Antithrombotic effect of LB-30057 (CI-1028), a new synthetic thrombin inhibitor, in a rabbit model of thrombosis: comparison with inogatran.

LB-30057 (CI-1028) is a novel, orally bioavailable, direct thrombin inhibitor with a Ki of 0.38 nM against human thrombin. The effects of LB-30057 on thrombus formation and hemostasis were evaluated in a veno-venous shunt model of thrombosis in rabbits, and compared with inogatran, another direct inhibitor of thrombin. Each compound was studied at 5 or 6 different doses with 5 or 6 rabbits in each group. After administration as a bolus i.v. injection followed by continuous infusion, both LB-30057 and inogatran dose-dependently inhibited thrombus formation, which was measured as an increase in time to occlusion (TTO) and a decrease in thrombus weight. Both compounds also improved vena caval blood flow and reduced the overall incidence of thrombotic occlusion. LB-30057 significantly prolonged TTO from 23 +/- 4 min (before dose) to 110 +/- 10 min at the highest dose (0.7 mg/kg + 47 microg/kg/min) (p < 0.001), and reduced thrombus weight from 57 +/- 2 mg to 15 +/- 5 mg (p < 0.001). Occlusive thrombus formed in only one of six rabbits that received the highest dose of LB-30057 (vs. 13/13 in the control group, p < 0.01). At the dose that produced the maximum antithrombotic effect (0.7 mg/kg + 47 microg/kg/min), LB-30057 increased aPTT and bleeding time approximately 2-and 2.5-fold above baseline, respectively. On a gravimetric basis, LB-30057 and inogatran displayed comparable in vivo antithrombotic efficacy. When compared to equally effective anti thrombotic doses of inogatran, LB-30057 caused less prolongation in aPTT, had no effect on PT, and tended to have less of effect on bleeding time. These results indicate that LB-30057 is an effective antithrombotic compound and it appears to have a better benefit/risk profile than inogatran in this experimental model.

CI-1011 lowers lipoprotein(a) and plasma cholesterol concentrations in chow-fed cynomolgus monkeys.

Lipoprotein(a) (Lp(a)), which is generated through the covalent association of apolipoprotein(a) (apo(a)) and apo B-100-LDL, is an independent risk factor for several vascular diseases. Therefore, there is interest in developing therapies for lowering Lp(a). This investigation was carried out to determine the effect of CI-1011, a potent lipid regulator in rodents, on Lp(a) and other lipid parameters in cynomolgus monkeys (Macaca fascicularis). Nine healthy male monkeys on a normal chow diet were orally treated with CI-1011 at 30 mg/kg per day for 3 weeks. Lp(a) and total cholesterol levels were significantly decreased after 1 week and maximally reduced to 68 and 73% of control levels, respectively, after 3 treatment weeks. The decreases in total cholesterol were mainly due to changes in low density lipoprotein (LDL). The LDL:HDL ratio decreased by 30%. Triglycerides were unaffected by treatment. Lp(a) and total cholesterol levels returned to pretreatment values after stopping treatment suggesting a direct effect of the compound on their inhibition. Further studies demonstrated that CI-1011 was effective at a low dose of 3 mg/kg per day after 1 week of administration. CI-1011 also decreased apo B-100 to 80% of control levels, but this change was not sufficient to account for the Lp(a) lowering. There was also no correlation between the changes in Lp(a) and apo B-100 levels. Treatment of cynomolgus monkey primary hepatocyte cultures with CI-1011 resulted in a dose-dependent inhibition of Lp(a) levels suggesting a direct hepatic effect of the compound. Western blot analysis of the samples showed that changes in Lp(a) were associated mainly with decreased apo(a) (47%), but not apo B-100 (17%). These results demonstrate that CI-1011 effectively decreases Lp(a) levels both in vivo and in vitro.

alpha-Substituted malonester amides: tools to define the relationship between ACAT inhibition and adrenal toxicity.

We prepared a series of alpha-substituted malonester amides that were evaluated for their ability to inhibit acyl-CoA:cholesterol O-acyl transferase activity in vitro and to lower plasma total cholesterol levels in a variety of cholesterol-fed animal models. Compounds of this series were also useful in examining the relationship between adrenal toxicity and ACAT inhibition. One compound from this series, 9f, was a potent inhibitor of ACAT in both the microsomal and cellular assays. It was also bioavailable as determined by both a bioassay and a HPLC-UV assay. This compound was evaluated in both guinea pig and dog models of adrenal toxicity and compared to tetrazole amide 15. In the most sensitive species, the dog, both of these compounds achieved good plasma levels; however, compound 9f caused adrenal necrosis, whereas compound 15 had no effect on the adrenal gland. This adds to the growing body of evidence that the adrenal toxicity observed with ACAT inhibitors may not be mechanism related.

Inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) as hypocholesterolemic agents: synthesis and structure-activity relationships of novel series of sulfonamides, acylphosphonamides and acylphosphoramidates.

Sulfoacetic acid, phosphoramidate, and phosphoramide analogs of the ACAT inhibitors, CI-999 and CI-1011 were synthesized. The structure-activity relationships of these compounds as ACAT inhibitors are described.

Heterocyclic ureas: inhibitors of acyl-CoA:cholesterol O-acyltransferase as hypocholesterolemic agents.

A series of diaryl-substituted heterocyclic ureas was prepared, and their ability to inhibit acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in cholesterol-fed animal models in vivo was examined. N-(2,6-Diisopropylphenyl)-N'-tetrazole or isoxazole-substituted heterocyclic ureas proved optimal. A carbon chain of 11-14 carbons substituted 1,3 with respect to the amine provided the optimal side chain. Substitution of the alkyl chain generally lowered activity. Tetrazole urea 2i dosed at 3 mg/kg lowered plasma total cholesterol (TC) 67% in an acute, cholesterol-fed (C-fed) rat model of hypercholesterolemia and 47% in C-fed dogs. Tetrazole 2i, dosed at 10 mg/kg, also lowered TC 52% and raised HDL cholesterol 113% in rats with pre-established hypercholesterolemia.

Heterocyclic amides: inhibitors of acyl-CoA:cholesterol O-acyl transferase with hypocholesterolemic activity in several species and antiatherosclerotic activity in the rabbit.

A series of heterocyclic amides were synthesized and evaluated as inhibitors of acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and for cholesterol lowering in cholesterol-fed rats. Compounds were evaluated for cell-based macrophage ACAT inhibition, bioactivity, and adrenal toxicity. Candidates were selected for evaluation in cholesterol-fed dogs and, ultimately, the injured cholesterol-fed rabbit model of atherosclerosis. The heterocyclic amides potently inhibited rabbit liver ACAT (IC50's = 0.014-0.11 microM), and the majority of compounds significantly lowered plasma cholesterol (42-68%) in an acute cholesterol-fed rat model at 3 mg/kg. The most efficacious compounds in the rat were evaluated for bioactivity in vivo and arterial ACAT inhibition in a cell-based macrophage ACAT assay. Two highly bioactive analogs, (+/-)-2-(3-dodecylisoxazol-5-yl)-2-phenyl-N-(2,4,6-trimethoxypheny l) acetamide (13a) and (+/-)-2-(5-dodecylisoxazol-3-yl)-2-phenyl-N-(2,4,6-trimethoxypheny l) acetamide (16a), were selected for further study and were found to be nontoxic in a guinea pig model of adrenal toxicity. Compounds 13a and 16a lowered total cholesterol in the cholesterol-fed rat, rabbit, and dog models of pre-established hypercholesterolemia. Compound 13a in the injured cholesterol-fed rabbit model of atherosclerosis was effective in slowing the development of cholesteryl ester-rich thoracic aortic lesions, reducing lesion coverage by 53% at a dose of 1 mg/kg.

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 17. Structure-activity relationships of several series of compounds derived from N-chlorosulfonyl isocyanate.

Several series of acyl-CoA:cholesterol O-acyltransferase inhibitors were prepared by the stepwise addition of nitrogen, oxygen, and sulfur nucleophiles to N-chlorosulfonyl isocyanate. The (aminosulfonyl)ureas 3-44 were the most potent inhibitors in vitro, with several compounds having IC50 values < 1 microM. Although the other series of compounds were not as potent in vitro, many compounds did display good in vivo activity in cholesterol-fed rats. Several of the oxysulfonyl carbamates (including CI-999, 115) showed excellent lipid-lowering activity in the chronic in vivo screen, demonstrating significant cholesterol lowering in a pre-established hypercholesterolemic state.

Bioisosterism in drug design: identification of and structure-activity relationships in a series of glycine anilide ACAT inhibitors.

To examine the effects of bioisosteric replacement on the biological activity of our previously disclosed disubstituted urea inhibitors of the enzyme acyl-CoA:cholesterol acyltransferase (ACAT), we prepared a series of N'-substituted and N',N'-disubstituted glycine anilides. These compounds were tested for the ability to inhibit ACAT in vitro and lower plasma total cholesterol in cholesterol-fed rats given a single high-fat, high-cholesterol meal. ACAT inhibitory potency was greatest in compounds containing 2,6-diisopropyl substituents in the anilide portion with the glycine nitrogen substituted by a 1,1-diphenylmethyl moiety. Small improvements in potency in vitro were obtained by substitution of electron donating groups in the 2-, 3- or 5-positions of the aryl rings of the 1,1-diphenylmethyl moiety, but not by substitution in the 4-position. In vitro potency was maintained, but not improved by acylation of the glycine nitrogen. Through a QSAR analysis of in vitro ACAT inhibition for this set of compounds, an equation could be derived which accounted for 85% of the variance in the dataset. An optimal clogp of 6.65 was found, comparable to that found for other series of ACAT inhibitors. In general, compounds from this series displayed inhibitory potency against ACAT in vitro and hypocholesterolemic activity in the in vivo rat model of hypercholesterolemia comparable to that found with the ureas.

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 11. Structure-activity relationships of several series of compounds derived from N-(chlorocarbonyl) isocyanate.

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.

Inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). 7. Development of a series of substituted N-phenyl-N'-[(1-phenylcyclopentyl)methyl]ureas with enhanced hypocholesterolemic activity.

We recently described our initial structure-activity relationship (SAR) studies on a series of N-phenyl-N'-aralkyl- and N-phenyl-N'-(1-phenylcycloalkyl)ureas as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). From this series of analogs, compound 1 (PD 129337) was identified as a potent inhibitor of ACAT with an IC50 value of 17 nM. It was also shown to dose-dependently lower plasma cholesterol in cholesterol-fed rats. However, further investigation led to the suggestion that this compound was poorly absorbed, due to a lack of efficacy when administered by gavage in an aqueous vehicle. To overcome this deficiency, we continued our SAR study on this novel series of ACAT inhibitors using an acute in vivo screen in which the compounds are administered to rats in an aqueous, CMC/Tween suspension vehicle. Modification of the N'-phenyl moiety by incorporating functional groups which were amenable to forming salts and/or polar groups to reduce lipophilicity led to the identification of several inhibitors which displayed excellent efficacy employing this protocol. Overall, substitution on the phenyl ring in the ortho, meta, or para positions led to inhibitors with only a slight decrease in potency in vitro compared to the parent unsubstituted compound. Bulkier groups in the para position tended to lower the ACAT inhibitory activity in vitro. Polar groups, such as carboxyl (33,34), lowered in vitro activity significantly, suggesting that polar-ionic interactions are disfavored for the enzyme activity. From this series, compound 28 was evaluated further in secondary in vivo screens. In a chronic cholesterol-fed rat model of hypercholesterolemia, compound 28 dose-dependently reduced nonHDL cholesterol and significantly elevated HDL cholesterol. It showed significantly greater aqueous solubility than the parent compound 1. However, it was shown to cause adrenal toxicity in guinea pigs. This led us to design a series of homologs (44-51) with increased basicity and lower lipophilicity. Some of these compounds were more potent ACAT inhibitors in vitro and demonstrated excellent hypocholesterolemic activity in vivo. Interestingly, compound 45, unlike 28, did not produce adrenal toxicity in guinea pigs and demonstrated excellent lipid-modulating activity in the chronic model of preestablished dyslipidemia in rats.

Inhibitors of acyl-CoA:cholesterol acyltransferase. 5. Identification and structure-activity relationships of novel beta-ketoamides as hypocholesterolemic agents.

A study of structure-activity relationships of substituted beta-ketoamide ACAT inhibitors I and II was performed. The results of this study suggest that whereas the beta-keto group was tolerated with no loss in activity, beta-hydroxy and oxime moieties led to significantly reduced activity in vitro and in vivo. The most potent inhibitor from the acyclic series (I) (11, IC50 = 0.006 microM) contained a C-13 alkyl chain. This compound reduced plasma total cholesterol by 38% and 66% at 3 and 30 mg/kg, respectively, in cholesterol-fed rats. Dimethylation alpha to the anilide core (5) and subsequent N-methylation of the amide NH (6) decreased in vitro potency significantly. It was also found that high potency was retained with inhibitors which incorporated the carbonyl into a lactam ring (II).

Effect of the ACAT inhibitor CI-976 on plasma cholesterol concentrations and distribution in hamsters fed zero- and low-cholesterol diets.

The overall objective of the present study was to determine if the ACAT inhibitor CI-976 can lower plasma cholesterol in hamsters fed zero or low, "human-like" levels of cholesterol. With a purified diet containing zero dietary cholesterol, CI-976 significantly lowered VLDL cholesterol (VLDL-C), but not total plasma cholesterol (TPC). When 0.06% cholesterol was added to this diet, reductions in both VLDL and LDL cholesterol (LDL-C) lowered TPC. Efficacy was still greater with 0.2% dietary cholesterol, but not potency. Mixing CI-976 into the purified diet resulted in greater decreases in VLDL-C compared to gavage administration, but LDL-C reductions with 0.2% cholesterol were optimal with gavage. With nonpurified, chow-based diets efficacy was markedly greater with diet-admix administration, regardless of the amount of dietary cholesterol. CI-976 inhibited cholesterol absorption with chow-based diets more potently compared to nonabsorbable agents (e.g., beta-sitosterol, tigogenin cellobioside), and the lowering of LDL-C was greatest when inhibition of cholesterol absorption was maximal. We conclude that the ACAT inhibitor CI-976 is efficacious in hamster models which utilize human-like levels of dietary cholesterol. Moreover, the data suggest that the pharmacologic responses to lipophilic ACAT inhibitors in the hamster, or even other lipid-regulating drugs, are likely to depend not only on the type of basal diet but also on the mode of drug administration.

Effects of quinapril, a new angiotensin converting enzyme inhibitor, on left ventricular failure and survival in the cardiomyopathic hamster. Hemodynamic, morphological, and biochemical correlates.

The effect of chronic therapy with quinapril on the temporal progression of left ventricular failure and survival was assessed in the CHF 146 cardiomyopathic (CM) hamster, which is an idiopathic model of congestive heart failure. Age-matched Golden Syrian (GS) hamsters served as normal controls. Quinapril was administered in the drinking water at average daily doses of 10.2, 112.4, and 222.4 mg/kg/day. In untreated CM hamsters, in vitro left ventricular performance progressively deteriorated with increasing age beginning at roughly 180 days. This decline in left ventricular performance was accompanied by a decrease in coronary flow and an increase in left ventricular volume. Administration of quinapril from 180 to 300 days of age prevented the decline of in vitro left ventricular contractile performance and coronary flow and also reduced the age-dependent increases in left ventricular volume. The cardioprotective effects of quinapril were observed at doses of 112.4 and 222.4 mg/kg/day but not at 10.2 mg/kg/day. Lung angiotensin converting enzyme activity was significantly inhibited by quinapril in GS and CM hamsters at 240 and 300 days of age at all dose levels. In contrast, significant inhibition of ventricular angiotensin converting enzyme activity was observed consistently at doses of 112.4 and 222.4 mg/kg/day quinapril but not at 10.2 mg/kg/day. In the survival protocol, CM and GS hamsters were treated with vehicle or quinapril (100 mg/kg/day) from 180 to 522 days of age. During the initial 210 days of treatment (from 180 to 390 days of age) 78.3% of the vehicle-treated CM hamsters died compared with 27.7% of quinapril-treated CM hamsters. Quinapril increased the median survival time of CM hamsters by 32.9% (112 days). It is concluded that chronic quinapril therapy exerts a significant cardioprotective effect and also increases survival.