GnRH Antagonists:  A New Generation of Long Acting Analogues Incorporatingp-Ureido-phenylalanines at Positions 5 and 6

Abstract

A series of antagonists of gonadotropin-releasing hormone (GnRH) of the general formula Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph/4Amf(P)-D4Aph/D4Amf(Q)-Leu-ILys-Pro-DAla-NH2 was synthesized, characterized, and screened for duration of inhibition of luteinizing hormone release in a castrated male rat assay. Selected analogues were tested in a reporter gene assay (IC50 and pA2) and an in vitro histamine release assay. P and Q contain urea/carbamoyl functionalities designed to increase potential intra- and intermolecular hydrogen bonding opportunities for structural stabilization and peptide/receptor interactions, respectively. These substitutions resulted in analogues with increased hydrophilicity and a lesser propensity to form gels in aqueous solution than azaline B [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Atz)-D4Aph(Atz)-Leu-ILys-Pro-DAla-NH2 with Atz = 3'-amino-1H-1',2',4'-triazol-5'-yl, 5], and in some cases they resulted in a significant increase in duration of action after subcutaneous (s.c.) administration. Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(L-hydroorotyl)-D4Aph(carbamoyl)-Leu-ILys-Pro-DAla-NH2 (acetate salt is FE200486) (31) and eight other congeners (20, 35, 37, 39, 41, 45-47) were identified that exhibited significantly longer duration of action than acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(Ac)-Leu-ILys-Pro-DAla-NH2] (6) when administered subcutaneously in castrated male rats at a dose of 50 microg in 100 microL of phosphate buffer. No correlation was found between retention times on a C18 reverse phase column using a triethylammonium phosphate buffer at pH 7.0 (a measure of hydrophilicity) or affinity in an in vitro human GnRH report gene assay (pA2) and duration of action. FE200486 was selected for preclinical studies, and some of its properties were compared to those of other clinical candidates. In the intact rat, ganirelix, abarelix, azaline B, and FE200486 inhibited plasma testosterone for 1, 1, 14, and 57 days, respectively, at 2 mg/kg s.c. in 5% mannitol (injection volume = 20 microL). Based on the information that 31, 33, 35 and 37 were significantly shorter acting than acyline or azaline B after intravenous administration (100 microg/rat), we surmised that the very long duration of action of the related FE200486 (for example) was likely due to unique physicochemical properties such as solubility in aqueous milieu, comparatively low propensity to form gels, and ability to diffuse at high concentrations in a manner similar to that described for slow release formulations of peptides. Indeed, in rats injected s.c. with FE200486 (2 mg/kg), plasmatic concentrations of FE200486 remained above 5 ng/mL until day 41, and the time after which they dropped below 3 ng/mL and plasma LH levels started rising until full recovery was reached at day 84 with levels of FE200486 hovering around 1 ng/mL. Additionally, FE200486 was less potent at releasing histamine from isolated rat mast cells than any of the GnRH antagonists presently described in preclinical reports.

Pharmacological Profile of a New, Potent, and Long-Acting Gonadotropin-Releasing Hormone Antagonist: Degarelix

“…Abarelix was found to decrease plasma testosterone in vivo with comparable potency to degarelix, consistent with their similar in vitro potency at the GnRH receptor (Jiang et al, 2001). When the durations of LH suppression induced by abarelix and degarelix were compared, degarelix, but not abarelix, exhibited a dose-dependent increase in duration of action.…”

“…We have developed a new series of potent and long-acting competitive antagonists for the GnRH receptor of the general formula, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph/Amf (P)-D-4Aph/-Amf (Q)-Leu-Ilys-Pro-D-Ala-NH 2 , showing high water solubility and low histamine-releasing properties (Jiang et al, 2001). The aim of the present study was to characterize the in vitro and in vivo pharmacological profile of a selected representative of this series, degarelix (FE200486: Ac-D-2Nal-D-4Cpa-D3Pal-Ser-4Aph (L-hydroorotyl)-D-4Aph (carbamoyl)-Leu-IlysPro-D-Ala-NH 2 ) in comparison with other recently developed GnRH antagonists such as abarelix (Cook and Sheridan, 2000), ganirelix (Gillies et al, 2000), cetrorelix (Reissmann et al, 2000), and azaline B (Rivier et al, 1995b).…”

Identification of new gonadotrophin-releasing hormone partial agonists

“…Recent substitutions attempting to improve the binding affinity of the partial agonists described here included modifications at positions 5 and 6 using some of the optimized substitutions used in the design of the latest generation of GnRH antagonists (Jiang et al 2001). Those analogues are all full antagonists.…”

In Vitro and In Vivo Human Metabolism of Degarelix, a Gonadotropin-Releasing Hormone Receptor Blocker

“…1) is a decapeptide that shows high affinity/ selectivity to human gonadotropin-releasing hormone receptors (Jiang et al, 2001). Degarelix binds to gonadotropin-releasing hormone receptors in the pituitary gland, which in turn, results in decreased secretion of luteinizing hormone and, consequently, decreased production of testosterone from the Leydig cells of the testes (Ortmann and Diedrich, 1999;Chengalvala et al, 2003).…”