Effect of progesterone and its synthetic analogues on the activity of mitochondrial permeability transition pore in isolated rat liver mitochondria.

The influence of progesterone and its synthetic analogues on the induction of the Ca(2+)-dependent mitochondrial permeability transition pore (MPTP) has been studied. The novel synthetic analogue of progesterone 17a-acetoxy-3b-butanoyloxy-6-methyl-pregna-4,6-diene-20-on (buterol) was compared with progesterone and medroxyprogesterone acetate (MPA). It was found that progesterone and buterol have opposite effects on the induction of MPTP opening by calcium ions. By contrast to progesterone, which decreased the calcium ion concentration necessary for pore opening, and MPA, which also, although at a lesser extent, activated the pore induction, buterol at a concentration of 20-100 microM blocked the pore opening and increased the calcium retention capacity of mitochondria more than twofold. The action of buterol is specific to the pore since it did not affect the respiration, whereas progesterone completely inhibited NAD-dependent respiration. MPA acted similar to progesterone but less effectively. The inhibitory effect of buterol was eliminated in the presence of carboxyatractyloside, which selectively binds the thiol groups of adenylate translocase and prevents the adenine nucleotide binding. These data indicate that buterol interacts with thiol groups, which explains its inhibitory effect not only on the mitochondrial pore but also on the transport system of xenobiotics in tumor cells in which buterol reduces the multidrug resistance.

Enhancement of 17alpha-hydroxyprogesterone production from progesterone by biotransformation using hydroxypropyl-beta-cyclodextrin complexation technique.

An enhancement of 17alpha-hydroxyprogesterone (17alpha-HP) production from progesterone by biotransformation using hydroxypropyl-beta-cyclodextrin (HPbetaCD) complexation together with aeration and sonication technique was demonstrated. The progesterone-hydroxypropyl-beta-cyclodextrin complex was prepared by co-evaporation method. The percentage yield of 17alpha-HP from P of 11.26+/-0.64% at 24h was observed in Curvularia lunata ATCC 12017. In the complex form of P, together with sonication at 40kHz for 5s and aeration, the yield of 17alpha-HP was increased to 72.92+/-4.28% which was about 6.5 and 1.3 times of that from the uncomplexed (P) and the complexed (PC), respectively without sonication and aeration. The increased aqueous solubility of P by complexation with HPbetaCD was the main factor which increased the yield of 17alpha-HP, while aeration had more effect on P than PC. Sonication did not significantly increased the yield of the product from both P and PC. When both aeration and sonication were used in the PC system, the product yield was increased significantly more than that from P. The result from this study can be applied for the biotransformation of other poor aqueous soluble precursors.

The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17.

A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s. This is illustrated here for the synthesis of hydroxylated steroids. This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providing electrons at an economically acceptable cost. To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+. Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography. Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450. Thus, high concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH. Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4). It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.