Intrauterine administration of CDB-2914 (Ulipristal) suppresses the endometrium of rhesus macaques.

BACKGROUND: Ulipristal (UPA; CDB-2914) is a progesterone receptor modulator with contraceptive potential. To test its effects when delivered by an intrauterine system (IUS), we prepared control and UPA-filled IUS and evaluated their effects in rhesus macaques. STUDY DESIGN: Short lengths of Silastic tubing either empty (n=3) or containing UPA (n=5) were inserted into the uteri of 8 ovariectomized macaques. Animals were cycled by sequential treatment with estradiol and progesterone. After 3.5 cycles, the uterus was removed. RESULTS: During treatment, animals with an empty IUS menstruated for a mean total of 11.66+/-0.88 days, while UPA-IUS treated animals bled for only 1+/-0.45 days. Indices of endometrial proliferation were significantly reduced by UPA-IUS treatment. The UPA exposed endometria were atrophied with some glandular cysts while the blank controls displayed a proliferative morphology without cysts. Androgen receptors were more intensely stained in the glands of the UPA-IUS treated endometria than in the blank-IUS treated controls. CONCLUSIONS: In rhesus macaques, a UPA-IUS induced endometrial atrophy and amenorrhea. The work provides proof of principle that an IUS can deliver effective intrauterine concentrations of Ulipristal.

An initial pharmacokinetic study with a Metered Dose Transdermal Systemfor delivery of the progestogen Nestorone as a possible future contraceptive.

BACKGROUND: Transdermal delivery of steroids is gaining popularity for contraception and hormone replacement therapy. This study aimed to test metered spray delivery of a precise dosage of Nestorone (NES) progestogen as a possible transdermal progestogen-only contraceptive. STUDY DESIGN: Six healthy postmenopausal volunteers, not recently using any hormonal therapies, comprise the sample for this study. Each subject was studied on two occasions with multiple blood sampling for assay of NES over a 24-h period: on the first occasion, after a single dosage of 3 x 90 microL NES sprays using a specially devised, precisely metered delivery device; on the second occasion, following the fifth in a series of five daily transdermal dosages of 3 x 90 microL of NES spray. Conventional pharmacokinetic parameters were calculated. NES was assayed in serum using a specific radioimmunoassay. RESULTS: Mean serum levels of NES peaked at around 20 h following dosing, and levels plateaued at 285-290 pmol/L after 4-5 days of daily spray application. All subjects achieved satisfactory serum levels, although substantial intersubject variation was noted. The apparent elimination half-life of NES after the last dose on Day 5 was 26.8 h. No unexpected adverse events were encountered. CONCLUSION: This early pharmacokinetic trial of a new transdermal steroid delivery system has demonstrated the feasibility of achieving serum levels of NES sufficient to block ovulation and potentially provide effective contraception.

Chronic toxicity and reversibility of antifertility effect of immunization against gonadotropin-releasing hormone in male rats and rabbits.

The chronic systemic toxicity of immunization with gonadotropin-releasing hormone, conjugated to tetanus toxoid (GnRH-TT), was investigated in male rats and rabbits in order to start Phase I clinical trials. Groups of rats and rabbits were immunized with GnRH-TT dissolved in aqueous adjuvant. The antigen was administered at weeks 0, 4, and 8, followed by boosters to maintain high antibody titers. At termination (8-9 months after first immunization), twenty rats and ten rabbits exhibiting the highest mean anti-GnRH titers and all the controls were selected for complete toxicological evaluation. In the rat study, a castrated control group was included for comparison with the immunized group. The hematological and serum chemistry parameters of immunized rats and rabbits were not affected in a significant manner. Most of the changes in serum chemistry of immunized rats were also found in castrated rats, indicating that the changes are most likely due to the withdrawal of androgenic support. The weights of the testes, epididymides, and sex accessory glands were lower in all immunized animals. There was significant atrophy of the germinal epithelium, which, however, sustained a population of Sertoli cells, spermatogonia, and pachytene spermatocytes. Other morphological changes in the prostate, seminal vesicles, pituitary, and mammary gland reflected the effect of androgen withdrawal. The decrease in the weight of liver, kidney, and heart seen in the immunized rats was also present in castrated rats and was not associated with any histopathological changes. The reversibility of immunization-induced infertility was investigated by mating the rats with normal females. Four months after the start of immunization, 9 out of 10 immunized rats were infertile whereas by nine months, all rats had regained fertility. Thus, it is concluded that immunization with GnRH-TT had no systemic toxicological effects in the adult male rats and rabbits for the period studied. The results also indicated that the GnRH-TT immunization had an antifertility effect in male rats. Fertility was restored following cessation of immunization and decline in anti-GnRH antibody titers.

Pharmacokinetics of 7 alpha-methyl-19-nortestosterone in men and cynomolgus monkeys.

Testosterone and its esters are widely used for androgen replacement therapy. In the prostate, testosterone ins 5 alpha-reduced to dihydrotestosterone (DHT), which leads to an amplification of its stimulatory activity in this and other tissues that have significant 5 alpha-reductase activity. While this amplification is essential during fetal development, it has potentially undesirable consequences during adult life. 7 alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that does not undergo 5 alpha reduction and is therefore being investigated for long-term clinical use because it is expected to be less stimulatory to the prostate. Since we anticipate using MENT acetate (MENT Ac) rather than MENT as the form of this androgen in humans, the bioavailability of MENT following the administration of MENT and MENT Ac was investigated in cynomolgus monkeys. Equimolar concentrations of MENT or MENT Ac were administered as a continuous subcutaneous infusion via Alzet osmotic pumps. Serum MENT levels were measured by radioimmunoassay (RIA) in blood samples collected daily for 4 days during steady state. The serum MENT levels were not significantly different in the two groups (11.3 +/- 1.6 vs. 13.1 +/- 1.2 nmol/L). This suggested that MENT Ac was rapidly converted to MENT in circulation. The hydrolysis of MENT Ac to MENT was confirmed by the in vitro incubation of MENT Ac with blood or plasma and the demonstration of MENT in products following separation by high-performance liquid chromatography (HPLC). Following the demonstration of the safety of MENT Ac in subchronic toxicity studies in rats and rabbits, a pharmacokinetic study was performed in men. In normal men, a single intravenous bolus of 500 micrograms of MENT led to peak serum MENT levels at 3 minutes after dosing (when the first samples were collected), followed by an exponential decline, reaching undetectable levels by 180 minutes. The average terminal half-life and the metabolic clearance rate (MCR) were calculated to be 40 minutes and 2,360 L/day, respectively. The results of the pharmacokinetic studies show that in both men and monkeys, the MCR of MENT is much faster than the values reported for testosterone. The faster MCR can be attributed, in part, to the finding that, in contrast to testosterone, MENT showed no binding to sex hormone binding globulin (SHBG).

Pharmacokinetics and pharmacodynamics of 7alpha-methyl-19-nortestosterone after intramuscular administration in healthy men.

7alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that is resistant to 5alpha-reductases and therefore less prone to over-stimulate the prostate. It is a good candidate for implant administration in long-term androgen replacement therapy for hypogonadal men or as part of a male contraceptive system. To investigate the pharmacokinetics of MENT after i.m. administration, single i.m. injections of 2, 4 or 8 mg of micronized MENT were given in aqueous suspension to 18 healthy men in two clinics. Blood was sampled frequently for 8 h and 1, 2, 3, 4 and 9 days after the injections. Serum MENT concentrations were determined by radioimmunoassay. Peak MENT concentrations were dose-dependent and were reached about 1-2 h after the injections. Doubling the dose of MENT resulted in an increase of 60% in peak serum MENT concentrations. The mean +/- SE clearance rate was 1790 +/- 140 l/day. The antigonadotrophic activity of MENT was investigated by giving six consecutive daily i.m. injections of 1, 2 or 4 mg of MENT to 24 healthy men in two clinics. Blood was sampled before each injection and up to 24 days after the last injection. Serum testosterone and gonadotrophin concentrations (determined by radioimmunoassay and fluoroimmunoassay respectively) decreased in a dose-dependent and statistically significant manner. The highest dose caused a 74% fall in testosterone, a 70% fall in luteinizing hormone, and a 57% fall in follicle stimulating hormone concentrations. MENT injections did not cause any side-effects. The results show that MENT is a potent antigonadotrophic agent in men.

Synthesis and progestational activity of 16-methylene-17 alpha-hydroxy-19-norpregn-4-ene-3,20-dione and its derivatives.

16-Methylene-17 alpha-hydroxy-19-norpregn-4-ene-3,20-dione 1 and its 17 alpha-acylated derivatives were synthesized. The length of the 17 alpha-side-chain ranges from C2-C6. As anticipated, compound 1 did not show any progestational activity or receptor binding activity; whereas, the acylated compounds, especially the butyrate, showed remarkable ability to bind to progesterone receptors. These compounds also showed progestational activity in an in vitro T47D cell culture assay in which progestins increase alkaline phosphatase activity and in an in vivo ovulation inhibition assay. All of the compounds synthesized were without estrogenic activities. The results showed that acylation of 16-methylene-17 alpha-hydroxy-19-norprogesterone can increase progestational activity. The progestational activities of these compounds varied with the 17 alpha-side chain.

Active immunization against LHRH alone or combined with LHRH-analogue treatment impedes growth of androgen-dependent prostatic carcinoma.

PROBLEM: To determine whether active immunization against LHRH can serve as treatment for androgen-dependent prostatic carcinoma. METHOD: Male rats of Copenhagen X Fisher strain, implanted with Dunning R-3327 prostatic carcinoma cells were either immunized against LHRH, treated with LHRH-antagonist, or received a combined treatment of active immunization against LHRH and LHRH-antagonist. RESULTS: Testicular histology was consistent with infertility in all treatment groups. The rate of tumor growth was inhibited by all three treatment regimens. Tumor size increased by 3.8 +/- 1.4 cm2 in the LHRH-antagonist group, 3.2 +/- 1.1 cm2 in the immunized group, and 1.0 +/- 0.4 cm2 in the combined treatment group, as compared to 8.2 +/- 2.6 cm2 in non-treated control group. CONCLUSION: LHRH-antagonist administration combined with immunization against LHRH appeared to exert a synergistic effect. This may be due to the blockade of prostatic LHRH-like receptors by the antagonist, while androgen depletion was rapidly achieved by LHRH-antagonist, and maintained by continued gonadotropin suppression caused by active immunization against LHRH once antagonist treatment had been discontinued.

Development of an antifertility vaccine for pets based on active immunization against luteinizing hormone-releasing hormone.

Male dogs and cats were immunized against LHRH in order to evaluate the feasibility of an immunological approach to pet contraception. In the first study, dogs were immunized with 100, 500, or 2500 micrograms of LHRH conjugated to tetanus toxoid. A significant decline in serum testosterone (T) levels was observed in all immunized dogs, reaching castration levels in some animals by Week 4 and remaining suppressed in all the immunized dogs through the course of the study. Testicular histology suggested arrest of spermatogenesis (infertility). The effects of "immunological castration" were reversible (study 2): steroidogenesis suppressed by "immunological castration" was restored as antibody titers declined. Effective antibodies were rapidly reinduced in dogs by a single injection of LHRH1-TT. In contrast, the level of antibodies induced in male cats (study 3) was not sufficient for "immunological castration." The conclusion was that active immunization against LHRH could provide a cost-effective, nonsurgical, reversible means to control the fertility of companion animals.

Effect of tarnish on copper release.

The rate of copper loss from bright and tarnished collars from Copper T Model TCu 380A IUDs has been investigated in amino acid solutions of pH 5.5 and 7.4 and in serum. In all three media, the tarnished collars quickly became bright and lost copper at the same rate as the initially bright collars. The single exception was when a high ratio of copper surface to serum was used. Under those conditions the tarnished collars initially became bright but after two days a black precipitate appeared on both the initially bright and tarnished collars and weight loss ceased. When a higher ratio of serum to copper surface was used, the pattern was one of continuing loss although at a lower rate than in the amino acid solutions. It is concluded that tarnish does not compromise the oxidation and dissolution of copper even in serum. Serum is considered a surrogate for uterine fluid.

Active immunization against LHRH: I. Effects of conjugation site and dose.

UNLABELLED: 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 micrograms, and three doses of LHRH1-TT (50 to 612 micrograms) 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.

Active immunization against gonadotropin-releasing hormone combined with androgen supplementation is a promising antifertility vaccine for males.

Male rats and rabbits were immunized against gonadotropin-releasing hormone (GnRH) conjugated to tetanus toxoid (GnRH10-TT) using only materials approved for humans. Testosterone (T)-releasing implants or the long-lasting T ester testosterone-17-trans-4-n-butyl-cyclohexane carboxylate (TE) was used as supplemental androgen for maintaining libido. Immunization against GnRH10-TT effectively suppressed fertility (spermatogenesis) in rats and rabbits. Neither T nor TE administration restored fertility. Both androgens were effective in maintaining normal libido in rats. TE, which is not hydrolyzed in rabbits, was less effective in maintaining normal ejaculatory behavior in this species. Active immunization against GnRH could be a convenient and cost-effective method of fertility control in males.

Formulation of a potential antipregnancy vaccine based on the beta-subunit of human chorionic gonadotropin (beta-hCG). IV. The role of adjuvants in booster injections.

Experiments were conducted in rabbits to determine the effect of adjuvant use on the antibody response following booster injections. The antigen used was in all cases the beta-subunit of human chorionic gonadotropin linked to tetanus toxoid (beta-hCG-TT). Adjuvants used were Al(OH)3, MDP analogs, and a streptococcus preparation, OK432. Primary vaccinations included Al(OH)3 adjuvant with or without supplementary adjuvants. In general, the greater the antibody response following primary vaccination, the greater the response following booster vaccination whether or not adjuvant was used in the booster. No increment in antibody titers was found by reason of including MDP analogs in booster vaccinations. OK432, in contrast, gave increased responses in booster injections which were in several cases statistically significant. The value of including Al(OH)3 in booster injections is not clear from the experimental data. In no case was the increment due to its inclusion large.

Formulation of a potential antipregnancy vaccine based on the beta-subunit of human chorionic gonadotropin (beta-hCG). I. Alternative macromolecular carriers.

The antibody response obtained after vaccinating rabbits with the beta-subunit of human chorionic gonadotropin (beta-hCG) linked to several protein and polysaccharide carriers was measured. In all but one preparation, carbodiimide was used to couple the beta-hCG to the carrier. Tetanus toxoid (TT) and cholera vaccine proved the most effective carriers among those examined. TT from different manufacturers proved to be greatly different in free amino group content and differed in ability to participate in the coupling reaction. Reasonably good replication of the coupling reaction was obtained with different production lots from the same manufacturer. Inferior antigenic response was obtained with the products of coupling beta-hCG to H. pertussis, influenza vaccine, polylysine, pneumococcus polysaccharide, or E. coli polysaccharide. The findings indicate TT and cholera vaccine to be especially effective in enhancing the antigenicity of a weakly antigenic peptide but point to significant differences in the TT from different manufacturers.

Formulation of a potential antipregnancy vaccine based on the beta-subunit of human chorionic gonadotropin (beta-hCG). II. Use of compounds of the muramyl dipeptide (MDP) family as adjuvants.

Earlier tests of an antipregnancy vaccine consisting of the beta-subunit of human chorionic gonadotropin (beta-hCG) linked by reaction with a carbodiimide reagent to tetanus toxoid (TT) and adsorbed on Al(OH)3 resulted in antibody responses that were judged inadequate in some women. Experiments were therefore conducted to evaluate the effectiveness of additional adjuvants in increasing the antibody response. Muramyl dipeptide (MDP) and several of its analogs were formulated with the vaccine and tested in rabbits. Some of the analogs, and notably N-acetyl-normuramyl-L-alanyl-D-isoglutamine, elicited substantial increments in the ability of the antisera to bind [125I]hCG and in its ability to neutralize hCG in the rat uterine weight assay. The effectiveness of these peptides was greatest when formulated in a water-in-oil emulsion. Increments of 10 fold were attained using a vegetable oil as the oil component. The MDP analogs were much less effective as adjuvants when formulated in oil-in-water emulsions or in aqueous suspensions of the antigen. It is concluded that selected MDP analogs incorporated in a water-in-vegetable oil emulsion can markedly increase the circulating antibody response to the beta-hCG-TT vaccine.

Formulation of a potential antipregnancy vaccine based on the beta-subunit of human chorionic gonadotropin (beta-hCG). III. Evaluation of various vehicles and adjuvants.

Rabbits were used to test the efficacy of several materials as supplementary adjuvants when administered as part of a vaccine formulation consisting of the beta-subunit of human chorionic gonadotropin linked to tetanus toxoid (beta-hCG-TT) and adsorbed on Al(OH)3. In the amounts used, Corynebacterium parvum, levamisole, thymic factor, and N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)propanediamine exhibited little adjuvant activity although the latter material elicited marginal increments when incorporated in liposomes. A Salmonella lipopolysaccharide preparation (SPLPS) and a streptococcal preparation (OK-432) each gave approximately 7-fold increments in titer. The SPLPS preparation was pyrogenic at the doses used. OK-432 was nonpyrogenic and did not cause other evident undesirable effects. It may therefore prove to be a useful adjuvant. It gave a nearly flat dose response curve over the range of 0.5 to 4.0 mg per rabbit. Incorporation of beta-hCG-TT on Al(OH)3 in a water-in-oil emulsion caused a moderate increase in titers. Incorporation into liposomes or an oil-in-water emulsion was not effective.

Studies on gossypol. I. Toxicity, antifertility, and endocrine analyses in male rats.

Toxicity, antifertility, and endocrine effects of gossypol have been studied in male rats. The animals grew nearly normally when administered a daily dose of 7.5 mg/kg orally for 12 wk. At 15 and 30 mg/kg/d doses growth rates were significantly reduced. Infertility occurred at all dose levels, but mating behavior was maintained throughout the period of treatment. Examination of the cauda epididymis after 6 wk at the 30-mg/kg/d dose and after 12 wk at the lower doses showed reductions in sperm numbers as compared with controls. All of the sperm from the groups receiving the 15- and 30-mg/kg/d doses were immotile; and at the 7.5-mg/kg/d dose level most sperm were immotile. Many of nonmotile sperm showed sharply bent tails or lacked tails. There was no significant change in the serum levels of testosterone, LH, or FSH in animals receiving 7.5 and 15 mg/kg/d of gossypol for 12 wk. However, at a dose of 30 mg/kg/d for 6 wk, the serum testosterone and LH concentrations were significantly reduced. FSH concentrations remained normal in all groups. A group of 5 animals receiving 15 mg/kg/d were allowed a recovery period of 6 wk after 12 wk of therapy and they evidenced complete recovery of fertility.

A synthetic peptide capable of eliciting antibodies that neutralize human chorionic gonadotropin.

Antisera generated to the human chorionic gonadotropin beta-subunit (hCGbeta) have been shown not only to neutralize the biologic activity of hCG but also to cross-react with human luteinizing hormone (hLH). In an attempt to reduce such cross-reactivity, a peptide fragment analogous to the amino acid sequence of the carboxylterminal 45 residues (101-145) of the hCGbeta-subunit with alpha-aminobutyric acid substituting for cysteine at position 110 was synthesized and tested for ability to produce antibodies interacting with hCG. Antisera were generated in rabbits to a conjugate of this peptide with tetanus toxoid emulsified with Freund's complete adjuvant. Antibody titers and specificity were assessed by the double-antibody technique. The results show that the antisera to the synthetic hCGbeta fragment bound 125I-labeled hCG and did not cross-react with hLH in the radioimmunoassay system. Most importantly, the antisera effectively neutralized the biologic activity of hCG as determined by the rat uterine weight assay.

Effect of estradiol on the early incorporation of (3H) thymidine into uterine DNA on immature mouse.

The incorporation of [3H]thymidine into uterine DNA was markedly depressed within 10 to 30 minutes after intraperitoneal administration of 17beta-estradiol to immature mouse. Maximum inhibition occurred about 6 hours after the hormone was administered. Uterine DNA content and the amount of [3H]thymidine incorporated into the acid-soluble fraction was not affected during the period of hormone-induced inhibition. Moreover, the in vitro incorporation of [3H]thymidine by isolated estradiol-treated mouse uterus was blocked. In contrast to the uterus, 17beta-estradiol did not influence the incorporation of thymidine into mouse liver DNA. Evidence is presented to show that the incorporation of thymidine into uterine DNA was blocked initially by 17beta-estradiol.