Evaluation of timefurone, a new anti-atherosclerotic drug, for its effects on lipoprotein cholesterol in male SEA Japanese quail and rats.

Timefurone was evaluated in several animal models for cholesterol-lowering and anti-atherosclerotic activity. In normal male rats, a dose-response study with timefurone (3, 10, 30, 50 and 100 mg/kg/day) was conducted for 7 days. Significant activity was observed only at 50 and 100 mg/kg/day, where very low and low density lipoprotein cholesterol [(VLDL + LDL)-C] and total-C levels were reduced (mean 27 and 20%). High density lipoprotein cholesterol (HDL-C) was lowered 24% by the high timefurone dose. Timefurone (10, 20, 50 and 100 mg/kg/day in the diet) was then examined in normocholesterolemic SEA japanese quail. beta-lipoprotein cholesterol (VLDL + LDL)-C was reduced at all doses (mean 58%), while alpha-lipoprotein cholesterol (HDL-C) was elevated by all doses of timefurone (mean 45%). Male weanling rats made moderately hypercholesterolemic represented a 3rd phase of timefurone (2.5, 5, 10, 20, 50, 100 mg/kg/day) testing. After 4 days of drug treatment, marked hypocholesterolemic activity was observed for (VLDL + LDL)-C (mean decrease 49%) and total-C (mean 33%). HDL-C levels were increased with 10 and 100 mg/kg/day doses. Timefurone (25 and 100 mg/kg/day in the diet) also caused a significant reduction in atherosclerotic development in hypercholesterolemic SEA japanese quail. Atherosclerotic involvement (determined by visual assessment of plaque), arterial weight, and arterial cholesterol (total and mg/g artery) were clearly lowered by both doses of timefurone. There was no evidence of significant drug toxicity in any of these experiments. On the basis of these data, timefurone has excellent therapeutic potential and additional study of the drug's hypocholesterolemic and anti-atherosclerotic properties appears warranted.

Activity of ethyl 5-(p-chlorophenoxy)-3-hydroxy-3-methylpentanoate, a new hypocholesterolemic compound, in male rats and SEA Japanese quail.

Ethyl 5-(p-chlorophenoxy)-3-hydroxy-3-methylpentanoate (HMP), a new hypocholesterolemic compound, was evaluated in male rats at dosage levels ranging from 25-800 mg/kg/day and in SEA Japanese quail at approximately 200 mg/kg/day. In the rat, the atherogenic lipoprotein cholesterol, that is, the combination of very low density plus low density lipoprotein cholesterol (VLDL-C + LDL-C), was reduced 20-27% at dosage levels over 100 mg/kg/day, while high density lipoprotein cholesterol (HDL-C) and total serum cholesterol (TSC) were significantly decreased 25-46%, respectively, at all dose levels of HMP. Liver weights with HMP treatment were significantly elevated (11-26%) in the rat. HMP was not active in the SEA Japanese quail, since an initial reduction in artery cholesterol could not be confirmed in subsequent tests.

Hypo-beta-lipoproteinemic agents. 1. Bicyclo[2.2.2]octyloxyaniline and its derivatives.

A new assay for agents which normalize beta-lipoproteins in cholesterol-cholic acid fed rats is described. Both lowering of serum cholesterol and of serum heparin precipitable lipoproteins (HPL) were measured at the end of the treatment period. Compounds which shifted the ratio of the decrease in favor of HPL are considered hypo-beta-lipoproteinemic. p-(1-Bicyclo[2.2.2]octyloxy)aniline and several of its derivatives proved active in this assay. The synthesis of these compounds is described.

Hypobetalipoproteinemic agents. 2. Compounds related to 4-(1-adamantyloxy)aniline.

While the previously used displacement reaction of sodim 1-adamantyl oxide on 4-fluoronitrobenzene was applicable to the preparation of 4-(1-adamantyloxy)aniline and several related compounds, certain derivatives were not easily accessible by this route. Thus the recently reported ortho alkylation of anilines and the dicyclohexylcarbodiimide-promoted coupling of 1-adamantanol with phenols were useful in the preparation of aromatic-substituted derivatives. Furthermore, addition of phenylmagnesium bromide to 1-cyanoadamantane provided entry to the 4-(1-adamantylmethyl)aniline series. 4-(1-Adamantyloxy)aniline (3) is herein reported to be a more potent hypobetalipoproteinemic agent than the previously reported bicyclooctyloxy analogue. Replacement of the oxygen atom of 3 with sulfur (74) or methylene (62), but not nitrogen (71), results in active compounds. In the oxygen series derived from 3, the widest scope of substitution on nitrogen resulting in activity is found. The N-ethoxycarbonyl (5), acetyl (6), methyl (12), ethyl (13), N-methyl-N-(2-hydroxyethyl) (19), N-methyl-N-formyl (22), N,N-dimethyl (26), pyrrolidine (14), and piperidine (15) derivatives are active. Aromatic ring substitution also provided the active 3-chloro (44b), 2-fluoro (41b, 42, and 43), and 2-methylthiomethyl (48) compounds. Thus these active compounds are identified for further development as hypobetalipoproteinemic agents.

Response to the hypobetalipoproteinemic agent adamantyloxyphenyl piperidine in hyperlipemic rats.

The hypobetalipoproteinemic activity of U-41,792 (1-[p-(1-adamantyloxy)-phenyl]-piperidine) is a marked and selective reduction of heparin precipitating lipoproteins (low density plus very low density lipoproteins) in cholesterol-cholic acid induced hypercholesterolemic rats. This activity consists of both a reduction in heparin precipitating lipoproteins (HPL) and an increase in high density lipoproteins that are not precipitated by heparin. The increase in high density lipoproteins is routinely noted by decreases in HPL/cholesterol ratios. The pattern of response following single 100 mg/kg doses of U-418792 was determined. After an I.V. dose was administered in a cottonseed oil emulsion, serum cholesterol levels were reduced, beginning at 8 hr after administration and persisting for 96 hr. Similar results, though delayed somewhat, were obtained after a single oral dose. Activity was accompanied by increases in weight and cholesterol content of livers. After multiple, daily, oral doses, liver weights, total lipids, and cholesterol contents were reduced. Hypobetalipopreteinmeic activity was enhanced by prolonged treatments as demonstrated by analyses of serum obtained weekly throughout 7 wk.

Selected animal models for systematic antiatherosclerotic drug development.

We have developed an integrated system for antiatherosclerosis drug development utilizing rats, SEA quail, and cynomolgus monkeys as animal models. In general, the way the system is presently functioning is that thousands of compounds per year are randomly screened for hypobeta- or hyperalphalipoproteinemic activity in rats, and hundreds of compounds per year are screened in SEA quail for antiatherosclerotic and hypocholesterolaric activity. A few selected compounds that have activity in both rats and quail are then tested for lipoprotein modifying activity in cynomolgus monkeys. Nontoxic compounds having very good lipoprotein modifying activity in the monkey will then be recommended for clinical trials in man. We do not anticipate that this battery of testing will guarantee activity in man, but are hoping that it will at least increase the probability for finding a truly effective antiatherosclerotic drug to control the human disease.

High volume screening procedures for hypobetalipoproteinemic activity in rats.

We describe high volume screening tests for hypobetalipoproteinemic agents in which compounds are administered orally to cholesterol-cholic acid fed (hypercholesterolemic( or normally fed weanling rats for 4 days. In these tests total serum cholesterol levels and heparin precipitating lipoproteins (HPL) are determined by automated analyses interfaced with a computer which eliminates all manual data reduction and provides necessary reports. The hypercholesterolemic rat test detects compounds which specifically reduce HPL (beta and pre beta lipoproteins) causing a decrease in the HPL: cholesterol ratio. Such activity is called hypobetalipoproteinemia. This activity is exhibited by bicyclo (2.2.2)-octyloxyaniline (U-26328) but not by any of the familiar hypocholesterolemic agents including clofibrats, lifibrats, nicotinic acid, probucol, triparanol, lentysine, D-throxine or the estrogens estrone and diethylstilbestrol.

Biological activity of a hypobetalipoproteinemic agent.

A new -ind of pharmacologic activity called hypobetalipoproteinemia is described. Iperationally the activity consists of a marked reduction of heparin precipitating lipoproteins (beta and/or pre-beta electrophoretic mobility) in hypercholesterolemic animals with a simultaneous decrease in the heparin precipitating lipoprotein: cholesterol ratio. As determined by ultracentrifugal fractionation of the lipoproteins from hypercholesterolemic rat serum, this activity consists of both a reduction in heparin precipitating lipoproteins and an increase high density lipoproteins that are not precipitated by heparin. Changes in composition were also induced in both lipoprotein fractions. The greatest changes were observed for free and esterified cholesterol, which were markedly reduced in the heparin precipitating lipoproteins and concomitantly increased in the high density lipoproteins. The hypobetalipoproteinemic agent exhibiting this activity is 1-[p-(1'-adamantyloxy) phenyl]-piperidine (U-41792). This agent is active in hypercholesterolemic rats, mice, quail, and pigeons.

Animal models for an integrated approach to the pharmacologic control of atherosclerosis.

A system of animal models potentially useful for the discovery and evaluation of new effective antiatherosclerotic agents is described. The models consist of a series of lipoprotein and atherosclerosis assays in rats, SEA Japanese quail and cynomolgus monkeys. SEA quail are particularly useful for detecting compounds that inhibit arterial cholesterol deposition. The use of this integrated system of models is illustrated with data on clofibrate, adamantyloxyaniline (a hypobetalipoproteinemic agent), and o,p'-DDD. Male SEA quail appear to be a quite satisfactory model for testing the effects of large numbers of compounds on atherosclerosis and are available in limited numbers to all qualified investigators in the field of atherosclerosis research for evaluation in their laboratories.

Utility of selected line (SEA) of the Japanese quail (Coturnix coturnix japonica) for the discovery of new anti-atherosclerosis drugs.

A special line of the Japanese quail (Coturnix coturnix japonica) was developed by selective breeding to screen for new drugs that are effective in either preventing or reversing atherosclerosis. By the fourth or fifth generation, approximately 95% of the males of the selected line developed aortic atherosclerosis in response to a 2% cholesterol atherogenic diet. The arterial cholesterol level was significantly elevated after 1 week on the diet and was 14 times the control level after 14 weeks. Significant macroscopic lesions developed as early as 4 weeks. Neither arterial cholesterol nor grossly visible fatty plaques regressed after return to a normal diet for 8 weeks. After 2 weeks on the atherogenic diet, both serum cholesterol and heparin precipitating lipoproteins were maximally elevated. Serum cholecterol returned to normal within 1 month after removal from the diet, and by 2 months, the heparin precipitating lipoproteins had almost returned to baseline values. The male of the selected line of Japanese quail was shown to be an economical and practical animal model for the screening of anti-atherosclerosis drugs; it is small, inexpensive, and readily develops atherosclerosis.