The in vitro dental plaque inhibitory properties of a series of N-[1-alkyl-4(1H)-pyridinylidene]alkylamines.

A series of novel N-[1-alkyl-4(1H)-pyridinylidene]alkylamine hydrohalides has been prepared and evaluated as inhibitors of dental plaque formation, in vitro. Several members of the series exhibited potency ca. 9-fold greater than that of chlorhexidine vs Streptococcus sobrinus 6715-13. The di-n-octyl analogue, 11 (pirtenidine), was found to be highly efficacious against several other oral plaque-forming microorganisms and is presently undergoing preclinical evaluation.

WIN 66306, a new neurokinin antagonist produced by an Aspergillus species: fermentation, isolation and physico-chemical properties.

WIN 66306 (1a), a cyclic peptide containing a novel amino acid, was isolated as a neurokinin antagonist from an Aspergillus species, labelled SC230. Conditions that maximized the production of 1a were developed, leading also to production of the related compound WIN 68577 (2) and rosellichalasin (3). Both 2 and 3 were more active in the rat NK1 than in the human NK1 receptor binding assay, while 1a was more active at the human receptor with an inhibitor affinity constant of 7 microM.

Benzomalvins, new substance P inhibitors from a Penicillium sp.

In the course of screening microbial broths for neurokinin receptor antagonists, a series of new benzodiazepines, benzomalvins A (1), B (2) and C (3), has been isolated from the culture broth of a fungus identified as a Penicillium sp. Benzomalvin A (1) showed inhibitory activity against substance P with Ki values of 12, 42 and 43 microM at the guinea pig, rat and human neurokinin NK1 receptors, respectively. Benzomalvins B (2) and C (3) were only weakly active. The structures of these compounds were determined by spectroscopic methods including MS measurements and NMR analysis.

WIN 64821, a novel neurokinin antagonist produced by an Aspergillus sp. I. Fermentation and isolation.

WIN 64821, a nonpeptide neurokinin antagonist, was isolated from a strain of Aspergillus sp., SC319. The compound was produced in different fermentation media with greatest yields observed when the culture was grown in a synthetic medium supplemented with L-tryptophan and L-phenylalanine. After 6 days fermentation, yields greater than 600 mg/liter were obtained. Two analogs of WIN 64821 were also identified in the culture extracts and subsequently tested for biological activity. WIN 64821 was the most potent compound isolated from this culture and exhibited activity as a substance P-binding inhibitor with submicromolar potency against the human neurokinin 1 receptor.

WIN 64821, a novel neurokinin antagonist produced by an Aspergillus sp. II. Biological activity.

WIN 64821, a secondary metabolite produced by Aspergillus sp. (ATCC 74177) was found to inhibit radiolabeled substance P (SP) binding in a variety of tissues, including those of human origin. This compound inhibited, in a competitive manner, the binding of SP with Ki values ranging from 0.24 microM in human astrocytoma U-373 MG cells to 7.89 microM in rat submaxillary membranes. Additionally, WIN 64821 was found to inhibit 125I-NKA binding to the NK2 receptor in human tissue at a concentration equivalent to its NK1 activity (0.26 microM). The inhibitory activity of WIN 64821 against an NK3 selective ligand, 3H-senktide, was found to be much weaker (Ki = 15.2 microM). WIN 64821 was also evaluated in NK1 functional assays and was found to be a competitive antagonist of SP-induced contractility in the guinea pig ileum (pA2 = 6.6) as well as an inhibitor of SP-induced 45Ca2+ efflux from human astrocytoma U-373 MG cells (IC50 = 0.6 microM). In a rat vas deferens model, WIN 64821 inhibited eledoisin-induced contractility with an IC50 of 3.4 microM indicating functional antagonism at the NK2 receptor. The data presented in this study provide biochemical, pharmacological and functional evidence supporting WIN 64821 as a competitive neurokinin antagonist.

Antimycins, inhibitors of ATP-citrate lyase, from a Streptomyces sp.

A related group of compounds belonging to the antimycin class of antibiotics was found in culture broth produced by a Streptomyces species. The group includes known antimycins A1, A2, A3 and A4, and new antimycins A7 and A8. These compounds inhibit ATP-citrate lyase with Ki values of 4 to 60 microM against the substrate magnesium citrate. The structures of the new antimycins were determined by spectroscopic analyses.

Microbicidal activity of octenidine hydrochloride, a new alkanediylbis[pyridine] germicidal agent.

The potential of octenidine hydrochloride (WIN 41464-2) as a topical microbicide was measured both by in vitro death kinetics and reductions in numbers of bacteria on the skin of cynomolgus monkeys. Semilogarithmic survival curves were plotted to measure the microbicidal activity of various concentrations of octenidine against Staphylococcus aureus. The microbicidal activity of octenidine was also determined for Staphylococcus epidermidis, Proteus mirabilis, Streptococcus pyogenes, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and Candida albicans. Death rates for the same microbial strains were compared with those obtained by using chlorhexidine gluconate. Octenidine concentrations of less than 1.5 microM (0.94 microgram/ml) caused a greater than 99% reduction of each microbial population within 15 min. Staphylococcus epidermidis was the most susceptible of the test organisms, and E. coli and C. albicans were the least susceptible. Octenidine was more active than chlorhexidine against each test strain. Skin-degerming activities of aqueous and formulated octenidine and formulated chlorhexidine were compared in single and multiple applications of these agents to the hand and foot surfaces of monkeys by using a glove-juice extraction procedure to measure the skin microflora. Aqueous octenidine, at a concentration of 0.2 to 1.6% reduced resident microflora populations from 90 to 99.98%, depending on the concentration and number of applications. Octenidine formulated at 2% in a surfactant-based vehicle exhibited significantly better skin-degerming activity than did either a nonmedicated vehicle or the Hibiclens brand of 4% chlorhexidine gluconate.

1'-(2-Phenyl-ethylene)-ditryptophenaline, a new dimeric diketopiperazine from Aspergillus flavus.

A new diketopiperazine dimer, 1'-(2-phenyl-ethylene)-ditryptophenaline [1], was isolated together with ditryptophenaline [2] from the fungus Aspergillus flavus. The structure of 1 was determined by analysis of spectroscopic data. In contrast to the structurally related substance-P antagonist WIN 64821 [3], both 1 and 2 are weak substance-P inhibitors, indicating that stereochemistry at the position of dimerization is an important determinant of biological potency for these molecules.

Detection of Salmonella enterotoxin using rabbit ileal loops.

The presence of an enterotoxin produced by Salmonella in broth culture has been demonstrated by using the rabbit ileal loop model. Response by the animal to enterotoxin in sterile culture supernatant fluids is enhanced when the intestinal lumen is washed with a mucolytic agent prior to the administration of toxin. Fluid secretion is untreated intestinal loops was also observed if enterotoxin was administered with a live, invasive Salmonella strain which did not evoke a secretory response. A limited survey of Salmonella isolated for clinical and food sources indicated the common occurrence of enterotoxin production, and stock cultures maintained the ability to produce the toxin. The host-adapted species which were tested varied in their ability to produce enterotoxin.

Role of lipids in augmenting the antibacterial activity of benzoyl peroxide against Propionibacterium acnes.

Antimicrobial activity and physicochemical properties of benzoyl peroxide (BP) were investigated to determine the mechanism of action for the compound as an antiacne agent. The MICs and MBCs against nine strains of Propionibacterium acnes ranged between 100 and 800 micrograms/ml in a nutrient broth system, with a median fourfold increase in activity demonstrated when lipid was added. The partition coefficient of BP in a 50:50 artificial skin lipid and water system was greater than 2,500, with the concentration of BP soluble in lipid measured at 1.12% and in water at 0.005%. When BP was incubated in the presence of a lipid mixture, reaction products were formed, with evidence that at least some of these compounds possessed antibacterial activity. These results suggest that BP reduces the P. acnes numbers in sebaceous follicles because of good lipid solubility and interaction with the lipid component, the latter property contributing to the antimicrobial activity of the compound in a high-lipid environment.

Production and partial purification of Salmonella enterotoxin.

By using a strain of Salmonella typhimurium, we detected the presence of an enterotoxin, as determined by the rabbit ileal loop assay, in various complex and defined media. The enterotoxin was concentrated by ultrafiltration of culture supernatant fluids and eluted in and adjacent to the void volume of a Sephadex G-100 column. This suggested that the enterotoxic factor was of a relatively high molecular weight, and additional evidence indicated it was heterogeneous in size. Further chromatography, using a diethylaminoethyl-cellulose anion exchanger, facilitated at least a 50-fold purification of the Salmonella enterotoxin.

Methylpendolmycin, an indolactam from a Nocardiopsis sp.

Methylpendolmycin, a new indole alkaloid with an N-methylisoleucine moiety incorporated in the nine-membered indolactam ring, has been isolated from an actinomycete culture of Nocardiopsis. Methylpendolmycin exhibited inhibition of phorbol ester binding to protein kinase C. Its structure was assigned on the basis of spectroscopic data.

Use of a hemadsorption technique to evaluate the stability of the hemagglutination reaction of Escherichia coli cultures possessing human colonization factor antigens.

Two strains of Escherichia coli, producing different colonization factor antigens (CFA), were monitored for the population density of CFA-producing bacteria after repeated subculture. The production of CFA was estimated by flooding agar plates containing isolated colonies with suspensions of human or bovine erythrocytes. The erythrocytes were suspended in a low-ionic-strength buffer and were fixed to CFA-positive colonies with a 1.0% tannic acid solution. Strain H-10407, possessing CFA/I fimbriae, showed a rapid loss of the hemagglutinin when subcultured, whereas strain CL-9699, producing CFA/II, was very stable. By using the hemadsorption assay, we could rapidly and easily distinguish CFA- positive colonies from the CFA-negative variants. A survey of additional E. coli strains demonstrated the utility and specificity of the hemadsorption technique used.

Analysis of parameters affecting the hemagglutination activity of Escherichia coli possessing colonization factor antigens: improved medium for observing erythrocyte agglutination.

The hemagglutination (HA) activity of two strains of Escherichia coli, each possessing different colonization factor antigens (CFA), was examined under different test conditions. The effects of ionic strength, temperature, pH, cations, and reaction surface on erythrocyte (RBC) agglutination were analyzed. Strain H-10407 (CFA/I) caused the agglutination of human, bovine, and chicken RBC, whereas strain CL-9699 (CFA/II) agglutinated only bovine and chicken RBC. The HA activity of both strains increased with decreasing ionic strength, pH, and temperature, the effects of temperature being negligible at low ionic strength. When accounting for ionic strength, the presence of Ca2+, Mg2+, Fe2+, or Fe3+ ions did not increase the HA activity of these bacteria. Optimum conditions for HA of reactive RBC by bacteria included low ionic strength (less than 50 mM) and slightly acidic pH (6.0 to 7.0). Use of a low-ionic-strength medium permitted application of microtitration methods to visualize the HA reactions. Storage of RBC in low-ionic-strength medium did not change their HA properties, and the use of this medium proved superior to saline in overcoming HA variation observed with different preparations of RBC.

Utilization of chlorhexidine gluconate to evaluate a nonhuman primate skin-degerming model.

We have developed a nonhuman skin-degerming model to predict the in vivo effectiveness of topical antimicrobial formulations. The model incorporates a balanced, randomized, complete block design and uses the hands of anesthetized cynomolgus monkeys as treatment sites to measure product effectiveness. Two different 4% chlorhexidine gluconate formulations were evaluated in the primate model and then retested in a human skin-degerming model of identical design. Statistical analysis of the data revealed no significant differences between the two models with regard to the response exhibited by each to the test formulations.

In vitro and in vivo activities of a new quinolone, WIN 57273, possessing potent activity against gram-positive bacteria.

The antibacterial activity of a new 7-dimethylpyridinyl quinolone, WIN 57273, was assessed by using in vitro and in vivo models. Agar inclusion and broth dilution in vitro tests revealed broad-spectrum activity against gram-positive and selected gram-negative organisms, with the greatest potency observed against the staphylococci. The MIC for 90% of coagulase-positive strains tested (MIC90) was less than or equal to 0.002 micrograms/ml; for the coagulase-negative strains the MIC90 was 0.008 micrograms/ml. Against enterococci the MIC90 was 0.06 micrograms/ml, with comparable activity observed against group A and group B streptococci as well as against the pneumococci. In general, the MIC90s for the gram-negative bacteria were less than or equal to 1 micrograms/ml. Exceptions were Serratia marcescens (MIC90, 16 micrograms/ml), Citrobacter freundii (MIC90, 4 micrograms/ml), and Pseudomonas aeruginosa (MIC90, 8 micrograms/ml). The greatest potency was observed against Haemophilus spp. and Neisseria spp., with MIC90s of 0.06 and 0.016 micrograms/ml, respectively. Broad-spectrum activity was also observed against anaerobes, with MIC90s ranging from 0.125 to 0.5 micrograms/ml among the species tested. The in vivo efficacy was determined by using a murine model by calculating the 50% protective doses against a lethal bacterial infection caused by strains of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Listeria monocytogenes, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The staphylocci were most susceptible, with 50% protective doses for all strains ranging from 0.1 to 0.7 mg/kg. With the exception of the Pseudomonas infection, which was refractory to treatment, animals that were part of the other infection models responded to less than 10 mg/kg. Equivalent activity was seen with the subcutaneous or the oral route of drug administration. WIN 57273 was significantly more potent than ciprofloxacin in treating gram-positive bacterial infections (2- to 20-fold) but was significantly less effective at treating gram-negative bacterial infections (30- to 300-fold).

Novel tricyclic benzothiazolo[2,3-c]thiadiazine antagonists of the platelet ADP receptor (P2Y(12)).

Novel non-nucleoside tricyclic platelet ADP receptor (P2Y(12)) antagonists have been discovered that bind reversibly and with high affinity to the platelet receptor. Condensation of various 2-aminobenzothiazoles with chlorosulfonylacetyl chloride affords these novel tricyclic heterocycles, which are novel and unpredicted products of this reaction.