Development of Superactive Analogs of FSH for Human Infertility

描述（由申请人提供）：大约10%的美国夫妇患有不孕症，这一领域的治疗药物市场非常大且增长迅速，特别是负责卵巢卵母细胞发育的主要激素，人类促卵泡激素（FSH）。虽然现有的尿用和重组的FSH产品已经相当成功，但目前对所有不孕妇女，特别是老年患者和对目前治疗反应相对迟钝的患者，改进的FSH类似物的治疗需求尚未得到满足。我们之前已经描述了第一个糖蛋白激素的超活性类似物，它大大增加了受体结合亲和力，以及体外和体内的生物效力和最大功效。在我们成功完成的2期SBIR研究中，我们已经达到或超过了所有目标，包括筛选额外的FSH类似物候选物，并确定用于临床试验的最佳FSH类似物TR 4401，使用多种体外和体内啮齿动物生物测定模型，包括经典的卵巢体重反应，以及卵母细胞，囊胚和胚胎移植产生的新生幼崽。在所有这些啮齿动物模型中，4401在与卵母细胞或胚胎质量相关的定量终点和定性终点的效力和功效方面都大大优于目前所有可用的重组卵泡刺激素制剂。此外，我们还利用模拟人类辅助生殖技术的方法，在两种人类不孕症模型、两种牛不孕症模型和恒河猴中，显示了TR4401对标准卵泡刺激素的卓越功效。我们还表明，在奶牛中注射一次TR4401可以产生与猪卵泡刺激素标准8次注射方案相当的超排卵，并且在重复给药后反应没有衰减。这些非人灵长类动物和牛的研究显示，TR4401在可能的最大剂量下没有卵巢过度刺激的证据，也没有任何免疫原性的证据，这是FDA对这种最小修改的，因此可能是安全的FSH类似物的唯一两个副作用。通过高效液相色谱验证的免疫分析，我们发现TR4401在啮齿类动物和猴子体内与标准FSH相比具有优越的药代动力学特性，这显然是延迟吸收的结果。我们还开发了稳定的中国仓鼠卵巢（CHO）细胞系，产生高水平的最终TR4401类似物；大型生物反应器生产方法的优化开发适合商业规模的新型、高容量净化方法；通过多种物理化学方法，包括碳水化合物分析，对纯化的类似物进行严格的定量和表征。在目前的申请中，根据FDA在我们与他们非常成功的Pre-IND会议上获得的具体指令，我们提出了FDA要求的所有步骤，包括详细甘特图上显示的具体时间表，以进一步开发这种新型FSH类似物。这些包括建立和表征主和工作CHO细胞库；另外生产两批TR4401（每批200毫克）：第一批在GLP条件下生产，第二批在GMP条件下生产；完成所有fda要求的疗效、特异性、稳定性、代谢、药代动力学、药效学和分析研究；所有fda要求的毒理学研究的表现，包括大鼠和家兔的两代生殖毒理学评估；提交IND以启动临床试验。我们已将TR4401授权给全球领先的兽医超排卵公司Bioniche， Inc.用于兽医用途，并可能选择共同开发用于人类的模拟物。

DESCRIPTION (provided by applicant): 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, human (h) follicle-stimulating hormone (FSH). Although available urinary and recombinant hFSH 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 successfully completed phase 2 SBIR study we have achieved or exceeded all the aims including screening additional FSH analog candidates and identifying the optimal FSH analog TR 4401 for clinical trials, using multiple in vitro and in vivo rodent bioassay models including the classic ovarian weight response as well as those of oocytes, blastocysts and newborn pups resulting from embryo transfer. In all these rodent models 4401 greatly outperformed all currently available recombinant FSH preparations both in potency and efficacy related to both quantitative endpoints as well as qualitative endpoints related to oocyte or embryo quality. In addition we have shown such superior efficacy of TR4401 to standard FSH in human cell lines with reduced FSH receptor expression representing two models of human infertility, in two bovine models of infertility and in Rhesus monkeys using methods emulating human assisted reproductive technology. We have also shown that one injection of TR4401 in cows could produce comparable superovulation to the standard 8-injection regimen of porcine FSH with no attenuation of response after repeated administration. These nonhuman primate and bovine studies showed no evidence of ovarian hyperstimulation by TR4401 at neither a presumably maximal dose, nor any evidence of immunogenicity, the only two side effects of concern to FDA for this minimally modified, and thus presumably safe, FSH analog. Using an HPLC-validated immunoassay we have discovered superior pharmacokinetic properties of TR4401 in comparison to standard FSH both in rodents and monkeys, apparently the result of delayed absorption. We have also achieved development of a stable Chinese Hamster Ovary (CHO) cell line producing high levels of the final TR4401 analog; optimization of large scale bioreactor production methods; development of novel, high capacity purification methods suitable for commercial scale-up; rigorous quantification and characterization of purified analogs by multiple physicochemical methods including carbohydrate analysis. In the current application, following specific directives from FDA obtained in our highly successful Pre-IND Meeting with them, we propose all steps, including specific timelines indicated on a detailed Gantt chart, required by FDA for further commercial development of this novel FSH analog. These include establishment and characterization of a master and working CHO cell bank; manufacturing of two additional large batches of TR4401 (>200 mg each): the first under GLP and the second under GMP compliant conditions; performance all FDA-required efficacy, specificity, stability, metabolic, pharmacokinetic, pharmacodynamic and analytic studies; performance of all FDA-required toxicology studies including two-generation reproductive toxicology assessment in rats and rabbits; and submission of IND to initiate clinical trials. We have licensed TR4401 to the worldwide leading veterinary superovulation company, Bioniche, Inc. for veterinary use, with a possible option to also co-develop the analog for human use.