Rosemarie B. Thau

[Ac-D-NAL(2)1,4FD-Phe2,D-Trp3,D-Arg6]-LHRH, a potent antagonist of LHRH, produces transient edema and behavioral changes in rats

Acute toxicity studies of [Ac-D-NAL(2)1,4FD-Phe2,D-Trp3,D-Arg6]-LHRH (LHRH-A), a potent antagonist of LHRH were performed. Subcutaneous administration of this peptide to rats induced transient edema of the face and extremities. This effect was maximal 3-5 h after peptide administration and subsided by 24 h. These effects were not seen with an LHRH agonist or two other antagonists. This side effects of LHRH-A was peculiar to rats and not observed in mice, rabbits and rhesus monkeys. Intravenous administration led within minutes to depression of spontaneous activity in rats and monkeys. We conclude that some LHRH antagonists produce species specific effects on vascular permeability and spontaneous activity.

Antagonists of luteinizing hormone releasing hormone bind to rat mast cells and induce histamine release

It was reported previously that administration of certain synthetic antagonists of LHRH to rats produced allergy-like symptoms that were attributed to their histamine releasing action. In the present study the interaction of LHRH analogs with rat peritoneal mast cells was investigatedin vitro. Potent antagonists of LHRH showed strongin vitro histamine releasing activity from rat peritoneal mast cells. Membrane preparations of rat pituitary glands showed specific binding of radioiodinated LHRH antagonist as well as LHRH agonist. However, rat peritoneal mast cells and membrane preparations from those cells bound antagonist but not the agonist. Furthermore, the LHRH antagonist did not bind to membranes prepared from tissues such as prostate, liver, kidney, and brain. Competitive displacement curves of the [125I]-antagonist with different LHRH analogs showed that the ability of the analogs to compete for binding sites on mast cells was related to their histamine releasing activity. We conclude that histamine release from rat mast cells induced by LHRH analogs is mediated by specific binding of the active peptides to cell membranes. Furthermore, using rat mast cells, the binding assay in conjunction with histamine releasing assay may be utilized to predict thein vivo histamine releasing potential of new LHRH peptides which are of clinical importance.

Active Immunization Against LHRH: I. Effects of Conjugation Site and Dose

ABSTRACT: Active immunization against LHRH is a promising method of contraception for men. In order to be acceptable, sufficient amounts of anti‐LHRH antibodies must be induced rapidly after vaccination. In previously reported animal studies, we found that it took considerable time (up to 5 months) to obtain antibody titers (AT) that were sufficiently high for complete suppression of spermatogenesis. The possibility of accelerating the immune response to LHRH by increasing the dose of immunogen was investigated in the male rat. Six doses of LHRH conjugated to tetanus toxoid (TT) in the 10 position (LHRH10‐TT), ranging from 2.5 to 612 μg, and three doses of LHRH1‐TT (50 to 612 μg) were tested. The magnitude of the immune response did not depend on the dose of the antigen, provided a threshold dose had been surpassed. Antigenicity of LHRH conjugated to TT at either the 1‐, 6‐, or 10‐position was compared in rats and rabbits. In both species LHRH1‐TT induced sufficient antibody concentrations to suppress pituitary gonadotropins (LH and FSH) and, subsequently, serum testosterone (T) levels faster than either the 6‐ or 10‐conjugates. Only materials permitted for use in humans were utilized in these experiments. Conclusion: Active immunization against LHRH conjugated to TT at the 1‐position has potential as a fast, convenient method of male contraception.

Effects of long-term immunization against LHRH and androgen treatment on gonadal function

The possibility of immunological suppression of spermatogenesis while normal libido is maintained by exogenous androgen supplementation was tested in male rats. Neither short- nor long-term treatment with androgen (testosterone-17-trans-4-N-butyl-cyclohexane carboxylate) alone influenced fertility. Active immunization against LHRH administered simultaneously with exogenous androgen supplement caused infertility in 100% of the tested animals, all of which displayed normal sexual behavior. The atrophy of the testes and accessory sex organs was reversible.

Radioimmunoassay of 7α-methyl-19-nortestosterone and investigation of its pharmacokinetics in animals

A method for the measurement of 7 alpha-methyl-19-nortestosterone (7MENT) in serum/plasma by radioimmunoassay (RIA) is described. The antiserum, raised against 7 alpha-methyl-19-nortestosterone-3-O-oxime-bovine serum albumin, had a low titer (final dilution = 1:4500) and low affinity (Ka = 1.17 x 10(9) l/mol) but showed little or no cross-reactivity with several of the steroids tested. The sensitivity of the RIA was 28.2 pg/ml and the mean recovery of added cold steroid was 86 to 100%. Intra- and inter-assay coefficients of variation ranged from 4.3 to 7.3% and 7.3 to 8.4%, respectively. This RIA was used to follow plasma 7MENT levels after a single i.v. injection of the steroid in rats and rabbits. The metabolic clearance rates (MCR) of 7MENT as determined from the plasma disappearance curve for rats and rabbits were 50 l/day and 336 l/day, respectively. The MCR of 7MENT in rats and rabbits lies in the same range as for testosterone. When compared to other nortestosterone derivatives such as norethisterone, 7MENT is metabolized relatively faster.

Species differences in the sensitivity to GnRH analogs

The effects of several GnRH agonists and antagonists with high biological activity, have been investigated in rats, mice, rabbits and monkeys. Striking differences exist in the response of different species to the antigonadal and antipituitary effects of these peptides. Of all the animals studied, the rat is the most sensitive. The magnitude of the response to GnRH agonists seems to depend on the sensitivity of the pituitary and the presence of GnRH receptors in the target organs. Findings from animal models require careful interpretation before predictions can be made regarding their possible effects in the human.

Influence of a levonorgestrel-containing contraceptive vaginal ring on plasma lipids and lipoproteins in cynomolgus monkeys

The effect of a contraceptive vaginal ring (CVR) containing levonorgestrel on plasma lipid and lipoprotein concentrations and characteristics was assessed in ten cynomolgus monkeys. The animals were fed a diet similar to the average American diet in fat (40% of calories) and cholesterol (0.2 mg/kcal) content. The objective of this study was to determine if changes in lipids and lipoproteins caused by progestogen administration parallel those seen in human females. A parallel pattern would recommend the cynomolgus monkey as a model for studying the effects of progestogens on the atherosclerotic process. Treatment with the CVR resulted in significant decreases in total plasma, VLDL + ILDL + LDL, and HDL cholesterol concentrations and a decrease in the percentage of HDL2 in total HDL. Plasma triglyceride concentrations were low throughout the study and consistent effects of the CVR were not seen. CVR treatment resulted in increases in TPC:HDL-C ratios and in the flotation rate of the LDL particle. The patterns of effects on HDL cholesterol, total plasma cholesterol, and HDL2 concentrations were similar to the progestogen-induced changes observed in human plasma lipids and lipoproteins. Based on these effects, the cynomolgus monkey appears to be a suitable model for the study of progestogen-induced changes in plasma lipids and lipoproteins and their consequent influences on coronary artery atherosclerosis.

Luteinizing hormone-releasing hormone (LHRH) and its analogs for contraception in women: a review.

In animals, LHRH agonists have multiple sites of action including the pituitary, the gonads, and the reproductive tract. In humans, the major antifertility action of this class of peptides is believed to be mediated via the pituitary. Studies in women have indicated that potent LHRH agonists can block ovulation when administered once daily. In the volunteers who have used these agents no serious side effects were observed, although some women experienced irregular bleeding or amenorrhea. It is anticipated that formal clinical trials could be conducted in the near future to determine the efficacy of continuous LHRH agonist administration. Early attempts to use an LHRH agonist to produce luteal insufficiency, luteolysis, or interruption of pregnancy have either been unsuccessful or the results are still too preliminary to ascertain whether these approaches warrant further trials. LHRH antagonists are believed to act by inhibiting the action of LHRH on the pituitary. Although some of these peptides are known to be active in women, very large doses have been required. Recently several investigators have produced LHRH antagonists with increased potency. In the near future, it should be possible to determine whether these peptides should be considered as potential contraceptives in men or in women.

Effects of Long‐Term Immunization Against the Beta‐Subunit of Ovine Luteinizing Hormone on Circulating Immune Complex Formation and on Arterial Changes in Rhesus Monkeys*

ABSTRACT: A major safety issue of contraceptive methods based on long‐term immunization is the possible effect of circulating immune complexes (CIC) on the arterial wall. We have measured CICs in 24 monkeys, immunized against the β‐subunit of ovine luteinizing hormone (oLHβ), emulsified with Freunds complete adjuvant, and in 7 nonimmunized controls by Raji assay, Clq assay, and an assay for rheumatoid factor. Eleven of the 24 immunized monkeys had CIC concentrations that were more than two standard deviations above the mean for controls in at least one of the assays. There was no correlation between antibody titer and CIC. Nine immunized and eight control animals on lowfat diets were killed to evaluate the effects of immunization on the artery wall. The cross‐sectional intimal area was measured at several sites from projected microscopic images using a sonic digitizer. No statistically significant differences between test and control groups were found. However, when we compared the upper half of the distribution of test and control animals, we found that the mean intimal area of the thoracic aorta of immunized monkeys was twice that of controls and that that of the abdominal aorta was three times as large. These data indicate that long‐term immunization against oLHβ induces CICs in rhesus monkeys. Small increases in the intimal area were found in about half of the immunized animals. The results of this study suggest the need for a larger, more definitive study in which the diet is manipulated so that plasma lipids mimic those of human females in Western society.

Impaired steroidogenesis in the luteal phase of the reproductive cycle and during pregnancy in rhesus monkeys immunized with the β-subunit of ovine luteinizing hormone

Monkeys immunized with the beta-subunit of ovine luteinizing hormone (oLH beta) develop antibodies which cross react with rhesus chorionic gonadotropin (rhCG) and luteinizing hormone (rhLH). Immunization causes shortened menstrual cycles and reduced fertility. Fertility can be restored by administration of medroxyprogesterone acetate (MPA) during the first 5 weeks of pregnancy. In the present study, we have measured the effects of circulating oLH beta-antibodies on peripheral estradiol, progesterone and 17 alpha OH-progesterone (17OH-P) concentrations throughout the menstrual cycle and during gestation in monkeys which became pregnant following MPA-treatment. Progesterone concentrations were markedly reduced during the luteal phase in cycling animals and the luteal phase of the cycle was significantly shorter as compared to non-immunized controls. Concentrations of estradiol and 17OH-P in the peripheral circulation were not affected by the oLH beta-antibodies. In immunized monkeys which became pregnant following MPA-treatment, progesterone and 17OH-P levels were consistently lower and estradiol concentrations were increased during the second and third trimesters. Our results show that circulating antibodies to oLH beta have multiple endocrinological effects. Corpus luteum function is impaired in cycling monkeys and during the early part of pregnancy. In addition, the pattern of steroid secretion remains abnormal in pregnant monkeys even after the luteal-placental shift.