke, transient ischemic attack, iary agents, e.g., lubricants, preservatives, stabilizers, wetting transient monocular blindness, asymptomatic carotid steno agents, emulsifiers, salts for influencing osmotic pressure, sis, PAOD, claudication, or limb ischemia. The methods com buffers, coloring, flavoring and/or aromatic Substances and prise assessing the level of a leukotriene metabolite (e.g., 40 the like which do not deleteriously react with the active LTE4, LTD4, LTB4) in the individual, wherein an increased agents. level of leukotriene metabolite is indicative of an increased The composition, if desired, can also contain minor risk. The level can be measured in any appropriate tissue or amounts of wetting or emulsifying agents, or pH buffering fluid sample, such as blood, serum, plasma, or urine. In one agents. The composition can be a liquid solution, Suspension, particular embodiment, the sample comprises neutrophils. 45 emulsion, tablet, pill, capsule, Sustained release formulation, The level of the leukotriene metabolite can be measured by or powder. The composition can be formulated as a Supposi standard methods, such as the methods described herein. For example, in one embodiment, production of a leukotriene tory, with traditional binders and carriers such as triglycer metabolite is stimulated in a first test sample from the indi ides. Oral formulation can include standard carriers such as vidual, using a calcium ionophore. The level of production is pharmaceutical grades of mannitol, lactose, starch, magne compared with a control level. The control level is a level that 50 sium Stearate, polyvinyl pyrollidone, sodium saccharine, cel is typically found in control individual(s), such as individual lulose, magnesium carbonate, etc. who are not at risk for MI, ACS, stroke or PAOD; alterna Methods of introduction of these compositions include, but tively, a control level is the level that is found by comparison are not limited to, intradermal, intramuscular, intraperitoneal, of disease risk in a population associated with the lowest band intraocular, intravenous, Subcutaneous, topical, oral and of measurement (e.g., below the mean or median, the lowest 55 intranasal. Other suitable methods of introduction can also quartile or the lowest quintile) compared to higher bands of include gene therapy (as described below), rechargeable or measurement (e.g., above the mean or median, the second, biodegradable devices, particle acceleration devices ('gene third or fourth quartile; the second, third, fourth or fifth quin guns) and slow release polymeric devices. The pharmaceu tile). A level of production of the leukotriene metabolite that tical compositions of this invention can also be administered is significantly greater than the control level, is indicative of 60 as part of a combinatorial therapy with other agents. an increased risk. Individuals at increased risk are candidates The composition can be formulated in accordance with the for treatments described herein. routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, composi Pharmaceutical Compositions tions for intravenous administration typically are solutions in 65 sterile isotonic aqueous buffer. Where necessary, the compo The present invention also pertains to pharmaceutical com sition may also include a solubilizing agent and a local anes positions comprising agents described herein, for example, thetic to ease pain at the site of the injection. Generally, the US 8,158,362 B2 77 78 ingredients are Supplied either separately or mixed togetherin wherein one dose is to deliver a leukotriene synthesis inhibi unit dosage form, for example, as a dry lyophilized powder or tor and one dose is to deliver a statin either in parallel or one water free concentrate in a hermetically sealed container Such following the other. as an ampule or Sachette indicating the quantity of active Agents assembled in a blister pack or other dispensing agent. Where the composition is to be administered by infu- 5 means is preferred. For the purpose of this invention, unit Sion, it can be dispensed with an infusion bottle containing dosage is intended to mean a dosage that is dependent on the sterile pharmaceutical grade water, saline or dextrose/water. individual pharmacodynamics of each agent and adminis Where the composition is administered by injection, an tered in FDA approved dosages in Standard time courses. ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. 10 Nucleic Acids of the Invention For topical application, nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible FLAP Nucleic Acids, Portions and Variants with topical application and having a dynamic viscosity pref erably greater than water, can be employed. Suitable formu In addition, the invention pertains to isolated nucleic acid lations include but are not limited to Solutions, Suspensions, 15 molecules comprising a human FLAP nucleic acid. The term, emulsions, creams, ointments, powders, enemas, lotions, "FLAP nucleic acid, as used herein, refers to an isolated Sols, liniments, salves, aerosols, etc., which are, if desired, nucleic acid molecule encoding FLAP polypeptide. The sterilized or mixed with auxiliary agents, e.g., preservatives, FLAP nucleic acid molecules of the present invention can be stabilizers, wetting agents, buffers or salts for influencing RNA, for example, mRNA, or DNA, such as cDNA and osmotic pressure, etc. The agent may be incorporated into a 20 genomic DNA. DNA molecules can be double-stranded or cosmetic formulation. For topical application, also suitable single-stranded; single stranded RNA or DNA can be either are sprayable aerosol preparations wherein the active ingre the coding, or sense Strand or the non-coding, or antisense dient, preferably in combination with a solid or liquid inert Strand. The nucleic acid molecule can include all or a portion carrier material, is packaged in a squeeze bottle or in admix of the coding sequence of the gene or nucleic acid and can ture with a pressurized volatile, normally gaseous propellant, 25 further comprise additional non-coding sequences such as e.g., pressurized air. introns and non-coding 3' and 5' sequences (including regu Agents described herein can be formulated as neutral or latory sequences, for example, as well as promoters, tran salt forms. Scription enhancement elements, splice donor/acceptor sites, Pharmaceutically acceptable salts include those formed etc.). with free amino groups such as those derived from hydro- 30 For example, a FLAP nucleic acid can consist of SEQID chloric, phosphoric, acetic, oxalic, tartaric acids, etc., and NOs: 1 or 3 or the complement thereof, or to a portion or those formed with free carboxyl groups such as those derived fragment of such an isolated nucleic acid molecule (e.g., from Sodium, potassium, ammonium, calcium, ferric hydrox cDNA or the nucleic acid) that encodes FLAP polypeptide ides, isopropylamine, triethylamine, 2-ethylamino ethanol, (e.g., a polypeptide such as SEQ ID NO: 2). In a preferred histidine, procaine, etc. 35 embodiment, the isolated nucleic acid molecule comprises a The agents are administered in a therapeutically effective nucleic acid molecule selected from the group consisting of amount. The amount of agents which will be therapeutically SEQ ID NOs: 1 or 3, or their complement thereof. effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can LTA4H Nucleic Acids, Portions and Variant be determined by Standard clinical techniques. In addition, in 40 vitro or in Vivo assays may optionally be employed to help In addition, the invention pertains to isolated nucleic acid identify optimal dosage ranges. The precise dose to be molecules comprising a human LTA4H nucleic acid. The employed in the formulation will also depend on the route of term, “LTA4H nucleic acid, as used herein, refers to an administration, and the seriousness of the symptoms, and isolated nucleic acid molecule encoding LTA4H polypeptide. should be decided according to the judgment of a practitioner 45 The LTA4H nucleic acid molecules of the present invention and each patient’s circumstances. Effective doses may be can be RNA, for example, mRNA, or DNA, such as cDNA extrapolated from dose-response curves derived from in vitro and genomic DNA. DNA molecules can be double-stranded or animal model test systems. or single-stranded; single stranded RNA or DNA can be either The invention also provides a pharmaceutical pack or kit the coding, or sense Strand or the non-coding, or antisense comprising one or more containers filled with one or more of 50 Strand. The nucleic acid molecule can include all or a portion the ingredients of the pharmaceutical compositions of the of the coding sequence of the gene or nucleic acid and can invention. Optionally associated with Such container(s) can further comprise additional non-coding sequences such as be a notice in the form prescribed by a governmental agency introns and non-coding 3' and 5' sequences (including regu regulating the manufacture, use or sale of pharmaceuticals or latory sequences, for example, as well as promoters, tran biological products, which notice reflects approval by the 55 Scription enhancement elements, splice donor/acceptor sites, agency of manufacture, use of sale for human administration. etc.). The pack or kit can be labeled with information regarding For example, an LTA4H nucleic acid can consist of SEQID mode of administration, sequence of drug administration NOs: 718 or 719 or the complement thereof, or to a portion or (e.g., separately, sequentially or concurrently), or the like. fragment of Such an isolated nucleic acid molecule (e.g., The pack or kit may also include means for reminding the 60 cDNA or the nucleic acid) that encodes LTA4H polypeptide patient to take the therapy. The pack or kit can be a single unit (e.g., a polypeptide such as SEQID NO: 720). In a preferred dosage of the combination therapy or it can be a plurality of embodiment, the isolated nucleic acid molecule comprises a unit dosages. In particular, the agents can be separated, mixed nucleic acid molecule selected from the group consisting of together in any combination, present in a single vial or tablet. SEQ ID NOs: 718 or 719, or their complement thereof. For example, a pack or kit of the invention may contain a 65 Additionally, the nucleic acid molecules of the invention single dose for delivery of both a leukotriene synthesis inhibi can be fused to a marker sequence, for example, a sequence tor and a statin concurrently, or contain two or more doses that encodes a polypeptide to assistin isolation or purification US 8,158,362 B2 79 80 of the polypeptide. Such sequences include, but are not lim encoding variant polypeptides such as analogues or deriva ited to, those that encode a glutathione-S-transferase (GST) tives of a FLAP polypeptide. Such variants can be naturally fusion protein and those that encode a hemagglutinin A (HA) occurring, Such as in the case of allelic variation or single polypeptide marker from influenza. nucleotide polymorphisms, or non-naturally-occurring. Such An "isolated nucleic acid molecule, as used herein, is one as those induced by various mutagens and mutagenic pro that is separated from nucleic acids that normally flank the cesses. Intended variations include, but are not limited to, gene or nucleic acid sequence (as in genomic sequences) addition, deletion and substitution of one or more nucleotides and/or has been completely or partially purified from other that can result in conservative or non-conservative amino acid transcribed sequences (e.g., as in an RNA library). For changes, including additions and deletions. Preferably the example, an isolated nucleic acid of the invention may be 10 nucleotide (and/or resultantamino acid) changes are silent or substantially isolated with respect to the complex cellular conserved; that is, they do not alter the characteristics or milieu in which it naturally occurs, or culture medium when activity of a FLAP polypeptide. In one preferred embodi produced by recombinant techniques, or chemical precursors ment, the nucleotide sequences are fragments that comprise or other chemicals when chemically synthesized. In some one or more polymorphic microsatellite markers. In another instances, the isolated material will form part of a composi 15 preferred embodiment, the nucleotide sequences are frag tion (for example, a crude extract containing other Sub ments that comprise one or more single nucleotide polymor stances), buffer system or reagent mix. In other circum phisms in a FLAP nucleic acid (e.g., the single nucleotide stances, the material may be purified to essential polymorphisms set forth in Table 13, below). homogeneity, for example as determined by PAGE or column The present invention also pertains to nucleic acid mol chromatography such as HPLC. In certain embodiments, an ecules which are not necessarily found in nature but which isolated nucleic acid molecule comprises at least about 50, 80 encode a LTA4H polypeptide (e.g., a polypeptide having an or 90% (on a molar basis) of all macromolecular species amino acid sequence comprising an amino acid sequence of present. With regard to genomic DNA, the term "isolated SEQ ID NO: 720), or another splicing variant of a LTA4H also can refer to nucleic acid molecules that are separated polypeptide or polymorphic variant thereof. Thus, for from the chromosome with which the genomic DNA is natu 25 example, DNA molecules that comprise a sequence that is rally associated. For example, the isolated nucleic acid mol different from the naturally occurring nucleic acid sequence ecule can contain less than about 5 kb, including but not but which, due to the degeneracy of the genetic code, encode limited to 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides a LTA4H polypeptide of the present invention are also the which flank the nucleic acid molecule in the genomic DNA of Subjects of this invention. The invention also encompasses the cell from which the nucleic acid molecule is derived. 30 nucleotide sequences encoding portions (fragments), or The nucleic acid molecule can be fused to other coding or encoding variant polypeptides such as analogues or deriva regulatory sequences and still be considered isolated. Thus, tives of a LTA4H polypeptide. Such variants can be naturally recombinant DNA contained in a vector is included in the occurring, Such as in the case of allelic variation or single definition of "isolated as used herein. Also, isolated nucleic nucleotide polymorphisms, or non-naturally-occurring. Such acid molecules include recombinant DNA molecules in het 35 as those induced by various mutagens and mutagenic pro erologous host cells, as well as partially or Substantially puri cesses. Intended variations include, but are not limited to, fied DNA molecules in solution. “Isolated nucleic acid mol addition, deletion and substitution of one or more nucleotides ecules also encompass in vivo and in vitro RNA transcripts of that can result in conservative or non-conservative amino acid the DNA molecules of the present invention. An isolated changes, including additions and deletions. Preferably the nucleic acid molecule or nucleic acid sequence can include a 40 nucleotide (and/or resultantamino acid) changes are silent or nucleic acid molecule or nucleic acid sequence that is syn conserved; that is, they do not alter the characteristics or thesized chemically or by recombinant means. Therefore, activity of a LTA4H polypeptide. In one preferred embodi recombinant DNA contained in a vector is included in the ment, the nucleotide sequences are fragments that comprise definition of "isolated as used herein. Also, isolated nucle one or more polymorphic microsatellite markers. In another otide sequences include recombinant DNA molecules in het 45 preferred embodiment, the nucleotide sequences are frag erologous organisms, as well as partially or Substantially ments that comprise one or more single nucleotide polymor purified DNA molecules in solution. In vivo and in vitro RNA phisms in a LTA4H nucleic acid (e.g., the single nucleotide transcripts of the DNA molecules of the present invention are polymorphisms set forth in Table 37, below). also encompassed by "isolated nucleotide sequences. Such Other alterations of the nucleic acid molecules of the isolated nucleotide sequences are useful in the manufacture 50 invention can include, for example, labeling, methylation, of the encoded polypeptide, as probes for isolating homolo intemucleotide modifications such as uncharged linkages gous sequences (e.g., from other mammalian species), for (e.g., methyl phosphonates, phosphotriesters, phosphoami gene mapping (e.g., by in situ hybridization with chromo dates, carbamates), charged linkages (e.g., phosphorothio Somes), or for detecting expression of the nucleic acid in ates, phosphorodithioates), pendent moieties (e.g., polypep tissue (e.g., human tissue), such as by Northern blot analysis. 55 tides), intercalators (e.g., acridine, psoralen), chelators, The present invention also pertains to nucleic acid mol alkylators, and modified linkages (e.g., alpha anomeric ecules which are not necessarily found in nature but which nucleic acids). Also included are synthetic molecules that encode a FLAP polypeptide (e.g., a polypeptide having an mimic nucleic acid molecules in the ability to bind to a des amino acid sequence comprising an amino acid sequence of ignated sequence via hydrogen bonding and other chemical SEQ ID NOS: 2), or another splicing variant of a FLAP 60 interactions. Such molecules include, for example, those in polypeptide or polymorphic variant thereof. Thus, for which peptide linkages Substitute for phosphate linkages in example, DNA molecules that comprise a sequence that is the backbone of the molecule. different from the naturally occurring nucleic acid sequence The invention also pertains to nucleic acid molecules that but which, due to the degeneracy of the genetic code, encode hybridize under high Stringency hybridization conditions, a FLAP polypeptide of the present invention are also the 65 Such as for selective hybridization, to a nucleic acid sequence Subjects of this invention. The invention also encompasses described herein (e.g., nucleic acid molecules which specifi nucleotide sequences encoding portions (fragments), or cally hybridize to a nucleic acid sequence encoding polypep US 8,158,362 B2 81 82 tides described herein, and, optionally, have an activity of the lar Biology”, John Wiley & Sons, (1998), which describes the polypeptide). In one embodiment, the invention includes determination of Washing conditions for moderate or low variants described herein which hybridize under high strin stringency conditions. Washing is the step in which condi gency hybridization conditions (e.g., for selective hybridiza tions are usually set So as to determine a minimum level of tion) to a nucleic acid sequence comprising a nucleic acid complementarity of the hybrids. Generally, starting from the sequence selected from the group consisting of SEQID NOs: lowest temperature at which only homologous hybridization 1, 3, 718 and 719 or the complement thereof. In another occurs, each C. by which the final wash temperature is embodiment, the invention includes variants described herein reduced (holding SSC concentration constant) allows an which hybridize under high stringency hybridization condi increase by 1% in the maximum extent of mismatching tions (e.g., for selective hybridization) to a nucleic acid 10 among the sequences that hybridize. Generally, doubling the sequence encoding an amino acid sequence of SEQID NO: 2. concentration of SSC results in an increase in T of -17°C. SEQ ID NO: 720 or a polymorphic variant thereof. In a Using these guidelines, the washing temperature can be deter preferred embodiment, the variant that hybridizes under high mined empirically for high, moderate or low Stringency, stringency hybridizations has an activity of a FLAP. In depending on the level of mismatch sought. another preferred embodiment, the variant that hybridizes 15 For example, a low stringency wash can comprise washing under high Stringency hybridizations has an activity of a in a solution containing 0.2xSSC/0.1% SDS for 10 minutes at LTA4H. room temperature; a moderate stringency wash can comprise Such nucleic acid molecules can be detected and/or iso washing in a prewarmed solution (42°C.) Solution containing lated by specific hybridization (e.g., under high Stringency 0.2xSSC/0.1% SDS for 15 minutes at 42° C.; and a high conditions). “Specific hybridization,” as used herein, refers to stringency wash can comprise washing in prewarmed (68°C.) the ability of a first nucleic acid to hybridize to a second solution containing 0.1xSSC/0.1% SDS for 15 minutes at 68° nucleic acid in a manner Such that the first nucleic acid does C. Furthermore, washes can be performed repeatedly or not hybridize to any nucleic acid other than to the second sequentially to obtain a desired result as known in the art. nucleic acid (e.g., when the first nucleic acid has a higher Equivalent conditions can be determined by varying one or similarity to the second nucleic acid than to any other nucleic 25 more of the parameters given as an example, as known in the acid in a sample wherein the hybridization is to be per art, while maintaining a similar degree of identity or similar formed). "Stringency conditions” for hybridization is a term ity between the target nucleic acid molecule and the primer or of art which refers to the incubation and wash conditions, e.g., probe used. conditions of temperature and buffer concentration, which The percent homology or identity of two nucleotide or permit hybridization of a particular nucleic acid to a second 30 amino acid sequences can be determined by aligning the nucleic acid; the first nucleic acid may be perfectly (i.e., sequences for optimal comparison purposes (e.g., gaps can be 100%) complementary to the second, or the first and second introduced in the sequence of a first sequence for optimal may share some degree of complementarity that is less than alignment). The nucleotides or amino acids at corresponding perfect (e.g., 70%, 75%, 85%. 95%). For example, certain positions are then compared, and the percent identity between high Stringency conditions can be used which distinguish 35 the two sequences is a function of the number of identical perfectly complementary nucleic acids from those of less positions shared by the sequences (i.e., 96 identity—it of iden complementarity. “High Stringency conditions”, “moderate tical positions/total it of positionsx100). When a position in stringency conditions' and “low stringency conditions' for one sequence is occupied by the same nucleotide or amino nucleic acid hybridizations are explained on pages 2.10.1- acid residue as the corresponding position in the other 2.10.16 and pages 6.3.1-6.3.6 in Current Protocols in 40 sequence, then the molecules are homologous at that position. Molecular Biology (Ausubel, F. M. et al., “Current Protocols As used herein, nucleic acid or amino acid “homology is in Molecular Biology”, John Wiley & Sons, (1998), the entire equivalent to nucleic acid or amino acid “identity. In certain teachings of which are incorporated by reference herein). The embodiments, the length of a sequence aligned for compari exact conditions which determine the stringency of hybrid son purposes is at least 30%, for example, at least 40%, in ization depend not only on ionic strength (e.g., 0.2xSSC, 45 certain embodiments at least 60%, and in other embodiments 0.1 xSSC), temperature (e.g., room temperature, 42°C., 68° at least 70%, 80%, 90% or 95% of the length of the reference C.) and the concentration of destabilizing agents such as sequence. The actual comparison of the two sequences can be formamide or denaturing agents such as SDS, but also on accomplished by well-known methods, for example, using a factors such as the length of the nucleic acid sequence, base mathematical algorithm. A preferred, non-limiting example composition, percent mismatch between hybridizing 50 of such a mathematical algorithm is described in Karlin et al., sequences and the frequency of occurrence of Subsets of that Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an sequence within other non-identical sequences. Thus, equiva algorithm is incorporated into the NBLAST and XBLAST lent conditions can be determined by varying one or more of programs (version 2.0) as described in Altschuletal. Nucleic these parameters while maintaining a similar degree of iden Acids Res. 25:389-3402 (1997). When utilizing BLAST and tity or similarity between the two nucleic acid molecules. 55 Gapped BLAST programs, the default parameters of the Typically, conditions are used such that sequences at least respective programs (e.g., NBLAST) can be used. In one about 60%, at least about 70%, at least about 80%, at least embodiment, parameters for sequence comparison can be set about 90% or at least about 95% or more identical to each at score=100, wordlength=12, or can be varied (e.g., W=5 or other remain hybridized to one another. By varying hybrid W=20). ization conditions from a level of stringency at which no 60 Another preferred, non-limiting example of a mathemati hybridization occurs to a level at which hybridization is first cal algorithm utilized for the comparison of sequences is the observed, conditions which will allow a given sequence to algorithm of Myers and Miller, CABIOS 4(1): 11-17 (1988). hybridize (e.g., selectively) with the most similar sequences Such an algorithm is incorporated into the ALIGN program in the sample can be determined. (version 2.0) which is part of the GCG sequence alignment Exemplary conditions are described in Krause, M. H. and 65 software package (Accelrys, Cambridge, UK). When utiliz S. A. Aaronson, Methods in Enzymology 200: 546-556 ing the ALIGN program for comparing amino acid (1991), and in, Ausubel, et al., “Current Protocols in Molecu sequences, a PAM120 weight residue table, a gap length US 8,158,362 B2 83 84 penalty of 12, and a gap penalty of 4 can be used. Additional The nucleic acid molecules of the invention such as those algorithms for sequence analysis are known in the art and described above can be identified and isolated using standard include ADVANCE and ADAM as described in Torellis and molecular biology techniques and the sequence information Robotti, Comput. Appl. Biosci. 10:3-5 (1994); and FASTA provided herein. For example, nucleic acid molecules can be described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA amplified and isolated using the polymerase chain reaction 85:2444-8 (1988). and synthetic oligonucleotide primers based on one or more In another embodiment, the percent identity between two of SEQ ID NOs: 1 or 3, or the complement thereof, or amino acid sequences can be accomplished using the GAP designed based on nucleotides based on sequences encoding program in the GCG Software package using either a BLO one or more of the amino acid sequences provided herein. See 10 generally PCR Technology: Principles and Applications for SUM63 matrix or a PAM250 matrix, and a gap weight of 12, DNA Amplification (ed. H. A. Erlich, Freeman Press, NY. 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet another N.Y., 1992): PCR Protocols: A Guide to Methods and Appli embodiment, the percent identity between two nucleic acid cations (Eds. Innis et al., Academic Press, San Diego, Calif., sequences can be accomplished using the GAP program in 1990); Mattila et al., Nucl. Acids Res. 19:4967 (1991); Eckert the GCG Software package using a gap weight of 50 and a 15 et al., PCR Methods and Applications 1:17 (1991): PCR (eds. length weight of 3. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. The present invention also provides isolated nucleic acid 4,683.202. The nucleic acid molecules can be amplified using molecules that contain a fragment or portion that hybridizes cDNA, mRNA or genomic DNA as a template, cloned into an under highly stringent conditions to a nucleic acid sequence appropriate vector and characterized by DNA sequence comprising SEQID NO: 1 or 3 or the complement of SEQID analysis. NO: 1 or 3, and also provides isolated nucleic acid molecules Other suitable amplification methods include the ligase that contain a fragment or portion that hybridizes under chain reaction (LCR) (see Wu and Wallace, Genomics 4:560 highly stringent conditions to a nucleic acid sequence encod (1989), Landegren et al., Science 241: 1077 (1988), transcrip ing an amino acid sequence of the invention or polymorphic tion amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA variant thereof. The nucleic acid fragments of the invention 25 86: 1173 (1989)), and self-sustained sequence replication are at least about 15, for example, at least about 18, 20, 23 or (Guatelli et al., Proc. Nat. Acad. Sci. USA 87: 1874 (1990)) 25 nucleotides, and can be 30, 40, 50, 100, 200 or more and nucleic acid based sequence amplification (NASBA). nucleotides in length. Longer fragments, for example, 30 or The latter two amplification methods involve isothermal reac more nucleotides in length, encoding antigenic polypeptides tions based on isothermal transcription, which produce both described herein are particularly useful. Such as for the gen 30 single stranded RNA (ssRNA) and double stranded DNA eration of antibodies as described below. (dsDNA) as the amplification products in a ratio of about 30 Probes and Primers or 100 to 1, respectively. In a related aspect, the nucleic acid fragments of the inven The amplified DNA can be labeled, for example, radiola tion are used as probes or primers in assays such as those beled, and used as a probe for screening a cDNA library described herein. “Probes’ or “primers' are oligonucleotides 35 derived from human cells, mRNA in Zap express, ZIPLOX or that hybridize in a base-specific manner to a complementary other Suitable vector. Corresponding clones can be isolated, Strand of nucleic acid molecules. Such probes and primers DNA can obtained following in vivo excision, and the cloned include polypeptide nucleic acids, as described in Nielsen et insert can be sequenced in either or both orientations by art al., (Science 254: 1497-1500 (1991)). recognized methods to identify the correct reading frame A probe or primer comprises a region of nucleic acid that 40 encoding a polypeptide of the appropriate molecular weight. hybridizes to at least about 15, for example about 20-25, and For example, the direct analysis of the nucleic acid molecules in certain embodiments about 40, 50 or 75, consecutive nucle of the present invention can be accomplished using well otides of a nucleic acid of the invention, such as a nucleic acid known methods that are commercially available. See, for comprising a contiguous nucleic acid sequence of SEQ ID example, Sambrook et al., Molecular Cloning, A Laboratory NOs: 1 or 3 or the complement of SEQID Nos: 1 or 3, or a 45 Manual (2nd Ed., CSHP, New York 1989); Zyskind et al., nucleic acid sequence encoding an amino acid sequence of Recombinant DNA Laboratory Manual. (Acad. Press, SEQID NO: 2 or polymorphic variant thereof. In preferred 1988)). Using these or similar methods, the polypeptide and embodiments, a probe or primer comprises 100 or fewer the DNA encoding the polypeptide can be isolated, nucleotides, in certain embodiments, from 6 to 50 nucle sequenced and further characterized. otides, for example, from 12 to 30 nucleotides. In other 50 Antisense nucleic acid molecules of the invention can be embodiments, the probe or primer is at least 70% identical to designed using the nucleotide sequences of SEQID NOs: 1 or the contiguous nucleic acid sequence or to the complement of 3 and/or the complement of one or more of SEQID NOs: 1 or the contiguous nucleotide sequence, for example, at least 3 and/or a portion of one or more of SEQID NOs: 1 or 3 or the 80% identical, in certain embodiments at least 90% identical, complement of one or more of SEQID NOs: 1 or 3 and/or a and in other embodiments at least 95% identical, or even 55 sequence encoding the amino acid sequences of SEQ ID capable of selectively hybridizing to the contiguous nucleic NOs: 2 or encoding a portion of one or more of SEQID NOs: acid sequence or to the complement of the contiguous nucle 1 or 3 or their complement. They can be constructed using otide sequence. Often, the probe or primer further comprises chemical synthesis and enzymatic ligation reactions using a label, e.g., radioisotope, fluorescent compound, enzyme, or procedures known in the art. For example, an antisense enzyme co-factor. 60 nucleic acid molecule (e.g., an antisense oligonucleotide) can Particularly useful probes and primers of the invention are be chemically synthesized using naturally occurring nucle those which hybridize to marker locations (e.g. in the FLAP otides or variously modified nucleotides designed to increase gene) and those that permit amplication (e.g. using PCR) of a the biological stability of the molecules or to increase the small DNA fragment that include a marker of interest, espe physical stability of the duplex formed between the antisense cially markers that form haplotypes of the invention, Kits 65 and sense nucleic acids, e.g., phosphorothioate derivatives containing one or two or three or more of Such probes and and acridine Substituted nucleotides can be used. Alterna primers are contemplated as aspects of the invention. tively, the antisense nucleic acid molecule can be produced US 8,158,362 B2 85 86 biologically using an expression vector into which a nucleic cell. This means that the recombinant expression vectors acid molecule has been Subcloned in an antisense orientation include one or more regulatory sequences, selected on the (i.e., RNA transcribed from the inserted nucleic acid mol basis of the host cells to be used for expression, which is ecule will be of an antisense orientation to a target nucleic operably linked to the nucleic acid sequence to be expressed. acid of interest). 5 Within a recombinant expression vector, “operably linked' or The nucleic acid sequences can also be used to compare “operatively linked' is intended to mean that the nucleic acid with endogenous DNA sequences in patients to identify one sequence of interest is linked to the regulatory sequence(s) in or more of the disorders related to FLAP, and as probes, such a manner which allows for expression of the nucleic acid as to hybridize and discover related DNA sequences or to sequence (e.g., in an in vitro transcription/translation system Subtract out known sequences from a sample. The nucleic 10 or in a host cell when the vector is introduced into the host acid sequences can further be used to derive primers for cell). The term “regulatory sequence' is intended to include genetic fingerprinting, to raise anti-polypeptide antibodies promoters, enhancers and other expression control elements using DNA immunization techniques, and as an antigen to (e.g., polyadenylation signals). Such regulatory sequences raise anti-DNA antibodies or elicit immune responses. Por are described, for example, in Goeddel, “Gene Expression tions or fragments of the nucleotide sequences identified 15 Technology'. Methods in Enzymology 185, Academic Press, herein (and the corresponding complete gene sequences) can San Diego, Calif. (1990). Regulatory sequences include those be used in numerous ways as polynucleotide reagents. For which direct constitutive expression of a nucleic acid example, these sequences can be used to: (i) map their respec sequence in many types of host cell and those which direct tive genes on a chromosome; and, thus, locate gene regions or expression of the nucleic acid sequence only in certain host nucleic acid regions associated with genetic disease; (ii) iden- 20 cells (e.g., tissue-specific regulatory sequences). It will be tify an individual from a minute biological sample (tissue appreciated by those skilled in the art that the design of the typing); and (iii) aid in forensic identification of a biological expression vector can depend on Such factors as the choice of sample. Additionally, the nucleotide sequences of the inven the host cell to be transformed and the level of expression of tion can be used to identify and express recombinant polypep polypeptide desired. The expression vectors of the invention tides for analysis, characterization or therapeutic use, or as 25 can be introduced into host cells to thereby produce polypep markers for tissues in which the corresponding polypeptide is tides, including fusion polypeptides, encoded by nucleic acid expressed, either constitutively, during tissue differentiation, molecules as described herein. or in diseased States. The nucleic acid sequences can addi The recombinant expression vectors of the invention can be tionally be used as reagents in the screening and/or diagnostic designed for expression of a polypeptide of the invention in assays described herein, and can also be included as compo- 30 prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. nents of kits (e.g., reagent kits) for use in the screening and/or coli, insect cells (using baculovirus expression vectors), yeast diagnostic assays described herein. cells or mammalian cells. Suitable host cells are discussed Vectors further in Goeddel, supra. Alternatively, the recombinant Another aspect of the invention pertains to nucleic acid expression vector can be transcribed and translated in vitro, constructs containing a nucleic acid molecule of SEQ ID 35 for example using T7 promoter regulatory sequences and T7 NOs: 1, 3,718 or 719 or the complement thereof (or a portion polymerase. thereof). Yet another aspect of the invention pertains to Another aspect of the invention pertains to host cells into nucleic acid constructs containing a nucleic acid molecule which a recombinant expression vector of the invention has encoding an amino acid of SEQID NO: 2, SEQID NO: 720 been introduced. The terms "host cell' and “recombinant host or polymorphic variant thereof. The constructs comprise a 40 cell are used interchangeably herein. It is understood that vector (e.g., an expression vector) into which a sequence of such terms refer not only to the particular subject cell but also the invention has been inserted in a sense or antisense orien to the progeny or potential progeny of Such a cell. Because tation. As used herein, the term “vector” refers to a nucleic certain modifications may occur in Succeeding generations acid molecule capable of transporting another nucleic acid to due to either mutation or environmental influences, such which it has been linked. One type of vector is a “plasmid', 45 progeny may not, in fact, be identical to the parent cell, but are which refers to a circular double stranded DNA loop into still included within the scope of the term as used herein. which additional DNA segments can be ligated. Another type A host cell can be any prokaryotic or eukaryotic cell. For of vector is a viral vector, wherein additional DNA segments example, a nucleic acid molecule of the invention can be can be ligated into the viral genome. Certain vectors are expressed in bacterial cells (e.g., E. coli), insect cells, yeast or capable of autonomous replication in a host cell into which 50 mammaliancells (such as Chinese hamster ovary cells (CHO) they are introduced (e.g., bacterial vectors having a bacterial or COS cells). Other suitable host cells are known to those origin of replication and episomal mammalian vectors). skilled in the art. Other vectors (e.g., non-episomal mammalian vectors) are Vector DNA can be introduced into prokaryotic or eukary integrated into the genome of a host cell upon introduction otic cells via conventional transformation or transfection into the host cell, and thereby are replicated along with the 55 techniques. As used herein, the terms “transformation' and host genome. Moreover, certain vectors, such as expression “transfection' are intended to refer to a variety of art-recog vectors, are capable of directing the expression of genes or nized techniques for introducing a foreign nucleic acid mol nucleic acids to which they are operably linked. In general, ecule (e.g., DNA) into a host cell, including calcium phos expression vectors of utility in recombinant DNA techniques phate or calcium chloride co-precipitation, DEAE-dextran are often in the form of plasmids. However, the invention is 60 mediated transfection, lipofection, or electroporation. intended to include Such other forms of expression vectors, Suitable methods for transforming or transfecting host cells Such as viral vectors (e.g., replication defective retroviruses, can be found in Sambrook, et al. (Supra), and other laboratory adenoviruses and adeno-associated viruses) that serve manuals. equivalent functions. For stable transfection of mammalian cells, it is known Preferred recombinant expression vectors of the invention 65 that, depending upon the expression vector and transfection comprise a nucleic acid molecule of the invention in a form technique used, only a small fraction of cells may integrate suitable for expression of the nucleic acid molecule in a host the foreign DNA into their genome. In order to identify and US 8,158,362 B2 87 88 select these integrants, a gene or nucleic acid that encodes a genic animals described herein can also be produced accord selectable marker (e.g., for resistance to antibiotics) is gen ing to the methods described in Wilmut et al., Nature 385: erally introduced into the host cells along with the gene or 810-813 (1997) and PCT Publication Nos. WO 97/07668 and nucleic acid of interest. Preferred selectable markers include WO 97/O7669. those that confer resistance to drugs, such as G418, hygro mycin and methotrexate. Nucleic acid molecules encoding a Polypeptides of the Invention selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or The present invention pertains to isolated polypeptides can be introduced on a separate vector. Cells stably trans encoded by FLAP nucleic acids (“FLAP polypeptides’), and fected with the introduced nucleic acid molecule can be iden 10 fragments and variants thereof, as well as polypeptides tified by drug selection (e.g., cells that have incorporated the encoded by nucleotide sequences described herein (e.g., selectable marker gene or nucleic acid will survive, while the other splicing variants). The present invention also pertains to other cells die). isolated polypeptides encoded by LTA4H nucleic acids A host cell of the invention, Such as a prokaryotic host cell (“LTA4H polypeptides’), and fragments and variants thereof, or eukaryotic host cell in culture can be used to produce (i.e., 15 express) a polypeptide of the invention. Accordingly, the as well as polypeptides encoded by nucleotide sequences invention further provides methods for producing a polypep described herein (e.g., other splicing variants)The term tide using the host cells of the invention. In one embodiment, "polypeptide' refers to a polymer of amino acids, and not to the method comprises culturing the host cell of invention a specific length; thus, peptides, oligopeptides and proteins (into which a recombinant expression vector encoding a are included within the definition of a polypeptide. As used polypeptide of the invention has been introduced) in a Suit herein, a polypeptide is said to be “isolated' or “purified able medium such that the polypeptide is produced. In when it is substantially free of cellular material when it is another embodiment, the method further comprises isolating isolated from recombinant and non-recombinant cells, or free the polypeptide from the medium or the host cell. of chemical precursors or other chemicals when it is chemi The host cells of the invention can also be used to produce 25 cally synthesized. A polypeptide, however, can be joined to nonhuman transgenic animals. For example, in one embodi another polypeptide with which it is not normally associated ment, a host cell of the invention is a fertilized oocyte or an in a cell (e.g., in a “fusion protein') and still be "isolated” or embryonic stem cell into which a nucleic acid molecule of the “purified.” A detailed discussion of the methods to make a invention has been introduced (e.g., an exogenous FLAP polypeptide of the invention is provided in International nucleic acid, or an exogenous nucleic acid encoding a FLAP 30 Application No. PCT/US03/32556, filed on Oct. 16, 2003, polypeptide). Such host cells can then be used to create non which is incorporated by reference herein in its entirety. human transgenic animals in which exogenous nucleotide sequences have been introduced into the genome or homolo Antibodies of the Invention gous recombinant animals in which endogenous nucleotide sequences have been altered. Such animals are useful for 35 Polyclonal and/or monoclonal antibodies that specifically studying the function and/or activity of the nucleic acid bindone form of the polypeptide or nucleic acid product (e.g., sequence and polypeptide encoded by the sequence and for a polypeptide encoded by a nucleic acid having a SNP as set identifying and/or evaluating modulators of their activity. As forth in Table 13), but not to another form of the polypeptide used herein, a “transgenic animal' is a non-human animal, or nucleic acid product, are also provided. Antibodies are also preferably a mammal, more preferably a rodent Such as a rat 40 provided which bind a portion of either polypeptide encoded or mouse, in which one or more of the cells of the animal by nucleic acids of the invention (e.g., SEQID NO: 1 or SEQ include a transgene. Other examples of transgenic animals ID NO: 3 or SEQ ID NO: 718 or SEQID NO: 719, or the include non-human primates, sheep, dogs, cows, goats, complement thereof), or to a polypeptide encoded by nucleic chickens and amphibians. A transgene is exogenous DNA acids of the invention that contain a polymorphic site or sites. which is integrated into the genome of a cell from which a 45 The invention also provides antibodies to the polypeptides transgenic animal develops and which remains in the genome and polypeptide fragments of the invention, or a portion of the mature animal, thereby directing the expression of an thereof, or having an amino acid sequence encoded by a encoded gene product in one or more cell types or tissues of nucleic acid molecule comprising all or a portion of SEQID the transgenic animal. As used herein, a "homologous recom NOs: 1, 3, 718 or 719, or the complement thereof, or another binant animal' is a non-human animal, preferably a mammal, 50 variant or portion thereof. A detailed discussion of the meth more preferably a mouse, in which an endogenous gene has ods to make the antibodies of the invention is provided in been altered by homologous recombination between the International Application No. PCT/US03/32556, filed on endogenous gene and an exogenous DNA molecule intro Oct. 16, 2003, which is incorporated by reference herein in its duced into a cell of the animal, e.g., an embryonic cell of the entirety. animal, prior to development of the animal. 55 Methods for generating transgenic animals via embryo Diagnostic Assays manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are The nucleic acids, probes, primers, polypeptides and anti described, for example, in U.S. Pat. Nos. 4.736,866 and bodies described herein can be used in methods of diagnosis 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan, Manipu 60 of a susceptibility to MI, ACS, stroke or PAOD, or to another lating the Mouse Embryo (Cold Spring Harbor Laboratory disease or condition associated with an MI gene. Such as Press, Cold Spring Harbor, N.Y., 1986). Methods for con FLAP or LTA4H, as well as in kits useful for diagnosis of a structing homologous recombination vectors and homolo susceptibility to MI, ACS, stroke or PAOD, or to another gous recombinant animals are described further in Bradley, disease or condition associated with FLAP or LTA4H. In one Current Opinion in BioTechnology 2:823-829 (1991) and in 65 embodiment, the kit useful for diagnosis of susceptibility to PCT Publication Nos. WO 90/11354, WO 91/01140, WO MI, ACS, stroke or PAOD, or to another disease or condition 92/0968, and WO93/04169. Clones of the non-human trans associated with FLAP or LTA4H comprises primers as US 8,158,362 B2 89 90 described herein, wherein the primers contain one or more of in a FLAP nucleic acid, LTA4H nucleic acid or contains a the SNPs identified in Table 13 or Table 37. nucleic acid encoding a particular splicing variant of a FLAP In one embodiment of the invention, diagnosis of Suscep nucleic acid or a particular splicing variant of a LTA4H tibility to MI, ACS, stroke or PAOD (or diagnosis of suscep nucleic acid. The probe can be any of the nucleic acid mol tibility to another disease or condition associated with FLAP 5 ecules described above (e.g., the nucleic acid, a fragment, a or LTA4H), is made by detecting a polymorphism in a FLAP vector comprising the nucleic acid, a probe or primer, etc.). or LTA4H nucleic acid as described herein. The polymor To diagnose a susceptibility to MI, ACS, stroke or PAOD phism can be an alteration in a FLAP or LTA4H nucleic acid, (or another disease or condition associated with FLAP), the Such as the insertion or deletion of a single nucleotide, or of test sample containing a FLAP nucleic acid is contacted with more than one nucleotide, resulting in a frame shift alteration; 10 at least one nucleic acid probe to form a hybridization sample. the change of at least one nucleotide, resulting in a change in A preferred probe for detecting mRNA or genomic DNA is a the encoded amino acid; the change of at least one nucleotide, labeled nucleic acid probe capable of hybridizing to mRNA resulting in the generation of a premature stop codon; the or genomic DNA sequences described herein. The nucleic deletion of several nucleotides, resulting in a deletion of one acid probe can be, for example, a full-length nucleic acid or more amino acids encoded by the nucleotides; the insertion 15 molecule, or a portion thereof. Such as an oligonucleotide of of one or several nucleotides, such as by unequal recombina at least 15, 30, 50, 100, 250 or 500 nucleotides in length and tion or gene conversion, resulting in an interruption of the Sufficient to specifically hybridize under Stringent conditions coding sequence of the gene or nucleic acid; duplication of all to appropriate mRNA or genomic DNA. For example, the or a part of the gene or nucleic acid; transposition of all or a nucleic acid probe can be all or a portion of one of SEQ ID part of the gene or nucleic acid; or rearrangement of all or a NOs: 1, 3, 718 or 719 or the complement thereof or a portion part of the gene or nucleic acid. More than one Such alteration thereof, or can be a nucleic acid encoding all or a portion of may be presentina single gene or nucleic acid. Such sequence one of SEQID NO: 2 or 720. Other suitable probes for use in changes cause an alteration in the polypeptide encoded by a the diagnostic assays of the invention are described above FLAP or LTA4H nucleic acid. For example, if the alteration is (see e.g., probes and primers discussed under the heading, a frame shift alteration, the frame shift can result in a change 25 “Nucleic Acids of the Invention'). in the encoded amino acids, and/or can result in the genera The hybridization sample is maintained under conditions tion of a premature stop codon, causing generation of a trun that are sufficient to allow specific hybridization of the cated polypeptide. Alternatively, a polymorphism associated nucleic acid probe to a FLAP nucleic acid or a LTA4H nucleic with a disease or condition associated with a FLAP or LTA4H acid. “Specific hybridization,” as used herein, indicates exact nucleic acid or a susceptibility to a disease or condition asso 30 hybridization (e.g., with no mismatches). Specific hybridiza ciated with a FLAP or LTA4H nucleic acid can be a synony tion can be performed under high Stringency conditions or mous alteration in one or more nucleotides (i.e., an alteration moderate stringency conditions, for example, as described that does not result in a change in the polypeptide encoded by above. In a particularly preferred embodiment, the hybridiza a FLAP nucleic acid or LTA4H nucleic acid). Such a poly tion conditions for specific hybridization are high Stringency. morphism may alter splicing sites, affect the stability or trans 35 Specific hybridization, if present, is then detected using port of mRNA, or otherwise affect the transcription or trans standard methods. If specific hybridization occurs between lation of the nucleic acid. A FLAP nucleic acid or a LTA4H the nucleic acid probe and FLAP nucleic acid in the test nucleic acid that has any of the alteration described above is sample, then the FLAP has the polymorphism, or is the splic referred to herein as an “altered nucleic acid.” ing variant, that is present in the nucleic acid probe. More than In a first method of diagnosing a susceptibility to MI, ACS, 40 one nucleic acid probe can also be used concurrently in this stroke or PAOD, hybridization methods, such as Southern method. Specific hybridization of any one of the nucleic acid analysis, Northern analysis, or in situ hybridizations, can be probes is indicative of a polymorphism in the FLAP nucleic used (see Current Protocols in Molecular Biology, Ausubel, acid, or of the presence of a particular splicing variant encod F. et al., eds. John Wiley & Sons, including all supplements ing the FLAP nucleic acid, and is therefore diagnostic for a through 1999). For example, a biological sample from a test 45 susceptibility to a disease or condition associated with FLAP subject (a “test sample') of genomic DNA, RNA, or cDNA, is (e.g., MI, ACS, stroke or PAOD). obtained from an individual Suspected of having, being Sus In addition, if specific hybridization occurs between the ceptible to or predisposed for, or carrying a defect for, a nucleic acid probe and LTA4H nucleic acid in the test sample, Susceptibility to a disease or condition associated with a then the LTA4H has the polymorphism, or is the splicing FLAP nucleic acid or a disease or condition associated with a 50 variant, that is present in the nucleic acid probe. More than LTA4H nucleic acid (the “test individual'). The individual one nucleic acid probe can also be used concurrently in this can be an adult, child, or fetus. The test sample can be from method. Specific hybridization of any one of the nucleic acid any source which contains genomic DNA, such as a blood probes is indicative of a polymorphism in the LTA4H nucleic sample, sample of amniotic fluid, Sample of cerebrospinal acid, or of the presence of a particular splicing variant encod fluid, or tissue sample from skin, muscle, buccal or conjunc 55 ing the LTA4H nucleic acid, and is therefore diagnostic for a tival mucosa, placenta, gastrointestinal tract or other organs. Susceptibility to a disease or condition associated with A test sample of DNA from fetal cells or tissue can be LTA4H (e.g., MI, ACS, stroke or PAOD). obtained by appropriate methods, such as by amniocentesis or In Northern analysis (see Current Protocols in Molecular chorionic villus sampling. The DNA, RNA, or cDNA sample Biology, Ausubel, F. et al., eds., John Wiley & Sons, supra) the is then examined to determine whether a polymorphism in an 60 hybridization methods described above are used to identify MI nucleic acid is present, and/or to determine which splicing the presence of a polymorphism or a particular splicing Vari variant(s) encoded by the FLAP or LTA4H is present. The ant, associated with a Susceptibility to a disease or condition presence of the polymorphism or splicing variant(s) can be associated with FLAP or LTA4H (e.g., MI, ACS, stroke or indicated by hybridization of the nucleic acid in the genomic PAOD). For Northern analysis, a test sample of RNA is DNA, RNA, or cDNA to a nucleic acid probe. A “nucleic acid 65 obtained from the individual by appropriate means. Specific probe.” as used herein, can be a DNA probe oran RNA probe; hybridization of a nucleic acid probe, as described above, to the nucleic acid probe can contain at least one polymorphism RNA from the individual is indicative of a polymorphism in a US 8,158,362 B2 91 92 FLAP nucleic acid, or of the presence of a particular splicing that contains a polymorphism associated with a Susceptibility variant encoded by a FLAP nucleic acid, a polymorphism in to a disease or condition associated with FLAP or LTA4H a LTA4H nucleic acid, or of the presence of a particular (e.g., MI, ACS, stroke or PAOD). An allele-specific oligo splicing variant encoded by a LTA4H nucleic acid and is nucleotide probe that is specific for particular polymorphisms therefore diagnostic for Susceptibility to a disease or condi in a FLAP or LTA4H nucleic acid can be prepared, using tion associated with FLAP or LTA4H (e.g., MI, ACS, stroke standard methods (see Current Protocols in Molecular Biol or PAOD). ogy, Supra). To identify polymorphisms in the nucleic acid For representative examples of use of nucleic acid probes, associated with Susceptibility to disease, a test sample of see, for example, U.S. Pat. Nos. 5.288,611 and 4,851,330. DNA is obtained from the individual. PCR can be used to Alternatively, a peptide nucleic acid (PNA) probe can be 10 amplify all or a fragment of a FLAP nucleic acid or LTA4H used instead of a nucleic acid probe in the hybridization nucleic acid, and their flanking sequences. The DNA contain methods described above. PNA is a DNA mimic having a ing the amplified FLAP or LTA4H nucleic acid (or fragment peptide-like, inorganic backbone, such as N-(2-aminoethyl) of the nucleic acid) is dot-blotted, using standard methods glycine units, with an organic base (A, G, C, Tor U) attached (see Current Protocols in Molecular Biology, Supra), and the to the glycine nitrogen via a methylene carbonyl linker (see, 15 blot is contacted with the oligonucleotide probe. The pres for example, Nielsen, P. E. et al., Bioconjugate Chemistry 5, ence of specific hybridization of the probe to the amplified American Chemical Society, p. 1 (1994). The PNA probe can FLAP or LTA4H is then detected. Specific hybridization of an be designed to specifically hybridize to a nucleic acid having allele-specific oligonucleotide probe to DNA from the indi a polymorphism associated with a Susceptibility to a disease vidual is indicative of a polymorphism in the FLAP or a or condition associated with FLAP (e.g., MI). Hybridization polymorphism in the LTA4H and is therefore indicative of a of the PNA probe to a FLAP nucleic acid as described herein susceptibility to a disease or condition associated with FLAP is diagnostic for the Susceptibility to the disease or condition. or LTA4H respectively (e.g., MI, ACS, stroke or PAOD). In another method of the invention, mutation analysis by An allele-specific primer hybridizes to a site on target DNA restriction digestion can be used to detect an altered nucleic overlapping a polymorphism and only primes amplification acid, or nucleic acids containing a polymorphism(s), if the 25 ofan allelic form to which the primer exhibits perfect comple mutation or polymorphism in the nucleic acid results in the mentarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 creation or elimination of a restriction site. A test sample (1989). This primer is used in conjunction with a second containing genomic DNA is obtained from the individual. primer which hybridizes at a distal site. Amplification pro Polymerase chain reaction (PCR) can be used to amplify a ceeds from the two primers, resulting in a detectable product FLAP nucleic acid or a LTA4H nucleic acid (and, if neces 30 which indicates the particular allelic form is present. A con sary, the flanking sequences) in the test sample of genomic trol is usually performed with a second pair of primers, one of DNA from the test individual. RFLP analysis is conducted as which shows a single base mismatch at the polymorphic site described (see Current Protocols in Molecular Biology, and the other of which exhibits perfect complementarity to a supra). The digestion pattern of the relevant DNA fragment distal site. The single-base mismatch prevents amplification indicates the presence or absence of the alteration or poly 35 and no detectable product is formed. The method works best morphism in the FLAP nucleic acid or LTA4H nucleic acid, when the mismatch is included in the 3'-most position of the and therefore indicates the presence or absence of the Suscep oligonucleotide aligned with the polymorphism because this tibility to a disease or condition associated with FLAP or position is most destabilizing to elongation from the primer LTA4H (e.g., MI, ACS, stroke or PAOD). (see, e.g., WO 93/224.56). Sequence analysis can also be used to detect specific poly 40 With the addition of such analogs as locked nucleic acids morphisms in the FLAP nucleic acid. A test sample of DNA (LNAs), the size of primers and probes can be reduced to as or RNA is obtained from the test individual. PCR or other few as 8 bases. LNAs are a novel class of bicyclic DNA appropriate methods can be used to amplify the nucleic acid, analogs in which the 2' and 4'positions in the furanose ring are and/or its flanking sequences, if desired. The sequence of a joined via an O-methylene (oxy-LNA), S-methylene (thio FLAP nucleic acid, or a fragment of the nucleic acid, or 45 LNA), or amino methylene (amino-LNA) moiety. Common cDNA, or fragment of the cDNA, or mRNA, or fragment of to all of these LNA variants is an affinity toward complemen the mRNA, is determined, using standard methods. The tary nucleic acids, which is by far the highest reported for a sequence of the nucleic acid, nucleic acid fragment, cDNA, DNA analog. For example, particular all oxy-LNA nonamers cDNA fragment, mRNA, or mRNA fragment is compared have been shown to have melting temperatures of 64° C. and with the known nucleic acid sequence of the nucleic acid, 50 74°C. when in complex with complementary DNA or RNA, cDNA (e.g., one or more of SEQ ID NOs: 1, 3, 718 or respectively, as oposed to 28°C. for both DNA and RNA for 719and/or the complement of SEQID NO: 1, 3,718 or 719), the corresponding DNA nonamer. Substantial increases in T. or a nucleic acid sequence encoding SEQID NO: 2, SEQID are also obtained when LNA monomers are used in combi NO: 720 or a fragment thereof) or mRNA, as appropriate. The nation with standard DNA or RNA monomers. For primers presence of a polymorphism in the FLAP or LTA4H indicates 55 and probes, depending on where the LNA monomers are that the individual has a susceptibility to a disease associated included (e.g., the 3' end, the 5'end, or in the middle), the T. with FLAP or LTA4H (e.g., MI, ACS, stroke or PAOD). could be increased considerably. Allele-specific oligonucleotides can also be used to detect In another embodiment, arrays of oligonucleotide probes the presence of polymorphism(s) in the FLAP nucleic acid or that are complementary to target nucleic acid sequence seg the LTA4H nucleic acid, through the use of dot-blot hybrid 60 ments from an individual, can be used to identify polymor ization of amplified oligonucleotides with allele-specific oli phisms in a FLAP nucleic acid or a LTA4H nucleic acid For gonucleotide (ASO) probes (see, for example, Saiki, R. et al., example, in one embodiment, an oligonucleotide array can be Nature 324:163–166 (1986)). An “allele-specific oligonucle used. Oligonucleotide arrays typically comprise a plurality of otide' (also referred to herein as an “allele-specific oligo different oligonucleotide probes that are coupled to a surface nucleotide probe') is an oligonucleotide of approximately 65 of a substrate in different known locations. These oligonucle 10-50 base pairs, for example, approximately 15-30 base otide arrays, also described as “GenechipsTM, have been pairs, that specifically hybridizes to a FLAP nucleic acid, and generally described in the art, for example, U.S. Pat. No. US 8,158,362 B2 93 94 5,143,854 and PCT patent publication Nos. WO 90/15070 vellet al., Cell 15:25 (1978); Geever, et al., Proc. Natl. Acad. and WO92/10092. These arrays can generally be produced Sci. USA 78:5081 (1981)); heteroduplex analysis; chemical using mechanical synthesis methods or light directed synthe mismatch cleavage (CMC) (Cotton et al., Proc. Natl. Acad. sis methods that incorporate a combination of photolitho Sci. USA 85.4397–4401 (1985)); RNase protection assays graphic methods and Solid phase oligonucleotide synthesis 5 (Myers, R. M. et al., Science 230:1242 (1985)); use of methods. See Fodor et al., Science 251:767-777 (1991); Pir polypeptides which recognize nucleotide mismatches, such rung et al., U.S. Pat. No. 5,143,854; (see also PCT Applica as E. coli mutS protein; allele-specific PCR, for example. tion WO 90/15070): Fodor et al., PCT Publication WO In one embodiment of the invention, diagnosis of a Suscep 92/10092; and U.S. Pat. No. 5,424,186, the entire teachings of tibility to a disease or condition associated with FLAP or each of which are incorporated by reference herein. Tech 10 LTA4H (e.g., MI, ACS, stroke or PAOD) can also be made by niques for the synthesis of these arrays using mechanical expression analysis by quantitative PCR (kinetic thermal synthesis methods are described in, e.g., U.S. Pat. No. 5,384, cycling). This technique utilizing TaqMan(R) can be used to 261, the entire teachings of which are incorporated by refer allow the identification of polymorphisms and whether a ence herein. In another example, linear arrays can be utilized. patient is homozygous or heterozygous. The technique can Once an oligonucleotide array is prepared, a nucleic acid of 15 assess the presence of an alteration in the expression or com interest is hybridized with the array and scanned for polymor position of the polypeptide encoded by a FLAP nucleic acid phisms. Hybridization and scanning are generally carried out or splicing variants encoded by a FLAP nucleic acid. The by methods described herein and also in, e.g., published PCT technique can likewise assess the presence of an alteration in Application Nos. WO92/10092 and WO95/11995, and U.S. the expression or composition of the polypeptide encoded by Pat. No. 5,424,186, the entire teachings of which are incor- 20 a LTA4H nucleic acid or splicing variants encoded by a porated by reference herein. In brief, a target nucleic acid LTA4H nucleic acid. Further, the expression of the variants sequence that includes one or more previously identified can be quantified as physically or functionally different. polymorphic markers is amplified using well-known ampli In another embodiment of the invention, diagnosis of a fication techniques, e.g., PCR. Typically, this involves the use susceptibility to MI, ACS, stroke or PAOD (or of another of primer sequences that are complementary to the two 25 disease or condition associated with FLAP or LTA4H) can Strands of the target sequence both upstream and downstream also be made by examining expression and/or composition of from the polymorphism. Asymmetric PCR techniques may a FLAP polypeptide, by a variety of methods, including also be used. Amplified target, generally incorporating a enzyme linked immunosorbent assays (ELISAs), Western label, is then hybridized with the array under appropriate blots, immunoprecipitations and immunofluorescence. A test conditions. Upon completion of hybridization and washing of 30 sample from an individual is assessed for the presence of an the array, the array is scanned to determine the position on the alteration in the expression and/or an alteration in composi array to which the target sequence hybridizes. The hybridiza tion of the polypeptide encoded by a FLAP nucleic acid, or a tion data obtained from the scan is typically in the form of polypeptide encoded by a LTA4H nucleic acid or for the fluorescence intensities as a function of location on the array. presence of a particular variant encoded by a FLAP or LTA4H In a reverse method, a probe, containing a polymorphism, can 35 nucleic acid. An alteration in expression of a polypeptide be coupled to a solid surface and PCR amplicons are then encoded by a FLAP or LTA4H nucleic acid can be, for added to hybridize to these probes. example, an alteration in the quantitative polypeptide expres Although primarily described in terms of a single detection sion (i.e., the amount of polypeptide produced); an alteration block, e.g., detection of a single polymorphism arrays can in the composition of a polypeptide encoded by a FLAP or include multiple detection blocks, and thus be capable of 40 LTA4H nucleic acid is an alteration in the qualitative analyzing multiple, specific polymorphisms. It will generally polypeptide expression (e.g., expression of an altered FLAP be understood that detection blocks may be grouped within a polypeptide, LTA4H polypeptide or of a different splicing single array or in multiple, separate arrays so that varying, variant). In a preferred embodiment, diagnosis of a suscepti optimal conditions may be used during the hybridization of bility to a disease or condition associated with FLAP is made the target to the array. For example, it may often be desirable 45 by detecting a particular splicing variant encoded by that to provide for the detection of those polymorphisms that fall FLAP variant, or a particular pattern of splicing variants. In within G-C rich stretches of a genomic sequence, separately another preferred embodiment, diagnosis of a Susceptibility from those falling in A-Trich segments. This allows for the to a disease or condition associated with LTA4H is made by separate optimization of hybridization conditions for each detecting a particular splicing variant encoded by that LTA4H situation. 50 variant, or a particular pattern of splicing variants. Additional uses of oligonucleotide arrays for detection of Both Such alterations (quantitative and qualitative) can also polymorphisms can be found, for example, in U.S. Pat. Nos. be present. An “alteration' in the polypeptide expression or 5,858,659 and 5,837,832, the entire teachings of which are composition, refers to an alteration in expression or compo incorporated by reference herein. Other methods of nucleic sition in a test sample, as compared with the expression or acid analysis can be used to detect polymorphisms in a 55 composition of polypeptide by a FLAP or LTA4H nucleic nucleic acid described herein, or variants encoded by a acid in a control sample. A control sample is a sample that nucleic acid described herein. Representative methods corresponds to the test sample (e.g., is from the same type of include direct manual sequencing (Church and Gilbert, Proc. cells), and is from an individual who is not affected by the Natl. Acad. Sci. USA 81:1991-1995 (1988); Sanger et al., disease or a Susceptibility to a disease or condition associated Proc. Natl. Acad. Sci., USA 74:5463-5467 (1977); Beavis et 60 with a FLAP or LTA4H nucleic acid. An alteration in the al. U.S. Pat. No. 5.288,644); automated fluorescent sequenc expression or composition of the polypeptide in the test ing; single-stranded conformation polymorphism assays sample, as compared with the control sample, is indicative of (SSCP); clamped denaturing gel electrophoresis (CDGE): a susceptibility to a disease or condition associated with denaturing gradient gel electrophoresis (DGGE) (Sheffield, FLAP or LTA4H (e.g., MI, ACS, stroke or PAOD). Similarly, V. C. et al., Proc. Natl. Acad. Sci. USA 86:232-236 (1989)), 65 the presence of one or more different splicing variants in the mobility shift analysis (Orita, M. et al., Proc. Natl. Acad. Sci. test sample, or the presence of significantly different amounts USA 86:2766-2770 (1989)), restriction enzyme analysis (Fla of different splicing variants in the test sample, as compared US 8,158,362 B2 95 96 with the control sample, is indicative of a susceptibility to a pared to the control sample; or both a difference in the amount disease or condition associated with a FLAP or LTA4H or level, and a difference in the composition, is indicative of nucleic acid. Various means of examining expression or com a susceptibility to a disease or condition associated with position of the polypeptide encoded by a FLAP or LTA4H FLAP (e.g., MI). nucleic acid can be used, including: spectroscopy, colorim The invention further pertains to a method for the diagnosis etry, electrophoresis, isoelectric focusing and immunoassays and identification of Susceptibility to myocardial infarction, (e.g., David et al., U.S. Pat. No. 4,376,110) such as immuno ACS, stroke or PAOD in an individual, by identifying an blotting (see also Current Protocols in Molecular Biology, at-risk haplotype in FLAP or by identifying an at-risk haplo particularly Chapter 10). For example, in one embodiment, an type in LTA4H. In one embodiment, the at-risk haplotype is antibody capable of binding to the polypeptide (e.g., as 10 described above), preferably an antibody with a detectable one which confers a significant risk of MI, ACS, stroke or label, can be used. Antibodies can be polyclonal, or more PAOD. In one embodiment, significance associated with a preferably, monoclonal. An intact antibody, or a fragment haplotype is measured by an odds ratio. In a further embodi thereof (e.g., Fab or F(ab')) can be used. The term “labeled”. ment, the significance is measured by a percentage. In one with regard to the probe orantibody, is intended to encompass 15 embodiment, a significant risk is measured as an odds ratio of direct labeling of the probe or antibody by coupling (i.e., at least about 1.2, including by not limited to: 1.2, 1.3, 1.4. physically linking) a detectable Substance to the probe or 1.5, 1.6, 1.7, 1.8, and 1.9. In a further embodiment, an odds antibody, as well as indirect labeling of the probe or antibody ratio of at least 1.2 is significant. In a further embodiment, an by reactivity with another reagent that is directly labeled. odds ratio of at least about 1.5 is significant. In a further Examples of indirect labeling include detection of a primary embodiment, a significant increase in risk is at least about 1.7 antibody using a fluorescently labeled secondary antibody is significant. In a further embodiment, a significant increase and end-labeling of a DNA probe with biotin such that it can in risk is at least about 20%, including but not limited to about be detected with fluorescently labeled streptavidin. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, Western blotting analysis, using an antibody as described 75%, 80%, 85%, 90%, 95, and 98%. In a further embodiment, above that specifically binds to a polypeptide encoded by an 25 a significant increase in risk is at least about 50%. In yet altered FLAP (e.g., by a FLAP having a SNP as shown in another embodiment, an at-risk haplotype has a p value Table 13) or an antibody that specifically binds to a polypep <0.05. It is understood however, that identifying whether a tide encoded by an altered LTA4H (e.g. by a LTA4H having a risk is medically significant may also depend on a variety of SNP as shown in Table 37), or an antibody that specifically factors, including the specific disease, the haplotype, and binds to a polypeptide encoded by a non-altered nucleic acid, 30 or an antibody that specifically binds to a particular splicing often, environmental factors. variant encoded by a nucleic acid, can be used to identify the The invention also pertains to methods of diagnosing a presence in a test sample of a particular splicing variant or of susceptibility to myocardial infarction, ACS, stroke or PAOD a polypeptide encoded by a polymorphic or altered FLAP or in an individual, comprising screening for an at-risk haplo altered LTA4H, or the absence in a test sample of a particular 35 type in the FLAP nucleic acid that is more frequently present splicing variant or of a polypeptide encoded by a non-poly in an individual Susceptible to myocardial infarction (af morphic or non-altered nucleic acid. The presence of a fected) or by screening for an at-risk haplotype in LTA4H, polypeptide encoded by a polymorphic or altered nucleic compared to the frequency of its presence in a healthy indi acid, or the absence of a polypeptide encoded by a non vidual (control), wherein the presence of the haplotype is polymorphic or non-altered nucleic acid, is diagnostic for a 40 indicative of susceptibility to myocardial infarction. Standard susceptibility to a disease or condition associated with FLAP techniques for genotyping for the presence of SNPs and/or or LTA4H, as is the presence (or absence) of particular splic microsatellite markers that are associated with myocardial ing variants encoded by the FLAP nucleic acid or the LTA4H infarction, ACS, stroke or PAOD can be used, such as fluo nucleic acid. rescent based techniques (Chen, et al., Genome Res. 9, 492 In one embodiment of this method, the level or amount of 45 (1999), PCR, LCR, Nested PCR and other techniques for polypeptide encoded by a FLAP nucleic acid in a test sample nucleic acid amplification. In a preferred embodiment, the is compared with the level or amount of the polypeptide method comprises assessing in an individual the presence or encoded by the FLAP in a control sample. A level or amount frequency of SNPs and/or microsatellites in the FLAP nucleic of the polypeptide in the test sample that is higher or lower acid that are associated with myocardial infarction, ACS, than the level or amount of the polypeptide in the control 50 stroke or PAOD, wherein an excess or higher frequency of the sample, Such that the difference is statistically significant, is SNPs and/or microsatellites compared to a healthy control indicative of an alteration in the expression of the polypeptide individual is indicative that the individual is susceptible to encoded by the FLAP, and is diagnostic for a susceptibility to myocardial infarction, ACS, stroke or PAOD. See Table 7 for a disease or condition associated with that FLAP. Alterna SNPs that comprise haplotypes that can be used as Screening tively, the composition of the polypeptide encoded by a FLAP 55 tools. See also Tables 13 and 37 that sets forth SNPs and nucleic acid in a test sample is compared with the composi markers for use as screening tools. tion of the polypeptide encoded by the FLAP in a control In one embodiment, the at-risk haplotype is characterized sample (e.g., the presence of different splicing variants). A by the presence of polymorphism(s) represented in Table 13. difference in the composition of the polypeptide in the test For example, SG13S99, where the SNP can be a “C” or a "T": sample, as compared with the composition of the polypeptide 60 SG13S25, where the SNP can be a “G” or an 'A'; SG13S377, in the control sample, is diagnostic for a Susceptibility to a where the SNP can be a “G” or an 'A'; SG13S106, where the disease or condition associated with that FLAP. In another SNP can be a “G” or an 'A'; SG13S114, where the SNP can embodiment, both the level or amount and the composition of be a “Toran “A: SG13S89, where the SNP can be a “G” or the polypeptide can be assessed in the test sample and in the an “A: SG13S30, where the SNP can be a “G” or a “T: control sample. A difference in the amount or level of the 65 SG13S32, where the SNP can be a “C” or an “A: SG13S42, polypeptide in the test sample, compared to the control where the SNP can be a “G” or an 'A'; and SG13S35, where sample; a difference in composition in the test sample, com the SNP can be a “G” or an 'A'. In addition, SG13A375, US 8,158,362 B2 97 98 where the SNP can be a “T, SG13S32, where the SNP can be SG13S89 and SG13S32 at the 13q12-12 locus. In one par an 'A', aand SG13S106, where the SNP can be a “G” or an ticular embodiment, the presence of the alleles G, T, G and A “A. at SG13S25, SG13S114, SG13S89 and SG13S32, respec Kits (e.g., reagent kits) useful in the methods of diagnosis tively (the A4 haplotype), is diagnostic of Susceptibility to comprise components useful in any of the methods described myocardial infarction, ACS, stroke or PAOD. In another herein, including for example, hybridization probes or prim embodiment, a haplotype associated with a susceptibility to ers as described herein (e.g., labeled probes or primers), myocardial infarction, ACS, stroke or PAOD comprises reagents for detection of labeled molecules, restriction marker SG13S375 at the 13q12-13 locus. In one particular enzymes (e.g., for RFLPanalysis), allele-specific oligonucle embodiment, the presence of T at SG13S375, (the HapC1 otides, antibodies which bind to altered or to non-altered 10 haplotype) is diagnostic of Susceptibility to to myocardial (native) FLAP polypeptide or a non-altered (native) LTA4H infarction, ACS, stroke or PAOD. In another embodiment, a polypeptide, means for amplification of nucleic acids com haplotype associated with a susceptibility to myocardial inf prising a FLAP or LTA4H, or means for analyzing the nucleic arction, ACS, stroke or PAOD comprises markers SG13S25 acid sequence of a nucleic acid described herein, or for ana and SG13S375 at the 13q12-13 locus. In one particular lyzing the amino acid sequence of a polypeptide as described 15 embodiment, the presence of T and G at SG13S375 and herein, etc. In one embodiment, a kit for diagnosing Suscep SG13S25, respectively (the HapC2 haplotype) is diagnostic tibility to MI, ACS, stroke or PAOD can comprise primers for of susceptibility to to myocardial infarction, ACS, stroke or nucleic acid amplification of a region in the FLAP nucleic PAOD. In another embodiment, a haplotype associated with a acid or LTA4H nucleic acid comprising an at-risk haplotype susceptibility to myocardial infarction, ACS, stroke or PAOD that is more frequently present in an individual having MI, comprises markers SG13S25, SG13S375 and SG13S32 at the ACS, stroke or PAOD or susceptible to MI, ACS, stroke or 13q12-13 locus. In one particular embodiment, the presence PAOD. The primers can be designed using portions of the of T, G and A at SG13S375, SG13S25 and SG13S32, respec nucleic acids flanking SNPs that are indicative of MI. In a tively (the HapC3 haplotype) is diagnostic of susceptibility to particularly preferred embodiment, the primers are designed to myocardial infarction, ACS, stroke or PAOD. In an addi to amplify regions of the LTA4H nucleic acid associated with 25 tional embodiment, a haplotype associated with a suscepti an at-risk haplotype for MI, as shown in Table 41 or Table 42, bility to myocardial infarction, ACS, stroke or PAOD com or more particularly the haplotype defined by the microsatel prises markers SG13S25, SG13S375, SG13S32 and lite markers and SNPs at the locus on chromosome 12q23. SG13S106 at the 13q12-13 locus. In one particular embodi In another particularly preferred embodiment, the primers ment, the presence of T, G, A and Gat SG13S375, SG13S25, are designed to amplify regions of the FLAP nucleic acid 30 SG13S32 and SG13S106, respectively (the HapC4-A) hap associated with an at-risk haplotype for MI, ACS, stroke or lotype) is diagnostic of susceptibility to to myocardial infarc PAOD, as shown in Table 7, or more particularly the haplo tion, ACS, stroke or PAOD. In one particularembodiment, the type defined by the following SNP markers: In one embodi presence of T, G, A and A at SG13S375, SG13S25, SG13S32 ment, a haplotype associated with a Susceptibility to myocar and SG13S106, respectively (the HapC4-B) haplotype) is dial infarction, ACS, stroke or PAOD comprises markers 35 diagnostic of susceptibility to to myocardial infarction, ACS, SG13S99, SG13S25, SG13S377, SG13S106, SG13S32 and Stroke or PAOD. SG13S35 at the 13q12-13 locus. In one particular embodi ment, the presence of the alleles T. G, G, G, A and G at Screening Assays and Agents Identified Thereby SG13S99, SG13S25, SG13S377, SG13S106, SG13S32 and SG13S35, respectively (the B6 haplotype), is diagnostic of 40 The invention provides methods (also referred to herein as susceptibility to myocardial infarction, ACS, stroke or PAOD. 'screening assays”) for identifying the presence of a nucle In another embodiment, a haplotype associated with a sus otide that hybridizes to a nucleic acid of the invention, as well ceptibility to myocardial infarction, ACS, stroke or PAOD as for identifying the presence of a polypeptide encoded by a comprises markers SG13S99, SG13S25, SG13S106, nucleic acid of the invention. In one embodiment, the pres SG13S30 and SG13S42 at the 13q12-13 locus. In one par 45 ence (or absence) of a nucleic acid molecule of interest (e.g., ticular embodiment, the presence of the alleles T.G, G, G and a nucleic acid that has significant homology with a nucleic A at SG13S99, SG13S25, SG13S106, SG13S30 and acid of the invention) in a sample can be assessed by contact SG13S42, respectively (the B5 haplotype), is diagnostic of ing the sample with a nucleic acid comprising a nucleic acid susceptibility to myocardial infarction, ACS, stroke or PAOD. of the invention (e.g., a nucleic acid having the sequence of In a third embodiment, a haplotype associated with a suscep 50 one of SEQ ID NOs: 1, 3, 718 or 719 or the complement tibility to myocardial infarction, ACS, stroke or PAOD com thereof, or a nucleic acid encoding an amino acid having the prises markers SG13S25, SG13S106, SG13S30 and sequence of SEQID NO: 2, SEQID NO: 720, or a fragment SG13S42 at the 13q12-13 locus. In one particular embodi or variant of such nucleic acids), understringent conditions as ment, the presence of the alleles G, G, G and A at SG13S25. described above, and then assessing the sample for the pres SG13S106, SG13S30 and SG13S42, respectively (the B4 55 ence (or absence) of hybridization. In a preferred embodi haplotype), is diagnostic of Susceptibility to myocardial inf ment, high Stringency conditions are conditions appropriate arction, ACS, stroke or PAOD. In a fourth embodiment, a for selective hybridization. In another embodiment, a sample haplotype associated with a susceptibility to myocardial inf containing a nucleic acid molecule of interest is contacted arction, ACS, stroke or PAOD comprises markers SG13S99, with a nucleic acid containing a contiguous nucleic acid SG13S25, SG13S114, SG13S89 and SG13S32 at the 13d 12 60 sequence (e.g., a primer or a probe as described above) that is 13 locus. In one particular embodiment, the presence of the at least partially complementary to a part of the nucleic acid alleles T, G, T, G and A at SG13S99, SG13S25, SG13S114, molecule of interest (e.g., a FLAP nucleic acid or a LTA4H SG13S89 and SG13S32, respectively (the A5 haplotype), is nucleic acid), and the contacted Sample is assessed for the diagnostic of Susceptibility to myocardial infarction, ACS, presence or absence of hybridization. In a preferred embodi stroke or PAOD. In a fifth embodiment, a haplotype associ 65 ment, the nucleic acid containing a contiguous nucleic acid ated with a susceptibility to myocardial infarction, ACS, sequence is completely complementary to a part of the stroke or PAOD comprises markers SG13S25, SG13S114, nucleic acid molecule of interest. US 8,158,362 B2 99 100 In any of these embodiments, all or a portion of the nucleic the presence of the agent relative to the activity in the absence acid of interest can be subjected to amplification prior to of the agent, indicates that the agent is an agent that enhances performing the hybridization. the activity. Similarly, a decrease in the level of polypeptide In another embodiment, the presence (or absence) of a activity in the presence of the agent, relative to the activity in polypeptide of interest, such as a polypeptide of the invention the absence of the agent, indicates that the agent is an agent or a fragment or variant thereof, in a sample can be assessed that inhibits polypeptide activity. In another embodiment, the by contacting the sample with an antibody that specifically level of activity of a polypeptide or derivative or fragment hybridizes to the polypeptide of interest (e.g., an antibody thereof in the presence of the agent to be tested, is compared Such as those described above), and then assessing the sample with a control level that has previously been established. A for the presence (or absence) of binding of the antibody to the 10 statistically significant difference in the level of the activity in polypeptide of interest. the presence of the agent from the control level indicates that In another embodiment, the invention provides methods the agent alters polypeptide activity. for identifying agents (e.g., fusion proteins, polypeptides, The present invention also relates to an assay for identify peptidomimetics, prodrugs, receptors, binding agents, anti ing agents which alter the expression of a FLAP nucleic acid bodies, Small molecules or other drugs, or ribozymes which 15 or a LTA4H nucleic acid (e.g., antisense nucleic acids, fusion alter (e.g., increase or decrease) the activity of the polypep proteins, polypeptides, peptidomimetics, prodrugs, recep tides described herein, or which otherwise interact with the tors, binding agents, antibodies, Small molecules or other polypeptides herein. For example, such agents can be agents drugs, or ribozymes; which alter (e.g., increase or decrease) which bind to polypeptides described herein (e.g., binding expression (e.g., transcription or translation) of the nucleic agent for members of the leukotriene pathway, such as FLAP acid or which otherwise interact with the nucleic acids binding agents or LTA4H binding agents); which have a described herein, as well as agents identifiable by the assays. stimulatory or inhibitory effect on, for example, activity of For example, a solution containing a nucleic acid encoding a polypeptides of the invention; or which change (e.g., enhance FLAP polypeptide (e.g., a FLAP nucleic acid) or a nucleic or inhibit) the ability of the polypeptides of the invention to acid encoding a LTA4H polypeptide (e.g., a LTA4H nucleic interact with members of the leukotriene pathway binding 25 acid) can be contacted with an agent to be tested. The Solution agents (e.g., receptors or other binding agents); or which alter can comprise, for example, cells containing the nucleic acid posttranslational processing of the leukotriene pathway or cell lysate containing the nucleic acid; alternatively, the member polypeptide, such as a FLAP polypeptide or a Solution can be another Solution that comprises elements LTA4H polypeptide (e.g., agents that alter proteolytic pro necessary for transcription/translation of the nucleic acid. cessing to direct the polypeptide from where it is normally 30 Cells not suspended in Solution can also be employed, if synthesized to another location in the cell. Such as the cell desired. The level and/or pattern of FLAP or LTA4H expres surface; agents that alter proteolytic processing such that sion (e.g., the level and/or pattern of mRNA or of protein more polypeptide is released from the cell, etc.) expressed, such as the level and/or pattern of different splic In one embodiment, the invention provides assays for ing variants) is assessed, and is compared with the level screening candidate or test agents that bind to or modulate the 35 and/or pattern of expression in a control (i.e., the level and/or activity of polypeptides described herein (or biologically pattern of the FLAP expression or LTA4H expression in the active portion(s) thereof), as well as agents identifiable by the absence of the agent to be tested). If the level and/or pattern in assays. Testagents can be obtained using any of the numerous the presence of the agent differ, by an amount or in a manner approaches in combinatorial library methods known in the that is statistically significant, from the level and/or pattern in art, including: biological libraries; spatially addressable par 40 the absence of the agent, then the agent is an agent that alters allel solid phase or solution phase libraries; synthetic library the expression of the FLAP nucleic acid or alters the expres methods requiring deconvolution; the “one-bead one-com sions f the LTA4H nucleic acid Enhancement of FLAP pound library method; and synthetic library methods using expression or LTA4H expression indicates that the agent is an affinity chromatography selection. The biological library activator of FLAP or LTA4H activity. Similarly, inhibition of approach is limited to polypeptide libraries, while the other 45 FLAP expression or LTA4H expression indicates that the four approaches are applicable to polypeptide, non-peptide agent is a repressor of FLAP or LTA4H activity. oligomer or Small molecule libraries of compounds (Lam, K. In another embodiment, the level and/or pattern of FLAP S., Anticancer Drug Des. 12:145 (1997)). polypeptide(s) or LTA4H polypeptide(s) (e.g., different splic In one embodiment, to identify agents which alter the ing variants) in the presence of the agent to be tested, is activity of a FLAP polypeptide or a LTA4H polypeptide, a 50 compared with a control level and/or pattern that have previ cell, cell lysate, or solution containing or expressing a the ously been established. A level and/or pattern in the presence polypeptide (e.g., SEQID NO: 2, SEQID NO; 720 or another of the agent that differs from the control level and/or pattern splicing variant thereof Such as a nucleic acid comprising a by an amount or in a manner that is statistically significant SNP as shown in Table 13 or 37), or a fragment or derivative indicates that the agent alters FLAP or LTA4H expression. thereof (as described above), can be contacted with an agent 55 In another embodiment of the invention, agents which alter to be tested; alternatively, the polypeptide can be contacted the expression of a FLAP nucleic acid or a LTA4H nucleic directly with the agent to be tested. The level (amount) of acid or which otherwise interact with the nucleic acids polypeptide activity is assessed (e.g., the level (amount) of described herein, can be identified using a cell, cell lysate, or polypeptide activity is measured, either directly or indi Solution containing a nucleic acid encoding the promoter rectly), and is compared with the level of activity in a control 60 region of the FLAP or LTA4H nucleic acid operably linked to (i.e., the level of activity of the FLAP or LTA4H polypeptide a reporter gene. After contact with an agent to be tested, the or active fragment or derivative thereof in the absence of the level of expression of the reporter gene (e.g., the level of agent to be tested). If the level of the activity in the presence mRNA or of protein expressed) is assessed, and is compared of the agent differs, by an amount that is statistically signifi with the level of expression in a control (i.e., the level of the cant, from the level of the activity in the absence of the agent, 65 expression of the reporter gene in the absence of the agent to then the agent is an agent that alters the activity of the be tested). If the level in the presence of the agent differs, by polypeptide. An increase in the level of polypeptide activity in an amount or in a manner that is statistically significant, from US 8,158,362 B2 101 102 the level in the absence of the agent, then the agent is an agent detect the interaction of a test agent with a polypeptide or a that alters the expression of the FLAP or LTA4H nucleic acid, polypeptide binding agent without the labeling of either the as indicated by its ability to alter expression of a nucleic acid test agent, the polypeptide, or the polypeptide binding agent. that is operably linked to the FLAP or LTA4H nucleic acid McConnell, H. M. et al., Science 257:1906-1912 (1992). As promoter. 5 used herein, a “microphysiometer” (e.g., CytosensorTM) is an Enhancement of the expression of the reporter indicates analytical instrument that measures the rate at which a cell that the agent is an activator of expression of the gene of acidifies its environment using a light-addressable potentio interest. Similarly, inhibition of the expression of the reporter metric sensor (LAPS). Changes in this acidification rate can indicates that the agent is a repressor of expression of the gene be used as an indicator of the interaction between ligand and of interest. In another embodiment, the level of expression of 10 polypeptide. the reporter in the presence of the test agent, is compared with Thus, these receptors can be used to screen for compounds a control level that has previously been established. A level in that are agonists for use in treating a disease or condition the presence of the agent that differs from the control level by associated with FLAP or LTA4H or a susceptibility to a an amount or in a manner that is statistically significant indi disease or condition associated with FLAP or LTA4H, or cates that the agent alters expression. 15 antagonists for studying a susceptibility to a disease or con Agents which alter the amounts of different splicing vari dition associated with FLAP (e.g., MI, ACS, stroke or ants encoded by a FLAP nucleic acid or a LTA4H nucleic acid PAOD). Drugs can be designed to regulate FLAP activation, (e.g., an agent which enhances expression of a first splicing that in turn can be used to regulate signaling pathways and variant, and which inhibits expression of a second splicing transcription events of genes downstream or of proteins or variant), as well as agents which stimulate activity of a first polypeptides interacting with FLAP (e.g., 5-LO). splicing variant and inhibit activity of a second splicing vari In another embodiment of the invention, assays can be used ant, can easily be identified using these methods described to identify polypeptides that interact with one or more above. polypeptides, as described herein. For example, a yeast two In one embodiments of the invention, assays can be used to hybrid system such as that described by Fields and Song assess the impact of a test agent on the activity of a polypep (Fields, S. and Song, O., Nature 340:245–246 (1989)) can be tide relative to a FLAP binding agent. For example, a cell that 25 used to identify polypeptides that interact with one or more expresses a compound that interacts with a FLAP nucleic acid FLAP polypeptides. In such a yeast two-hybrid system, vec (herein referred to as a “FLAP binding agent', which can be tors are constructed based on the flexibility of a transcription a polypeptide or other molecule that interacts with a FLAP factor that has two functional domains (a DNA binding nucleic acid, such as a receptor, or another molecule, such as domain and a transcription activation domain). If the two 5-LO) is contacted with a FLAP in the presence of a test 30 domains are separated but fused to two different proteins that agent, and the ability of the test agent to alter the interaction interact with one another, transcriptional activation can be between the FLAP and the FLAP binding agent is deter achieved, and transcription of specific markers (e.g., nutri mined. Alternatively, a cell lysate or a solution containing the tional markers such as His and Ade, or color markers such as FLAPbinding agent, can be used. An agent which binds to the lacz) can be used to identify the presence of interaction and FLAP or the FLAP binding agent can alter the interaction by 35 transcriptional activation. For example, in the methods of the interfering with, or enhancing the ability of the FLAP to bind invention, a first vector is used which includes a nucleic acid to, associate with, or otherwise interact with the FLAP bind encoding a DNA binding domain and also a FLAP or LTA4H ing agent. polypeptide of the invention, splicing variant, or fragment or In other embodiments of the invention, assays can be used derivative thereof, and a second vector is used which includes to assess the impact of a testagent on the activity of a polypep a nucleic acid encoding a transcription activation domain and tide relative to a LTA4H binding agent. For example, a cell also a nucleic acid encoding a polypeptide which potentially that expresses a compound that interacts with a LTA4H may interact with the FLAP or LTA4H polypeptide, splicing nucleic acid (herein referred to as a “LTA4H binding agent'. variant, or fragment or derivative thereof (e.g., a FLAP which can be a polypeptide or other molecule that interacts polypeptide binding agent or receptor, or a LTA4H polypep with a LTA4H nucleic acid, such as a receptor, or another tide binding agent or receptor). Incubation of yeast containing molecule) is contacted with a LTA4H in the presence of a test 45 the first vector and the second vector under appropriate con agent, and the ability of the test agent to alter the interaction ditions (e.g., mating conditions such as used in the Match between the LTA4H and the LTA4H binding agent is deter makerTM system from Clontech (Palo Alto, Calif. USA)) mined. Alternatively, a cell lysate or a solution containing the allows identification of colonies that express the markers of LTA4H binding agent, can be used. An agent which binds to interest. These colonies can be examined to identify the the LTA4H or the LTA4H binding agent can alter the interac 50 polypeptide(s) that interact with the polypeptide of interest or tion by interfering with, or enhancing the ability of the fragment or derivative thereof. Such polypeptides may be LTA4H to bind to, associate with, or otherwise interact with useful as agents that alter the activity or expression of a FLAP the LTA4H binding agent. polypeptide or alter the activity or expression of a LTA4H Determining the ability of the test agent to bind to a nucleic polypeptide, as described above. acid or a nucleic acid binding agent can be accomplished, for In more than one embodiment of the above assay methods example, by coupling the test agent with a radioisotope or 55 of the present invention, it may be desirable to immobilize enzymatic label Such that binding of the test agent to the either the FLAP or LTA4H, the FLAP or LTA4H binding polypeptide can be determined by detecting the labeled with agent, or other components of the assay on a solid Support, in '*I, S, ''C or H, either directly or indirectly, and the order to facilitate separation of complexed from uncom radioisotope detected by direct counting of radioemmission plexed forms of one or both of the polypeptides, as well as to or by Scintillation counting. Alternatively, test agents can be 60 accommodate automation of the assay. Binding of a test agent enzymatically labeled with, for example, horseradish peroxi to the polypeptide, or interaction of the polypeptide with a dase, alkaline phosphatase, or luciferase, and the enzymatic binding agent in the presence and absence of a test agent, can label detected by determination of conversion of an appropri be accomplished in any vessel Suitable for containing the ate substrate to product. It is also within the scope of this reactants. Examples of Such vessels include microtiter plates, invention to determine the ability of a test agent to interact 65 test tubes, and micro-centrifuge tubes. In one embodiment, a with the polypeptide without the labeling of any of the inter fusion protein (e.g., a glutathione-S-transferase fusion pro actants. For example, a microphysiometer can be used to tein) can be provided which adds a domain that allows a US 8,158,362 B2 103 104 FLAP or LTA4H nucleic acid or a FLAP or LTA4H binding tacted directly with the agent to be tested. The level (amount) agent to be bound to a matrix or other Solid Support. of binding agent activity is assessed (either directly or indi In one embodiment, modulators of expression of nucleic rectly), and is compared with the level of activity in a control (i.e., the level of activity in the absence of the agent to be acid molecules of the invention are identified in a method tested). If the level of the activity in the presence of the agent wherein a cell, cell lysate, or Solution containing a nucleic differs, by an amount that is statistically significant, from the acid encoding a FLAP polypeptide or a LTA4H polypeptide is level of the activity in the absence of the agent, then the agent contacted with a test agent and the expression of appropriate is an agent that alters the activity of the member of the leu mRNA or polypeptide (e.g., splicing variant(s)) in the cell, kotriene pathway. An increase in the level of the activity cell lysate, or solution, is determined. The level of expression relative to a control, indicates that the agent is an agent that of appropriate mRNA or polypeptide(s) in the presence of the 10 enhances (is an agonist of) the activity. Similarly, a decrease test agent is compared to the level of expression of mRNA or in the level of activity relative to a control, indicates that the polypeptide(s) in the absence of the test agent. The test agent agent is an agent that inhibits (is an antagonist of) the activity. can then be identified as a modulator of expression based on In another embodiment, the level of activity in the presence of this comparison. For example, when expression of mRNA or the agent to be tested, is compared with a control level that has polypeptide is greater (statistically significantly greater) in previously been established. A level of the activity in the 15 presence of the agent that differs from the control level by an the presence of the testagent than in its absence, the test agent amount that is statistically significant indicates that the agent is identified as a stimulator or enhancer of the mRNA or alters the activity. polypeptide expression. Alternatively, when expression of the This invention further pertains to novel agents identified by mRNA or polypeptide is less (statistically significantly less) the above-described screening assays. Accordingly, it is in the presence of the test agent than in its absence, the test within the scope of this invention to further use an agent agent is identified as an inhibitor of the mRNA or polypeptide identified as described herein in an appropriate animal model. expression. The level of mRNA or polypeptide expression in For example, an agent identified as described herein (e.g., a the cells can be determined by methods described herein for testagent that is a modulating agent, an antisense nucleic acid detecting mRNA or polypeptide. molecule, a specific antibody, or a polypeptide-binding In yet another embodiment, the invention provides meth agent) can be used in an animal model to determine the ods for identifying agents (e.g., fusion proteins, polypeptides, 25 efficacy, toxicity, or side effects of treatment with such an peptidomimetics, prodrugs, receptors, binding agents, anti agent. Alternatively, an agent identified as described herein bodies, Small molecules or other drugs, or ribozymes) which can be used in an animal model to determine the mechanism alter (e.g., increase or decrease) the activity of a member of of action of Such an agent. leukotriene pathway binding agent, Such as a FLAP binding Furthermore, this invention pertains to uses of novel agents agent (e.g., 5-LO) or a LTA4H binding agent, as described 30 identified by the above-described screening assays for treat herein. For example, such agents can be agents which have a ments as described herein. In addition, an agent identified as stimulatory or inhibitory effect on, for example, the activity described herein can be used to alteractivity of a polypeptide of a member of leukotriene pathway binding agent, such as a encoded by a FLAP nucleic acid or a LTA4H nucleic acid, or to alter expression of a FLAP nucleic acid or LTA4H nucleic FLAP binding agent or a LTA4H binding agent; which acid, by contacting the polypeptide or the nucleic acid (or change (e.g., enhance or inhibit) the ability a member of 35 contacting a cell comprising the polypeptide or the nucleic leukotriene pathway binding agents, (e.g., receptors or other acid) with the agent identified as described herein. binding agents) to interact with the polypeptides of the inven The present invention is now illustrated by the following tion; or which alter posttranslational processing of the mem Examples, which are not intended to be limiting in any way. ber of leukotriene pathway binding agent, (e.g., agents that The teachings of all references cited are incorporated herein alter proteolytic processing to direct the member of the leu in their entirety. kotriene pathway binding agent from where it is normally 40 synthesized to another location in the cell. Such as the cell EXAMPLE 1. Surface; agents that alter proteolytic processing such that more active binding agent is released from the cell, etc.). Identification of Gene and Haplotypes Associated For example, the invention provides assays for Screening with MI candidate or test agents that bind to or modulate the activity of 45 a member of the leukotriene pathway (orenzymatically active A genome wide scan of 296 multiplex Icelandic families with 713 MI patients was performed as described in detail in portion(s) thereof), as well as agents identifiable by the International Application No. PCT/US2005/03312 (Publica assays. As described above, test agents can be obtained using tion No. WO2005/075022) incorporated herein by reference any of the numerous approaches in combinatorial library in its entirety. Through the Suggestive linkage to a locus on methods known in the art, including: biological libraries; 50 chromosome 13q12-13 for female MI patients and early onset spatially addressable parallel solid phase or Solution phase MI patients, and haplotype association analysis, the gene libraries; synthetic library methods requiring deconvolution; encoding the 5-lipoxygenase activating protein (FLAP) was the “one-bead one-compound library method; and synthetic identified, and a 4-SNP haplotype within the gene was deter library methods using affinity chromatography selection. The mined to confer a near 2-fold risk of MI. Male patients biological library approach is limited to polypeptide libraries, 55 showed strongest association to the at-risk haplotype. Inde while the other four approaches are applicable topolypeptide, pendent confirmation of FLAP association to MI was non-peptide oligomer or Small molecule libraries of com obtained in a British cohort of patients with sporadic MI. pounds (Lam, K. S. Anticancer Drug Des., 12:145 (1997)). These findings Support FLAP as the first specific gene iso In one embodiment, to identify agents which alter the lated that confers substantial risk of the complex trait of MI. activity of a member of the leukotriene pathway (such as a FLAPbinding agent, LTA4H binding agent oran agent which 60 Linkage Analysis binds to a member of the leukotriene pathway (a “binding agent)), a cell, cell lysate, or Solution containing or express A genome wide scan was performed in search of MI Sus ing a binding agent (e.g., 5-LO, or a leukotriene pathway ceptibility genes using a framework set of around 1000 mic member receptor, or other binding agent), or a fragment (e.g., rosatellite markers. The initial linkage analysis included 713 an enzymatically active fragment) or derivative thereof, can 65 MI patients who fulfilled the WHO MONICA research crite be contacted with an agent to be tested; alternatively, the ria (The World Health Organization MONICA Project, WHO binding agent (or fragment or derivative thereof) can be con MONICA Project Principal Investigators...J Clin Epidemiol US 8,158,362 B2 105 106 41, 105-14 (1988)) and were clustered in 296 extended fami None of these analyses yielded a locus of genome-wide lies. The linkage analysis was also repeated for early onset, significance. However, the most promising LOD score male and female patients separately. Description of the num (LOD=2.86) was observed on chromosome 13q12-13 for ber of patients and families in each analysis are provided in Table 1. 5 female MI patients at the peak marker D13S289 (data not shown). This locus also had the most promising LOD score TABLE 1. (LOD=2.03) for patients with early onset MI. After increas ing the information on identity-by-descent sharing to over Number of patients that cluster into families and the corresponding number of families used in the linkage analysis 90% by typing 14 additional microsatellite markers in a 30 10 centiMorgan (cM) region around D13S289, the LOD score Number of Number of Number of Genotyped Phenotype patients families pairs relatives from the female analysis dropped to 2.48 (P value=0.00036), while the highest LOD score remained at D13S289. All MI patients 713 296 863 1741 Males 575 248 724 1385 Females 140 56 108 366 15 Microsatellite Association Study Early onset 194 93 1S6 739 The 7.6 Mb region that corresponds to a drop of one in Genotyped relatives were used to increase the information on IBD sharing among the patients in the linkage analysis LOD score in the female MI linkage analysis, contains 40 known genes (Table 2). TABLE 2 Genes residing within the One LOD drop region of the chromosome 13q12-13 linkage peak.

LL Symbol LL gene name

USP12L1 ubiquitin specific protease 12 like 1 ribosomal protein L21 general transcription factor IIIA mitochondrial translational initiation factor 3 PDZ domain containing ring finger 1 hypothetical protein MGC9850 polymerase (RNA) I polypeptide D, 16 kDa GS homeobox 1 insulin promoter factor 1, homeodomain transcription factor caudal type homeo box transcription factor 2 ims-related tyrosine kinase 3 LOC255967 hypothetical protein LOC255967 FLT1 ims-related tyrosine kinase 1 (vascular endothelial growth factor? vascular permeability factor receptor) C13orf12 chromosome 13 open reading frame 12 LOC283537 hypothetical protein LOC283537 KIAAO774 KIAAO774 protein SLC7A1 Solute carrier family 7 (cationic amino acid transporter, y- system), member 1 ubiquitin-like 3 hypothetical protein MGC2599 similar to katanin pé0 subunit A 12599 high-mobility group box. 1 D13S106E highly charged protein ALOXSAP arachidonate 5-lipoxygenase-activating protein FLJ14834 hypothetical protein FLJ14834 hypothetical protein MGC40178 heat shock 105 kDa 110 kDa protein 1 beta 3-glycosyltransferase-like similar to G protein coupled receptor affecting testicular descent (H.

LOC196549 similar to hypothetical protein FLJ20897 3CDNA73 hypothetical protein CG003 breast cancer 2, early onset hypothetical gene CG018 PROO297 PROO297 protein LOC88523 CGO16 CGO12 hypothetical gene CG012 CGO3O hypothetical gene CG030 CGOOS hypothetical protein from BCRA2 region APRIN androgen-induced proliferation inhibitor KL Klotho STARD13 START domain containing 13 RFC3 replication factor C (activator 1) 3,38 kDa US 8,158,362 B2 107 108 To determine which gene in this region most likely con TABLE 3-continued tributes to MI, 120 microsatellite markers positioned within this region were typed, and a case-control association study Locations of all genotyped SNPs in NCBI build 34 of the human genome was performed using 802 unrelated MI patients and 837 assembly population-based controls. The association study was also repeated for each of the three phenotypes that were used in the SNP name Build34 start linkage study, i.e. early onset, male and female MI patients. SG13S1 29O86224 SG13S2 29O87473 The initial association analysis was performed when the SG13S367 29O88090 average spacing between microsatellite markers was approxi SG13S10 29.088473 mately 100 kb. This analysis revealed several extended hap 10 SG13S3 29O89044 lotypes composed of 4 and 5 microsatellite markers that were SG13S368 29O89886 significantly associated with female MI (see FIGS. 1 and 2. SG13S4 29O90997 and Tables 13 and 14). A region common to all these extended SG13SS 29091307 haplotypes, is defined by markers DG13S166 and D13S1238. SG13S90 29091780 This region included only one gene, the FLAP nucleic acid SG13S6 29092536 15 SG13S371 29.093.964 sequence. The two marker haplotype involving alleles 0 and SG13S372 290942.59 –2 for markers DG13S166 and D13S1238, respectively, was SG13S373 29096688 found in excess in patients. SG13S375 29096874 This was the first evidence that the FLAP gene might be SG13S376 29.096962 involved in the pathogenesis of myocardial infarction. SG13S25 29097553 SG13S377 29.101.96S Subsequent haplotype analysis that included more micro SG13S100 291.04271 satellite markers (n=120) in the candidate region on chromo SG13S95 29106329 some 13q12-13, now with a marker density of 1 microsatellite SG13S191. 29.107830 marker per 60 kb, showed decreased significance of the origi SG13S106 29.108579 nal haplotype association. However, the haplotype associa SG13S114 2911 0096 tion analysis using increased density of markers again 25 SG13S121 2.9112174 pointed towards the FLAP gene. This analysis strongly Sug SG13S122 2.9112264 gested that a 300 kb region was involved in the susceptibility SG13S43 2.9112455 SG13S192 291.16308 of myocardial infarction. The haplotype that showed associa SG13S88 291. 164O1 tion to all MI with the lowest P value (0.00009) covered a SG13S137 291.18118 region that contains 2 known genes, including the gene 30 SG13S86 291.1881S encoding arachidonate 5-lipoxygenase-activating protein SG13S87 291. 18873 (FLAP) and a gene with an unknown function called highly SG13S39 291.1974O charged protein. However, the haplotype association to SG13S26 2.91222S3 female MI in this region was less significant (Pvalue=0.005) SG13S27 2.9122283 than for all MI patients and to our Surprise, the most signifi SG13S29 29.123643 cant haplotype association was observed for male MI patients 35 SG13S89 29124441 SG13S96 291.24906 (P value=0.000002). This male MI haplotype was the only SG13S30 2912S840 haplotype that remained significant after adjusting for all SG13S97 291291.39 haplotypes tested. SG13S32 291.30547 In view of the association results described above, FLAP SG13S41 29134045 was an attractive candidate and therefore efforts were focused 40 SG13S42 291.35877 on this gene. SG13S34 29.1371OO SG13S35 29.1381.17 Screening for Polymorphisms in FLAP and Linkage SG13S181 291.38633 Disequilibrium Mapping SG13S184 291.39435 SG13S188 29.1408OS 45 To determine whether variations within the FLAP gene significantly associate with MI and to search for causal varia tions, the FLAP gene was sequenced in 93 patients and 93 In addition to the SNPs, a polymorphism consisting of a controls. The sequenced region covers 60 kb containing the monopolymer A repeat that has been described in the FLAP FLAP gene, including the 5 known exons and introns and the promoter region was typed (Koshino, T. et al., Mol Cell Biol 26 kb region 5' to the first exon and 7 kb region 3' to the fifth 50 Res Commun 2, 32-5 (1999)). exon. In all, 144 SNPs were identified, of those 96 were The linkage disequilibrium (LD) block structure defined excluded from further analysis either because of low minor by the 48 SNPs that were selected for further genotyping is allele frequency or they were completely correlated with other SNPs and thus redundant. Only one SNP was identified shown in FIG. 6. A strong LD was detected across the FLAP within a coding sequence (exon 2). This SNP did not lead to 55 region, although it appears that at least one recombination amino acid substitution. The locations of these SNPs in the may have occurred dividing the region into two strongly NCBI human genome assembly, build 34, are listed in Table correlated LD blocks. 3. Haplotype Association to MI TABLE 3 To perform a case-control association study the 48 selected 60 SNPs and the monopolymer A repeat marker were genotyped Locations of all genotyped SNPs in NCBI build 34 of the human genome assembly in a set of 779 unrelated MI patients and 628 population based controls. Each of the 49 markers were tested individu SNP name Build34 start ally for association to the disease. Three SNPs, one located 3 SG13S381 290833SO 65 kb upstream of the first exon and the other two 1 and 3 kb SG13S366 29083.518 downstream of the first exon, showed nominally significant association to MI (Table 4). US 8,158,362 B2 109 110 TABLE 4

SNP allelic association in the MI cohort

Phenotype Marker Allele P value RR # Pat. 96 Pat. # Ctrl 96 Ctrl All patients SG13S106 G O.OO44 129 681 72.O 530 66.6 SG13S100 A. O.O2O 1.29 388 69.6 377 63.9 SG13S114 T O.O21 1.21 764 70.O 602 65.8 Males SG13S106 G O.OO37 13S 422 72.9 530 66.6 SG13S100 A. O.0099 1.36 292 70.7 377 63.9 SG13S114 T O.O26 1.24 477 70.4 602 65.8 Early onset SG13S100 A. O.0440 143 99 71.7 377 63.9 Nominally significant SNP association with corresponding number of patients (#Pat.) and controls (#Ctrl), RR refers to relative risk.

However, after adjusting for the number of markers tested, 15 excess in the patients were tested. The resulting P values were these results were not significant. A search was then con adjusted for all the haplotypes tested by randomizing the ducted for haplotypes that show association to the disease patients and controls (see Methods). using the same cohorts. For computational reasons, the search Several haplotypes were found that were significantly was limited to haplotype combinations constructed out of associated to the disease with an adjusted P value less that two, three or four SNPs and only haplotypes that were in 0.05 (Table 5). TABLE 5 SNP haplotypes that significantly associate with Icelandic MI patients

A.

A. T G A

T G A.

A.

A. A. A. G

G

A A. A. A. C

C

G s G A.

A A. US 8,158,362 B2 111 112 TABLE 5-continued SNP haplotypes that significantly associate with Icelandic MI patients SG13S27 SG13S89 SG13S96 SG13S32 SG13S41 SG13S42 SG13S34 SG13S188 P value P value Pat. fr Ctrl. fra. RR D'c

G A. OOOOOO23 O.OOS O.158 O.095 80 OO A. OOOOOO3O O.OO6 O.158 O.095 .78 OO A. r OOOOOO32 O.OO7 0.157 O.O94 .79 OO A. OOOOOO46 OO12 O.158 O.083 2.07 O.89 A. OOOOOO47 O.O12 O.154 O.093 .78 OO A. OOOOOOSS OO15 O.147 O.087 81 OO A. r OOOOOO61 O.O17 0.157 O.083 2.07 O.89 G A. OOOOOO63 0.017 0.157 O.O84 2.04 O.89 A. OOOOOO7O O.O21 0.157 O.096 .76 OO A. A. OOOOOO75 O.O22 O.149 O.O89 .78 OO A. OOOOOO83 0.024 O.208 O.139 62 0.99 A. OOOOOO84 O.O26 O.145 O.074 2.14 O.88 A. OOOOOO84 O.O26 O.139 O.O82 82 OO G A. OOOOOO91 O.O28 O.156 O.096 .75 OO A. T OOOOOO94 O.O28 O.210 O.141 61 O.99 A. OOOOO1 OO O.O28 O.156 O.096 .74 OO A. A. OOOOO101 O.O28 O.215 O.133 80 O.81 A. OOOOO10S 0.028 0.157 O.O84 2.03 O.89 A. OOOOO108 O.O29 O.214 O.133 .78 O.81 A. A. OOOOO11O O.O3O O.146 O.07S 2.10 O.88 A. OOOOO112 O.O3O O.212 0.144 60 1.OO r OOOOO113 O.O3O O.151 O.O81 2.03 0.78 A. OOOOO118 O.O31 O.156 O.096 .73 1.00 A. r OOOOO126 O.O34 O.212 O.131 79 0.79 G A. OOOOO129 O.O3S O.211 0.144 S9 1.OO G A. OOOOO134 O.O3S O.156 O.O84 2.01 O.89 A. OOOOO136 0.036 O.211 O.143 60 1.OO A. OOOOO137 0.036 O.156 O.O85 2.OO O.89 A. OOOOO148 O.O37 O.151 O.O81 2.01 O.78 r OOOOO15O O.O37 O.160 O.O99 .73 0.87 A. OOOOO15O O.O37 O.130 O.066 2.13 O.90 r OOOOO154 O.O39 O.152 O.O94 .73 0.93 A. A. OOOOO154 O.O40 0.155 O.097 .70 100 A. OOOOO157 0.040 O.141 O.O85 .76 1.OO A. OOOOO158 0.040 O.152 O.O84 94 O.90 G A. OOOOO163 O.O40 O.210 O.143 S9 O.99 A. OOOOO166 0.041 O.200 O.134 61 O.92 G A. OOOOO168 0.042 O.213 O.133 .76 O.81 A. OOOOO168 0.042 O.156 O.O84 2.OO O.89 A. OOOOO171 O.O42 O.211 O.136 .7O O.81 A. OOOOO183 0.043 O.192 O.128 62 0.85 A. OOOOO184 O.O43 O.212 O.132 .77 0.81 A. r OOOOO193 O.O46 O.328 O.251 46 0.99 G r OOOOO194 O.O46 0.175 O.115 64 O.98 A. OOOOO2O2 (0.048 O.210 O.136 .7O O.81 OOOOO209 O.O49 O.151 O.O82 2.00 0.76 Single test P values. P values adjusted for all the SNP haplotypes tested. Measure of correlation with Haplotype A4.

The most significant association was observed for a four TABLE 6-continued SNP haplotype spanning 33 kb, including the first four exons of the gene, with a nominal P value of 0.0000023 and an - Association of the A4 haplotype to MI, Stroke and PAOD adjusted P value of 0.005. This haplotype, labelled A4, has Phenotype (n) Frq. Pat. RR PAR P-value P-value haplotype frequency of 15.8% (carrier frequency 30.3%) in patients versus 9.5% (carrier frequency 17.9%) in controls Males (356) O.126 1.36 O.06S 0.057 ND (Table 6). Females (221) O. 114 122 O.O41 O.31 ND 55 “P value adjusted for the number of haplotypes tested. TABLE 6 Not done. Excluding known cases of MI. Shown is the FLAPA4haplotype and corresponding number Association of the A4 haplotype to MI.MI, Stroke and PAOD attributedof patients risk (n), (PAR) haplotype and P frequency values. in patients (Frq. pat), relative risk (RR), population The A4 haplotype is defined by the following Phenotype (n) Frq. Pat. RR PAR P-value P-value SNPs: SG13S25, SG13S114, SG13S89 and SG13S32 (Table 5). The same controls (n = 628) are used for the association analysis in MI, stroke and PAOD as well as for the male, female MI (779) O.158 18O O.13S OOOOOO23 O.OOS 60 and early onset analysis. The A4 haplotype frequency in the control cohort is 0.095. Males. Ea 8. 2. 8. gol N.i The relative risk conferred by The A4 haplotype compared Early onset (358) O.138 1.53 O.O94 O.OOS8 ND to otherhaplotypes constructed out of the same SNPs, assum St. t 9. 5. 9. g NR ing a multiplicative model, was 1.8 and the corresponding R Go 0.141 155 0.098 00074 N 65 population attributable risk (PAR) was 13.5%. As shown in PAOD (577) O.122 1.31 O.OS6 O.061 ND Table 6, the A4 haplotype was observed in higher frequency in male patients (carrier frequency 30.9%) than in female US 8,158,362 B2 113 114 patients (carrier frequency 25.7%). All the other haplotypes (15-16%). The haplotypes in series B and A are strongly that were significantly associated with an adjusted P value correlated, i.e. the haplotypes in B define a subset of the less than 0.05, were highly correlated with the A4 haplotype haplotypes in A. Hence, haplotypes B are more specific than and should be considered variants of that haplotype (Table 5). Table 6 shows the results of the haplotype A4 association 5 A. Haplotypes A are however more sensitive, i.e. they capture study using 779 MI patients, 702 stroke patients, 577 PAOD more individuals with the putative mutation, as is observed in patients and 628 controls. First and second degree relatives the population attributable risk which is less for B than for A. were excluded from the patient cohorts. All known cases of Furthermore, these haplotypes show similar risk ratios and MI were removed from the stroke and PAOD cohorts before 10 allelic frequency for early-onset patients (defined as onset of testing for association. A significant association of the A4 first MI before the age of 55) and for both gender. In addition, haplotype to stroke was observed, with a relative risk of 1.67 analyzing various groups of patients with known risk factors, (P value=0.000095). In addition, it was determined whether the A4 haplotype was primarily associated with a particular Such as hypertension, high cholesterol, Smoking and diabetes, did not reveal any significant correlation with these haplo Sub-phenotype of stroke, and found that both ischemic and 15 hemorrhagic stroke were significantly associated with the A4 types, indicating that the haplotypes in the FLAP gene rep haplotype (Table 22, below). resent an independent genetic susceptibility factor for MI. TABLE 7 The selected SNP haplotypes and the corresponding p-values p-val RR #aff aff, frc. carr. fra. #con con. fra. PAR SG13S99 SG13S25 B4 4.8OE-05 2.08 903 0.106 O.2 619 O.OS4 O.11 G B5 2.4OE-05 2.2 91O O.101 O.19 623 O.049 O.11 T G B6 18OE-06 2.22 913 0.131 O.24 623 O.O63 O.14 T G A4 5.1OE-06 1.81 919 0.159 O.29 623 O.09S O.14 G AS 2.6OE-06 1.91 920 O.15 O.28 624 O.O85 O.14 T G

B4 G A. B5 G A. B6 A4 AS g

Relative risk (RR), number of patients (Haff), allelic frequency in patients (aff frc.), carrier frequency in patients (carr, frc), number of controls (icon), allelic frequency in controls (con, frc), population attributable risk (PAR). The patients used for this analysis were all unrelated within 4 meioses.

More Variants of Haplotype A4 Haplotype Association to Female MI Two correlated series of SNP haplotypes were observed in Before we had typedall the SNPs that eventually lead to the excess in patients, denoted as A and B in Table 7. The length identification of A4 haplotype we performed a haplotype of the haplotypes varies between 33 and 69 Kb, and the association analysis that included 437 female MI patients, haplotypes cover one or two blocks of linkage disequilibrium. 1049 male MI patients, and 811 controls that had been geno Both series of haplotypes contain the common allele G of the typed with several SNPs and 3 microsatellite markers. These SNPSG13S25. Allhaplotypes in the A series contain the SNP markers were all located within 300 kb region encompassing SG13S114, while all haplotypes in the B series contain the 50 the FLAP gene. In a case-control study of the MI patients SNPSG13S106. In the B series, the haplotypes B4, B5, and using these data, several haplotypes were found, that were B6 have a relative risk (RR) greater than 2 and with allelic significantly over-represented in the female MI patients com frequencies above 10%. The haplotypes in A series have pared to controls (see Table 8). All these haplotypes were slightly lower RR and lower p-values, but higher frequency highly correlated with each other. TABLE 8 haplotypes in the FLAP region (FLAP and flanking nucleotide sequences) that were associated with female MI. US 8,158,362 B2 115 116 TABLE 8-continued haplotypes in the FLAP region (FLAP and flanking nucleotide sequences) that were associated with female MI.

C T O T A. T C T O T T G C T O G T C T O G T C T O G T C T O A. T C T O A. T C T O G A. T A. C T O G T A. C T O T A. C T O T T C A. T O G T A. C T O A. T A. C A. T O T SG13S184 D13S1238 DG13S2605 p-val Naff aff, fra N ctrl ctrl. fra rel risk PAR info

G -2 1.3OE-OS 455 O.108 811 O.048 2.4 O.122 O.615 -2 O 7.61E-06 455 O.06S 812 O.O2 3.45 0.091 0.615 -2 O 8.82E-O6 455 O.06S 812 O.O2 3.47 0.092 O.6O2 -2 O 9.31E-06 455 O.06S 812 O.O2 3.39 O.O89 0.611 G -2 O 6.91E-O6 455 O.063 812 O.019 3.54 0.09 0.624 -2 O 9.76E-06 455 O.063 812 O.019 3.S1 O.O89 0.606 G -2 1.09E-OS 455 O.063 8 O.019 3.41 O.O86 0.618 -2 O 11 OE-05 455 O.063 812 O.019 3.44 0.087 0.611 G -2 O 111E-05 455 O.063 812 O.018 3.56 0.086 O.S89 G -2 1.22E-05 455 O.063 8 O.018 3.6 0.087 0.577 G -2 O 1.26E-05 455 O.063 812 O.O2 3.35 0.088 O.629 G -2 O 8.59E-06 455 O.062 812 O.018 3.53 O.O85 0.62 G -2 1.2OE-05 455 O.062 8 O.019 3.42 0.086 O.617 G -2 1.21E-05 455 O.062 8 O.019 3.43 O.O86 0.619 G -2 7.93E-06 455 O.061 8 O.O16 3.95 O.O88 OS 62 G -2 1.09E-OS 455 O.061 8 O.O17 3.85 0.09 O.S6 G -2 S.OOE-06 455 O.O6 8 O.O15 4.11 O.O87 O.S76 G -2 1.31E-OS 455 O.O6 8 O.O17 3.66 0.085 OS86 G -2 8.53E-06 455 O.O59 8 O.O16 3.85 O.O85 0.593 G -2 9.63E-06 455 O.OS8 8 O.O15 4.03 0.085 OS6S

Table 9 shows two haplotypes that are representative of 40 Table 10 shows that these same haplotypes were also over these female MI risk haplotypes. The relative risk of these represented in male MI patients compared to controls, haplotypes were 2.4 and 4, and they were carried by 23% and although with lower relative risk. It should be noted that after 13% of female MI patients, respectively. typing and analysing more SNPs in the FLAP region. TABLE 9

Two representative variants of the female MI “at risk haplotypes

Female MI C T O T T G -2 6.38E-06 C T O G -2 2.74E-OS

Naff aff, fra N ctrl ctrl. fra rel risk PAR info

Female MI 454 O.O59 809 O.O15 40S 0.086 O.S77 447 O. 106 809 O.048 2.33 0.116 0.623

P-val: p-value for the association. N aff; Number of patients used in the analysis, Aff frc: haplotype frequency in patients, N ctrl: number of controls used in the analysis. Ctrl, frc: Haplotype frequency in controls, Rel risk: Relative risk of the haplotype. PAR: population attributable risk. Info: information content.

US 8,158,362 B2 125 126 -continued

NCBI NCBI end build34: build34: minor Start position Start allele position in in on chr. on chr. marker public minor frequency SEQID SEQID 13 (bp) 13 (bp) exon name alias1 alias2 SNP Variation allele (%) NO: 1 NO: 1 291451.43 29145143 SG13S411 FLA331395 AG A. 1.54 213143 213143 29145171 29145.171 SG13S9 FLA331423 C.T C 16.37 213171 213171 29145221 29145221 SG13S412 FLA331473 rS4769062 AG A. 7.42 21.3221 213221 2914526S 2914526S SG13S413 FLA331517 rS4238.138 C.T T 1.91 21326S 21326S

TABLE 1.4 Extended 4 microsatellite marker haplotypes a markers: DOS. Trfragilpere Length p val RR N af Pal P. ca. N. ct Pal P. ca. Alleles Markers O.88 4.71 6.23 428 721 O.O11 O.O22 O -12

O.82 INF 438 O.062 720 O O O 4 14

0.67 6.98 1991 435 O.059 721 O.OO2 O.OO3 8 6

0.767 4.85 26.72 436 O.048 721 O.OO2 (0.004 O O 12

0.515 193E INF 422 0.048 721 O O 2 O

O.864 1.68E INF 424 O.048 717 O O O 2 -16

O.927 5.38E. INF 435 720 O O 4 2 14

Alleles it's: For SNP alleles A = 0, C = 1, G = 2, T = 3; for microsatellite alleles: the CEPH sample (Centre d’Etudes du Polymorphisme Humain, genomics repository) is used as a reference, as described above, Length = length of haplotype in Mb. P-val = p-value. RR = Relative risk. Naf = Number of patients, Pal = allelic frequency of haplotype. Pca = carrier frequency of haplotype, Nct = number of controls. Alleles = alleles in the haplotype. Markers = markers in the haplotype,

TABLE 1.5 Extended 5 microsatellite marker haplotypes 5 markers: pos. Tr-fragilpere

Length p val RR N af Pal P. ca. N. ct Pal P. ca. Alleles Markers O.851 7.45 E-06 1543 413 O.O34 0.067 715 O.OO2 O.OOS O 18 O O

O.964 8.07 E-06 INF O O O -12

O.964 2.38 E-06 INF 437 0.026 O.OS2 720 O O O 6

US 8,158,362 B2 129 130 TABLE 15-continued Extended 5 microsatellite marker haplotypes 5 markers: pos. Tr-fragilpere Length p-val RR N af Pal P ca. Nct Pal P ca. Alleles Markers 3S163 S290 3S1061 O.683 6.58E-06 INF 437 O.O29 O.OS6 721 O O O 3S172 3S1103 3S166 S1287 3S1061 O.767 2.85E-06 32.43 436 0.044 0.087 721 O.OO1 O.OO3 O 3S1101 3S166 S290 S1287 3S1061 O.86S 9.58E-06 18.39 451 O.O23 O.O4S 739 O.OO1 O.OO3 O S289 3S166 3S163 S1287 3S293 O.865 S.O8E-06 INF 453 O.O19 O.O38 739 O O O S289 DG13S166 DG13S163 DG13S1061 DG13S293 O.927 102E-07 27.6S 437 O.O37 0.073 721 O.OO1 O.OO3 4 O 2 14 3 DG13S1103 DG13S166 D13S1287 DG13S1061 DG13S301 Length = length of haplotype in Mb. P-val = p-value. RR = Relative risk, Naf = Number of patients, Pal = allelic frequency of haplotype. Pca = carrier frequency of haplotype, Nct = number of controls. Alleles = alleles in the haplotype. Markers = markers in the haplotype

EXAMPLE 2 p-0.05). The results are consistent with a disease hypothesis 40 that increased expression of the 5-LO plays a role in the Relationship Between Polymorphism in the pathogenesis of MI. 5-Lipoxygenase Promoter and MI TABLE 16 A family of mutations in the G-C rich transcription factor binding region of the 5-LO gene has previously been identi- 45 Risk P fied. These mutations consist of deletion of one, deletion of Naff Fraaff N ctrl Frq ctrl Ratio value two, or addition of one Zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation MI patients 1112 O.87O1 734 O.8SO 1803 0.048 start site where there are normally 5 Sp1 binding motifs in Independent 969 O.872O 734 O.8SO 2013 O.O37 tandem. These naturally occurring mutations in the human 50 Males 646 (0.8740 734 O.8SO 2232 O.O39 5-LO gene promoter have been shown to modify transcription Females 465 0.8645 734 O.8SO 1249 0.18O factor binding and reporter gene transcription. The capacity Age of S22 O.8745 734 O.8SO 2286 0.046 of the mutant forms of DNA to promote transcription of CAT reporter constructs have been shown to be significantly less onset < 60 than that of the wild type DNA (J. Clin. Invest. Volume 99, 55 Males 353 0.8768 734 O.8SO 2542 O.OS3 Number 5, March 1997, 1130-1137). Females 169 O.8698 734 O.8SO 1779 0.2O2 To test whether 5-LO is associated with theatherosclerotic PAOD patients 748 0.8763 734 O.8SO 2492 O.O22 diseases, particularly myocardial infarction (MI) in the Independent 703 0.8755 734 O.8SO 2400 0.027 human population, this promoter polymorphism, consisting Males 473 0.8774 734 O.8SO 2613 O.O33 of variable number of tandem Sp1/Egr-1 binding sites, was 60 Females 275 O.8745 734 O.8SO 2289 0.092 genotyped in 1112 MI patients, 748 patients with PAOD, and Stroke patients 541 O.8743 734 O.8SO 2262 0.046 541 stroke patients. Males 326 O.8758 734 O.8SO 2427 OO67 The results, shown in Table 16, demonstrate that the wild Females 215 O.8721 734 O.8SO 2O19 O.144 type allele (which represents the allele with the most active Cardio/Large V 2O2 O.8861 734 O.8SO 3719 O.O38 promoter and thus with the highest expression of the 5-LO 65 Cardioembolic 114 O.8772 734 O.8SO 2592 O.16S mRNA. J. Clin. Invest. Volume 99, Number 5, March 1997, Large Vessel 88 O.8977 734 O.8SO S474 0.053 1130-1137) is significantly associated with MI (RR=1.2, US 8,158,362 B2 132 TABLE 16-continued Table 17 (below) shows that the female MI “at risk” hap lotype was more associated with female MI patients who have Risk P high LTE4 levels (top 50th percentile), than with female MI Naff Fraaff N ctrl Frq ctrl Ratio value patients who have low levels of LTE4 (bottom 50th percen Small Vessel 77 O.8831 734 O.85O1 1.2791. O.163 5 tile). Hemorrhagic 27 O.9259 734 O.85O1 2.2O3S O.O81 In addition, haplotype analysis, comparing female MI Single sided p-values: Fisher exact test, patients with high levels of LTE4 with female patients with Naff= number of affected individuals; low levels, showed that those with high levels had increased Frq aff= frequency of the wild type allele of the promoter polymorphism in the affected group; prevalence of the “at risk” haplotype by 1.6 fold (see Table N.ctrl= number of controls; 10 Frq ctrl= frequency of the wild type allele of the promoter polymorphism in the control 18). Although the association did not rise to the level of group; statistical significance, the results show clearly that the “at risk” haplotypes are enriched in the MI patient group that has EXAMPLE 3 high levels of LTE4. The carrier frequency of the “at risk” 15 haplotypes are 12% and 20%, respectively, in the whole Elevated Lite4 Biosynthesis in Individuals with the female MI group, but go up to 15% and 24%, respectively, in Flap Mi-Risk Haplotype the female MI group that has high levels of LTE4. Corre spondingly, the carrier frequency of the "at risk” haplotypes Based on the known function of the end products of the decrease to 8% and 18%, respectively, in the group of female leukotriene pathway and based on our 5-LO association data, MI that has low levels of LTE4 (Note carrier frequencies are the association of the FLAP haplotype with MI is best explained by increased expression and/or increased function twice the disease allele frequency times 1 minus the disease of the FLAP gene. In other words, those individuals that have allele frequency plus the square of the disease allele fre a “at risk” FLAP haplotype have either, or both, increased quency). amount of FLAP, or more active FLAP. This would lead to Note that LTE4 may simply reflect the leukotriene synthe increased production of leukotrienes in these individuals. 25 sis rate of the leukotriene synthetic pathway upstream of the To demonstrate this theory, LTE4, a downstream leukot key leukotriene metabolite involved in MIrisk. For example, riene metabolite, was measured in patient serum samples as leukotriene B4 is probably more likely than leukotriene E4 to described in detail in International Application No. PCT/ be involved in the inflammatory aspects of MI plaques but US2005/03312 (Publication No. WO2005/075022) incorpo since B4 has a short halflife, it is difficult to measure reliably rated herein by reference in its entirety. in serum samples, while E4 has long term stability. TABLE 17

Association of the at risk haplotypes for female MI, with female MI who also have high levels of LTE4 (>50 pg/ml (roughly the upper 50th percentile).

High LTE4

T O T T G -2 T O G -2 Low LTE4

T O T T G -2 T O G -2

p-val Naff aff, fra N ctrl ctrl. fra rel risk PAR info

High LTE4

3.72E-06 221 0.075 809 O.O14 S.S1 O.115 O.S6S 2.3OE-OS 220 O.122 809 O.046 2.89 0.154 O.608 Low LTE4

4.65E-02 185 O.040 809 O.O15 2.67 0.048 O.S11 2.88E-O2 182 O.O87 809 O.048 1.89 O.O8 O.622

P-val: p-value for the association. N aff; Number of patients used in the analysis. Aff, frc; haplotype frequency in patients, N ctrl: number of controls used in the analysis, Ctrl, frc: Haplotype frequency in controls, Rel risk: Relative risk of the haplotype. PAR: population attributable risk. Info: information content, Less association was found between the at risk haplotype for female MI, with femaleMI who also have low levels of LTE4 (<50 pg/ml). US 8,158,362 B2 133 134 TABLE 1.8 Association between haplotypes that were most significantly associated with female MI, and serum LTE4 levels.

High vs low LTE4 C T O T G -2 C T O G -2 p-val Naff aff, fra N ctrl ctrl. fro rel risk PAR info High vs low LTE4 1.61E-O1 221 O.O84 18S O.OS4 1.61 O.O63 O.689 1.2OE-O1 220 O.13 182 O.O88 1.54 O.O89 O.686 P-val: p-value for the association. N aff; Number of patients used in the analysis, Aff frc: haplotype frequency in patients, N ctrl: number of controls used in the analysis. Ctrl, frc: Haplotype frequency in controls, Rel risk: Relative risk of the haplotype. PAR: population attributable risk. Info: information content. Here, the group of affected individuals were defined as female MIpatients with high serum LTE4 (higher than 50 pg/ml) and the control group is defined as female MI patients with low serum LTE4 (below 50 pg/ml).

EXAMPLE 4 3) C-reactive protein levels correlate with serum leukotriene E4; and 4) Patients with MI-risk FLAP haplotypes have Elevated LTE4 Correlated with Elevated C-Reactive higher levels of serum leukotriene E4 and CRP. Based on Protein (CRP) 25 these data, it was hypothesized that serum leukotriene E4 levels correlate with MI risk. The relationship between the increased production of leu To test this hypothesis, LTE4, a downstream leukotriene kotrienes and the inflammatory marker CRP, a well estab metabolite, was measured in 488 female MI patient and 164 lished risk factor for MI, was then explored. As shown in FIG. control serum samples. The LTE4 levels for the patients were 4, a significant positive correlation was found between serum 30 higher than that for the controls using a one-sided Wilcoxon LTE4 levels and serum CRP levels. rank-Sum test. The p-value of the difference was 0.0092, thus confirming our hypothesis. Therefore, elevated leukotriene EXAMPLE 5 E4 represents a risk factor for MI. Serum or plasma LTE4 levels may be used to profile the MIrisk for individuals to aid Assessment of Level of CRP in Patients with At-Risk 35 in deciding which treatment and lifestyle management plan is Haplotype best for primary or secondary MI prevention. In the same way other leukotriene metabolites may be used to risk profile for The level of CRP in female patients with female MI at-risk MI. haplotypes was assessed, in order to assess whether there was a presence of a raised level of inflammatory marker in the 40 EXAMPLE 7 presence of the female MI at-risk haplotype. Results are shown in Table 19. Although the association did not rise to the Increased LTB4 Production in Activated Neutrophils level of statistical significance, it was demonstrated that the From MI Patients average CRP was elevated in those patients with the at-risk haplotype versus those without it. 45 A principal bioactive product of one of the two branches of the 5-LO pathway is LTB4. To determine whether the patients TABLE 19 with past history of MI have increased activity of the 5-LO pathway compared to controls, the LTB4 production in iso All female patients lated blood neutrophils was measured before and after stimu 50 lation in vitro with the calcium ionophore, ionomycin as O Mean CRP SECRP described in detail in International Application No. PCT/ affecteds: With haplotype. 155 4.91 8.7 US2005/03312 (Publication No. WO2005/075022) incorpo Not with haplotype. 218 4.35 6.13 rated herein by reference in its entirety. No difference was detected between the LTB4 production in resting neutrophils 55 from MI patients or controls (results not shown). In contrast, EXAMPLE 6 the LTB4 generation by neutrophils from MI patients stimu lated with the ionophore was significantly greater than by Elevated Serum LTE-4 Levels in MI Patients Versus neutrophils from controls at 15 and 30 minutes, respectively. Controls Moreover, the observed increase in the LTB4 release was 60 largely accounted for by male carriers of haplotype A4. The end products of the leukotriene pathway are potent whose cells produced significantly more LTB4 than cells inflammatory lipid mediators that can potentially contribute from controls (Pvalue=0.0042) (Table 20). As shown in Table to development of atherosclerosis and destabilization of ath 20, there was also a heightened LTB4 response in males who erosclerotic plaques through lipid oxidation and/or proin do not carry Hap A but of borderline significance. This could flammatory effects. Examples one through five show that: 1) 65 be explained by additional variants in the FLAP gene that MI correlates with genetic variation at FLAP; 2) MI corre have not been uncovered, or alternatively in other genes lates with high expression promoter polymorphism at 5-LO; belonging to the 5-LO pathway, that may account for upregu US 8,158,362 B2 135 136 lation in the LTB4 response in some of the patients without TABLE 20-continued the FLAP at-risk haplotype. As shown in Table 20, differ ences in LTB4 response were not detected in females. How LTB4 levels after ionomycin stimulation of isolated neutrophils ever, due to a small sample size this cannot be considered conclusive. Taken together, the elevated levels of LTB4 pro After 15 Minutes After 30 Minutes duction of stimulated neutrophils from male carriers of the Phenotype (n) Mean (SD) P value Mean (SD) P value at-risk haplotype Suggest that the disease associated variants in the FLAP gene increase FLAP's response to factors that Carriers (6) 4.59 (0.80) O.90 4.75 (0.82) O.85 stimulate inflammatory cells, resulting in increased leukot Non-carriers (7) 4.94 (1.04) O.34 5.12 (0.96) O.25 10 Mean+ SD of log-transformed values of LTB4 levels of ionomycin-stimulated neutrophils riene production and increased risk for MI. from MIpatients and controls. Results are shown for two time points; 15 and 30 minutes. The results for males and females and for MImale and female carriers and non-carriers of the at-risk haplotype HapA are shown separately, Two-sided p values corresponding to a TABLE 20 standard two-sampletest of the difference in the mean values between the MIpatients, their various sub-cohorts and the controls are shown, LTB4 levels after ionomycin stimulation of isolated neutrophils' 15 EXAMPLE 8 After 15 Minutes After 30 Minutes Haplotypes Associated with MI also Confer Risk of Stroke and PAOD Phenotype (n) Mean (SD) P value Mean (SD) P value Because stroke and PAOD are diseases that are closely Controls (35) 4.53 (1.00) 4.67 (0.88) related to MI (all occur on the basis of atherosclerosis), it was Males (18) 4.61 (1.10) 4.68 (1.07) examined whether the SNP haplotype in the FLAP gene that confers risk to MI also conferred risk of stroke and/or PAOD. Females (17) 4.51 (0.88) 4.67 (0.62) The at risk haplotype (A4 haplotype)can be defined by the MI (41) 5.18 (1.09) O.O11 5.24 (1.06) O.O16 following 4 SNPs: SG13S25 with allele G, SG13S114 with Carriers (16) 5.26 (1.09) O.O27 5.27 (1.09) O.OS1 25 allele T, SG13S89 with allele G, and SG13S32 with allele A. Non-carriers (24) 5.12 (1.08) O.040 5.22 (1.03) O.O3S Table 21 shows that the haplotype A4 increases the risk of MI males (28) 5.37 (1.10) O.OO33 5.38 (1.09) O.OO76 having a stroke to a similar extent as it increases the risk of having an MI. The at risk haplotype is carried by 28% of Carriers (10) 5.66 (1.04) O.OO42 5.58 (1.12) O.O13 stroke patients and 17% of controls, meaning that the relative Non-carriers (18) 5.20 (1.09) O.O39 5.26 (1.05) O.O41 risk of having stroke for the carriers of this haplotype is 1.7 MI females (13) 4.78 (0.95) O46 4.95 (0.92) O.36 30 (p-value=5.810'). Although not as significant, the at risk haplotype also confers risk of having PAOD. TABLE 21 p-val r #aff aff, fra. #con con. frc. info SG13S25 MI haplotypes All MI patients

A4 5.3E-07 1.80 1407 O.16 614 O.09 O.82 G 1.OE-04 1.87 1388. O.10 612 O.O6 O.67 G Males MI

A4 2.5E-08 2.OO 864 O.17 614 O.09 O.82 G 11E-OS 2.12 852 O.11 612 O.O6 O.67 G Females MI

A4 1.9E-O2 44 543 O.13 614 O.09 0.73 G 7.9E-02 4S S36 0.08 612 O.O6 O.6O G Replication in stroke All stroke patients

A4 5.8E-O6 .73 1238 0.15 614 O.09 O.8O G B4 2.3E-04 83 1000 0.10 612 O.O6 O.71 G Males stroke

A4 11E-O6 .91 710 O.17 614 O.09 0.79 G B4 3.1E-OS 2.11 S74 0.11 612 O.O6 0.72 G Females stroke

A4 9.9E-03 49 S28 0.13 614 O.10 O.74 G 6.3E-O2 47 426 O.08 612 O.O6 O.70 G All stroke excluding MI 8.4E-05 6S 1054 0.15 614 O.09 O.78 G Males stroke excluding MI 6.4E-05 .78 573 0.16 614 O.09 0.75 G Females stroke excluding MI 1.2E-O2 49 481 0.14 614 O.10 0.72 G Cardioembolic stroke 6.6E-04 87 248 0.16 614 O.10 O.74 G Cardioembolic stroke excluding MI 3.8E-O2 56 191 0.14 614 O.10 O.70 G Large vessel stroke 8.OE-02 47 150 0.13 614 O.09 O.83 G Large vessel stroke excluding MI 2.9E-O1 31 114 O.12 614 O.09 O.8O G Small vessel stroke 7.2E-04 2.05 166 0.18 614 O.09 O.71 G Small vessel stroke excluding MI 1.OE-04 2.31 152 O.20 614 O.10 O.71 G Hemorrhagic stroke 4.4E-02 73 97 0.15 614 O.09 0.72 G Hemorrhagic stroke excluding MI 3.9E-O2 .78 92 O.16 614 O.09 O.71 G Unknown cause stroke 1.3E-04 .88 335 0.16 614 O.09 0.75 G US 8,158,362 B2 137 138 TABLE 21-continued Unknown cause stroke excluding MI 6.5E-04 1.82 297 0.16 614 O.09 0.72 MI and stroke together All patients Best haplo A4 4.1E-07 1.75 2659 0.15 614 O.09 O.82 B4 4.1E-05 1.85 220S 0.10 612 O.O6 O.70 Males

A4 1.4E-08 1.93 1437 0.17 614 O.09 O.82 B4 2.OE-06 2.11 1290 O.11 612 O.O6 O.70 Females

A4 3.6E-O3 1.47 1024 O.13 614 O.09 O.77 B4 2.8E-O2 148 91S O.08 612 O.O6 O.66 Patients with both MI and stroke

A4 6.1E-05 2.10 184 O.18 614 O.09 O.86 G Replication in PAOD All PAOD patients 3.6E-O2 1.31 920 O.12 614 O.10 O.84 Males PAOD 18E-O2 140 S8O. O.13 614 O.10 O.84 Females PAOD 3.7E-O1 1.17 340 O.11 614 O.10 O.83 All PAOD excluding MI 1.1E-01 1.24 750 0.12 614 O.10 O.83 Males PAOD excluding MI 8.3E-O2 1.30 461 O.12 614 O.10 O.83 Males PAOD excluding MI and 8.7E-O2 1.32 388 O.12 614 O.10 O.83 stroke

SG13S89 SG13S30 MI haplotypes All MI patients

A4 G B4 G Males MI

A4 G B4 G Females MI

A4 G B4 G Replication in stroke All stroke patients

A4 G B4 G Males stroke

A4 G B4 G Females stroke

A4 G G All stroke excluding MI G Males stroke excluding MI G Females stroke excluding MI G Cardioembolic stroke G Cardioembolic stroke excluding MI G Large vessel stroke G Large vessel stroke excluding MI G Small vessel stroke G Small vessel stroke excluding MI G Hemorrhagic stroke G Hemorrhagic stroke excluding MI G Unknown cause stroke G Unknown cause stroke excluding MI G MI and stroke together All patients Best haplo A4 G B4 G Males

A4 G B4 G Females

G G US 8,158,362 B2 139 140 TABLE 21-continued Patients with both MI and stroke

A4 T G A. Replication in PAOD All PAOD patients T G Males PAOD T G Females PAOD T G All PAOD excluding MI T G Males PAOD excluding MI T G Males PAOD excluding MI and T G stroke

The patient cohorts used in the association analysis shown 15 encryption system as previously described (Gulcher, J. R. in Table 21 may include first and second degree relatives. Kristjansson, K., Gudbartsson, H. & Stefansson, K., Eur J Table 21, discussed above, shows the results of the haplo Hum Genet 8, 739-42 (2000)). type A4 association study using 779 MI patients, 702 stroke In addition, in an independent linkage study of male patients, 577 PAOD patients and 628 controls. First and sec patients with ischemic stroke or transient ischemic attack, ond degree relatives were excluded from the patient cohorts. linkage to the same locus was observed with a LOD score of All known cases of MI were removed from the stroke and 1.51 at the same peak marker (FIG. 5), further suggested that PAOD cohorts before testing for association. A significant a cardiovascular Susceptibility factor might reside at this association of the A4 haplotype to stroke was observed, with locus. a relative risk of 1.67 (P value=0.000095). In addition, it was determined whether the A4 haplotype was primarily associ 25 EXAMPLE 9 ated with a particular Sub-phenotype of stroke, and found that both ischemic and hemorrhagic stroke were significantly Haplotype Association to FLAP in a British Cohort associated with the A4 haplotype (Table 22). In an independent study, it was determined whether vari 30 ants in the FLAP gene also have impact on risk of MI in a TABLE 22 population outside Iceland. The four SNPs, defining the A4 haplotype, were typed in a cohort of 750 patients from the Association of the A4 haplotype to subgroups of stroke United Kingdom who had sporadic MI, and in 728 British Phenotype (n) Pat. Frq. RR PAR P-value population controls. The patients and controls come from 3 Stroke (702) O.149 1.67 O-116 O.OOOO95 35 separate study cohorts recruited in Leicester and Sheffield. Ischemic (484) O.148 1.65 O.113 O.OOOS3 No significant differences were found in the frequency of the TIA (148) O.137 1.51 O.O90 O.OS8 haplotype between patients and controls (16.9% versus Hemorrhagic (68) O.167 1.91 O.153 O.O24 15.3%, respectively). However, when an additional 9 SNPs, Excluding known cases of MI. distributed across the FLAP gene, were typed in the British 40 cohort and searched for other haplotypes that might be asso Finally, the A4 haplotype was less significantly associated ciated with MI, two SNPs showed association to MI with a with PAOD (Table 21). It should be noted that similar to the nominally significant P value (data not shown). Moreover, stronger association of the A4 haplotype to male MI com three and four SNP haplotype combinations increased the risk pared to female MI, it also shows stronger association to male of MI in the British cohort further and the most significant stroke and PAOD (Table 21). 45 association was observed for a four SNP haplotype with a nominal P value=0.00037 (Table 23). Study Population TABLE 23 The stroke and PAOD cohorts used in this study have Association of the HapB haplotype to British MI patients previously been described (Gretarsdottir, S. et al. Nat Genet 50 35, 131-8 (2003); Gretarsdottir, S. et al., Am J Hum Genet 70, Phenotype (n) Frq. Pat. RR PAR P-value P-value 593-603 (2002); Gudmundsson, G. et al., Am J Hum Genet MI (750) 0.075 1.9S O.O72 O.OOO37 O.O46 70, 586-92 (2002)). For the stroke linkage analysis, geno Males (546) 0.075 1.97 O.O72 O.OOO93 ND types from 342 male patients with ischemic stroke or TIA that Females (204) O.O73 1.90 O.O68 O.O21 ND were linked to at least one other male patient within and 55 P value adjusted for the number of haplotypes tested using 1,000 randomization tests. including 6 meioses in 164 families were used. For the asso Shown are the results for HapB that shows the strongest association in British MI cohort, ciation studies 702 patients with all forms of stroke (n=329 HapB is defined by the following SNPs: SG13S377, SG13S114, SG13S41 and SG13S35 (that have the following alleles AAA and G, respectively. In all three phenotypes shown the females and n=373 males) and 577 PAOD patients (n=221 same set of n = 728 British controls is used and the frequency of HapB in the control cohort is 0.040, Number of patients (n), haplotype frequency in patients (Frq pat), relative risk females and n=356 males) were analysed. Patients with (RR) and population attributed risk (PAR). stroke or PAOD that also had MI were excluded. Controls 60 used for the stroke and PAOD association studies were the This was called haplotype HapB. The haplotype frequency same as used in the MISNP association study (n=628). of HapB is 7.5% in the MI patient cohort (carrier frequency The study was approved by the Data Protection Commis 14.4%), compared to 4.0% (carrier frequency 7.8%) in con sion of Iceland and the National Bioethics Committee of trols, conferring a relative risk of 1.95 (Table 23). This hap Iceland. Informed consent was obtained from all study par 65 lotype remained significant after adjusting for all haplotypes ticipants. Personal identifiers associated with medical infor tested, using 1000 randomisation steps, with an adjusted P mation and blood samples were encrypted with a third party value=0.046. No other SNP haplotype had an adjusted P US 8,158,362 B2 141 142 value less than 0.05. The two at-risk haplotypes A4 and HapB haplotype that spans the FLAP gene is associated with MI in appear to be mutually exclusive with no instance where the a British cohort. Suggestive linkage to chromosome 13q12 same chromosome carries both haplotypes. 13 was observed with several different phenotypes, including female MI, early onset MI of both sexes, and ischemic stroke British Study Population or TIA in males. However, Surprisingly, the strongest haplo type association was observed to males with MI or stroke. The method of recruitment of 3 separate cohorts of British Therefore, there may be other variants or haplotypes within subjects has been described previously (Steeds, R., Adams, the FLAP gene, or in other genes within the linkage region, M., Smith, P., Channer, K. & Samani, N.J., Thromb Haemost that also may confer risk to these cardiovascular phenotypes. 79,980-4 (1998); Brouilette, S., Singh, R. K., Thompson, J. 10 R., Goodall, A. H. & Samani, N.J., Arterioscler Thromb Vasc These data also show that the at-risk haplotype of the FLAP Biol 23, 842-6 (2003)). In brief, in the first two cohorts a total gene has increased frequency in all Subgroups of stroke, of 547 patients included those who were admitted to the including ischemic, TIA, and hemorrhagic stroke. Of interest coronary care units (CCU) of the Leicester Royal Infirmary, is that the A4 haplotype confers significantly higher risk of MI Leicester (July 1993-April 1994) and the Royal Hallamshire 15 and stroke than it does of PAOD. This could be explained by Hospital, Sheffield (November 1995-March 1997) and satis differences in the pathogenesis of these diseases. Unlike fied the World Health Organization criteria for acute MI in PAOD patients who have ischemic legs because of athero terms of symptoms, elevations in cardiac enzymes or electro Sclerotic lesions that are responsible for gradually diminish cardiographic changes (Nomenclature and criteria for diag ing blood flow to the legs, the MI and stroke patients have nosis of ischemic heart disease. Report of the Joint Interna suffered acute events, with disruption of the vessel wall Sud tional Society and Federation of Cardiology/World Health denly decreasing blood flow to regions of the heart and the Organization taskforce on standardization of clinical nomen brain. clature. Circulation 59, 607-9 (1979)). A total of 530 control Association was not found between the A4 haplotype and subjects were recruited in each hospital from adult visitors to MI in a British cohort. However, significant association to MI patients with non-cardiovascular disease on general medical, 25 was found with a different variant spanning the FLAP gene. Surgical, orthopaedic and obstetric wards to provide Subjects The fact that different haplotypes of the gene are found con likely to be representative of the source population from ferring risk to MI in a second population is not surprising. A which the subjects originated. Subjects who reported a his common disease like MI associates with many different tory of coronary heart disease were excluded. mutations or sequence variations, and the frequencies of these In the third cohort, 203 subjects were recruited retrospec 30 disease associated variants may differ between populations. tively from the registries of 3 coronary care units in Leicester. Furthermore, the same mutations may be seen arising on All had suffered an MI according to WHO criteria before the different haplotypic backgrounds. age of 50 years. At the time of participation, patients were at In summary, it has been found that: MI correlates with least 3 months from the acute event. The control cohortcom genetic variation at FLAP:MI correlates with high expression prised 180 subjects with no personal or family history of 35 promoter polymorphism at 5-LO; patients with female MI premature coronary heart disease, matched for age, sex, and at-risk FLAP haplotypes have higher levels of serum LTE4: current Smoking status with the cases. Control Subjects were LTE4 levels correlate with CRP levels in serum; and patients recruited from 3 primary care practices located within the with MI at-risk FLAP haplotypes have elevated CRP. In addi same geographical area. In all cohorts subjects were white of tion, we have shown that isolated neutrophils from MI Northern European origin. 40 patients, produce more LTB4 when stimulated with ionomy cin compared to controls. Taken together, these results show Discussion that increased leukotriene synthesis is a risk factor for MI, and that this risk is driven in part by variants in FLAP and 5-LO These results show that variants of the gene encoding genes and are captured in part by measurement of levels of FLAP associate with increased risk of MI and stroke. In the 45 serum LTE4 and CRP. Furthermore, the SNP haplotype in the Icelandic cohort, a haplotype that spans the FLAP gene is FLAP gene that confers risk to MI also confers risk of stroke carried by 30% of all MI patients and almost doubles the risk and/or PAOD. of MI. These findings were subsequently replicated in an independent cohort of stroke patients. In addition, another Markers Utilized Herein TABL E 24 Basepair position of microsatellite markers (start and stop of the amplimers in NCBI sequence assembly build 34 and primer SSCUSICS (forward and reverse). basepair basepair Marker start stop ale forward primer reverse primer position position

DG13S2393 CCTTTGCTTTGTTCCTATTTCTTT TCCCATTGCCCAGAGTTAAT 228.314 O122831787 (SEQ ID NO. 4) (SEO ID NO. 5)

DG13S2O7 O TCCTCATGTCTTCACC TAGAAGC CCACTCATGAGGGAGCTGTT (SEQ ID NO. 6) (SEO ID NO. 7)

DG13S2O71 TGTCACAGGCACACACTCTCT GAGTATGGCTGCTGCTCCTC 23 O6697.323 O67076 (SEQ ID NO. 8) (SEQ ID NO. 9)

US 8,158,362 B2 163 164 TABL 24- Continued Basepair position of microsatellite markers (start and stop of the amplimers in NCBI sequence assembly build 34 and primer sequences (forward and reverse) . basepair basepair Marker start stop ale forward primer reverse primer position position

DG13S2OO AATGGATGGATAC CTCCTTATCA CTCATTGTGGCTTTCTGTGC 4O7373.374. Of 3757 O (SEQ ID NO. 490) (SEQ ID NO. 491)

DG13S1.98 GTACCCACACCTCACCAAGC CGTAGCTCACATTCCCAACA 4081-1813 40812O59 (SEQ ID NO. 492) (SEQ ID NO. 493)

DG13S215 GGCGAGTGAAAGAGAGGACA GGGTGGTAATTCCCAGATGA 4 O871695 4 O871992 (SEQ ID NO. 494) (SEO ID NO. 495)

DG13S221. TCTGCAACAGCCAGAATCAA TGTCTGTTGGCAACTTTCTGTC 411 Offf3 4.1108117 (SEQ ID NO. 496) (SEO ID NO. 497)

DG13S219 AGGTGAACCCAGTCCAGCTA TCTTAGGCAAAGGAGCCAGT 41127591. 41127734 (SEQ ID NO. 498) (SEQ ID NO. 499)

D13S1270 ACATGAGCACTGGTGACTG GGCCT CAAATGTTTTAAGCA 41161654. 411.61831 (SEO ID NO. 5 OO) (SEQ ID NO. 501)

DG13S225 TTCTGGGTGTTCGCTATTCC TTTCCTGTCCAGTCCTGACC 41212951. 4121331 O (SEQ ID NO. 5O2) (SEO ID NO. 503)

D13S1276 GTTTTGCAGGTCTAGGTCACAC AGGATAGCTTGAGCCCG 41213917 41214 O9 O (SEQ ID NO. 504) (SEO ID NO. 505)

All references cited herein are incorporated by reference in 30 Patient Population their entirety. While this invention has been particularly All patients in the study had a history of MI and were shown and described with references to preferred embodi carriers of specific MI-associated haplotypes in the FLAP ments thereof, it will be understood by those skilled in the art and/or the LTA hydrolase genes (See U.S. patent application that various changes inform and details may be made therein Ser. No. 10/944.272 (now abandoned; published as U.S. without departing from the scope of the invention encom 2005/0272051A1) and PCT Application No. PCT/2004/ passed by the appended claims. 35 030582, incorporated by reference in its entirety. The recruit ment process included individuals who had previously par ticipated in a study of the genetics of MI (Helgadottir et al., EXAMPLE 10 Nat. Genet. 2004:36(3):233-9. 2004). Apart from FLAP, the LTA, hydrolase gene also shows significant association to 40 MI in Iceland and baseline mRNA expression of the LTA, Randomized, Placebo-Controlled, Crossover Clinical hydrolase gene is greater in MI patients than in control Sub Trial Demonstrates Inhibition of FLAP Reduced jects; that is subjects with at-risk variants in either the FLAP Biomarkers of Risk of Myocardial Infarction or LTA hydrolase genes are at increased risk of Sustaining MI. Thus, carriers of either the FLAP or LTA hydrolase at-risk haplotypes were recruited and their haplotypes were The 5-lipoxygenase pathway, through FLAP leads to the 45 confirmed by analysis of DNA from blood sample collected production of leukotriene B, one of the most potent chemok in the study. ine mediators of arterial inflammation. The experiments Nine Single Nucleotide Polymorphism (SNP) markers described in Example 7 showed that MI patients make more were genotyped to define the at-risk haplotypes. These SP LTB than do controls. Hence, it appears that the at-risk markers are set out in Table 25 below and are described in variant upregulates the leukotriene pathway. A clinical trial 50 detail in Example 1. SNPs genotyping within the FLAP and was carried out to demonstrate that patients with the genetic LTA hydrolase genes was performed using SNP-based Taq variation in FLAP that predisposes to MI could benefit from man platform (ABI) as described in Helgadottir et al., 2004 inhibiting FLAP, with the FLAP inhibitor DG-031. In the March;36(3):233-9. The haplotypes carried by each indi short term study, changes in levels of biomarkers that are vidual were estimated using the program NEMO (version associated with risk of MI were scored as evidence of changes 1.01) and 902 in-house population controls, as described in in the risk of MI. Gretarsdottir et al., Nat Genet 35:131-8, 2003. TABLE 25 Genotypes used to derive FLAP and LTA hydrolase at-risk haplotypes. Haplotype Allele SNP Allele SNP Allele SNP 1 A3 (FLAP gene) G SG-13S25 T SG13S114 A SG13S32 2 AF (FLAP gene) G SG-13S25 T SG13S114 3 NA3 (FLAP gene) A SG13S122 C SG13S32 C SG13S8 4 HF (LTA-OH gene) A SG12S25 C SG12S223 5 GF (LTA-OH gene) A SG12S225 T SG12S233