INHIBITION OF PITUITARY GONADOTROPIN-SECRETION BY THE GONADOTROPIN-RELEASING-HORMONE ANTAGONIST ANTIDE .2. DEVELOPMENT OF AN INVITRO BIOASSAY FOR CHARACTERIZATION OF PHARMACOKINETICS AND PHARMACODYNAMICS OF ANTIDE IN CIRCULATION

Previous data from this laboratory revealed a rapid and unexpectedly long inhibition of pituitary gonadotropin secretion in ovariectomized monkeys after a single high dose injection of the GnRH antagonist antide. This extended action of antide may correlate with an extended presence of antide in the peripheral circulation. We have reported on use of a RRA for antide in serum; however, during such a prolonged presence in the body, the possibility of catabolic loss of biological activity remained to be analyzed. In the present study, we have developed an in vitro pituitary cell bioassay for antide to investigate the pharmacokinetics and possible mechanism(s) contributory to its long action. Dispersed anterior pituitary cells from adult female rats were plated (48 h; 5 x 10(5) cells/well), washed, and incubated with 0.024-6 ng antide for 24 h. Media were removed, and cells were washed twice and then incubated with GnRH (1 x 10(-8) M) plus antide standards or serum samples for 4 h. Before antide injection into long term ovaiectomized monkeys, peripheral GnRH antagonist levels were undetectable. One day after a single injection (3.0 mg/kg, sc, in 50% propylene glycol-water), the level of antide was 31 +/- 13 ng/ml (n = 3). Thereafter, antide levels declined slowly and were still detectable (> 1.4 ng/ml) in two of three monkeys 31 days after injection. After iv administration (3.0 mg/kg; n = 2), peripheral antide levels followed a similar pharmacokinetics profile and declined slowly. Detectable antide concentrations were still present 36 days after single iv injection in both monkeys. The circulating half-lives of antide were 1.7 and 14.5 days for the first and second phases, respectively. Peripheral LH levels were suppressed to the limits of detectability within 1 day and slowly recovered to pretreatment levels within 30 +/- 5 days after sc or iv antide treatment. The ratio of bioactive antide to antide levels measured by RRA was similar throughout the study (x = 1.24 +/- 0.09; range, 0.40-2.22), although there was a trend toward an increased B/R ratio at the end of the study. In summary, we have developed an in vitro bioassay using cultured rat pituitary cells to measure biologically active antide concentrations in peripheral circulation after sc and iv treatments The prolonged action of antide on pituitary gonadotropin secretion in vivo is apparently due to the continued presence of biologically active antide in circulation after a single injection. The sustained presence of antide in peripheral blood probably reflects its entry into circulation from a peripheral depot site(s), whether given sc or iv, as well as its long t1/2 for clearance. The pharmacokinetic curves for antide in serum were similar in bioassay and RRA during an extended but reversible interval of LH suppression.