Chronic Toxicity and Reversibility of Antifertility Effect of Immunization against Gonadotropin-Releasing Hormone in Male Rats and Rabbits

TOXICOLOGICAL SCIENCES 53, 92–99 (2000) Copyright © 2000 by the Society of Toxicology Chronic Toxicity and Reversibility of Antifertility Effect of Immunization against Gonadotropin-Releasing Hormone in Male Rats and Rabbits Narender Kumar, 1 Toyin Savage, William DeJesus, Y. Y. Tsong, A. Didolkar, and K. Sundaram Center for Biomedical Research, Population Council, 1230 York Avenue, New York, New York 10021 Received June 3, 1998; accepted March 25, 1999 gonadotropins from the pituitary, which in turn regulates gonadal function (Garner et al., 1990). Active immunization with GnRH conjugated to tetanus toxoid (GnRH-TT) has been shown to neutralize GnRH leading to inhibition of gonadal function in animals (Chappel et al., 1980; Fraser et al., 1974; Giri et al., 1990; Ladd et al., 1989, 1994; Upadhyay et al., 1989). A male contraceptive method based on this approach has been proposed (Awoniyi et al., 1992a; Ladd et al., 1988). The method involves immunization with GnRH-TT and administration of an androgen supplement to maintain sexual behavior and other androgen-supported functions (Awoniyi et al., 1992b; Ladd et al., 1988). Immunization against GnRH may also be useful for treating sex steroid-dependent abnormal growth of mammary glands and prostate (Jayashankar et al., 1989; Ravdin and Jordan, 1988). In order to initiate clinical trials, chronic systemic toxicity studies of GnRH-TT were carried out in male rats and rabbits. To be acceptable, a contraceptive method must also be reversible. In a separate study, the reversibility of GnRH-TT induced infertility was investigated in rats. The chronic systemic toxicity of immunization with gonadotropin-releasing hormone, conjugated to tetanus toxoid (GnRHTT), 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 antiGnRH antibody titers. Key Words: immunization, GnRH, toxicity, reversibility, pituitary response. MATERIALS AND METHODS Test and Control Articles Synthetic (Gln 1)-GnRH was provided by the Salk Institute, La Jolla, CA. It was conjugated to tetanus toxoid (Wyeth Laboratories, Marietta, PA) at the N-terminal amino acid (Gln) using 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide and was designated as GnRH-TT. The test article was an off-white, lyophilized powder containing 0.73 mg GnRH-TT per mg of powder. The conjugate, when analyzed by high-performance-liquid chromatography on BioRad TSK-250 column showed one broad peak with a shoulder. Based on radioimmunoassay, there were 3.38 moles of (Gln 1) GnRH per mole of (Gln 1) GnRH-TT. GnRH-TT was dissolved in an aqueous adjuvant, PBS-PT (0.01 M phosphate buffered saline containing 2.5% Pluronic L 121 and 0.2% Tween 80). The adjuvant was used as control vehicle. GnRH-TT and the adjuvant were prepared as per GMP regulations. Animals and Husbandry Crl:CD 威 Br COBS 威 male (BW 300 –325 g) and female rats (BW 200 –225 g) were purchased from Charles River Laboratories (NY). New Zealand White rabbits (BW 3.0 –3.5 kg) were purchased from Hazelton Dutchland Laboratories, Inc. (NY). The animals were housed at the Laboratory Animal Research Center (LARC) of the Rockefeller University in New York City according to the NIH guidelines outlined in the Public Health Service Policy on Humane Gonadotropin-releasing hormone (GnRH), a hypothalamic peptide hormone, is critical for the synthesis and secretion of 1 To whom correspondence should be addressed. Fax: (212) 327-7678. E-mail: . 92 CHRONIC TOXICITY OF GnRH-TT VACCINE Care and Use of Laboratory Animals. Rats were given food and water ad libitum and housed under standard conditions with a photo period of 12L:12D. The rats were housed 2 per cage for the chronic toxicity study. Rats for fertility testing were housed singly in hanging wire-bottom cages. Rabbits were housed singly and given a restricted diet of approximately 125 g per day, in order to prevent an excessive increase in body weight. Water was allowed ad libitum. Experimental Design Toxicity study in rats. A total of 90 male rats were used for this study. After an acclimatization period, rats were randomly allocated: 60 to the vehicle control, Group 1; the treatment group (Group 2, 60 rats) or the castrated group (Group 3, 10 rats). The rats in Group 3 were bilaterally castrated on the day of initiation of the treatment, to compare with immunized rats. The test article (GnRH-TT in adjuvant), and vehicle (adjuvant) were administered by intramuscular injection in the lateral part of the thigh. A total of 200 ␮g GnRH-TT suspended in 0.5 ml of the adjuvant was administered to rats in the treatment group. For the primary immunization phase, rats were injected 3 times at 4-week intervals. A booster injection was given to all rats in Group 2, 12 weeks after the last injection, in order to maintain high antibody titers. Control rats received a similar number of injections with the vehicle. The total duration of the study was 8 months. All signs of ill health and/or behavior changes (aggressiveness or lethargy) in response to treatment we (...truncated)