Biochemical characterization of cholesteryl ester transfer protein inhibitors

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.     

Modulating cholesteryl ester transfer protein activity maintains efficient pre-��-HDL formation and increases reverse cholesterol transport

The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-β-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-β-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [³H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [³H]neutral sterols and [³H]bile acids, whereas all compounds increased plasma HDL-[³H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-β-HDL formation, which may be required to increase reverse cholesterol transport.     

Cholesteryl ester transfer protein inhibition, high-density lipoprotein metabolism and heart disease risk reduction

PURPOSE OF REVIEW: Cholesteryl ester transfer protein (CETP) inhibitors (JTT-705 and torcetrapib) are currently in clinical testing, and significantly raise high-density lipoprotein (HDL) cholesterol levels. Low HDL cholesterol is a significant independent predictor of coronary heart disease (CHD) and HDL raising has been associated with coronary heart disease risk reduction, but there is debate about whether CETP inhibition will reduce coronary heart disease risk. RECENT FINDINGS: It has been documented in transgenic mouse models that apolipoprotein (apo) C-I inhibits CETP, and that high mono-unsaturated fat diets prevent the normal stimulation of CETP activity by dietary cholesterol. In rabbits, torcetrapib markedly decreases clearance of HDL cholesteryl ester via an indirect pathway, but has no effect on total plasma cholesteryl ester clearance. In humans, torcetrapib raises HDL apoA-I by modestly decreasing its fractional catabolic rate, while having a very profound effect on raising HDL cholesterol and large alpha-1 migrating HDL particles by more than 50%, with no effect on fecal cholesterol excretion. When JTT-705 at 600 mg/day was given to hypercholesterolemic patients already on pravastatin 40 mg/day, the combination was well tolerated and increases in HDL cholesterol of 28% were noted. SUMMARY: In our view, CETP inhibitors in combination with statins will be profoundly beneficial in reducing human atherosclerosis, primarily because they normalize HDL particles and prevent the transfer of cholesteryl ester from HDL to atherogenic lipoproteins.     

Evacetrapib is a novel, potent, and selective inhibitor of cholesteryl ester transfer protein that elevates HDL cholesterol without inducing aldosterone or increasing blood pressure

Cholesteryl ester transfer protein (CETP) catalyses the exchange of cholesteryl ester and triglyceride between HDL and apoB containing lipoprotein particles. The role of CETP in modulating plasma HDL cholesterol levels in humans is well established and there have been significant efforts to develop CETP inhibitors to increase HDL cholesterol for the treatment of coronary artery disease. These efforts, however, have been hampered by the fact that most CETP inhibitors either have low potency or have undesirable side effects. In this study, we describe a novel benzazepine compound evacetrapib (LY2484595), which is a potent and selective inhibitor of CETP both in vitro and in vivo. Evacetrapib inhibited human recombinant CETP protein (5.5 nM IC(50)) and CETP activity in human plasma (36 nM IC(50)) in vitro. In double transgenic mice expressing human CETP and apoAI, evacetrapib exhibited an ex vivo CETP inhibition ED(50) of less than 5 mg/kg at 8 h post oral dose and significantly elevated HDL cholesterol. Importantly, no blood pressure elevation was observed in rats dosed with evacetrapib at high exposure multiples compared with the positive control, torcetrapib. In addition, in a human adrenal cortical carcinoma cell line (H295R cells), evacetrapib did not induce aldosterone or cortisol biosynthesis whereas torcetrapib dramatically induced aldosterone and cortisol biosynthesis. Our data indicate that evacetrapib is a potent and selective CETP inhibitor without torcetrapib-like off-target liabilities. Evacetrapib is currently in phase II clinical development.     

Description of the torcetrapib series of cholesteryl ester transfer protein inhibitors, including mechanism of action

We have identified a series of potent cholesteryl ester transfer protein (CETP) inhibitors, one member of which, torcetrapib, is undergoing phase 3 clinical trials. In this report, we demonstrate that these inhibitors bind specifically to CETP with 1:1 stoichiometry and block both neutral lipid and phospholipid (PL) transfer activities. CETP preincubated with inhibitor subsequently bound both cholesteryl ester and PL normally; however, binding of triglyceride (TG) appeared partially reduced. Inhibition by torcetrapib could be reversed by titration with both native and synthetic lipid substrates, especially TG-rich substrates, and occurred to an equal extent after long or short preincubations. The reversal of TG transfer inhibition using substrates containing TG as the only neutral lipid was noncompetitive, suggesting that the effect on TG binding was indirect. Analysis of the CETP distribution in plasma demonstrated increased binding to HDL in the presence of inhibitor. Furthermore, the degree to which plasma CETP shifted from a free to an HDL-bound state was tightly correlated to the percentage inhibition of CE transfer activity. The finding by surface plasmon resonance that torcetrapib increases the affinity of CETP for HDL by approximately 5-fold likely represents a shift to a binding state that is nonpermissive for lipid transfer. In summary, these data are consistent with a mechanism whereby this series of inhibitors block all of the major lipid transfer functions of plasma CETP by inducing a nonproductive complex between the transfer protein and HDL.     

Crystal Structures of Cholesteryl Ester Transfer Protein in Complex with Inhibitors

Human plasma cholesteryl ester transfer protein (CETP) transports cholesteryl ester from the antiatherogenic high-density lipoproteins (HDL) to the proatherogenic low-density and very low-density lipoproteins (LDL and VLDL). Inhibition of CETP has been shown to raise human plasma HDL cholesterol (HDL-C) levels and is potentially a novel approach for the prevention of cardiovascular diseases. Here, we report the crystal structures of CETP in complex with torcetrapib, a CETP inhibitor that has been tested in phase 3 clinical trials, and compound 2, an analog from a structurally distinct inhibitor series. In both crystal structures, the inhibitors are buried deeply within the protein, shifting the bound cholesteryl ester in the N-terminal pocket of the long hydrophobic tunnel and displacing the phospholipid from that pocket. The lipids in the C-terminal pocket of the hydrophobic tunnel remain unchanged. The inhibitors are positioned near the narrowing neck of the hydrophobic tunnel of CETP and thus block the connection between the N- and C-terminal pockets. These structures illuminate the unusual inhibition mechanism of these compounds and support the tunnel mechanism for neutral lipid transfer by CETP. These highly lipophilic inhibitors bind mainly through extensive hydrophobic interactions with the protein and the shifted cholesteryl ester molecule. However, polar residues, such as Ser-230 and His-232, are also found in the inhibitor binding site. An enhanced understanding of the inhibitor binding site may provide opportunities to design novel CETP inhibitors possessing more drug-like physical properties, distinct modes of action, or alternative pharmacological profiles.     

Cholesteryl-ester transfer protein enhances the ability of high-density lipoprotein to inhibit low-density lipoprotein oxidation

Therapeutic strategies to increase high-density lipoprotein (HDL) to treat or prevent vascular disease include the use of cholesteryl-ester transfer protein (CETP) inhibitors. Here, we show, to the best of our knowledge for the first time, that addition of CETP to HDL enhances the ability of HDL to inhibit low-density lipoprotein oxidation by ～ 30% for total HDL and HDL(2) (both P < 0.05) and 75% for HDL(3) (P < 0.01). Therefore, CETP inhibition may be detrimental to the antiatherosclerotic properties of HDL, and these findings may partly explain the failure of the CETP inhibitor, torcetrapib, treatment to retard vascular disease despite large increases in HDL, in addition to its "off target" toxicity, a property which appears not to be shared by other members of this class of CETP inhibitor currently under clinical trial. Further, detailed studies are urgently required.     

Role of CETP inhibitors in the treatment of dyslipidemia

PURPOSE OF REVIEW: This review summarizes novel human data on cholesteryl ester-transfer protein (CETP) and atherosclerosis and the possible use of CETP inhibitors in the treatment of dyslipidemia. In addition, it will underline that therapeutic targeting of the high-density lipoprotein (HDL) metabolism entails more than simply observing changes in cholesterol levels of this lipoprotein. RECENT FINDINGS: Two pharmacological small-molecule inhibitors of CETP, JTT-705 and torcetrapib, have recently been shown to effectively raise HDL cholesterol in humans without serious side effects when either used as a monotherapy or combined with statins that lower low-density lipoprotein cholesterol. Importantly, prospective data from the Epic-Norfolk study furthermore indicate that elevated CETP concentration in conjunction with elevated triglyceride levels are associated with increased odds for cardiovascular events. Data from the Diabetic Atherosclerosis Intervention Study furthermore show that elevated CETP concentration is associated with increased progression of coronary atherosclerosis in patients with type 2 diabetes who use fenofibrate. SUMMARY: Long-term studies will have to show whether CETP inhibition decreases the risk of atherosclerotic disease in dyslipidemic patients. Increased CETP activity might be detrimental under hypertriglyceridemic conditions which is of importance when considering that a large proportion of patients at increased risk from coronary artery disease exhibit elevated triglyceride levels. Studies into the effects of CETP inhibition in hypertriglyceridemic patients therefore seem warranted. Awaiting the first data on the effect of CETP inhibition on surrogate endpoints for atherosclerosis, this review furthermore outlines that the complexity of HDL metabolism will necessitate a wide variety of studies on many aspects of this intriguing lipoprotein.     

Inhibition of CETP activity by torcetrapib reduces susceptibility to diet-induced atherosclerosis in New Zealand White rabbits

Cholesteryl ester transfer protein (CETP) inhibitors increase high density lipoprotein-cholesterol (HDL-C) in animals and humans, but whether CETP inhibition will be antiatherogenic is still uncertain. We tested the CETP inhibitor torcetrapib in rabbits fed an atherogenic diet at a dose sufficient to increase HDL-C by at least 3-fold (207 +/- 32 vs. 57 +/- 6 mg/dl in controls at 16 weeks). CETP activity was inhibited by 70-80% throughout the study. Non-HDL-C increased in both groups, but there was no difference apparent by the study's end. At 16 weeks, aortic atherosclerosis was 60% lower in torcetrapib-treated animals (16.4 +/- 3.4% vs. 39.8 +/- 5.4% in controls) and aortic cholesterol content was reduced proportionally. Sera from a separate group of rabbits administered torcetrapib effluxed 48% more cholesterol from Fu5AH cells than did sera from control animals, possibly explaining the reduced aortic cholesterol content. Regression analyses indicated that lesion area in the torcetrapib-treated group was strongly correlated with the ratio of total plasma cholesterol to HDL-C but not with changes in other lipid or lipoprotein levels. CETP inhibition with torcetrapib retards atherosclerosis in rabbits, and the reduced lesion area is associated with increased levels of HDL-C.     

HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis

High-density lipoprotein (HDL) has been identified as a potential target in the treatment of atherosclerotic vascular disease. The failure of torcetrapib, an inhibitor of cholesteryl ester transfer protein (CETP) that markedly increased HDL levels in a clinical trial, has called into doubt the efficacy of HDL elevation. Recent analysis suggests that failure may have been caused by off-target toxicity and that HDL is functional and promotes regression of atherosclerosis. New studies highlight the central importance of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 in reducing macrophage foam cell formation, inflammation, and atherosclerosis. A variety of approaches to increasing HDL may eventually be successful in treating atherosclerosis.     

Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 μl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-β-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique.     

Apolipoprotein CI is a physiological regulator of cholesteryl ester transfer protein activity in human plasma but not in rabbit plasma

Plasma cholesteryl ester transfer protein (CETP) activity is high in rabbits, intermediate in humans, and nondetectable in rodents. Human apolipoprotein CI (apoCI) was found to be a potent inhibitor of CETP. The aim of this study was to compare the ability of rabbit and human apoCI to modulate the interaction of CETP with HDLs and to evaluate to which extent apoCI contributes to plasma cholesteryl ester transfer rate in normolipidemic humans and rabbits. Rabbit apoCI gene was cloned and sequenced, rabbit and human apoCI were purified to homogeneity, and their ability to modify the surface charge properties and the CETP inhibitory potential of HDL were compared. It is demonstrated that unlike human apoCI, rabbit apoCI does not modulate cholesteryl ester transfer rate in total plasma. Whereas both human and rabbit apoCI readily associate with HDL, only human apoCI was found to modify the electrostatic charge of HDL. In humans, both CETP and apoCI at normal, physiological levels contribute significantly to the plasma cholesteryl ester transfer rate. In contrast, CETP is the sole major determinant of cholesteryl ester transfer in normolipidemic rabbit plasma as a result of the inability of rabbit apoCI to change HDL electronegativity.     

ApoA-II expression in CETP transgenic mice increases VLDL production and impairs VLDL clearance

Apolipoprotein (apo)A-II is a major high density lipoprotein (HDL) protein; however, its role in lipoprotein metabolism is largely unknown. Transgenic (Tg) mice that overexpress human apoA-II present functional lecithin: cholesterol acyltransferase deficiency, HDL deficiency, hypertriglyceridemia and, when fed an atherogenic diet, increased non-HDL cholesterol and increased susceptibility to atherosclerosis. In contrast to humans, mice do not present cholesteryl ester transfer protein (CETP) activity in plasma. To study the in vivo interaction of these two proteins, we crossbred human apoA-II and CETP-Tg mice. CETP x apoA-II-Tg mice fed an atherogenic diet, compared with CETP-Tg mice presented a 2-fold decrease in HDL cholesterol and a quantitatively similar increase in total plasma cholesterol and percentage of free cholesterol, non-HDL cholesterol, and free fatty acids, together with a remarkable 112-fold increase in plasma triglycerides. Plasma triglycerides in CETP x apoA-II-Tg mice were mainly associated with very low density lipoproteins (VLDL), which were also enriched in protein content, and resulted from a combination of higher production rate compared with both of their progenitors and non-Tg control mice, and decreased catabolism compared only with CETP-Tg mice. These results show CETP x apoA-II-Tg mice to be a good model with which to study mechanisms leading to VLDL overproduction and suggest that CETP and, in particular apoA-II, may play a role in the regulation of VLDL metabolism.     

Human apolipoprotein C-I accounts for the ability of plasma high density lipoproteins to inhibit the cholesteryl ester transfer protein activity

The aim of the present study was to identify the protein that accounts for the cholesteryl ester transfer protein (CETP)-inhibitory activity that is specifically associated with human plasma high density lipoproteins (HDL). To this end, human HDL apolipoproteins were fractionated by preparative polyacrylamide gradient gel electrophoresis, and 30 distinct protein fractions with molecular masses ranging from 80 down to 2 kDa were tested for their ability to inhibit CETP activity. One single apolipoprotein fraction was able to completely inhibit CETP activity. The N-terminal sequence of the 6-kDa protein inhibitor matched the N-terminal sequence of human apoC-I, the inhibition was completely blocked by specific anti-apolipoprotein C-I antibodies, and mass spectrometry analysis confirmed the identity of the isolated inhibitor with full-length human apoC-I. Pure apoC-I was able to abolish CETP activity in a concentration-dependent manner and with a high efficiency (IC(50) = 100 nmol/liter). The inhibitory potency of total delipidated HDL apolipoproteins completely disappeared after a treatment with anti-apolipoprotein C-I antibodies, and the apoC-I deprivation of native plasma HDL by immunoaffinity chromatography produced a mean 43% rise in cholesteryl ester transfer rates. The main localization of apoC-I in HDL and not in low density lipoprotein in normolipidemic plasma provides further support for the specific property of HDL in inhibiting CETP activity.     

Evidence for a role of CETP in HDL remodeling and cholesterol efflux: Role of cysteine 13 of CETP

Cholesteryl ester transfer protein (CETP), a key regulator of high-density lipoprotein (HDL) metabolism, induces HDL remodeling by transferring lipids between apolipoprotein B-containing lipoproteins and HDL, and/or by promoting lipid transfer between HDL subparticles. In this study, we investigated the mechanism as to how CETP induces the generation of lipid-poor particles (pre-β-HDL) from HDL, which increases ATP-binding cassette transporter 1-mediated cholesterol efflux. This CETP-dependent HDL remodeling is enhanced by the CETP modulator dalcetrapib both in plasma and isolated HDL. The interaction of dalcetrapib with cysteine 13 of CETP is required, since this effect was abolished when using mutant CETP in which cysteine 13 was substituted for a serine residue. Other thiol-containing compounds were identified as CETP modulators interacting with cysteine 13 of CETP. In order to mimic dalcetrapib-bound CETP, mutant CETP proteins were prepared by replacing cysteine 13 with the bulky amino acid tyrosine or tryptophan. The resultant mutants showed virtually no CETP-dependent lipid transfer activity but demonstrated preserved CETP-dependent pre-β-HDL generation. Overall, these data demonstrate that the two functions of CETP i.e., cholesteryl ester transfer and HDL remodeling can be uncoupled by interaction of thiol-containing compounds with cysteine 13 of CETP or by introducing large amino acid residues in place of cysteine 13.     

Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice

The human cholesteryl ester transfer protein (CETP) facilitates the exchange of neutral lipids among lipoproteins. In order to evaluate the effects of increased plasma CETP on lipoprotein levels, a human CETP minigene was placed under the control of the mouse metallothionein-I promoter and used to develop transgenic mice. Integration of the human CETP transgene into the mouse genome resulted in the production of active plasma CETP. Zinc induction of CETP transgene expression caused depression of serum cholesterol due to a significant reduction of high density lipoprotein cholesterol. There was no change in total cholesterol content in very low and low density lipoproteins. However, there was a decrease in the free cholesterol/cholesteryl ester ratio in plasma and in all lipoprotein fractions of transgenic mouse plasma, suggesting stimulation of plasma cholesterol esterification. The results suggest that high levels of plasma CETP activity may be a cause of reduced high density lipoproteins in humans.     

Mechanism of inhibition defines CETP activity: a mathematical model for CETP in vitro

Because cholesteryl ester transfer protein (CETP) inhibition is a potential HDL-raising therapy, interest has been raised in the mechanisms and consequences of CETP activity. To explore these mechanisms and the dynamics of CETP in vitro, a mechanistic mathematical model was developed based upon the shuttle mechanism for lipid transfer. Model parameters were estimated from eight published experimental datasets, and the resulting model captures observed dynamics of CETP in vitro. Simulations suggest the shuttle mechanism yields behaviors consistent with experimental observations. Three key findings predicted from model simulations are: 1) net CE transfer activity from HDL to VLDL and LDL can be significantly altered by changing the balance of homoexchange versus heteroexchange of neutral lipids via CETP; 2) lipemia-induced increases in CETP activity are more likely caused by increases in lipoprotein particle size than particle number; and 3) the inhibition mechanisms of the CETP inhibitors torcetrapib and JTT-705 are significantly more potent than a classic competitive inhibition mechanism with the irreversible binding mechanism having the most robust response. In summary, the model provides a plausible representation of CETP activity in vitro, corroborates strong evidence for the shuttle hypothesis, and provides new insights into the consequences of CETP activity and inhibition on lipoproteins.     

Antisense oligonucleotide inhibition of cholesteryl ester transfer protein enhances RCT in hyperlipidemic,CETP transgenic,LDLr-/- mice

Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.     

Familial cholesteryl ester transfer protein deficiency is associated with triglyceride-rich low density lipoproteins containing cholesteryl esters of probable intracellular origin

The net transfer of core lipids between lipoproteins is facilitated by cholesteryl ester transfer protein (CETP). We have recently documented CETP deficiency in a family with hyperalphalipoproteinemia, due to a CETP gene splicing defect. The purpose of the present study was to characterize the plasma lipoproteins within the low density lipoprotein (LDL) density range and also the cholesteryl ester fatty acid distribution amongst lipoproteins in CETP-deficient subjects. In CETP deficiency, the conventional LDL density range contained both an apoE-rich enlarged high density lipoprotein (HDL) (resembling HDLc), and also apoB-containing lipoproteins. Native gradient gel electrophoresis revealed clear speciation of LDL subclasses, including a distinct population larger in size than normal LDL. Anti-apoB affinity-purified LDL from the CETP-deficient subjects were shown to contain an elevated triglyceride to cholesteryl ester ratio, and also a high ratio of cholesteryl oleate to cholesteryl linoleate, compared to their own HDL or to LDL from normal subjects. Addition of purified CETP to CETP-deficient plasma results in equilibration of very low density lipoprotein (VLDL) cholesteryl esters with those of HDL. These data suggest that, in CETP-deficient humans, the cholesteryl esters of VLDL and its catabolic product, LDL, originate predominantly from intracellular acyl-CoA:cholesterol acyltransferase (ACAT). The CETP plays a role in the normal formation of LDL, removing triglyceride and transferring LCAT-derived cholesteryl esters into LDL precursors.     

Dalcetrapib and anacetrapib increase apolipoprotein E-containing HDL in rabbits and humans

The large HDL particles generated by administration of cholesteryl ester transfer protein inhibitors (CETPi) remain poorly characterized, despite their potential importance in the routing of cholesterol to the liver for excretion, which is the last step of the reverse cholesterol transport. Thus, the effects of the CETPi dalcetrapib and anacetrapib on HDL particle composition were studied in rabbits and humans. The association of rabbit HDL to the LDL receptor (LDLr) in vitro was also evaluated. New Zealand White rabbits receiving atorvastatin were treated with dalcetrapib or anacetrapib. A subset of patients from the dal-PLAQUE-2 study treated with dalcetrapib or placebo were also studied. In rabbits, dalcetrapib and anacetrapib increased HDL-C by more than 58% (P < 0.01) and in turn raised large apo E-containing HDL by 66% (P < 0.001) and 59% (P < 0.01), respectively. Additionally, HDL from CETPi-treated rabbits competed with human LDL for binding to the LDLr on HepG2 cells more than control HDL (P < 0.01). In humans, dalcetrapib increased concentrations of large HDL particles (+69%, P < 0.001) and apo B-depleted plasma apo E (+24%, P < 0.001), leading to the formation of apo E-containing HDL (+47%, P < 0.001) devoid of apo A-I. Overall, in rabbits and humans, CETPi increased large apo E-containing HDL particle concentration, which can interact with hepatic LDLr. The catabolism of these particles may depend on an adequate level of LDLr to contribute to reverse cholesterol transport.