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.

No abstract

Neuropeptide Y (NPY) is known to be involved in the central regulation of appetite, sexual behavior, and reproductive function. Whereas central administration of NPY strongly stimulates feeding in satiated animals, diet restriction or other unfavorable metabolic situations, such as diabetes, produce enhanced NPY gene expression and NPY release in the hypothalamus. Numerous studies have indicated that acute central administration of NPY results in various actions on LH secretion in the rat, either stimulatory or inhibitory. We recently demonstrated that chronic infusion of NPY into the lateral ventricle of adult intact female rats profoundly inhibited both the gonadotropic and somatotropic axes, with disruption of estrous cyclicity. Furthermore, we showed that central chronic infusion of NPY delayed sexual maturation in female rats. To analyze the effects of the same type of chronic NPY treatment on the pituitary-testicular axis, 45-day-old Sprague-Dawley male rats were implanted with stainless steel cannulas in the right lateral ventricle. Ten days later, Alzet osmotic minipumps were filled with different NPY solutions, adjusted to deliver 6, 18, or 36 micrograms/day, connected to the intracerebroventricular (icv) cannula, and sc implanted dorsally. The effects of these treatments were evaluated over 7 days. In one case, rats were castrated 5 days after initiation of NPY treatment, and the effect of castration was evaluated 2 days later. Chronic icv infusion of NPY produced the expected dose-related increases in food intake from 33.0 +/- 0.9 (basal) to 53.4 +/- 3.3 g/day (18 micrograms NPY/day) and body weight gain (5.7 +/- 0.7 to 10.5 +/- 1.2 d/day). As in female rats, this orexigenic action of NPY resulted in a significant dose-related decrease in pituitary weight, from 12.4 +/- 0.7 to 9.9 +/- 0.4 mg. The 7-day NPY infusion produced highly significant decreases in seminal vesicle weight (853 +/- 77 to 230 +/- 31 mg) and testis weight (3.82 +/- 0.09 to 3.18 +/- 0.15 g; P = 0.003). Plasma levels of testosterone (231 +/- 71 to 48 +/- 13 ng/dl), LH (20.7 +/- 3.7 to 9.1 +/- 1.2 ng/ml), and FSH (282 +/- 17 to 190 +/- 18 ng/ml) were markedly decreased at the 18 micrograms/day dosage, as also demonstrated for the 36 micrograms/day dosage. None of these effects was observed if vehicle was infused into the lateral ventricle instead of the NPY solution. When bilateral orchidectomy was performed 5 days after initiation of the NPY infusion (18 micrograms/day), the immediate LH and FSH rises usually seen after castration were seriously blunted.(ABSTRACT TRUNCATED AT 400 WORDS)

The ability of hCG and LH to induce testosterone (T) secretion by Leydig cells is well documented. However, the influence of the pulsatile nature of LH secretion, with varying frequency and amplitude, on T production in vivo is less clear. In our earlier studies on the relationship between pulsatile LH release and T secretion in adult male rats, no simple causality was observed. The recent availability of rat recombinant (rec) LH prompted us to study the effects of one and of three i.v. pulses of different doses of rat recLH on T secretion in adult male rats rendered hypogonadotropic by treatment with the GnRH antagonist cetrorelix. One or three supraphysiological pulses of 1.0 microg of rat recLH produced similar maximal T responses. In contrast, high physiological LH pulses (0.1 microg) produced discrete T-response peaks, whereas multiple low pulses of LH (0.03 microg) were needed before a T response was achieved. The T stimulation was greatly diminished after an LH pulse of 0.1 microg if rats had been treated on the previous day with pulses of 0.03 vs. 0.1 microg rat recLH, apparently because of prolonged LH deprivation and lack of Leydig cell priming due to the GnRH antagonist treatment. The novel preparation of rat recLH provides a physiologically relevant tool for studying the complex relationship between pulsatile LH release and T secretion in male rats.