S. M. Khomutov

Dissolution of a Mixture of Steroids in Cyclodextrin Solutions: a Model Description

Cyclodextrins (CDs) are widely used in pharmacology as biocompatible and nontoxic carriers of biologically active agents. The use of various drugs in the form of CD-based inclusion complexes (of the host – guest type) increases the solubility and stability of the parent drugs, facilitates delivery of the hydrophobic components to the target organ, and reduces undesired side effects [1]. The ideal partners for CD-based inclusion complexes are steroidal structures [2]. However, the use of several steroids forming a common complex with CD [3] in pharmaceutical preparations poses a question of the possible mutual influence and the resulting solubility under the conditions of competitive binding. The purpose of this study was to establish the laws of dissolution for a mixture of steroids in CD solutions. For this purpose, we have developed a simple but effective model of solubility for such steroid mixtures.

Synthesis of 3β-hydroxy-androsta-5,7-dien-17-one from 3β-hydroxyandrost-5-en-17-one via microbial 7α-hydroxylation

The synthesis of 3beta-hydroxy-androsta-5,7-dien-17-one from 3beta-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA) via microbial 7alpha-hydroxylation has been accomplished. At the first stage, 3beta,7alpha-dihydroxy-androst-5-en-17-one was obtained in high yield (71.2%) using a strain of Gibberella zeae VKM F-2600, which was first applied for DHEA conversion. The further route included the substitution of 7alpha-hydroxyl group with chlorine followed by a dehydrochlorination stage, and required minimal purifications of the intermediate products. The steroids obtained at every step were characterized by TLC,1H NMR, MS, UV- and IR-spectrometry. The combination of microbial and chemical steps ensured 54.6% yield of the target 3beta-hydroxy-androsta-5,7-dien-17-one from DHEA and can be applied for obtaining novel vitamin D derivatives.

Estimation of cyclodextrin affinity to steroids

A nonlinear spectrometric method for determination of the stability constant (KS) for cyclodextrin complex with steroid was developed. The method is based on calculation of the parameters of competitive cyclodextrin complexation by simultaneous fitting of two types of curves. Those of the first type are the dependencies of absorbance of methyl orange solution on the cyclodextrin concentration, the second type being the absorption curves of displacement of the dye, by steroid, from the cyclodextrin complex. With the method proposed, KS values were calculated with standard deviation less than 10%. This method is validated by determination of KS values using the phase‐solubility technique. For neutral steroid molecules, the effect of pH on KS was found to be insignificant. KS values for the cyclodextrin‐dye complex were determined for randomly methylated β‐cyclodextrin, 2‐hydroxypropyl‐β‐cyclodextrin, carboxymethyl‐β‐cyclodextrin and sulfobutylether‐β‐cyclodextrin. More hydrophobic steroids were characterised by higher KS values. Anionic β‐cyclodextrins showed high affinity for the steroids studied. Simple equipment and sufficient computing allowed recommendation of the method for express estimation of cyclodextrins affinity for hydrophobic substrates.

The inhibitory effect of cyclodextrin on the degradation of 9α-hydroxyandrost-4-ene-3,17-dione by Mycobacterium sp. VKM Ac-1817D

The inhibitory effect of methylated β-cyclodextrin (mCD) on steroid degradation was studied using the degradation of 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) by Mycobacterium sp. VKM Ac-1817D as a model process. The formation of the [9-OH-AD–mCD] complex was shown by 1H NMR-spectroscopy. The biodegradation of 9-OH-AD by whole and disrupted cells was carried out at 30°C in aqueous solutions with or without mCD. Enzyme kinetic parameters were calculated by non-linear regression of the Michaelis–Menten plot. The complexation of 9-OH-AD and mCD was evaluated via the stability constant for the [9-OH-AD–mCD] complex. The Vmax and KM values calculated for the free (non-complex) steroid in mCD solutions corresponded to steroid degradation in the absence of mCD. The inclusion complex [9-OH-AD–mCD] was shown to be resistant to enzymatic degradation. The inference is made that the ‘‘guest–host’’ molecular complexation with cyclodextrin can be used for the control of steroid bioconversions.

Formation of hydroxylated steroid lactones from dehydroepiandrosterone by Spicaria fumoso-rosea F-881

The transformation of dehydroepiandrosterone by Spicaria fumoso-rosea VKM F-881 produced 7α- and 7β-hydroxy-dehydroepiandrosterone, 3β,7α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one, and 3β,7β-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one. The yield of the main product—3β,7β-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one—was 49.5–72 mol % at substrate loadings of 5–20 g/L. Lactone formation proceeded through 7α- and 7β-hydroxy derivatives of dehydroepiandrosterone. The structure of the products was determined by mass spectrometry, 1H-NMR spectroscopy, and 13C-NMR spectroscopy. The proposed microbiological method for producing steroid lactones opens prospects for the synthesis of novel steroid compounds.

Obtaining of 11α-Hydroxyandrost-4-ene-3,17-dione from Natural Sterols

Two-step one-pot microbial transformation enables obtaining of valuable steroids that are difficult to produce chemically. Here we describe a method for obtaining 11α-hydroxyandrost-4-ene-3,17-dione (11α-HAD) from cheap and available natural sterols (phytosterols or cholesterol).11α-HAD is a primary adrenal steroid in mammals and also a key precursor in the syntheses of halogenated corticoids. Conventional routes for its obtaining are based on chemical synthesis, or microbial hydroxylation of androst-4-ene-3,17-dione (AD). AD in turn is produced primarily with microbial biotransformation of natural sterols by some actinobacteria.Consequent bioconversions of sterols using two microbial strains in one bioreactor vessel without separation and purification of AD provides high yield of 11α-HAD. At the first fermentation step, phytosterol is converted to AD with Mycobacterium neoaurum NRRL 3805B, or relative strains, to yield about 70% (mol/mol). At the second step, AD is almost fully (98%) hydroxylated at the position 11α with Aspergillus ochraceus VKM F-830, or other suitable organisms, in the same bioreactor. At the average, 30% (w/w) of the high-purity crystalline 11α-HAD can be obtained.The method can be exploited for production of 11α-HAD for practical use.

Transformation of wood industry waste into key intermediates for pharmaceutical substances: Biotechnological study

Selective microbial conversion of plant sterols allows direct production of 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD), a key intermediate in the synthesis of important pharmaceutical substances from the steroid glucocorticoid group. As substrates for bioconversion, sterol-enriched wastes of local pulp-and-paper industry, particularly, unsaponifiable tall pitch residue and its derivatives can be used. A series of simple and effective one- or two-stage fractionation procedures yielded the derivatives containing 51–88 wt % of convertible sterols. The analysis of the bioconversion dynamics of the sterol-enriched samples demonstrated that while the 9-OH-AD accumulation rates were 2–2.5 times lower than with commercial phytosterol, acceptable molar yields of 53–57% of the product were obtained. The obtained results make it possible to optimize the technological chain starting from the primary products of processing of renewable raw materials (by-products of wood processing) and ending with the desired steroid pharmaceutical substances via 9-OH-AD.