Cooperative Contraceptive Research Center

Population Council, New York NY

Investigators

Linked publications & trials

Abstract

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of this proposal seeks to develop AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to be used as a male contraceptive in humans. AF-2364 appears to be a potential male contraceptive by inducing premature germ cell loss from the seminiferous epithelium, and by interfering with cell adhesion function between Sertoli and germ cells via several putative signal transduction pathways. More importantly, AF-2364 has no toxicity in tests for acute toxicity, mutagenicity, and genotoxicity conducted by licensed toxicologists. However, a mild toxicity of AF-2364 was detected in a recently completed sub-chronic toxicity study in male rats (50 mg/kg body weight/day for 29 days; n=20 rats with 10 males and 10 females). However, if the effective dosing of AF-2364 can be reduced by 2,000-fold by developing a novel delivery vehicle so that it can be targeted specifically to the testes, this may resolve the mild toxicity issue associated with AF-2364. Interestingly, it has been shown that less than 0.035% of the orally administered AF-2364 can indeed reach the testes and as little as 10 ?g, if targeted entirely to the testes per adult rat at -300 mg body weight, is sufficient to induce transient infertility in treated rats. If AF-2364 can be conjugated to a FSH mutant recombinant protein, which lacks the hormonal activity while maintaining its receptor binding activity (note: FSH receptors reside exclusively on Sertoli cells in mammalian testes), it can become a novel delivery vehicle for AF-2364. Preliminary studies have unequivocally shown that this approach is feasible since using an AF-2364-FSH mutant conjugate, we required less than 1/2,000 of the original effective dose to induce germ cell depletion from the seminiferous epithelium of the rat testes, inducing aspermatogenesis in the treated rats. In this proposal, the PL will prepare a second-generation FSH mutant construct with appropriate mutations and deletions on the a and p subunits, by site-directed mutagenesis and deletion, using polymerase chain reactions. This will be used as a drug carrier by conjugating a single AF-2364 per ? subunit at the serine (Ser) residue at its N-terminus without altering the conformation of the recombinant FSH protein. This conjugate will be tested for its ability to bind to FSH receptors in Sertoli cell membranes and to induce intracellular cAMP levels in Sertoli cells in vitro to assess its hormonal activity. The efficacy of this AF-2364-FSH mutant conjugate will be tested in vivo, monitoring its effects on cell adhesion function by immunoblotting, fluorescent microscopy, electron microscopy and an in vitro cell adhesion assay. These studies will develop not only a vehicle to deliver AF-2364 to the testes, but one that can be used to target therapeutic agent(s) to the testes for treatment of diseases. [unreadable] [unreadable]