Anne Perez

Mechanisms underlying off-target effects of the cholesteryl ester transfer protein inhibitor torcetrapib involve L-type calcium channels.

Objective The increased mortality observed with the cholesteryl ester transfer protein inhibitor torcetrapib is partly due to increased aldosterone production and blood pressure. The mechanisms underlying these effects were investigated. Methods Cytochrome P450 subunit 11B2 (aldosterone synthase), extracellular signal-regulated kinase (p44/42) and voltage-gated Ca2+channel alpha subunit mRNA profiling, aldosterone production, cytosolic calcium and RNA interference were assessed in adrenocarcinoma human cells (H295R). Telemetry was conducted in spontaneously hypertensive rats. Results Torcetrapib and angiotensin II (Ang II) but not dalcetrapib (a structurally different cholesteryl ester transfer protein inhibitor) elevated both cytochrome P450 subunit 11B2 mRNA and aldosterone production in H295R cells at 6 h. At days 1–5, torcetrapib produced a sustained increase of cytochrome P450 subunit 11B2 mRNA, unlike Ang II. Although torcetrapib and Ang II potentiated the effect of 25-OH cholesterol and raised pregnenolone levels, torcetrapib increased neither cytosolic Ca2+ at 5 min nor extracellular signal-regulated kinase1/2 phosphorylation, suggesting initially divergent pathways. Unlike Ang II, torcetrapib steroidogenesis was not affected by Ang II type 1 receptor antagonism or voltage-gated T-type Ca2+ channel antagonism, but was blocked by several L-type Ca2+channel antagonists. In unbiased genome-wide screening, Ang II and torcetrapib modulated an overlapping but distinct set of genes in H295R cells. Torcetrapib, but not Ang II, upregulated mRNA levels of the L-type Ca2+ channel alpha 1C subunit. In spontaneously hypertensive rat, torcetrapib had a potent hypertensive effect mediated by the L-type Ca2+ channel. Conclusion The unique steroidogenic and hypertensive side effects of torcetrapib may be linked and involve voltage-gated L-type Ca2+ channels. Structurally unrelated cholesteryl ester transfer protein inhibitors such as dalcetrapib do not share this effect.

The Nuclear Receptors FXR and LX alpha Potential Targets for the Development of Drugs Affecting Lipid Metabolism and Neoplastic Diseases

The orphan nuclear receptors FXR and LXRalpha have become challenging targets for the discovery of new therapeutic agents. Bile acids and hydroxysterol intermediates are the respective natural ligands of these two structurally and functionally closely related receptors. Both FXR and LXRalpha; are thought to play a major role in the control of cholesterol catabolism by regulating the expression of cholesterol 7alpha-hydroxylase, the rate limiting enzyme of bile acid synthesis. Reverse cholesterol transport might also be affected by FXR and LXR since they control the expression of PLTP and CETP, two proteins involved in the transfer of phospholipid, cholesterol and cholesteryl esters among plasma lipoproteins. A new class of potent synthetic activators of FXR, the 1,1-bisphosphonate esters, has been discovered which up regulate the Intestinal Bile Acid Binding Protein gene (I-BABP) as demonstrated for chenodeoxycholic acid, however there are no known synthetic activators yet identified for LXRalpha. The evaluation of FXR as a potential target for the development of drugs affecting plasma cholesterol can take advantage of the fact that the activators of FXR (farnesol, bile acids and the 1,1-bisphosphonate esters) have been studied in various in vitro and in vivo models. Administration of chenodeoxycholic acid to animals and man did not result in the increase in plasma cholesterol expected from a decrease in cholesterol 7alpha-hydroxylase expression. Like farnesol, the 1,1-bisphosphonate esters increase the rate of degradation of HMGCoA reductase and have the unexpected property of inducing hypocholesterolemia in normal animals. The natural and synthetic FXR agonists trigger differentiation, inhibit cell proliferation and are potent inducers of apoptosis. The 1,1-bisphosphonate ester SR-45023A (Apomine) is presently being developed as an antineoplastic drug.

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

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

MARCO, a macrophage scavenger receptor highly expressed in rodents, mediates dalcetrapib-induced uptake of lipids by rat and mouse macrophages

Dalcetrapib (RO4607381/JTT-705), an agent that targets cholesteryl ester transfer protein, is in development for prevention of cardiovascular events. In vitro studies were performed to identify receptors that mediate an off-target effect of dalcetrapib observed in preclinical models: increased lipid uptake into the lamina propria of the small intestine and into mesenteric lymph node macrophages. Uptake of oxidized low-density lipoprotein (LDL) cholesterol or dalcetrapib-treated chylomicrons was quantitated by triglyceride assay or fluorescent labeling in primary macrophages and the cell lines CHO, J774A.1 (mouse macrophages) and THP-1 (human macrophages). Quantitative reverse-transcriptase polymerase chain reaction and immunoblotting measured candidate receptor expression. Lectin-like oxidized LDL receptor (LOX-1) and scavenger receptor type AI (SR-AI) were excluded as candidate receptors based on lack of association between their expression and uptake of dalcetrapib-treated lipids. In J774A.1 cells, uptake of dalcetrapib-treated chylomicrons was increased by LPS and associated with expression of MAcrophage Receptor with COllagenous domain (MARCO). MARCO was expressed at very low levels in human macrophages and was not inducible by LPS. The MARCO receptor may account for the variable species susceptibility towards dalcetrapib-mediated chylomicron uptake by macrophages.