Microbiological N-deoxygenation and C-oxygenation of pioglitazone-N-oxide in a single fermentation.

Oxygenation of pioglitazone-N-oxide by a microorganism isolated from soil was accompanied by N-deoxygenation to produce the pioglitazone metabolites 5-[4-[2-[5-(1-hydroxyethyl)-2-pyridyl]ethoxy]benzyl]-2,4-thiazolidinedio ne and 5-[4-[2-(5-acetyl-2-pyridyl)ethoxy]benzyl]2,4-thiazolidinedione. The oxygenating/deoxygenating organism has been characterized as Streptomyces hygroscopicus strain 02179 (UC 11099). The culture has been deposited with Agricultural Research Service, USDA, with accession number NRRL 18975.

Purification of lincosaminide O-nucleotidyltransferase from Streptomyces coelicolor Müller.

An enzyme (lincosaminide O-nucleotidyltransferase) that catalyzes 3-(5'-ribonucleotidylation) of pirlimycin and several other lincosaminide antibodies has been purified approximately 35-fold from cell-free extracts of Streptomyces coelicolor Müller NRRL 3532 (UC 5240). The crude enzyme was prepared using lysozyme and was treated with MnCl2 and (NH4)2SO4. Final purification was achieved by anion exchange chromatography. The pirlimycin reaction product was verified as being pirlimycin-3-(5'-adenylate) by NMR spectroscopy and MS. As a result of purification, this lincosaminide nucleotidylating and inactivating enzyme was separated from the macrolide phosphorylating enzyme also present in the cell-free extract.

Effects of trospectomycin on serum sensitivity of Escherichia coli UC 9451.

Trospectomycin sulfate, a chemically synthesized analog of spectinomycin, exhibits a broad range of activity against both aerobes and anaerobes, including the etiological agents of sexually transmitted diseases. Its activity in vitro against Escherichia coli is considered only moderate. At subinhibitory levels, however, trospectomycin induced changes in a pathogenic strain of E. coli, UC 9451, which significantly increased its sensitivity to serum lysis. This strain of E. coli shows high-level resistance to serum in vitro, typically growing twofold within a 45-min incubation period. Following exposure to one-fifth the MIC of trospectomycin, greater than 99% of the bacteria were killed in 25% serum within 15 min. Surviving bacteria were static in this level of serum for over 3 h. Killing was due to lysis mediated by both the classical and alternative complement pathways. The bacteria exposed to trospectomycin were enlarged in both diameter and length, but they still grew at rates comparable to those of untreated bacteria. No other visible morphological changes could be directly related to the increase in serum sensitivity. The profile of outer membrane proteins obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical for trospectomycin-treated or untreated bacteria. However, the relative proportion of four major outer membrane proteins varied considerably.

Inhibition of cyclooxygenase activity and platelet aggregation by epoxyeicosatrienoic acids. Influence of stereochemistry.

Certain epoxyeicosatrienoic acids (EETs) that were not cyclooxygenase substrates were effective cyclooxygenase inhibitors. Both (+/-)-14,15-cis-EET and (+/-)-8,9-cis-EET inhibited purified enzyme at concentrations from 1 to 50 microM; (+/-)-11,12-cis-EET was ineffective at concentrations below 100 microM. For the case of 14,15-cis-EET, only the (14R,15S)-stereoisomer was active. Other isomers including (14S,15R)-cis-EET, (14R,15R)-trans-EET, (14S,15S)-trans-EET, and the erythro and threo vicinal 14,15-diols were inactive. In addition to their effects on isolated enzyme preparations, cyclooxygenase activity in platelet suspensions, reflected by thromboxane B2 formation, was also inhibited by (14R,15S)-cis-EET and (+/-)-8,9-cis-EET but not by the other isomers. Thus potency and stereospecificity requirements were maintained for cyclooxygenase within intact platelets. Unlike the stereospecific inhibition of the cyclooxygenase enzyme, platelet aggregation induced by arachidonic acid was inhibited by all EET isomers at concentrations from 1 to 10 microM with no evident stereospecificity. Inhibition of aggregation was not uniformly associated with inhibition of thromboxane B2 formation; ordinarily, these two parameters correlate closely. This dissociation was not maintained for another biochemical process involved in platelet activation. For instance, there was a uniform correlation between inhibition of phosphorylation of a 40-kDa platelet protein and inhibition of aggregation. Our results suggest that effects of EET may originate from either stereospecific or nonspecific mechanisms. Definition of such mechanisms may be important to appreciate any physiological relevance of these substances.

Albumin-eicosanoid interactions. A model system to determine their attributes and inhibition.

A model system was developed to (a) reflect the chemical attributes of the microenvironment involved in albumin-eicosanoid interactions and (b) determine the effects of other ligands on these interactions. Albumin-dependent modulation of prostaglandin stability was chosen as the basis for this system. 15-Ketoprostaglandin E2 (PGE2) was evaluated as a model ligand because under special conditions it decomposes with formation of a visible chromophore. Human serum albumin, in a concentration-dependent fashion, catalyzed the dehydration of 15-keto-PGE2 with the concurrent generation of this chromophore (lambda max = 505 nm, epsilon = 35,000). Since chromophore production from 15-keto-PGE2 in albumin-free solution occurs only at pH greater than 10, the results suggest that albumin-eicosanoid interactions involve a microenvironment with alkaline attributes. The effect of other ligands on albumin-15-keto-prostaglandin E2 interactions was determined by monitoring their ability to inhibit the spectral component of these interactions. Inhibition correlated with an affinity for specific binding sites on albumin. At mole ratios of ligand/albumin below 1, only phenylbutazone, its analogs, and warfarin inhibited chromophore development. Other ligands including fatty acids, steroids, tryptophan, and drugs with an affinity for other binding sites were ineffective inhibitors.

Sodium 5-(3'-pyridinylmethyl)benzofuran-2-carboxylate (U-63557A), a new, selective thromboxane synthase inhibitor: intravenous and oral pharmacokinetics in dogs and correlations with ex situ thromboxane B2 production.

The pharmacokinetics of a new, selective thromboxane synthase inhibitor, sodium 5-(3'-pyridinylmethyl)benzofuran-2-carboxylate were determined for single dose, bolus intravenous injections (1, 3, and 10 mg/kg); for continuous 24 hr infusions (10 and 30 micrograms/kg/min); and for oral doses of gelatin encapsulated powdered drug (3, 10, and 30 mg/kg). Drug disappeared biexponentially after intravenous administration, and plasma concentrations were proportional to the dose. Absorption of drug occurred rapidly after its oral administration; peak plasma levels occurred 1-2 hours after ingestion, and circulating drug was detectable within 30 minutes. For all experiments, inhibition of cellular thromboxane B2 production, ex situ, corresponded with plasma drug levels and its reactivation corresponded with disappearance of the drug indicating that it was not accumulated by platelets.

Fluorometric determination of trifluoromethyl-substituted 2,3-bis(4-methoxyphenyl)indoles in biological materials.

Interest in the 5-, 6-, and 7-trifluoromethyl-substituted 2,3-bis(4-methoxyphenyl)indoles as potent, orally active anti-inflammatory agents required a method for their determination in serum, urine, and feces to permit studies of their absorption, metabolism, and excretion. A simple, rapid, sensitive, and specific procedure was developed based on an ethyl acetate extraction of alkaline specimens and subsequent fluorometric analysis of ethanolic solutions of the extract residues. The method is sensitive to 0.1 microgram/ml, 0.5 microgram/ml, and 0.7 microgram/100 mg of these compounds in serum, urine, and feces, respectively. Overall mean recoveries and standard deviations of the 5-, 6-, and 7-trifluoromethyl-substituted compounds from biological samples were 102.2 +/- 3.0, 102.2 +/- 5.4, and 100.5 +/- 5.7%, respectively. The procedure was applied successfully to absorption studies with 2,3-bis(4-methoxyphenyl)-7-(trifluoromethyl)indole in the dog.