Enhancement of microbial hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Metarhizium anisopliae using nano-liposome technique.

The introduction of 11α-hydroxy to 13-ethyl-gon-4-ene-3,17-dione (GD) by microbial transformation is a key step in the synthesis of oral contraceptive desogestrel, while low substrate solubility and uptake into cells are tough problems influencing biotransformation efficiency greatly. Nano-liposome technique was used in the hydroxylation of GD by Metarhizium anisopliae. The substrate GD was processed to be GD-loaded nano-liposomes (GNLs) with high stability and encapsulation efficiency, and then applied in microbial hydroxylation by M. anisopliae. The results proved that the yield of the main product 11α-hydroxy-13-ethyl-gon-4-ene-3,17-dione (HGD) tripled compared to regular solvent dimethylformamide dispersion method at 2 g/l of substrate feeding concentration, and the HGD conversion rate showed no obvious reduction when the substrate feeding concentration increased from 2 to 6 g/l, which indicated the improvement of GNL addition method on biotransformation. Furthermore, the main byproduct changed from 6ß-hydroxy derivative of GD (with similar polarity to HGD) to 6ß,11α-dihydroxy derivative, which benefits the following purification of HGD from fermentation broth. These advantages suggest a great potential for the application of nano-liposome technique in microbial steroid transformation.

15α-Hydroxylation of a steroid (13-ethyl-gon-4-en-3,17-dione) by Penicillium raistrickii in an ionic liquid/aqueous biphasic system.

Biphasic processes are used in whole-cell biotransformation to overcome the low water solubility of substrates and products as well as their inhibitory effects on the biocatalyst. Commercially available [NTf(2)]- and [PF(6)]-based ionic liquids (ILs) were used in a biphasic system for the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione by Penicillium raistrickii. With the substrate at 5 g l(-1) and a volume ratio of IL to buffer, buffer pH and cell density at, 1:9, 6.5, 16.8 g(DW) l(-1), respectively, the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione was achieved with a yield of 70 % after 72 h using [BMIm][NTf(2)] in a 50 ml biphasic system. This is compared to a 30 % yield in a monophasic aqueous system. This suggests the potential industrial application of ILs-based biphasic systems for steroid biotransformation.

Molecular properties and preclinical pharmacology of JNJ-1250132, a steroidal progesterone receptor modulator that inhibits binding of the receptor to DNA in vitro.

Progesterone receptor modulators have diverse potential therapeutic uses, including the treatment of endometriosis, uterine fibroids and breast cancer. Here we describe the molecular properties and preclinical pharmacology of a new steroidal progestin antagonist, JNJ-1250132. The compound is a high affinity ligand for the progesterone receptor, possessing cross-reactivity with other steroid receptors comparable to that of steroidal antagonists such as mifepristone. It inhibits progestin-inducible alkaline phosphatase gene expression in T47D human breast cancer cells, and also inhibits their in vitro proliferation. It inhibits gestation in rats and progesterone-dependent endometrial transformation in rabbits with efficacies comparable to mifepristone. Like mifepristone, it is a glucocorticoid antagonist in vivo. In cell-free DNA binding assays, the compound inhibits binding of the human progesterone receptor to a progesterone response element, and thus is similar to onapristone in this regard. In contrast, as judged by proteolytic analysis, JNJ-1250132 induces a receptor conformation more similar to that induced by mifepristone, which promotes receptor binding to DNA. Therefore, JNJ-1250132 has unique effects on the progesterone receptor that may translate into a novel clinical profile.

Mutagenic and carcinogenic metabolites of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one.

Microsomal metabolites of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one (Structure I) were separated by high-pressure liquid chromatography, and their structures were established on the basis of their ultraviolet and mass spectra, together with considerations of their general chemical properties. This was assisted by comparisons with metabolites formed in the same way from the synthetic 15-hydroxy (Structure III), 16-hydroxy (Structure II), and 11-hydroxymethyl (Structure IV) derivatives, which themselves occur as metabolites of Structural I. Products derived from attack at the two benzo-ring double bonds occurred, but no K-region products were found. Only metabolites having a non-bay region 3,4-dihydrodiol system were mutagenic and bound to DNA after in vitro microsomal activation, and it was concluded that the 3,4-dihydro-3,4-diol (Metabolite e) was the main form and that the 3,4-diols of the monools (Structure II to IV) were minor proximate forms of this carcinogen. In a two-stage experiment, the synthetic 16-ol (Structure II) was shown to be almost as carcinogenic as was Structure I itself in mice; the 15-ol (Structure III) and 11-hydroxymethyl derivative (Structure IV) were much less active. The same order was also observed in the mutagenicity of these compounds in the Ames test.

Repair of DNA adducts of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one in mouse tissues and its relation to tumor induction.

The formation and repair of DNA adducts of the carcinogen, 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one, have been studied in three mouse tissues, liver, lung, and skin. Following a single i.m. dose of the carcinogen, DNA adduct formation was observed in all three tissues, the highest being found in liver DNA, the tissue resistant to tumor formation by this carcinogen. In skin and lung, the tissues which were susceptible to tumor formation, binding was approximately one-half that found in liver. Detailed analysis by high-pressure liquid chromatography of the adducts formed in each of the three tissues revealed no major qualitative differences in the eight adduct peaks. In vivo removal of the labeled adducts was studied over a 14-day period following initial treatment, and adducts were analyzed at each time point by high-pressure liquid chromatography. In skin and lung, active removal of the major adducts could not be measured above the normal rate of DNA turnover. By contrast, in the liver, where the rate of DNA turnover was slower, adducts were removed relatively rapidly with a half-life of approximately 2.5 days. Only one minor adduct present in both skin and liver was removed more rapidly than were the major adducts. The results suggest that the persistence of carcinogen-DNA adducts coupled with a relatively high rate of cell division may be related to tissue-specific carcinogenesis by this polycyclic ketone.

Strain-specific tumorigenesis in mouse skin induced by the carcinogen, 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one, and its relation to DNA adduct formation and persistence.

The incidence of skin tumors has been studied in three strains of mice, namely, TO, C57BL, and DBA/2, after treatment with the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one. After either a single dose followed by croton oil promotion or a continual dose of the carcinogen, tumors were observed in the TO and C57BL strains, with the TO mice having the shorter mean latent period. The DBA/2 mice, however, appeared to be resistant to tumor formation by either treatment. To understand the mechanism of resistance, several criteria have been investigated. Metabolism of the carcinogen was assessed in terms of the total DNA adduct formation and the pattern of individual adducts after separation by high-pressure liquid chromatography, and no major differences between the three strains was found. Similarly, the rates of disappearance of the individual adducts when measured over 14 days posttreatment were not strain specific. Persistent binding of the carcinogen after 2 months was found in all three strains and could be reduced markedly if croton oil was administered throughout this period. The ability of the phorbol esters to cause biochemical changes in both sensitive and resistant strains was indicated by the induction of ornithine decarboxylase in each of the three strains after treatment with either croton oil or its active component, 12-O-tetradecanoylphorbol-13-acetate.

Binding characteristics of progesterone and antiprogestin ZK 98.299 in human endometrial and myometrial cytosol.

The binding of ZK 98.299, a synthetic progesterone antagonist, with human endometrium and myometrium cytosol was studied and compared with that of progesterone. Progesterone showed specific saturable binding to its receptors in both endometrium and myometrium. ZK 98.299 and progesterone were mutually competitive for binding to progesterone receptors; however, the relative binding affinity of ZK 98.299 was 16% that of progesterone. ZK 98.299 exchanged the progesterone-labelled receptor sites. [3H]ZK 98.299 showed specific binding which was linearly related to the cytosol protein concentration. The binding was not saturable at 15 nM of ligand. The binding capacity and binding affinity of ZK 98.299 receptor was less than that of progesterone. Progesterone also partially displaced the binding of [3H]ZK 98.299. This study suggest that ZK 98.299 and progesterone both bind to the same protein. However, whether ZK 98.299 binds to progesterone receptors alone or even to other functionally related sites is not known. It appears that ZK 98.299 when present in higher concentration than progesterone would be an effective receptor ligand.

Antiprogestin ZK-98.299 and progesterone display differential binding characteristics in the human myometrial cytosol.

To investigate whether the synthetic progesterone antagonist ZK-98.299 binds to progesterone receptor or also has distinct binding sites, the binding characteristics of ZK-98.299 were compared with those of progesterone in the human myometrial cytosol. [3H]ZK-98.299 and [3H]progesterone showed specific binding in the myometrial cytosol and the binding of each radiolabelled ligand could be displaced with the respective ligand in a dose-response manner. However, while the binding of [3H]progesterone could be completely blocked with progesterone or ZK 98.299, the binding of [3H]ZK-98.299 could not be displaced more than 50%. The non-specific binding of [3H]ZK-98.299 was very high as compared to that of [3H]progesterone. Using [3H]progesterone, the relative binding affinity (RBA) of progesterone was more than that of ZK 98.299, whereas using [3H]ZK-98.299 the RBA of ZK 98.299 exceeded that of progesterone. Treatment of myometrial cytosol with increasing concentrations of -SH-modifying agents (iodoacetamide (IA) 0-10 mM or N-ethylmaleimide (NEM) 0-1000 nM) decreased the binding of progesterone by over 80%, whereas similar treatment did not have appreciable effect on the binding of [3H]ZK-98.299. Although both preformed ligand-receptor complexes were relatively stable in the presence of IA and NEM, the [3H]progesterone-receptor complex was more sensitive as compared to the [3H]ZK-98.299-receptor complex. The addition of 20 mM molybdate in the cytosol had a protective effect against the -SH-modifying agents. [3H]ZK-98.299 and [3H]progesterone-receptor complexes also showed differential stability when incubated at elevated temperatures (25 degrees C and 37 degrees C), [3H]ZK-98.299-binding sites being more thermolabile as compared to [3H]progesterone binding sites. Prior occupation of the receptor by the two ligands gave the complexes the ability to resist an elevated temperature of 25 degrees C. Moreover, molybdate stabilized both the liganded and unoccupied receptors at 25 degrees C. When the ligand-receptor complexes were applied onto a prefocused polyacrylamide gel, the progesterone and ZK-98.299-receptor complexes were resolved and focused at pH 7.2 and 8.4, respectively. The results of this study suggest that although progesterone and ZK-98.299 are mutually competitive for binding to progesterone receptor, ZK-98.299 also has distinct binding sites.

Interaction of rat liver glucocorticoid receptor with a newly synthesized antisteroid ZK98299.

Steroid receptor antagonists are important biochemical probes for understanding the mode of steroid hormone action. We have studied the interaction between rat liver glucocorticoid receptor and a newly synthesized antisteroid ZK98299 (13-antigestagen; [11-beta-(4-dimethylaminophenyl)-17a-hydroxy-17 beta-(3- hydroxypropyl)-13 alpha-methyl-4,9-gonadien-3-one]). Glucocorticoid receptor from freshly prepared hepatic cytosol bound [3H]ZK98299 with affinity approximately equal to that of [3H]triamcinolone acetonide. The binding of both steroids reached a maximum at 4 h at 0 degrees C. Both ligands were able to compete for the steroid binding site but progesterone, estradiol and dihydrotestosterone (DHT) failed to compete for the [3H]ZK98299 and [3H]triamcinolone acetonide binding. While [3H]ZK98299 binding to glucocorticoid receptor could occur in the presence of iodoacetamide and N-ethylmaleimide (NEM), [3H]triamcinolone acetonide binding capacity was completely abolished following such treatments. The [3H]ZK98299-receptor complexes sedimented as 9 S and 4 S molecules under control (4 degrees C) and receptor transforming (23 degrees C) conditions, and exhibited a faster rate of dissociation at 23 degrees C when compared with [3H]triamcinolone acetonide-receptor complexes. These results indicate that ZK98299 interacts with hepatic glucocorticoid receptor. The differential effects of iodoacetamide and NEM on the interaction of glucocorticoid receptor with ZK98299 and triamcinolone acetonide, and the faster rate of dissociation of [3H]ZK98299-receptor complexes suggest that treatment with these agents (NEM and iodoacetamide) results in distinct conformational changes in glucocorticoid receptor structure with respect to triamcinolone acetonide and ZK98299 binding. Alternatively, ZK98299 may be interacting with a site which is distinct from one which accepts triamcinolone acetonide.

Involvement of cytochrome P-450 in the 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Penicillium raistrickii.

The 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione (GD) with different subcellular fractions of Penicillium raistrickii i 477 was investigated. Cytochrome P-450 was shown to be involved in this reaction. The steroid transformation was inhibited by carbon monoxide, metyrapone, p-CMB, iodoacetamide, N-methylmaleimide and several metal ions. The 15alpha-hydroxylase was observed to be dependent on nicotinamide-adenine dinucleotide phosphate (NADPH) replaceable by NaIO4, and the activity was enhanced by a NADPH-regenerating system, indicating the involvement of the NADPH-cytochrome c (P-450) reductase. This was further confirmed by the inhibition of the hydroxylase activity in the presence of cytochrome c. No effect was observed in the presence of azide and antimycin A. Solubilized microsomes gave an absorption maximum at 453 nm in carbon monoxide difference spectrum, and showed a Type-I GD-binding spectrum typically for cytochrome P-450 interaction with substrate. First results about the inducibility of the enzymes involved in the 15alpha-hydroxylation of GD are shown.

Type II antagonists impair the DNA binding of steroid hormone receptors without affecting dimerization.

Two types of steroid antagonists exert their activity by different mechanisms when bound to the cognate receptor. Type I anti-progestins, such as RU486, induce DNA binding of the human progesterone receptor (hPR), while no hPR/DNA complexes were seen in gel shift assays in the presence of the type II anti-progestin ZK98,299 or RU50,331. ZK98,299-liganded hPR exerted significantly less tight nuclear binding than receptor complexes formed with RU486. Also a type II anti-glucocorticoid (RU43,044) was detected which completely abrogated DNA binding of its cognate receptor in vivo. In keeping with the existence of two different classes of anti-progestins, agonist- or RU486-induced hyperphosphorylation of the two hPR isoforms present in the T47D breast cancer cells was not induced by ZK98,299. This lack of hyperphosphorylation was, however, not the cause but most likely the consequence, of the reduced ability of the hPR/ZK98,299 complex to interact with DNA. No "mixed-ligand" heterodimers were formed in vitro between hPR isoform A bound to ZK98,299 and R5020-liganded isoform B, but nuclear co-translocation studies indicated that ZK98,299 efficiently induced hPR dimerization in vivo.

The metabolism and activation of 15,16-dihydrocyclopenta[a]phenanthren-17-one by cytochrome P-450 proteins.

The in vitro metabolism and activation to mutagens of 15,16-dihydrocyclopenta[a]phenanthren-17-one (CPP-17-one) were investigated using hepatic preparations from rats pretreated with prototype inducers of the cytochrome P-450-dependent mixed-function oxidases. Aroclor 1254-induced microsomes were the most effective metabolisers of this compound, the major metabolites being oxidation products of the bay region A ring. To a lesser extent hydroxylation of the non-aromatic D ring occurred, the products being the 15- and 16-hydroxyderivatives. Oxidation of the A ring was also achieved with microsomes from benzo[a]pyrene-treated rats but not with those from rats treated with clofibrate, phenobarbitone, isoniazid, dexamethasone and CPP-17-one itself, where the metabolites were primarily the oxidation products of the D ring. When CPP-17-one was used as a promutagen in the Ames test, only microsomes from Aroclor 1254-treated rats could elicit a positive mutagenic response. When 3,4-dihydrodihydroxy-CPP-17-one, the precursor of the ultimate mutagen, was used as the promutagen, a positive response was observed with microsomes from Aroclor 1254- and benzo[a]pyrene-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiprogestins and iatrogenic glucocorticoid resistance.

The antiglucocorticoid action of the antiprogestin RU 38486 has interfered with its successful clinical application in long-term treatment. Several new antiprogestins (Org 31710, Org 31806 and ZK 98299) have recently been developed with the aim to eliminate this side-effect. We have used a human lymphocyte proliferation assay to estimate the antiglucocorticoid potency of RU 38486 and the newer antiprogestins. In this assay 100 nmol/L RU 38486 shifted the dexamethasone inhibition curve by at least one order of magnitude. The other antiprogestins showed no effect at 100 nmol/L. RU 38486 (30 nmol/L) was able to antagonize 1000 nmol/L dexamethasone. The other antiprogestins showed only slight effects even at 1000 nmol/L. We conclude that the new antiprogestins have antiglucocorticoid effects that are one to two orders of magnitude lower than that of RU 38486. This may make them more suitable than RU 38486 for application in long-term antiprogestin treatment.

The identification of some human metabolites of norgestrel, a new progestational agent.

PIP: Norgestrel 1 (racemic 13-beta-ethyl-17-alpha-hydroxygon-4-en-3-one) a progestational agent with an angular ethyl group between Rings C and D, was studied by mass spectrometry to discover its structural characteristics. Synthesis of postulated metabolites of Norgestrel 1 for use in identification is described and structural formulas are given. Urine was used as a source to characterize fractions via mass spectra, and the fraction spectra are listed. The major metabolite was 13-beta-ethyl-17-alpha-ethynl-5-beta-gonan-3-alpha-1m-beta-diol 8c.^ieng

The influence of dihydrodiol conformation on the metabolic activation of cyclopenta[a]phenanthrenes.

The present study was undertaken in order to rationalise the apparent biological inactivity of 15,16-dihydro-6-methylcyclopenta[a]phenanthren-17- one (4) when other methyl isomers of 15,16-dihydrocyclopenta[a]phenanthren- -17-one, e.g. the 11-methyl derivative (2), display appreciable tumorigenicity. In vitro metabolism of the 6-methyl-ketone-17-one (4) demonstrated that its principal metabolite was the 3,4-dihydro-3,4-diol (3,4-dihydroxy-6-methyl-3,4,15,16- tetrahydrocyclopenta[a]phenanthren-17-one) (5) which, in the case of the active 11-methyl derivative, is the proximate genotoxin. Thus the inactivity of this 6-methyl-17-ketone cannot be ascribed to lack of formation of the 3,4-dihydro-3,4-diol, the precursor of the 3,4-diol-1,2-epoxides (the ultimate mutagens in this series). However, the 6-methyl-3,4-dihydro-3,4-diol exists in a pseudo-diaxial rather than a pseudo-diequatorial conformation characteristic of the 3,4-dihydro-3,4-diols of the other members of the series. It is therefore suggested that a diequatorial conformation in the dihydrodiol is essential to the metabolic activation of the cyclopenta[a]phenanthren-17-ones.

Metabolic activation of cytochrome P-450/P-448 in the yeast Saccharomyces cerevisiae.

The strains D6 and JD1 of the yeast Saccharomyces cerevisiae were used to assay the genetic activity of several compounds, benzo[a]pyrene, 15,16-dihydro-11-methyl-cyclopenta[a]phenanthren-17-one, 2-naphthylamine and cyclophosphamide, which require metabolic activation by cytochromes P-450 and P-448 to produce genetically active chemical species. Cells from both strains were harvested from cultures grown in low concentrations of glucose and switched to growth in high glucose containing media. Treatments under these conditions resulted in increased sensitivity of the test systems without the presence of an exogenous S9 mix and the presence of S9 was found not to enhance this sensitivity. The yeasts used under these treatment conditions showed a P-450/P-448 type metabolism.

Microbial transformation of 13-ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14, 17-dione to its 17-beta hydroxy derivative by Pichia farinosa in pilot plant fermentors.

Parameters for microbial transformation of 13-ethyl-3-methoxy-8, 14-seco-gona-1,3,5 (10), 9(11)-tetraene-14,17-dione to its 17 beta-hydroxy derivative by P. farinosa have been standardised in pilot plant fermentors. The yield of the pure crystalline compound was 80%.

Mass spectrometry of cyclopenta[alpha]phenanthrenes. Metabolites formed in vitro and in vivo from the carcinogen 15,16-dihydro-11-methyl-cyclopenta[alpha]phenanthren-17-one.

The mass spectra of a number of synthetic cyclopenta[alpha]phenanthrenes are described, and identification of an in vitro metabolite of a carcinogen of this series is reported. The correlation of the mass spectra of the major urinary metabolite, its dehydration product and its triacetate with the structure assigned to the metabolite is described.

High skin tumour initiating activity of the metabolically derived trans-3,4-dihydro-3,4-diol of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one.

Eight main metabolites of the parent carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthrene-17-one (I) were assayed for their ability to initiate skin tumours in T.O. mice after topical application in two-stage experiments with croton oil used as the promoter. All were less active than 1 with the exception of the trans-3,4-dihydro-3,4-diol which was more than ten times as active. This diol is therefore confirmed as the proximate carcinogen, a conclusion reached previously on different evidence.