A potent, nonpeptidyl 1H-quinolone antagonist for the gonadotropin-releasing hormone receptor.

Extensive development of the structure-activity relationships of a screening lead determined three important pharmacophores for gonadotropin-releasing hormone (GnRH) receptor antagonist activity. Incorporation of the 3,4,5-trimethylphenyl group at the 3-position, 2-(2(S)-azetidinyl)ethoxy group at the 4-position, and N-4-pyrimidinylcarboxamide at the 6-position of the quinolone core resulted in the identification of 4-(2-(azetidin-2(S)-yl)ethoxy)-7-chloro-2-oxo-3-(3,4,5-trimethylphenyl)-1,2-dihydroquinoline-6-carboxylic acid pyrimidin-4-ylamide (1) as a potent antagonist of the GnRH receptor. A 10(4)-fold increase in in vitro binding affinity is observed for the GnRH receptor as compared to the initial screening lead. Compound 1 exhibits nanomolar binding activity and functional antagonism at the human receptor and is 7-fold less active at the rhesus receptor. Intravenous administration of compound 1 to rhesus monkeys results in a significant decrease of the serum levels of downstream hormones, luteinizing hormone (79% decrease in area under the curve) and testosterone (92% decrease in area under the curve), at a dose of 3 mg/kg. Quinolone 1 is a potent nonpeptidyl antagonist for the human GnRH receptor that is efficacious for the suppression of luteinizing hormone and testosterone in primates.

Administration of a nonpeptidyl growth hormone secretagogue, L-163, 255, changes somatostatin pattern, but has no effect on patterns of growth hormone-releasing factor in the hypophyseal-portal circulation of the conscious pig.

The activity of the growth hormone secretagog, L-163,255, on growth hormone (GH), growth hormone-releasing factor (GRF), and somatostatin (SRIF) levels was evaluated in a porcine model of hypophyseal portal blood (HPB) collection. Young, castrated pigs had HPB and jugular blood collected for approximately 300 min. The blood collection was divided into discrete periods: baseline (BL) approximately 180 min; GH response period (RSP) approximately 90 min; and positive control period following a GRF bolus, 30 min. RSP was divided into a dominant response period (DOM) and a tail (TL). The spontaneous relationship between HPB GRF and SRIF and peripheral GH during BL has been reported (Proc Soc Exp Biol Med 217:188-196, 1998). The apex of the GH pulse resulting from L-163,255 administration was nonrandomly associated (P < 0.05) with descending periods of SRIF troughs. Frequency and amplitude of GRF and SRIF pulses, and frequency and depth of SRIF troughs were not different between BL and the beginning of DOM (the 20-30 min of GH increase). GH AUC was significantly greater (P < 0.05) for DOM compared to BL and TL, and for TL compared to BL. GRF AUC tended to be greater (P < 0.1) for RSP compared to BL, but the majority of the increase was in the TL period. There were no significant differences in the SRIF AUCs between the sampling periods. Furthermore, in a separate experiment, fos activity (a marker of neuronal activation) in the hypothalamus of pigs was examined after either L-163,255 (1x or 4x), isotonic saline (control), or hypertonic saline (positive control) administration. There were no differences in fos activity in the GRF, SRIF, or CRH immunopositive neurons between L-163,255 treatment and control. The pituitaries of the L-163,255-treated pigs showed marked fos activation compared to the controls. In conclusion, L-163,255 in pigs has its primary effect at the level of the anterior pituitary.

Hypophyseal-portal concentrations of growth hormone-releasing factor and somatostatin in conscious pigs: relationship to production of spontaneous growth hormone pulses.

A method of collecting hypophyseal portal blood (HPB) in conscious pigs was used to show the relationship between GRF and somatostatin (SRIF) concentration and peripheral GH response. Six male castrate pigs (approximately 63 kg body weight) had HPB and jugular blood collected individually for an average of 175 min each. Twenty-seven spontaneous GH pulses were detected in the 1050 min of total HPB collection. Of the associations examined, the only significant finding was that GH pulse maxima occurred nonrandomly within periods of SRIF descent (63%; P = 0.005). Although 48% (13/27) of GH pulse maxima were associated with an ascent in portal GRF concentration, these associations were not determined to be nonrandom (P = 0.14). Only 7 of 27 (26%) GH pulse maxima were associated with an ascent in portal GRF concentration and a descent in SRIF concentration occurring simultaneously. A saline infusion given approximately 120 min after beginning blood collection resulted in an increase in SRIF pulse frequency and a decrease in GH-AUC and GRF-AUC. The cause of this saline effect is unknown, but it may have been related to acclimation of the pigs to the blood collection procedure. These data show the complexity of the relationship between SRIF and GRF concentrations and GH secretion and may indicate a close relationship with SRIF in GH pulse generation in the pig. In addition, these data support the hypothesis that, in the pig, mediation of GH release cannot be explained simply by antagonism between GRF and SRIF.

Transorbital approach to the porcine pituitary.

A transorbital approach to the pituitary gland is described in domestic swine weighing between 40 and 70 kg. A transpalpebral eye exenteration is performed and the optic canal is enlarged caudally, using a bone drill. An operating microscope is used to improve visualization of the surgical site as the pituitary stalk and anterior pituitary are exposed to the level of the optic chiasm. This approach exposes the pituitary sufficiently to perform either a hypophyseal stalk transection or a hypophysectomy or to implant cannulas for hypothalamic-hypophyseal portal blood sampling. This technique has been performed in more than 50 pigs without major complications. Postoperative recovery has been rapid and uneventful. The transorbital approach is a significant refinement of the frontal craniotomy and cerebral elevation technique previously described in the pig, and results in shortened surgery time, minimal brain manipulation, and greatly decreased morbidity.