Bifunctional gonadotropin-releasing hormone antagonist-progesterone analogs with increased efficacy and duration of action.
Bifunctional gonadotropin-releasing hormone antagonist-progesterone analogs with increased efficacy and duration of action.
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DOI：
10.1210/en.2004-1481
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发表时间：
2006
期刊：
影响因子：
4.8
通讯作者：
K. Ratcliffe;H. Fraser;R. Sellar;J. Rivier;R. Millar
中科院分区：
文献类型：
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作者：
K. Ratcliffe;H. Fraser;R. Sellar;J. Rivier;R. Millar
GnRH肽类似物广泛用于治疗各种临床病症。然而，它们具有较差的口服活性并且表现出快速代谢清除，因此需要注射和贮库制剂。由于类固醇激素与血浆蛋白结合，我们探索了将羟基化的孕酮与GnRH类似物结合以减少肽的代谢清除的可能性。通过半琥珀酸桥将[D-Lys 6]GnRH激动剂与α 11-羟基孕酮的α 11-羟基偶联可使家兔静脉注射后的血浆半衰期延长3.6倍，同时保持高结合亲和力，从而提供了概念证明。然后用通过C21-羟基与GnRH拮抗剂的6位（缀合物A和B）和7位（缀合物C和D）缀合的21-羟孕酮合成5种GnRH拮抗剂。在第五种化合物中，缺少前两个氨基酸的GnRH拮抗剂的NH 2末端通过C21-羟基与21-羟基孕酮缀合（缀合物E）。所有五种类似物均以相对高的亲和力（264-1020 nM）与豚鼠孕酮结合球蛋白结合。此外，所有五种缀合物在刺激T47 D乳腺癌细胞系中的孕酮反应元件驱动的氯霉素乙酰转移酶报告基因中保留了高的孕激素活性。通过D-赖氨酸6的ε-氨基官能团的缀合（缀合物A和B）产生对人GnRH受体具有高结合亲和力的化合物（15和7 nM），与未缀合的GnRH拮抗剂（4和26 nM）的结合亲和力相当。通过Lys 7的ε-氨基官能团（缀合物C和D）或N-末端截短的拮抗剂的NH 2末端（缀合物E）的缀合产生低结合亲和力的化合物。与未缀合的拮抗剂（1.3和122 nM）相比，缀合物A和B也表现出对表达人GnRH受体的COS-7细胞中磷酸肌醇产生的GnRH刺激的高功能性拮抗作用（2.6和16 nM）。根据它们差的受体结合亲和力，缀合物C、D和E具有差的功能性拮抗作用。在雌性绒猴中进行的初步剂量探索研究显示，0.25 mg剂量对孕酮有短暂抑制作用，0.5 mg和1.0 mg剂量对孕酮的抑制作用延长至12和17 d。在成年雄性绒猴中注射缀合物A（0.5mg sc）迅速抑制血浆睾酮水平，其保持抑制至少3天。与此相反，未结合的母体拮抗剂单独或与孕酮抑制睾酮仅为8小时至1天。研究结果表明，孕激素与GnRH拮抗剂的结合传递了血浆结合和孕激素特性，并增加了它们在体内的疗效和作用持续时间。这些新的GnRH拮抗剂显示出作为激素依赖性疾病的治疗剂和作为避孕药的前景。
GnRH peptide analogs are widely used to treat diverse clinical conditions. However, they have poor oral activity and exhibit rapid metabolic clearance, thus requiring injection and depot formulation. Because steroid hormones are bound to plasma proteins, we explored the possibility of conjugating hydroxylated progesterones to GnRH analogs to reduce metabolic clearance of the peptides. Conjugation of [D-Lys6]GnRH agonist to the alpha11-hydroxyl of alpha11-hydroxyl progesterone via a hemi-succinate bridge increased the plasma half-life after iv injection in rabbits by 3.6-fold while retaining high binding affinity, thus providing proof of concept. Five GnRH antagonists were then synthesized with 21-hydroxyprogesterone conjugated via C21-hydroxyl to positions six (conjugates A and B) and position seven (conjugates C and D) of GnRH antagonists. In the fifth compound the NH2 terminus of a GnRH antagonist lacking the first two amino acids was conjugated via the C21-hydroxyl to 21-hydroxyprogesterone (conjugate E). All five analogs bound to guinea pig progesterone binding globulin with relatively high affinities (264-1020 nM). Moreover, all five conjugates retained high progestogenic activity in stimulating a progesterone-response-element-driven chloramphenicol acetyltransferase reporter gene in the T47D breast cancer cell line. Conjugation via the epsilon-amino function of D-Lys6 (conjugates A and B) produced compounds with high binding affinity for the human GnRH receptor (15 and 7 nM) comparable to that of the unconjugated GnRH antagonists (4 and 26 nM). Conjugation via the epsilon-amino function of Lys7 (conjugates C and D) or the NH2 terminus of an N-terminally truncated antagonist (conjugate E) produced compounds of low binding affinity. Conjugates A and B also exhibited high functional antagonism of GnRH stimulation of inositol phosphate production in COS-7 cells expressing the human GnRH receptor (2.6 and 16 nM) compared with the unconjugated antagonists (1.3 and 122 nM). In accordance with their poor receptor binding affinity, conjugates C, D, and E had poor functional antagonism. Preliminary dose-finding studies in female marmosets showed transitory progesterone inhibition by 0.25 mg and prolonged suppression of 12 and 17 d by 0.5- and 1.0-mg doses. Injection of conjugate A in adult male marmosets (0.5 mg sc) rapidly suppressed plasma testosterone levels, which remained suppressed for at least 3 d. In contrast, the unconjugated parent antagonist alone or with progesterone suppressed testosterone for only 8 h to 1 d. The findings demonstrate that conjugation of progesterone to GnRH antagonists conveys plasma binding and progestogenic properties and increases their efficacy and duration of action in vivo. These new GnRH antagonists show promise as therapeutic agents for hormone-dependent diseases and as contraceptives.