Throughout spermatogenesis, developing germ cells at different stages of their development must attach tothe seminiferous epithelium via specialized cell junctions at the Sertoli-germ cell interface. As such,disruption of germ cell adhesion, even transiently, can lead to germ cell loss from the epithelium, resulting ininfertility. Studies completed during the past grant period have shown that Adjudin [formerly called AF-2364, 1-(2,4-dichlorobenzyl)-7H-indazole-3-carbohydrazide] is a promising candidate for male contraceptionsince it effectively depletes germ cells, particularly elongating/elongate spermatids, round spermatids, andspermatocytes, but not spermatogonia, from the epithelium in adult rats. More important, studies performedby licensed toxicologists according to FDA guidelines to assess the acute toxicity, mutagenicity, andgenotoxicity of Adjudin have shown that it is safe for its further development. In a subsequent subchronictoxicity study, however, it was shown that Adjudin has a narrow margin between its safety and efficacy. Tocircumvent this issue, Adjudin was conjugated to an FSH mutant in which the intrinsic hormonal activity ofthe mutant was stripped without compromising its FSH receptor binding activity. Most importantly, itsefficacy was significantly improved. The P.I. has now proposed studies to develop techniques for GMPproduction of this Adjudin-FSH mutant conjugate in collaboration with an industrial partner, and to developalternative administrative routes, such as a gel patch or nasal spray for its absorption instead of parentaladministration, using technologies established in the field and at the Population Council. Once the efficacyand bioavailability of the conjugate are established, its safety issue will be carefully evaluated by subchronictoxicity studies in rats and dogs to assess the margin between its safety and efficacy. Furthermore,contemporary techniques of biochemistry, molecular biology and cell biology will be used to continue theongoing research in this laboratory to probe the molecular mechanism(s) of action of Adjudin including itscellular effects on Sertoli and germ cells in the seminiferous epithelium. We will also identify the cellulartarget(s) of Adjudin in the testis, including mapping the phosphorylation site(s) in integrin, since its activationlikely triggers the Adjudin-induced germ cell loss from the testis. In short, this proposal will continue theproductive research in the P.l.'s laboratory, which will lead to a Phase 1 clinical study of Adjudin.
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