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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2016 Mar 10;36(5):994–1002. doi: 10.1161/ATVBAHA.115.306680

CETP inhibition with Anacetrapib Decreases Fractional Clearance Rates of HDL Apolipoprotein A-I and Plasma CETP

Gissette Reyes-Soffer 1,a, John S Millar 2,a, Colleen Ngai 1, Patricia Jumes 3, Ellie Coromilas 1, Bela Asztalos 4, Amy O Johnson-Levonas 3, John A Wagner 3, Daniel S Donovan 1, Wahida Karmally 1, Rajasekhar Ramakrishnan 1, Stephen Holleran 1, Tiffany Thomas 1, Richard L Dunbar 2, Emil M deGoma 2, Hashmi Rafeek 5, Amanda L Baer 2, Yang Liu 3, Michael E Lassman 3, David E Gutstein 3, Daniel J Rader 2, Henry N Ginsberg 1
PMCID: PMC4911016  NIHMSID: NIHMS764488  PMID: 26966279

Abstract

Objective

Anacetrapib, an inhibitor of cholesteryl ester transfer protein (CETP) activity, increases plasma concentrations of HDL-C, apoA-I, apoA-II, and CETP. The mechanisms responsible for these treatment-related increases in apolipoproteins and plasma CETP are unknown. We performed a randomized, placebo-controlled, double-blind, fixed-sequence study to examine the effects of anacetrapib on the metabolism of HDL apoA-I and apoA-II and plasma CETP.

Approach and Results

Twenty-nine participants received atorvastatin 20mg/day plus placebo for four weeks, followed by atorvastatin plus anacetrapib 100 mg/day for 8 weeks (ATV-ANA). Ten participants received double placebo for four weeks followed by placebo plus anacetrapib for 8 weeks (PBO-ANA). At the end of each treatment, we examined the kinetics of HDL apoA-I, HDL apoA-II and plasma CETP after D3-leucine administration as well as 2D gel analysis of HDL subspecies. In the combined ATV-ANA and PBO-ANA groups, anacetrapib treatment increased plasma HDL-C (63.0%, P < 0.001) and apoA-I levels (29.5%, P < 0.001). These increases were associated with reductions in HDL apoA-I fractional clearance rate (FCR) (18.2%, P = 0.002) without changes in production rate (PR). Although the apoA-II levels increased by 12.6% (P < 0.001), we could not discern significant changes in either apoA-II FCR or PR. CETP levels increased 102% (P < 0.001) on anacetrapib due to a significant reduction in the FCR of CETP (57.6%, P < 0.001) with no change in CETP PR.

Conclusion

Anacetrapib treatment increases HDL apoA-I and CETP levels by decreasing the fractional clearance rate of each protein.

Clinical Trial Registration

URL: http://www.clinicaltrials.gov. Unique identifier: NCT00990808

Keywords: HDL metabolism, CETP metabolism, CETP inhibitor, apoA-I, apoA-II

Introduction

Elevated levels of low density lipoprotein (LDL) cholesterol (C) and decreased concentrations of high density lipoprotein (HDL)-C predict increased risk of cardiovascular disease (CVD).1 Reducing LDL-C with statins decreases the risk of CVD events.2 However, it remains to be demonstrated if raising HDL-C will produce similar benefits.3 Cholesterol ester transfer protein (CETP) is a plasma glycoprotein that mediates net exchange of cholesteryl ester (CE) from HDL for triglyceride in apolipoprotein B (apoB)-containing lipoproteins, including chylomicrons, very low density lipoproteins (VLDL) and LDL.4 Inhibition of CETP increases HDL-C levels by reducing the transfer of CE from HDL to atherogenic apoB-containing lipoproteins.5

Increasing HDL-C via inhibition of CETP as a means of enhancing reverse cholesterol transport to reduce CVD has come under scrutiny6 after failures to show benefit in large outcome studies. In ILLUMINATE, torcetrapib, which increased HDL-C in the range of 60 – 100% and lowered LDL-C by up to 20%, actually increased CVD events,7 a result likely associated with off-target effects on blood pressure and adrenal synthesis of mineralo- and glucocorticoids.8 In dal-OUTCOMES, dalcetrapib, which did not have off-target effects and raised HDL-C about 30%, there was no effect on CVD.9 More recently, ACCELERATE, a CVD outcome study with evacetrapib,10 another CETP inhibitor without off-target effects, was stopped early due to apparent insufficient efficacy of the drug, although no specific data has been presented yet. Two additional CETP inhibitors under development, anacetrapib and TA-8995, do not appear to have the off target effects of torcetrapib and increase HDL-C by up to 140 – 180%.11,12 The REVEAL study determining the effect of anacetrapib on CVD outcomes is ongoing, while TA-8995 is at an earlier stage of development.13,14 As a result, the controversy regarding the effect of pharmacologic inhibition of CETP on reverse cholesterol transport continues.15

A previous study of the CETP inhibitor torcetrapib, administered as monotherapy, significantly increased plasma concentrations of apoA-I and apoA-II by reducing their fractional clearance rates (FCR) from plasma.16,17 However, there was no discernable effect of torcetrapib on apoA-I FCR when it was added to atorvastatin, although power was limited.16 In the present study, we determined the effects of anacetrapib on the metabolism of HDL apoA-I and apoA-II with or without atorvastatin therapy. Inhibition of CETP activity also increases plasma levels of CETP although the mechanism responsible for this increase is unknown.18 We report, for the first time, the effects of a CETP inhibitor on the metabolism of CETP.

Materials and Methods are available in the online-only Data Supplement

Results

Thirty-nine participants completed this study. Ten were in PBO-ANA and 29 were in ATV-ANA. There were 4 (40%) and 9 (31%) females in PBO-ANA and ATV-ANA, respectively. Mean age was 50.6 ± 11.3 years in the PBO-ANA group and 46.7 ± 10.4 in the ATV-ANA group. BMI ranged from 20.5 to 37.7 kg/m2 across the two groups. Screening lipid levels were similar in both groups (Table 1). There were no significant differences in baseline demographic parameters between the ATV-ANA and PBO-ANA groups.

Table 1. Baseline Demographic and Lipids.

Characteristic ATV-ANA (N=29) PBO-ANA (N=10) P-values (ATV-ANA vs. PBO-ANA)* All participants (N=39)
Age (y)
Mean ± SD 46.7 ± 10.1 50.6 ± 11.3 0.3091 47.7 ± 10.4
Range (28.0, 67.0) (28.0, 66.0) (28.0, 67.0)
Sex, n (%)
Male 20 (69.0) 6 (60.0) 0.6040 26 (66.7)
Female 9 (31.0) 4 (40.0) 13 (33.3)
Body Weight (kg)
Mean ± SD 89.04 ± 16.83 80.26 ± 15.82 0.1575 86.79 ± 16.82
Range (55.20, 128.40) (54.70, 107.60) (54.70, 128.40)
Body mass index (kg/m2)
Mean ± SD 30.3 ± 4.8 27.6 ± 4.0 0.1296 29.6 ± 4.7
Range (20.5, 37.7) (21.0, 32.9) (20.5, 37.7)
Prior statin use, n (%)
Atorvastatin 20 mg 1 (3.4) 0 1 (2.6)
Simvastatin 5 mg 1 (3.4) 0 1 (2.6)
Simvastatin 20 mg 2 (6.9) 0 2 (5.1)
Simvastatin 40 mg 1 (3.4) 0 1 (2.6)
Concomitant use of anti-hypertensives, n (%)
Beta blocker 1 (3.4) 0 1 (2.6)
Concomitant use of hormone replacements, n (%)
Estrogens 1 (3.4) 0 1 (2.6)
Thyroid replacement 1 (3.4) 0 1 (2.6)
Total 1 (3.4) 0 1 (2.6)
Total Cholesterol (mg/dL)
Mean ± SD 215.8 ± 31.6 207.0 ± 27.0 0.4030 213.5 ± 30.4
Triglycerides (mg/dL)
Median (IQR) 117.0 (84.0, 142.0) 120.0 (100.0, 185.0) 0.2253 118.0 (84.0, 146.0)
LDL-C (mg/dL)
Mean ± SD 139.7 ± 26.9 130.9 ± 20.5 0.4715 137.5 ± 25.5
HDL-C (mg/dL)
Mean ± SD 50.5 ± 13.9 45.6 ± 12.5 0.3293 49.2 ± 13.6
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*

P-values are for null hypothesis that there is no difference between the two population means for ATV-ANA and PBO-ANA. For the continuous variables (age, body weight, body mass index and lipid values), P-values are from equal variance two sample two-tailed T-test. For gender, P-value is from Chi-Square test for proportion. SD, Standard Deviation; ATV-ANA, atorvastatin-anacetrapib; PBO-ANA, placebo-anacetrapib

Effects of anacetrapib on lipids and lipoproteins are presented in Table 2. At the end of the statin or placebo run-in period, plasma lipid levels differed between ATV-ANA and PBO-ANA because of the administration of atorvastatin, 20 mg/day, in the ATV-ANA group. In the ATV-ANA group, 8 weeks of anacetrapib treatment on a statin background resulted in an increase in HDL-C (68.1%; P<0.001), and a decrease in LDL-C (38.0%; P<0.001), with no changes in TG. In the PBO-ANA group, HDL-C increased (53.5%; P=0.055) and LDL-C decreased (34.5%; P=0.039). TG levels also decreased in PBO-ANA (24.5%; P=0.012). Plasma apoA-I concentrations increased in the ATV-ANA (29.3%) and PBO-ANA (29.7%) groups after anacetrapib treatment (P<0.001 for both the ATV-ANA and PBO-ANA groups). Plasma apoA-II levels also increased in both the ATV-ANA (11.9%; P<0.001) and the PBO-ANA (13.3%; P=0.008) groups.

Table 2. Plasma Lipid and Apolipoprotein Values.

ATV-ANA (N=29) PBO-ANA (N=10) All Participants (N=39)
ATV ANA+ATV % change
from baseline
(95% CI)		 P-
value		 PBO ANA % change from baseline (95% CI) P-value ATV or
PBO		 ANA or
ANA+ATV		 % change
from baseline
(95% CI)		 P-
value
Plasma Cholesterol, (mg/dL) 162.8 (13.02) 167.6 (21.90) 3.0 (-3.73, 10.11) 0.385 208.5 (17.19) 188.1 (26.38) -9.8 (-20.27, 2.05) 0.099 184.3 (17.28) 177.6 (23.30) -3.6 (-10.17, 3.38) 0.294
Plasma Triglyceride, (mg/dL) 89.4 (38.37) 90.6 (41.99) 1.3 (-9.67, 13.69) 0.816 120.8 (59.31) 91.2 (48.28) -24.5 (-39.08, -6.47) 0.012 103.9 (44.49) 90.9 (42.95) -12.5 (-22.55, -1.23) 0.032
Plasma HDL-C*, (mg/dL) 48.0 (19.00) 82.0 (35.00) 68.1 (48.70, 92.01) <.001 43.0 (20.00) 91.5 (61.00) 53.5 (-0.06, 164.36) 0.055 48.0 (20.00) 86.0 (48.00) 63.0 (45.90, 91.30) <.001
Plasma LDL-C*, (mg/dL) 89.5 (16.00) 53.0 (25.00) -38.0 (-47.57, -27.62) <.001 134.0 (23.00) 79.5 (41.00) -34.5 (-54.69, -14.36) 0.039 93.0 (39.00) 58.0 (33.00) -37.3 (-46.06, -28.73) <.001
Plasma apoA-I (mg/dL) 132.6 (17.69) 171.4 (21.96) 29.3 (21.33, 37.82) <.001 127.5 (19.74) 165.29 (28.39) 29.7 (16.33, 44.53) <.001 130.0 (18.05) 168.3 (23.40) 29.5 (21.59, 37.90) <.001
Plasma apoA-II (mg/dL) 37.8 (11.29) 42.4 (15.35) 11.9 (6.05, 18.15) <.001 38.0 (14.50) 43.1 (15.87) 13.3 (3.49, 24.11) 0.008 37.9 (12.00) 42.7 (15.29) 12.6 (6.83, 18.75) <.001
LCAT Activity, (nmol/mL/hr) 147.9 (27.34) 100.6 (31.33) -32.0 (-37.78, -25.60) <.001 185.3 (26.65) 120.6 (30.44) -34.9 (-44.09, -24.21) <.001 165.6 (28.73) 110.2 (31.81) -33.5 (-39.07, -27.31) <.001
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Abbreviations: atorvastatin (ATV), anacetrapib (ANA), placebo (PBO)

Mixed model analysis performed on log scale for the endpoints that satisfy normality. Geometric Mean (% CV) displayed.

*

Non-parametric method used. Median (IQR) on raw scale displayed. Hodges-Lehmann estimate (95% CI) back transformed from log scale displayed under % Change from baseline. P-value from Wilcoxon signed rank test.

Data are missing from two participants in PBO-ANA, PBO and one subject in each of ATV-ANA, ANA+ATV and PBO-ANA, ANA for Total Cholesterol, Triglycerides, HDL Cholesterol and LDL Cholesterol.

We isolated HDL by ultracentrifugation to determine the effects of anacetrapib treatment on lipid composition.19 The cholesterol content in isolated HDL increased by 85.2% in the ATV-ANA group (P<0.001) and by 97.8% in the PBO-ANA group (P<0.001); cholesteryl ester (CE), which accounted for >80% of the total cholesterol content in the HDL fractions, increased by 81.0% in the ATV-ANA group (P<0.001) and by 89.8% in the PBO-ANA group (P<0.001). HDLTG content decreased by 53.7% in the ATV-ANA group (P<0.001) and by 46.6% in the PBO-ANA group (P<0.001). The cholesterol esterification rate was decreased in both groups (ATV-ANA by 32.0%, P<0.001, and PBO-ANA by 34.9%, P<0.001).

The metabolism of HDL apoA-I and apoA-II was examined at the end of the 4 week run-in period with placebo or statin and after addition of anacetrapib for 8 weeks (Figure 1, Table 3). The increase in apoA-I pool size during anacetrapib treatment was due to a significant decrease in the HDL apoA-I FCR (15.7% in the ATV-ANA and 20.7% in the PBO-ANA group; P=0.009 and P=0.029, respectively). There were no significant changes in the PR of apoA-I in either group. Despite small but significant increases in plasma apoA-II levels in each group, we did not observe significant changes in either FCRs or PRs of apoA-II.

Figure 1.

Figure 1

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HDL apoA-I and HDL apoA-II kinetic parameters for participants in ATV-ANA, PBO-ANA, and combined groups at the end of each treatment period. All data are shown as geometric mean values. Unadjusted raw p-values: §p<0.05; ‡p<0.01; †p<0.001. Abbreviations: atorvastatin (ATV), anacetrapib (ANA), fractional catabolic rate (FCR), placebo (PBO), pool size (PS), production rate (PR). % CV displayed at base of each bar.

Table 3. ApoA-I and ApoA-II Kinetic Parameters.

ATV-ANA (N=29) PBO-ANA (N=10) All Participants (N=39)
ATV ANA+ATV % change
from baseline
(95% CI)		 P-
value		 PBO ANA % change
from baseline
(95% CI)		 P-value ATV or
PBO		 ANA or
ANA+ATV		 % change
from baseline
(95% CI)		 P-
value
HDL apoA-I PS, (mg) 5,113 (26.19) 6,643 (26.69) 29.9 (22.88, 37.39) <.001 4,362 (18.75) 5,792 (21.32) 32.8 (20.75, 46.03) <.001 4,723 (25.29) 6,203 (25.90) 31.4 (24.31, 38.79) <.001
HDL apoA-I FCR, (pools/day) 0.17 (28.49) 0.14 (36.25) -15.7 (-25.68, -4.39) 0.009 0.19 (46.25) 0.15 (71.13) -20.7 (-35.50, -2.42) 0.029 0.18 (33.64) 0.15 (46.01) -18.2 (-27.56, -7.69) 0.002
HDL apoA-I PR, (mg/kg/day) 9.9 (27.42) 11.0 (36.66) 11.1 (-1.51, 25.28) 0.085 10.4 (45.36) 10.9 (53.41) 5.5 (-13.41, 28.57) 0.585 10.1 (32.36) 11.0 (40.77) 8.3 (-3.57, 21.54) 0.173
HDL apoA-II PS, (mg) 1,480 (23.76) 1,666 (25.41) 12.6 (7.48, 18.00) <.001 1,332 (22.48) 1,538 (23.16) 15.5 (6.67, 25.05) <.001 1,404 (23.63) 1,601 (24.81) 14.1 (8.91, 19.42) <.001
HDL apoA-II FCR, (pools/day) 0.17 (35.80) 0.17 (33.56) 0.46 (-15.47, 19.39) 0.957 0.17 (60.60) 0.14 (85.95) -16.4 (-37.01, 11.08) 0.210 0.17 (42.40) 0.16 (49.83) -8.3 (-22.35, 8.23) 0.295
HDL apoA-II PR, (mg/kg/day) 2.8 (36.04) 3.2 (34.86) 13.5 (-3.46, 33.41) 0.121 2.9 (57.49) 2. 8 (69.99) -3.2 (-25.76, 26.34) 0.808 2.9 (41.60) 3.0 (45.33) 4.8 (-10.27, 22.49) 0.542
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Abbreviations: atorvastatin (ATV), anacetrapib (ANA), placebo (PBO)

Mixed model analysis performed on log scale for the endpoints that satisfy normality. Geometric Mean (% CV) displayed.

We determined the distribution of HDL subpopulations by two-dimensional gel electrophoresis.20,21 In both groups there were marked increases in both the proportion and absolute amount of apoA-I in alpha 1 HDL (Table 4 and Supplemental Table I). Absolute concentrations of apoA-I in prebeta-1 particles increased proportionately at the end of anacetrapib treatment in both groups, without any changes in the percent of apoA-I in prebeta-1 HDL particles. There were parallel increases in both the absolute amount and proportion of apoA-II in alpha-1 HDL particles (Supplemental Tables II and III, respectively). Concomitant with increases in large alpha-1 HDL particles, there were, in general, reductions in the proportion of apoA-I and apoA-II in the smaller, alpha-2, alpha-3, and alpha 4 HDL subfractions in both groups.

Table 4. ApoA-I Mass (mg/dL) in HDL Subclasses.

ATV-ANA (N=29) PBO-ANA (N=10) All Participants (N=39)
ATV ANA+ATV % change
from baseline
(95% CI)		 P-
value		 PBO ANA % change
from baseline
(95% CI)		 P-
value		 ATV or
PBO		 ANA or
ANA+ATV		 % change
from baseline
(95% CI)		 P-
value
preβ-1 13.1 (49.65) 16.0 (42.62) 22.0 (2.97, 44.62) 0.023 12.6 (44.52) 16.1 (47.75) 27.8 (-3.94, 70.01) 0.090 12.9 (47.76) 16.1 (43.29) 24.9 (5.77, 47.44) 0.010
preβ-2 5.0 (40.71) 7.6 (36.72) 52.6 (34.12, 73.72) <.001 5.5 (42.52) 8.4 (23.45) 51.9 (22.21, 88.71) <.001 5.3 (40.84) 8.0 (33.64) 52.3 (34.17, 72.77) <.001
α-1 20.6 (47.15) 51.8 (52.90) 151.7 (113.02, 197.32) <.001 17.2 (40.74) 44.2 (76.19) 157.7 (94.78, 240.91) <.001 18.8 (45.85) 47.9 (58.79) 154.7 (116.38, 199.70) <.001
α-2 42.4 (23.66) 42.2 (27.94) -0.6 (-11.41, 11.58) 0.919 38.0 (35.81) 38.7 (19.89) 1.8 (-16.17, 23.61) 0.854 40.2 (27.14) 40.4 (26.07) 0.6 (-10.14, 12.63) 0.915
α-3 19.2 (27.33) 15.5 (25.78) -18.8 (-26.61, -10.23) <.001 21.2 (26.07) 18.0 (16.88) -14.8 (-28.11, 0.87) 0.062 20.1 (27.06) 16.7 (24.51) -16.9 (-24.66, -8.25) <.001
α-4* 9.5 (4.40) 8.9 (5.40) -13.8 (-29.52, 7.35) 0.202 10.0 (2.10) 11.0 (2.80) 7.9 (-10.89, 28.34) 0.275 9.7 (4.20) 9.2 (5.30) -6.9 (-21.17, 9.19) 0.482
preα-1 3.8 (39.53) 8.9 (50.53) 136.8 (99.77, 180.75) <.001 2.8 (61.62) 7.5 (62.53) 170.2 (103.04, 259.61) <.001 3.2 (47.31) 8.2 (53.60) 153.0 (114.21, 198.74) <.001
preα-2* 4.6 (1.60) 4.3 (1.70) -7.6 (-20.50, 6.09) 0.292 3.5 (2.90) 3.7 (2.80) -0.2 (-40.18, 33.69) 0.984 4.2 (2.00) 4.2 (1.70) -5.5 (-19.68, 7.38) 0.363
preα-3* 1.9 (1.10) 1.4 (0.60) -30.1 (-42.26, -15.04) <.001 2.0 (0.50) 1.9 (0.90) -16.8 (-45.51, 10.00) 0.193 1.9 (1.00) 1.5 (0.70) -27.5 (-38.21, -13.93) <.001
preα-4* 1.1 (0.60) 0.8 (0.55) -26.8 (-45.53, -4.26) 0.017 1.0 (0.40) 0.9 (0.50) -7.4 (-38.76, 38.87) 0.695 1.1 (0.60) 0.9 (0.50) -21.1 (-37.45, -2.90) 0.024
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Abbreviations: atorvastatin (ATV), anacetrapib (ANA), placebo (PBO)

Mixed model analysis performed on log scale for the endpoints that satisfy normality. Geometric Mean (% CV) displayed.

*

Non-parametric method used. Median (IQR) on raw scale displayed. Hodges-Lehmann estimate (95% CI) back transformed from log scale displayed under % Change from baseline. P-value from Wilcoxon signed rank test.

CETP mass increased 108% in the ATV-ANA group (P<0.001) and 91.2% in the PBO-ANA group (P=0.002; Table 5). This increase was caused by a significant reduction in the FCR of CETP (59.2% in the ATV-ANA group and 53.6% in PBO-ANA group; P<0.001 and P=0.002, respectively), with no change in CETP PR in either group (Figure 2 and Table 5). We did not find a correlation between changes in CETP mass and changes in HDL cholesterol levels. The impact of the change in CETP FCR on CETP mass is evident from the relatively strong correlation observed between changes in FCR and changes in CETP mass from Period 1 to Period 2 (r=0.66; P<0.0001). The FCRs for CETP at the end of Period 1 were 0.44 pools/day and 0.48 pools/day for the ATV-ANA and PBO-ANA groups, respectively. These FCRs are similar to the FCR for LDL apoB (0.44 pools/day) in these same participants during statin or placebo treatment.22 In contrast, the FCRs for CETP at the end of anacetrapib treatment were 0.18 pools/day and 0.25 pools/day, which are closer to the FCR of apoA-I observed in these participants (Table 3). There was a significant correlation between anacetrapib-induced changes in apoA-I FCR and changes in CETP FCR (r=0.40; P=0.037).

Table 5. CETP Kinetic Parameters.

ATV-ANA (N=29) PBO-ANA (N=10) All Participants (N=39)
ATV ANA+ATV % change
from baseline
(95% CI)		 P-
value		 PBO ANA % change
from baseline
(95% CI)		 P-
value		 ATV or
PBO		 ANA or
ANA+ATV		 % change
from baseline
(95% CI)		 P-
value
Plasma CETP mass*, (microgm/mL) 1.7 (0.50) 3.0 (2.50) 107.9 (63.30, 156.73) <.001 2.0 (0.40) 3.2 (2.90) 91.2 (22.48, 168.33) 0.002 1.8 (0.60) 3.0 (3.10) 101.7 (65.79, 141.53) <.001
Plasma CETP PS*, (microgm) 6,918 (3148.20) 12,312 (12684.60) 108.3 (63.35, 158.73) <.001 6,300 (3589.20) 13,084 (9043.65) 91.1 (24.58, 169.82) 0.002 6,789 (3277.80) 12,312 (10602.90) 99.9 (66.39, 141.46) <.001
Plasma CETP FCR*, (pools/day) 0.44 (0.09) 0.18 (0.08) -59.2 (-64.07, -52.66) <.001 0.48 (0.19) 0.25 (0.17) -53.6 (-63.55, -38.01) 0.002 0.44 (0.13) 0.19 (0.11) -57.6 (-62.67, -51.71) <.001
Plasma CETP PR, (microgm/kg/day) 35.8 (29.93) 31.1 (54.16) -13.1 (-25.84, 1.82) 0.081 44.1 (25.04) 39.2 (75.67) -11.0 (-32.05, 16.57) 0.387 39.7 (29.97) 34.9 (59.96) -12.1 (-24.80, 2.84) 0.105
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Abbreviations: atorvastatin (ATV), anacetrapib (ANA), placebo (PBO)

Mixed model analysis performed on log scale for the endpoints that satisfy normality. Geometric Mean (% CV) displayed.

*

Non-parametric method used. Median (IQR) on raw scale displayed. Hodges-Lehmann estimate (95% CI) back transformed from log scale displayed under % Change from baseline. P-value from Wilcoxon signed rank test.

Figure 2.

Figure 2

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Plasma CETP kinetic parameters for participants in ATV-ANA, PBO-ANA, and combined groups at the end of each treatment period. Pool size (PS) and fractional catabolic rate (FCR) data are shown as median values, and production rate (PR) is shown as geometric mean values. Unadjusted raw p values: ‡p<0.01; †p<0.001. Abbreviations: atorvastatin (ATV), anacetrapib (ANA), placebo (PBO). Interquartile range displayed at base of each bar for PS and FCR; % CV displayed at base of each bar for PR.

Previous studies in CETP deficient participants identified apoA-I lipoproteins in the LDL density range.23 We measured apoA-I levels in the LDL density range (1.019-1063 g/mL) during statin or placebo treatment and found that they were usually below the lower limit of the assay (<3mg/dL) or, if present, were very low (Supplemental Table IV). However, we did observe a greater number of participants with measureable or higher levels of apoA-I in this density range after anacetrapib treatment.

Discussion

HDL is the most complex, heterogeneous, protein-enriched, and lipid-poor class of lipoproteins, varying in density (d: 1.063 to 1.210 g/L), in diameter (7nm to 13nm), in the number of apoA-I per particle (2 to 4), and in the presence or absence of other proteins and apolipoproteins. The study of HDL metabolism and functionality has been challenging due to dynamic nature of its lipid components, which can be catabolized or transferred between lipoproteins, and the rapid exchanges of its apolipoproteins between its subspecies and other lipoprotein classes. Studies conducted over the past 4 decades, with either exogenous or endogenous labeling of apoA-I, have demonstrated that, in general, low levels of apoA-I are associated with increased FCRs of the protein, although some individuals with low apoA-I levels do have defects in the secretion of the protein into plasma.24-26

The FCR of HDL apoA-I is affected mainly by size, density, and core lipid composition; larger, more buoyant HDL are cleared more slowly than smaller, denser HDL.26-28 The physical-biochemical characteristics of HDL result from the actions of lipoprotein lipase (LpL), hepatic lipase (HL), endothelial lipase (EL), LCAT, and CETP. In this study, anacetrapib treatment increased HDL cholesterol dramatically in both groups while decreasing HDL-TG. These changes were paralleled by dramatic shifts in the size distribution of HDL toward larger, alpha1 particles and a significant decrease in the FCR of apoA-I. In accord with our results, individuals completely deficient in CETP have large, mainly alpha1-sized, CE-enriched HDL with increased apoA-I levels and very low apoA-I FCRs.29-31

Our results in the PBO-ANA group are similar to those reported by Brousseau et al for torcetrapib monotherapy.16 However, whereas Brousseau et al did not observe a reduction in apoA-I FCR in participants taking both torcetrapib and atorvastatin,16 we observed comparable effects of anacetrapib on apoA-I FCR in participants receiving either placebo or statin as background treatment. Brousseau et al found increased apoA-I PRs when torcetrapib was added to atorvastatin;16 we saw no change in apoA-I PR in our study when comparing atorvastatin alone with atorvastatin plus anacetrapib. The reasons for these differences are unclear; they may be related to differences between torcetrapib and anacetrapib, or may simply reflect the fact that the torcetrapib study was substantially smaller.

As noted above, we found marked absolute and relative increases in apoA-I containing alpha1 particles with, in general, decreases in apoA-I alpha 2, 3, and 4 subclasses. Brousseau et al reported similar effects of torcetrapib on the distribution of apoA-I across HDL subpopulations, with increased apoA-I in the large alpha1 subpopulation and decreases in the smaller alpha 3 subclass.16 These metabolic and compositional changes appear to be direct effects of CETP inhibition; anacetrapib did not change the activities of HL or LpL in PBO-ANA and ATV-ANA groups.22 Additionally, the cholesterol esterification rate, which plays a key role in the maturation of HDL particles, fell by about 30% during anacetrapib therapy.

Importantly, the proportion of apoA-I particles in the pre-beta range did not change during anacetrapib treatment on either ATV or placebo background; in fact, the absolute level of pre-beta 1 apoA-I particles increased during anacetrapib treatment. Pre-beta apoA-I HDL particles are important acceptors of free cholesterol from cells via the ABCA1 transporter.32 Our finding that the absolute amount of pre-beta apoA-I-containing HDL is increased during anacetrapib treatment is consistent with prior studies of anacetrapib in hamsters33 and supports the view that inhibition of CETP may increase cholesterol efflux capacity, as has been suggested.34 The finding that anacetrapib increased RCT in hamsters is consistent with this view.35 The mechanisms by which CETP inhibition with anacetrapib increases prebeta HDL remain to be established.

Previous studies of apoA-I metabolism during CETP inhibition have focused on apoA-I within HDL.16 Ikewaki et al isolated apoE-rich lipoproteins, containing apoA-I without apoB, from the d=1.019-1.063 gm/ml fraction of plasma from patients with CETP deficiency; these apoE-rich lipoproteins were referred to as HDL1.29 Therefore, we determined if pharmacologic inhibition of CETP affected the presence of apoA-I in the LDL density range. Anacetrapib-mediated inhibition of CETP significantly increased the number of individuals with measurable apoA-I within the isolated LDL density range fractions, both on the background of placebo and atorvastatin treatment. It is likely that this apoA-I is on a large HDL1-like HDL particle. The relevance of large HDL1-like particles in human biology is currently unknown.

Although the apoA-II pool size increased significantly (but modestly) by 13% during anacetrapib treatment (compared to the 31% increase in apoA-I pool size), we did not observe significant changes in either FCR or PR of apoA-II. Our results differ from those of Brousseau et al, who observed a lowering of apoA-II FCR between 10-20% during torcetrapib therapy, dependent on the dose used.17 The reason for this difference in results is unclear, although the participants studied by Brousseau et al had much lower baseline HDL-C levels than our study subjects. We did observe a larger reduction of apoA-II FCR in the PBO-ANA group, but this was not statistically significant. The effects of CETP inhibition on apoA-II subpopulations were similar in the two studies, with a shift of apoA-II from small to larger alpha subclasses. It is notable that CETP inhibition has a substantially greater effect on apoA-I turnover and pool size than on apoA-II, which suggests that CETP inhibition may preferentially affect the LpA-I HDL particles that contain apoA-I but not apoA-II. Further studies are needed to explore this interesting implication.

For the first time, the effects of CETP inhibition on the kinetics of CETP were examined. The regulation of CETP gene expression and control of plasma CETP concentration by its synthesis have been well defined in animal models.36,37 Large cohort studies have identified non-coding genetic variants that affect plasma CETP levels38 most likely through affecting transcription of the CETP gene. The impact of the synthesis and fractional clearance of CETP on its plasma concentrations has not been reported in humans. Our current findings indicate that treatment with a CETP inhibitor is associated with a marked increase in plasma levels of the protein due to a decrease in the FCR without a change in PR. Of note, the FCR of CETP was similar to that of LDL apoB during treatment with placebo or atorvastatin alone.22 This finding is not consistent with in vitro data where CETP was found mainly in the lipoprotein-free region of untreated plasma.23 Anacetrapib forms a complex with CETP and HDL,23 a model concordant with our finding that, during anacetrapib treatment, the FCR for CETP was much closer to the FCR of apoA-I, and that changes in CETP FCR on anacetrapib were strongly related to changes in apoA-I FCR. Validation of the model23 will require further studies.

A limitation of this study is the fixed sequence protocol. Thus, although the study was double-blind regarding placebo plus anacetrapib versus atorvastatin plus anacetrapib, it was single-blind regarding the administration of anacetrapib, which the investigators knew was during the second treatment period. Because of the extremely long half-life of anacetrapib, a cross-over design was not possible and a parallel arm study would have meant increasing the size of the study by 3- to 4-fold. A “study effect” or investigator-mediated bias cannot, therefore, be ruled out, but seems unlikely considering the magnitude of changes in plasma HDL and LDL cholesterol levels. In conclusion, anacetrapib, a CETP inhibitor, significantly increased plasma levels of HDL-C, apoA-I, and, to a lesser extent, apoA-II. The increase in apoA-I levels resulted from a significant reduction in the FCR of apoA-I without a change in PR; the increase in apoA-II, which was smaller, was not clearly associated with changes in either FCR or PR of the protein. Concomitant with the changes in plasma levels, there was a shift of HDL subclasses from smaller to larger species, but the absolute mass of both the pre-beta1 and pre-beta2 subclasses increased as well. CETP mass doubled during treatment with anacetrapib and this change resulted from a significant reduction in the FCR of the protein with no change in synthesis. During the placebo period, the FCR of CETP was similar to that of LDL apoB; during anacetrapib treatment, the FCR of CETP was similar to that of apoA-I, suggesting that CETP may be associated with HDL or apoA-I during treatment with anacetrapib. These results enhance our understanding of the effects of potent CETP inhibition on HDL apolipoprotein kinetics and subclass distribution, and for the first time provide a clear explanation for the increase in plasma CETP mass with CETP inhibition. We must note, however, that our studies do not inform directly the issue of how CETP inhibitors effect RCT, which is determined by net efflux of cholesterol from peripheral tissues to the liver and then to the intestine for excretion. The fact that free-living individuals with increased HDL-C levels have reduced FCRs26 and reduced risk for CVD supports the hypothesis that treatments to lower the FCR of apoA-I while raising HDL-C will be beneficial. We await the results of the REVEAL study, the last remaining clinical trial testing this hypothesis.

Supplementary Material

Methods
Supplement

Significance.

We conducted detailed analyses of the effects of anacetrapib, a CETP inhibitor presently being studied in a large CVD outcomes trial, on the metabolism of apoA-I, apoA-II, and CETP. ApoA-I levels increased 30% during anacetrapib treatment due to a significant decrease in the FCR of apoA-I. Importantly, the reductions in the fractional turnover of apoA-I were associated with an increase in both pre-beta as well as larger alpha HDL subfractions. We also determined, for the first time, the turnover of plasma CETP. In the combined background of placebo or atorvastatin, the FCR of CETP was 0.44 pools/day and this decreased to 0.19 pools/day after anacetrapib treatment. The reduction in FCR resulted in a doubling of CETP levels in blood with no change in production. The FCR of CETP on placebo or atorvastatin was similar to that of LDL apoB whereas it was similar to HDL apoA-I during anacetrapib treatment.

Acknowledgments

The authors would like to acknowledge laboratory assistance from Marianna Pavlyha BS, and the inpatient and outpatient nursing staff (CRC) and Bionutrition unit of the Irving Institute for Clinical and Translational Science Award (IICTR) at Columbia University Medical Center and University of Pennsylvania. Editorial assistance was provided by Sheila Erespe MS and Jennifer Rotonda PhD (Merck & Co., Inc., Kenilworth, NJ, USA).

Funding source: Merck & Co., Inc., Kenilworth, NJ provided financial support for the conduct of the study. Additional support for instrumentation for this work at the Perelman School of Medicine at the University of Pennsylvania was obtained from the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR000003. Further support for instrumentation for this work at the Irving Center for Clinical Research at the College of Physicians & Surgeons of Columbia University was obtained from the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR000040.

Footnotes

Authors: GRS, JSM, CN, PJ, BA, AOJL, JAW, DSD, WK, RR, SH, TT, RLD, EMDG, HR, ALB, YL, MEL, DEG, DJR, and HNG are responsible for the work described in this paper. All authors were involved in at least one of the following: conception, design, acquisition, analysis, statistical analysis, and interpretation of data in addition to drafting the manuscript and/or revising/reviewing the manuscript for important intellectual content. All authors provided final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Disclosures: PJ, AOJ-L, JAW, YL, MEL, and DEG are or were employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA and may own stock and/or hold stock options in the Company. JSM received grant support and honoraria from Merck. GRS reports grants and non-financial support from Merck during the conduct of the study. RLD and DSD report grants from Merck during the conduct of the study. HNG reports grants from Merck during the conduct of the study and personal fees from Merck outside the submitted work and is a member of Merck Scientific Advisory Boards. DJR is a member of the Merck Scientific Advisory Board. ALB, CN, BA, WK, RR, SH, TT, EMD, and HR have no disclosures.

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Supplementary Materials

Methods
NIHMS764488-supplement-Methods.pdf (297.5KB, pdf)
Supplement
NIHMS764488-supplement-Supplement.pdf (173.9KB, pdf)

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