The synergistic hypocholesterolemic activity of the potent cholesterol absorption inhibitor, ezetimibe, in combination with 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors in dogs.

Ezetimibe (SCH 58235) and SCH 48461 are potent cholesterol absorption inhibitors, which cause significant decreases in plasma cholesterol levels in cholesterol-fed animals and in humans with hypercholesterolemia. These compounds selectively block intestinal uptake and absorption of cholesterol. These cholesterol absorption inhibitors cause modest, inconsistent reductions in plasma cholesterol levels in animals fed cholesterol-free chow diets. Although, these compounds block cholesterol absorption and increase neutral sterol excretion, chow-fed animals compensate for the loss of biliary cholesterol by increasing hepatic cholesterol synthesis. Therefore, we determined the effect of SCH 48461 and ezetimibe in combination with 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors in chow-fed dogs. A synergistic reduction in plasma cholesterol was observed in chow-fed dogs given SCH 48461 (0.1 mg/kg/d) and the HMG CoA reductase inhibitor, lovastatin (5 mg/kg/d). Neither SCH 48461 nor lovastatin alone affected plasma cholesterol levels. Their combination for 14 days caused a 36% reduction in plasma cholesterol levels from 129 mg/dL to 83 mg/dL (P <.05). Ezetimibe (0.007 mg/kg/d) also caused synergistic reductions in plasma cholesterol levels in chow-fed dogs when combined with HMG CoA reductase inhibitors for 2 weeks (5 mg/kg lovastatin -50%; 2.5 mg/kg pravastatin -41%; 5 mg/kg fluvastatin -60%, and -30% with low doses of simvastatin and atorvastatin 1 mg/kg). The combination of this class of cholesterol absorption inhibitors with an HMG CoA reductase inhibitor should be very effective clinically at reducing plasma cholesterol levels, even with reduced dietary intake of cholesterol.

GR231118 (1229U91) and other analogues of the C-terminus of neuropeptide Y are potent neuropeptide Y Y1 receptor antagonists and neuropeptide Y Y4 receptor agonists.

GR231118, BW1911U90, Bis(31/31')[[Cys31, Trp32, Nva34] neuropeptide Y(31-36)] (T-190) and [Trp-Arg-Nva-Arg-Tyr]2-NH2 (T-241) are peptide analogs of the C-terminus of neuropeptide Y that have recently been shown to be antagonists of the neuropeptide Y Y1 receptor. In this study, the activity of these peptides at each of the cloned neuropeptide Y receptor subtypes is determined in radioligand binding assays and in functional assays (inhibition of forskolin-stimulated cAMP formation). GR231118 is a potent antagonist at the human and rat neuropeptide Y Y1 receptors (pA2 = 10.5 and 10.0, respectively; pKi = 10.2 and 10.4, respectively), a potent agonist at the human neuropeptide Y Y4 receptor (pEC50 = 8.6; pKi = 9.6) and a weak agonist at the human and rat neuropeptide Y Y2 and Y5 receptors. GR231118 also has high affinity for the mouse neuropeptide Y Y6 receptor (pKi = 8.8). Therefore, GR231118 is a relatively selective neuropeptide Y Y1 receptor antagonist, but has appreciable activity at the neuropeptide Y Y4 and Y6 receptors as well. BW1911U90, T-190 and T-241 are moderately potent neuropeptide Y Y1 receptor antagonists (pA2 = 7.1, 5.8 and 6.5, respectively; pKi = 8.3, 6.5 and 6.8, respectively) and neuropeptide Y Y4 receptor agonists (pEC50 = 6.8, 6.3 and 6.6, respectively; pKi; 8.3, 7.7 and 8.3, respectively). These data suggest that the C-terminus of neuropeptide Y and related peptides is sufficient for activation of the neuropeptide Y Y4 receptor, but is not sufficient for activation of the neuropeptide Y Y1 receptor. Because BW1911U90, T-190 and T-241 are significantly less potent at the cloned human neuropeptide Y Y1 receptor than at the neuropeptide Y receptor in human erythroleukemia cells, these cells may express a novel neuropeptide Y receptor with high affinity for these peptides.

Wiedendiol-A inhibits cholesteryl ester binding to its transfer protein.

AIM: To study the wiedendiol-A (W-A) inhibition mechanism of plasma cholesteryl ester (CE) transfer protein (CETP) on the transfer of CE. METHODS: Using gel filtration method. RESULTS: W-A at 30 mumol.L-1 inhibited association of CE with CETP by 76% and CETP transfer activity by 81%. In addition, W-A enhanced binding of TP2, a monoclonal antibody with a CETP C-terminal epitope which is involved in CE binding, to CETP, suggesting a W-A-induced conformational change at the epitope for increased TP2 binding. When CETP activity was measured by varying high-density lipoproteins (HDL) concentration, the apparent Vmax of CE transfer was inhibited by 74% and 83% in the presence of W-A at 14 and 25 mumol.L-1, respectively, while the apparent K(m) of HDL for CETP did not change. CONCLUSION: W-A action is mediated through interaction between W-A and CETP, but not through those between W-A and lipoproteins.

Genomic organization and functional characterization of the mouse GalR1 galanin receptor.

Galanin mediates diverse physiological functions in digestive, endocrine, and central nervous systems through G-protein-coupled receptors. Two galanin receptors have been cloned but the gene structures are unknown. We report genomic and cDNA cloning of the mouse GalR1 galanin receptor and demonstrate that the coding sequence is uniquely divided into three exons encoding the N-terminal portion through the fifth transmebrane domain, the third intracellular loop, and the sixth transmembrane domain through the C-terminus. Functional analysis of the encoded cDNA revealed active ligand binding and intracellular signaling. The expression is detected in brain, spinal cord, heart and skeletal muscle.

2-Azetidinone cholesterol absorption inhibitors: structure-activity relationships on the heterocyclic nucleus.

A series of azetidinone cholesterol absorption inhibitors related to SCH 48461 ((-)-6) has been prepared, and compounds were evaluated for their ability to inhibit hepatic cholesteryl ester formation in a cholesterol-fed hamster model. Although originally designed as acyl CoA: cholesterol acyltransferase (ACAT) inhibitors, comparison of in vivo potency with in vitro activity in a microsomal ACAT assay indicates no correlation between activity in these two models. The molecular mechanism by which these compounds inhibit cholesterol absorption is unknown. Despite this limitation, examination of the in vivo activity of a range of compounds has revealed clear structure-activity relationships consistent with a well-defined molecular target. The details of these structure-activity relationships and their implications on the nature of the putative pharmacophore are discussed.

Inhibitors of acyl CoA:cholesterol acyltransferase.

Conformational restriction of previously disclosed acyclic (diphenylethyl)diphenylacetamides led to the discovery of several potent inhibitors of acyl CoA:cholesterol acyltransferase (ACAT). cis-[2-(4-Hydroxyphenyl)-1-indanyl]diphenylacetamide (4a) was the most potent ACAT inhibitor identified (IC50 = 0.04 microM in an in vitro rat hepatic microsomal ACAT assay, ED50 = 0.72 mg/kg/day in cholesterol-fed hamster.

Amides of piperidine, morpholine and piperazine substituted 1-phenylethylamines: inhibitors of acylCoA:cholesterol acyltransferase (ACAT) activity in vitro and in vivo.

Amides of some substituted 1,2-diarylethylamines have been shown to exhibit potent acylCoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) inhibitory activity in vitro in microsomal ACAT assays but show poor in vivo activity in a cholesterol-fed hamster model. In an effort to design ACAT inhibitors that are potent in both our in vitro and in vivo assays a series of amides of piperidine, morpholine and piperazine substituted 1-phenylethylamines were synthesized. Compounds of this series were found to be very potent inhibitors of ACAT in a microsomal ACAT assay and also exhibited potent activity in a cholesterol-fed hamster model.

Hypocholesterolemic activity of a novel inhibitor of cholesterol absorption, SCH 48461.

The amount of cholesterol that circulates in the plasma as lipoproteins can be affected by the balance of cholesterol metabolism within and between the intestines and liver. In the present report, we describe a novel hypocholesterolemic agent and document its pharmacological effects in animal models of hypercholesterolemia. The oral administration of (3R,4S)-1,4-bis-(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone (SCH 48461) reduced plasma cholesterol concentrations in cholesterol-fed hamsters, rats and rhesus monkeys with ED50s of 1, 2 and 0.2 mg/kg per day, respectively, SCH 48461 was also highly effective in reducing hepatic cholesteryl ester accumulation in cholesterol-fed hamsters and rats after 7 days of treatment. In one 3 week study, rhesus monkeys were fed a 0.25% cholesterol/22% saturated fat diet with or without SCH 48461. At the end of the 3 week period the control group's VLDL + LDL-cholesterol increased to 180 Mg/dl from a baseline of approximately 65 mg/dl while plasma apolipoprotein B levels had doubled. Animals treated daily with 1 mg/kg SCH 48461 maintained their baseline levels of VLDL + LDL-cholesterol, HDL-cholesterol, and plasma apolipoproteins B and A-I. After 3 weeks the diets of the two groups were switched. Within 1 week SCH 48461 (1 mg/kg per day) rapidly reversed the elevated VLDL + LDL-cholesterol levels of the previous control group to near baseline values. SCH 48461 exerted its hypocholesterolemic effect through the inhibition of cholesterol absorption. A dose of 10 mg/kg per day inhibited cholesterol absorption in cholesterol-fed hamsters by 68% while a similar reduction was achieved in chow-fed monkeys with 3 mg/kg per day. This latter dose inhibited cholesterol absorption in cholesterol-fed monkeys by 95%. Treatment of cholesterol-fed monkeys with 10 mg/kg per day SCH 48461 significantly increased fecal neutral sterol excretion (52 vs. 32 mg/kg) but had no effect on acidic sterol excretion. Using a 2-h absorption model in cholesterol-fed hamsters, SCH 48461 caused a 46% inhibition of unesterified [14C]cholesterol accumulation in the intestinal wall and a 90% inhibition of cholesteryl ester formation at a dose of 10 mg/kg. Similar data were observed when the plasma radioactivity was assessed, indicating inhibition of both free (61%) and esterified (85%) cholesterol appearance. In contrast, CI-976, a potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, did not affect the uptake of free cholesterol into the intestines while inhibiting cholesterol esterification (98% inhibition).(ABSTRACT TRUNCATED AT 400 WORDS)

Substituted (1,2-diarylethyl)amide acyl-CoA:cholesterol acyltransferase inhibitors: effect of polar groups on in vitro and in vivo activity.

Substituted (1,2-diarylethyl)amides have been prepared and evaluated for their ability to inhibit microsomal acyl-CoA:cholesterol acyltransferase activity in vitro and to lower hepatic cholesteryl ester content in vivo in a cholesterol-fed hamster. Simple unsubstituted (diarylethyl)amides were potent inhibitors in vitro but showed poor activity in vivo. Introduction of polar groups at specific locations on the diarylethylamine moiety decreased in vitro activity but increased in vivo activity. Both effects were highly structure dependent, suggesting specific interactions which were mediating activity in each model. Optimization of these opposing effects led to compounds which were potent in both models.

The effect of acyl CoA: cholesterol acyltransferase inhibition on the uptake, esterification and secretion of cholesterol by the hamster small intestine.

Acyl CoA: cholesterol acyltransferase (ACAT) inhibitors are known to inhibit cholesterol absorption and are under investigation to reduce hypercholesterolemia. These studies examine the effect of an ACAT inhibitor 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)-dodecanamide (PD128042) on the uptake, metabolism and secretion of cholesterol by the hamster intestinal wall in a short-term model. Preliminary studies in this model indicated that the uptake of 14C-cholesterol and its subsequent esterification 2 hr postoral dosing occurs primarily in the duodenal and jejunal segments of the small intestine and most of the radiolabeled cholesterol and cholesteryl ester in the plasma was associated with chylomicrons. In both single- and multiple-dose studies, PD128042 (50 mg kg-1 day-1) did not inhibit intestinal uptake of [14C]-cholesterol but [14C]-cholesteryl ester formation was inhibited. The free [14C]-cholesterol appearing in plasma was not affected despite a large reduction in [14C]-cholesteryl ester. In contrast, cholestyramine (1 g kg-1 day-1) inhibited the uptake of the radiolabeled free cholesterol and the appearance of cholesteryl ester in the intestine and plasma. The effects of PD128042 on cholesterol and cholesteryl ester mass associated with scraped intestinal mucosa were consistent with the effects observed with the use of the radiolabeled cholesterol. In addition, PD128042 did not affect the uptake of appearance of radiolabeled triglyceride in the intestinal wall after oral gavage of 3H-trioleoylglycerol. Taken together, the data suggest that ACAT inhibition reduces cholesterol absorption by limiting cholesteryl ester incorporation into chylomicrons and has no effect on the intestinal processing of free cholesterol to be secreted into plasma.

Demonstration of a direct effect on hepatic acyl CoA: cholesterol acyl transferase (ACAT) activity by an orally administered enzyme inhibitor in the hamster.

Orally active inhibitors of acyl CoA:cholesterol acyl transferase (ACAT), such as Lederle CL277082 (LE), are known to reduce plasma and hepatic cholesteryl ester levels, although the mechanisms are not well understood. Several groups have reported the inhibition of cholesterol absorption upon oral ACAT inhibitor administration. In this study, we used 7-day dietary and drug treatments of hamsters to examine the possible effects of LE on hepatic ACAT. ACAT assays were performed using liver homogenates in the absence and presence of a saturating level of exogenously added cholesterol. LE (100 mg/kg/day) treatment of chow or 0.5% cholesterol-fed animals caused reductions in ACAT activity without additional cholesterol as compared with non-treated animals. When a saturating level of cholesterol was added to the assays, reductions in ACAT activity upon LE treatment of chow- or cholesterol-fed animals were also observed. Treatment of cholesterol-fed animals with cholestyramine in the diet reduced ACAT activity in the absence of added cholesterol. However, ACAT activities similar to those of non-treated animals were observed at a saturating level of cholesterol. This latter effect demonstrates that inhibition of cholesterol absorption reduces cholesterol delivery to the liver but does not reduce cholesterol esterifying capacity since cholestyramine is not absorbed and has no direct effect on the liver. The decreased ACAT activity in homogenates from LE-treated animals could also be mimicked in a dose-dependent manner by the addition of exogenous LE to liver homogenates from non-treated animals. These results indicate that hepatic ACAT activity is regulated by the availability of free cholesterol, and that orally administered LE has a direct effect on hepatic ACAT activity in the liver. In addition, the data are consistent with LE activity in the liver as being responsible, in part, for the reduced hepatic and plasma cholesteryl esters in treated animals.

Effects of acyl-CoA: cholesterol O-acyltransferase inhibition on cholesterol absorption and plasma lipoprotein composition in hamsters.

1. The ACAT inhibitors, CL 277082 and SA 58-035 were administered for 7 days to hamsters fed diets containing 0.5% cholesterol. 2. Both agents inhibited cholesterol absorption, decreased hepatic. VLDL and IDL cholesterol esters, plasma HDL and HDL apoE and A-I. 3. In addition, CL 277082 treatment produced significant decreases in plasma cholesterol, VLDL apoB and plasma IDL. 4. The cholesteryl esters in VLDL and LDL but not HDL were more polyunsaturated in CL 277082 treated animals. 5. These results support the hypothesis that ACAT inhibition in the cholesterol fed hamster results in an inhibition of dietary cholesterol absorption, thus limiting the cholesterol supply required for the hepatic production of triglyceride-rich lipoproteins.

Rabbit heart fatty acid-binding protein. Isolation, characterization, and application of a monoclonal antibody.

A fatty acid-binding protein (FABP) was purified from rabbit heart and characterized with respect to size, isoelectric point, and tissue distribution. This protein was found in red muscle, diaphragm, and aorta, as well as in the heart. Amino acid composition of rabbit heart FABP differed only slightly from the human and rat proteins. Rabbit heart FABP was shown to bind two molecules of fatty acid. A monoclonal antibody was developed and used to demonstrate the feasibility of a one-step purification with affinity chromatography. Cross-reactivity was found between the human protein and the rabbit antibody, and an immunoassay was developed to human heart FABP. Levels of human heart FABP in the plasma of patients with acute myocardial infarction were significantly elevated (83 +/- 9 micrograms/ml) compared with patients with pulmonary edema (52 +/- 7 micrograms/ml) and normal volunteers (28 +/- 5 micrograms/ml; p less than 0.05, mean +/- SEM).

Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis.

The ability of purified rat liver and heart fatty acid binding proteins to bind oleoyl-CoA and modulate acyl-CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart fatty acid binding protein was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver fatty acid binding protein has a single binding site acyl-CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl-CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver fatty acid binding protein stimulated acyl-CoA production, whereas that from heart did not stimulate production over control values. 14C-labeled fatty acid-fatty acid binding protein complexes were prepared, incubated with membranes, and acyl-CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl-CoA in the presence of liver fatty acid binding protein but in the presence of heart fatty acid binding protein, only 45% of the fatty acid was converted. Liver but not heart fatty acid binding protein bound the acyl-CoA formed and removed it from the membranes. The amount of product formed was not changed by additional membrane, enzyme cofactors, or incubation time. Additional liver fatty acid binding protein was the only factor found that stimulated product formation. Acyl-CoA hydrolase activity was also shown in the absence of ATP and CoA. These studies suggest that liver fatty acid binding protein can increase the amount of acyl-CoA by binding this ligand, thereby removing it from the membrane and possibly aiding transport within the cell.

Binding of lysophosphatidylcholine to the rat liver fatty acid binding protein.

In this study, lysophosphatidylcholine (lysoPC) was shown to bind to a fatty acid binding protein isolated from rat liver. To demonstrate the binding, lysoPC was incorporated into multilamellar liposomes and incubated with protein. For comparison, binding of both lysoPC and fatty acid to liver fatty acid binding protein, albumin, and heart fatty acid binding protein were measured. At conditions where palmitic acid bound to liver fatty acid binding protein and albumin at ligand to protein molar ratios of 2:1 and 5:1, respectively, lysoPC binding occurred at molar ratios of 0.4:1 and 1:1. LysoPC did not bind to heart fatty acid binding protein under conditions where fatty acid bound at a molar ratio of 2:1. Competition experiments between lysoPC and fatty acid to liver fatty acid binding protein indicated separate binding sites for each ligand. An equilibrium dialysis cell was used to demonstrate that liver fatty acid binding protein was capable of transporting lysoPC from liposomes to rat liver microsomes, thereby facilitating its metabolism. These studies suggest that liver fatty acid binding protein may be involved in the intracellular metabolism of lysoPC as well as fatty acids, and that functional differences may exist between rat liver and heart fatty acid binding protein.

Effect of surface composition on triolein hydrolysis in phospholipid vesicles and microemulsions by a purified acid lipase.

Sonicated dispersions of egg yolk phosphatidylcholine and triolein as vesicles and microemulsions have been used as substrates for the assay of a purified acid lipase. Previous studies have also shown that triolein localized in the surface phase of emulsions is the preferred substrate. In this study, we examined enzyme activity following several surface modifications using both vesicles and microemulsions. When the acidic phospholipids phosphatidylserine and phosphatidic acid were incorporated into both vesicles and microemulsions at up to 10 mol % of the total phospholipid, a dose-dependent reduction in the apparent Km was observed. Using the vesicles as substrate, a dose-dependent decrease in Vmax was also observed. Agarose gel electrophoresis was used to verify suspected changes in net particle charge. Analogous inclusion of phosphatidylethanolamine, sphingomyelin, or cholesterol did not affect kinetic parameters. Addition of oleic acid to sonication mixtures produced vesicles with a decreased apparent Km and Vmax, but triolein hydrolysis in microemulsions was not significantly altered. Triolein-containing vesicles prepared by using dimyristoyl- or dipalmitoylphosphatidylcholine were hydrolyzed maximally at the gel liquid-crystalline transition temperatures of the appropriate phospholipid. Differential scanning calorimetry was used to verify the temperatures of transition in these vesicles. The results indicate that acid lipase activity is influenced by the charge or physical state of the surface phase of model substrates and suggest that degradation of core components of naturally occurring substrates such as lipoprotein may be influenced by chemical changes on the surface of these particles.

Hydrolysis of triolein in phospholipid vesicles and microemulsions by a purified rat liver acid lipase.

An acid lipase was purified from rat liver lysosomes. Lipase purification involved affinity chromatography, gel filtration, and stabilization of the purified preparation using ethylene glycol and Triton X-100. A molecular weight of 67,000-69,000 was determined independently using density gradient centrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel filtration. To study enzyme action, model substrates were prepared by incorporating radiolabeled triolein into either unilamellar vesicles or microemulsions. Substrates were prepared by cosonicating aqueous dispersions of lecithin and triolein. Formation of vesicles or emulsions depended on the relative amount of each lipid and on sonication conditions. Vesicles were prepared at molar ratios between 70:1 and 26:1 (lecithin:triolein) and the microemulsion preparation at a molar ratio of 1:1. The substrate particles were of similar size (220-250 A) as determined by Bio-Gel A-15m chromatography. Hydrolysis of triolein contained in vesicles or emulsions was similar with respect to pH, temperature, and reaction products. Kinetic studies on vesicles with increasing triolein content showed progressively greater Vmax values (0-0.6 mumol/min/mg), and Vmax for the emulsion was 3.1 mumol/min/mg. Addition of human very low or low density lipoprotein produced a dose-dependent inhibition with both substrates. The results show that synthetically prepared microemulsions are stable and effective substrates for the acid lipase and indicate that surface-oriented triolein is hydrolyzed in both preparations.