SBIR-STTR Award

Our previous structure-function studies resulted in the development of the first analogs of glycoprotein hormones with major increases in receptor binding affinity and bioactivity ("super active analogs"). More recently, we have further optimized human luteinizing hormone (LH) and human follicle-stimulating hormone (FSH) analogs. However, in contrast to human thyroid-stimulating hormone (TSH) analogs, we have not yet studied bioactivity of these analogs in vivo. Such animal studies with therapeutically relevant end-points are critical to demonstrate potential therapeutic utility of newly developed LH and FSH analogs. Since the gonadotropin in vivo bioassays require higher quantities of hormones than that obtainable from transient transfection experiments we propose in Aim 1 to develop cell lines producing high levels of candidate super active analogs and produce quantities sufficient for initial animal studies. This Aim will consist of construction of dicistronic vectors with amplifiable markers and mutated subunit cDNAs, amplification process, production and concentration of unpurified analogs. In Aim 2 we propose to perform initial experiments using currently established rodent in vivo bioassays modified to study the effect of LH and FSH on oocyte production, ovarian weight and steroidogenesis. The Aim 2 will also include respective LH and FSH bioassays with four selected analogs, two unmodified hormones and two mock controls. The results of such in vivo bioassays will be essential in the final selection of candidate analogs for high-level production and purification, as well as subsequent phase 2 primate and toxicology studies.

Thesaurus Terms:
drug design /synthesis /production, follicle stimulating hormone, hormone analog, luteinizing hormone, protein purification bioassay CHO cell, laboratory rat, transfection /expression vector

Infertility affects about 10% of American couples, and there is a very large and rapidly growing market for therapeutics in this field, particularly the primary hormone responsible for ovarian oocyte development, follicle-stimulating hormone (FSH). Although available urinary and recombinant FSH products have been quite successful, there is currently an unmet therapeutic need for improved FSH analogs for all infertile women and particularly for older patients and those relatively unresponsive to current therapies. We have previously described the first superactive analogs of glycoprotein hormones that considerably increase receptor binding affinity as well as both in vitro and in vivo biopotency and maximal efficacy. During our Phase I support at Trophogen, Inc. we have identified a number of promising LH and FSH superactive analog candidates in several in vitro and in vivo models, including rodent oocyte quantity and quality. We now wish under Phase II support to focus our efforts on further in vivo testing of our lead hFSH analog candidate and two alternates versus standard recombinant FSH in various physiologically and therapeutically relevant rodent and nonhuman primate models. Specifically, in rodents we propose to employ the classic Steelman Pohley assay, various assays of in vitro fertilization and embryo development and, in selected cases, embryo transfer to surrogate females, assessment of pregnancy rates and the quantity and quality of newborn pups. Such studies will be performed in immature, mature and aging rodents as well as in those with models of genetic or chemically induced infertility. In nonhuman primates we propose to do a paired study of analog vs standard FSH using methods emulating human ART. We also propose to develop additional stable cell lines with the lead FSH analog candidates and optimize further fibrous bed bioreactor production as well as purification methods. In addition we plan to characterize selected purified analogs by multiple physicochemical methods relating to both protein and carbohydrate structure