Single-dose pharmacokinetics and pharmacodynamics of anacetrapib, a potent cholesteryl ester transfer protein (CETP) inhibitor, in healthy subjects

Abstract

AIMS

Anacetrapib is an orally active and potent inhibitor of CETP in development for the treatment of dyslipidaemia. These studies endeavoured to establish the safety, tolerability, pharmacokinetics and pharmacodynamics of rising single doses of anacetrapib, administered in fasted or fed conditions, and to preliminarily assess the effect of food, age, gender and obesity on the single-dose pharmacokinetics and pharmacodynamics of anacetrapib.

METHODS

Safety, tolerability, anacetrapib concentrations and CETP activity were evaluated.

RESULTS

Anacetrapib was rapidly absorbed, with peak concentrations occurring at ∼4 h post-dose and an apparent terminal half-life ranging from ∼9 to 62 h in the fasted state and from ∼42 to ∼83 h in the fed state. Plasma AUC and C_max appeared to increase in a less than approximately dose-dependent manner in the fasted state, with an apparent plateau in absorption at higher doses. Single doses of anacetrapib markedly and dose-dependently inhibited serum CETP activity with peak effects of ∼90% inhibition at t_max and ∼58% inhibition at 24 h post-dose. An E_max model best described the plasma anacetrapib concentration vs CETP activity relationship with an EC₅₀ of ∼22 nm. Food increased exposure to anacetrapib; up to ∼two–three-fold with a low-fat meal and by up to ∼six–eight fold with a high-fat meal. Anacetrapib pharmacokinetics and pharmacodynamics were similar in elderly vs young adults, women vs men, and obese vs non-obese young adults. Anacetrapib was well tolerated and was not associated with any meaningful increase in blood pressure.

CONCLUSIONS

Whereas food increased exposure to anacetrapib significantly, age, gender and obese status did not meaningfully influence anacetrapib pharmacokinetics and pharmacodynamics.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Inhibition of cholesteryl ester transfer protein (CETP) is considered a potential new mechanism for the treatment of dyslipidaemia. Whereas several studies have described the effects of the CETP inhibitors, torcetrapib (Pfizer) and dalcetrapib (Roche), on lipids and lipoproteins, few studies have characterized the effect of rising single doses on the pharmacokinetics and pharmacodynamics (CETP activity) of an individual CETP inhibitor or have described the effects of intrinsic and extrinsic factors on the pharmacokinetics and pharmacodynamics of these agents.

WHAT THIS STUDY ADDS

• We have characterized the exposure/response relationship for the inhibition of CETP activity over a wide exposure range. We have shown that single doses of anacetrapib produced marked and dose-dependent inhibition of serum CETP activity with peak effects of ∼90% inhibition at t_max and persistent inhibition at 24 h post-dose. We have also shown that whereas food increased exposures to anacetrapib significantly and variably, age, gender and obese status did not meaningfully influence the pharmacokinetics and pharmacodynamics of anacetrapib.

Introduction

Lowering LDL-cholesterol (LDL-C) remains a primary therapeutic goal for reducing cardiovascular (CV) risk. Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) are effective in lowering LDL-C and are the cornerstone of therapy for reducing CV risk [1–3]. However, patients still remain at an unacceptably high residual risk for cardiovascular events, underscoring the need for pharmacotherapeutic options that can result in incremental LDL-C-lowering while producing beneficial effects on other targets that may influence the dyslipidaemic state, including but not limited to high-density lipoprotein-cholesterol (HDL-C) [4].

One mechanism being investigated as a potential approach to reduce LDL-C while raising HDL-C is the cholesteryl ester transfer protein (CETP) pathway [5]. CETP is a plasma protein that catalyzes the exchange of cholesteryl esters and triglycerides (TG) between HDL and the atherogenic apolipoprotein (apo) B-containing lipoproteins, especially very low density lipoprotein (VLDL). A clinical outcomes study with the investigational CETP inhibitor developed by Pfizer, torcetrapib in high risk patients, the ‘Investigation of Lipid Level Management to Understand Its Impact in Atherosclerotic Events’ (ILLUMINATE), was stopped early due to an excess in mortality due to both cardiovascular and noncardiovascular reasons [6]. Thus, there is no proven evidence that inhibiting CETP activity reduces CV risk in mortality and morbidity CV outcome trials. It is presumed that developmental CETP inhibitors which do not share the off-target liabilities of torcetrapib such as increased blood pressure and aldosterone, and altered serum electrolyte concentrations, could shed light on the clinical significance of this mechanism.

Anacetrapib (MK-0859; Merck & Co., Inc., Whitehouse Station, NJ) is an orally active, potent inhibitor of CETP currently in phase III development for the treatment of dyslipidaemias including primary hypercholesterolaemia and mixed hyperlipidaemia which increase susceptibility to atherosclerotic cardiovascular disease [7]. Anacetrapib inhibits the CETP mediated transfer of cholesteryl ester (IC₅₀ 16 nm) and triglyceride (IC₅₀ 29 nm), and increases HDL in 6-Tg(CETP) transgenic mice expressing the cynomolgus monkey CETP gene [7]. In nonclinical experiments, anacetrapib did not increase blood pressure in animals which do not contain CETP and in transgenic mice expressing the cynomologus CETP gene, and did not stimulate aldosterone release in vitro, at concentrations up to 10 µm, whereas a positive control angiotensin II (0.1 µm) did [8].

The purpose of this study, representing the first introduction of anacetrapib to humans, was to evaluate the pharmacokinetics, pharmacodynamics and tolerability of single oral doses of anacetrapib in healthy subjects. Understanding the effect of a single dose administration of anacetrapib to elderly obese and non-obese men and women and to healthy young women of non-childbearing potential, all of whom comprising a large segment of the initial target population, is important for the design of Phase IIb studies in these populations. Therefore, another purpose of this study was to examine the safety, tolerability, pharmacokinetics and pharmacodynamics of single oral doses of anacetrapib in healthy elderly male and female subjects, healthy young obese subjects and young female volunteers (women of non-childbearing potential). The effect of food on the pharmacokinetics of anacetrapib was also investigated.

Methods

Study participants

All study participants provided written informed consent prior to enrolment. The protocols were approved by the Ethical Review Committee of University Ghent, Ghent, Belgium (Study 1), the Southern Institutional Review Board, Miami, Florida (Study 2) and the Independent Institutional Review Board, Plantation, Florida (Study 3), and were conducted in accordance with the guidelines on good clinical practice and with ethical standards for human experimentation established by the Declaration of Helsinki. Subjects were in good general health according to routine medical history, physical examination, vital signs, and laboratory data.

Study design

Study 1

A double-blind, randomized, placebo-controlled study was performed in two stages. In an alternating-panel design, rising single oral doses of anacetrapib (n= 6/panel) or matching placebo (n= 2/panel), as a liquid-filled capsule (LFC) formulation, were administered after an overnight fast (2–1000 mg) and with a standard high fat breakfast, 827 kcal, 57% fat (25–800 mg) in healthy male volunteers aged 18–45 years. In a three-period, randomized, crossover manner, subjects were randomized to receive a single oral dose of a 125 mg anacetrapib or placebo capsule administered with 240 ml of water in the fasted state, following a low fat breakfast, and following a high fat breakfast. The same six subjects received anacetrapib in periods 1–3 and the same two subjects received placebo, and there was a washout period of at least 10 days between the study periods.

Study 2

In an open-label, randomized, crossover manner, eight male subjects were randomized, on day 1, to the sequence of treatments in which they received a single 250 mg dose of anacetrapib administered as a LFC with a standard high fat breakfast, either as 2 × 125 mg or as 5 × 50 mg, separated by a washout interval of 5 days between treatments (day 1 through day 5). In another cohort of subjects, done as an open-label, four-period assessment (with two two-period crossover parts), eight male subjects were randomized in two periods, to a sequence of two treatments, in which they received a single 50 mg dose of anacetrapib administered as a LFC with a standard high fat breakfast, either as 1 × 50 mg or as 5 × 10 mg, separated by a washout interval of 5 days between treatments (day 1 through day 5). After completion of the first crossover portion of the study, a second crossover design was implemented on day 12 (i.e. 7 days after the previous dose of study medication). In this segment, subjects received a single 50 mg dose of anacetrapib as a LFC after an overnight fast as 1 × 50 mg or as a LFC after an overnight fast as 5 × 10 mg, separated by a washout period of 5 days between treatments (day 12 through day 17).

Study 3

A double-blind, randomized, placebo-controlled, four-panel single-dose study was performed in a total of 32 subjects in four panels. Subjects received a single 125 mg dose of anacetrapib or placebo, formulated as a LFC, after an overnight fast. The populations included in each panel were eight healthy elderly (65 ≤ age ≤ 80 years) male subjects (n= 6 active, n= 2 placebo) with body mass index of 18.5 ≤ BMI ≤ 27.0 kg m⁻², eight healthy elderly (65 ≤ age ≤ 80 years) female subjects (n= 6 active, n= 2 placebo) with body mass index of 18.5 ≤ BMI ≤ 27.0 kg m⁻², eight healthy young (18 ≤ age ≤ 45 years) female subjects (n= 6 active, n= 2 placebo) with body mass index of 18.5 ≤ BMI ≤ 27.0 kg m⁻², and eight healthy young obese (18 ≤ age ≤ 45 years) male and female subjects (four of each gender, n= 6 active, n= 2 placebo) with body mass index of 30.0 ≤ BMI ≤ 35.0 kg m⁻².

Safety and tolerability

In all studies, safety was assessed by physical examinations, and by monitoring vital signs, orthostatic vital signs, 12-lead electrocardiograms (including an assessment of QTc and PR interval), and safety laboratory measurements comprising routine haematology, serum chemistry and urinalysis performed pre-study, at various time points post-dosing and at post-study. Adverse experiences were monitored throughout the study. Furthermore, specifically in study 1 where rising single doses were given for the first time in human subjects, blood pressure (systolic and diastolic) was carefully monitored by an automatic blood pressure monitor, and measurements taken at 15 min intervals beginning 1 h prior to administration of anacetrapib. Baseline blood pressure (systolic and diastolic) was the average of the blood pressure readings taken at the end of each of the three 15 min periods just prior to dosing. For orthostatic measurements, subjects stood upright from a semi-recumbent position for 2 min prior to measurement. Pre-dose, 1 and 4 h post-dose measurements were taken after semi-recumbent vital signs were obtained.

Anacetrapib pharmacokinetics and pharmacodynamics

Blood samples were obtained pre-dose and at selected time points up to 216 h post-dose for determination of anacetrapib plasma concentrations and serum CETP activity, using methods that have been previously described [9].

Measurement of anacetrapib plasma concentrations

A sensitive, specific, and validated HPLC-MS/MS assay was used for the determination of anacetrapib in human plasma over the concentration range of 1–1000 ng ml⁻¹. The LLOQ (lower limit of quantification) was defined as the lowest concentration on the standard curve that can be measured with a precision better than 15% CV and an accuracy within ±15% of the nominal concentration. The LLOQ was 1 ng ml⁻¹ (using 0.25 ml of plasma), the inter-assay precision was <5% at all concentrations within the standard curve range, and the assay accuracy was 95–103% of nominal. The method to quantify anacetrapib urine concentrations was based on liquid-liquid extraction in a 96-well format of drug from acidified Tween 20-treated urine. Drug and internal standard were chromatographed using HPLC and detected by MS/MS in the positive ionization mode using a heated nebulizer interface and monitoring their precursor to product ion combinations in multiple reaction monitoring (MRM) mode. The limit of reliable quantification for the assay was 10 ng ml⁻¹ when 0.5 ml of urine is processed. The dynamic range of the assay was 10 ng ml⁻¹ to 2000 ng ml⁻¹. Plasma concentration vs time data were analyzed by noncompartmental pharmacokinetic methods using WinNonlin (Enterprise Version 5.0.1, Pharsight Corporation, Mountain View, California) to determine the pharmacokinetic parameter estimates. Pharmacokinetic parameters (apparent terminal half-life t_1/2, area under the plasma concentration-time curve AUC_0–∞, maximum observed plasma concentration C_max and its corresponding t_max, C_{24 h}) were determined using non-compartmental methods using established methods. Pseudo SD, calculated as (interquartile distance)/1.35, is reported for the pharmacokinetic parameter apparent terminal half-life values that are reported as harmonic mean [10].

Measurement of CETP activity

Serum samples from blood collected without anticoagulant were prepared at the clinical site and stored at or below −70°C until analysis. Enzyme activity was assessed by incubating serum in a reaction mixture containing a native lipoprotein used as acceptor and a synthetic donor particle similar in size and density to HDL which contains a core of fluorescently labeled cholesterol ester (CE) and a fluoresence quenching agent. As a molecule of CE is removed from the donor and transferred to acceptor by CETP, it escapes quench and becomes fully fluorescent. The assay measures the increase in fluoresence over time as a readout of CETP activity. Both donor and acceptor particles used in the CETP activity assay were prepared by Merck Basic Research. Synthetic donor particles contain Dioleoyl Phosphatidyl choline (DOPC), Bodipy-CE (Molecular Probes C-3927), Triolein, Dabcyl dicetyl amide, and apoHDL. Native lipoproteins from human blood were used as acceptor particles. The release of fluorescence from the donor particles was measured using an excitation setting of 480 nm and emission of 511 nm over a 59 min interval. The change in fluorescence between each 45 s interval was averaged over 20 min to calculate the slope for each individual sample. Enzyme activity was defined as the mean slope in relative fluorescent units/second (RFU s⁻¹) from 10 to 30 min at 37°C.

Statistical methods and analysis

Natural log-transformed pharmacokinetic parameter values were analyzed with linear models, from which means and confidence intervals were generated for the comparisons of interest. To evaluate the effect of age, gender and obesity, an analysis of variance (anova) model was used having factors for gender (male, female), age (elderly, young), obesity (obese, non-obese), and gender-by-age-by-obesity interaction. A two-sided 90% confidence interval for the true mean difference was calculated using the mean square error from the anova and referencing a t-distribution. These confidence limits were then exponentiated to obtain a 90% CI for the true geometric mean ratio. To assess the effect of capsule units, models having fixed effects for sequence, treatment, and period and a random effect for subject were used.

PK/PD(CETP) analysis

Data from the studies described in this manuscript together with other multiple-dose PK and PD data [9] were used to construct a PK/CETP activity relationship [unpublished data]. Population PK model development was performed using NONMEM, version V (Globomax, Hanover, MD). A first-order conditional estimation with interaction method was used. Diagnostic plots, point and interval estimates of parameters, and the minimum value of the objective function were used to guide model building and to assess goodness-of-fit. CETP model development was carried out using the NLME package as implemented in SPLUS version 8.0 (Insightful, Seattle, USA) and the Markov Chain-Monte Carlo methods available in WinBUGS. Using the developed PK model, population estimates of anacetrapib plasma concentrations were computed for each CETP assessment time point. A model of CETP total activity was developed using the predicted anacetrapib concentrations. Because of the effects of anacetrapib on CETP mass after multiple-dose administration (not generated as part of studies described in this manuscript) the model for CETP total activity was developed as the product of CETP specific activity and CETP mass in the collective dataset (data on file).

Results

Pharmacokinetics

Figure 1 illustrates the plasma anacetrapib concentration vs time profile and Table 1 summarizes the key anacetrapib pharmacokinetic parameters after rising single oral doses of anacetrapib, administered after an overnight fast (2–1000 mg) and after a high fat breakfast (25–800 mg). The pharmacokinetics of anacetrapib were characterized by less than dose-proportional increases in exposure in both fasted and fed conditions. However, administration with food significantly increased exposure. As part of the rising single dose design, the effect of a high fat meal on the pharmacokinetics of anacetrapib was studied preliminarily at the 25 mg dose (Figure 1). At this dose, the fed/fasted geometric mean ratios (GMR) for AUC_0–∞, C_max, and C_{24 h} were 3.67, 3.96, and 2.70, respectively. The apparent t_1/2 for anacetrapib ranged from approximately 9–62 h in the fasted state (Table 1a) and from approximately 42–83 h in the fed state (Table 1b). Median t_max values across doses ranged from 3 to 6 h. The fraction of the anacetrapib dose excreted unchanged in urine was negligible (all concentrations below the limit of quantification).

Figure 1.

Mean plasma anacetrapib concentration–time (A), CETP activity–time (B) and PK/CETP activity (C) profiles after administration of rising single oral doses in healthy male subjects.

Table 1.

Summary of the pharmacokinetic parameters for anacetrapib following rising single dose administration to healthy subjects, administered after an overnight fast (A) or with a meal (B)

(A) Fasted pharmacokinetic parameters
	Geometric mean (CV%), n= 6
						125 mg	125 mg
	2 mg	5 mg	10 mg	25 mg	50 mg	(2 × 50-mg + 2 × 10-mg + 5 × 1 mg)	(1 × 125-mg)	250 mg	500 mg	1000 mg
AUC0–∞	0.26	0.68	2.0	2.9	5.3	9.9	4.0	14.0	15.7	13.6
(µm h)	(52)	(30)	(42)	(16)	(36)	(24)	(50)	(17)	(63)	(28)
CL/F	12.1	11.6	7.7	13.5	14.8	19.8	49.6	27.9	49.9	118.9
(l h−1)	(52)	(30)	(42)	(16)	(36)	(24)	(50)	(17)	(63)	(28)
Cmax (nm)	24	56	145	246	414	731	168	860	788	889
	(33)	(28	(37)	(7)	(28)	(29)	(41)	(32)	58	(24)
tmax (h)*	4	4	4	4	4	4	4	4	4	4
	(4–8)	(4–4)	(2–4)	(2–4)	(2–4)	(2–4)	(4–4)	(2–4)	(2–8)	(4–4)
Terminal	9.3	21.0	28.9	28.7	34.5	46.1	55.7	44.2	60.0	61.9
t1/2 (h)†	(6)	(7)	(5)	(6)	(8)	(14)	(14)	(26)	(10)	(19)
C24 h (nm)	3.2	6.7	21	27	46	69	32	102	102	83
	(13)	(13)	(20)	(7)	(17)	(10)	(19)	(9)	(25)	(12)
(B) Fed pharmacokinetic parameters
		Geometric mean (CV%), n= 6
Dose	25 mg	100 mg	125 mg	125 mg	200 mg	400 mg	800 mg
Meal	High fat	High fat	High fat	Low fat	High fat	High fat	High fat
AUC0–∞	10.7	34.2	23.9	9.0	44.0	82.0	117.4
(µm h)	(37)	(35)	(52)	(40)	(11)	(57)	(25)
CL/F	3.7	4.6	8.2	21.7	7.2	7.6	10.7
(l h−1)	(37)	(35)	(52)	(40)	(11)	(57)	(25)
Cmax (nm)	972	2421	1498	561	3959	5064	9140
	(32)	(32)	(81)	(28)	(48)	(42)	(21)
tmax (h)*	3	4	4	4	4	6	4
	(2–4)	(4–8)	(4–12)	(2–4)	(2–4)	(4–18)	(2–4)
Terminal	42.2	62.0	54.4	50.2	50.9	61.5	82.6
t1/2 (h)†	(11)	(6)	(11)	(9)	(21)	27	(48)
C24 h (nm)	72	216	144	65	249	494	668
	(16)	(23)	(20)	(18)	(7)	(44)	(19)

^*Median (range);

^†Harmonic mean (pseudo SD).

Since food increased exposure based on preliminary assessment, a crossover assessment was performed at a single 125 mg (1 × 125 mg) dose, administered after an overnight fast, with a low-fat meal and with a high-fat meal. Table 2 summarizes the effect of food on the pharmacokinetic parameters of anacetrapib. Food increased anacetrapib exposure significantly and variably, regardless of the type of meal (Table 2). Interestingly, anacetrapib exposure at the 125 mg fasted dose used in this panel (administered as 1 × 125 mg, one capsule) appeared in general lower than those associated with the 125 mg fasted dose from the rising single dose cohorts (administered as 2 × 50 mg, 2 × 10 mg, and 5 × 1 mg for a total of nine capsules). Specifically, exposures were ∼2.5-fold higher for AUC_0–∞ and ∼4.4-fold higher for C_max for the dose administered as nine capsules as compared with the dose administered as one capsule.

Table 2.

Comparison of pharmacokinetic parameters of anacetrapib following a single dose of 125 mg, administered in the fasted state, or with a high-fat or low-fat meal

	n	Geometric mean* (95% CI)			GMR* (90% CI)
125 mg high fat	125 mg low fat	125 mg fasted	High fat/fasted	Low fat/fasted
AUC0–∞ (µm h)	6	23.9 (15.8, 36.1)	9.0 (6.0, 13.7)	4.0 (2.6, 6.0)	6.03 (4.11, 8.85)	2.28 (1.56, 3.35)
Cmax (nm)	6	1498.2 (965.2, 2325.7)	561.3 (361.6, 871.4)	167.8 (108.1, 260.4)	8.93 (5.68, 14.03)	3.35 (2.13, 5.26)
C24 h (nm)	6	144.1 (96.1, 216.2)	65.5 (43.6, 98.2)	31.7 (21.1, 47.5)	4.55 (3.24, 6.39)	2.07 (1.47, 2.90)

^*Model-based least squares estimate.

To assess whether the increased exposure seen with the dose associated with the higher number of capsules was due to higher surfactant volume in the liquid-filled capsule, a study was performed at two doses, 50 mg and 250 mg, in fasted and high fat meal states. At the 250 mg dose, the impact of five capsules (2479.5 µl) vs two (894.8 µl) capsules was assessed. At the 50 mg dose, the impact of five capsules (2679.5 µl) vs one (495.9 µl) was assessed. The results are summarized in Table 3. Plasma anacetrapib AUC_0–∞ was approximately 50% higher for the 50 mg five capsule treatment as compared with the 50 mg one capsule treatment. Similarly, plasma AUC_0–∞ was approximately 20% higher for the 250 mg five capsule treatment compared with the 250 mg two capsule treatment period. Similar changes were observed in C_max and C_{24 h} (Table 3), but t_max and t_1/2 did not appear to be altered by the number of capsules administered (data not shown). The magnitude of the capsule effect appeared to be diminished in the fed state as compared with the fasted state.

Table 3A.

Comparison of pharmacokinetics parameters of anacetrapib following single dose 2 × 125 mg or 5 × 50 mg anacetrapib with a high fat meal

PK parameter	n	Geometric mean* (95% CI)		GMR* (90% CI)_
2 × 125 mg fed	5 × 50 mg fed	5 × 50 mg/2 × 125 mg
AUC0–∞ (µm h)	8	42.6 (30.6, 59.3)	51.1 (36.7, 71.2)	1.20 (1.00, 1.44)
Cmax (nm)	8	4387.6 (2945.9, 6534.7)	5716.8 (3838.4, 8514.5)	1.30 (1.05, 1.62)
C24 h (nm)	8	147.1 (103.3, 209.5)	165.4 (116.1, 235.6)	1.12 (0.90, 1.41)

Table 3B.

Comparison of pharmacokinetics parameters of anacetrapib following single dose 1 × 50 mg or 5 × 10 mg anacetrapib with a high fat meal

Parameter	Dose	n†	Geometric mean* (95% CI)		GMR* (90% CI)	Ratio of food effects* (90% CI)
Fed	Fasted	Fed/fasted	5 × 10 mg/1 × 50 mg
AUC0–∞ (µm h)	1 × 50 mg	7	13.8 (9.4, 20.2)	6.2 (4.2, 9.0)	2.23 (1.48, 3.37)	0.90 (0.64, 1.26)
5 × 10 mg	7	21.3 (14.7, 30.8)	10.6 (7.2, 15.8)	2.00 (1.33, 3.01)
Cmax (nm)	1 × 50 mg	7	1722.5 (988.4, 3001.7)	160.2 (92.7, 276.8)	10.75 (4.92, 23.49)	0.40 (0.21, 0.75)
5 × 10 mg	7	2099.3 (1250.7, 3523.5)	493.6 (274.5, 887.9)	4.25 (1.96, 9.21)
C24 h (nm)	1 × 50 mg	7	52.9 (39.3, 71.2)	25.8 (19.3, 34.6)	2.05 (1.37, 3.07)	0.88 (0.64, 1.23)
5 × 10 mg	7	77.4 (58.6, 102.2)	42.7 (31.2, 58.3)	1.81 (1.22, 2.70

^*Model-based least squares estimate.

^†Excludes one subject who discontinued after period 1.

Table 4 summarizes the effect of age, gender and obesity on the pharmacokinetic parameters of anacetrapib following administration of a single oral dose of 125 mg.

Table 4.

Comparison of pharmacokinetic parameters of anacetrapib following single-dose 125 mg to elderly and young non-obese male and female subjects and young obese male and female subjects

Demographic group	n	AUC0–∞ (µm h)	Cmax (nm)	C24 h (nm)	Terminal t1/2 (h)
Geometric mean (95% CI)*
Non-obese elderly males	6	3.4	266.6	29.5	38.7
		(2.4, 4.7)	(194.5, 365.6)	(20.5, 42.4)	(8.9)
Non-obese elderly females	6	3.0	239.9	29.6	30.5
		(2.2, 4.2)	(174.9, 328.9)	(20.6, 42.7)	(7.2)
		4.0	167.8	31.7	55.7
Non-obese young males	6
		(2.8, 5.6)	(122.4, 230.1)	(22.0, 45.6)	(14.3)
Non-obese young females	6	2.9	214.4	29.1	36.5
		(2.1,4.1)	(156.4, 294.0)	(20.2, 41.9)	(11.5)
Obese young males	3	3.4	247.2	30.9	38.2
		(2.1, 5.5)	(158.2, 386.3)	(18.5, 51.8)	(23.0)
Obese young females	3	2.5	158.8	26.1	41.1
		(1.6, 4.0)	(101.6, 248.1)	(15.6, 43.7)	(12.5)
GMR (90% CI)*
Non-obese young female vs male		0.74	1.28	0.92	–
		(0.50, 1.10)	(0.88, 1.85)	(0.60, 1.41)	–
Non-obese elderly vs young		0.94	1.33	0.97	–
		(0.71, 1.24)	(1.03, 1.73)	(0.72, 1.32)	–
Young obese vs young non-obese		0.86	1.04	0.94	–
		(0.61, 1.21)	(0.76, 1.44)	(0.65, 1.35)	–

^*Model-based least squares estimate (CI) for AUC_0–∞, C_max, and C_{24 h}, and harmonic mean (pseudo SD) for terminal t_1/2.

The pharmacokinetics of anacetrapib were similar across the different demographic groups (Table 4).

Pharmacodynamics

The mean serum CETP activity vs time profiles after administration of rising single oral doses of anacetrapib, after an overnight fast and with a high fat breakfast are shown in Figure 1B. A dose-related increase in percent inhibition of serum CETP activity was observed over the anacetrapib dose range. Anacetrapib administered with a high fat meal inhibited serum CETP activity with peak maximum inhibition of approximately 80% reached at approximately 4 h post-dose and approximately 20% or greater inhibition retained at trough (24 h post-dose) (Figure 1B). In addition, there was a dose-dependent increase in CETP concentrations (data not shown). The extent of inhibition of CETP activity across various demographic groups were similar. The pharmacokinetic-pharmacodynamic relationship between plasma concentrations of anacetrapib after an overnight fast and inhibition of serum CETP activity was best described using a E_max model with random effects on the baseline and maximum effect. Based on a collective dataset which included data from the studies described herein as well as those published [9], the PK/CETP relationship indicated that at the maximum effect, anacetrapib reduced CETP specific activity by 83% (80–85%) with 4.8% intersubject variability, and half of this reduction was achieved at a trough concentration of 0.014 µg ml⁻¹ (0.012–0.016 µg ml⁻¹), which is ∼22 nm (Figure 1C). Review of individual plots of CETP activity vs time indicated an absence of hysteresis (data not shown).

Safety and tolerability

Anacetrapib was generally well tolerated and there were no serious adverse events, clinical or laboratory adverse experiences. There were no clinical adverse experiences reported in studies 2 and 3. The listing of adverse events in study 1 is summarized in Table S1. There were no clinically significant abnormalities found in routine serum chemistry, CBC, urinalysis, ECG, physical examinations and vital signs. All adverse events were transient and were mostly mild or moderate in intensity. There were no clinically meaningful changes in systolic or diastolic blood pressure (Figure S1). A total of three subjects withdrew consent from two of the three studies, all for personal reasons.

Discussion

The studies reported here represent the first clinical data for anacetrapib obtained in humans. Collectively, these data support the proof of target engagement attained with anacetrapib based on potent and sustained inhibition of CETP activity. They also indicate that anacetrapib has pharmacokinetic and pharmacodynamic properties that support a once-daily dosing regimen, and that anacetrapib is generally well tolerated in healthy subjects with no demonstrable increases in blood pressure.

Anacetrapib exhibits a complex pharmacokinetic profile, being influenced by such factors as the fat content in meals, surfactant concentrations (as a function of capsule number) in capsules and dose. These effects are elaborated in the following sections.

Anacetrapib was absorbed after oral administration with a biphasic elimination profile that indicated a fairly long-terminal elimination phase. Whereas approximately 80–90% of the AUC was evident in the first 36–48 h, there was a flat elimination phase that was suggestive of a peripheral compartment. This observed half-life of anacetrapib was shorter as compared with the longer half-life observed for another CETP inhibitor, torcetrapib; as long as 373 h for total radioactivity and 211 h for torcetrapib have been observed [11]. An increase in half-life of anacetrapib with increasing doses (Table 1) was also observed. The exact mechanism for the increase in half-life is unclear and warrants further investigation.

Anacetrapib exposure increased with increasing dose, albeit less than dose proportionally. Furthermore, absorption of anacetrapib appeared saturable and dependent on fat content in meals with a high fat breakfast significantly and variably increasing anacetrapib exposures. Notably, both the mean and range of increases were correspondingly lower in magnitude with a low-fat breakfast, suggesting anacetrapib absorption was influenced by type of meal. Anacetrapib was negligibly present in the urine, an expected finding based on negligible urinary excretion in nonclinical experiments [12].

In the studies described here, anacetrapib was formulated in a surfactant liquid-filled capsule (LFC). Because anacetrapib was administered for the first time in the rising dose panel, flexibility in dosing was prospectively planned requiring various dosing units to administer a given dose. When interpreting our results, we found that the number of capsules administered also appeared to influence the exposure to anacetrapib, both in the presence and absence of a meal, with a higher number of units to administer a given dose resulting in apparent higher exposure. We hypothesized that the presence of a higher volume of the surfactant in the formulation by virtue of the number of capsule units administered may have contributed to increased absorption. The highest gain in exposure was with the 125 mg dose administered as nine capsules, with which there was nearly 15-fold higher surfactant volume. Both a low fat and a high fat meal increased exposure to anacetrapib after a single 125 mg oral dose (one capsule unit), with the low fat meal producing up to ∼two-fold increase in AUC_0–∞ and up to ∼three-fold increase in C_max and the high fat meal producing up to ∼six-fold increase in AUC_0–∞ and up to ∼nine-fold increase in C_max. It is not known whether the greater magnitude of mean effect observed with a high fat meal at the 125 mg dose was due to a dose-dependent meal effect or due to lower number of capsule units.

The observed effects of food on the clinical pharmacokinetics of anacetrapib appear in general on the higher end in magnitude and variability when compared with those reported for other developmental candidates as well as those marketed [13, 14]. The observed effects, however, are smaller than those reported with torcetrapib, whose mean increases in exposure with food ranged from 20 to 30-fold [15]; however, neither the variability in the effect or the type of meal has been disclosed. Food also increased the exposure to dalcetrapib by ∼two-fold. However, the effects were in general lower than those for anacetrapib or torcetrapib [16]. The effects observed for anacetrapib suggest that the observed increases with food may be due in part, at least, to increased solubility and absorption in the presence of food and surfactant volume and due to likely low oral bioavailability. The exact mechanism by which food increases anacetrapib absorption is unclear and warrants further study. The results of this study in conjunction with later studies [9, 17, 18] were instrumental in providing guidance for dosing anacetrapib with a meal in dose range finding studies.

Our data also indicated an uncertain interplay between capsule units and meal on whether the two variables produce incremental or independent effects [19]. To investigate this issue further, we studied the influence of multiple capsule units for a given dose on the relative bioavailability of anacetrapib when administered as the liquid-filled capsule (LFC) formulation. Two different dose levels were evaluated under conditions that provide maximum bioavailability one at the end of the linear range (i.e. 250 mg) and other at the middle of the linear range (i.e. 50 mg). At the 250 mg dose, the impact of five capsules (2480 µl) vs two (895 µl) capsules was assessed, the volume of surfactant varying ∼three-fold. At the 50 mg dose, the impact of five capsules (2680 µl) vs 1 (496 µl) was assessed, the volume of surfactant varying ∼five-fold. The magnitude of the capsule effect appeared to be diminished in the fed state representing maximum bioavailability, as compared with the fasted state. The observed data are not inconsistent with the physicochemical properties of anacetrapib. Specifically, anacetrapib is a poorly water soluble compound with a high log D, and hence formulated in imwitor-tween surfactant within a LFC formulation. Thus, it is not surprising that increased solubility (and hence, better absorption) can result with approaches that can increase solubility in the physiological milieu. In recent years, surfactant filled gelatin capsules have been used as a method to improve the bioavailability of hydrophobic drug substances [20]. Our results suggest that in the fasted state there may have been a capsule effect when multiple units of capsules are administered. To mitigate any potential difficulties in the interpretation of pharmacokinetic data as a result of the number of capsules per dose, this study provided guidance for later studies in that a fixed drug loading was subsequently used.

In order to understand the complex pharmacokinetic profile of anacetrapib, we pooled pharmacokinetic data across several clinical studies and developed a population pharmacokinetic model [21, 22]. A two-compartment pharmacokinetic model with a first-order absorption and an absorption lag time reasonably described the plasma concentration vs. time data for anacetrapib [21, 22]. The final model was parameterized in terms of systemic clearance (CL), intercompartmental clearance (Q), central and peripheral compartment volumes (V₂ and V₃), and bioavailability (F) [21, 22]. As only CL and F included intersubject variability, the individual parameter estimates, Ka_i, V_2i, Q_i, and V_3i, remain unchanged from the population estimates, Ka_TV, 2_TV, Q_TV, and V_3TV. Based on the collective data (which includes data from the studies described herein), the intersubject variability of anacetrapib on clearance was assessed to be ∼31% [21, 22]. Furthermore, the bioavailability was described as a function of the effect of meal and capsule units, and described by the relationship, , where F_1i is the bioavailability in subject i, F_eff is the effect of the number of capsules and meal type on bioavailability, and DG₁ and DG₂ are covariate effects. The intersubject variability on bioavailability was assessed to be ∼46% [22]. Food and formulation effects on bioavailability were dose-dependent [22]. For a 100 mg dose administered as a tablet, for example, the typical value of bioavailability for fasted subjects is 0.35 [22]. The expression, DG₂= 1 − C50/(C50 +n_CAP), best describes the relationship between number of capsules and bioavailability, where 50 is the number of capsules for half maximal effect on bioavailability and n_CAP is the number of capsules dosed [22] An increase in the number of capsules increases bioavailability by an E_max-like relationship for fed subjects and increases bioavailability exponentially for fasted subjects (when n_CAP > 5.5 capsules). A two-capsule dose, for example, increases bioavailability by ∼25% in fed subjects and by ∼35% in fasted subjects. In contrast, for a six-capsule dose, the increase in fasted subjects reaches ∼113% as compared with a 50% increase in fed subjects [22].

There was no meaningful effect of age, gender and obese status on the pharmacokinetics of anacetrapib, following single doses. Further, the apparent terminal half-life values across the different demographic groups were similar. It has been speculated that for torcetrapib, given its lipophilicity and long-terminal half-life properties, that the compound may accumulate in adipose tissue, and hence exhibit a longer terminal half-life in obese subjects [11]. This was attributed to their low recovery of dose in humans and preclinical whole-body autoradioluminography studies in Long-Evans rats, which demonstrated sequestration of radioactivity in adipose tissue. In contrast, the apparent terminal half-life of anacetrapib was consistent in all demographic groups evaluated.

Anacetrapib demonstrated significant inhibition of serum CETP activity in a dose related fashion. Anacetrapib, at doses of 25 mg (fasted) and above resulted in CETP activity being inhibited by ∼75% and above at peak (4 h post-dose), and at doses of 125 mg (fasted) and above or 25 mg (high fat meal) and above resulted in CETP activity being inhibited by ∼50% or greater at 24 h post-dose trough. Together with pharmacokinetic data suggesting a long apparent terminal half-life, anacetrapib appeared to exhibit a pharmacokinetic and pharmacodynamic profile that was amenable to once-daily dosing regimen. When anacetrapib concentrations were modeled with CETP activity, the relationship was best described by a E_max relationship, with an EC₅₀ value of ∼22 nm. This EC₅₀ value was consistent with the in vitro inhibition data for anacetrapib [7]. Interestingly, anacetrapib appeared to increase variably but modestly CETP concentrations after single doses, which was consistent with observations with multiple dose anacetrapib data [9], and other CETP inhibitors [23]. Because inhibition of CETP activity was significant and sustained, it was assumed that this would translate into marked alterations in lipids and lipoproteins. Consistent with this hypothesis, anacetrapib produced potent HDL-C raising and LDL-C lowering effects in humans in later studies, up to as much as ∼130% increase in HDL-C and ∼60% lowering of LDL-C [9, 17, 18]. The sustained and predictable effects on CETP activity, a key target engagement biomarker of interest, provided pharmacologic proof of concept for anacetrapib in humans. Inhibition of CETP activity was independent of age, gender, and obese status.

Administration of single doses of anacetrapib across a dose range 2 mg to 1000 mg did not result in serious adverse experiences or discontinuations due to adverse experiences. Clinical adverse experiences with anacetrapib were similar to those observed with placebo and were generally mild, transient and resolved without treatment. Anacetrapib was not associated with any clinically meaningful treatment-related adverse effects as assessed by measurement of blood cell counts, transaminase and serum creatinine concentrations, vital sign, or electrocardiographic parameters such as QTc- or PR-interval prolongation.

Blood pressure was carefully monitored in these initial studies. There was no demonstrable increase in systolic or diastolic blood pressure, at peak concentrations ∼14 µm, a finding that was confirmed later in a 24 h ambulatory blood pressure study [17].

In summary, the studies described in this report established the pharmacological proof of concept for anacetrapib. Anacetrapib also exhibited an apparent terminal half-life and an inhibitory effect on serum CETP activity of a duration and magnitude that is amenable to a once-daily dosing regimen. Whereas food markedly influenced anacetrapib pharmacokinetics, there was no meaningful effect of age, gender and obese status on the pharmacokinetics of anacetrapib. Anacetrapib was generally well tolerated across the dose range studied in healthy volunteers.

Competing interests

Authors who are Merck & Co., Inc. employees may have stock or stock options in the company.

Source of support: This study was funded by Merck & Co., Inc

We acknowledge the assistance of Michele Green at Pharsight Corporation in the PK/CETP work and the assistance of Julie Stone in preliminary statistical and pharmacokinetic calculations for one of the studies. We thank the study volunteers for their participation in this study and the other members of the study group, including study coordinators and pharmacists. Portions of the data contained in this manuscript have been presented at the 2007 Drugs Affecting Lipid Metabolism Symposium in New York, NY.

Supporting information

Additional Supporting Information may be found in the online version of this article:

Figure S1

Effect of single 400 mg and 800 mg doses of anacetrapib, administered with a high fat meal, on systolic (A) and diastolic (B) blood pressure

Table S1

Listing of drug-related clinical adverse experiences

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.