John L. McGuire

Progestational and androgenic receptor binding affinities and in vivo activities of norgestimate and other progestins

The progestational and androgenic in vitro receptor binding affinity and the in vivo activity of norgestimate was compared with that of its metabolites and other progestins. The relative binding affinities (RBAs) of norgestimate and its 17-deacetylated metabolite for rabbit uterine progestin receptors were similar to that of progesterone (P); those of 3-keto norgestimate and levonorgestrel were about five times that of P; those of gestodene and 3-keto desogestrel were about nine times that of P. The RBAs of norgestimate, P, and 3-keto norgestimate for rat prostatic androgen receptors were from 0.003 to 0.025 times that of dihydrotestosterone (DHT); those of 3-keto desogestrel, gestodene, and levonorgestrel were from 0.118 to 0.220 times that of DHT. The order of receptor level selectivity represented by the ratio of androgen:progestin IC50 values (with a greater ratio value reflecting a better selectivity) was norgestimate greater than P = 3-keto norgestimate greater than 17-deacetylated norgestimate greater than 3-keto desogestrel greater than gestodene greater than levonorgestrel. In vivo studies demonstrated similar profiles for norgestimate and its 17-deacetylated metabolite. These latter two steroids were equally potent as progestins in stimulating rabbit endometrium, and compared with the other progestins, both steroids exhibited minimal androgenicity as measured by the stimulation of rat prostate growth. In conclusion, these studies, as well as previous preclinical and clinical studies, provide evidence of the selectivity of norgestimate based on minimal androgenicity, indicating an improvement over other progestins used in oral contraceptives.

A comparison of the potencies and activities of progestogens used in contraceptives

The potencies and activities of six progestational agents, norethindrone, levonorgestrel, desogestrel, medroxyprogesterone acetate (MPA), progesterone (P) and norgestimate have been evaluated using standard laboratory bioassays. The endocrine activities measured are those most closely related to the clinically important actions of contraceptives. Relative potencies varied with parameter measured, route of administration and species showing clearly that each progestogen is a distinct pharmacological entity. The order of potency using oral administration for either ovulation inhibition or endometrial stimulation in rabbits was desogestrel greater than levonorgestrel greater than MPA greater than norgestimate greater than norethindrone. Levonorgestrel was more androgenic than desogestrel, and P, norethindrone, norgestimate and MPA were essentially devoid of androgen activity. This profile demonstrates clear differences in the potencies and activities of these progestogens and in their selectivity for target organs.

Experimental evidence for failure to implant as a mechanism of infertility associated with endometriosis

The effect of endometriosis on pregnancy, from ovulation through day 14 of pregnancy, was studied in an animal model previously developed and validated with the use of the rabbit. Endometrial tissue was implanted surgically in rabbits and allowed to grow for 11 weeks without hormonal supplementation. The animals were artificially inseminated with semen from bucks with established fertility and human chorionic gonadotropin was administered to induce ovulation. The animals were put to death 1, 4, 8, or 14 days later. The number of corpora lutea and fertilized ova was not affected through day 4. However, on days 8 and 14, a significant reduction in the number of normal fetuses was observed. In a second experiment peritoneal fluid from animals with endometriosis was transferred to normal rabbits 1 day before artificial insemination. A significant reduction in the number of normal fetuses was observed. These studies suggest that failure of nidation due to the maternal environment may be a major factor in infertility associated with endometriosis.

Pharmacologic and pharmacokinetic characteristics of norgestimate and its metabolites

Biotransformation, pharmacologic, and pharmacokinetic studies of norgestimate and its metabolites indicate that 17-deacetyl norgestimate, along with the parent drug, contributes to the biologic response. The postulated metabolic pathway, which is based on the identification of urinary products had indicated that three metabolites of norgestimate, 17-deacetyl norgestimate, 3-keto norgestimate, and levonorgestrel, might participate in the response. The pharmacologic evaluation of these metabolites demonstrates that only 17-deacetyl norgestimate has a pharmacologic profile consistent with that of norgestimate, and significant concentrations of this metabolite have been measured in the serum of women after the administration of norgestimate. These studies indicate that 17-deacetyl norgestimate contributes to the pharmacologic response to norgestimate.

Smooth muscle contraction as a model to study the mediator role of endogenous lipoxygenase products of arachidonic acid

In the lung, the contraction of smooth muscle, or bronchospasm, is generally caused by an immunologic insult resulting in mast cell degranulation and the release of histamine, slow reacting substances, and other mediators of inflammation (1). Although the immediate response is bronchospasm, continued activation of this sequence of events results in a chronic inflammatory disease. In the uterus, numerous conditions can result in smooth muscle contraction. One major pathophysiological syndrome associated with increased uterine tone and severe rhythmic contraction is primary dysmenorrhea (2). In this disease state, prostaglandins have been shown to play a major role in these contractions (3,4), and inhibitors of cyclooxygenase have proven beneficial in clinical practice (5). Both dysmenorrhea and cervical ripening have been likened to inflammatory reactions due to varying degrees of vasodilation, invasion by inflammatory cells, proliferation of fibroblasts and smooth muscle contraction (6,7). Metabolism of arachidonic acid (AA) via cyclooxygenase to prostaglandins and thromboxanes and via lipoxygenase to hydroxyeicosatetraenoic acids (HETEs) and leukotrienes is an integral part of both the acute and chronic inflammatory reaction in the lung or uterus. The material reviewed here examines the effect of endogenous leukotrienes on both the lung and uterus and suggests that other smooth muscles and pathophysiological states may be more involved with the lipoxygenase pathway of AA metabolism than previously believed.

Preclinical evaluation of norgestimate, a progestin with minimal androgenic activity

Norgestimate is a novel progestin with highly selective progestational activity and minimal androgenicity. In rabbits, norgestimate binds to uterine progestin receptors, stimulates the endometrium, and inhibits ovulation. Norgestimate acts directly on target organs, stimulating rabbit endometrium when injected into the uterine cavity and inhibiting luteinizing hormone-releasing hormone-stimulated luteinizing hormone release in dispersed rat pituitary cells in culture. Norgestimate has no estrogenic activity, and like other progestins, it suppresses the action of estrogen. Unlike some other progestins, it is relatively free of androgenic activity. Norgestimate and its 17-deacetylated metabolite demonstrate very poor affinity for androgen receptors compared with levonorgestrel and gestodene and do not exhibit androgenic activity when measured as the stimulation of prostatic growth in immature rats. Norgestimates lack of affinity for human sex hormone-binding globulin is further evidence of its minimal androgenicity.

THE AFFINITY OF NORGESTIMATE FOR UTERINE PROGESTOGEN RECEPTORS AND ITS DIRECT ACTION ON THE UTERUS

Norgestimate is a new orally active progestational agent. Studies were conducted to demonstrate that norgestimate is active pharmacologically without requiring biotransformation to an active metabolite. In in vitro studies, norgestimate exhibited a relatively high affinity for the rabbit uterine progestogen receptor. To demonstrate that norgestimate was not being degraded to a biologically active entity, which was binding to receptor sites in the cytosol preparation, the stability of 14C-norgestimate in the preparation was determined. Following the incubation of 14C-norgestimate with the cytosol fraction used in the receptor assay, the labeled material was extracted and analyzed by reverse phase high performance liquid chromatography. 14C-Norgestimate was recovered from these incubation mixtures, confirming that norgestimate was available to bind to the progestogen receptor. In in vivo studies, norgestimate stimulated the endometrium in immature rabbits when applied directly to the uterus, again suggesting that bio-transformation to an active metabolite is not required for expression of norgestimates pharmacological activity. These in vitro and in vivo studies, when considered with previously reported studies, show that norgestimate is a unique progestogen.

Development of an animal model for quantitatively evaluating effects of drugs on endometriosis

The present study was conducted to induce endometriosis in an experimental animal model in which the condition and its response to pharmacologic agents could be quantified. Endometriosis was induced in New Zealand White rabbits by transplanting endometrial sections into various sites throughout the peritoneum. After 7 weeks, the mean implant weight increased in concomitant controls from 10.3 to 89.0 mg. In the next 8 weeks, endometrial implant weight increased to 163.6 mg. Daily subcutaneous administration of a luteinizing hormone-releasing hormone agonist, histrelin, or oral administration of danazol, reduced the ectopic implant weight within 8 weeks to 21.7 and 46.0 mg, respectively. In a group of animals that were bilaterally ovariectomized, implant weight decreased significantly in the same 8-week period to 22.4 mg. Furthermore, histologic analysis of the endometriomas showed that ovariectomy, histrelin, or danazol treatment reduced the number of endometrial glands and atrophied the stroma. We conclude that this animal model represents an excellent method for quantitative evaluation of potential therapeutic agents for endometriosis.

The pharmacological profile of norgestimate, a new orally active progestin

A pharmacological profile of a new progestin norgestimate (ORF 10131; d-13 beta-ethyl-17alpha-ethynyl-17 beta-acetoxy-gon-4-en-3-one oxime) based on dose-response assays is presented. The test systems included progestational response of immature female rabbit uteri pregnancy maintenance in ovariectomized animals progestin receptor binding antigonadotropic activity antiovulatory activity uterotropic (estrogenic) response vaginal cornification estrogen receptor binding androgen-anabolic activity and inhibition of vaginal cornification. Norgestimate is a moderately potent progestin as measured by the classic Clauberg Assay. Oral potency is about 1/5 of d-norgestrel. It binds to progesterone receptors in vitro and maintains pregnancy in ovariectomized rats. Norgestimate inhibits ovulation in rats with a potency of at least 800 times that of d-norgestrel. Androgenicity is slightly less than d-norgestrel. Norgestimate is nonestrogenic is nonuterotropic is unable to induce cornification of the vagina or bind estrogen receptors. Compared with d-norgestrel norgestimate has marked antiestrogenic activity and is a potent inhibitor of estrogen-induced vaginal cornification. Norgestimate is being studied clinically in combination with ethinyl estradiol as a new low-dose oral contraceptive.

Influence of ovarian steroids on prostaglandin- and leukotriene-induced uterine contractions

The mammalian uterus is capable of metabolizing arachidonic acid via the lipoxygenase pathway, and the uterus responds to lipoxygenase products. We postulated that progesterone influences the production of leukotrienes in the uterus in a way similar to that in which estradiol influences prostaglandin production. Uterine contractions were measured in actively sensitized guinea pigs throughout the estrous cycle and in ovariectomized, hormonally primed, sensitized guinea pigs. Antigen challenge stimulated uterine contractions (caused by prostaglandins) that increased throughout the estrous cycle to a maximum in day 15, when estradiol is at its peak. Pretreatment with indomethacin abolished uterine contractions except on day 9 of the cycle, when progesterone levels are at their highest. Day 9 contractions were blocked by FPL 55712, a selective receptor antagonist of leukotrienes. These findings were confirmed in ovariectomized/sensitized guinea pigs. Our data suggest that endogenous synthesis of leukotrienes in the uterus may be directly related to the rise of progesterone.