Dilip Amin

A novel endothelial-derived lipase that modulates HDL metabolism.

High-density lipoprotein (HDL) cholesterol levels are inversely associated with risk of atherosclerotic cardiovascular disease. At least 50% of the variation in HDL cholesterol levels is genetically determined, but the genes responsible for variation in HDL levels have not been fully elucidated. Lipoprotein lipase (LPL) and hepatic lipase (HL), two members of the triacylglyerol (TG) lipase family, both influence HDL metabolism and the HL (LIPC) locus has been associated with variation in HDL cholesterol levels in humans. We describe here the cloning and in vivo functional analysis of a new member of the TG lipase family. In contrast to other family members, this new lipase is synthesized by endothelial cells in vitro and thus has been termed endothelial lipase (encoded by the LIPG gene). EL is expressed in vivo in organs including liver, lung, kidney and placenta, but not in skeletal muscle. In contrast to LPL and HL, EL has a lid of only 19 residues. EL has substantial phospholipase activity, but less triglyceride lipase activity. Overexpression of EL in mice reduced plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I. The endothelial expression, enzymatic profile and in vivo effects of EL suggest that it may have a role in lipoprotein metabolism and vascular biology.

Restenosis Following Angioplasty in the Swine Coronary Artery Is Inhibited By an Orally Active PDGF-Receptor Tyrosine Kinase Inhibitor, RPR101511A

BACKGROUND Platelet-derived growth factor (PDGF), a purported mediator of arterial response to injury, stimulates proliferation, chemotaxis, and matrix production by activation of its membrane receptor tyrosine kinase. Because these activities underlie restenosis, inhibition of the PDGF-receptor tyrosine kinase (PDGFr-TK) is postulated to decrease restenosis. METHODS AND RESULTS RPR101511A is a novel compound which selectively and potently inhibits the cell-free and in situ PDGFr-TK and PDGFr-dependent proliferation and chemotaxis in vascular smooth muscle cells (VSMC). To evaluate the effect of RPR101511A (30 mg. kg-1. d-1 BID for 28 days following PTCA) on coronary restenosis, PTCA was performed in hypercholesterolemic minipigs whose left anterior descending (LAD) coronary artery had been injured by overdilation and denudation, yielding a previously existing lesion. Angiographically determined prePTCA minimal lumen diameters (MLD) were similar in vehicle and RPR101511A-treated pigs (1.98+/-0.09 versus 2.01+/-0.08 mm) and increased to the same extent in the 2 groups following successful PTCA (2.30+/-0.06 versus 2.52+/-0.13). At termination, there was an average 50% loss of gain in the vehicle-treated group but no loss of gain with RPR101511A (2.16+/-0. 05 versus 2.59+/-0.11, P<0.001). Morphometric analysis of the LAD showed that RPR101511A caused a significant decrease in total intimal/medial ratio (0.96+/-0.58 versus 0.67+/-0.09, P<0.05). CONCLUSIONS RPR101511A, which acts by inhibition of the PDGFr-TK, completely prevented angiographic loss of gain following PTCA and significantly reduced histological intimal hyperplasia.

RG 12561 (dalvastatin): a novel synthetic inhibitor of HMG-CoA reductase and cholesterol-lowering agent.

RG 12561 (dalvastatin) is a prodrug which converts to its open hydroxyacid form in the body. The Na salt of RG 12561 (RG 12561-Na) is a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. It competitively inhibits rat liver HMG-CoA reductase with an IC50 value of 3.4 nmol/l. In the same assay, the IC50 values for other potent HMG-CoA reductase inhibitors, lovastatin-Na and pravastatin, were 2.3 and 8.9 nmol/l, respectively. In Hep G2 liver cells, RG 12561-Na, lovastatin-Na and pravastatin inhibited cholesterol biosynthesis from radiolabeled octanoate with IC50 values of 4 and 5 nmol/l and 1.1 mumol/l, respectively. In a rat ex vivo assay, orally administered RG 12561, lovastatin and pravastatin inhibited cholesterol biosynthesis in liver slices with ED50 values of 0.9, 0.5 and 12 mg/kg, respectively. In cholestyramine-fed hamsters, RG 12561 (0.1% in food for 18 days) reduced LDL cholesterol, whereas HDL was slightly increased. The reductions in the LDL/HDL ratio for RG 12561, RG 12561-Na, lovastatin and lovastatin-Na were 35, 76, 88 and 88%, respectively. At a higher dose, RG 12561 (0.4% in food) reduced serum cholesterol, LDL and LDL/HDL by 84, 97 and 91%, respectively. In WHHL rabbits, RG 12561 and lovastatin (5 mg/kg, b.i.d., 12 days) reduced serum cholesterol by 17 and 16%, respectively. These results demonstrate that RG 12561 is a potent cholesterol-lowering agent.

RPR 101821, a new potent cholesterol-lowering agent: inhibition of squalene synthase and 7-dehydrocholesterol reductase

RPR 101821 (trans-2-[4-(benzoxazol-2-yl)phenylmethoxy] amino cyclohexane hydrochloride) is a potent cholesterol-lowering agent in rodents and marmoset. The compound inhibited rat liver microsomal squalene synthase (IC50 = 1 nM) and 7-dehydrocholesterol (7DHC) reductase (IC50 = 1 μM; Lewis et al. 1995). When RPR 101821 (10 mg/kg), the 7DHC reductase inhibitor BM 15.766 (4[2-[4-(4-chlorocinnamyl)piperazine-1-yl]ethyl] benzoic acid; 10 mg/kg) or the HMG-CoA reductase inhibitor lovastatin (30 mg/kg) was given orally to rats at −29 h, − 21 h and − 5 h, serum cholesterol was reduced by 56%, 46% or 15%, respectively. The reduction in cholesterol with RPR 101821 was associated with an accumulation of 7DHC in serum, suggesting an inhibition of 7DHC reductase. In the presence of BM 15.766, RPR 101821 reduced the serum accumulation of 7DHC in a dose-dependent manner, with complete inhibition at 30 mg/kg, p.o. In Balb-cJ mice, RPR 101821 and lovastatin (50 mg/kg, b.i.d., p.o., for 14 days) lowered serum cholesterol by 67% and 2%, respectively. In marmosets, RPR 101821 and lovastatin (both at a dose of 10 mg/kg, p.o., b.i.d., for 7 days) reduced cholesterol by 28% and 19%, respectively.In summary, RPR 101821 is an orally effective potent cholesterol-lowering agent in rodents and a small primate species. The suggested mechanism of hypocholesterolemic effect is the inhibition of squalene synthase and 7DHC reductase.

Enzyme inhibition during the conversion of squalene to cholesterol

Two separate enzymatic assays were developed in order to test the selectivity of inhibitors in cholesterol biosynthesis. One assay detects inhibition of delta 5.7-sterol delta 7-reductase, the enzyme involved in the conversion of 7-dehydrocholesterol to cholesterol. Delta 5.7-Sterol delta 7-reductase was inhibited by both RPR 101821, a protonated cyclohexylamine, and BM 15.766, a piperazine derivative, with IC50 values of 1 microM. The second assay detects accumulation of any of five intermediates (squalene oxide, squalene dioxide, lanosterol, desmosterol, and 7-dehydrocholesterol) upon inhibition of enzymes catalyzing reactions in the conversion of squalene to cholesterol. In this assay, inhibition data were most accurate when control assays exhibited a conversion of squalene to cholesterol in the order of 50%. The time required to attain 50% conversion of squalene to cholesterol was 6 h. Given a high inhibitor to substrate concentration ratio and the possible values of Ki, kon, and koff for the reaction between enzymes and inhibitor to form enzyme-inhibitor complexes, it was predicted that in the presence of inhibitors, intermediate accumulation could still be observed after 6 h incubation. The experimental results were in agreement with this prediction.

Stent-induced restenosis in the swine coronary artery is inhibited by a platelet-derived growth factor receptor tyrosine kinase inhibitor, TKI963.

Activities of vascular smooth muscle cells (SMCs) such as proliferation, migration, and matrix production contribute to restenosis following clinical interventions of angioplasty and stent placement. Because activation of platelet-derived growth factor (PDGF)-receptor tyrosine kinase (PDGFr-TK) influences these processes and promotes restenosis, TKI963, an inhibitor of the PDGFr-TK was discovered, and its efficacy was evaluated in blocking stent-induced restenosis as analyzed by intravascular ultrasound (IVUS). TKI963, a low-molecular-weight compound, inhibited the cell-free PDGF&bgr;r-TK with a Ki value of 56 ± 14 n M. TKI963 also inhibited PDGF-dependent events in human aortic SMCs (e.g., in situ PDGFr autophosphorylation, mitogenesis, chemotaxis, and collagen production with median inhibitory concentration values of approximately 300 n M) without affecting the activity of a series of membrane receptor tyrosine kinases and intracellular serine/threonine kinases. In vivo, stent-induced restenosis in the swine coronary artery was reduced by oral administration of TKI963 (1.25, 2.5, and 5 mg/kg BID, for 28 days). Late lumen cross-sectional area (CSA) loss, plaque CSA growth, and plaque volume in the stent determined by IVUS were dose-relatedly decreased (33–62% at 1.25 mg/kg BID to 66–92% at 5 mg/kg BID, depending on the parameter) compared with controls. TKI963 treatment of ≤1 week following stent placement had no effect on the prevention of restenosis. TKI963, a selective, orally bioavailable inhibitor of the PDGFr-TK, dose-relatedly reduced stent-induced restenosis and did so by inhibiting PDGF-dependent activities that occur as late events following stent placement.

Lovastatin is hypertriglyceridemic in syrian golden hamsters

The effects of a potent HMG CoA reductase inhibitor, lovastatin, was studied in male Syrian Golden hamsters. Lovastatin (0.1% in food for 6 days) increased hamster serum triglycerides by 12-fold with 2.4-fold increase in serum cholesterol. On continuous treatment serum triglyceride and cholesterol levels gradually decreased to below control values by 12-18 days. When hamsters were fed a mixture of lovastatin and Na-mevalonate no increase in serum triglyceride was observed. [14C]Cholesterol synthesis was increased by 266-fold in livers of hamsters fed lovastatin for 6 days. The increased synthesis of endogenous mevalonate metabolites may be a reason for the decrease in triglyceride levels after 6 days in our studies. The present study suggests that a mevalonate metabolite(s) is necessary for normal triglyceride metabolism in hamsters.

Inhibitors of HMG-CoA reductase. Biological effects of A 6-[2-[2-(4-fluoro-3-methylphenyl)-4-substituted cyclohexe-1-en-1-yl]ethenyl]-4-hydroxy-3,4,5,6-tetrahydro-2h-pyran-2-one

Abstract We report on a series of potent inhibitors of HMG-CoA reductase (HMGR) that were designed to examine the biological consequences that result from molecular changes in RG 12561. The introduction of functional groups on the cyclohexene nucleus of RG 12561 and the resulting inhibitor-enzyme interactions at remote binding sites of HMGR are discussed. Cellular membrane permeability may also contribute to potency. The HMGR inhibitory activity was measured utilizing solubilized enzyme. The compounds were evaluated as inhibitors of sterol biosynthesis in vitro using liver slices and in vivo after oral administration to the rat.