RP 64477: a potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase with low systemic bioavailability.

RP 64477 (N-butyl-3-(p-decyloxybenzamido)-4-(methylthio)benzamide) has been shown to be a potent inhibitor of the cholesterol esterifying enzyme Acyl-coenzyme A:cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) in intestinal, hepatic, adrenal, and arterial tissue preparations obtained from a range of animal species. Drug concentrations producing 50% inhibition of enzyme activity (IC50 values) ranged from 14-283 nM. Inhibition by RP 64477 in a rabbit intestinal enzyme preparation was shown to be non-competitive with respect to the substrate oleoyl-CoA. In whole cell assays using human intestinal (CaCo-2), hepatic HepG2) and monocytic (THP-1) cell lines, RP 64477 inhibited ACAT activity with IC50s of 113, 503, and 180 nM, respectively. RP 64477 (0.03% w/w by diet) reduced significantly cholesterol absorption in cholesterol/cholic acid-fed rats from 94+/- 8% to 65 +/- 4%. In cholesterol-fed rabbits, cholesterol absorption was reduced from 72 +/- 5% to 50 +/-5% and 44 +/- 5% at dose levels of 10 and 30 mg kg-1 b.i.d., respectively. Plasma cholesterol levels were reduced dose-dependently in both cholesterol/cholic-acid-fed rats and cholesterol-fed rabbits. Neither cholesterol absorption nor plasma cholesterol levels were reduced significantly in animals maintained on standard laboratory diets. Pharmacokinetic studies indicated that RP 64477 were very poorly absorbed following oral administration to rats. Plasma levels of drug were < 2 ng mL-1 following a dose of 2000 mg kg-1 p.o.. When radiolabelled RP 64477 was administered orally, limited absorption was indicated by the overwhelming elimination of radioactivity in the faces (96.4% of administered material) coupled with low renal clearance (0.6% of dose) and biliary excretion (0.05% of dose). In conclusion, this work shows that RP 64477 is a potent inhibitor of ACAT obtained from a range of animal species and man. Inhibition of cholesterol absorption and hypocholesterolaemic activity has been demonstrated in rats and rabbits maintained on diets supplemented with cholesterol. Pharmacokinetic studies indicate low systemic exposure to RP 64477 as a result of limited absorption of this drug.

Acyl-CoA:cholesterol O-acyl transferase (ACAT) inhibitors. 1. 2-(Alkylthio)-4,5-diphenyl-1H-imidazoles as potent inhibitors of ACAT.

A potent, bioavailable ACAT inhibitor may have beneficial effects in the treatment of atherosclerosis by (i) reducing the absorption of dietary cholesterol, (ii) reducing the secretion of very low density lipoproteins into plasma from the liver, and (iii) preventing the transformation of arterial macrophages into foam cells. We have found that a mevalonate derivative 2, which contains a 4,5-diphenyl-1H-imidazol-2-yl moiety, inhibits rat hepatic microsomal ACAT in vitro and produces a significant hypocholesterolemic effect in the cholesterol-fed rat. Structure-activity relationships for analogues of 2 demonstrate that the 4,5-diphenyl-1H-imidazole moiety is a pharmacophore for inhibition of rat microsomal ACAT.

Hypolipidemic 2-[4-(1,1-dimethylethyl)phenyl]-4H-3,1-benzoxazin-4-ones. Structure-activity relationships of a novel series of high-density lipoprotein elevators.

The preparation and plasma lipid altering characteristics of a series of 4H-3,1-benzoxazin-4-ones are described. Hypocholesterolemic, hypotriglyceridemic, and high-density-lipoprotein elevating properties are found for derivatives bearing a 4-(1,1-dimethylethyl)phenyl group at the 2-position, and this activity is displayed in both hypercholesterolemic and in normolipidemic rats when the ring system is substituted at position 6 with hydrogen, methyl, chloro, or iodo groups, and is optimal when the 6-position is substituted by a bromine atom. Evidence is presented suggesting that a metabolite or degradation product is responsible for the changes in lipoprotein concentration observed with active molecules of this type. Synthesis of anticipated degradation products of the active molecules gave products displaying the expected in vivo activity, but no improvement in the narrow therapeutic margin of the best compound, 6-bromo-2-[4-(1,1-dimethylethyl)phenyl]-4H-3,1-benzoxazin-4-one, was obtained.

Radioimmunoassay of chemically modified insulins.

The reactions between four insulin antisera and eighteen insulin derivatives with modifications at the A1, B1 and B29 positions have been studied using a standard double-antibody radioimmunoassay procedure. The derivatives studied had: a) single modifications at A1, B1 or B29,; b) modifications at two sites with or without a crosslink between them; c) modifications at all three sites with or without a crosslink. Analysis of the results showed a clear difference in the reactivity of the antisera. One antiserum (GP5) was highly sensitive to modifications of the B1 residue and another (Ab1) was sensitive to A1 and B29 modifications. Thus, immunological potencies of insulin analogues derived on the basis of these reactions with the antisera give widely varying results. These antisera were used in discriminatory radioimmunoassays of chemically modified insulins in biological fluids for estimation of in vivo hypoglycaemic potencies by an infusion technique, where the knowledge of the specificity of the antisera was useful in assessing the immunological identity of immunoreactive material in plasma with the analogue infused.

Studies on the relationship between the molecular structure and the catabolism of insulin.

The catabolism of insulins modified at the A1, B1 or B29 positions or containing a synthetic crosslink between the A1 and B29 positions has been studied in vivo and in vitro. The metabolic clearance rates (MCR) of insulin, proinsulin and chemically modified insulins have been measured by a priming-dose constant infusion technique in greyhounds. Insulins modified at A1 and B29, particularly the crosslinked materials, had markedly lowered MCR's whilst B1 analogues did not differ from insulin. Proinsulin and the A1-B29 crosslinked materials showed a markedly lowered degradability by glutathione-insulin transhydrogenase.

Biological properties of chemically modified insulins. I. Biological activity of proinsulin and insulin modified at A1-glycine and B29-lysine.

Beef insulin, pork proinsulin and four derivatives of beef insulin modified at the A1-B29 site on the molecular surface have been studied. Three derivatives had a synthetic crosslink between the A and B chains. Previous studies with these materials [2, 3 and 5] had demonstrated in vivo bioactivities which were much higher than those displayed in vitro. This paper reports experiments which explain this discrepancy. The analogues were administered at equimolar rates to anaesthetised greyhounds by a priming-dose constant infusion technique and the plasma concentrations achieved were estimated by radioimmunoassay. Proinsulin and the modified insulins were metabolised more slowly than insulin. Biopotency values, which related fall in plasma glucose concentration to the total administered dose of analogue, agreed broadly with published results of conventional in vivo bioassays. On the other hand, calculation of potency in relation to the serum concentration of analogue actually achieved, yielded results which agreed more closely with in vitro assay data. We conclude that for these analogues, reported discrepancies between in vitro and in vivo biopotencies can be largely explained by the different rates at which these materials are metabolised.