Separating the Mechanism-Based and Off-Target Actions of Cholesteryl Ester Transfer Protein Inhibitors With CETP Gene Polymorphisms
Background— Cholesteryl ester transfer protein (CETP) inhibitors raise high-density lipoprotein (HDL) cholesterol, but torcetrapib, the first-in-class inhibitor tested in a large outcome trial, caused an unexpected blood pressure elevation and increased cardiovascular events. Whether the hypertensive effect resulted from CETP inhibition or an off-target action of torcetrapib has been debated. We hypothesized that common single-nucleotide polymorphisms in the CETP gene could help distinguish mechanism-based from off-target actions of CETP inhibitors to inform on the validity of CETP as a therapeutic target.
Methods and Results— We compared the effect of CETP single-nucleotide polymorphisms and torcetrapib treatment on lipid fractions, blood pressure, and electrolytes in up to 67 687 individuals from genetic studies and 17 911 from randomized trials. CETP single-nucleotide polymorphisms and torcetrapib treatment reduced CETP activity and had a directionally concordant effect on 8 lipid and lipoprotein traits (total, low-density lipoprotein, and HDL cholesterol; HDL2; HDL3; apolipoproteins A-I and B; and triglycerides), with the genetic effect on HDL cholesterol (0.13 mmol/L, 95% confidence interval [CI] 0.11 to 0.14 mmol/L) being consistent with that expected of a 10-mg dose of torcetrapib (0.13 mmol/L, 95% CI 0.10 to 0.15). In trials, 60 mg of torcetrapib elevated systolic and diastolic blood pressure by 4.47 mm Hg (95% CI 4.10 to 4.84 mm Hg) and 2.08 mm Hg (95% CI 1.84 to 2.31 mm Hg), respectively. However, the effect of CETP single-nucleotide polymorphisms on systolic blood pressure (0.16 mm Hg, 95% CI −0.28 to 0.60 mm Hg) and diastolic blood pressure (−0.04 mm Hg, 95% CI −0.36 to 0.28 mm Hg) was null and significantly different from that expected of 10 mg of torcetrapib.
Conclusions— Discordance in the effects of CETP single-nucleotide polymorphisms and torcetrapib treatment on blood pressure despite the concordant effects on lipids indicates the hypertensive action of torcetrapib is unlikely to be due to CETP inhibition or shared by chemically dissimilar CETP inhibitors. Genetic studies could find a place in drug-development programs as a new source of randomized evidence for drug-target validation in humans.
Received March 17, 2009; accepted October 20, 2009.
Higher concentrations of high-density lipoprotein (HDL) cholesterol are associated with a lower risk of coronary heart disease (CHD) independent of low-density lipoprotein (LDL) cholesterol.1 HDL particles have antiatherogenic actions in vitro, and experimental elevation of HDL cholesterol concentration in some animal models attenuates atheroma formation.2,3 Inhibitors of cholesteryl ester transfer protein (CETP), which mediates exchange of lipids between HDL particles and other lipoproteins, are a new class of drugs developed for their ability to raise HDL cholesterol. However, when the combination of a CETP inhibitor (torcetrapib) and a statin (atorvastatin) was compared with atorvastatin alone in the Investigation of Lipid Level Management to Understand Its Impact in Atherosclerotic Events (ILLUMINATE) trial,4 the Data Safety Monitoring Board terminated the trial prematurely because of an unexpectedly higher rate of both cardiovascular and noncardiovascular events in the torcetrapib-treated patients.
Clinical Perspective on p 62
Whether the higher rate of cardiovascular events from torcetrapib treatment was a mechanism-based effect of CETP inhibition, which would be shared by other members of the same drug class, or an idiosyncratic (or off-target) action of the torcetrapib molecule is uncertain. It is important to distinguish between the two, because at least 2 other CETP inhibitors, anacetrapib and dalcetrapib, are in advanced stages of drug development.5–7 Torcetrapib treatment has been associated with consistent and substantial elevations in blood pressure,4,8–10 perhaps secondary to a mineralocorticoid-like effect, which could have contributed to the increased risk of cardiovascular events.11 Although it has been proposed that the other CETP inhibitors do not share this blood pressure–elevating effect,5,12 this is based on evidence from nonrandomized animal experiments and short-term dose-ranging studies in humans, both of which have limitations. Large, randomized outcome trials of anacetrapib or dalcetrapib would provide a definitive answer but could expose the trial participants to a potential hazard should the hypertensive effect be mechanism based rather than off target. On the other hand, the failure to further evaluate other members of this class in randomized trials could lead to the abandonment of a potentially valuable preventive therapy.
An alternative way of obtaining randomized evidence on the efficacy and safety of CETP inhibition in humans without the recruitment of new trial participants, prospective follow-up, or exposure to a drug is to study the effect of carriage of common alleles of the human CETP gene associated with reduced CETP levels and activity.13 Genetic association studies are a type of natural randomized trial, because maternal and paternal alleles assort at random at conception.14,15 In effect, a study of alleles of the CETP gene that reduce CETP activity is akin to a very long-term randomized intervention trial of a “clean” CETP inhibitor, free from the off-target effects of individual drug molecules. We therefore compared the effect of torcetrapib and carriage of common CETP alleles on lipids and lipoproteins, blood pressure, and other markers of cardiovascular risk in a large-scale, international, collaborative analysis to ascertain whether the increase in blood pressure seen in the clinical trials of torcetrapib was mechanism based or off target.
Search Strategy and Selection Criteria
Randomized Controlled Trials
Randomized controlled trials evaluating the effect of torcetrapib on markers of cardiovascular risk or clinical outcomes were identified from PubMed and EMBASE up to the end of November 2007 with the use of the US National Library of Medicine’s Medical Subject Headings and the free-text terms “torcetrapib” or “CETP inhibitor” in combination with “randomized controlled trial.” For inclusion in the main analyses, studies had to be randomized, parallel-design studies in adults that examined the effect of treatment with torcetrapib (alone or in combination) with a suitable comparator. Studies were included if they had been published as full-length articles or letters in peer-reviewed journals in any language. Randomized studies were further subdivided into shorter dose-finding studies of <1 year’s duration and longer clinical trials of >1 year’s duration and analyzed separately.
PubMed and EMBASE were searched up to November 2007 for studies in humans evaluating any polymorphism in the CETP gene. The search included the Medical Subject Headings and free-text terms “cholesteryl ester transfer protein” or “CETP” in combination with “polymorphism*,” “mutation*,” “allele*,” “gene*,” “Taq1B,” “−629C>A,” or “I405V,” with no limits or restrictions. We supplemented information from published studies with unpublished genetic data obtained through a large collaborative network of investigators that allowed access to information on a wider range of traits of interest, enabled more precise estimation of genetic effect sizes, and minimized the scope for reporting and publication bias. (For further details, see the online-only Data Supplement.)
Generation of Tabular Data
Two of the authors (A.D.H. and R.S.) extracted data, and disagreements were resolved by discussion with a third author (J.P.C.). For randomized controlled trials, information was extracted on treatment regimen and comparator, as well as pretreatment and posttreatment measures of a wide range of cardiovascular risk markers (see the online-only Data Supplement for further details). The relationship between torcetrapib dose and effect on these variables, if available, was also recorded from dose-ranging studies. For genetic studies, study-level information was either extracted from published studies by 2 authors or requested from principal investigators (see the online-only Data Supplement).
Randomized Clinical Trials of Torcetrapib
The effect of torcetrapib on different lipid fractions, blood pressure, and other cardiovascular traits was assessed by calculation of the difference in the change in mean values between active and control arms. Study-specific estimates were weighted by the inverse of the variance and pooled by random-effects meta-analysis to generate summary estimates.
Primary analyses were based on the CETP gene variants commonly referred to as TaqI B (rs708272) and −629C>A (rs1800775), which were the most widely typed variants. The 2 are in linkage disequilibrium (r2 measure of association 0.73 in individuals of European descent16; online-only Data Supplement Figure I), which allows information on the 2 variants to be treated jointly in a pooled analysis. Additional analyses involved the I405V variant (rs5882). For continuous outcomes, the mean difference and 95% confidence interval (CI) by genotype category were obtained from each study and then pooled with a random-effects model to obtain a summary mean difference and 95% CI. Individuals homozygous for the common TaqIB (or −629C) allele served as the reference group throughout, and this group was designated B1B1, with heterozygous individuals and individuals homozygous for either rare allele designated B1B2 and B2B2, respectively, to preserve the convention introduced in prior studies. For binary outcomes, results were expressed as an odds ratio and 95% CI. To assess the robustness of the findings, stratified analyses were conducted according to study-level characteristics. In a subset of studies, predefined stratified analysis of individual-level data was performed to investigate the effect of CETP genotype on HDL cholesterol by quartiles of systolic, diastolic, and pulse pressure and by LDL cholesterol quartile to gain insight into the potential for effect modification by blood pressure–lowering or cholesterol-lowering medications. Deviation from Hardy-Weinberg equilibrium was assessed in each study. Heterogeneity was assessed with a χ2 test. The I2 measure17 and 95% CI were used to describe the extent of variability across studies. Additional information on the statistical analysis is provided in the online-only Data Supplement. All analyses were conducted with Stata 9.0 (StataCorp LP, College Station, Tex).
Consistency Between CETP Gene Effects and Equivalent Torcetrapib Dose
To determine the consistency of the observed effect of CETP genotype on cardiovascular traits with the expected effects for a comparable dose of torcetrapib, the shape of the dose–effect relationship for torcetrapib was evaluated from dose-ranging trials by use of the reported continuous outcomes HDL, HDL2, and HDL3, as well as apolipoprotein A-I (apoA-I) and apolipoprotein B (apoB). Despite careful searching, no quantitative information on the relationship between torcetrapib dose and blood pressure from these trials was available in a form that could be used in the analysis. Having confirmed a linear dose–response relationship for the available variables (Figures 1A through 1C), we used the summary effect of a 60-mg dose of torcetrapib on HDL cholesterol (the measure with the most data) from the meta-analysis of randomized trials and the summary effect of CETP genotype on HDL cholesterol from the meta-analysis of genetic studies (1) to express the effect of carriage of the B2 variant as a torcetrapib dose equivalent and (2) to estimate the effect of this dose of torcetrapib on blood pressure and other traits. A simulation model that incorporated the variance in the effect estimates of the genotype and drug effects was used to obtain the CIs (see online-only Data Supplement for details). The observed gene effect was compared with the effect of a comparable dose of torcetrapib by means of a z test.18 More details are provided in the online-only Data Supplement.
Randomized Controlled Trials of Torcetrapib
Dose–Response Relationship of Torcetrapib on HDL
Three studies (median size 40 participants, range 19 to 162 participants) with a mean study duration of 5.3 (standard deviation 3.1) weeks enabled the exploration of the effect of different doses of torcetrapib on HDL cholesterol and its subfractions (HDL2 and HDL3).19–21 Over the dose range studied (10 to 240 mg daily), torcetrapib produced a linear, dose-dependent increase in HDL cholesterol (P<0.001 from meta-regression), HDL2 (P=0.03), and HDL3 (P=0.003), with no evidence of a threshold effect (Figures 1A through 1C).
Effect of Torcetrapib on Lipid Profile, Blood Pressure, and Biomarkers
Four randomized trials (range 752 to 15 067 participants) with a mean duration of 21 (standard deviation 6) months that involved 17 911 participants in aggregate with a mean age of 55.4 (standard deviation 6.9) years evaluated the effect of torcetrapib 60 mg daily (in combination with atorvastatin) versus atorvastatin alone and were included in the main analysis.4,8–10 Torcetrapib 60 mg daily increased HDL cholesterol by 0.78 mmol/L (95% CI 0.68 to 0.87 mmol/L), apoA-I by 0.30 g/L (95% CI 0.30 to 0.31 g/L), and total cholesterol by 0.18 mmol/L (95% CI 0.10 to 0.25 mmol/L). The same dose reduced LDL cholesterol by 0.54 mmol/L (95% CI −0.64 to −0.43 mmol/L), triglycerides by 0.12 mmol/L (95% CI −0.18 to −0.07 mmol/L), and apoB by 0.11 g/L (95% CI −0.11 to −0.10 g/L; Table 1; Figure IIa in the online-only Data Supplement). A pooled analysis of all 17 911 participants from the 4 trials indicated that torcetrapib 60 mg daily led to a mean increase in systolic blood pressure of 4.47 mm Hg (95% CI 4.10 to 4.84 mm Hg) and an increase in diastolic blood pressure of 2.08 mm Hg (95% CI 1.84 to 2.31 mm Hg). In the ILLUMINATE trial, the elevation in blood pressure was accompanied by a decrease in plasma potassium, an increase in sodium, and an increase in aldosterone concentration4 (Table 2). In 3 trials4,9,10 that included 17 159 participants, there was no effect of torcetrapib on C-reactive protein concentration (online-only Data Supplement Table I).
Study Details and CETP Polymorphisms Evaluated
A total of 31 studies (online-only Data Supplement references S1 to S39) and 67 687 individuals a mean of 55.8 (standard deviation 9.6) years old contributed information on at least 1 continuous outcome. Twenty-three studies with 60 316 individuals provided previously unpublished data. Of the unpublished studies, 21 studies (50 908 individuals) provided data on the rs708272 (Taq1B) polymorphism, and 2 studies (8535 participants) provided data only on the rs1800775 (−629C>A) polymorphism. Where studies provided data on both −629C>A and Taq1B, the latter was used for the primary analysis. Seven studies (21 353 individuals) also provided data on the rs5882 (I405V) polymorphism (online-only Data Supplement references S8, S10, S15, S16, S18–S20, S22, S25, S32, and S33), and these results are provided in the online-only Data Supplement. Study details are provided in online-only Data Supplement Tables II and III, respectively.
Effect of CETP Genotypes on CETP Concentration, CETP Activity, and Lipids
Six studies in individuals of European ancestry (5340 participants) provided information on the effect of CETP genotype on CETP concentration (online-only Data Supplement references S7, S8, S15, S28, S30, and S31), and 2 studies (858 participants; online-only Data Supplement references S15 and S18–S20) provided information on the effect on CETP activity. A further 5 studies (1867 participants) contributed data from individuals of Japanese origin (online-only Data Supplement Figure III and references S34 through S39). A graded effect of genotype on CETP concentration and activity was evident in both populations. People of European ancestry who were homozygous for the B2 allele had lower CETP concentrations (−0.47 μg/mL, 95% CI −0.67 to −0.26 μg/mL) and lower CETP activity (−17.00 nmol · mL−1 · h−1, 95% CI −18.52 to −15.49 nmol · mL−1 · h−1) than people homozygous for the B1 allele (online-only Data Supplement Figures IIIa and IIIb). In 31 studies with 67 687 participants, B2-homozygous individuals had higher concentrations of HDL cholesterol (0.13 mmol/L, 95% CI 0.11 to 0.14 mmol/L; Figure 2). The link between genotype and HDL cholesterol was consistent in analyses stratified by study size, sex, presence of CHD, and ancestry and across quartiles of LDL cholesterol, systolic and diastolic blood pressure, and pulse pressure (online-only Data Supplement Figures IIIc and IV). In addition, B2-homozygous individuals exhibited higher concentrations of total cholesterol (0.05 mmol/L, 95% CI 0.03 to 0.07 mmol/L) and apoA-I (0.06 g/L, 95% CI 0.05 to 0.08 g/L) and lower concentrations of LDL cholesterol (−0.03 mmol/L, 95% CI −0.05 to −0.01 mmol/L), triglycerides (−0.06 mmol/L, 95% CI −0.10 to −0.02 mmol/L), and apoB (0.02 g/L, 95% CI −0.03 to −0.01 g/L). In 2 studies, individuals homozygous for the B2 allele had higher circulating concentrations of both the larger HDL2 particles (0.03 mmol/L, 95% CI 0.01 to 0.04 mmol/L) and smaller HDL3 particles (0.06 mmol/L, 95% CI 0.02 to 0.11 mmol/L; Table 1). Heterozygous subjects exhibited lipid and lipoprotein concentrations approximately intermediate between those found in homozygous subjects, consistent with an additive effect of each copy of the variant allele (Table 1; per-allele data available on request). The effect of variant CETP alleles on lipid and lipoprotein profile thus reproduced the direction of effect of treatment with torcetrapib in clinical trials for 8 separate lipid and lipoprotein traits (Table 1; Figure 3A; online-only Data Supplement Figures 2a and 2b). Using a simulation model and assuming a linear dose–response relationship (Figure 1), we estimated that the effect on HDL in B2-homozygous individuals corresponded to a dose of torcetrapib of 9.7 mg (95% CI 8.18 to 11.41 mg), and for heterozygous individuals, it corresponded to a dose of 4.5 mg (95% CI 3.71 to 5.38 mg), ie, to a torcetrapib dose of approximately 10 and 5 mg, respectively (Figure 3B).
Effect of CETP Genotypes on Blood Pressure and Electrolytes
Twenty-two studies (58 948 individuals) provided information on CETP genotypes and systolic and diastolic blood pressure, including previously unpublished information from 20 studies (54 936 individuals). CETP genotype had no effect on systolic and diastolic blood pressure; the mean differences in comparisons between homozygous subjects were 0.16 mm Hg (95% CI −0.28 to 0.60 mm Hg) and −0.04 mm Hg (95% CI −0.36 to 0.28 mm Hg) for systolic and diastolic blood pressure, respectively. Mean differences in systolic and diastolic blood pressure between heterozygous individuals (B1B2) and those homozygous for the B1 allele were −0.27 mm Hg (95% CI −0.64 to 0.10 mm Hg) and −0.23 mm Hg (95% CI −0.43 to −0.04 mm Hg), respectively (Figure 4A). The null findings were again consistent in analyses stratified by study size, sex, presence of preexisting CHD, ancestral origin, and allele types (Figures 4A and 4B; online-only Data Supplement Figures Va and Vb). The expected effect on blood pressure of a 10-mg daily dose of torcetrapib was estimated to be 0.72 mm Hg (95% CI 0.60 to 0.87 mm Hg) and 0.33 mm Hg (95% CI 0.27 to 0.41 mm Hg) for systolic and diastolic blood pressure, respectively, assuming a linear relationship between torcetrapib dose and blood pressure, and this was significantly different from the observed genetic effect on blood pressure (Figures 5A and 5B). Unlike torcetrapib treatment, CETP genotype was not associated with serum sodium, potassium, or creatinine concentration or with urinary sodium or potassium concentration (Table 2; Figures 5C and 5D). Individuals with variant CETP alleles were also no more likely to receive antihypertensive medications (odds ratio 0.98, 95% CI 0.80 to 1.21; online-only Data Supplement Table I).
Effect of CETP Genotypes on Variables Unrelated to CETP Inhibition
There was no link between CETP genotypes and variables unrelated to CETP function, including age, body mass index, or smoking habit (online-only Data Supplement Table I). There was also no consistent association with blood glucose or with C-reactive protein concentration, consistent with data from clinical trials of torcetrapib (online-only Data Supplement Table I).
Main Findings and Interpretation
We found concordance in the effect of common variants in the CETP gene and pharmacological inhibition of CETP by torcetrapib on 8 continuous lipid and lipoprotein markers evaluated in both randomized trials and genetic studies (HDL cholesterol, HDL2, HDL3, LDL cholesterol, triglycerides, total cholesterol, apoA-I, and apoB). The only continuous traits for which the effect of genotype and drug were consistently discordant were systolic and diastolic blood pressure and the electrolytes sodium and potassium. This large-scale randomized evidence in humans supports the interpretation that the blood pressure–elevating effect of torcetrapib (and the connected effect on electrolytes) is mechanistically unrelated to CETP inhibition. The findings have important implications, specifically for the development of other CETP inhibitors and more generally for the potential use of genetic variants to inform drug development.
Other Sources of Evidence on the Same Question
Our interpretation that the hypertensive effect of torcetrapib is off target receives additional support from other lines of evidence. First, treatment with the CETP inhibitors anacetrapib and dalcetrapib has not been associated with blood pressure elevation, although the studies thus far have been relatively small in size and of short duration.5,7 Second, torcetrapib (but not anacetrapib) has been reported to cause a blood pressure increase in several animal models,12 including species that do not express CETP. Third, a recent study22 indicated that torcetrapib treatment elevates aldosterone concentration, with corresponding effects on sodium and potassium concentration, and these electrolyte changes were not observed in a short-term dose-ranging study of anacetrapib.7 These findings, from the separate lines of investigation, each with differing limitations and sources of error, provide reassurance that the hypertensive effect of torcetrapib is off target and therefore unlikely to be shared by other CETP inhibitors.
CETP Inhibition and Prevention of CHD
The higher blood pressure among individuals in the torcetrapib arm of the ILLUMINATE trial might explain the higher rate of cardiovascular events, but there may also be other explanations. CETP inhibition might interfere with reverse cholesterol transport and generate an HDL particle of abnormal size and function,23 a mechanism-based adverse effect. Prior small mechanistic studies have suggested torcetrapib treatment increased the concentration of both large HDL2 and small HDL3 particles but that the effect on HDL2 was proportionately greater. However, this differential effect was only seen at a dose of torcetrapib 4 times as large as the dose used in the large-scale clinical trials.19 Genetic data on the effect of CETP genotype on HDL subtype were limited, but in the present analysis, there was no clear evidence of a differential effect of CETP genotype on HDL subclasses. Although we have focused here on the effect of CETP genotypes on lipids, lipoproteins, and blood pressure to make direct comparison of the effect of pharmacological CETP inhibition and carriage of CETP alleles, a recent meta-analysis of studies that included 27 196 coronary cases and 55 338 controls and a genome-wide analysis from the Women’s Genome Health Study both provided support for the CETP variants studied here being protective against CHD events.24,25 Although this protective effect has not been consistent across all studies,26 there has been no consistent signal for an increase in CHD risk from carriage of these alleles.
Although the findings are robust, our interpretation requires consideration in light of certain theoretical and practical limitations of the genetic approach we have used. CETP alleles are of much smaller effect than the most widely studied dose of torcetrapib, so it might be argued that the failure to detect an association between genotype and a continuous marker such as blood pressure could have arisen because of inadequate power, or perhaps the effect on blood pressure requires a suprathreshold degree of CETP inhibition. We attempted to maximize power and minimize the potential for a type II error by establishing a large genetic collaboration that included a substantial amount of previously unpublished information. Blood pressure was an outcome that had been widely recorded in the studies included in the present analysis (22 studies and 59 948 individuals) but was not widely reported, and so the findings should not be prone to bias. Although the investigation of the effect of CETP polymorphism on blood pressure was not the primary aim of any of the studies included here, blood pressure measurement was performed with validated devices and widely accepted methods. The study was also sufficiently powered to detect a blood pressure signal of the size expected of a 5- to 10-mg dose of torcetrapib (see the online-only Data Supplement). Indeed, 3 of these studies (14 109 individuals) contributed to the recent whole-genome analysis of blood pressure loci that identified single-nucleotide polymorphisms (SNPs) that altered blood pressure by ≈1 mm Hg/0.5 mm Hg, close to the effect size being sought in the present analysis.27,28 With the available sample size, we also detected an effect of CETP genotype on triglycerides that was similar in size to that which would have been expected for blood pressure were this effect mechanism based (online-only Data Supplement Figure IIa). We also triangulated the findings from randomized controlled trials with the genetic data (ie, we compared the expected effect of a 5- and 10-mg dose of torcetrapib with the observed genetic effect) rather than focusing solely on statistical tests in the genetic associations. Taken together, these analyses suggest that the null findings in relation to blood pressure are neither biased nor explained by inadequate sample size. Although we were unable to exclude a hypothetical nonlinear (threshold) relationship between CETP inhibition by torcetrapib and blood pressure because none of the dose-ranging studies of torcetrapib reported quantitative data on the dose–response effect in a form that could be extracted for analysis, the effect of torcetrapib on all lipid and lipoprotein traits evaluated was linear over the dose range studied. We therefore made the assumption that this was also true for blood pressure.
The randomized allocation of alleles in genetic studies differs from the randomized drug intervention in a clinical trial in that assignment of genotype occurs at conception and produces an effect across a lifetime, rather than in mid to late adulthood, when most randomized controlled trials are conducted. It is conceivable, therefore, that an adverse effect of a common genetic variant on blood pressure from early life may have led to developmental compensation by other systems.15 If this were the case, a null association of CETP genotype with blood pressure seen in genetic studies might lead to unreliable inference on the likely effect of modification of CETP activity by a drug. However, there was no evidence that such developmental compensation was operating in the case of any of the 8 lipid traits we studied, for which both the lifelong effect of the genetic exposure and the shorter-term effect of the drug were consistent.
Although the precise functional alleles at the CETP locus have yet to be identified with certainty, the −629C>A (rs1800775) and I405V (rs5885) alleles are either likely to be functional themselves or to be in sufficiently strong linkage disequilibrium with functional variant(s) so as to be valid tools for this type of analysis. The −629C>A variant has been shown to alter binding of Sp transcription factors.29 The Taq1B allele (rs708272) is intronic and less likely to be functional itself, but it is in strong linkage disequilibrium with several promoter polymorphisms (including the −629C>A variant), and as the present analyses show, it exhibits very strong association with multiple lipid traits. It is important to be clear, however, that for the analyses we have conducted, it is not necessary for functional alleles to have been delineated precisely provided that an effect of the alleles studied on the traits of interest can be demonstrated robustly.30 Although there are likely to be other variants in and around the CETP gene that are also associated with CETP activity and lipids, some because they are causal and some because they are simply associated with causal SNPs by linkage disequilibrium, the use of a single SNP in this region does not compromise the analysis, provided it can be demonstrated that it provides a reliable index of CETP activity and differences in the lipid traits of interest (which we have demonstrated), and on the assumption that the SNP is in linkage disequilibrium with a causal SNP rather than causal itself, that the main analyses are grouped according to subjects of similar ancestry to ensure that the linkage disequilibrium relationships are consistent across studies. Moreover, SNPs at the CETP locus, including rs1800775 (−629C>A) and rs708272 (Taq1B) studied here, have emerged as among the strongest associated signals with HDL cholesterol in recent genome-wide association studies25,31–33 (online-only Data Supplement Figure I).
Wider Implications of This Work
We used the principle that allelic variants in a gene encoding a specific drug target can be used to model the mechanism-based effect of modifying the same target pharmacologically. In the present analysis, this was applied to help distinguish the mechanism-based from off-target actions of a drug molecule in advanced development. However, further research should now address whether this principle could be exploited at earlier phases in the drug-development pathway to help, for example, with the validation of a promising new target or to assemble a panel of biomarkers of efficacy to test in clinical trials. The directional concordance of the effect of HMGCR SNPs in genetic studies and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (statin) treatment on LDL cholesterol and CHD risk in clinical trials lends additional support to the potential utility of this approach. There is likely to be wide availability of genetic tools for this purpose, because the majority of drug targets are proteins, and regulatory genetic variants acting in cis, located within 100 kb of genes, appear to be a common feature of the human genome.34
In summary, a novel large-scale genetic approach has provided evidence that the hypertensive effect of torcetrapib is likely an off-target action. This provides reassurance that this particular adverse effect of torcetrapib is unlikely to be shared by other chemically dissimilar CETP inhibitors, but further drug development will be required to assess whether these other agents and the CETP inhibitor class of drugs in general are likely to be efficacious in the prevention of CHD events with an acceptable risk–benefit profile. Further research should investigate whether genetic studies could find use in drug-development programs as a new source of randomized evidence for drug-target validation in humans.
Sources of Funding
Dr Sofat is supported by a British Heart Foundation (Schillingford) clinical training fellowship (FS/07/011). Dr Hingorani holds a British Heart Foundation senior fellowship (FS 05/125). Dr Smeeth holds a Wellcome Trust senior research fellowship. Dr Humphries holds a British Heart Foundation Chair in Cardiovascular Genetics; he and Dr Talmud are supported by British Heart Foundation PG04/110/17827. Dr Marmot is supported by a Medical Research Council research professorship. Dr Lawlor is funded in part by a United Kingdom Department of Health career scientist award. Dr Deanfield holds the British Heart Foundation Vandervell Chair in Cardiology of the Young. The Whitehall II study has been supported by grants from the Medical Research Council; Economic and Social Research Council; British Heart Foundation; Health and Safety Executive; Department of Health; US National Heart, Lung, and Blood Institute (HL36310), National Institutes of Health: US National Institute on Aging (AG13196), National Institutes of Health; US Agency for Health Care Policy and Research (HS06516); and the John D and Catherine T MacArthur Foundation Research Networks on Successful Midlife Development and Socio-economic Status and Health. Samples from the English Longitudinal Study of Ageing DNA Repository received support under a grant (AG1764406S1) awarded by the National Institute on Aging. The English Longitudinal Study of Ageing was developed by a team of researchers based at the National Centre for Social Research, University College London and the Institute of Fiscal Studies. The data were collected by the National Centre for Social Research. The developers and funders of the English Longitudinal Study of Ageing and the Archive do not bear any responsibility for the analyses or interpretations presented here. EPIC-Norfolk is supported by the Medical Research Council and Cancer Research UK. The British Women’s Heart and Health Study is funded by the Department of Health Policy Research Programme and the British Heart Foundation. The UK Medical Research Council provide support for the MRC Centre where Drs Lawlor and Smith work. The views expressed in the publication are those of the authors and not necessarily those of any of the funding bodies.
Dr Hingorani is a member of the editorial board of the Drug and Therapeutics Bulletin, has provided nonremunerated advice to GlaxoSmithKline and London Genetics, and has received honoraria for speaking at educational meetings on cardiovascular risk that have been donated in whole or in part to charity. Dr Arca was on the Pfizer advisory board for torcetrapib.
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The inverse relationship between high-density lipoprotein cholesterol and risk of coronary heart disease suggests that therapeutic elevation of high-density lipoprotein cholesterol may provide an effective means of prevention of coronary heart disease. Pharmacological inhibition of cholesteryl ester transfer protein (CETP) leads to elevation in high-density lipoprotein cholesterol, but torcetrapib (the first-in-class CETP inhibitor) increased the risk of cardiovascular events in the ILLUMINATE trial (Investigation of Lipid Level Management to Understand Its Impact in Atherosclerotic Events), which may have resulted from an unexpected blood pressure–elevating effect of this agent. We used common genetic polymorphisms in the CETP gene to distinguish whether the hypertensive action of torcetrapib was mechanism based or off target, because a genetic study of these variants can be considered to be a type of natural randomized trial of a “clean” low-dose CETP inhibitor with no off-target actions. Common CETP gene polymorphisms and torcetrapib treatment had concordant effects on 8 lipid and lipoprotein markers, including high-density lipoprotein cholesterol, but CETP gene variants had no effect on blood pressure. The blood pressure–elevating effect of torcetrapib appears to be an off-target action that is unlikely to be shared by chemically dissimilar CETP inhibitors. Genetic studies could be used in drug-development programs as a new source of randomized evidence for drug-target validation in humans.
↵*Drs Sofat and Hingorani contributed equally to this article.
Guest Editor for this article was Mary Cushman, MD, MSc.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.865444/DC1.