Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.

Control of the interaction of cholesterol ester-rich lipoproteins with arterial receptors.

Incorporation of 125I-labeled cholesterol ester rich lipoproteins from cholesterol fed rabbits into normal rabbit aorta in vitro was inhibited by heparin, lecithin, and collagenase and by succinylation of the lipoprotein. Aortic uptake of lipoprotein was increased by neuraminidase, proteases, lipase, and beta-glucuronidase. These results suggest that it may be possible to control atherogenesis by controlling the interaction of atherogenic lipoproteins with their arterial receptor.

The mechanism of action of the hypocholesterolemic drug p-(1-adamantyloxy)-aniline.

The effect of p-(1-adamantyloxy)-aniline (AOA) on the biliary secretion of cholesterol, bile salts, and phospholipids was studied in hypercholesterolemic rats. A second study was conducted using hypercholesterolemic rats with 14C-labeled cholesterol pools for the purpose of determining the effects of AOA on cholesterol metabolism. Treatment with AOA (100 mg/kg, orally) daily for 1 week resulted in the increased secretion of biliary cholesterol, but did not affect the secretion of bile salts or phospholipids. This treatment also resulted in an increase in the fecal excretion of 14C-labeled neutral and acidic sterols, and in reductions of both the total radioactivity in the liver and liver cholesterol. The data presented support the conclusion that the hypocholesterolemic action of AOA is due to the increased secretion of cholesterol into the bile and to the increased fecal excretion of cholesterol and bile salts.

Evaluation of timefurone, a new anti-atherosclerotic drug, for its effects on lipoprotein cholesterol in male cynomolgus monkeys fed an atherogenic diet for 18 months.

Forty male cynomolgus monkeys were fed a nutritionally complete diet containing butter and 0.5% cholesterol for 18 months to ensure development of atherosclerosis. Timefurone was administered daily at 10 mg/kg/day. Lipoprotein cholesterol parameters were measured every 4 weeks and clinical chemistries were done at approximately 8-week intervals. Low density lipoprotein cholesterol [LDL-C] was significantly reduced 24-45% at all time periods and total-C was lowered 17-23% at weeks 12, 16, and 24-40 in the timefurone group. Very low density lipoprotein cholesterol [VLDL-C] was increased 68-156% from weeks 40-78 and triglycerides [TG] were significantly elevated 52-220% on weeks 4-16, 24, 28, and 36-78 by timefurone. Timefurone caused small but significant changes in several clinical chemistry parameters including: creatinine, total bilirubin, albumin, glucose, serum glutamic-oxalacetic transaminase, and serum glutamic-pyruvic transaminase during the test. Significant reductions in arterial cholesterol were observed in thoracic aorta (-24%) and carotid arteries (-29%) in treated monkeys when compared to placebo. Arterial cholesterol in treated monkeys was positively correlated to LDL-C (R = 0.54, p less than or equal to 0.05). Timefurone, therefore, appears to have a significant beneficial effect against the development of atherosclerosis in cholesterol-fed male monkeys and possesses excellent potential for clinical experimentation.

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.

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.

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.

Silent allelic variants of a T-cell receptor V beta 12 gene are present in diverse human populations.

Amino acid substitutions in variable regions of the T-cell receptor (TCR) can alter T-cell reactivity; however, relatively little is known about the extent of allelic variation in human TCR coding sequences. In the present studies, coding region variation in the human TCR V beta 12.2 gene was examined in detail. Virtually the entire V beta 12.2 coding region was screened for nucleotide substitutions by single-stranded conformational polymorphism analysis. Four alleles were identified in a sample population of 90 unrelated people from diverse genetic backgrounds. Three of the alleles were common, with estimated frequencies of 0.32, 0.47, and 0.20. Sequence analyses revealed that variation between the alleles was confined to three single-base differences in codons 24, 31, and 45; none of these changes altered the amino acid sequence. No evidence for other coding region differences in this gene were found. This analysis suggests that coding region variation in V beta 12.2 is limited, and amino acid sequence is highly conserved.

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.

New animal model for atherosclerosis research.

Japanese quail were investigated for their utility as a model for the discovery and evaluation of anti-atherosclerosis compounds. Although they possessed suitable characteristics for a screening animal, their development of atherosclerosis was too variable to make them a practical model. A search was conducted to find a means to make quail uniformly atherosclerotic. To this end a line of quail susceptible to experimental atherosclerosis (SEA) were selectively bred. Thus, the SEA Japanese quail is a new animal model for atherosclerosis research.

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.

Produced by selective breeding of Japanese quail animal model for experimental atherosclerosis.

The Japanese quail (Coturnix coturnix japonica) was bred selectively to produce a strain highly susceptible to experimental atherosclerosis. A population was produced where aortic atherosclerosis is contracted by 99% of the fourth generation males and 83% of the femlaes. Forty-three percent of the males exhibited severe atherosclerosis making this line of Japanese quail a suitable model for discovering and testing anti-atherosclerosis compounds. This feature is augmented by other features such as size, disposition, and abundance which qualify them as suitable experimental subjects. A second line of Japanese quail was bred to be resistant to dietary-induced atherosclerosis. This strain may be a useful research tool for characterizing the etiology of atherosclerosis.

[Allelic polymorphism in the coding region of human T cell receptor V beta 12 gene].

Sequence substitutions in T cell antigen receptor (TCR) beta chains have been shown to have a profound impact upon the fine specificity of T lymphocytes. It is, therefore, of interest to characterize allelic variations in the TCR gene segments that assemble to encode TCR molecules. In the present report, the sensitive technique of single stranded conformational polymorphism (SSCP) was used to screen for allelic sequence variations in the coding region of TCR V beta 12 gene. TCR V beta 12 specific primers were used to PCR amplify and 32P label a 273bp segment of the variable (V) gene segment. Sequence differences between different allelic forms were detected by denaturation of the DNA and separation on a non-denaturing, high resolution acrylamide gel. Using this rapid approach, four alleles of the TCR V beta 12 gene were detected in a population of 90 ethnically diverse individuals. Correct Mendelian segregation of the four alleles was demonstrated in family studies (n = 23) and allele frequencies were 0.4722, 0.3166, 0.2056, and 0.0056 respectively. Sequence analysis revealed sequence differences of 1-3 nucleotides involving three separate codons. However, predicted amino acid sequences of all four alleles are identical. These data strongly suggest that the TCR V beta 12 protein sequence is highly conserved.