Ultrastructural, fertility, and spermicidal studies with isomers and derivatives of gossypol in male hamsters.

The effects of fourteen new, orally administered synthetic analogs of gossypol on testicular ultrastructure and fertility in hamsters and the spermicidal properties of these compounds, as well as of the optical isomers of gossypol against hamster and human sperm in vitro, are reported in this study. Test compounds were administered to adult male hamsters by daily gavage for 9 weeks at doses ranging from 15 to 50 mg/kg. The results of this study have demonstrated that the fourteen new gossypol analogs evaluated herein are not effective as male antifertility agents and their in vitro activity or lack of activity as spermicides is unrelated to their in vivo contraceptive potential. In addition, the results of the study suggest that (1) the isopropyl moiety of the gossypol molecule, like the aldehyde group, is essential for its mechanism of action and (2) the pathognomonic defect in the mitochondrial sheath induced by gossypol appears to be related to its unique activity as a male antifertility agent. The significance of these findings is discussed.

Comparative anti-fertility effects of gossypol enantiomers in male hamsters.

The enantiomers of gossypol have been tested as male oral anti-fertility agents in hamsters. As determined by fertility tests the (-) isomer was fully active at half the effective dose of the racemate, whereas the (+) isomer exerted no anti-fertility effect.

Development and use of a radioimmunoassay for D-(-)-norgestrel 17 beta-cyclopentanecarboxylate.

The synthesis of the 6 alpha-carboxymethylmercapto BSA and homologous histamine conjugate of D-(-)-norgestrel 17 beta-cyclopentanecarboxylate is reported. Using the BSA conjugate as an immunogen for the development of antibody in the rabbit and the 125I-histamine conjugate as the radioligand, a radioimmunoassay (RIA) for the ester was developed. Serum profiles of the free alcohol and ester were determined following IV or IM injection in macaques. Peak values for the ester (about 12 ng/mL) were observed 2 min following an IV bolus of 0.5 mg in one rhesus monkey. Blood levels dropped rapidly within the first 30 min and were barely detectable at 24 h. Serum levels of the free alcohol rose to a peak at 30 min and then declined slowly to very low values by 24 h. Following IM injection of 20 mg in cynomolgus monkeys, peak levels of the ester were observed within a few days while the free alcohol reached a maximum about day 30. Serum concentrations of D-(-)-norgestrel had fallen to about 0.4 ng/mL 160 days post-injection when levels of the ester fell below 0.2 ng/mL.

(-)-Gossypol: an active male antifertility agent.

The enantiomers of gossypol have been resolved by preparative HPLC of diastereomeric Schiff's base derivatives on a chiral bonded phase. Whereas (+)-gossypol has previously been reported to be inactive, (-)-gossypol is now shown to be active as a male oral antifertility agent in hamsters.

PIP: This paper describes the successful separation of (-)-gossypol from the racemate and demonstrates that in hamsters, it is indeed the component possessing male oral antifertility activity. Young, adult male hamsters were randomly divided into 4 groups of 5, and were treated with the agent orally, once a day, for 40 days. Each male was cohabited with a new set of 2 adult, virgin females for 1 week and the females, in turn, were checked daily for the presence of sperm in the vagina. After 37 days of treatment with (-)-gossypol, only 2 out of 5 males were fertile, and a further loss of fertility was apparent during the next cohabitation period. Results show that gossypol-acetic acid is more effective in reducing fertility at a dose level of 16 mg/kg/day than either of the other drugs tested. However, it is also shown to be more toxic as expressed by reduced body weight gain of the hamsters tested. Overall, based on the limited dosing schedule employed, the minus isomer appears about twice as potent as the racemic mixture.

Biopharmaceutic stability of [D-Trp6, des-Gly10]-LHRH ethyl amide in corn oil.

When sperm-positive female rats were treated with freshly prepared formulations of [D-Trp6, des-Gly10]-LHRH ethyl amide (LRA) in corn oil, the effects on pregnancy occurred in a dose-related manner. When the same formulation was retested after five-and-a-half years of storage under refrigeration, the results were essentially identical. Therefore, the biopharmaceutic stability of corn oil formulations of LRA appears to be 100% following five-and-a-half years of storage at 4 degrees C.

Evaluation of the Subhuman Primate Pregnancy Test Kit for the detection of early pregnancy in the rhesus monkey (Macaca mulatta).

The performance of The Subhuman Primate Pregnancy Test Kit was evaluated for routine detection of early (days 19-21) pregnancy in the rhesus monkey. Out of 123 confirmed matings, 19 resulted in pregnancy. In the pregnant animals the kit had an accuracy of 73.7%. In the nonpregnant females the accuracy was higher, 88.5%. False positives were encountered in ovariectomized females as well as adult intact males.

Effect of dose on the pharmacokinetics of levonorgestrel in the rat during the rapid elimination phase following subcutaneous administration.

Adult female rats were given a single, subcutaneous injection of 8, 16 or 32 micrograms of levonorgestrel (LNG). Blood samples were collected at various time intervals and serum concentrations of LNG were determined by radioimmunoassay. The patterns of temporal decline in LNG concentrations in the three dose groups indicated that the pharmacokinetics of LNG during the post-absorptive, rapid-elimination (beta) phase in the rat may be dose-dependent. Half-life, Co and AUC increased with the dose and -beta decreased as the dose increased. Mathematical relationships have been presented which can be used to predict the four parameters as well as concentrations of LNG at any given time after subcutaneous administration during the beta-phase for a given dose in the range of 8-32 micrograms. Significance of dose-dependent pharmacokinetic studies is discussed.

Pharmacokinetics of levonorgestrel in the rat.

Levonorgestrel was administered intravenously as a bolus to adult female rats and blood samples were collected at various time intervals. Serum concentrations of levonorgestrel were measured by radioimmunoassay. Analysis of data for two- and three-compartment open models indicated that in the rat, as in the human and the rabbit, a tri-exponential equation provided a better fit of the data. The half-lives for the alpha, beta and gamma phases were 10.1 min, 42.7 min and 23.1 hours, respectively. These values were closer to those reported for women than were the half-lives reported for the rabbit. The alpha and the beta phases appeared to last for 51 min and 1.3 hours, respectively, and the gamma phase was longer than 45 hours.

Long-acting contraceptive agents: structure activity relationships in a series of norethisterone and levonorgestrel esters.

A large number of esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) and levonorgestrel (D-(-)-13 beta-ethyl-17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one) were synthesized and tested for biological activity. The test employed in these studies was the duration of estrus suppression in cycling mature rats. In the norethisterone series several esters exhibited duration of activity comparable to that of norethisterone enanthate. In the levonorgestrel series the butanoic, cyclobutylcarboxylic and cyclopropylcarboxylic esters were longer acting than medroxyprogesterone acetate (17 alpha-acetoxy-6 alpha-methylpregn-4-ene-3,20-dione) when prepared as aqueous microcrystalline suspensions.

PIP: A large number of esters of norethisterone (17alpha-ethynyl-17beta-hydroxyestr-4-en-3-one) and levonorgestrel (D-(-)-13beta-ethyl-17alpha-ethynyl-17beta-hydroxygon-4-en-3-one) were synthesized and tested for biological activity. The test employed in these studies was the duration of estrus suppression in cycling in mature rats. In the norethisterone series, several esters exhibited duration of activity comparable to that of norethisterone enanthate. In the levonorgestrel series, the butanoic, cyclobutylcarboxylic, and cyclopropylcarboxylic esters were longer acting than medroxyprogesterone acetate (17alpha-acetoxy-6alpha-methylpregn-4-ene-3,20-dione) when prepared as aqueous microcrystalline suspensions.

Concentrations of oestradiol-17beta and progesterone in the peripheral plasma of the cynomolgus monkey (Macaca fascicularis) in relation to the length of the menstrual cycle and its component phases.

Ninety-three female cynomolgus monkeys were monitored throughout 647 menstrual cycles; 93.2% of these cycles were 22--37 days long and were normally distributed, with a mean of 29.2 days. Menstrual bleeding generally lasted for 3--5 days and was not related to the length of the cycle. The levels of progesterone and oestradiol-17beta in the plasma were measured during the menstrual cycle in 30 monkeys. The concentration of oestradiol-17beta reached a mid-cycle peak on day 11 or 12 of the cycle. The interval between the beginning of the cycle and the oestrogen peak was constant; the interval between the oestrogen peak and the end of the cycle increased or decreased with the cycle length. The level of progesterone began to rise at about the time of the oestrogen peak and remained raised for longer periods as the length of the cycle increased. The length of the menstrual cycle, therefore, appeared to be determined by the duration of the increase in the level of progesterone or by the life of the corpus luteum in the luteal phase. The relationship between the lengths of the cycle and the luteal phase can be defined by the expression: cycle length = 12.6 + 0.96 X length of luteal phase (correlation coefficient = 0.875).

Prostaglandins E and F in uterine venous plasma in relation to peripheral plasma levels of progesterone and 20alpha-hydroxyprogesterone in the rat throughout pregnancy and parturition.

Prostaglandins E and F in uterine venous plasma and progesterone (P) and 20alpha-hydroxyprogesterone (20alpha-OH-P) in peripheral plasma were measured by radioimmunoassays throughout pregnancy and parturition in the rat. E Prostaglandins are low (approx. 2 ng/ml) and maintain a more or less constant level throughout most of the pregnancy except just before parturition when they rise to 3.8 ng/ml on day 20. F Prostaglandin levels are always higher than E prostaglandins and show distinct peaks around day 5 (5 ng/ml), day 11 (7 ng/ml), and before parturition (8.4 ng/ml). Progesterone levels are higher than 20alpha-OH-P levels throughout most of the pregnancy (day 6-20); however, during early pregnancy (day 1-5) and before parturition more 20alpha-OH-P than P is present in peripheral blood. The possible role of uterine venous prostaglandin levels in altering the 20alpha-OH-P/P ratio during pregnancy and parturition is discussed.