Impact of drug distribution into adipose on tissue function: The cholesteryl ester transfer protein (CETP) inhibitor anacetrapib as a test case.

Anacetrapib is an inhibitor of cholesteryl ester transfer protein (CETP) previously under development as a lipid-modifying agent that reduces LDL-cholesterol and increases HDL-cholesterol in hypercholesterolemic patients. Anacetrapib demonstrates a long terminal half-life and accumulates in adipose tissue, which contributes to a long residence time of anacetrapib. Given our previous report that anacetrapib distributes into the lipid droplet of adipose tissue, we sought to understand whether anacetrapib affected adipose function, using a diet-induced obese (DIO) mouse model. Following 20 weeks of treatment with anacetrapib (100 mg/kg/day), levels of the drug increased to approximately 0.6 mmol/L in white adipose tissue. This level of anacetrapib was not associated with any impairment in adipose functionality as evidenced by a lack of any reduction in biomarkers of adipose functionality (plasma adiponectin, leptin, insulin; adipose adiponectin, leptin mRNA). In DIO wild-type (WT) mice treated with anacetrapib for 2 weeks and then subjected to 30% food restriction during washout to induce weight loss (18%) and fat mass loss (7%), levels of anacetrapib in adipose and plasma were not different between food restricted and ad lib-fed mice. These data indicate that despite deposition and long-term residence of ~0.6 mmol/L levels of anacetrapib in adipose tissue, adipose tissue function appears to be unaffected in mice. In addition, these data also indicate that even with severe caloric restriction and acute loss of fat mass, anacetrapib does not appear to be mobilized from the fat depot, thereby solidifying the role of adipose as a long-term storage site of anacetrapib.

Characterization of Anacetrapib Distribution into the Lipid Droplet of Adipose Tissue in Mice and Human Cultured Adipocytes.

Anacetrapib is an inhibitor of cholesteryl ester transfer protein (CETP), associated with reduction in LDL cholesterol and increase in HDL cholesterol in hypercholesterolemic patients. Anacetrapib was not taken forward into filing/registration as a new drug for coronary artery diease, despite the observation of a ∼9% reduction in cardiovascular risk in a large phase III cardiovascular outcomes trial (REVEAL). Anacetrapib displayed no adverse effects throughout extensive preclinical safety evaluation, and no major safety signals were observed in clinical trials studying anacetrapib, including REVEAL. However, anacetrapib demonstrated a long terminal half-life in all species, thought to be due, in part, to distribution into adipose tissue. We sought to understand the dependence of anacetrapib's long half-life on adipose tissue and to explore potential mechanisms that might contribute to the phenomenon. In mice, anacetrapib localized primarily to the lipid droplet of adipocytes in white adipose tissue; in vitro, anacetrapib entry into cultured human adipocytes depended on the presence of a mature adipocyte and lipid droplet but did not require active transport. In vivo, the entry of anacetrapib into adipose tissue did not require lipase activity, as the distribution of anacetrapib into adipose was-not affected by systemic lipase inhibition using poloaxamer-407, a systemic lipase inhibitor. The data from these studies support the notion that the entry of anacetrapib into adipose tissue/lipid droplets does not require active transport, nor does it require mobilization or entry of fat into adipose via lipolysis.

Evaluation of CETP activity in vivo under non-steady-state conditions: influence of anacetrapib on HDL-TG flux.

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.

Discovery and optimization of orally active cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors.

The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.

A new class of prolylcarboxypeptidase inhibitors, part 1: discovery and evaluation.

A new structural class of potent prolylcarboxypeptidase (PrCP) inhibitors was discovered by high-throughput screening. The series possesses a tractable SAR profile with sub-nanomolar in vitro IC(50) values. Compared to prior inhibitors, the new series demonstrated minimal activity shifts in pure plasma and complete ex vivo plasma target engagement in mouse plasma at the 20 h post-dose time point (po). In addition, the in vivo level of CNS and non-CNS drug exposure was measured.

A new class of prolylcarboxypeptidase inhibitors, part 2: the aminocyclopentanes.

A series of potent inhibitors of prolylcarboxypeptidase (PrCP) was developed by modifying a lead structure that was discovered by high-throughput screening. The tert-butyl pyrrolidine was replaced by an aminocyclopentane to reduce the metabolic liabilities of the original lead. The compounds demonstrated sub-nanomolar in vitro IC(50) values, minimal activity shifts in pure plasma and improved pharmacokinetics. Complete ex vivo plasma target engagement was achieved with low brain exposure at the 20 h time point following p.o. dosing in a mouse. The results indicate that the aminocyclopentanes are useful tools for studying the therapeutic potential of peripheral (non-CNS) PrCP inhibition.

Discovery of aminoheterocycles as potent and brain penetrant prolylcarboxypeptidase inhibitors.

Efforts were dedicated to develop potent and brain penetrant prolylcarboxypeptidase (PrCP) inhibitors by replacing the amide group of original leads 1 and 2 with heterocycles. Aminopyrimidines including compound 32a were identified to display good PrCP inhibitory activity (32a, IC(50)=43 nM) and impressive ability to penetrate brain in mice (brain/plasma ratio: 1.4).

Discovery of benzodihydroisofurans as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase inhibitors.

A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure-activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.

Discovery of a new class of potent prolylcarboxypeptidase inhibitors derived from alanine.

Efforts to modify the central proline portion of lead compound 4 lead to the discovery of novel prolylcarboxypeptidase (PrCP) inhibitors. Especially, replacement with alanine afforded compound 19 displaying more potent human and mouse PrCP inhibitory activity than 4 and an overall comparable profile.

The discovery of non-benzimidazole and brain-penetrant prolylcarboxypeptidase inhibitors.

Novel prolylcarboxypeptidase (PrCP) inhibitors with nanomolar IC(50) values were prepared by replacing the previously described dichlorobenzimidazole-substituted pyrrolidine amides with a variety of substituted benzylamine amides. In contrast to prior series, the compounds demonstrated minimal inhibition shift in whole serum and minimal recognition by P-glycoprotein (P-gp) efflux transporters. The compounds were also cell permeable and demonstrated in vivo brain exposure. The in vivo effect of compound (S)-6e on weight loss in an established diet-induced obesity (eDIO) mouse model was studied.

SAR studies on the central phenyl ring of substituted biphenyl oxazolidinone-potent CETP inhibitors.

SAR studies of the substitution effect on the central phenyl ring of the biphenyl scaffold were carried out using anacetrapib (9a) as the benchmark. The results revealed that the new analogs with substitutions to replace trifluoromethyl (9a) had a significant impact on CETP inhibition in vitro. In fact, analogs with some small groups were as potent or more potent than the CF(3) derivative for CETP inhibition. Five of these new analogs raised HDL-C significantly (>20mg/dL). None of them however was better than anacetrapib in vivo. The synthesis and biological evaluation of these CETP inhibitors are described.

Biphenyl-substituted oxazolidinones as cholesteryl ester transfer protein inhibitors: modifications of the oxazolidinone ring leading to the discovery of anacetrapib.

The development of the structure-activity studies leading to the discovery of anacetrapib is described. These studies focused on varying the substitution of the oxazolidinone ring of the 5-aryloxazolidinone system. Specifically, it was found that substitution of the 4-position with a methyl group with the cis-stereochemistry relative to the 5-aryl group afforded compounds with increased cholesteryl ester transfer protein (CETP) inhibition potency and a robust in vivo effect on increasing HDL-C levels in transgenic mice expressing cynomolgus monkey CETP.

Discovery of benzimidazole pyrrolidinyl amides as prolylcarboxypeptidase inhibitors.

A series of benzimidazole pyrrolidinyl amides containing a piperidinyl group were discovered as novel prolylcarboxypeptidase (PrCP) inhibitors. Low-nanomolar IC(50)'s were achieved for several analogs, of which compound 9b displayed modest ex vivo target engagement in eDIO mouse plasma. Compound 9b was also studied in vivo for its effect on weight loss and food intake in an eDIO mouse model and the results will be discussed.

2-Arylbenzoxazoles as CETP inhibitors: raising HDL-C in cynoCETP transgenic mice.

We describe structure-activity studies leading to the discovery of 2-arylbenzoxazole 3, the first in a series to raise serum high-density lipoprotein cholesterol levels in transgenic mice. Replacement of the 4-piperidinyloxy moiety with piperazinyl provided a more synthetically tractable lead, which upon optimization resulted in compound 4, an excellent inhibitor of cholesteryl ester transfer protein function with good pharmacokinetic properties and in vivo efficacy.

Design of a novel class of biphenyl CETP inhibitors.

A new class of CETP inhibitors was designed and prepared. These compounds are potent both in vitro and in vivo. The most active compound (12d) has shown an ability to raise HDL significantly in transgenic mouse PD model.

2-Arylbenzoxazoles as CETP inhibitors: substitution and modification of the alpha-alkoxyamide moiety.

The development of a series of 2-arylbenzoxazole alpha-alkoxyamide and beta-alkoxyamine inhibitors of cholesteryl ester transfer protein (CETP) is described. Highly fluorinated alpha-alkoxyamides proved to be potent inhibitors of CETP in vitro, and the highly fluorinated 2-arylbenzoxazole beta-alkoxyamine 4 showed a desirable combination of in vitro potency (IC(50)=151 nM) and oral bioavailability in the mouse.

Discovery of a highly potent, selective, and bioavailable soluble epoxide hydrolase inhibitor with excellent ex vivo target engagement.

4-Substituted piperidine-derived trisubstituted ureas are reported as highly potent and selective inhibitors for sEH. The SAR outlines approaches to improve activity against sEH and reduce ion channel and CYP liability. With minimal off-target activity and a good PK profile, the benchmark 2d exhibited remarkable in vitro and ex vivo target engagement. The eutomer entA-2d also elicited vasodilation effect in rat mesenteric artery.

Discovery of 3,3-disubstituted piperidine-derived trisubstituted ureas as highly potent soluble epoxide hydrolase inhibitors.

3,3-Disubstituted piperidine-derived trisubstituted urea entA-2b was discovered as a highly potent and selective soluble epoxide hydrolase (sEH) inhibitor. Despite the good compound oral exposure, excellent sEH inhibition in whole blood, and remarkable selectivity, compound entA-2b failed to lower blood pressure acutely in spontaneously hypertensive rats (SHRs). This observation further challenges the premise that sEH inhibition can provide a viable approach to the treatment of hypertensive patients.

A strategy of employing aminoheterocycles as amide mimics to identify novel, potent and bioavailable soluble epoxide hydrolase inhibitors.

Distinct from previously reported urea and amide inhibitors of soluble epoxide hydrolase (sEH), a novel class of inhibitors were rationally designed based on the X-ray structure of this enzyme and known amide inhibitors. The structure-activity relationship (SAR) study was focused on improving the sEH inhibitory activity. Aminobenzisoxazoles emerged to be the optimal series, of which a potent human sEH inhibitor 7t was identified with a good pharmacokinetics (PK) profile. The strategy of employing aminoheterocycles as amide replacements may represent a general approach to develop mimics of known hydrolase or protease inhibitors containing an amide moiety.

Discovery of spirocyclic secondary amine-derived tertiary ureas as highly potent, selective and bioavailable soluble epoxide hydrolase inhibitors.

Spirocyclic secondary amine-derived trisubstituted ureas were identified as highly potent, bioavailable and selective soluble epoxide hydrolase (sEH) inhibitors. Despite good oral exposure and excellent ex vivo target engagement in blood, one such compound, rac-1a, failed to lower blood pressure acutely in spontaneously hypertensive rats (SHRs). This study posed the question as to whether sEH inhibition provides a robust mechanism leading to a significant antihypertensive effect.