Preparation of 3-ketodesogestrel metabolites by microbial transformation and chemical synthesis.

Specific microbial reactions were used for the preparation of metabolites of 3-ketodesogestrel (13-ethyl-17 beta-hydroxy-11-methylene-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one, the active from of the progestagen desogestrel. Clostridium paraputrificum transformed 3-ketodesogestrel (KDG) to the 5 beta-dihydro and tetrahydro metabolites 13-ethyl-17 beta-hydroxy-11-methylene-18,19-dinor-5 beta, 17 alpha-pregnan-20-yn-3-one and 13-ethyl-11-methylene-18,19-dinor-5 beta, 17 alpha-pregnan-20-yne-3 alpha, 17 beta-diol, respectively. The epimeric compound 13-ethyl-11-methylene-18,19-dinor-5 beta, 17 alpha-pregnan-20-yne-3 beta, 17 beta-diol was obtained by chemical reduction of the 3-oxo compound. Mycobacterium smegmatis converted KDG to metabolites of the 5 alpha H-series: 13-ethyl-17 beta-hydroxy-11-methylene-18,19-dinor-5 alpha, 17 alpha-pregnan-20-yn-3-one, 13-ethyl-11-methylene-18,19-dinor-5 alpha, 17 alpha-pregnan-20-yne-3 alpha, 17 beta-diol and 13-ethyl-11-methylene-18,19-dinor-5 alpha, 17 alpha-pregnan-20-yne-3 beta, 17 beta-diol. The ring A-aromatized analog of KDG 13-ethyl-11-methylene-18,19-dinor-17 alpha-pregna-1,3,5(10)-trien-20-yne-3,17 beta-diol was obtained by microbial 1-dehydrogenation with Rhodococcus rhodochrous. Additionally, chemical syntheses of the microbially obtained KDG metabolites listed above were carried out. These included Birch reduction, reduction of KDG with sodium borohydride in aqueous pyridine and in methanol, reduction of KDG with potassium selectride in tetrahydrofuran, and dehydrogenation of KDG with cupric-II bromide in acetonitrile. The problems encountered in chemical syntheses favor the microbial procedures. The compounds were characterized by mass spectra (MS), IR, and circular dichroism (CD). Complete assignments of 1H and 13C chemical shifts were made using homo- and heteronuclear 2-DN-NMR spectroscopy. Chromatographic [gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC)] data of all the prepared KDG metabolites are presented.

Steroid metabolism with intestinal microorganisms.

As a result of the metabolic activities of numerous anaerobic microorganisms with sterols, bile acids and steroid hormones as substrates in connection with the enterohepatic circulation of these compounds, the intestine may be considered as an "endocrine" active site or organ. The review summarizes transformations of steroids by anaerobic intestinal bacteria, the physiological and supposed pathophysiological meaning thereof. The aim is to recommend further investigation in this field with respect to both the elucidation of the reactions and biological responses.

Bioconversion of steroids by Cochliobolus lunatus--II. 11 beta-hydroxylation of 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate in dependence of the inducer structure.

The 11 beta-hydroxylase of the filamentous fungus Cochliobolus lunatus m 118 was induced with the substrate 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate (11 beta-deoxyprednisolone 17-acetate) itself, substrate analogues, different pregnane compounds, sterols, intermediates of microbial sterol side-chain degradation or bile acids, together with 24 different steroids in a standardized test system. The resulting 11 beta-hydroxylation rate, leading to prednisolone 17-acetate and prednisolone, respectively, was determined and compared with the hydroxylation rate of non-induced cultures. The transformation yield strongly depended on the inducer structure. The microbial sterol side-chain degradation intermediates (20S)-20-hydroxymethylpregn-4-en-3-one and the corresponding pregna-1,4-diene compound caused the highest induction effects (induction factors 5.1 and 4.9, respectively). The metabolism of (20S)-20-hydroxymethylpregna-1,4-dien-3-one during the cultivation was elucidated. The induction effect decreased with the rising oxidation of the inducer. The significant increase of the 11 beta-hydroxylation rate of 1-dehydro-pregnane substrates by specific induction allows alternative pathways to glucocorticoid partial syntheses.

Evaluation of patient-specific computer-generated drug information monographs.

A computerized Medication Awareness Reporting System (MARS) generated by a Sperry PC microcomputer with an Epson FX-80+ printer was evaluated on four adult units (163 beds) of a 353-bed community hospital. MARS is a drug database containing clinical information monographs on 200 commonly prescribed medications. Information provided through the MARS system includes mechanism of action, dosage and administration guidelines, adverse effects and contraindications. In addition, pharmacists can add written information to each MARS report. Patient-specific MARS reports were generated on all patients admitted to the four units during the 16-week study period. Copies of the reports were placed into each patient's chart and into the patient's medication Kardex. Reports were updated daily by pharmacists who provide decentralized service to the units. Physicians, pharmacists, and nurses who encountered the MARS reports were surveyed at the end of the 16-week period. The results indicated that nurses used MARS more than physicians or pharmacists. The groups that valued the MARS reports as a source of drug information the least were the pharmacists and the physicians. MARS, or a drug information system similar to MARS, can be provided with relatively little cost for equipment and materials. However, the use of MARS as described in this study may be limited due to the time required to provide this service. More efficient methods of delivering MARS may enhance its use in the hospital setting. Currently, MARS may be more appropriate for use in long-term care facilities and home health agencies.

Steroid transformation with immobilized microorganisms. VI. The reverse reaction of steroid-1-dehydrogenases from different micoorganisms in immobilized state.

Whole cells of Nocardia erythropolis, N. opaca and Mycobacterium phlei containing 4-en-3-oxosteroid: (acceptor)-1-en-oxidoreductase activities were immobilized by adsorption on DEAE-cellulose and silica and by entrapment in polyacrylamide gel. The obtained biocatalysts were used in an anaerobic continuous column process to transform 1,4-dien-3-oxo-steroids into 4-en-3-oxo- steroids. The half life of steroid-1(2)-reductase activity of the N. erythropolis was found to be up to 15 days. The DEAE-cellulose-adsorbed cells showed a slightly higher relative activity than free cells. The influence of substrate concentration and the action of dimethylformamide were also investigated.

Degradation of steroids by microorganisms. XVIII. The reversibility of steroid-1-dehydrogenation during microbial side chain degradation of sterols by Nocardia.

The aerobic side chain degradation of sterols often leads to mixtures of 1,4-diene and 4-ene compounds. By an additional anaerobic step a practically complete reverse reaction of the steroid-1-dehydrogenation occurs yielding only the corresponding 4-ene compound.

[Steroid-transforming enzymes from microorganisms. XI. Reversibility of the dehydrogenation reaction of the steroid-1-dehydrogenase from Nocardia opaca]. / Steroidumwandelnde Enzyme aus Mikroorganismen. XI. Reversibilität der Dehydrierungsreaktion der Steroid-1-Dehydrogenase aus Nocardia opaca.

Highly purified preparations of the 4-en-3-oxosteroid: (acceptor)-1-en-oxidoreductase from Nocardia opaca have been investigated in both types of reactions: 1.2-dehydrogenation of the 4-en-3-oxo-derivative and 1.2-hydrogenation of the 1.4-dien-3-oxo-derivative. It was not possible to separate the hydrogenating activity from the dehydrogenating activity by affinity chromatography, disc electrophoresis, SDS-electrophoresis, and isoelectric focusing techniques. The pure enzyme preparation is discussed as only one FAD depending protein acting in response to the system as a dehydrogenase as well as a reductase.

[Steroid-transforming enzymes from microorganisms. X. Enrichment of a 4-en-3-oxosteroid-5 alpha-reductase from Mycobacterium smegmatis as well as separation and enrichment of the apoenzyme by means of affinity chromatography]. / Steroidumwandelnde Enzyme aus Mikroorganismen. X. Anreicherung einer 4-En-3-oxosteroid-5 alpha-Reduktase aus Mycobacterium smegmatis sowie Abtrennung und Anreicherung des Apoenzyms mit Hilfe der Affinitätschromatographie.

The 4-en-3-oxosteroid-5 alpha-reductase from Mycobacterium smegmatis was bound biospecifically on the affinant containing an immobilized testosterone ligand. The enzyme obtained by elution with ethylene glycol and urea in a 32 fold purity has a S. A. of 8.73 X 10(-3) microM androstenedione min-1 mg-1. The coenzyme (FAD) could be separated from the immobilized enzyme substrate complex on the affinity matrix, in the presence of (NH4)2SO4 at pH 3.0. After elution of the apoenzyme 97% of the initial enzyme activity was obtained by incubation with FAD. The reactivated enzyme results in a 40-fold enrichment.