Biotransformation into 11α-hydroxyprogesterone glucosides by glucosyltransferase.

Recently, studies on the steroidal hormone activity in the brain have attracted attention, and the influences of the varied glucosides and their artificial derivatives have been discussed; additionally, it has been suggested that glucosides are the synthetic precursors of glucuronide as a label molecule. However, glucosides are formed with 11α-hydroxyprogesterone (1), which is important as a blood pressure regulator, but anti-androgen activity remains unknown. Using UDP-glucosyltransferase, glucoside synthesis was successful in linking ß-d-glucopyranose and ß-d-laminaribiose to 11α oxygen of 1 at a high conversion ratio, and full assignment structure was analyzed for the two glucosides by high-resolution quadrupole-time flight electrospray ionization-mass spectrometry, 1D (1H and 13C) NMR and 2D (COSY, ROESY, HSQC-DEPT and HMQC) NMR. Furthermore, the bioactivity of 1 and two 11α-hydroxyprogesterone glucosides [11α-(ß-d-glucopyranosyl)oxyprogesterone, 2, and 11α-(ß-d-laminaribiosyl)oxyprogesterone, 3] was tested in vitro. On rotenone-induced PC12 cells, the two 11α-hydroxyprogesterone glucosides (2 and 3) showed superior neuroprotective effects and increased cellular ATP levels compared with those of 1.

An efficient biotransformation of progesterone into 11α-hydroxyprogesterone by Rhizopus microsporus var. oligosporus.

Rhizopus microsporus var. oligosporus is a fungus that belongs to the Mucoraceae family that is used for the preparation of some soy-fermented foods. Microbial biotransformation of progesterone by R. microsporus var. oligosporus afforded some monohydroxylated and dihydroxylated metabolites. The main product was purified using chromatographic methods and identified as 11α-hydroxyprogesterone on the basis of its spectroscopic features. Time course studies by high-performance thin-layer chromatography demonstrated that this fungi efficiently hydroxylated progesterone at the 11α-position for 3 days with a yield of 76.48%, but beyond this time, the microorganism transformed 11α-hydroxyprogesterone into dihydroxylated metabolites. 11α-Hydroxyprogesterone is widely used as a precursor in the synthesis of hydrocortisone and other steroidal anti-inflammatory agents.

Protein Engineering of the Progesterone Hydroxylating P450-Monooxygenase CYP17A1 Alters Its Regioselectivity.

The CYP171 enzyme is known to catalyse a key step in the steroidogenesis of mammals. The substrates progesterone and pregnenolone are first hydroxylated at the C17 position, and this is followed by cleavage of the C17-C20 bond to yield important precursors for glucosteroids and androgens. In this study, we focused on the reaction of the bovine CYP17A1 enzyme with progesterone as a substrate. On the basis of a created homology model, active-site residues were identified and systematically mutated to alanine. In whole-cell biotransformations, the importance of the N202, R239, G297 and E305 residues for substrate conversion was confirmed. Additionally, mutation of the L206, V366 and V483 residues enhanced the formation of the 16α-hydroxyprogesterone side product up to 40 % of the total product formation. Furthermore, residue L105 was found not to be involved in this side activity, which contradicts a previous study with the human enzyme.

Characterization of new crystalline forms of hydroxyprogesterone caproate.

A systematic polymorph screening process was conducted on the steroid hydroxyprogesterone caproate, which had only one previously described orthorhombic crystalline form (A), in order to fully elucidate its solid state properties. Cooling, anti-solvent and evaporative techniques largely reproduced the same polymorph, but slurries in various solvents over two days produced a new triclinic form (B). Experiments at different temperatures in ethyl acetate or isopropyl alcohol confirmed this was an enantiotropic system with a transition temperature of approximately 30°C. DSC was used to confirm the transition of Form B to Form A below the melting point. Form B was the thermodynamically stable form at room temperature, and 8% less soluble in a non-aqueous solvent mixture. In viscous solvents used commercially to dissolve the oil-soluble steroid for injection, solutions near the solubility limit can remain supersaturated after exposure to cooler temperatures for months. In resolving the crystalline structure of Form A, a third conformational polymorph was detected that exists only at -133 to -143°C; this monoclinic form was designated Form C, and converts back to Form A upon warming to room temperature. These studies have increased the understanding of this drug and how the polymorphs may affect its physical stability in different dosage forms.

Blockage of progestin physiology disrupts ovarian differentiation in XX Nile tilapia (Oreochromis niloticus).

Previous studies indicated that maturation inducing hormone, 17α, 20ß-Dihydroxy-4-pregnen-3-one (DHP), probably through nuclear progestin receptor (Pgr), might be involved in spermatogenesis and oogenesis in fish. To further elucidate DHP actions in teleostean ovarian differentiation, we analyzed the expression of pgr in the ovary of Nile tilapia (Oreochromis niloticus), and performed RU486 (a synthetic Pgr antagonist) treatment in XX fish from 5 days after hatching (dah) to 120 dah. Tilapia Pgr was abundantly expressed in the follicular cells surrounding oocytes at 30 and 90 dah. Continuous RU486 treatment led to the blockage of oogenesis and masculinization of somatic cells in XX fish. Termination of RU486 treatment and maintenance in normal condition resulted in testicular differentiation, and estrogen compensation in RU486-treated XX fish successfully restored oogenesis. In RU486-treated XX fish, transcript levels of female dominant genes were significantly reduced, while male-biased genes were evidently augmented. Meanwhile, both germ cell mitotic and meiotic markers were substantially reduced. Consistently, estrogen production levels were significantly declined in RU486-treated XX fish. Taken together, our data further proved that DHP, possibly through Pgr, might be essential in the ovarian differentiation and estrogen production in fish.

Stability of hydroxyprogesterone caproate alone and in a compounded pharmaceutical product.

PURPOSE: The chemical stability of hydroxyprogesterone caproate in powder form and in a pharmaceutical formulation under different experimental conditions was evaluated. METHODS: Pure hydroxyprogesterone caproate was subjected to hydrolysis, photolysis, and thermal degradation. The content of hydroxyprogesterone caproate in pharmaceutical products was evaluated after using two different sterilization methods and after exposure to light. Hydroxyprogesterone caproate and its degradation products were analyzed using a validated reverse-phase high-performance liquid chromatographic method. Variables examined included specificity, accuracy, precision, linearity, theoretical plate numbers, signal:noise ratio, resolution between any two peaks, and relative standard deviation of the peak response. Statistical analysis was performed with Stata software, version 11 (StataCorp, College Station, TX). Mean values and standard deviations were calculated. The level of significance was set at p < 0.05. RESULTS: Components of hydroxyprogesterone caproate and organic impurities in pharmaceutical products were scanned with wide ultraviolet wavelength from 200 to 400 nm. In powder form, hydroxyprogesterone caproate was stable when exposed to high temperatures and light. Considerable degradation of hydroxyprogesterone caproate was observed in alkaline solution, with the major degradation product being hydroxyprogesterone. Much less degradation of hydroxyprogesterone caproate was observed in acidic conditions over 72 hours. The content of hydroxyprogesterone caproate in pharmaceutical products was not altered by the sterilization methods (filtration or heat sterilization) used and after exposure to light. CONCLUSION: Hydroxyprogesterone caproate in powder form and in a pharmaceutical formulation under different experimental conditions appeared to be fairly stable in the presence of strong acid, high temperatures, and light.

A direct stereoselective preparation of a fish pheromone and application of the zinc porphyrin tweezer chiroptical protocol in its stereochemical assignment.

A two-step stereoselective preparation of a goldfish pheromone, 17α,20ß-dihydroxy-4-pregnen-3-one, is reported from the readily available cortexolone in 64% overall yield. The (20S)-epimer was also synthesized in three steps from cortexolone with an overall yield of 47%. A microscale chiroptical technique based on a host/guest complexation mechanism between the substrate and a dimeric metalloporphyrin host (tweezer) was used to confirm the stereochemical assignment, while Density Functional Theory (DFT) calculations were employed to explain the high stereoselectivity induced by the 17α-hydroxyl and C18-methyl groups.

Qualitative and quantitative measures of various compounded formulations of 17-alpha hydroxyprogesterone caproate.

OBJECTIVE: 17-alpha hydroxyprogesterone caproate (17-OHPC) is available both as an Food and Drug Administration (FDA)-approved medication and as a product prepared for individual patients by compounding pharmacies. Compounding pharmacies may omit the preservative that is used in the FDA-approved formulation or use an alternate preservative and may dispense 17-OHPC in containers that differ from the FDA-approved product. The objective of this study was to assess the stability and the microbiologic and pyrogen status of 17-OHPC formulations under various compounding and dispensing conditions. STUDY DESIGN: 17-OHPC was prepared by a local compounding pharmacy. The formulations that were prepared included 1 identical to the FDA-approved product with benzyl alcohol as a preservative, 1 with benzalkonium chloride as a preservative, and 1 without a preservative. These various formulations were dispensed into either single-dose 1-mL plastic syringes or glass vials or 10-mL glass vials. The concentration of 17-OHPC and microbial and pyrogen status were evaluated at various time intervals over the ensuing 19 weeks. RESULTS: The concentration of 17-OHPC did not change over the duration of study, regardless of the dispensing medium that was used or the absence or presence of any preservatives. The preparations remained microbe- and pyrogen-free during the study period, regardless of the dispensing medium that was used or the absence of presence of any preservatives. CONCLUSION: Products that contained 17-OHPC tested in this study were quite stable over the 19-week period of study in different dispensing containers and in the absence or presence of a different preservative. The compounded products remained sterile and pyrogen-free during the period of observation.

Synthesis of deuterium labeled NMDA receptor inhibitor - 20-Oxo-5ß-[9,12,12-(2)H(3)]pregnan-3α-yl-L-glutamyl 1-ester.

20-Oxo-5ß-[9,12,12-(2)H(3)]pregnan-3α-yl-l-glutamyl 1-ester 11 was synthesized as an internal standard for quantification of a neuroprotective NMDA receptor ligand, 20-oxo-5ß-pregnan-3α-yl-l-glutamyl 1-ester 18 and its metabolites, in plasma and tissue. 11α-Hydroxy-progesterone (1) was reduced under basic conditions to yield the corresponding 5ß-steroid. Protection of the 3- and 20-oxo groups and oxidation of the 11α-hydroxy group was then followed by a deuterium exchange, conducted under basic conditions using deuterated methanol. Next, the carbonyl moiety at C-11 was reduced and the 11α-hydroxyl group removed through utilization of the Barton-McCombie reaction. Subsequent deprotection of the 3- and 20-acetals and stereoselective reduction of the 3-oxo group gave the desired trideuterated pregnanolone (8). This was coupled with protected glutamic acid, which was then deprotected to yield [9,12,12-(2)H(3)]-pregnanolone glutamate (11) with >99% isotopic purity.

Preparation and preliminary bioevaluation of 99mTc(CO)3-11ß-progesterone derivative prepared via click chemistry route.

INTRODUCTION: Progesterone receptors (PRs) overexpressed in breast cancers serve as potential targets for developing radiotracers for use in nuclear medicine. Hence, suitably derivatized progesterone can be envisaged as a potential vector for targeting overexpression of receptors in breast cancer. In the present article, we report the preparation of a (99m)Tc(CO)(3)-progesterone triazole using the Cu(I)-catalyzed novel click chemistry route. Preliminary evaluation of the radiolabeled derivative has been carried out in binding studies with MCF 7 cell lines. METHODS: 11-Hydroxyprogesterone has been synthetically derivatized to 11-azidoprogesterone. Subsequently, the cycloaddition reaction between progesterone azide and propargyl glycine was carried out to prepare 1,4-bifunctionalized progesterone triazole analogue. The clicked progesterone triazole derivative was radiolabeled with (99m)Tc and characterized by HPLC. The chemical characterization of (99m)Tc(CO)(3)-progesterone triazole has been carried out by preparing its corresponding rhenium complex using the [NEt(4)](2)[Re(CO)(3)Br(3)] precursor. While in vitro studies were carried out in MCF7 cell lines, in vivo distribution studies were performed in female Swiss mice. RESULTS: The radiolabeled complex could be prepared in >95% radiochemical yield as determined by HPLC. In vitro studies of (99m)Tc(CO)(3)-progesterone complex in MCF7 cell lines overexpressing receptors for breast cancer showed binding up to 30%. In vivo distribution studies in female Swiss mice have shown uterine uptake of 0.41 (0.06) % ID/g at 3 h postinjection (pi) and retention therein till 24 h pi. CONCLUSION: The present study demonstrates a novel and facile route for preparation of (99m)Tc-labeled progesterone complex using click chemistry. This strategy can be further extended towards preparation of radiolabeled complexes of other steroidal derivatives.

Control of steroid 21-oic acid synthesis by peroxisome proliferator-activated receptor alpha and role of the hypothalamic-pituitary-adrenal axis.

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes.

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring. All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized. GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed. We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.

Derivatization of 11-alpha-hydroxyprogesterone using phosphoramidite chemistry.

The well-known phosphoramidite chemistry routinely used in solid-phase oligonucleotide synthesis is exploited here in the preparation of steroid conjugates in solution. The applicability of the method is tested by conjugating one nucleosidic and one non-nucleosidic phosphoramidite building block to 11-alpha-hydroxyprogesterone. The suitability of one of the conjugates synthesized (5) for a competitive binding assay is also demonstrated.

[Chemical constituents in aerial part of Reineckea carnea].

OBJECTIVE: To study the chemical constituents in the aerial part of Reineckea carnea. METHOD: The compounds were isolated by extraction, silica gel, gel, and reversed-phase silica gel coloum chromatography, and high-performance liquid chromatography. The structures were identified by various spectroscopic methods including 1D and 2D NMR spectrum, MS, IR, etc. RESULT: Six compounds were isolated and identified as 1alpha, 3beta-dihydroxy-5beta-pregn-16-en-20-one-3-O-beta-D-glucopyranoside (1), syringaresinol-beta-D-glucoside (2), sophoraflavone B (3), stigmast-5, 22-dien-3-O-beta-D-glucopyranoside (4), daucosterol (5), a-D-glucose (6). CONCLUSION: Compound 1 was a new compound, coumpounds 2-6 were obtained from the plant for the first time.

Biocatalytic synthesis of 4-pregnen-20,21-diol-3-one, a selective inhibitor of human 5alpha-reductase type II.

Biocatalysis, the conversion of substrates into valuable products by the use of enzymes, has some striking advantages in comparison to standard organic chemistry for drug synthesis. By biocatalysis, substrates that contain several identical reactive groups at different positions can be converted with high regio-selectivity and enantio-selectivity. In this study, an E. coli isolate (E132) was identified which was able to convert the steroid desoxycorticosterone into the product 4-pregnen-20,21-diol-3-one in real terms. The product was purified from the cell culture supernatant by HPLC and its structure was demonstrated by mass spectrometry and NMR spectroscopy. It was tested on inhibition of human 5alpha-reductases type I and type II. At a concentration of 10 microM, inhibition was 49.0% for type I and 81.8% for type II, whereas there was no inhibition of human aromatase (CYP19) at 20 microM and human 17alpha-hydroxylase-C17,20-lyase (CYP17) at 2.5 microM detectable. The IC50 value of 4-pregnen-20,21-diol-3-one for human 5alpha-reductase type II was determined to be 1.56 microM.

Steroid extraction in a microchannel system--mathematical modelling and experiments.

The continuous ethyl acetate extraction of progesterone and 11alpha-hydroxyprogesterone, a reactant and the product of the biotransformation step involved in corticosteroid production, was studied in a microchannel at different flow velocities. In addition, non-steady state batch extraction without mixing was performed and modelled in order to verify the theoretically predicted parameters. In order to analyze experimental data and to forecast microreactor performance, a three-dimensional mathematical model with convection and diffusion terms was developed considering the velocity profile for laminar flow of two parallel phases in a microchannel at steady-state conditions. For the numerical solution of a complex equation system, non-equidistant finite differences were used. Very good agreement between model calculations and experimental data was achieved without any fitting procedure. Due to the efficient phase separation and high extraction yields obtained, the micro scale extraction units were found to be a promising tool for the development of an integrated system of 11alpha-hydroxylation of progesterone by Rhizopus nigricans in the form of pellets.

[Influence of the structure of synthetic gestagens on their binding to progesteron receptors in the endometrium].

Chemical modification of progesterone molecule leads to changes both in the gestagenic activity of new derivatives and in their specific binding with progesterone receptors. The passage from esters (acetomepregenole, butagest) to the corresponding OH-forms such as 17a-acetoxy-3b-hydroxy-6-methyl-pregna-4,6-dien-20-one (ABMP)is accompanied by an increase in the binding with progesterone receptors in vitro. The translocation of a double bond from endocyclic (N6-N7) to exocyclic position (methylene group at N6 in ABMP) has no significant effect on the ability to binding with progesterone receptors.

Validation of the accuracy of the perturbation peak method for determination of single and binary adsorption isotherm parameters in LC.

An analytical validation of the precision and accuracy of the perturbation peak (PP) method for determination of single and competitive thermodynamic isotherm parameters was performed using frontal analysis as a reference. The isotherm parameters of 11alpha-hydroxyprogesterone were determined in an achiral system and the isotherm parameters of (+)-methyl L-mandelate and (-)-methyl D-mandelate were determined in a chiral system, both for the single components and for the competitive binary mixture. The experimental errors in the PP method using different injection techniques were investigated, and we devised a new injection technique for the determination of competitive isotherm parameters that considerably reduced the experimental errors and also made both perturbation peaks detectable. We showed that the PP method with the new injection technique can be used to determine isotherm parameters directly from a racemic mixture. These parameters agreed well with those determined using several enantiomer ratios. Elution-band profiles simulated using the isotherm parameters showed excellent agreement with experimental profiles.

Microbial hydroxylation of progesterone with Acremonium strictum.

Microbial hydroxylation of progesterone occurred in the culture of Acremonium strictum PTCC 5282 to produce two hydroxylated pregnene-like steroids. The metabolites were purified and characterized using spectroscopic methods and identified as 15alpha-hydroxyprogesterone and 15alpha-hydroxydeoxycorticosterone.