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For the last 30 years fibrates have been widely prescribed to treat human dyslipidemia. However, the primary mechanism by which they lower plasma lipid levels is still unknown. Studies with transgenic mice have suggested that changes in apoC-III expression levels have a dramatic influence on plasma triglyceride levels. These results suggested that fibrates could reduce lipid levels by lowering apoC-III gene expression. In the current studies, we sought to determine whether the selected fibrates, bezafibrate, clofibrate, fenofibrate, and gemfibrozil, could reduce hepatic apoC-III mRNA and plasma apoC-III levels. Chow-fed rats were orally gavaged daily with a dosing vehicle alone or with 100 mg/kg of each of the fibrates for 1 week and in addition with gemfibrozil for 2 weeks. Bezafibrate and fenofibrate lowered plasma triglyceride by approximately half and dramatically reduced hepatic apoC-III mRNA and plasma apoC-III levels. In contrast, clofibrate did not reduce plasma triglyceride levels and only partially reduced apoC-III mRNA and plasma protein levels. Gemfibrozil strongly reduced plasma triglyceride levels and had an intermediate but significant effect on apoC-III mRNA and plasma apoC-III levels. Some of the fibrates, especially gemfibrozil also reduced plasma apoC-II levels, an effect that could contribute to the observed triglyceride-lowering effect. In addition, the ratio of plasma apoE to plasma apoC-II plus apoC-III was strongly and inversely correlated with plasma triglyceride levels. As plasma apoE levels were not reduced in gemfibrozil-treated animals, this could also have contributed to the triglyceride-lowering effect of this fibrate. Fibrate-mediated triglyceride lowering was not the result of a decreased apoB or VLDL production and, therefore, suggested an enhanced VLDL remnant catabolism. Our results suggest that the mechanism by which fibrates lower plasma triglycerides is by reducing the level of hepatic apoC-III expression.

Eight male cynomolgus monkeys (Macaca fascicularis) on a normal chow diet were orally administered gemfibrozil daily using a weekly rising dose protocol for 3 weeks (50, 125, and 200 mg/kg per day). At these drug doses, Lp[a] levels were reduced: 83.7% +/- 3.2 (SEM), (P < 0.024); 63.7% +/- 4.1 (P < 0.013); and 36.2% +/- 1.1 (P < 0.002), respectively, of pretreatment values. Lp[a] reduction was directly related to blood gemfibrozil concentration (range 36-428 microM, r = 0.969) and occurred without concomitant changes in apolipoprotein B. Three weeks posttreatment Lp[a] levels returned to pretreatment values. A specific ribonuclease protection assay demonstrated that liver apolipoprotein[a] (apo[a]) mRNA expression was decreased in all animals to an average of 19.1% +/- 3.0 (P < 0.0026), of pretreatment values after the 200 mg/kg treatment, whereas, albumin, apolipoprotein A-I, apolipoprotein E, and glyceraldehyde-3-phosphate dehydrogenase mRNAs were unchanged. Lp[a] levels were unaffected by gemfibrozil in HepG2 cells permanently transfected with an apo[a] 10-kringle cDNA construct containing partial 5'- and 3'-untranslated sequences and under control of a constitutive CMV promoter. However, both Lp[a] and apo[a] mRNA in primary cynomolgus monkey hepatocytes were coordinately lowered in a dose-dependent fashion by gemfibrozil. Thus, Lp[a] can be regulated by gemfibrozil at the level of apo[a] mRNA expression.

The presence of plasma cholesteryl ester transfer protein (CETP) activity may be atherogenic, and, therefore, strategies to inhibit its activity or production may result in a beneficial effect on lipoprotein profiles and the disease process. The current report describes 4-phenyl-5-tridecyl-4H-1,2,4- triazole-3-thiol (PD 140195), a novel CETP inhibitor. The concentration-dependent inhibition of CETP by PD 140195 and the inhibitory monoclonal antibody (Mab) TP2 is demonstrated in a variety of in vitro assay systems. Molecular models of PD 140195 suggest a spatial mimicry of the cholesteryl ester structure. Despite the structural similarity, kinetic studies with a fluorescent cholesteryl ester analog suggest that the inhibition of transfer is not competitive. PD 140195 also selectively inhibited cholesteryl ester but not triglyceride transfer, while the Mab TP2 blocked CETP transfer of both. Studies were carried out to determine whether CETP inhibition observed in vitro could also be demonstrated in vivo. When PD 140195 was intravenously infused to anesthetized rabbits (up to 20 mg/kg), only transient CETP inhibition was observed. In vitro reconstitution studies in the presence of bovine serum albumin resulted in marked reduction of PD 140195 inhibitory activity. Thus, the low activity of PD 140195 in whole plasma probably results from binding to other plasma proteins.

Five series of compounds (4-9) derived from N-(chlorocarbonyl) isocyanate have been synthesized and evaluated for their ability to inhibit the enzyme acyl-CoA:cholesterol O-acyltransferase and lower plasma cholesterol levels in cholesterol-fed rats. Structure-activity relationships indicate that the imino dicarboxylates (6 and 7) and the oxycarbonyl thiocarbamates (8) are the most potent and efficacious series. In these series, the combination of a 2,6-diisopropylphenyl group and an aliphatic alkyl group with a chain length between 6 and 14 carbon atoms gives good activity in vitro and in vivo. In addition, a hydrogen donor is required to maintain good in vitro activity, and the acidic proton on the central nitrogen in these series appears to be important for in vivo activity.

In the present report we describe a simple and practical method to assess CETP activity in a defined system by use of microemulsions containing a fluorescent cholesteryl ester analog. The microemulsions are stable, simple to prepare, and can be made to defined composition. Initial transfer rates are easily determined by monitoring changes in fluorescence. We have used the fluorescent cholesteryl ester analog, cholesteryl 4,4-difluoro-5,7-dimethyl-4-boro-3 alpha, 4 alpha-diaza-3-indacenedodecanoate (BODIPY-CE), to demonstrate the utility of this assay. The assay takes advantage of the concentration-dependent self-quenching of BODIPY-CE, when this analog is incorporated into microemulsions. We have used this new assay to demonstrate fluorescent lipid transfer facilitated by rabbit and human d > 1.21 g/ml plasma fraction and recombinant human CETP. A known inhibitory monoclonal antibody (Mab) to human CETP blocked BODIPY-CE transfer in a dose-dependent manner. We have also used BODIPY-CE microemulsions to measure CETP activity in whole plasma.