6
Animals as Models for Studying Antiprogestins

Newly synthesized compounds are generally screened by using assays that assess their in vitro binding affinities for the progesterone receptor. Usually, these compounds are also screened for binding to other steroid hormone receptors. In general, compounds that have high binding affinity for the progesterone receptor are assessed further for their agonist or antagonist activity by using animal models (Van Look and von Hertzen, Appendix B12). Papers presented in Appendix B of this report provide a history of the development of antiprogestins including the use of animal models (Baulieu, Appendix B1); provide an overview and perspective of the use of animal models in general to predict the pharmacologic effects in humans (Van Look and von Hertzen, Appendix B12); address the applicability of the primate model to humans (Hodgen, Appendix B11); address animal and cell culture models for their relevance to evaluating the treatment potential of these compounds for breast cancer and mammary tumors (Henderson, IOM workshop; Horwitz, Appendix B9); describe species specificity of receptor binding and the effect of amino acid substitutions within the progestin-receptor-binding site (Weigel, Appendix B2); and discuss research on antiprogestin effects on estrogen-receptor levels and induction of labor in primates versus humans (Baird, Appendix B4; Ulmann and Silvestre, Appendix B6). The committee has concluded from these discussions that results in most animal models appear to have been fairly predictive of the effects of antiprogestins in humans despite the fact that there are major differences between humans and other species, including nonhuman primates, in terms of the pharmacokinetics of the antiprogestins and the type of placentation (Van Look and von Hertzen,



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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda 6 Animals as Models for Studying Antiprogestins Newly synthesized compounds are generally screened by using assays that assess their in vitro binding affinities for the progesterone receptor. Usually, these compounds are also screened for binding to other steroid hormone receptors. In general, compounds that have high binding affinity for the progesterone receptor are assessed further for their agonist or antagonist activity by using animal models (Van Look and von Hertzen, Appendix B12). Papers presented in Appendix B of this report provide a history of the development of antiprogestins including the use of animal models (Baulieu, Appendix B1); provide an overview and perspective of the use of animal models in general to predict the pharmacologic effects in humans (Van Look and von Hertzen, Appendix B12); address the applicability of the primate model to humans (Hodgen, Appendix B11); address animal and cell culture models for their relevance to evaluating the treatment potential of these compounds for breast cancer and mammary tumors (Henderson, IOM workshop; Horwitz, Appendix B9); describe species specificity of receptor binding and the effect of amino acid substitutions within the progestin-receptor-binding site (Weigel, Appendix B2); and discuss research on antiprogestin effects on estrogen-receptor levels and induction of labor in primates versus humans (Baird, Appendix B4; Ulmann and Silvestre, Appendix B6). The committee has concluded from these discussions that results in most animal models appear to have been fairly predictive of the effects of antiprogestins in humans despite the fact that there are major differences between humans and other species, including nonhuman primates, in terms of the pharmacokinetics of the antiprogestins and the type of placentation (Van Look and von Hertzen,

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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda Appendix B12). Some of the similarities and differences are discussed below. Antiprogestins bind to progesterone-receptor preparations from a variety of species, including rat, rabbit, calf, marmoset, bonnet monkey, and human, but they do not bind to chicken or hamster progesterone receptors (Baulieu, Appendix B1; Weigel, Appendix B2; Van Look and von Hertzen, Appendix B12). In the latter cases, the lack of mifepristone (RU 486) binding to the progesterone receptor has been attributed to a single amino acid change in the hormone-binding domain—the replacement of a glycine by a cysteine at positions 575 and 722 for the chicken and hamster, respectively. All receptors that bind mifepristone, including glucocorticoid and androgen receptors, have a glycine residue at a corresponding position in the hormone-binding domain (Van Look and von Hertzen, Appendix B12). Both the chicken and the hamster progesterone receptors can be modified by a substitution of glycine (but not methionine or leucine) for this key cysteine, and the mutated receptor will bind mifepristone. Conversely, replacing the key glycine in the human progesterone receptor with cysteine renders the receptor incapable of binding mifepristone (Baulieu, Appendix B1). This exquisite sensitivity of progestin-receptor binding to a single amino acid substitution suggests that studies of the molecular actions of antagonists that have potential clinical applications should be conducted by using human steroid receptors, since receptors from other species may respond somewhat differently (Weigel, Appendix B2). When using animal models, it is important to recognize that there are marked species differences in plasma proteins that can bind to the steroid hormones and to the antihormones. For example, no animal species appears to have the high-affinity binding protein a1-acid glycoprotein, which is found in the human (Baulieu, Appendix B1; Van Look and von Hertzen, Appendix B12). This plasma glycoprotein binds to some, but not all, antiprogestins, and appears to affect clearance rates of these compounds. For example, a1-acid glycoprotein strongly binds to mifepristone and probably lilopristone, but not to onapristone. Research suggests that the low clearance of mifepristone is exacerbated by this tight protein binding and is reflected in the long half-life of mifepristone (20 to 24 hours) versus that for onapristone (2 to 4 hours) (Van Look and von Hertzen, Appendix B12). The pregnant guinea pig model has been used extensively for the study of abortifacient potency and the mechanism of action of antiprogestins. Studies using this model led to the development and testing of the sequential treatment regimen of mifepristone followed by prostaglandin. However, studies in the guinea pig model have not always correlated with those in humans. For example, in the pregnant guinea pig model, a marked synergism was demonstrated between antipro-

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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda gestins and the antiestrogen, tamoxifen, in inducing abortion. This was not seen in one study in women (Van Look and von Hertzen, Appendix B12). However, the study in women may not have been expected to demonstrate synergy due to the drug regimens tested. The concentration of mifepristone used in the human study was a maximal, or near maximal, dose. By definition, synergism could not be demonstrated under these circumstances, and lower (submaximal) doses would have to be utilized to evaluate the potential synergistic action properly. Cook of Research Triangle Institute (RTI) presented information at the Institute of Medicine workshop on a new RTI compound that has a basic structure similar to other antiprogestins, but exhibits agonist rather than antagonist effects in the rabbit. Chwalisz of Schering AG pointed out that the rabbit progesterone receptor was anomalous and many antiprogestins that exhibit antagonist effects in other animals often exhibit agonist effects in rabbits. Therefore, the rabbit does not appear to be a good model for evaluating antiprogestins for potential application in humans. Most of the control systems that govern reproductive function in the higher primates are fundamentally different from those in other mammals. This holds true for the control of ovulation, the recognition and maintenance of pregnancy, and the initiation of labor. The rhesus monkey has been a good model for the human in the context of neuroendocrine control of the menstrual cycle and ovulation; however, it differs markedly in the control of pregnancy. Progesterone metabolism is totally different from that in the human. In the rhesus monkey, progesterone is not converted to pregnanediol, and its concentration during pregnancy does not rise much above luteal phase except in the last few days before parturition (Neill et al., 1969). In this regard, the rhesus monkey behaves like sheep, cow, and other ungulates. Despite the differences described above, Hodgen (Appendix B11), in reviewing the primate model for the study of antiprogestins, suggested that data from macaques (both rhesus and cynomolgus monkeys) and humans are quite similar. His comparisons focused on the noncompetitive antiestrogenic activity of progesterone antagonists, the dose-dependent blockade by mifepristone of the proliferative action of estradiol on the endometrium, and the elevation of estrogen receptors in the endometrium induced by mifepristone (Hodgen, Appendix B11). Some differences between primates and humans, however, have been noted. For example, Spitz and coworkers (1993; and Danforth et al., 1989) found that intermittent mifepristone was more effective at inhibiting ovulation in the monkey than in women. Furthermore, Frydman and coworkers (1991) found mifepristone to be more effective at inducing labor in women at the end of the third trimester than had been previously reported in monkeys.

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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda Ulmann and Silvestre (Appendix B6) reported on studies in ewes (Burgess, 1992) and monkeys (Wolf et al., 1989) showing that mifepristone induces uterine contractions and enhances the myometrial sensitivity to oxytocin at term. Newborn animals from mothers treated with mifepristone were normal. Very limited data in both animals and humans are available to corroborate this finding, and more studies are necessary on this use (see also Chapter 3). Extensive discussions of the use of animal models in evaluating potential effects of new antiprogestins on breast cancer are presented in the paper by Horwitz (Appendix B9). Progesterone agonists increase the incidence of spontaneous mammary tumors in dogs and mice and, at physiologic levels, increase the growth of established tumors in some species (Horwitz, Appendix B9). Various animal models of hormone-dependent mammary cancer have been used to study the antiproliferative properties of progesterone antagonists and estrogen antagonists. These include rats that have chemically induced tumors and mice bearing transplanted tumor lines (Horwitz, Appendix B9). In these animal models, combined treatment with mifepristone and antiestrogens or gonadotropin-releasing hormone agonists produces high rates of tumor remission (Bakker et al., 1990). Other antiprogestins have been shown to have similar effects (Horwitz, Appendix B9). However, these models have limitations. For example, the nude mouse model has low progesterone-receptor levels and, despite hormone dependence, was found to be resistant to antiprogestins. Another difference is that the time of induction of breast cancer is weeks in rodent models versus years in human. This may explain why hormonal effects in cancer induction are more obvious or exaggerated in animal models than in humans. In women, only preliminary clinical studies have been reported on the potential use of antiprogestins in the treatment of advanced breast cancer, and there has been no report of the combined use of antiprogestins and antiestrogens as studied in the animal models described above (see Chapter 4). Although data from animal models are promising, long-term comparative human studies will be necessary to establish whether antiprogestins, or antiprogestins in combination with antiestrogens, might form a treatment modality for human breast cancer. In conclusion, as for many drugs, animal models have been useful in understanding the mechanism of action and evaluating the potential of treatment modalities with antiprogestins. As would be expected, animal models are not always accurate predictors of the results in human beings. However, in the antiprogesterone data presented to and reviewed by the committee, particularly on mifepristone, animal models have been extremely useful in providing clues to the documented

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Clinical Applications of Mifepristone (RU 486) and other Antiprogestins: Assessing the Science and Recommending a Research Agenda beneficial effects of these compounds and as leads for potential alternate uses described elsewhere in this report. REFERENCES Bakker, G.H., Setyono-Han, B., Portengen, H., et al. Treatment of breast cancer with different antiprogestins: Preclinical and clinical studies. Journal of Steroid Biochemistry and Molecular Biology 37:789–794, 1990. Burgess, K.M., Jenkin, G., Ralph, M.M. et al. Effect of the antiprogestin RU 486 on uterine sensitivity to oxytocin in ewes in late pregnancy. Journal of Endocrinology 134:353–360, 1992. Danforth, D.R., Dubois, C., Ulmann, A., et al. Contraceptive potential of RU 486 by ovulation inhibition. III. Preliminary observations on once weekly oral administration. Contraception 40:195–200, 1989. Frydman, R., Baton, C., Lelaidier, C., et al. Mifepristone for induction of labour. Lancet 337:488–489, 1991. Neill, J.D., Johansson, E.D., and Knobil, E. Patterns of circulating progestone concentrations during the fertile menstrual cycle and the remainder of gestation in the rhesus monkey. Endocrinology 84:45–48, 1969. Spitz, I.M. and Bardin, C.W. Clinical pharmacology of RU 486—An antiprogestin and antiglucocorticoid. Contraception, in press (also see: RU 486—A modulator of progestin and glucocorticoid action. New England Journal of Medicine, in press). Spitz, I.M., Croxatto, H.B., Salvatierra, A.M., et al. Intermittent RU 486 administration to normal women. Fertility and Sterility 1993 (in press). Wolf, J.P., Sinosich M., Anderson T.L., et al. Progesterone antagonist (RU 486) for cervical dilation, labor induction and delivery in monkeys: Effectiveness in combination with oxytocin. American Journal of Obstetrics and Gynecology 160:45–47, 1989.

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