Development of Superactive Analogs of FSH for Human Infertility

描述(申请人提供)：大约10%的美国夫妇受到不孕症的影响，这一领域的治疗药物有一个非常巨大且快速增长的市场，特别是负责卵巢卵母细胞发育的主要激素，人(H)卵泡刺激素(FSH)。尽管现有的尿液和重组hFSH产品已经相当成功，但目前对于所有不孕妇女，特别是老年患者和对当前治疗相对无效的患者，对改进的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超活性的糖蛋白激素类似物，它显著增加了受体结合亲和力，以及体外和体内的生物潜力和最大疗效。在我们成功完成第二阶段SBIR研究期间，我们已经达到或超过了所有目标，包括筛选额外的FSH类似物候选对象和确定用于临床试验的最佳FSH类似物TR4401，使用多种体外和体内啮齿动物生物测定模型，包括经典的卵巢重量反应以及来自胚胎移植的卵母细胞、囊胚和新生幼崽的反应。在所有这些啮齿动物模型中，4401在与数量终点和与卵母细胞或胚胎质量有关的质量终点方面的效力和效果都大大优于所有现有的重组FSH制剂。此外，我们还在人类细胞系中显示了TR4401优于标准FSH的疗效，FSH受体表达减少代表了两种人类不孕症模型，在两种牛不孕症模型中，以及在使用模仿人类辅助生殖技术的方法的恒河猴中。我们还表明，在奶牛体内注射TR4401可以产生与标准的猪FSH 8次注射方案相当的超数排卵，而且重复给药后反应不会减弱。这些非人灵长类动物和牛的研究表明，没有证据表明TR4401在推测的最大剂量下刺激卵巢过度刺激，也没有任何证据表明免疫原性，这是FDA担心的仅有的两个副作用，这种经过最小限度修改的FSH类似物因此可能是安全的。使用高效液相验证的免疫分析，我们发现TR4401在啮齿动物和猴子身上比标准FSH具有更好的药代动力学特性，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，生产高水平的最终TR4401类似物；优化了大型生物反应器的生产方法；开发了适合商业放大的新型、高容量的纯化方法；通过包括碳水化合物分析在内的多种物理化学方法对纯化的类似物进行了严格的定量和表征。在目前的申请中，根据FDA在我们与IND前非常成功的会议上获得的具体指示，我们建议FDA进一步商业化开发这种新型FSH类似物所需的所有步骤，包括详细甘特图上指示的具体时间表。这些措施包括建立和鉴定一个主要的和正在运行的CHO细胞库；再生产两个大批量的TR4401(&gt；200毫克)：第一个在GLP下，第二个在符合GMP的条件下；进行所有FDA要求的有效性、特异性、稳定性、代谢、药代动力学、药效学和分析研究；进行FDA要求的所有毒理学研究，包括对大鼠和兔的两代生殖毒理学评估；以及将IND提交启动临床试验。我们已将TR4401授权给全球领先的兽医超数排卵公司Bioniche，Inc.用于兽医，并可能选择共同开发供人类使用的类似物。

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.