GnRH antagonists: A new generation of long acting analogues incorporating p-ureido-phenylalanines at positions 5 and 6

A series of antagonists of gonadotropin-releasing hormone (GnRH) of the gen eral 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. Se lected analogues were tested in a reporter gene assay (IC50 and pA(2)) and an in vitro histamine release assay. P and Q contain urea/carbamoyl functio nalities designed to increase potential intra- and intermolecular hydrogen bonding opportunities for structural stabilization and peptide/receptor int eractions, respectively. These substitutions resulted in analogues with inc reased hydrophilicity and a lesser propensity to form gels in aqueous solut ion than azaline B [Ac-D2Nal-D4Cpa-n3Pal-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 (sc) administration. Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(L-hydroorot yl)-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-D 4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(Ac)-Leu-ILys-Pro-DAla-NH2] (6) when administe red subcutaneously in castrated male rats at a dose of 50 mug in 100 muL Of phosphate buffer No correlation was found between retention times on a C-1 8 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 (pA(2)) and duration of action. FE200486 was selected for precli nical studies, and some of its properties were compared to those of other c linical candidates. In the intact rat, ganirelix, abarelix, azaline B, and FE200486 inhibited plasma testosterone for 1, 1, 14, and 57 days, respectiv ely, at 2 mg/kg sc in 5% mannitol (injection volume = 20 muL). Based on the information that 31, 33, 35 and 37 were significantly shorter acting than acyline or azaline B after intravenous administration (100 mug/rat), we sur mised that the very long duration of action of the related FE200486 (for ex ample) was likely due to unique physicochemical properties such as solubili ty in aqueous milieu, comparatively low propensity to form gels, and abilit y to diffuse at high concentrations in a manner similar to that described f or slow release formulations of peptides. Indeed, in rats injected sc with FE200486 (2 mg/kg), plasmatic concentrations of FE200486 remained above 5 n g/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 w ith 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.