Inhibition Of Cholesteryl Ester Transfer Protein Activity Research Articles

Quantitative Prediction of Human Pharmacokinetics and Pharmacodynamics of CKD519, a Potent Inhibitor of Cholesteryl Ester Transfer Protein (CETP).

CKD519, a selective inhibitor of cholesteryl ester transfer protein(CETP), is undergoing development as an oral agent for the treatment of primary hypercholesterolemia and mixed hyperlipidemia. The aim of this study was to predict the appropriate efficacious dose of CKD519 for humans in terms of the inhibition of CETP activity by developing a CKD519 pharmacokinetic/pharmacodynamic (PK/PD) model based on data from preclinical studies. CKD519 was intravenously and orally administered to hamsters, rats, and monkeys for PK assessment. Animal PK models of all dose levels in each species were developed using mixed effect modeling analysis for exploration, and an interspecies model where allometric scaling was applied was developed based on the integrated animal PK data to predict the human PK profile. PD parameters and profile were predicted using in vitro potency and same-in-class drug information. The two-compartment first-order elimination model with Weibull-type absorption and bioavailability following the sigmoid Emax model was selected as the final PK model. The PK/PD model was developed by linking the interspecies PK model with the Emax model of the same-in-class drug. The predicted PK/PD profile and parameters were used to simulate the human PK/PD profiles for different dose levels, and based on the simulation result, the appropriate efficacious dose was estimated as 25 mg in a 60 kg human. However, there were some discrepancies between the predicted and observed human PK/PD profiles compared to the phase I clinical data. The huge difference between the observed and predicted bioavailability suggests that there is a hurdle in predicting the absorption parameter only from animal PK data.

Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in Rabbits.

CETP (cholesteryl ester transfer protein) plays an important role in lipoprotein metabolism; however, whether inhibition of CETP activity can prevent cardiovascular disease remains controversial. We generated CETP knockout (KO) rabbits by zinc finger nuclease gene editing and compared their susceptibility to cholesterol diet-induced atherosclerosis to that of wild-type (WT) rabbits. On a chow diet, KO rabbits showed higher plasma levels of high-density lipoprotein (HDL) cholesterol than WT controls, and HDL particles of KO rabbits were essentially rich in apolipoprotein AI and apolipoprotein E contents. When challenged with a cholesterol-rich diet for 18 weeks, KO rabbits not only had higher HDL cholesterol levels but also lower total cholesterol levels than WT rabbits. Analysis of plasma lipoproteins revealed that reduced plasma total cholesterol in KO rabbits was attributable to decreased apolipoprotein B-containing particles, while HDLs remained higher than that in WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. Apolipoprotein B-depleted plasma isolated from CETP KO rabbits showed significantly higher capacity for cholesterol efflux from macrophages than that from WT rabbits. Furthermore, HDLs isolated from CETP KO rabbits suppressed tumor necrosis factor-α-induced vascular cell adhesion molecule 1 and E-selectin expression in cultured endothelial cells. These results provide evidence that genetic ablation of CETP activity protects against cholesterol diet-induced atherosclerosis in rabbits.

WITHDRAWN: Cold-water extract of Korean Red Ginseng exhibits potent inhibitory effects against cholesteryl ester transfer protein activity and fructose-mediated glycation along with lipid-lowering activity in hyperlipidemic zebrafish

Abstract Background Red ginseng (RG) is widely used to treat complications of diabetes and atherosclerosis because of its antioxidative and anti-inflammatory activities. Methods In order to investigate the antiatherosclerotic and antisenescence activities of water extracts of RG powder, the lyophilized extract was treated to human macrophage, dermal cells, and brain glioma cells, and supplemented to hyperlipidemic zebrafish. Results Aqueous extracts of RG showed potent inhibitory activity against fructose-mediated glycation and protective effect against low-density lipoprotein (LDL) oxidation. RG extracts also showed potent antiatherogenic activities, demonstrated inhibitory activity against cholesteryl ester transfer protein (CETP), and inhibited cellular uptake of oxidized LDL into macrophages. As in vivo test, zebrafish consumed a high cholesterol diet (HCD) [final, 4% in diet (wt/wt)] with or without RG [final, 10% or 20% (wt/wt)]. The 10% and 20% RG groups showed reductions of 5% and 3%, respectively, in weight and height compared to the HCD group. Plasma total cholesterol level was significantly reduced in both the 10% and 20% RG groups, and glutamic–pyruvic transaminase activity was reduced in the 20% RG group compared to the HCD group. Finally, plasma from zebrafish that consumed 20% RG showed the lowest plasma CETP activity with the least hepatic inflammation and fatty liver change. Conclusion Aqueous extracts of RG suppressed the incidence of atherosclerosis via strong antioxidant potential, prevention of apolipoprotein A-I glycation, uptake of oxLDL, and inhibition of CETP activity. Consumption of RG in hyperlipidemic zebrafish model exhibited potent lipid-lowering and atheroprotective activities with the least fatty liver change.

Anti-atherosclerosis effect of different doses of CETP vaccine in rabbit model of atherosclerosis.

To evaluate atheroprotective effects of different doses of cholesteryl ester transfer protein (CETP) vaccine, three doses of Tetanus toxoid-CETP (TT-CETP) peptide including 10, 50 and 100/rabbit, termed FA10, FA50, FA100, respectively, were administered in rabbit model of atherosclerosis. Animals were vaccinated subcutaneously (S.C.) with 100μl of vaccine in presence of complete Freund's adjuvant (CFA) for the first administration. Rabbits were boosted 4 times at 3 weeks intervals with the same peptide dose formulated in incomplete Freund's adjuvant (IFA). Animals were fed with diet supplemented with 2% cholesterol from week 11 to week 19. Anti-TT-CETP specific antibody and CETP activity in sera were determined. Therapeutic response was examined by tracking plasma lipoprotein levels (HDL-C, LDL-C and total cholesterol), and pathologic observation of intima/media thickness at the site of aortic lesions. All TT-CETP vaccine doses generated strong anti TT-CETP antibody response. CETP activity reduced in rabbits vaccinated with FA100 (P=0.031). FA100 showed significant increase in level of HDL-C rather than control group (P=0.006). However, no significant reduction were found in atherosclerotic lesion when compared to control. Inhibition of CETP activity and increased HDL-C were found with FA100, but the vaccine failed to prevent aortic lesion development in immunized rabbits when compared to control. Our result supports the hypothesis stated that CETP may not be an attractive therapeutic target for the prevention of cardiovascular disease.

A molecular dynamics investigation on the inhibition mechanism of cholesteryl ester transfer protein by Anacetrapib

A decrease in high-density lipoprotein (HDL) level and an increase in atherogenic low-density lipoprotein (LDL) concentration in plasma are involved in the progress of atherosclerosis and incidence of cardiovascular diseases. Cholesteryl ester transfer protein (CETP) passes cholesteryl ester and triglyceride molecules through a hydrophobic tunnel between lipoproteins and via that decreases the HDL/LDL ratio in plasma. Thus, inhibition of CETP activity by specific inhibitors could diminish atherosclerosis occurrence. Anacetrapib as a specific reversible inhibitor of CETP by stabilizing CETP–HDL complex significantly increases the HDL/LDL ratio in plasma. For better understanding the mechanism of CETP inhibition by Anacetrapib, after docking Anacetrapib against a CETP structure, molecular dynamics simulation of CETP and CETP–Anacetrapib in an explicit water cage was performed. Analyses of acquired trajectories show that Anacetrapib increases fluctuation of residues 421–442 of CETP through hydrophobic interactions and thus disrupts the integrity of an α-helix (helix B) structure in the C-terminal concave surface. Creation of a hinge by defeat of the helix B in the C-terminal of CETP, where CETP interacts with LDL, results in missing of an α-helix (residues 436–440) and conversion of banana-shaped CETP to a hemisphere conformation. According to the obtained results, bending of the CETP structure and conversion of the secondary structure in the C-terminal domain in the presence of Anacetrapib may destabilize CETP–VLDL complex and through that shift the dynamics of CETP to the formation of the CETP–HDL complex.

How anacetrapib inhibits the activity of the cholesteryl ester transfer protein? Perspective through atomistic simulations.

Cholesteryl ester transfer protein (CETP) mediates the reciprocal transfer of neutral lipids (cholesteryl esters, triglycerides) and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity and thereby raise high density lipoprotein (HDL)-cholesterol and decrease low density lipoprotein (LDL)-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of cholesteryl ester to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The novel insight gained in this study has potential use in the development of new molecular agents capable of preventing the progression of cardiovascular diseases.

Effects of the cholesteryl ester transfer protein inhibitor evacetrapib on lipoproteins, apolipoproteins and 24-h ambulatory blood pressure in healthy adults.

ObjectivesWe investigated the safety, tolerability, pharmacokinetics and pharmacodynamics of evacetrapib.MethodsHealthy volunteers received multiple daily doses of evacetrapib (10–600 mg) administered for up to 15 days in a placebo-controlled study.Key findingsMean peak plasma concentrations of evacetrapib occurred at 4–6 h and terminal half-life ranged 24–44 h. Steady state was achieved at approximately 10 days; all subjects had undetectable levels of evacetrapib 3 weeks after their last dose. The trough inhibition of cholesteryl ester transfer protein (CETP) activity was 65 and 84% at 100 and 300 mg, respectively. At the highest dose (600 mg), evacetrapib significantly inhibited CETP activity (91%), increased HDL-C (87%) and apo AI (42%), and decreased LDL-C (29%) and apo B (26%) relative to placebo. For the highest dose tested, levels of evacetrapib, CETP activity, CETP mass, HDL-C and LDL-C returned to levels at or near baseline after a 2-week washout period. Evacetrapib at the highest dose tested did not produce any significant effect on 24-h ambulatory systolic or diastolic blood pressure.ConclusionsMultiple doses of evacetrapib potently inhibited CETP activity, leading to substantial elevations in HDL-C and lowering of LDL-C. Evacetrapib was devoid of clinically relevant effects on blood pressure and mineralocorticoid levels.

Anacetrapib and dalcetrapib differentially alters HDL metabolism and macrophage-to-feces reverse cholesterol transport at similar levels of CETP inhibition in hamsters

Cholesteryl ester transfer protein (CETP) inhibitors dalcetrapib and anacetrapib differentially alter LDL- and HDL-cholesterol levels, which might be related to the potency of each drug to inhibit CETP activity. We evaluated the effects of both drugs at similar levels of CETP inhibition on macrophage-to-feces reverse cholesterol transport (RCT) in hamsters. In normolipidemic hamsters, both anacetrapib 30mg/kg QD and dalcetrapib 200mg/kg BID inhibited CETP activity by ~60%. After injection of 3H-cholesteryl oleate labeled HDL, anacetrapib and dalcetrapib reduced HDL-cholesteryl esters fractional catabolic rate (FCR) by 30% and 26% (both P<0.001 vs. vehicle) respectively, but only dalcetrapib increased HDL-derived 3H-tracer fecal excretion by 30% (P<0.05 vs. vehicle). After 3H-cholesterol labeled macrophage intraperitoneal injection, anacetrapib stimulated 3H-tracer appearance in HDL, but both drugs did not promote macrophage-derived 3H-tracer fecal excretion. In dyslipidemic hamsters, both anacetrapib 1mg/kg QD and dalcetrapib 200mg/kg BID inhibited CETP activity by ~65% and reduced HDL-cholesteryl ester FCR by 36% (both P<0.001 vs. vehicle), but only anacetrapib increased HDL-derived 3H-tracer fecal excretion significantly by 39%. After 3H-cholesterol labeled macrophage injection, only anacetrapib 1mg/kg QD stimulated macrophage-derived 3H-tracer appearance in HDL. These effects remained weaker than those observed with anacetrapib 60mg/kg QD, which induced a maximal inhibition of CETP and stimulation of macrophage-derived 3H-tracer fecal excretion. In contrast, dalcetrapib 200mg/kg BID reduced macrophage-derived 3H-tracer fecal excretion by 23% (P<0.05 vs. vehicle). In conclusion, anacetrapib and dalcetrapib differentially alter HDL metabolism and RCT in hamsters. A stronger inhibition of CETP may be required to promote macrophage-to-feces reverse cholesterol transport in dyslipidemic hamsters.

Glycation of Apolipoprotein C1 Impairs Its CETP Inhibitory Property: Pathophysiological Relevance in Patients With Type 1 and Type 2 Diabetes

OBJECTIVE Apolipoprotein (apo)C1 is a potent physiological inhibitor of cholesteryl ester transfer protein (CETP). ApoC1 operates through its ability to modify the electrostatic charge at the lipoprotein surface. We aimed to determine whether the inhibitory ability of apoC1 is still effective in vivo in patients with diabetes and whether in vitro glycation of apoC1 influences its electrostatic charge and its CETP inhibitory effect. RESEARCH DESIGN AND METHODS ApoC1 concentrations and CETP activity were measured in 70 type 1 diabetic (T1D) patients, 113 patients with type 2 diabetes, and 83 control subjects. The consequences of in vitro glycation by methylglyoxal on the electrostatic properties of apoC1 and on its inhibitory effect on CETP activity were studied. An isoelectric analysis of apoC1 was performed in patients with T1D and in normolipidemic-normoglycemic subjects. RESULTS An independent negative correlation was found between CETP activity and apoC1 in control subjects but not in patients with diabetes. HbA1c was independently associated with CETP activity in T1D patients. In vitro glycation of apoC1 modified its electrostatic charge and abrogated its ability to inhibit CETP activity in a concentration-dependent manner. The isoelectric point of apoC1 in T1D patients was significantly lower than that in control subjects. CONCLUSIONS The ability of apoC1 to inhibit CETP activity is impaired in patients with diabetes. Glycation of apoC1 leads to a change in its electrostatic properties that might account, at least in part, for a loss of constitutive CETP inhibition and an increase in plasma CETP activity in patients with diabetes.

Study of Cholesteryl Ester Transfer Protein (CETP) I405v Genotype and Its Association with Lipid Fractions in Myocardial Infarction Patients: A Case Control Study.

We determine the significant relation of HDL cholesterol and total cholesterol/HDL cholesterol between CETP I405V genotypes and activity of CETP. CETP is an essential for transfer of cholesterol ester to the liver from peripheral tissues which facilitating its transfer to TG rich VLDL. Reduction activity of CETP I405V may associate with genotypes of CETP I405V. This study is undertaken to assess the presence and impact of CETP I405V genotype in our population. In this study 100 acute myocardial infarction patients and 100 normal age & sex matched healthy individuals were included. Serum Lipid profile was estimated by using universal standard methods whereas CETP I405V genotype was studied by ARMS PCR. There is presence of CETP 405Val genotype both in patient as well as in control group. RESULTs show that HDL cholesterol (p<0.0001) and ratio of total cholesterol/HDL cholesterol are significantly (p<0.0043) associated with Val/Val genotype. In addition to that the CETP I405V genotype is associated with inhibition of CETP activity with higher HDL-C level and decreased total cholesterol/HDL cholesterol ratio. Our results show that the CETP I405V genotypes are very much significantly determinant of HDL cholesterol in patients with CHD.

Abstract 69: Anacetrapib Dose-dependently Decreases Atherosclerosis Development and Adds to the Beneficial Effects of Atorvastatin in APOE*3Leiden.CETP Mice

Introduction The residual risk of cardiovascular disease that remains after statin treatment has triggered the search for a secondary treatment target. Epidemiological studies propose HDL-cholesterol (HDL-C) as a possible candidate. Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from atheroprotective HDL to atherogenic (V)LDL. In human intervention trials, the CETP inhibitor anacetrapib decreases (V)LDL-C by 30-40% and increases HDL-C by 40-140%. Hypothesis Complete inhibition of CETP activity may result in adverse effects as compared to partial inhibition due to the appearance of a dysfunctional HDL. We, therefore, evaluated the effect of a broad treatment window of anacetrapib-induced CETP inhibition with partial to full inhibition, as well as the combination of atorvastatin and anacetrapib on atherosclerosis development in APOE*3Leiden.CETP mice. Methods Female mice were fed a Western-type diet containing 0.1% cholesterol without or with incremental dosages of anacetrapib (0.03; 0.3; 3; 30 mg/kg/d), atorvastatin (2.4 mg/kg/d) or a combination of anacetrapib (0.3 mg/kg/d) and atorvastatin (2.4 mg/kg/d) for 20 weeks. Effects on plasma lipids, CETP activity and levels, as well as atherosclerotic lesion size and severity were assessed. Results Anacetrapib dose-dependently reduced CETP activity (-60% to -100%, P&lt;0.001) and increased CETP levels (+13% to +31%, P&lt;0.05), thereby decreasing nonHDL-C (-23% to -44%, P&lt;0.001) and increasing HDL-C (+32% to +88%, P&lt;0.001). Atorvastatin decreased CETP activity (-29%, P&lt;0.001) and nonHDL-C (-36%, P&lt;0.001). Anacetrapib dose-dependently decreased atherosclerotic lesion size (-36%, P&lt;0.05 to -92%, P&lt;0.001) and the percentage severe lesions. Anacetrapib added to the effects of atorvastatin by further reducing nonHDL-C (-36%, P&lt;0.001), increasing HDL-C (+72%, P&lt;0.001) and decreasing lesion size (-86%, P&lt;0.001) and severity. Results on lesion composition are pending. Conclusions Anacetrapib dose-dependently decreases atherosclerosis development and adds to the beneficial effects of atorvastatin in APOE*3Leiden.CETP mice. Total blockage of CETP activity does not reveal adverse effects as compared to partial blockage.

Abstract 9560: Clinical Trial Simulations of Dyslipidemic Patients in a Mechanistic Model of Cardiovascular Disease Predict Little Impact on CHD Events by CETP Inhibitors

The Entelos® CV PhysioLab® platform, a mechanistic model of CHD, quantitatively predicts cholesterol metabolism and plaque growth in the coronary artery over time through the dynamic simulation of relevant biological pathways. Statistical methodologies are used to translate predicted changes in circulating lipids and plaque characteristics to predictions of CHD risk. Such predictions can be made for approved or novel therapeutics over time in virtual populations representing a diverse set of patient populations that can be created. PK and PD effects of cholesteryl ester transfer protein (CETP) inhibitors, torcetrapib (60 mg qd), dalcetrapib (600 mg qd) and anacetrapib (100mg qd) were implemented in the platform, calibrated to published data and administered to simulated patients to predict effects on lipoprotein profile and the mean hazard ratio (HR) relative to the same simulated patients on a statin treated background (statin treated mean LDL-C 84 ± 15 mg/dL, HDL-C 44 ± 9 mg/dL). An additional effect of the reported increase on blood pressure was implemented for torcetrapib simulations only. Predicted changes in lipoproteins and 4 year HRare shown in the table. When the mechanistic effects of the reported increase in systolic blood pressure with torcetrapib are added to the lipoprotein changes that result from appropriate CETP inhibition, simulations predict an increased HR similar to that seen in the ILLUMINATE study. Dalcetrapib simulations do not predict any clinically meaningful changes in HR. Finally, despite an additional decrease in LDL-C and the largest increase in HDL-C, anacetrapib simulations predict a very small reduction in HR. Simulations also define a subpopulation within whom the inhibition of CETP activity is more beneficial with the general characteristics of high TG and low HDL-C. 1Additional effect on blood pressure was included in simulations

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.

Cholesteryl ester transfer protein and its inhibition.

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that facilitates the transfer of cholesteryl esters from the atheroprotective high density lipoprotein (HDL) to the proatherogenic low density lipoprotein cholesterol (LDL) and very low density lipoprotein cholesterol (VLDL) leading to lower levels of HDL but raising the levels of proatherogenic LDL and VLDL. Inhibition of CETP is considered a potential approach to treat dyslipidemia. However, discussions regarding the role of CETP-mediated lipid transfer in the development of atherosclerosis and CETP inhibition as a potential strategy for prevention of atherosclerosis have been controversial. Although many animal studies support the hypothesis that inhibition of CETP activity may be beneficial, negative phase III studies on clinical endpoints with the CETP inhibitor torcetrapib challenged the future perspectives of CETP inhibitors as potential therapeutic agents. The review provides an update on current understanding of the molecular mechanisms involved in CETP activity and its inhibition.

La elevación del colesterol unido a lipoproteínas de alta densidad: perspectiva futura. La CETP como diana terapéutica

Cholesteryl ester transfer protein (CETP) facilitates transfer of neutral lipids between lipoprotein classes. CETP plays a key role in cholesterol homeostasis, especially in the redistribution of cholesterol among particles and in reverse cholesterol transport. Consequently, modifying the activity of this protein could influence the development and progression of atheromatosis. The effects of inhibiting CETP include reductions in lowdensity lipoprotein (LDL)-cholesterol and cholesterol in triglyceride-rich particles and increases in high-density lipoprotein (HDL)-cholesterol, apolipoprotein A-1 and the size of LDL and HDL particles. The induction of expression of CETP activity in mice increases susceptibility to atherosclerosis, while inhibition of CETP activity in rabbits tends to reduce its development. However, the role of CETP inhibition in atheromatosis in humans remains to be established.

Control of cholesteryl ester transfer protein activity by sequestration of lipid transfer inhibitor protein in an inactive complex

Lipid transfer inhibitor protein (LTIP) is a physiologic regulator of cholesteryl ester transfer protein (CETP) function. We previously reported that LTIP activity is localized to LDL, consistent with its greater inhibitory activity on this lipoprotein. With a recently described immunoassay for LTIP, we investigated whether LTIP mass is similarly distributed. Plasma fractionated by gel filtration chromatography revealed two LTIP protein peaks, one coeluting with LDL, and another of approximately 470 kDa. The 470 kDa LTIP complex had a density of 1.134 g/ml, indicating approximately 50% lipid content, and contained apolipoprotein A-I. By mass spectrometry, partially purified 470 kDa LTIP also contains apolipoproteins C-II, D, E, J, and paraoxonase 1. Unlike LDL-associated LTIP, the 470 kDa LTIP complex does not inhibit CETP activity. In normolipidemic subjects, approximately 25% of LTIP is in the LDL-associated, active form. In hypercholesterolemia,this increases to 50%, suggesting that lipoprotein composition may influence the status of LTIP activity. Incubation (37 degrees C) of normolipidemic plasma increased active, LDL-associated LTIP up to 3-fold at the expense of the inactive pool. Paraoxon inhibited this shift by 50%. Overall, these studies show that LTIP activity is controlled by its reversible incorporation into an inactive complex. This may provide for short-term fine-tuning of lipoprotein remodeling mediated by CETP.

Lipoprotein inflammatory properties and serum amyloid A levels but not cholesterol levels predict lesion area in cholesterol-fed rabbits

Rabbits on a 1% cholesterol diet received injections of vehicle with or without D-4F or L-4F. After 1 month, the percent of aorta with atherosclerotic lesions was 24 +/- 15% (vehicle), 10 +/- 6% (D-4F) (P < 0.01 vs. vehicle), and 13 +/- 9% (L-4F) (P < 0.05 vs. vehicle). Inflammatory indexes for HDL and LDL were determined by measuring monocyte chemotactic activity after adding rabbit lipoproteins to human endothelial cells. HDL-inflammatory index (HII) and LDL-inflammatory index (LII), respectively, were 1.39 +/- 0.24; 1.35 +/- 0.29 (vehicle), 0.67 +/- 0.26; 0.63 +/- 0.38 (D-4F) (P < 0.001 vs. vehicle), and 0.67 +/- 0.2; 0.68 +/- 0.32 (L-4F) (P < 0.01 vs. vehicle). Serum amyloid A (SAA) levels were 95 +/- 39, 8 +/- 22, and 7 +/- 19 mug/ml, respectively, for vehicle, D-4F, and L-4F (P < 0.001 vs. vehicle). There was no correlation between lesion area and total plasma or HDL-cholesterol levels. In contrast, there was a positive correlation with HII, LII, and SAA (P = 0.002, P = 0.0026, P = 0.0079, respectively). HII correlated closely with SAA levels (r = 0.6616; r(2) = 0.4377, P < 0.0001). Thus, HII, LII, and SAA are better predictors of lesion area than are total plasma or HDL-cholesterol levels in cholesterol-fed rabbits.

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.

Molecular Mechanism of the Blockade of Plasma Cholesteryl Ester Transfer Protein by Its Physiological Inhibitor Apolipoprotein CI

Genetically engineered mice demonstrated that apolipoprotein (apo) CI is a potent, physiological inhibitor of plasma cholesteryl ester transfer protein (CETP) activity. The goal of this study was to determine the molecular mechanism of the apoCI-mediated blockade of CETP activity. Kinetic analyses revealed that the inhibitory property of apoCI is independent of the amount of active CETP, but it is tightly dependent on the amount of high density lipoproteins (HDL) in the incubation mixtures. The electrostatic charge of HDL, i.e. the main carrier of apoCI in human plasma, is gradually modified with increasing amounts of apoCI, and the neutralization of apoCI lysine residues by acetylation produces a marked reduction in its inhibitory potential. The inhibitory property of full-length apoCI is shared by its C-terminal alpha-helix with significant electrostratic properties, whereas its N-terminal alpha-helix with no CETP inhibitory property has no effect on HDL electronegativity. Finally, binding experiments demonstrated that apoCI and to a lower extent its C-terminal alpha-helix are able to disrupt CETP-lipoprotein complexes in a concentration-dependent manner. It was concluded that the inhibition of CETP activity by apoCI is in direct link with its specific electrostatic properties, and the apoCI-mediated reduction in the binding properties of lipoproteins results in weaker CETP-HDL interactions and fewer cholesteryl ester transfers.

Total syntheses of (+)-chloropuupehenone and (+)-chloropuupehenol and their analogues and evaluation of their bioactivities.

Tetracyclic pyrans (+)-chloropuupehenone (1) and (+)-chloropuupehenol (5) and its C8-R-isomer (+)-3 were synthesized via a one-pot condensation of 1-chloro-2-lithio-3,5,6-tris(tert-butyldimethylsilyloxy)benzene (8) with (4aS,8aS)-3,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethylnaphthalene-1-carboxaldehyde (7). The major condensation product, (4aS,6aR,12bS)-2H-9,10-bis(tert-butyldimethylsilyloxy)-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene (4), after desilylation provided tetracyclic pyran (+)-(4aS,6aR,12bS)-2H-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene-9,10-diol (3). At a dosage of 42 mg/rat over 8 h, pyran diol 3 inhibited the intestinal absorption of cholesterol by 71% in rats. Tetracyclic pyran 4 was also converted to o-quinone 28, which inhibited cholesteryl ester transfer protein (CETP) activity and L1210 leukemic cell viability with IC(50) values of 31 and 2.4 microM, respectively. Diol (+)-5 inhibited CETP activity with an IC(50) value of 16 microM. The minor condensation product, (4aS,6aS,12bS)-2H-9,10-bis(tert-butyldimethylsilyloxy)-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene (6), was transformed into (+)-5 and (+)-1. A stepwise stereoselective synthesis of (+)-1 was also developed utilizing an oxyselenylation ring-closure reaction. The synthetic sequence also produced four biologically active naturally occurring drimanic sesquiterpenes, (+)-drimane-8alpha,11-diol (34), (-)-drimenol (38), (+)-albicanol (39), and (-)-albicanal (31) as intermediates.