# Patent

## Title
Dalcetrapib for therapeutic use

## Abstract

The present invention provides genotyping methods and compositions for selecting patients with cardiovascular disease who will benefit from treatment with HDL-raising or HDL mimicking agent, in particular with a CETP inhibitor/modulator.

## Background
RELATED APPLICATIONS
This application is a continuation of International Patent Application No. PCT/EP2014/055790, filed Mar. 24, 2014, and claims benefit of priority under 35 USC 119(a) to European Patent Application No. 13161386.1 filed Mar. 27, 2013, the contents of which are incorporated herein by reference.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 28, 2015, is named SequenceListing.txt and is 6,505 bytes in size.
FIELD OF THE INVENTION
The field of the invention relates to the treatment or prophylaxis of subject with cardiovascular disorder.
BACKGROUND
Although 20 years ago, one treatment fits all was the approach taken which led to formidable “blockbuster” drugs. Today with the sequencing of the human genome and advances in molecular profiling technologies, approaches to drug development are taking a more stratified or personalized approach. These advances increasingly allow for classification of individuals into subpopulations that are at risk of a specific disease, respond to a specific treatment, don't respond to a specific treatment or are at high risk of an adverse event when treated. As such genetic tests can be used to inform diagnosis, prognosis and treatment selection. Numerous studies have shown a relationship between genotype and response to pharmaceutical therapies. This approach has been widely embraced over the past years, particularly in oncology where numerous personalized medicine approaches have been successfully developed and have provided major improvement in clinical outcomes.
In cardiovascular disorders the stratification of the population by genotype for a specific therapeutic intervention has been limited. One of the objectives of the present invention is to demonstrate that the population suffering from cardiovascular disorders might behave differently and consequently may respond differently to a specific treatment. Lowering LDL is an important therapeutic strategy in the management of cardiovascular disease. Indeed statin drugs, which lower LDL, such as Crestor®, Lipitor®, Pravachol®, and Zocar® are widely used and among the most prescribed drugs. For some time it has also been generally accepted that increasing HDL could also be therapeutic in cardiovascular disease. Several drugs HDL-raising drugs have been developed including: niacin and CETP inhibitors such as torcetrapib, anacetrapib, evacetrapib and dalcetrapib.
Cholesterylester transfer protein (CETP) also called plasma lipid transfer protein is a hydrophobic glycoprotein that is synthesized in several tissues but mainly in the liver. CETP promotes bidirectional transfer of cholesteryl esters and triglyceride between all plasma lipoprotein particles. The first evidence the affect of CETP activity on plasma lipoproteins was provided by observations in people with genetic deficiencies of CETP. The first CETP mutation was identified in Japan in 1989 as a cause of markedly elevated HDL-C. Ten mutations associated with CETP deficiency have since been identified in Asians and one in Caucasians. It was found in Japan that 57% of subjects with levels of HDL-C>100 mg/dL have mutations of the CETP gene. In addition, 37% of Japanese with levels HDL-C between 75-100 mg/dL have mutations of the CETP gene. Subsequently, studies of animals treated with an anti-CETP antibody showed that CETP inhibition resulted in a substantial increase in the concentration of HDL-C. Consistent with these observations in CETP deficient patients and rabbits treated with an anti-CETP antibody, it has since been found that treatment of humans with CETP inhibitor drugs increases the concentration of HDL cholesterol and apoA-I (the major apolipoprotein in HDLs). Numerous epidemiologic studies have correlated the effects of variations in CETP activity with coronary heart disease risk including studies of human mutations (Hirano, K. I. Yamishita, S. and Matsuzawa Y. (2000) Curr. Opin. Lipido. 11(4), 389-396).
Atherosclerosis and its clinical consequences, including coronary heart disease (CHD), stroke and peripheral vascular disease represents an enormous burden on health care systems internationally. Drugs that inhibit CETP (CETP inhibitors) have been under development for some time with the expectation that they will be useful for treating or preventing atherosclerosis. A number of classes of CETP inhibitor drugs have been shown to increase HDL, decrease LDL in humans and to have therapeutic effects for treating atherosclerosis and cardiovascular disease including dalcetrapib, torcetrapib, anacetrapib, evacetrapib, BAY 60-5521 and others (Table 1).







TABLE 1







Overview of Lead CETP Inhibitor Drugs and Clinical Status









Structure
Compound
Clinical phase









Torcetrapib
Phase III  discontinued in 2006


 






Anacetrapib
Phase III


 






Dalcetrapib
Phase III  trial halted May 2012


 






BAY 60-5521
Phase I









However there is evidence that these drugs may not be safe and effective in all patients. The clinical trial for torcetrapib was terminated in Phase III due to incidence of mortality in patient to whom torcetrapib and atorvastatin were administered concomitantly compared to patients treated with atorvastatin alone. The clinical trial for dalcetrapib was also halted in Phase III in this case due to a lack of efficacy relative to statins alone. Additional CETP inhibitors are still being pursued in clinical trials and earlier stage development. In general treatment strategies using CETP inhibitors that provide better efficacy, reduced off-target effects would be clinically beneficial. There is a need for biomarkers, methods and approaches for predicting response to CETP inhibitors and accessing risk of adverse events associated with administration of CETP inhibitors.
CETP inhibitors are useful for the treatment and/or prophylaxis of atherosclerosis, peripheral vascular disease, dyslipidemia, hyperbetalipoproteinemia, hypoalphalipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial hypercholesterolemia, cardiovascular disorders, angina, ischemia, cardiac ischemia, stroke, myocardial infarction, reperfusion injury, angioplastic restenosis, hypertension, and vascular complications of diabetes, obesity or endotoxemia.
Clinical trials have shown that patient response to treatment with pharmaceuticals is often heterogeneous. There a pressing need to improve drug development, clinical development and the therapeutic impact of drugs for individuals or sub-populations of patients. SNPs can be used to identify patients most suited to therapy with particular pharmaceutical agents (this is often termed “pharmacogenomics”). Similarly, SNPs can be used to exclude patients from certain treatment due to the patient's increased likelihood of developing toxic side effects or their likelihood of not responding to the treatment. Pharmacogenomics can also be used in pharmaceutical research to assist the drug development and selection process. Linder et al, Clinical Chemistry 43:254 (1997); Marshall, Nature Biotechnology 15: 1249 (1997); International Patent Application WO 97/40462, Spectra Biomedical; and Schafer et al, Nature Biotechnology 16:3 (1998).
The dalcetrapib mortality and morbidity trial (dal-OUTCOMES) was a double-blind, randomized, placebo-controlled, parallel group, multi-centre study in stable CHD patients recently hospitalized for acute coronary syndrome (ACS). The study was conducted to test the hypothesis that CETP inhibition will reduce the risk of recurrent cardiovascular events in patients with recent ACS by raising levels of HDL-C through CETP inhibition. Eligible patients entered a single-blind placebo run-in period of approximately 4 to 6 weeks to allow for patients to stabilize and for completion of planned revascularization procedures. At the end of the run-in period, eligible patients in stable condition were randomized in a 1:1 ratio to 600 mg of dalcetrapib or placebo on top of evidence-based medical care for ACS. Dalcetrapib is an inhibitor of cholesterol-ester transfer protein (CETP). It has been shown to induce dose-related decreases in CETP activity and increases in HDL-C levels in several animal species and in humans. Decreasing CETP activity, through different approaches, has demonstrated anti-atherosclerotic effects in several animal models. The trial was interrupted in May 2012 by the DSMB on grounds of futility. The dal-OUTCOMES study resulted in unexpected observations related to cardiovascular disease progression. Despite a marked increase in HDL-c, patients on treatment did not show a significant reduction in cardiovascular events and the study was terminated.
SUMMARY
Following the termination of dal-OUTCOMES study, it was hypothesized that a subgroup of the patients under study were responding differently to dalcetrapib and that dalcetrapib could be having a significant therapeutic effect in a sub-population of patients. A pharmacogenomic study of the dal-OUTCOMES study population was conducted to study the inter-individual variation in dalcetrapib response and to identify genetic markers for predicting therapeutic response to dalcetrapib, or other CETP inhibitors, for patient stratification and for treatment selection.
The present invention provides genotyping methods, reagents and compositions for selecting individuals who can benefit from treatment with HDL-raising or HDL mimicking agent, in particular with a CETP inhibitor/modulator, in particular wherein the individuals have cardiovascular disorder. The invention also provides methods of treating patients with a cardiovascular disorder comprising genotyping and selection of patients who will benefit from treatment with HDL-raising or HDL mimicking agent, in particular with a CETP inhibitor/modulator. Surprisingly the pharmacogenomic study of the dal-OUTCOMES patient cohort found single nucleotide polymorphisms (SNPs), genetic markers, associated with an individual's response to dalcetrapib and useful for predicting therapeutic response to HDL-raising or HDL mimicking agent (in particular a CETP inhibitor/modulator) and in treating patients with HDL-raising or HDL mimicking agent (in particular CETP inhibitor/modulator).
Genetic markers detected in the genotyping methods of the invention include: 15 SNPs that occur in the Adenylate Cyclase Type 9 (ADCY9) gene on chromosome 16, rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119 and rs13337675, in particular rs1967309, which is strongly associated (P=4.11.10-8). with response to an HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator.
Other gene markers of the invention include a SNP in the ADCY9 gene that are either in linkage disequilibrium with rs1967309 or provided an association signal with P<0.05 and may provide useful surrogate biomarkers of rs1967309. In one embodiment a surrogate biomarker, consisting of a SNP inherited in linkage disequilibrium with rs1967309, is detected and the genotype of rs1967309 is inferred.
The present invention relates to methods of genotyping and treating patients with HDL-raising drugs, in particular a CETP inhibitor. Three genotypes at rs1967309 are predictive of an individual's response to an HDL-raising drug, in particular a CETP inhibitor: AA, AG and GG. Of these the AA genotype is associated with a improved therapeutic response in patients treated with an HDL-raising drug, the AG genotype is associated with a partial response and the GG genotype is associated with a lack of response (non-response). For the purpose of the present invention: patients who carry the AA genotype can benefit from treatment with an HDL-raising drug; patients who carry the AG genotype can benefit from treatment with an HDL-raising drug and patients who carry the GG genotype cannot benefit from treatment with an HDL-raising drug. Two genotypes at rs1967309, AA and AG indicate a therapeutic response to a CETP inhibitor, in particular dalcetrapib, in patients with cardiovascular disorder. In particular, AA genotype for rs1967309 is indicative of greater therapeutic response to a CETP inhibitor, in particular dalcetrapib, in patients with cardiovascular disorder.
The present invention relates to nucleic acid molecules containing polymorphisms or gene variants, variant proteins encoded by these nucleic acid molecules, reagents for detecting the polymorphic nucleic acid molecules, and methods of using the nucleic acid molecules and proteins as well as methods of using reagents for their detection (e.g., primers and probes for use in the genotyping methods of the invention).
In one embodiment the invention provides: methods of detecting the gene variants of the invention and detection reagents, such as probes or primers, for use in these methods.
The invention specifically provides, genetic markers associated with therapeutic response to a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator, and synthetic nucleic acid molecules (including DNA and RNA molecules) containing the gene variants of the invention. The invention further provides variant proteins encoded by nucleic acid molecules containing such gene variants, antibodies to the encoded variant proteins, computer-based and data storage systems containing the novel gene variant or SNP information, methods of detecting these SNPs in a test sample, methods of identifying individuals who respond therapeutically when administered a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator, based on the presence or absence of one or more of the gene variants of the invention or the detection of one or more encoded variant products {e.g., variant mRNA transcripts or variant proteins), and methods of treating individuals with a cardiovascular disease who carry one of more of the gene variants of the invention.
Exemplary embodiments of the present invention further provide methods for selecting or formulating a treatment regimen (e.g., methods for determining whether or not to administer HDL-raising or HDL mimicking agent, in particular CETP inhibitor/modulator treatment to an individual).
Various embodiments of the present invention also provide methods for selecting individuals to whom a HDL-raising or HDL mimicking agent (in particular CETP inhibitor/modulator) can be therapeutically administered based on the individual's genotype, and methods for selecting individuals for participation in a clinical trial of a HDL-raising or HDL mimicking agent (in particular a CETP inhibitor/modulator) based on the genotypes of the individuals (e.g., selecting individuals to participate in the trial who are most likely to respond positively and/or excluding individuals from the trial who are unlikely to respond positively to treatment based on their genotype(s), in particular their genotype is AA at rs1967309, or selecting individuals who are unlikely to respond positively for participation in a clinical trial of alternative drug that may benefit them.
The nucleic acid molecules of the invention can be inserted in an expression vector, to produce a variant protein in a host cell. Thus, the present invention also provides for a vector comprising a SNP-containing nucleic acid molecule of the invention, genetically-engineered host cells containing the vector, and methods for expressing a recombinant variant protein using such host cells. In another specific embodiment, the host cells, SNP-containing nucleic acid molecules, and/or variant proteins can be used as targets in a method for screening or identifying therapeutic agents that are HDL-raising or HDL mimicking agent (in particular CETP inhibitor/modulator).
Exemplary SNPS of ADCY9 that can be determined/evaluated in the herein provided method for identification of an improved response to dalcetrapib are those where the mutation results in a change in the nucleotide sequence at position 4,062,592 and 4,065,583 (genome assembly GRCh37.p5) also known as single nucleotide polymorphisms with identifiers rs12595857 and rs1967309 respectively, as shown in SEQ. ID. NO.1 and 2.
The present invention is based on the identification of genetic polymorphisms that are predictive of an increased likelihood that treatment with a HDL-raising or HDL mimicking agent, in particular CETP inhibitor/modulator will benefit patients with cardiovascular disorders.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: SNPsrs1967309 and rs12595857 of the invention are strongly associated with a reduction of cardiovascular events (primary composite event or unanticipated coronary revascularization) in patients treated with the CETP inhibitor dalcetrapib. The figures show the results of a genome-wide association study (GWAS) with samples from the treatment arm of the dal-OUTCOMES study. Panel A shows a Manhattan plot for logistic regression with a strong signal in the ADCY9 gene region on chromosome 16. Each dot represents a P value for the comparison of participants who experienced cardiovascular events during treatment versus those who did not and adjusted for gender and 5 principal components for genetic ancestry. Panel B shows P values for single-nucleotide polymorphisms (SNPs) in the ADCY9 region. There is a strong association between cardiovascular events during treatment and the rs1967309 SNP, and neighboring SNP rs12595857 which is in linkage disequilibrium with rs1967309. The x axis shows the SNP position on chromosome 16 (National Center for Biotechnology Information assembly GRCh37.p5). The left y axis shows the negative login of P values for the comparison between cardiovascular events versus no events, as described in Panel A. The right y axis shows the recombination rate on chromosome 16. The diamonds show the degree of linkage disequilibrium (LD) in the samples as estimated from the reference CEU samples from HapMap.
FIG. 2: Frequency of cardiovascular events (dal-OUTCOMES primary composite event or unanticipated coronary revascularization) by study termination in the dalcetrapib and placebo treatment arms separately and by rs1967309 genotypes in the ADCY9 gene. Percentages of events are reported with 95% CI.
FIG. 3: Cumulative incidence of cardiovascular events (dal-OUTCOMES primary composite event or unanticipated coronary revascularization) for the dalcetrapib treatment arm and the placebo arm separately and stratified by the three genotypes at the rs1967309 SNP in the ADCY9 gene (GG, AG, AA).
FIG. 4A: Shows changes in lipid levels according to genotype during 24 months of treatment. Mean±SE (mg/dL) of change of lipid values from baseline to 1 month by genotype groups of the ADCY9 SNP rs1967309 for the dalcetrapib treatment arm. P values are shown for univariate statistics between change in lipid and genotypes.
FIG. 4B: Shows changes in lipid levels according to genotype during 24 months of treatment. Mean±95% CI for absolute values of LDL cholesterol during the follow up period of the dal-OUTCOMES trial for patients in the treatment arm. P value for multivariate mixed regression model.
FIG. 5: MDS plot showing the first two dimensions (C1, C2) from 76,854 SNPs for 6297 individuals from the genetic study of dal-Outcomes and 83 CEU founder, 186 JPT-CHB and 88 YRI founder from the 1000 genome data set.
FIG. 6: Plot of the cumulative variance explained by the first ten components from the principal component analysis from 76,854 SNPs for 6297 individuals from the genetic study of dal-Outcomes and 83 CEU founder, 186 JPT-CHB and 88 YRI founder from the 1000 genome data set.
FIG. 7: Quantile-quantile (QQ) plot of observed-log 10 P values versus the expectation under the null for the genomewide association of SNPs with MAF≧0.05. The shaded region is the 95% concentration band formed by calculating the 2.5th and 97.5th percentiles of the distribution under the null hypothesis. Dots represent the ranked P values from the logistic regression in PLINK for the comparison of dal-Outcomes participants in the treatment arm who experienced cardiovascular events during treatment versus those who did not and adjusted for gender and 5 principal components for genetic ancestry.
FIG. 8: Heat plot showing the linkage disequilibriumpattern (r2) at the ADCY9 gene around the strongly associated SNP rs1967309. Blocks 6 7, 8 and 9 show a region that is in high linkage disequilibrium with rs1967309 spanning from position chr16: 4049365 to chr16: 4077178 (assembly GRCh37/hg19) from SNP rs12935810 to SNP rs13337675.



DETAILED DESCRIPTION
Various features and embodiments of the present invention are disclosed herein, however other features of the invention, modifications and equivalents will be apparent to a person skilled in the relevant art, based on the teachings provided. The invention described is not limited to the examples and embodiments provided, various alternatives equivalents will be appreciate by those skilled in the art. As used herein, the singular forms “a”, “an” and “the” include the plural unless the context clearly dictates otherwise. For example, “a” cell will also include “cells”.
An “allele” is defined as any one or more alternative forms of a given gene. In a diploid cell or organism the members of an allelic pair (i.e. the two alleles of a given gene) occupy corresponding positions (loci) on a pair of homologous chromosomes and if these alleles are genetically identical the cell or organism is said to be “homozygous”, but if genetically different the cell or organism is said to be “heterozygous” with respect to the particular gene.
A “gene” is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product and may include untranslated and untranscribed sequences in proximity to the coding regions. Such non-coding sequences may contain regulatory sequences needed for transcription and translation of the sequence or introns etc. or may as yet to have any function attributed to them beyond the occurrence of the SNP of interest.
“genotyping” refers to the determination of the genetic information an individual carries at one or more positions in the genome. For example, genotyping may comprise the determination of which allele or alleles an individual carries for a single SNP or the determination of which allele or alleles an individual carries for a plurality of SNPs. For example, at rs1967309 the nucleotides may be a A in some individuals and a G in other individuals. Those individuals who have a A at the position have the A allele and those who have a G have the G allele. In a diploid organism the individual will have two copies of the sequence containing the polymorphic position so the individual may have a A allele and a G allele or alternatively, two copies of the A alleles or two copies of the G allele. Those individuals who have two copies of the G allele are homozygous for the G allele, those individuals who have two copies of the A allele are homozygous for the A allele, and those individuals who have one copy of each allele are heterozygous. The alleles are often referred to as the A allele, often the major allele, and the B allele, often the minor allele. The genotypes may be AA (homozygous A), BB (homozygous B) or AB (heterozygous). Genotyping methods generally provide for identification of the sample as AA, BB or AB.
The term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others.
“HDL-raising or HDL mimicking agent” refers to compounds which increase HDL levels by either one of the following mechanisms: CETP inhibition/modulation, PPAR agonism, LXR agonism, HM74 agonism (niacin receptor) thyrotropin hormone receptor agonism, Inhibitors of lipases and HDL catabolismApoA1 inducers, compounds which provide at least one of the HDL athero-protective activities such as compounds that would increase cellular lipid efflux (cholesterol and/or phospholipids), have antioxidant and anti-inflammatory activities. In particular HDL mimicking agent is ApoA1 and ApoA1 derivatives (such as apoA1 Milano, ApoA1 Paris) and other analogues, reconstituted HDL containing ApoA1 and or ApoAII and the appropriate lipids such as phospholipids. ApoE, derivatives, analogues, and peptidomimetics of amphipathic lipoproteins. Examples of “HDL-raising or HDL mimicking agent” are niacin, fibrates, glitazone, dalcetrapib, anacetrapib, evacetrapib, DEZ-001 (formerly known as TA-8995)(Mitsubishi Tanabe Pharma), ATH-03 (Affris), DRL-17822 (Dr. Reddy's), DLBS-1449 (Dexa Medica), RVX-208 (Resverlogix), CSL-112 (Cls Behring), CER-001 (Cerenis), ApoA1-Milnano (Medicine Company). Particular examples of “HDL-raising or HDL mimicking agent” are niacin, fibrates, glitazone, dalcetrapib, anacetrapib, evacetrapib, torcetrapib preferably niacin, fibrates, glitazone, dalcetrapib, anacetrapib or evacetrapib. More particularly HDL-raising or mimicking agent is selected from a CETP inhibitor/modulator. Examples of CETP inhibitor/modulators are dalcetrapib, anacetrapib, evacetrapib, DEZ-001 (formerly known as TA-8995)(Mitsubishi Tanabe Pharma), ATH-03 (Affris), DRL-17822 (Dr. Reddy's), DLBS-1449 (Dexa Medica). More particularly examples of CETP inhibitor/modulators are dalcetrapib, anacetrapib, evacetrapib and torcetrapib, preferably dalcetrapib, anacetrapib and evacetrapib. Most particularly the HDL-raising or mimicking agent according to the invention would refer to a CETP inhibitor/modulator, especially when the CETP inhibitor/modulator is dalcetrapib.
“CETP inhibitor/modulator” refers to a compound which decreases CETP activity (assessed by standard transfer assays) by inhibiting CETP and/or inducing conformational changes of the CETP polypeptide once bound to the CETP polypeptide. The CETP conformational changes of the CETP polypeptide allow CETP activity to proceed between HDL particles and increase its recycling/turnover bay increasing the production of nascent pre-beta HDL formation. Preferably the CETP inhibitor/modulator refers to all compounds that would bind to cysteine 13 of the CETP polypeptide. More preferably, the “CETP inhibitor/modulator” is selected from S-[2-[1-(2-ethylbutyl)cyclohexylcarbonylamino]-phenyl]2-methylthiopropionate, 1-(2-Ethyl-butyl)-cyclohexanecarboxylic acid (2-mercapto-phenyl)-amide and or bis [2-[1-(2-ethylbutyl)cyclohexylcarbonylamino]phenyl]disulfide. Most preferably, “CETP inhibitor/modulator” is S-[2-[1-(2-ethylbutyl)cyclohexylcarbonylamino]-phenyl]2-methylthiopropionate as a prodrug or 1-(2-Ethyl-butyl)-cyclohexanecarboxylic acid (2-mercapto-phenyl)-amide as its active metabolite.
“Anacetrapib” refers to ((4S,5R)-5-[3,5-bis(trifluoromethyl)phenyl]-3-{[4′-fluoro-2′-methoxy-5′-(propan-2-yl)-4-(trifluoromethyl)[1,1′-biphenyl]-2-yl]methyl}-4-methyl-1,3-oxazolidin-2-one) also known as MK 0859, CAS 875446-37-0 or a compound of formula (XA).




Anacetrapib as well as methods of making and using the compound, are described in WO2006/014413, WO2006/014357, WO2007005572.
“Evacatrapib” refers Trans-4-({(5S)-5-[{[3,5-bis(trifluoromethyl)phenyl]methyl}(2-methyl-2H-tetrazol-5-yl)amino]-7,9-dimethyl-2,3,4,5-tetrahydro-1H-benzazepin-1-yl}methyl)cyclohexanecarboxylic acid also known as LY2484595, CAS1186486-62-3 or a compound of formula (XB)




Evacetrapib as well as methods of making and using the compound are described in WO2011002696.
“Torcetrapib” refers to (2R,4S)-4-[(3,5-bistrifluoromethylbenzyl)methoxycarbonylamino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic acid ethyl ester, also known as CP-529,414, CAS 262352-17-0 or a compound of formula (XC)




Torcetrapib as well as methods of making and using the compound are described in WO0017164 or WO0140190.
“BAY 60-5521” refers to (5S)-5-Quinolinol, 4-cyclohexyl-2-cyclopentyl-3-[(S)-fluoro[4-(trifluoromethyl)phenyl]methyl]-5,6,7,8-tetrahydro-7,7-dimethyl-, also known as CAS 893409-49-9 or a compound of formula (XD)




BAY 60-5521 as well as methods of making and using the compound are described in WO2006063828.
“Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented or delayed.
The term “polymorphism” “polymorphism site” Polymorphic site” or “single nucleotide polymorphism site” (SNP site) or “single nucleotide polymorphism” refers to a location in the sequence of a gene which varies within a population. A polymorphism is the occurrence of two or more forms of a gene or position within a gene “allele”, in a population, in such frequencies that the presence of the rarest of the forms cannot be explained by mutation alone. Preferred polymorphic sites have at least two alleles. The implication is that polymorphic alleles confer some phenotype variability on the host. Polymorphism may occur in both the coding regions and the noncoding region of genes. Polymorphism may occur at a single nucleotides site or may involve an insertion or a deletion. The location of such a polymorphism may be identified by its nucleotide position in the gene, on the chromosome or on the transcriptor by the amino acids that is altered by the nucleotide polymorphism. Individual polymorphisms are also assigned unique identifiers (“Reference SNP”, “refSNP” or “rs#”) known to one of skill in the art and used, e.g., in the Single Nucleotide Polymorphism Database (dbSNP) of Nucleotide Sequence Variation available on the NCBI website.
The terms “linkage disequilibrium” or “in linkage disequilibrium” or “LD” refers to the non-random association of alleles in a collection of individuals, in other words it is the preferential segregation of a particular polymorphic form with another polymorphic form at a different chromosomal location more frequently than expected by chance. By opposition the alleles that co-occur at expected frequencies are said to be in ‘linkage equilibrium”.
The “rs” prefix refers to a SNP in the database found at the NCBI SNP database world wide web address: ncbi.nlm.nih.gov/snp/?term. The “rs” numbers are the NCBI rsSNP ID form.
The term “sample” includes any biological sample taken from a patient or individual including a cell, tissue sample or body fluid. For example, a sample may include a skin sample, a cheek cell sample, saliva or blood cells. A sample can include, without limitation, a single cell, multiple cells, fragments of cells, an aliquot of a body fluid, whole blood, platelets, serum, plasma, red blood cells, white blood cells, endothelial cells, tissue biopsies, synovial fluid and lymphatic fluid. In particular “sample” refers to blood cells.
The term “therapeutic agents” refers to agents capable of treating or preventing cardiovascular disorder. As used herein, an agent “capable of treating or preventing cardiovascular disorder” refers to a molecule that is capable of treating and/or preventing a cardiovascular disorder in humans and/or in a cellular or animal model of said cardiovascular disorder.
An “improved response polymorphism”, “improved response genotype” or “responsive genotype” as used herein refers to an allelic variant or genotype at one or more polymorphic sites within the ADCY9 gene as described herein (for example, rs1967309/AA), which predicts that a subject will respond therapeutically and benefit from treatment with an HDL-raising or HDL mimicking agent (which may be measured by decreased number of cardiovascular events) as compared to an allelic variant or genotype or polymorphism (for example, rs1967309/AG or, rs1967309/GG) which predicts that a subject will respond less to HDL-raising or HDL mimicking agent administration. “Reduced response” “partial response”, “non response” or “lack of therapeutic efficacy”, may be measured by a relative increase in number of cardiovascular events relative to subjects having an “improved response genotype”. Alternately, “improved response”, “responder” or “therapeutic efficacy” may be measured by a relative decrease in number of cardiovascular events relative to subjects that carry polymorphisms associated with “non response” or “partial response” to a HDL-raising or HDL mimicking agent. In particular rs12595857/GG, rs1967309/AA, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG, rs8061182/AA are improved response genotypes. More particularly, rs1967309/AA is improved response genotype.
“Cardiovascular events” as used herein refers to cardiovascular death, non-fatal myocardial infarction (MI), non-fatal stroke of ischemic origin, hospitalization for unstable angina and coronary revascularization.
“Oligonucleotides” as used herein are variable length nucleic acids or polynucleotides. Such oligonucleotides may be useful as probes, primers and in the manufacture of microarrays (arrays) for the detection and/or amplification of specific nucleic acids. Such DNA or RNA strands may be synthesized by the sequential addition (5′-3′ or 3′-5′) of activated monomers to a growing chain, which may be linked to an insoluble support. Numerous methods are known in the art for synthesizing oligonucleotides for subsequent individual use or as a part of the insoluble support, for example in arrays (BERNFIELD M R. and ROTTMAN F M. J. Biol. Chem. (1967) 242(18):4134-43; SULSTON J. et al. PNAS (1968) 60(2):409-415; GILLAM S. et al. Nucleic Acid Res. (1975) 2(5):613-624; BONORA G M. et al. Nucleic Acid Res. (1990) 18(11):3155-9; LASHKARI D A. et al. PNAS (1995) 92(17):7912-5; MCGALL G. et al. PNAS (1996) 93(24): 13555-60; ALBERT T J. et al. Nucleic Acid Res. (2003) 31(7):e35; GAO X. et al. Biopolymers (2004) 73(5):579-96; and MOORCROFT M J. et al. Nucleic Acid Res. (2005) 33(8):e75). In general, oligonucleotides are synthesized through the stepwise addition of activated and protected monomers under a variety of conditions depending on the method being used. Subsequently, specific protecting groups may be removed to allow for further elongation and subsequently and once synthesis is complete all the protecting groups may be removed and the oligonucleotides removed from their solid supports for purification of the complete chains if so desired.
The term “genotype” refers to the genetic constitution of an organism, usually in respect to one gene or a few genes or a region of a gene relevant to a particular context (i.e. the genetic loci responsible for a particular phenotype). In particular, the specific combination of alleles at a given position in a gene, such as for example, the genotypes AA, AG, or GG which are possible genotypes of the rs1967309 SNP.
A “phenotype” is defined as the observable characters of an organism. Table 1 shows a genotype correlation for ADCY9 SNP with values representing an indication of responsiveness to treatment of cardiovascular disorders with HDL-raising or HDL mimicking agent.
The term “biomarker” as used herein refers to a sequence characteristic of a particular variant allele (polymorphic site, such as a SNP) or wild-type allele. Biomarker also refers to a peptide or epitope encoded by a particular variant or wild-type allele.
The term “surrogate marker” as used herein refers to a genetic variant, including a SNP, that is present in linkage disequilibrium with an improved response genotype of the invention, in particular rs1967309 AA.
The term ‘genetic marker’ as used herein refers to variants of polymorphic sites of a particular gene that are associated with response to a HDL-raising or HDL mimicking agent, in particular CETP inhibitor/modulator. In particular ‘genetic marker’ as used herein refers to variants of polymorphic sites in the ADCY9 gene that are associated with response to a HDL-raising or HDL mimicking agent, in particular CETP inhibitor/modulator.
In certain methods described herein one or more biomarkers are used to identify or select individuals who will benefit from treatment with a HDL-raising or HDL mimicking agent, in particular CETP inhibitor/modulator. A SNP biomarker for use in the invention can be predictive of either a therapeutic response (R) to treatment or non-response to treatment (NR). Table 2 shows genotypes observed in the dal-Outcomes cohort, present at the polymorphic site rs1967309, which can be used as a biomarker to predict response to dalcetrapib or HDL-raising or HDL mimicking agent, in particular to other CETP inhibitor/modulator. Each genotype shown in Table 2 or 3 alone or in combination with genotypes at other polymorphic sites can be used as a biomarker to predicting response to a HDL-raising or HDL mimicking agent, in particular to a CETP inhibitor/modulator).







TABLE 2







genetic markers and predicted response to treatment with


HDL-raising or HDL mimicking agent











SNP
Genotype
Responsiveness to treatment







rs1967309
AA
R



rs1967309
AG
PR



rs1967309
GG
NR







R: Responsive



PR: Partial Responsive



NR: Non Responsive













TABLE 3







genetic markers and predicted response to treatment with


HDL-raising or HDL mimicking agent











SNP
Genotype
Responsiveness to treatment







rs12595857
AA
NR



rs12595857
AG
PR



rs12595857
GG
R







R: Responsive



PR: Partial Responsive



NR: Non Responsive













TABLE 4







genetic markers and predicted response to treatment with


HDL-raising or HDL mimicking agent











SNP
Genotype
Responsiveness to Treatment







rs111590482
AA
NR




AG
R




GG
R



rs11647828
AA
NR




AG
PR




GG
R



rs12935810
GG
R




GA
NR




AA
NR



rs13337675
AA
NR




AG
PR




GG
PR



rs17136707
AA
NR




AG
PR




GG
R



rs2239310
AA
NR




AG
PR




GG
R



rs2283497
CC
NR




CA
PR




AA
R



rs2531967
GG
NR




GA
PR




AA
R



rs3730119
GG
NR




GA
PR




AA
R



rs4786454
GG
NR




GA
PR




AA
R



rs74702385
GG
NR




GA
R




AA
R



rs8049452
GG
R




GA
PR




AA
NR



rs8061182
AA
R




AG
PR




GG
NR







R: Responsive



PR: Partial Responsive



NR: Non Responsive






Both rs1967309 and rs12595857 are located in an intronic (non-coding) region of the ADCY9 gene in a region that is concordant with having regulatory activity on the ADCY9 gene expression.
In certain methods described herein individuals who will respond therapeutically to treatment with a HDL-raising or HDL mimicking agent, in particular with a CETP inhibitor/modulator are identified and selected for treatment using the genotyping methods of the invention. In particular patients who carry one or more of the following improved response genotypes are selected for treatment in the methods of the invention: rs12595857/GG, rs1967309/AA, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG, rs8061182/AA. More particularly, patients who carry rs12595857/GG or rs1967309/AA genotypes are selected for treatment in the methods of the invention: Most particularly, patients who carry rs1967309/AA genotypes are selected for treatment in the methods of the invention:
In another embodiment the invention provides, a method for identifying a subject benefiting from a HDL-raising or HDL mimicking agent, the method comprising determining a genotype of said subject (e.g., genotyping) at one or more of polymorphic sites in the ADCY9 gene.
In another embodiment the invention provides, a method for determining an individual's responsiveness to a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor, the method comprising determining a genotype of said subject (e.g., genotyping) at one or more of rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675, rs12920508, rs12599911, rs2531971 or rs2238448, using one or more of the primers or probes disclosed herein.
In another embodiment the invention provides, a method for determining an individual's responsiveness to a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor, the method comprising determining a genotype of said subject (e.g., genotyping) at one or more of rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967 or rs3730119, rs13337675, using one or more of the primers or probes disclosed herein.
In a particular embodiment, the invention provides the method herein described wherein the polymorphic sites comprises one or more of the following sites elected from the group consisting of: rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675, rs12920508, rs12599911, rs2531971 or rs2238448, particularly wherein the polymorphic site is elected from the group consisting of rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119 and rs13337675, more particularly wherein the polymorphic site is rs1967309 or rs12595857, more particularly wherein the polymorphic site is rs1967309, in particular wherein the corresponding genotype comprises AA.
In a particular embodiment, the invention provides methods of genotyping one or more polymorphic sites selected from the group consisting of: rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675, rs12920508, rs12599911, rs2531971 or rs2238448, particularly wherein the polymorphic site is elected from the group consisting of rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119 and rs13337675 more particularly wherein the polymorphic site is rs1967309 or, rs12595857, more particularly wherein the polymorphic site is rs1967309.
In a particular embodiment, the invention provides a method wherein a subject carrying one or more of rs12595857/GG, rs1967309/AA, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG, rs8061182/AA benefits from treatment with an HDL-raising or HDL mimicking agent, particularly wherein the HDL-raising or HDL mimicking agent is a CETP inhibitor/modulator, and more particularly wherein the HDL-raising or HDL mimicking agent is S-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester. In a particular embodiment, the invention provides the method wherein a HDL-raising or HDL mimicking agent is administered to say subject.
In a particular embodiment, the invention provides a method wherein a subject carrying one or more of rs12595857/GG, rs1967309/AA, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG, rs8061182/AA is treated with an HDL-raising or HDL mimicking agent, particularly wherein the HDL-raising or HDL mimicking agent is a CETP inhibitor/modulator, and more particularly wherein the HDL-raising or HDL mimicking agent is S-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester.
In particular embodiments, the invention provides a method wherein the subject, has a cardiovascular disorder, in particular wherein the cardiovascular disorder is selected from the group consisting of atherosclerosis, peripheral vascular disease, dyslipidemia, hyperbetalipoproteinemia, hypoalphalipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial-hypercholesterolemia, angina, ischemia, cardiac ischemia, stroke, myocardial infarction, reperfusion injury, angioplastic restenosis, hypertension, and vascular complications of diabetes, obesity or endotoxemia in a mammal, more particularly wherein the cardiovascular disorder is selected from the group consisting of cardiovascular disease, coronary heart disease, coronary artery disease, hypoalphalipoproteinemia, hyperbetalipoproteinemia, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, hypertriglyceridemia, hyperlipidoproteinemia, peripheral vascular disease, angina, ischemia, and myocardial infarction.
In another embodiment the invention provides a method of treating a cardiovascular disorder in a subject in need thereof, the method comprising:

    
    
        (a) selecting a subject having an improved response genotype at one or more of the following sites: rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675, rs12920508, rs12599911, rs2531971 or rs2238448;
        (b) administering to said subject a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator.
    
    


In another embodiment the invention provides a method of treating a cardiovascular disorder in a subject in need thereof, the method comprising:

    
    
        (a) selecting a subject having an improved response genotype at one or more of the following sites: rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675;
        (b) administering to said subject a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator.
    
    


In a particular embodiment the invention provides a method of treating a cardiovascular disorder in a subject in need thereof, the method comprising:

    
    
        (a) selecting a subject having an improved response polymorphism at rs1967309, in particular wherein the subject has AA genotype at rs1967309;
        (b) administering to said subject a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator.
    
    


In another embodiment the invention provides a method of treating a cardiovascular disorder in a subject in need thereof, the method comprising:

    
    
        (a) genotyping a subject at one or more of the following sites: rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675, rs12920508, rs12599911, rs2531971 or rs2238448;
        (b) administering to said subject a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator.
    
    


In another embodiment the invention provides a method of treating a cardiovascular disorder in a subject in need thereof, the method comprising:

    
    
        (c) genotyping a subject at one or more of the following sites: rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675;
        (d) administering to said subject a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator.
    
    


The invention also provides a method of treating a patient comprising:

    
    
        a. analyzing a patient sample for the presence of one or more genetic markers selected from the group consisting of rs12595857/GG, rs1967309/AA, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG and rs8061182/AA and
        b. treating patients who carry one or more of said genetic markers with a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator.
    
    


In a particular embodiment, the invention provides the method wherein a genotype is determined at one or more sites selected from: rs1967309, rs12595857.
In particular a method of determining and individuals responsiveness to a HDL-raising drug comprising:

    
    
        a. obtaining a sample from the individual, wherein the sample comprising genetic material;
        b. contacting the sample with a reagent, generating a complex between the reagent and a genetic marker selected from Table 7;
        c. detecting the complex to obtain a dataset associated with the sample and
        d. analyzing the dataset to determine the presence or absence of a genetic marker.
    
    


The complex between the reagent and a genetic marker generated in the genotyping methods provided can be generated by either polymerase chain reaction (PCR) or DNA sequencing.
The invention provides reagents for genotyping a genetic marker selected from the group consisting of rs12595857/GG; rs1967309/AA; rs111590482/AG; rs111590482/GG; rs11647828/GG; rs12935810/GG; rs17136707/GG; rs2239310/GG; rs2283497/AA; rs2531967/AA; rs3730119/AA; rs4786454/AA; rs74702385/GA; rs74702385/AA; rs8049452/GG; rs8061182/AA; rs1967309/GA, rs12595857/AG, rs13337675/AG, rs13337675/GG, rs17136707/AG, rs2239310/AG, rs2283497/CA, rs2531967/GA, rs3730119/GA, rs4786454/GA, rs8049452/GA, rs8061182/AG, rs12595857/GG, rs1967309/AA, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG, rs8061182/AA, rs12935810/GA, rs12935810/AA, rs11647828/AA, rs2531967/GG, rs3730119/GG, rs2239310/AA, rs12595857/AA, rs111590482/AA, rs74702385/GG, rs1967309/GG, rs2283497/CC, rs8061182/GG, rs17136707/AA, rs8049452/AA, rs4786454/GG, rs13337675/AA and rs11647828/AG, in particular a primer or a probe.
In a particular embodiment the primer comprising strand of DNA that is 15 to 30 nucleotides in length and hybridizes under high stringency conditions to a region of chromosome 16 adjacent to a rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675, rs12920508, rs12599911, rs2531971 or rs2238448.
In a particular embodiment the primer comprising strand of DNA that is 15 to 30 nucleotides in length and hybridizes under high stringency conditions to a region of chromosome 16 adjacent to a rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119 or rs13337675.
In another embodiment the reagent is a primer comprising strand of DNA that is 15 to 30 nucleotides in length and hybridizes under high stringency conditions to a region of chromosome 16 overlapping with rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675, rs12920508, rs12599911, rs2531971 or rs2238448.
In another embodiment the reagent is a primer comprising strand of DNA that is 15 to 30 nucleotides in length and hybridizes under high stringency conditions to a region of chromosome 16 overlapping with rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119 or rs13337675.
In another embodiment the probe comprising that is 15 to 30 nucleotides in length and hybridizes under high stringency conditions to a region of chromosome 16 overlapping with rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675, rs12920508, rs12599911, rs2531971 or rs2238448.
In another embodiment the probe comprising that is 15 to 30 nucleotides in length and hybridizes under high stringency conditions to a region of chromosome 16 overlapping with rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119 or rs13337675.
In another embodiment the probe comprising that is 15 to 30 nucleotides in length and hybridizes under high stringency conditions an oligonucleotide selected from SEQ. ID. NO. 1 to SEQ. ID. NO 15.
In a particular embodiment, the invention provides the method wherein HDL-raising or HDL mimicking agents a CETP inhibitor/modulator, in particular, wherein HDL-raising or HDL mimicking agent is S-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester.
In yet another embodiment, the invention provides a use of HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator, in the manufacture of a medicament for the treatment of cardiovascular disorder, wherein the subjects treated have an improved response genotype at one or more of the following sites: rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119 or rs13337675.
In a particular embodiment, the invention provides the use as defined herein, wherein the subject treated have an improved response polymorphism at rs1967309.
In another embodiment, the invention provides a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator for use in the treatment of cardiovascular disorder, wherein the subjects treated carry one or more genetic markers selected from: rs12595857/GG; rs1967309/AA; rs111590482/AG; rs111590482/GG; rs11647828/GG; rs12935810/GG; rs17136707/GG; rs2239310/GG; rs2283497/AA; rs2531967/AA; rs3730119/AA; rs4786454/AA; rs74702385/GA; rs74702385/AA; rs8049452/GG; rs8061182/AA; rs1967309/GA, rs12595857/AG, rs13337675/AG, rs13337675/GG, rs17136707/AG, rs2239310/AG, rs2283497/CA, rs2531967/GA, rs3730119/GA, rs4786454/GA, rs8049452/GA, rs8061182/AG, rs12595857/GG, rs1967309/AA, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG, rs8061182/AA, rs12935810/GA, rs12935810/AA, rs11647828/AA, rs2531967/GG, rs3730119/GG, rs2239310/AA, rs12595857/AA, rs111590482/AA, rs74702385/GG, rs1967309/GG, rs2283497/CC, rs8061182/GG, rs17136707/AA, rs8049452/AA, rs4786454/GG, rs13337675/AA and rs11647828/AG.
In a particular embodiment, the invention provides the HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator for use in the treatment of cardiovascular disorder, wherein the subject treated carries an improved response genotypers1967309. In a particular embodiment, the invention provides the HDL-raising or HDL mimicking agent as herein defined wherein the genotype is AA.
In a particular embodiment, the invention provides an HDL-raising or HDL mimicking agent as herein described, wherein the cardiovascular disorder is selected from the group consisting of atherosclerosis, peripheral vascular disease, dyslipidemia, hyperbetalipoproteinemia, hypoalphalipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial-hypercholesterolemia, angina, ischemia, cardiac ischemia, stroke, myocardial infarction, reperfusion injury, angioplastic restenosis, hypertension, and vascular complications of diabetes, obesity or endotoxemia in a mammal.
In a particular embodiment, the invention provides the HDL-raising or HDL mimicking agent as herein described, wherein the cardiovascular disorder is selected from the group consisting of cardiovascular disease, coronary heart disease, coronary artery disease, hypoalphalipoproteinemia, hyperbetalipoproteinemia, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, hypertriglyceridemia, hyperlipidoproteinemia, peripheral vascular disease, angina, ischemia, and myocardial infarction.
In a particular embodiment, the invention provides the HDL-raising or HDL mimicking agent as herein described, wherein the HDL-raising or mimicking agent is and HDL-raising agent, particularly a CETP inhibitor/modulator, more particularly is-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester.
In another embodiment, the invention provides S-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester for treating patient with cardiovascular disorder, who carries an improved response genotype, in particular wherein the genotype is rs12595857/GG, rs1967309/AA, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG or rs8061182/AA, more particularly wherein the genotype is rs1967309/AA.
In a particular embodiment, the invention provides S-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester for treating a patient with a cardiovascular disorder who carries an improved response genotype, wherein the cardiovascular disorder is selected from the group consisting of atherosclerosis, peripheral vascular disease, dyslipidemia, hyperbetalipoproteinemia, hypoalphalipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial-hypercholesterolemia, angina, ischemia, cardiac ischemia, stroke, myocardial infarction, reperfusion injury, angioplastic restenosis, hypertension, and vascular complications of diabetes, obesity or endotoxemia in a mammal.
In a particular embodiment, the invention provides S-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester for treating a patient with a cardiovascular disorder who carries an improved response genotype, wherein the cardiovascular disorder is selected from the group consisting of cardiovascular disease, coronary heart disease, coronary artery disease, hypoalphalipoproteinemia, hyperbetalipoproteinemia, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, hypertriglyceridemia, hyperlipidoproteinemia, peripheral vascular disease, angina, ischemia, and myocardial infarction.
In another embodiment, the invention provides a method of predicting whether a cardiovascular disorder patient has an increased likelihood of benefiting from treatment with a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator, comprising screening a sample isolated from said patient for a genetic marker in the Adenylate Cyclase Type 9 gene (ADCY9) selected from rs12595857/GG, rs1967309/AA, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG, rs8061182/AA, wherein the patient has an increased likelihood of benefiting from said treatment with an HDL-raising or HDL mimicking agent. In a particular embodiment the genetic marker screened is selected from rs12595857/GG; rs1967309/AA. More particularly the genetic marker screened is rs1967309/AA.
In a further embodiment, the invention provides a method of selecting a patient cardiovascular disorder as likely to respond to a therapy comprising HDL-raising or HDL mimicking agent, the method comprising:

    
    
        (a) detecting an AA genotype at rs1967309 in a sample from the patient,
        (b) selecting the patient as more likely to respond to a therapy comprising HDL-raising or HDL mimicking agent when rs1967309 with AA genotype is detected in the sample from the patient.
    
    


In a particular embodiment, the invention provides the method herein described, wherein the presence of an AA genotype at rs1967309 in a reference sample indicates that the patient is more likely to respond to the therapy with a HDL-raising or HDL mimicking agent.
In a particular embodiment, the invention provides the method herein described which further comprises c) selecting the therapy comprising a HDL-raising or HDL mimicking agent.
In a particular embodiment, the invention provides the method herein described wherein detecting rs1967309 is done by detecting rs1967309 in a sample from the patient, contacting the sample with a reagent that binds to rs1967309, thereby forming a complex between the reagent and rs1967309, detecting the complex formed, and thereby detecting rs1967309,
In another embodiment, the invention provides a method for determining the prognosis of a clinical response in a human patient to a HDL-raising or HDL mimicking agent, wherein the presence of at least one polymorphism in the ADCY9 gene of that patient which is associated with a delayed, partial sub-optimal or lacking clinical response to said HDL-raising or mimicking agent is determined, wherein at least one, wherein at least one first polymorphism rs1967309 is determined.
In a particular embodiment, the invention provides the method herein described, wherein the polymorphism is determined by a genotyping analysis.
In a particular embodiment, the invention provides the method herein described, wherein the genotyping analysis comprises a microarray analysis or a mass-spectrometric analysis or the use of polymorphism-specific primers and/or probes, in particular a primer extension reaction.
In a particular embodiment, the invention provides the method herein described, wherein HDL-raising or HDL mimicking agent is a HDL-raising agent, in particular a CETP inhibitor/modulator, more particularly S-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester.
In a particular embodiment, the “CETP inhibitor/modulator” is thioisobutyric acid S-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester, also known as S-[2-([[1-(2-ethylbutyl)-cyclohexyl]-carbonyl]amino)phenyl]2-methylpropanethioate, dalcetrapib or a compound of formula I




S-[2-([[1-(2-ethylbutyl)cyclohexyl]carbonyl]amino)phenyl]2-methylpropanethioate has been shown to be an inhibitor of CETP activity in humans (de Grooth et al., Circulation, 105, 2159-2165 (2002)) and rabbits (Shinkai et al., J. Med. Chem., 43, 3566-3572 (2000); Kobayashi et al., Atherosclerosis, 162, 131-135 (2002); and Okamoto et al., Nature, 406 (13), 203-207 (2000)). S-[2-([[1-(2-ethylbutyl)cyclohexyl]carbonyl]amino)phenyl]2-methylpropanethioate has been shown to increase plasma HDL cholesterol in humans (de Grooth et al., supra) and in rabbits (Shinkai et al., supra; Kobayashi et al., supra; Okamoto et al., supra). Moreover, S-[2-([[1-(2-ethylbutyl)cyclohexyl]carbonyl]amino)phenyl]2-methylpropanethioate has been shown to decrease LDL cholesterol in humans (de Grooth et al., supra) and rabbits (Okamoto et al., supra). S-[2-([[1-(2-ethylbutyl)cyclohexyl]carbonyl]amino)phenyl]2-methylpropanethioate, as well as methods of making and using the compound, are described in EP1020439, Shinkai et al., J. Med. Chem. 43:3566-3572 (2000) or WO 2007/051714, WO 2008/074677 or WO2011/000793.

In a preferred embodiment the CETP inhibitor/modulator (e.g. compound of formula I) is a solid in crystalline or amorphous form, or more preferably in crystalline form. In a particular embodiment S-[2-([[1-(2-ethylbutyl)-cyclohexyl]-carbonyl]amino)phenyl]2-methylpropanethioate is in crystalline form A as disclosed in WO2012/069087. Form A is characterized by an X-ray powder diffraction pattern having peaks at about 7.9°, 8.5°, 11.7°, 12.7°, 17.1°, 18.0°, 18.5°, 20.2°, 22.1°, 24.7°±0.2°, particularly by an XRPD peaks observed at an angle of diffraction 2Theta of 7.9°, 11.7°, 17.1°, 18.5° (±0.2°).
Other CETP inhibitors known in the art and useful in the present invention include: evacetrapib, anacetrapib and torcetrapib, particularly evacetrapib and anacetrapib.
Accordingly, the invention provides a method for the treatment or prophylaxis of a cardiovascular disorder in a mammal, which method comprises administering to a mammal (preferably a mammal in need thereof) a therapeutically effective amount of the pharmaceutical composition. The mammal preferably is a human (i.e., a male or female human). The human can be of any race (e. g., Caucasian or Oriental).
The cardiovascular disorder preferably is selected from the group consisting of atherosclerosis, peripheral vascular disease, dyslipidemia, hyperbetalipoproteinemia, hypoalphalipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial-hypercholesterolemia, angina, ischemia, cardiac ischemia, stroke, myocardial infarction, reperfusion injury, angioplastic restenosis, hypertension, and vascular complications of diabetes, obesity or endotoxemia in a mammal. More preferably, the cardiovascular disorder is selected from the group consisting of cardiovascular disease, coronary heart disease, coronary artery disease, hypoalphalipoproteinemia, hyperbetalipoproteinemia, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, hypertriglyceridemia, hyperlipidoproteinemia, peripheral vascular disease, angina, ischemia, and myocardial infarction.
In certain embodiments of the present invention, the subject are administered between 100 mg to 600 mg of S-[2-([[1-(2-ethylbutyl)-cyclohexyl]-carbonyl]amino)phenyl]2-methylpropanethioate. In particular, the subject are administered between 150 mg to 450 mg of S-[2-([[1-(2-ethylbutyl)-cyclohexyl]-carbonyl]amino)phenyl]2-methylpropanethioate. More particularly, the subject are administered between 250 mg to 350 mg of S-[2-([[1-(2-ethylbutyl)-cyclohexyl]-carbonyl]amino)phenyl]2-methylpropanethioate. Most particularly, the subject are administered between 250 mg to 350 mg of S-[2-([[1-(2-ethylbutyl)-cyclohexyl]-carbonyl]amino)phenyl]2-methylpropanethioate.
In another embodiment of the present invention, the subject for pediatric use are administered between 25 mg to 300 mg of S-[2-([[1-(2-ethylbutyl)-cyclohexyl]-carbonyl]amino)phenyl]2-methylpropanethioate. In particular the subject for pediatric use are administered 75 mg to 150 mg of S-[2-([[1-(2-ethylbutyl)-cyclohexyl]-carbonyl]amino)phenyl]2-methylpropanethioate.
The CETP inhibitor can be administered to the mammal at any suitable dosage (e. g., to achieve a therapeutically effective amount). For example, a suitable dose of a therapeutically effective amount of Compound I for administration to a patient will be between approximately 100 mg to about 1800 mg per day. A desirable dose is preferably about 300 mg to about 900 mg per day. A preferred dose is about 600 mg per day.
Genotyping Methods
Identification of the particular genotype in a sample may be performed by any of a number of methods well known to one of skill in the art. For example, identification of the polymorphism can be accomplished by cloning of the allele and sequencing it using techniques well known in the art. Alternatively, the gene sequences can be amplified from genomic DNA, e.g. using PCR, and the product sequenced. Numerous methods are known in the art for isolating and analyzing a subject's DNA for a given genetic marker including polymerase chain reaction (PCR), ligation chain reaction (LCR) or ligation amplification and amplification methods such as self sustained sequence replication. Several non-limiting methods for analyzing a patient's DNA for mutations at a given genetic locus are described below.
DNA microarray technology, e.g., DNA chip devices and high-density microarrays for high-throughput screening applications and lower-density microarrays, may be used. Methods for microarray fabrication are known in the art and include various inkjet and microjet deposition or spotting technologies and processes, in situ or on-chip photolithographic oligonucleotide synthesis processes, and electronic DNA probe addressing processes. The DNA microarray hybridization applications has been successfully applied in the areas of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs). Additional methods include interference RNA microarrays and combinations of microarrays and other methods such as laser capture micro-dissection (LCM), comparative genomic hybridization (CGH) and chromatin immunoprecipitation (ChiP). See, e.g., He et al. (2007) Adv. Exp. Med. Biol. 593: 117-133 and Heller (2002) Annu Rev. Biomed. Eng. 4: 129-153. Other methods include PCR, xMAP, invader assay, mass spectrometry, and pyrosequencing (Wang et al. (2007) Microarray Technology and Cancer Gene Profiling Vol 593 of book series Advances in Experimental Medicine and Biology, pub. Springer New York).
Another detection method is allele specific hybridization using probes overlapping the polymorphic site and having about 5, or alternatively 10, or alternatively 20, or alternatively 25, or alternatively 30 nucleotides around the polymorphic region. For example, several probes capable of hybridizing specifically to the allelic variant or genetic marker of interest are attached to a solid phase support, e.g., a “chip”. Oligonucleotide probes can be bound to a solid support by a variety of processes, including lithography. Mutation detection analysis using these chips comprising oligonucleotides, also termed “DNA probe arrays” is described e.g., in Cronin et al. (1996) Human Mutation 7′:244.
In other detection methods, it is necessary to first amplify at least a portion of the gene prior to identifying the allelic variant. Amplification can be performed, e.g., by PCR and/or LCR or other methods well known in the art.
In some cases, the presence of the specific allele in DNA from a subject can be shown by restriction enzyme analysis. For example, the specific nucleotide polymorphism can result in a nucleotide sequence comprising a restriction site which is absent from the nucleotide sequence of another allelic variant.
In a further embodiment, protection from cleavage agents (such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine) can be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA heteroduplexes (see, e.g., Myers et al. (1985) Science 230: 1242). In general, the technique of “mismatch cleavage” starts by providing duplexes formed by hybridizing a probe, e.g., RNA or DNA, which is optionally labeled, comprising a nucleotide sequence of the allelic variant of the gene with a sample nucleic acid, obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as duplexes formed based on base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions. Alternatively, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine whether the control and sample nucleic acids have an identical nucleotide sequence or in which nucleotides they are different. See, for example, U.S. Pat. No. 6,455,249; Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Meth. Enzymol. 217:286-295.
Alterations in electrophoretic mobility may also be used to identify the particular allelic variant. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766; Cotton (1993) Mutat. Res. 285: 125-144 and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids are denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence; the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using R A (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In another preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
The identity of the allelic variant or genetic marker may also be obtained by analyzing the movement of a nucleic acid comprising the polymorphic region in polyacrylamide gels containing a gradient of denaturant, which is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265: 1275).
Examples of techniques for detecting differences of at least one nucleotide between 2 nucleic acids include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide probes may be prepared in which the known polymorphic nucleotide is placed centrally (allele-specific probes) and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324: 163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotide hybridization techniques are used for the detection of the nucleotide changes in the polymorphic region of the gene. For example, oligonucleotide probes having the nucleotide sequence of the specific allelic variant are attached to a hybridizing membrane and this membrane is then hybridized with labeled sample nucleic acid. Analysis of the hybridization signal will then reveal the identity of the nucleotides of the sample nucleic acid.
Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the allelic variant of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucl. Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238 and Newton et al. (1989) Nucl. Acids Res. 17:2503). This technique is also termed “PROBE” for PRobeOligo Base Extension. In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell. Probes 6: 1).
In another embodiment, identification of the allelic variant or genetic marker is carried out using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Pat. No. 4,998,617 and in Laridegren, U. et al. Science 241: 1077-1080 (1988). The OLA protocol uses two oligonucleotide probes which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is linked to a separation marker, e.g., biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-8927). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA. A variation of the OLA method as described in Tobe et al. (1996) Nucleic Acids Res. 24: 3728 each allele specific primers is labeled with a unique hapten, i.e. digoxigein and florescein and each OLA reaction is detected using hapten specific antibodies labeled with reporter enzymes.
The invention provides methods for detecting a single nucleotide polymorphism (SNP) in ADCY9. Because single nucleotide polymorphisms are flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single variant nucleotide and it is unnecessary to determine a complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of SNPs.
The single base polymorphism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g., in U.S. Pat. No. 4,656,127. According to the method, a primer complementary to the allelic sequence immediately 3′ to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.
A solution-based method may also be used to determine the identity of the nucleotide of the polymorphic site (WO 91/02087). As above, a primer is employed that is complementary to allelic sequences immediately 3′ to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.
An alternative method is described in WO 92/15712. This method uses mixtures of labeled terminators and a primer that is complementary to the sequence 3′ to a polymorphic site. The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated. The method is usually a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.
Many other primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komher, J. S. et al. (1989) Nucl. Acids. Res. 17:7779-7784; Sokolov, B. P. (1990) Nucl. Acids Res. 18:3671; Syvanen, A.-C, et al. (1990) Genomics 8:684-692; Kuppuswamy, M. N. et al. (1991) Proc. Natl. Acad. Sci. USA 88: 1143-1147; Prezant, T. R. et al. (1992) Hum. Mutat. 1: 159-164; Ugozzoli, L. et al. (1992) GATA 9: 107-112; Nyren, P. et al. (1993) Anal. Biochem. 208: 171-175). These methods all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at a polymorphic site.
Moreover, it will be understood that any of the above methods for detecting alterations in a gene or gene product or polymorphic variants can be used to monitor the course of treatment or therapy.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits, such as those described below, comprising at least one probe, primer nucleic acid, or reagent which may be conveniently used for genotyping, e.g., analyzing a genetic marker present in the ADCY9 gene to determine whether an individual has an increased likelihood of benefiting from treatment with HDL-raising or mimicking agent including a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator. In particular the genetic markers are as described herein.
Primers or probes of the present invention, for use as reagents for genotyping genetic markers present in the ADCY9 gene, comprise a synthetic nucleotide sequence that is complimentary to and hybridizes with a contiguous sequence within the ADCY9 gene, of preferably 12 to 30 nucleotides, adjacent to or encompassing one or more SNPs selected from rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119 and rs13337675, preferably rs1967309. In other aspects a primer comprises 100 or fewer nucleotides, in certain aspects from 12 to 50 nucleotides or from 12 to 30 nucleotides. The primer is at least 70% identical to the contiguous sequence or to the complement of the contiguous nucleotide sequence, preferably at least 80% identical, and more preferably at least 90% identical. A primer or probe of the invention is preferably 15-50 nucleotides in length comprising a region of 15 to 20 nucleotides that is complimentary to a sequence selected from SEQ. ID. NO 1-15, in particular is complementary to sequence SEQ. ID No 1. The degree of complimentary between a probe or primer and SEQ. ID. NO. 1-15 maybe 100%, 95%, 90%, 85%, 80% or 75%.
Oligonucleotides, including probes and primers, “specific for” a genetic allele or genetic marker bind either to the polymorphic region of a gene or bind adjacent to the polymorphic region of the gene. For oligonucleotides that are to be used as primers for amplification, primers are adjacent if they are sufficiently close to be used to produce a polynucleotide comprising the polymorphic region. In one embodiment, oligonucleotides are adjacent if they bind within about 1-2 kb, e.g. less than 1 kb from the polymorphism. Specific oligonucleotides are capable of hybridizing to a sequence, and under suitable conditions will not bind to a sequence differing by a single nucleotide.
Oligonucleotides of the invention, whether used as probes or primers, can be detectably labeled. Labels can be detected either directly, for example for fluorescent labels, or indirectly. Indirect detection can include any detection method known to one of skill in the art, including biotin-avidin interactions, antibody binding and the like. Fluorescently labeled oligonucleotides also can contain a quenching molecule. Oligonucleotides can be bound to a surface. In some embodiments, the surface is silica or glass. In some embodiments, the surface is a metal electrode.
Probes can be used to directly determine the genotype of the sample or can be used simultaneously with or subsequent to amplification. The term “probes” includes naturally occurring or recombinant single- or double-stranded nucleic acids or chemically synthesized nucleic acids. They may be labeled by nick translation, Klenow fill-in reaction, PCR or other methods known in the art. Probes of the present invention, their preparation and/or labeling are described in Sambrook et al. (1989) supra. A probe can be a polynucleotide of any length suitable for selective hybridization to a nucleic acid containing a polymorphic region of the invention. Length of the probe used will depend, in part, on the nature of the assay used and the hybridization conditions employed.
Labeled probes also can be used in conjunction with amplification of a polymorphism. (Holland et al. (1991) Proc. Natl. Acad. Sci. USA 88:7276-7280). U.S. Pat. No. 5,210,015 describes fluorescence-based approaches to provide real time measurements of amplification products during PCR. Such approaches have either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double-stranded DNA present, or they have employed probes containing fluorescence-quencher pairs (also referred to as the “TaqMan®” approach) where the probe is cleaved during amplification to release a fluorescent molecule whose concentration is proportional to the amount of double-stranded DNA present. During amplification, the probe is digested by the nuclease activity of a polymerase when hybridized to the target sequence to cause the fluorescent molecule to be separated from the quencher molecule, thereby causing fluorescence from the reporter molecule to appear. The TaqMan® approach uses a probe containing a reporter molecule—quencher molecule pair that specifically anneals to a region of a target polynucleotide containing the polymorphism.
Probes can be affixed to surfaces for use as “gene chips.” Such gene chips can be used to detect genetic variations by a number of techniques known to one of skill in the art. In one technique, oligonucleotides are arrayed on a gene chip for determining the DNA sequence of a by the sequencing by hybridization approach, such as that outlined in U.S. Pat. Nos. 6,025,136 and 6,018,041. The probes of the invention also can be used for fluorescent detection of a genetic sequence. Such techniques have been described, for example, in U.S. Pat. Nos. The probes of the invention also can be used for fluorescent detection of a genetic sequence. Such techniques have been described, for example, in U.S. Pat. Nos. 5,968,740 and 5,858,659. A probe also can be affixed to an electrode surface for the electrochemical detection of nucleic acid sequences such as described in U.S. Pat. No. 5,952,172 and by Kelley, S. O. et al. (1999) Nucl. Acids Res. 27:4830-4837. One or more probes for detecting the SNP of the invention (Table 2, 3, 4 or 5, in particular Table 2) can be affixed to a chip and such a device used to predict response to HDL-raising or HDL mimicking agent, in particular CETP inhibitor/modulator and select an effective treatment for an individual with cardiovascular disease. It is conceivable that probes for detecting the SNP of the invention could be included on a chip with a variety of other probes for uses other than predicting response to a HDL-raising or HDL mimicking agent, in particular CETP inhibitor/modulator.
Additionally, synthetic oligonucleotides used as probes or primers may be modified to become more stable. Exemplary nucleic acid molecules which are modified include uncharged linkages such as phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564 and 5,256,775). Primers and probes of the invention can include for example, labeling methylation, inter-nucleotide modification such as pendent moieties (e.g., polypeitides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids). Also included are synthetic molecules that mimic nucleotide acid molecules in the ability to bind to a designated sequence by hydrogen bonding and other chemical interactions, including peptide linkages that substitute for phosphate linkages in the nucleotide backbone.
The invention relates to synthetic oligonucleotide molecules, primers and probes that hybridize under high stringency hybridization conditions to naturally occurring oligonucleotides described herein, gene markers of the ADCY9 gene. Oligonucleotides can be detected and/or isolated by specific hybridization, under high stringency conditions. “High stringency conditions” are known in the art and permit specific hybridization of a first oligonucleotide to a second oligonucleotide where there is a high degree of complimentarity between the first and second oligonucleotide. For the genotyping methods disclosed herein this degree of complimentarity is between 80% and 100% and preferably between 90% and 100%
The SNP of the invention can also be detected from pre-existing data, such as whole genome sequence data present in a data base. The invention provides a computer implemented method of querying genomic data to determine a genotype for predicting the response of a patient to a CETP inhibitor and treating said patient accordingly i.e. treating responder patients with a CETP inhibitor.
Sample nucleic acid of the invention for use in the genotyping methods, treatment selection or methods of treatment can be obtained from any cell type or tissue of a subject. For example, a subject's bodily fluid, a sample, (e.g. blood) can be obtained by known techniques. Alternatively, nucleic acid tests can be performed on dry samples (e.g., hair or skin) More particularly, the genotyping methods, treatment selection or methods of treatment will use blood cell type.
The invention described herein relates to methods and reagents for determining and identifying the allele present on the ADCY9gene at rs1967309 or rs12595857, or any other genetic variant in linkage disequilibrium with those two SNPs such as displayed in FIG. 8, spanning from position chr16:4049365 to chr16:4077178 (assembly GRCh37/hg19. In particular the invention also relates to methods and reagents for determining and identifying the allele present in the ADCY9 gene rs1967309, rs12595857, rs2239310, rs11647828, rs8049452, rs12935810, rs74702385, rs17136707, rs8061182, rs111590482, rs4786454, rs2283497, rs2531967, rs3730119, rs13337675, more particularly at rs1967309 or rs12595857, and most particularly at rs1967309.
As set forth herein, the invention also provides treatment selection methods comprising detecting one or more genetic markers present in the ADCY9 gene. In some embodiments, the methods use probes or primers comprising nucleotide sequences which are complementary to a polymorphic region of ADCY9. Accordingly, the invention provides kits comprising probes and primers for performing the genotyping methods of the invention.
In some embodiments, the invention provides a kit for determining whether a patient with a cardiovascular disorder has an increased likelihood of benefiting from treatment with a HDL-raising or HDL mimicking agent, in particular a CETP inhibitor/modulator. Such kits contain one of more of the reagents, in particular primers or probes, described herein and instructions for use. As an example only, the invention also provides kits for determining whether a patient with cardiovascular disorder has an increased likelihood of benefiting from treatment with thioisobutyric acid S-(2-{[1-(2-ethyl-butyl)-cyclohexanecarbonyl]-amino}-phenyl) ester comprising a first oligonucleotide and a second oligonucleotides specific for a AA polymorphism in the ADCY9 rs1967309 SNP.
The kit can comprise at least one probe or primer which is capable of specifically hybridizing to the polymorphic region of ADCY9 and instructions for use. The kits usually comprise at least one of the above described nucleic acids. Kits for amplifying at least a portion of ADCY9 generally comprise two primers, at least one of which is capable of hybridizing to the allelic variant sequence. Such kits are suitable for detection of genotype by, for example, fluorescence detection, by electrochemical detection, or by other detection.
Yet other kits of the invention comprise at least one reagent necessary to perform the assay.
For example, the kit can comprise an enzyme. Alternatively the kit can comprise a buffer or any other necessary reagent.
The kits can include all or some of the positive controls, negative controls, reagents, primers, sequencing markers, probes and antibodies described herein for determining the subject's genotype in the polymorphic region of ADCY9.
The following example is intended merely to illustrate the practice of the present invention and is not provided by way of limitation.
The present invention refers to the following nucleotide and amino acid sequences:
The sequences provided herein are available in the NCBI database and can be retrieved from world wide web address: ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such “variants” are genetic variants. On NCBI database the Nucleotide sequence encoding homo sapiens Adenylate Cyclase Type 9 (ACDY9) is available.
Homo sapiens Adenylate Cyclase Type 9 (ADCY9), RefSeqGene on chromosome 16
NCBI Reference Sequence: NCBI accession number NG_011434.1
Homo sapiens chromosome 16 genomic contig, GRCh37.p10 Primary Assembly
NCBI Reference Sequence: NCBI accession number NT_010393.16
The intronic sequences for homo sapiens ACDY9 gene SNPs providing the “rs” designation, alleles and corresponding SEQ ID number designations is disclosed in Table 4a. The polymorphisms are identified in bold and underlined text.







TABLE 4a







ACDY9 SNPs and respective intronic sequence











SEQ.




SNP rs
ID.
Intronic



ID
NO.:
sequence1
HGVS Names





rs1967309
20
TTAACCTATTTATTT
NC_000016.9:




CTTTCAACCCT[C/T]
g.4065583A > G




AGCCCAGATCCTAA
NG_011434.1:




CCTTCGGTAAG
g.105604T > C




(Map to Genome
NM_001116.3:




Build 37.3)
c.1694-8024T > C





NT_010393.16:




 
g.4005583A > G


 


rs12595857
 2
CATTGATTTTAAAC
NC_000016.9:




CTCAACAACAGC[A/
g.4062592G > A




G]ATGTCTTTTATCA
NG_011434.1:




GCTTAATTTTAC
g.108595C > T




(Map to Genome
NM_001116.3:




Build 37.3)
c.1694-5033C > T





NT_010393.16:




 
g.4002592G > A





1Source from NCBI Genome reference Build 37.3













TABLE 5







List of genetic variants in gene ADCY9 on chr16   


which have provided evidence of association 


(P < 0.05) with response to treatment with 


dalcetrapib from the GWAS study with reference   


sequence from the genotyping chip used for the


experiment (Illumina OMNI2.5S):













Position
SNPrs


SEQ.



(GRCh37/
identifier


ID


Chr.
hg19)
(NCBI)
P value
Sequence1,2
NO.





16
4,065,583
rs1967309
4.11E−08
TTCATGCACCCAG
 1






CAGACTAAATGT







TTACTGAGTACTT







ACCGAAGGTTAG







GATCTGGGCT[A/







G]AGGGTTGAAA







GAAATAAATAGG







TTAAAAAAGAAA







AAAAGCCACCTA







GGTGACTTTCACT







C1



 


16
4,062,592
rs12595857
4.53E−07
TTAATATGATTTC
 2






TTATATTCTTTCC







TGGTTATCCATTG







ATTTTAAACCTCA







ACAACAGC[A/G]







ATGTCTTTTATCA







GCTTAATTTTACA







AAGGCTACAGAGA







GGGGTGGGCATT







TCCTAATGG2



 


16
4,060,661
rs2239310
1.29E−06
CCTGTGTGGAGC
 3






CCATTACCTGAA







GAGGGGCCAAGA







GGACAAGCAGGT







ATGACTATGGTC







[A/G]GGCGTGCC







AAGTCCCAGGACA







AGGAAGGACGGG







TGCTCCAGGAAG







CACAGGAGGGGG







CAT2



 


16
4,051,513
rs11647828
2.76E−06
TACCGGATGGCA
 4






GTGAGCAGGGAG







GCTCACCTGGATC







ATTTGGTGAAGG







TGGCATCTGCC







[T/C]GGTTTGTC







CACTGTGAAGTTC







CTATTCCTACCCC







GCCCCCCACCTTT







CTTTTTTGAGATG2



 


16
4,076,094
rs8049452
6.63E−06
ACTTAACTATTTG
 5






TTGGGTGAATAT







AGAAATGAATGA







ATGAATGGATGG







ATGAGCAGATA







[T/C]ATCAAGAA







GTTAATTCACAAA







TTAAAGCCCATTA







TGAAACTAAAGTA







GAGGCTGGGCGC







G1



 


16
4,049,365
rs12935810
2.98E−05
ACCCGTGAACAA
 6






GTCGGGCCCCCA







TCCACGCAATATC







TGCAGTCTCGACT







GTATGATCTC[A/







G]TCCTTTGCAGCC







ACACTGTGAGGC







AGCAATGATCAT







TCCGCAGACGGC







CACAGACTCCAG2



 


16
4,065,495
rs74702385
8.87E−05
GACGACACCCAG
 7






CACACCCAGCAC







ACCCAGCACACC







AGCGAACAGCCC







ACCAGGTGCTAT







[T/C]GCTGTCAT







TCATTTGCTCATT







CGCTCGTTCATGC







ACCCAGCAGACTAA







ATGTTTACTGAG1



 


16
4,076,047
rs17136707
9.11E−05
AAAACAGTGCTC
 8






CAAAGGCAAAGA







AATAGCAAAGAC







AGAAGTAAGGCA







CTTAACTATTTG







[T/C]TGGGTGAAT







ATAGAAATGAAT







GAATGAATGGAT







GGATGAGCAGAT







ACATCAAGAAGT







TAA1



 


16
4,070,333
rs8061182
1.51E−04
GGCAGCTATGTA
 9






GGAAGCAGTGAA







GATCCACATCCTT







CCTTATTGGTGAA







AGGAATGAAT[T/







C]GGAAACAGAA







AGTTCTTTTTTAC







CTTTATTAAATAA







ACGTGAAGTCAT







AAGAACTACTAA2



 


16
4,064,368
rs11159048
1.64E−04
AGACTTTGTCTCA
10




2

AAAAAGAAAAAA







AAAAAAAAAGAA







GTCCCAAATAAT







AAAATATGAGA[T/







C]GGATTTATGG







AAGAAAGTGAAA







GAAACAAAGGGT







AGGCACCTTGCCT







GTTTAATTTGATC1



 


16
4,076,136
rs4786454
1.98E−04
TGGATGGATGAG
11






CAGATACATCAA







GAAGTTAATTCA







CAAATTAAAGCC







CATTATGAAACT







[A/G]AAGTAGAGG







CTGGGCGCGGTG







GATCACGCCTAT







AATCCCAGCACTT







TGGGAGGTCAAG







GC2



 


16
4,066,061
rs2283497
8.87E−04
TGTGATATGATG
12






GTCATATCATAGC







ACAGGGCTGTTG







TGAGGATTAAAT







GAGTTGATTCA[T/







G]GTAAACAGGG







ACATCCGAAAAA







GGGAAAGACGGT







GCTTGTCCTGAGA







ACAGCTGTGAAT







G1



 


16
4,052,486
rs2531967
1.11E−03
AGGTGAGTGGCC
13






TTAAAGGGGAAG







GAGAAACCTTTT







GAAAGCAGGACA







GGTCCTCTCTGA







[A/G]TCATCCCCG







TATGGGTAAATCT







ACATCACTAGCTT







CATTACTGACTGG







TCCATGTAGAAA1



 


16
4,057,603
rs3730119
0.0108
CAGGTATGTCTTC
14






AAACCTATGATG







GATAAAAGTTAC







AGTCAGCACAGA







TTGAAAGCACC[A/







G]TCTGTTGAAAC







GCAGCTCCGTCTT







GCTCTCTGGAGA







GGACTCACTCCTG







GAAAGTTGAGA2



 


16
4,077,178
rs13337675
0.0377
TGTAACCAAGTA
15






ACCAATGGTAAA







CCTCTACAGGGT







ATTAAGGCTCCA







GAAAATTCTCTA







[A/G]TCAGCCACT







TGCTCCTGCTCGAG







CCTGCTCCCACTC







CGTGGAGTGTAC







TTTCATTTCAGT1





Chr: chromosome number; P value: for association with cardiovascular events (primary composite event or unanticipated coronary revascularization) in patients treated with the CETP inhibitor dalcetrapib;


1Reference sequence from the 1000 Genomes public database, as presented in the ILLUMINA annotation file for the OMNI 2.5S Chip HumanOmni25Exome-8v1_A.csv;


2Reference sequence from the dbSNP public database version 131 from NCBI, as presented in the ILLUMINA annotation file for the OMNI 2.5S Chip HumanOmni25Exome-8v1_A.csv.













TABLE 6







List of additional genetic variants in gene ADCY9 on chr16:
















Distance




SEQ




(bp)




ID


Variation
Location1
from a
r21
D′1
Column2
HGV Names2
NO.

















rs12920508
16:4066891
1308
0.952954
1
TTTGGGGTG
NC_000016.9:g.4066891G > C
16







ACGAAAATG
NC_000016.9:g.4066891G > T








TAAAATTA[C/
NG_011434.1:g.104296C > A








G/T]GTTGTG
NG_011434.1:g.104296C > G








GTGATGGTT
NM_001116.3:c.1694-








GCACAACAC
9332C > A








C
NM_001116.3:c.1694-









9332C > G









NT_010393.16:g.4006891G >









C









NT_010393.16:g.4006891G >









T



 


rs12599911
16:4062436
3147
0.908417
1
GAATAACCA
NC_000016.9:g.4062436G > T
17







CACACATGG
NG_011434.1:g.108751C > A








ACCCTGGG
NM_001116.3:c.1694-








[G/T]CCAAG
4877C > A








TTCATTAGA
NT_010393.16:g.4002436G >








ATGGCTCTTT
T



 


rs2531971
16:4051261
14322
0.840627
0.973493
AAGACAGAG
NC_000016.9:g.4051261C > A
18







GAACCCCCA
NG_011434.1:g.119926G > T








TAGGCTGG
NM_001116.3:c.1884 + 








[G/T]GGTGAG
6108G > T








CAGGGGGCA
NT_010393.16:g.3991261C >








TGAGGGCTA
A








A




 


rs2238448
16:4059439
6144
0.840582
0.973467
TGTCCAACT
NC_000016.9:g.4059439T > C
19







ATTTCTTTCT
NG_011434.1:g.111748A > G








TTCTTTT[C/
NM_001116.3:c.1694-








T]TGAGATGGG
1880A > G








GGTCTCACT
NT_010393.16:g.3999439T >








GTGTTGG
C





References:


a. rs1967309


1Location r2 and D′ values from the 1000 Genomes public database


2Reference sequence & HGV Names from the dbSNP public database version 137 from NCBI






EXAMPLES
Example 1
Dal-OUTCOMES trial (NC20971) was a double blind, randomized, placebo-controlled, parallel group, multi-center, phase III study to assess the safety and efficacy of the CETP inhibitor dalcetrapib in patients recently hospitalized for an Acute Coronary Syndrome (ACS). At time of the interim analysis the study included 15871 randomized patients, distributed over two treatment arms: placebo (7933 patients) and dalcetrapib (600 mg daily; 7938 patients). The study has shown no evidence of reduction of the event rate in the primary efficacy endpoint in the dalcetrapib arm compared to the placebo arm. The dal-OUTCOMES study details can be found in G. Schwartz et al., N. Engl. J. Med. 367; 22, 2012.\
Genotyping:
Whole genome analysis was performed in GLP-environment at the Beaulieu-Saucier Pharmacogenomics Centre. The Infinium® HumanOmni25Exome-8v1_A BeadChip (Illumina, San Diego, Calif.) including 2,567,845 genomic markers was used and processed according to the manufacturer's specifications. Approximately 200 ng of genomic DNA was whole-genome amplified, fragmented, and hybridized to locus-specific probes bound to the surface of each BeadChip. DNA was genotyped using a fluorescently labelled single base extension assay. BeadChips were scanned and analyzed using the Illumina® iScan Reader. Infinium® process controls were monitored and all results were within manufacturer's specifications. Scanned images were analyzed using Illumina's GenomeStudio® version 2011.1 with the GenTrain 2.0 cluster algorithm, using a No-Call threshold of 0.15, without manual cluster adjustment and using the manufacturer's Illumina® HumanOmni25Exome-8v1_A cluster file. Genotype data files were produced in three instalments of comparable size as data became available. Three genotyping files in PLINK format generated by GenomeStudio® were combined and transformed to binary PLINK format.
Genotyping completion rate for samples and SNPs was set to 98%. SNPs with genotyping plate bias (based on the 96 well plates used to dilute DNA samples) were flagged but not removed as the effect of genetic ancestry could not be excluded. Pairwise IBD was used to conduct close familial relationship checks. We flagged and removed all but one pair-member of related pairs and sample duplicates (IBS2*ratio>0.80) based on a selection of uncorrelated SNPs (r2<0.1). The pairwise IBS matrix was used as a distance metric to identify cryptic relatedness among samples and sample outliers by multi-dimensional scaling (MDS). The first two MDS components of each subject were plotted including the genotypes of HapMap CEU, JPT-CHB, and YRI data (keeping only founder individuals). Outliers from the main Caucasian cluster were flagged and removed by k-nearest neighbour. (Supplementary FIG. 1) The scree plot and the cumulative explained variance were computed using the smartpca program of the EIGENSOFT suite (version 3.0). Options used were a numoutlieriter (maximum number of outlier removal iterations) of 0 (turned off) and altnormstyle (normalization formula) equal to NO. 4 (Supplementary FIG. 2)
Statistical Methods
A statistical analysis plan was developed at the Beaulieu-Saucier Pharmacogenomics Centre and the final version approved prior to genotyping completion in October 2012. Statistical tests performed on the genetic data were two-sided and adjusted to account for the multiple testing of SNPs. The genome-wide significance threshold of p<5·10−8 was used as the acceptance threshold for significant findings. Results with p<1·10−6 were considered as potential candidates. Multivariate models included sex as a covariate and the first 5 principal components (PC) of population structure to avoid confounding by population structure. The analysis set was defined as participants to the dalcetrapib treatment arm, with validation in the placebo arm to confirm absence of effect and to test for gene by treatment arm interaction in the combined set.
Genome-wide association tests with frequent genetic variants (MAF≧0.05) were conducted using PLINK software version 1.07. All results with a P value≦10−6 were validated in SAS software. The 1-degree of freedom additive genetic test was used. Genotypes were coded as 0, 1 or 2, according to the count of minor alleles. In the presence of covariates, the P value for the additive genetic test is adjusted for the covariate such




  
    log
    ⁡
    
      (
      
        r
        
          1
          -
          r
        
      
      )
    
  
  =
  
    
      b
      0
    
    +
    
      
        b
        1
      
      ⁢
      add
    
    +
    
      
        Σ
        j
      
      ⁢
      
        b
        j
      
      ⁢
      
        cov
        j
      
    
  




or log(event rate)=b0+b1add+Σjbjcovj and the null (H0) under the additive test is of b1=0. The genome-wide analysis was first conducted using a logistic regression model using PLINK software for occurrence of events versus non-occurrence. All results from the logistic regression test that provided a P value≦10−6 were validated in SAS software v.9.3 (SAS Institute Inc., Cary, N.C., USA) and tested using Cox proportional hazards regression. The results from the Cox regression were pre-defined to be more appropriate for the study objectives than those of the logistic regression and were planned to be reported as primary results. A Cox proportional hazards regression model was also used to test for gene-by-treatment interaction according to: log(event rate) ̃genotype+treatment arm+genotype*treatment arm+sex+PC, using both treatment and placebo arm samples.

The intronic variant rs1967309 in the Adenylate Cyclase Type 9 gene, ADCY9, was significantly associated with events (Cox proportional hazards p=2.4·10−8). The genetic variant at this position has a minor allele frequency of 0.45, and the additive genetic effect of one allele has a HR=0.65 (95% CI 0.55, 0.76) for events in the dal-Outcomes treatment arm. The gene by treatment arm interaction p-value is 0.0013, and there was no detectable genetic effect of the variant in the placebo arm alone (p=0.25). Sensitivity analysis with adjustment for statin are consistent (p=5.4·10−8). A neighbouring SNP in linkage disequilibrium (r2=0.86), rs12595857, has correlated results. Stratification by genotypes shows that homozygotes AA at rs1967309 have a HR=0.40 (95% CI 0.28, 0.57) for events in the dal-Outcomes treatment arm as compared to reference GG homozygotes and heterozygotes AG have a HR=0.68 (95% CI 0.55, 0.84). In the subgroup of homozygotes AA at rs1967309, treatment with dalcetrapib versus placebo had a HR=0.61 (95% CI 0.41, 0.92).







TABLE 7







Hazard Ratio (HR) fortime to events (first occurrence of CHD


death, MI, hospitalization for ACS, resuscitated cardiac arrest,


atherothrombotic stroke or unanticipated coronary


revascularization) for patients in the dalcetrapib treatment arm


only, comparing heterozygous and homozygous patients for the


variant allele to patients homozygous for the common allele,


for SNPs identified to be associated with events in the ADCY9 gene.












Genotype



SNP rs identifier
N patients
comparison
HR (95% CI)*














rs12935810
2844
GA vs GG
1.3
(1.02, 1.65)


rs12935810
2844
AA vs GG
1.87
(1.42, 2.48)


rs11647828
2822
AG vs AA
0.74
(0.6, 0.92)


rs11647828
2822
GG vs AA
0.46
(0.33, 0.63)


rs2531967
2834
GA vs GG
0.75
(0.61, 0.93)


rs2531967
2834
AA vs GG
0.5
(0.29, 0.86)


rs3730119
2845
GA vs GG
0.78
(0.62, 0.99)


rs3730119
2845
AA vs GG
0.49
(0.22, 1.1)


rs2239310
2841
AG vs AA
0.71
(0.58, 0.87)


rs2239310
2841
GG vs AA
0.43
(0.29, 0.63)


rs12595857
2838
AG vs AA
0.7
(0.57, 0.87)


rs12595857
2838
GG vs AA
0.45
(0.33, 0.62)


rs111590482
2783
AG vs AA
0.64
(0.49, 0.84)


rs111590482
2783
GG vs AA
0.15
(0.02, 1.1)


rs74702385
2845
GA vs GG
0.63
(0.48, 0.83)


rs74702385
2845
AA vs GG
0.14
(0.02, 1)


rs1967309
2842
GA vs GG
0.68
(0.56, 0.84)


rs1967309
2842
AA vs GG
0.4
(0.28, 0.57)


rs2283497
2845
CA vs CC
0.83
(0.67, 1.02)


rs2283497
2845
AA vs CC
0.53
(0.37, 0.76)


rs8061182
2843
AG vs AA
1.23
(0.98, 1.56)


rs8061182
2843
GG vs AA
1.81
(1.37, 2.39)


rs17136707
2841
AG vs AA
0.62
(0.47, 0.81)


rs17136707
2841
GG vs AA
0.16
(0.02, 1.15)


rs8049452
2845
GA vs GG
1.26
(0.99, 1.6)


rs8049452
2845
AA vs GG
1.98
(1.51, 2.61)


rs4786454
2845
GA vs GG
0.69
(0.54, 0.87)


rs4786454
2845
AA vs GG
0.37
(0.15, 0.9)


rs13337675
2843
AG vs AA
0.84
(0.68, 1.03)


rs13337675
2843
GG vs AA
0.7
(0.43, 1.14)





*Cox proportional hazards regression without adjustment for covariates stratified for each SNP by genotype with the homozygous genotype of common allele as the reference group.













TABLE 8







Hazard Ratio (HR) fortime to events (first occurrence of CHD death, MI,


hospitalization for ACS, resuscitated cardiac arrest, atherothrombotic


stroke or unanticipated coronary revascularization) on the dalcetrapib


treatment arm versus the placebo arm stratified by genotypes of SNPs


identified to be associated with cardiovascular events in the ADCY9 gene:
















N








patients
N patients


SNP rs


with
without

Predicted


identifier
Genotype
N patients
events
events
HR (95% CI)*
response
















rs12935810
GG
1848
225
1623
0.79 (0.61, 1.03)
R


rs12935810
GA
2861
385
2476
1.03 (0.84, 1.26)
NR


rs12935810
AA
1038
177
861
1.28 (0.95, 1.73)
NR


rs11647828
AA
1789
276
1513
1.33 (1.05, 1.68)
NR


rs11647828
AG
2830
385
2445
0.97 (0.8, 1.19)
PR


rs11647828
GG
1087
121
966
0.61 (0.42, 0.87)
R


rs2531967
GG
3396
497
2899
1.12 (0.94, 1.33)
NR


rs2531967
GA
2010
258
1752
0.86 (0.67, 1.1)
PR


rs2531967
AA
324
32
292
0.67 (0.33, 1.34)
R


rs3730119
GG
3934
559
3375
1.08 (0.92, 1.28)
NR


rs3730119
GA
1655
211
1444
0.86 (0.66, 1.13)
PR


rs3730119
AA
160
18
142
0.48 (0.18, 1.27)
R


rs2239310
AA
2322
370
1952
1.16 (0.95, 1.42)
NR


rs2239310
AG
2690
343
2347
0.96 (0.78, 1.19)
PR


rs2239310
GG
733
75
658
 0.6 (0.37, 0.95)
R


rs12595857
AA
1741
282
1459
1.27 (1.01, 1.61)
NR


rs12595857
AG
2867
385
2482
0.95 (0.78, 1.16)
PR


rs12595857
GG
1131
119
1012
 0.7 (0.48, 1)
R


rs111590482
AA
4301
632
3669
1.04 (0.89, 1.22)
NR


rs111590482
AG
1252
135
1117
0.85 (0.6, 1.19)
R


rs111590482
GG
81
5
76
0.26 (0.03, 2.3)
R


rs74702385
GG
4393
646
3747
1.05 (0.9, 1.23)
NR


rs74702385
GA
1266
137
1129
0.84 (0.6, 1.18)
R


rs74702385
AA
89
5
84
0.26 (0.03, 2.36)
R


rs1967309
GG
1984
322
1662
1.27 (1.02, 1.58)
NR


rs1967309
GA
2796
368
2428
0.94 (0.77, 1.16)
PR


rs1967309
AA
961
97
864
0.61 (0.41, 0.92)
R


rs2283497
CC
2098
304
1794
1.23 (0.98, 1.54)
NR


rs2283497
CA
2765
377
2388
0.99 (0.81, 1.21)
PR


rs2283497
AA
885
107
778
0.57 (0.38, 0.85)
R


rs8061182
AA
2018
257
1761
0.79 (0.62, 1.01)
R


rs8061182
AG
2800
381
2419
0.99 (0.81, 1.21)
PR


rs8061182
GG
923
148
775
1.55 (1.11, 2.15)
NR


rs17136707
AA
4423
649
3774
1.06 (0.91, 1.24)
NR


rs17136707
AG
1241
135
1106
 0.8 (0.57, 1.13)
PR


rs17136707
GG
80
4
76
0.37 (0.04, 3.54)
R


rs8049452
GG
1930
239
1691
0.78 (0.6, 1)
R


rs8049452
GA
2832
384
2448
0.97 (0.8, 1.19)
PR


rs8049452
AA
983
165
818
1.55 (1.14, 2.12)
NR


rs4786454
GG
3973
588
3385
1.06 (0.9, 1.25)
NR


rs4786454
GA
1615
186
1429
0.89 (0.67, 1.19)
PR


rs4786454
AA
161
14
147
0.52 (0.17, 1.54)
R


rs13337675
AA
3250
467
2783
1.07 (0.89, 1.28)
NR


rs13337675
AG
2145
284
1861
0.89 (0.71, 1.13)
PR


rs13337675
GG
345
36
309
1.03 (0.53, 1.97)
PR





*Cox proportional hazards regression for treatment effects stratified by genotype groups, without covariate adjustment

## Claims

1. A method for treating a cardiovascular disorder, comprising administering an effective amount of dalcetrapib to a subject in need thereof and known to carry one or more of the following improved response genotypes: rs1967309/AA, rs1967309/AG, rs12595857/GG, rs12595857/AG, rs111590482/AG, rs111590482/GG, rs11647828/AG, rs12935810/GG, rs17136707/GG, rs17136707/AG, rs2239310/GG, rs2239310/AG, rs2283497/AA, rs2283497/CA, rs2531967/AA, rs2531967/GA, rs3730119/AA, rs3730119/GA, rs4786454/AA, rs4786454/GA, rs74702385/GA, rs74702385/AA, rs8049452/GG, rs8049452/GA, rs8061182/AA, rs8061182/AG, rs11647828/GG, and rs13337675/AG.

2. The method of claim 1, wherein the one or more improved response genotypes are: rs1967309/AA, rs12595857/GG, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG, and rs8061182/AA.

3. The method of claim 1, wherein the one or more improved response genotypes is rs12595857/GG.

4. The method of claim 1, wherein the one or more improved response genotypes is rs1967309/AA.

5. The method of claim 1, wherein the one or more improved response genotypes are rs1967309/AA and 1259587/GG.

6. The method of claim 1, wherein the one or more improved response genotypes are rs1967309/AA and 1259587/GG.

7. The method of claim 1, wherein the effective amount of dalcetrapib is about 100 mg to about 1800 mg per day.

8. The method of claim 1, wherein the effective amount of dalcetrapib is about 300 mg to about 900 mg per day.

9. The method of claim 1, wherein the effective amount of dalcetrapib is about 600 mg per day.

10. A method for reducing the risk of a cardiovascular event, comprising administering an effective amount of dalcetrapib to a subject in need thereof and known to carry one or more of the following improved response genotypes: rs1967309/AA, rs1967309/AG, rs12595857/GG, rs12595857/AG, rs111590482/AG, rs111590482/GG, rs11647828/AG, rs12935810/GG, rs17136707/GG, rs17136707/AG, rs2239310/GG, rs2239310/AG, rs2283497/AA, rs2283497/CA, rs2531967/AA, rs2531967/GA, rs3730119/AA, rs3730119/GA, rs4786454/AA, rs4786454/GA, rs74702385/GA, rs74702385/AA, rs8049452/GG, rs8049452/GA, rs8061182/AA, rs8061182/AG, rs11647828/GG, and rs13337675/AG.

11. The method of claim 10, wherein the one or more improved response genotypes are: rs1967309/AA, rs12595857/GG, rs111590482/AG, rs111590482/GG, rs11647828/GG, rs12935810/GG, rs17136707/GG, rs2239310/GG, rs2283497/AA, rs2531967/AA, rs3730119/AA, rs4786454/AA, rs74702385/GA, rs74702385/AA, rs8049452/GG, and rs8061182/AA.

12. The method of claim 10, wherein the one or more improved response genotypes is rs1967309/AA.

13. The method of claim 10, wherein the effective amount of dalcetrapib is about 100 mg to about 1800 mg per day.

14. The method of claim 10, wherein the effective amount of dalcetrapib is about 300 mg to about 900 mg per day.

15. The method of claim 10, wherein the effective amount of dalcetrapib is about 600 mg per day.