Analytical studies of some agents for fertility regulation (Volume 2)

This Thesis describes three pieces of work, all of which were commissioned by the World Health Organization Special Programme in Human Reproduction which are concerned with aspects of the chemistry of agents for the regulation of fertility. The first two topics are related to agents causing infertility in the male, whilst the third is related to a method of contraceptive delivery in the female. Gossypol is a chiral natural product with phenolic and aldehyde functionality, occurring in the cotton plant. The literature on the isolation, identification, chemistry and biological properties of gossypol is reviewed in this Thesis. Following reports by Chinese scientists that racemic gossypol causes reversible infertility in male animals and human beings, it became clear from further studies in both China and in Western countries that toxic side effects were seen on prolonged dosing. In an attempt to lower the toxicity of this interesting antispermatogenic agent, attention turned to studies of the individual enantiomers, which were first separated in Matlin' s group by derivatisation with a chiral amine and chromatography of the resulting diastereomeric Schiff's bases on a non-chiral (normal or reverse) phase. Following hydrolysis and fractional crystallization, 40 g of chemically and optically pure (-)-gossypol was prepared and micronized for a toxicology study. Extracts of Tripterygium wilfordii L. Hook have been used in Traditional Chinese Medicine for many centuries for the treatment of inflammatory and dermatological disorders. Recent studies in China have revealed that men undergoing prolonged treatment with tabletted extracts of T. wilfordii suffered a reversible loss of fertility and tests in male rats have shown that this is related to a dramatic reduction in sperm density and motility. This Thesis gives a literature review of all the known constituents of T. wilfordii and the biological studies which have been reported. Furthermore, it describes a detailed bioassay-directed sub-fractionation of both T. wilfordii tablets and of crude extracts of the plant, obtained from China. This work led to the isolation and identification of a series of diterpene epoxides, including the known triptolide and tripdiolide and some isomers of the latter, one of which is a new compound, which show varying degrees of activity as oral anti-spermatogenic agents in male rats. Extensive use was made of analytical and preparative HPLC and of spectroscopic techniques, including high field NMR and mass spectrometry, in accomplishing the identification of the antifertility constituents of T.wilfordii. In the final part of the Thesis, a literature review is presented of the design, mode of action and clinical studies of vaginal rings. These are slow-release drug delivery systems in which a contraceptive steroid is contained within a polymer ring and is released by diffusion processes at a slow rate for prolonged periods of time. The Thesis then describes experimental studies in which suitable, constant eluent conditions and analytical procedures were developed and successfully applied. Release rates were measured for two types of newly fabricated rings, designed to release either progesterone or levonorgestrel at a near constant rate for periods of three months. For both steroids, it was possible to show that release could be described mathematically by a simple diffusion-distance model and that reproducible in vitro release rates could be obtained for devices in which a fixed loading of steroid is dispersed within a polymer core surrounded by an outer polymer sheath. Suitable dimensions were selected for the production of rings providing an initial release rate of 5 mg/day of progesterone and quality control was carried out on four batches of these rings fabricated by Dow Corning Ltd for WHO clinical trials. Similarly, suitable ring dimensions were selected for the production of rings providing an initial release rate of 20 ug/day of levonorgestrel and the production of these rings for clinical use in Britain and other countries is now being undertaken by Roussel Laboratories