Inhibition of cholesterol ester transfer protein by CGS 25159 and changes in lipoproteins in hamsters

doi:10.1016/S0021-9150(96)05981-3

Atherosclerosis

Volume 128, Issue 1, 3 January 1997, Pages 59-66

https://doi.org/10.1016/S0021-9150(96)05981-3 Get rights and content

Abstract

As a result of screening, several isoflavans were identified to be antagonists of cholesterol ester transfer protein (CETP) activity. The present study evaluates CGS 25159, a synthetic isoflavan, as a putative inhibitor of CETP activity of human and hamster plasma. Determined by [³H]CE transfer from HDL to VLDL+LDL fraction or by fluorescent-CE transfer assay, CGS 25159 inhibited CETP in both human plasma bottom fraction (d=1.21 g/ml) and in plasma from Golden Syrian Hamsters with an IC₅₀<10 μM. The compound also inhibited (IC₅₀≈15 μM) the reciprocal transfer of triglycerides in the incubated whole plasma from normal and hyperlipidemic hamsters. When orally administered to normolipidemic hamsters, CGS 25159 (10 mg/kg, 4 days) reduced plasma transfer activity by 35–60%. Treatment with CGS 25159 (10 and 30 mg/kg, p.o.) resulted in dose dependent and time dependent changes in CETP activity. After two weeks of treatment at 10 mg/kg, the changes in VLDL+LDL cholesterol, total triglycerides and HDL cholesterol were −22±4.6*, −23±7.5 and +10±2.8%, respectively. The corresponding changes at 30 mg/kg were −28±5.5*, −38±6.8* and +29±4.4*%, (*, P, 0.05; mean±S.E.M., n=6). A single spin gradient density ultracentrifugation of plasma lipoproteins from treated animals showed an increase in HDL cholesterol and a redistribution to larger HDL particles. These data support the contention that pharmacological down regulation of CETP activity could result in favorable changes in lipoprotein profile.

Introduction

Cholesterol ester transfer protein (CETP) facilitates transfer of cholesterol ester from high density lipoprotein (HDL) to acceptor Apoprotein B containing lipoproteins and a reciprocal transfer of triglycerides to HDL 1, 2. The metabolic consequence of high or low CETP activity and their effects on plasma lipoprotein composition have now been intensely studied [3]. Several studies 4, 5have demonstrated an accelerated transfer of HDL cholesterol ester to very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) in hyperlipidemic plasma. Low levels of HDL cholesterol and increased CETP activity, independent of triglyceride levels, have been demonstrated in familial hypercholesterolemia [6]and in patients with angiographic evidence of coronary artery disease [7]. The importance of CETP in the regulation of plasma HDL cholesterol concentrations have gained credence from studies of familial CETP deficiency [8], in rabbits or hamsters in which CETP has been inhibited 9, 10and in murine transgenic models 11, 12. Thus a growing body of evidence has established that CETP plays a central role in cholesterol transport and that the plasma lipoprotein profile can be favorably altered by targeting CETP.

CGS 25159 [3-(3-fluorophenyl)-3,4-dihydro-2H-1-benzopyran-6,7-diol] is a synthetic isoflavan of the structure shown in Fig. 1. The compounds of this chemical class exhibit valuable pharmacological properties [13]. They, for example, improve hemorrheological parameters, inhibit platelet aggregation and phosphodiesterase, have calcium antagonist activity and improve peripheral oxygenation.

The purpose of the present study was to evaluate CGS 25159 as an inhibitor of CETP and its effects on lipoprotein composition in an animal model that is amenable to drug discovery. Golden Syrian hamsters are considered a suitable model for human lipid metabolism [14]. Hamsters mimic the response of humans to diet and develop atherosclerotic lesions. This model is also responsive to inhibition of CETP as shown by an increase in HDL cholesterol on treatment with neutralizing monoclonal antibodies [10]. Hamsters were therefore used to investigate the biochemical and pharmacological profile of CGS 25159.

Section snippets

Human plasma

Blood was obtained from normolipidemic healthy volunteers recruited from the personnel of the CIBA Corporation of Summit, NJ. Blood was collected from donors in tubes containing EDTA (1 mg/ml) after a 14 h overnight fast under standardized conditions. Plasma was isolated by centrifugation at 1750×g for 10 min at 4°C. Lipoproteins were separated from the plasma and the bottom fraction (d=1.21 g/ml) was isolated by the sequential ultracentrifugation method as previously described [15].

Experimental animals

All animal

In vitro effects of CGS 25159 on CETP

In vitro effects of CGS 25159 on plasma cholesteryl ester transfer activity was determined by two separate methods. In the assay using isolated human lipoproteins as substrates, the transfer of [³H]cholesterol oleate from HDL to VLDL+LDL was determined. In the assay, CGS 25159 inhibited CETP activity of both human plasma bottom fraction (d>1.21 g/ml) and hamster plasma (Fig. 2). The effects of the CGS 25159 were dose dependent and both activities were inhibited with an IC₅₀ of ≈10 μM. In the

Discussion

The present study has identified CGS 25159 as an inhibitor of CETP activity of human and hamster plasma. The potency of the compound is dependent on CE acceptor and/or donor substrates in the in vitro assay. The compound, for example, displays potent effects in the assay in which human VLDL is the acceptor and fluorescent CE containing liposomes serve as the donor. On the other hand, CGS 25159 shows moderate inhibitory activity against CETP in the incubated whole plasma assay (Fig. 7).

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