Diamine containing VLA-4 antagonists.

Design and synthesis of a library as potential VLA-4 antagonists has been accomplished, based around a proposed pharmacophoric model. Compounds possessing submicromolar potency were identified and structure-activity relationships were seen across the library. Further derivatisation produced compounds with IC(50)'s <10 nmol for inhibiting the VLA-4 mediated binding of fibronectin to RAMOS cells, providing an ideal starting point for a lead optimisation Programme.

Selective ET(A) antagonists. 5. Discovery and structure-activity relationships of phenoxyphenylacetic acid derivatives.

The fifth paper in this series describes the culmination of our investigations into the development of a potent and selective ET(A) receptor antagonist for the treatment of diseases mediated by ET-1. Receptor site mapping of several ET(A) antagonists prepared previously identified a common cationic binding site which prompted synthesis of phenoxyphenylacetic acid derivative 13a, which showed good in vitro activity (IC(50) 59 nM, rat aortic ET(A)). Optimization of 13a led to the identification of 27b, which exhibited an IC(50) of 4 nM. Although this did not translate into the expected in vivo potency, a compound of comparable in vitro activity, 27a (RPR118031A), showed a far better pharmacokinetic profile and in vivo potency (75 micromol/kg) and was duly proposed and accepted as a development candidate.

Selective endothelin A receptor antagonists. 3. Discovery and structure-activity relationships of a series of 4-phenoxybutanoic acid derivatives.

The third in this series of papers describes our further progress into the discovery of a potent and selective endothelin A (ETA) receptor antagonist for the potential treatment of diseases in which a pathophysiological role for endothelin has been implicated. These include hypertension, ischemic diseases, and atherosclerosis. In earlier publications we have outlined the discovery and structure-activity relations of two moderately potent series of nonpeptide ETA receptor antagonists. In this paper, we describe how a pharmacophore model for ETA receptor binding was developed which enabled these two series of compounds to be merged into a single class of 4-phenoxybutanoic acid derivatives. The subsequent optimization of in vitro activity against the ETA receptor led to the discovery of (R)-4-[2-cyano-5-(3-pyridylmethoxy)phenoxy]-4-(2-methylphenyl)b utanoi c acid (12m). This compound exhibits low-nanomolar binding to the ETA receptor and a greater than 1000-fold selectivity over the ETB receptor. Data are presented to demonstrate that 12m is orally bioavailable in the rat and is a functional antagonist in vitro and in vivo of ET-1-induced vasoconstriction.

Selective endothelin A receptor antagonists. 4. Discovery and structure-activity relationships of stilbene acid and alcohol derivatives.

This publication describes the synthesis and optimization of a novel series of stilbene endothelin antagonists. Analysis of the SAR established for previous papers in this series prompted the design and synthesis of (Z)-4-phenyl-5-(3-benzyloxyphenyl)pent-4-enoic acid 3 which was found to be a moderately active inhibitor of the binding of [125I]ET-1 to ETA receptors with an IC50 of 6 microM. More interestingly, the intermediate compound (E)-2-phenyl-3-(3-benzyloxyphenyl)propenoic acid 5 was equiactive with 3. Optimization of 5 resulted in the preparation of (E)-2-phenyl-3-(2-cyano-5-(thien-3-ylmethoxy))phenylprope noic acid 18 (RPR111723) which had an IC50 in the binding assay of 80 nM on the ETA receptor and a pKB of 6.5 in the functional assay, measured on rat aortic strips. Reduction of the acid group of 5 gave the first nonacidic ETA antagonist in our series, (E)-2-phenyl-3-(3-benzyloxyphenoxy)prop2-enol 6 with an IC50 of 20 microM. Optimization of 6 resulted in the preparation of 2-(2-methylphenyl)-3-(2-cyano-5-(thien-3-ylmethyl)phenyl)pro p-2-enol 33 with an IC50 of 300 nM on the ETA receptor.

Hypolipidaemic properties of a potent and bioavailable alkylsulphinyl-diphenylimidazole ACAT inhibitor (RP 73163) in animals fed diets low in cholesterol.

RP 73163 ((S)-2-[5-(3,5-dimethyl-l-pyrazolyl)pent-l-yl)-sulphinyl]-5, 6-diphenylimidazole) has been shown to be a potent and specific inhibitor of acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26; ACAT) in vitro using the tissues of experimental animals as sources of the enzyme. The concentrations of RP 73163 required to produce 50% inhibition of ACAT activity (IC50 values) in microsomal preparations ranged from 86 nM for rat liver to 370 nM for rabbit intestine. In whole cell assays using human hepatic (HepG2), intestinal (Caco2), and monocytic (THP-1) cell lines, RP 73163 inhibited ACAT activity with IC50 values of 266, 158, and 314 nM, respectively. The addition of RP 73163 (0.03-1.0 microM) to the medium of cultured HepG2 cells produced a concentration-dependent decrease in apolipoprotein B (apoB) secretion. The compound has high systemic bioavailability. Using a bioassay, a concentration of active inhibitor equivalent to 29 microM of parent compound was present in plasma 1 hr after oral administration of RP 73163 (50 mg.kg-1). In rats that had been fed a basal diet ad libitum or starved for 18 hr prior to blood sampling, the administration of RP 73163 (50 mg.kg-1 b.i.d. for 7 days) reduced plasma triglyceride levels by 50% without affecting the concentration of cholesterol. This hypotriglyceridaemic effect was associated with reductions in plasma very-low-density-lipoprotein (VLDL) and low-density-lipoprotein (LDL) levels. RP 73163 decreased the rate of VLDL secretion by 24% in Triton WR-1339-treated rats that had been fasted overnight but did not affect the secretion rate in animals fed ad libitum, indicating that ACAT was only important in regulating VLDL secretion under certain nutritional conditions. RP 73163 reduced the accumulation of intraperitoneally administered [3H]leucine into the plasma VLDL-apoB pool in both fed and fasted states. The results suggest that, in fed animals at least, an increase in the clearance of VLDL from the bloodstream may contribute to the hypolipidaemic activity of the compound. In rabbits with casein-induced endogenous hypercholesterolaemia, RP 73163 specifically reduced the levels of cholesterol carried by LDL. In conclusion, the hypolipidaemic actions of RP 73163, a potent and systemically bioavailable ACAT inhibitor, are consistent with a reduction in the secretion of apoB containing lipoproteins by hepatic tissue and possibly with an increase in the clearance of these particles.

Acyl-CoA:Cholesterol O-acyltransferase (ACAT) inhibitors. 2. 2-(1,3-Dioxan-2-yl)-4,5-diphenyl-1H-imidazoles as potent inhibitors of ACAT.

The second in this series of papers concerns our further investigations into the search for a potent bioavailable acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitor suitable for the treatment of atherosclerosis. The design, synthesis, and structure-activity relationship for a series of ACAT inhibitors based on the 2-(1,3-dioxan-2-yl)-4,5-diphenyl-1H-imidazole pharmacophore are described. Compounds such as 13a bearing simple alkyl or hydroxymethyl substituents at the 5-position of the 1,3-dioxane ring are potent bioavailable inhibitors of the rat hepatic microsomal enzyme in vitro (IC50 < 100 nM) but are only weak inhibitors of the human hepatic enzyme. We have found however that 1,3-dioxanes substituted at the 5-cis position with pyrazolylalkyl or aminoalkyl groups are potent inhibitors in vitro of human macrophage ACAT, the potency depending on the nature of the terminal heterocycle and the length of the alkyl chain. An ex vivo bioassay herein demonstrates that potent inhibitors such as 13t (IC50 = 10 nM) which contain lipophilic terminal heterocycles do not appear to be systematically available. Less potent but more water soluble compounds such as 13h (IC50 = 60 nM) and 13n (IC50 = 70 nM) are absorbed following oral dosing and achieve plasma levels significantly in excess of their IC50 for ACAT inhibition. These compounds are therefore possible candidates for further investigation as oral antiatherosclerotic agents.

Prevalence of HTLV types I and II among drug users in King County, Washington.

We investigated the prevalence of human T-cell lymphotropic virus (HTLV) types I and II among drug users entering treatment in King County, Washington, between 1988 and 1990. Of 762 injection-drug users, 81 (10.6%) were HTLV-positive; of 89 noninjection-drug users, 2 (2%) were HTLV-positive. Most (95.8%) of those typed) were HTLV-II-positive. The relationship between HTLV and demographic and behavioral characteristics was further evaluated among injection-drug users. The prevalence rates for HTLV increased 25-fold from the youngest age group (15 to 24 years) to the oldest (older than 45 years), after adjusting for race. After adjustment for age, American Indians or Alaska Natives were 7.9 times, blacks 6.2 times, Asians or Pacific Islanders 4.7 times, and Hispanics 4.1 times as likely as whites to be HTLV-positive. The prevalence of HTLV among heroin injectors was more than double that observed among injectors of other drugs after adjusting for age, although this association was only marginally significant. The strong association between HTLV prevalence and age suggests that HTLV-II (the predominant virus) has been endemic among King County injection-drug users for some time. Its relatively high prevalence indicates that there is both an opportunity and a need to further investigate the epidemiologic and clinical implications of HTLV-II infection.

Structure-activity relationships of dihydrofolate reductase inhibitors.

Structure-activity relationships of antibacterial dihydrofolate reductase inhibitors are reviewed. A short introduction is followed by a more detailed review of developments since 1980. The design of site directed inhibitors based on data from X-ray crystallographic, NMR spectroscopy and molecular graphic studies is discussed. The development of quantitative structure-activity relations is surveyed from a historical point of view and these are contrasted with more recent alternative approaches. Conclusions are reached regarding possible future developments.

Risk factors for HIV infection among injection drug users: results of blinded surveys in drug treatment centers, King County, Washington 1988-1991.

Among injection drug users (IDUs) entering drug treatment in King County, Washington between 1988 and 1991, we investigated HIV seroprevalence in relationship to demographic, sexual, and drug-use characteristics. Eighty-two of 3,039 (2.7%) IDUs tested HIV positive. Gay or bisexual men had the highest HIV prevalence (37.1%), followed by lesbian or bisexual women (8.3%), heterosexual men (2.3%), and heterosexual women (1.5%). American Indians were more likely to be infected with HIV than were whites. Those with no permanent address were more likely to be infected than those with an address. Unexpectedly, the prevalence of HIV infection among amphetamine injectors (13.1% of 168) was higher than among those who did not report using amphetamines. After adjustment for sexual orientation, HIV prevalence was four times higher among primary amphetamine injectors and three times higher among secondary amphetamine injectors than among injectors of other drugs. The basis for the strong association observed between HIV infection and a history of injection of amphetamines is not known and should be clarified through further research that obtains more detailed information on IDUs.

The role of exposure to animals in the etiology of Campylobacter jejuni/coli enteritis.

To determine the role of animals as possible sources for human infection with Campylobacter jejuni/coli, 218 human cases of Campylobacter enteritis diagnosed among members of Group Health Cooperative of Puget Sound, King County, Washington, from April 1982 through September 1983 were compared with 526 controls, randomly selected from Group Health Cooperative members. All subjects were questioned regarding animal exposures one week prior to illness (cases) or interview (controls). There was no increase in risk for C. jejuni/coli enteritis associated with contact with various animals. However, exposure to diarrheic animals was associated with a fourfold increase in the risk of C. jejuni/coli enteritis (odds ratio = 4.3, 95% confidence interval (CI) 1.9-9.7). Adjustment for potential confounding factors by logistic regression analysis yielded an odds ratio of 3.3 (95% CI 1.2-7.5) associated with such exposure. An estimated 6.3% of cases of C. jejuni/coli enteritis was attributed to exposure to diarrheic animals.

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.

Antimicrobial activity against Staphylococcus aureus of semisynthetic injectable streptogramins: RP 59500 and related compounds.

Pristinamycin displays unique antibacterial properties due to the synergy between its two components, pristinamycin I and pristinamycin II. Because this antibiotic is not water-soluble, its administration is restricted to the oral route, and its therapeutic potential is thereby limited. Novel water-soluble derivatives of the naturally-occurring antibiotic pristinamycin were obtained by modifications of its two major components. The modifications included regioselective and stereoselective substitution alpha to the carbonyl group in the 4-oxo-pipecolic acid residue of pristinamycin IA (PIA) and stereoselective conjugate addition to the double bond of the dehydroproline ring in pristinamycin IIA (PIIA). We report here the in-vitro and in-vivo activities of some representative water-soluble derivatives of pristinamycin IA and pristinamycin IIA against Staphylococcus aureus reference strains, sensitive or resistant to methicillin and/or macrolides.

Animal exposures and antibodies to Toxoplasma gondii in a university population.

To determine the risk of toxoplasma infection to individuals exposed to cats in a research institution, we compared the prevalence of toxoplasma antibodies with exposure to cats in university employees. Of 116 employees tested, 42 (36 percent) had toxoplasma antibodies as determined by the indirect fluorescent antibody test. Women and individuals aged 35 years or more had a greater prevalence of antibodies. The antibody prevalence by occupation was 72.1 percent for physicians and those with doctorates, 45.3 percent for animal and veterinary technicians, 33.3 percent for research technicians, 28.2 percent for administrative staff, 25.0 percent for graduate students and fellows, and 13.4 percent for veterinarians. There was no significant positive association between exposure to cats and the prevalence of toxoplasma antibodies. A follow-up of seronegative employees, 6 and 18 months later, revealed no seroconversions indicative of acute toxoplasma infection. We concluded that there was no significant risk of toxoplasma infection in university employees exposed to cats.

Antifolate and antibacterial activities of 5-substituted 2,4-diaminoquinazolines.

A series of 5-substituted 2,4-diaminoquinazolines (3) has been synthesized and evaluated as inhibitors of the enzyme dihydrofolate reductase (DHFR) from both bacterial and mammalian sources. The best compounds (e.g. 53) show good activity against Escherichia coli DHFR, but there is no significant selectivity for the bacterial over the mammalian enzyme. The structure-activity relationships for enzyme inhibition appear to be complex and not amenable to simple analysis; a hypothesis to explain the observed qualitative structure-activity relationships is proposed. The inhibitory activities of the compounds against the growth of intact bacterial cells in vitro closely parallel those for the inhibition of the isolated bacterial enzymes, suggesting that their antifolate action is responsible for their antibacterial effects. Five of the compounds were tested for their ability to cure a systemic E. coli infection in the mouse, but they showed no therapeutic effects at their maximum tolerated doses.

Campylobacter jejuni enteritis associated with raw goat's milk.

During a three-week period in July 1983, six cases of Campylobacter jejuni enteritis in King County, Washington were associated with a dairy that produced raw goat's milk. Four patients consumed the dairy's milk, and the other two patients comprised an employee of the dairy and her infant son. Two case-control studies confirmed that, at the time the cases occurred, consumption of the dairy's milk was a risk factor for C. jejuni enteritis in King County. C. jejuni was isolated from the intestinal tract of three of the dairy's goats. Two of the three isolates, as well as those from five of the patients (all of those tested), were Lior serotype 36. That serotype was not encountered among 14 other C. jejuni isolates from King County during the period of the outbreak, including three isolates from goats at another inspected dairy. The study shows that raw goat's milk may transmit C. jejuni infection from animals to humans, as other investigators have shown for unpasteurized cow's milk.

A survey of Campylobacter and other bacterial contaminants of pre-market chicken and retail poultry and meats, King County, Washington.

As part of a larger study to determine the flow of Campylobacter and Salmonella from food animals to humans during 1982-83, 1,936 swabs were collected for bacteriologic study from pre-market chickens, retail poultry, and other retail meats as well as from equipment and work surfaces used to process such foods. Of the 297 samples collected in a poultry processing plant, 56.6 per cent were positive for Campylobacter jejuni/coli (CJC), as were 23.1 per cent of the 862 retail chicken, and 17.2 per cent of the 29 retail game hen samples. CJC was found infrequently in retail turkey, pork, and beef samples. Contamination of retail and pre-market chicken with CJC appeared to increase as the week progressed, and in pre-market chicken, later in the day. Less than 5 per cent of the retail samples of poultry, beef, and pork were found to contain Yersinia or Salmonella. However, Salmonella was cultured from 14.8 per cent of the swabs taken from the processing plant with 68 per cent of 44 Salmonellas being isolated concurrently with CJC. Tetracycline resistance which was plasmid-mediated was the most common antibiotic resistance observed, and was carried by 23.8 per cent of all CJC isolates. Overall, 38.8 per cent of all CJC isolates were resistant to ampicillin, erythromycin, streptomycin, or tetracycline, either singly or in combination.

The role of poultry and meats in the etiology of Campylobacter jejuni/coli enteritis.

To determine the role of meats as possible sources of infection leading to Campylobacter jejuni/coli (CJC) enteritis, 218 cases and 526 controls were selected from the King County Group Health Cooperative (GHC) population from April 1982 through September 1983. All subjects were interviewed regarding food consumption one week prior to case onset. Consumption of chicken and cornish game hen were both associated with more than a doubling of the risk of CJC enteritis: for chicken (relative risk = 2.4, 95% CI = 1.6-3.6), and for game hen, (RR = 3.3, 95% CI = 1.1-9.8). The consumption of raw or rare chicken was even more strongly associated (RR = 7.6, 95% CI = 2.1-27.6). Strains of CJC bearing R factors for tetracycline were equally as likely as tetracycline-susceptible strains to have been acquired from chicken and game hens. Processed turkey sandwich meats (RR = 1.7, 95% CI = 1.0-2.9) raw or rare fish (RR = 4.0, 95% CI = 1.1-14.5) and shellfish (RR = 1.5, 95% CI = 1.1-2.1) were the only other meats reported to have been eaten significantly (p less than .05) more often by cases than by controls. These data along with the results of bacteriologic sampling of meats from King County retail food markets during the same period suggest that ingestion of contaminated chicken is a primary source of CJC enteritis, contributing to approximately half of the cases.

Breast cancer in relation to patterns of oral contraceptive use.

A total of 112 white females residents of King County, Washington, aged 35-54 years, who had received a first diagnosis of invasive breast cancer between July 1977 add August 1978, were investigated concerning prior use of oral contraceptives. Their responses were compared with those of a random sample of 469 demographically comparable women from the same population. Overall, oral contraceptive use in cases and controls was similar. However, use of oral contraceptives in preparous women was more common among cases than controls, with the estimated risk of breast cancer associated with such use being 2.2 times that of nonusers (90% confidence interval = 1.1-4.6). This relationship could be explained only in part by the effect of oral contraceptives in postponing or preventing childbirth. The association of breast cancer with use of oral contraceptives prior to ever giving birth has been observed in three studies, including this one, suggesting that the susceptibility of breast tissue to hormonal factors that influence the development of malignancy may be altered by having been exposed to the events of pregnancy.