treatment with a progesterone antagonist zk 98.299 delays endometrial development without blocking...

Clinical Article

Treatment with a Progesterone Antagonist ZK 98.299 Delays Endometrial Development without Blocking Ovulation in Bonnet Monkeys

P.C. Ishwad, R.R. Katkam, I.N. Hinduja, K. Chwalisz,l W. Elger,’ and C.P. Puri

Institute for Research in Reproduction (ICMR), Parel, Bombay 400012,

India, and ‘Department of Experimental Gynecology and Fertility Control,

Schering Ag, Berlin, Germany

The effects of an antiprogestin ZK 98.299 (onapristone) on serum levels of estradiol and progesterone, and on the endometrial morphology were studied in adult bonnet monkeys. Twelve animals having menstrual cycles of normal duration (24 to 30 days) were randomly distributed into 4 equal groups. The animals in Group 1 were treated (SC.) with the vehicle (benzyl benzoate : castor oil, 1 :lO), and in Groups 2, 3 and 4 with 5 mg, 10 mg, or 20 mg ZK 98.299 once-a-week, respectively. Treatment was initiated on day 1 of the menstrual cycle and each animal in Groups 1, 2 and 3 was treated for two consecutive cycles. Since the treatment cycle length of animals in Group 4 was considerably prolonged, they were treated for one menstrual cycle only. Endometrial biopsy was taken around day 20 of the second treatment cycle of first three groups and around day 50 of the 4th group of animals, Treatment with vehicle or 5 mg ZK 98.299 had no significant effect on the menstrual cycle length. Treatment with 10 mg dose had no effect in two animals and prolonged the cycle length in one, whereas, further increase in the dose to 20 mg prolonged the cycle length in all the animals. The duration of menses was generally reduced. Treat- ment with vehicle or different doses of ZK 98.299 had no effect

Submitted for publication February 10, 1993; accepted for publication May 10, 1993. Address correspondence to: C. P. Puri, Institute for Research in Reproduction (ICMR), Parel, Bombay 400012, India.

0 1993 Butterworth-Heinemann Contraception 1993:48, July 57

Clinical Article

on ovulation. In animals treated with 5 or 10 mg dose, the pattern of mid cycle rise in serum estradiol levels and progesterone levels during the luteal phase of both treatment cycles were comparable to those of vehicle-treated animals and were suggestive of normal ovulatory cycles. On the other hand, in animals treated with the higher dose (20 m&week), progesterone levels during the luteal phase were significantly reduced and were indicative of luteal insufficiency. The hormonal data during the treatment period of this group of animals was suggestive of two distinct ovarian cycles indicating that the menstrual bleeding during the treatment period was probably very scanty. Treatment with ZK 98.299 impaired the endometrial development in a dose-dependent manner. In vehicle-treated animals, the endometrium had large and tortous glands with secretions. Treatment with ZK 98.299 caused atrophic changes in the glands as well as in the stroma. The height of the epithelial cells was markedly decreased and they became small and inactive. This study, therefore, suggests that treatment with low doses of antiprogestin ZK 98.299 at weekly intervals does not block folliculogenesis or ovulation, but has an inhibitory effect on the endometrium. This study opens up a possibility of development of antiprogestins as a contraceptive agent.

Keywords: Antiprogestin; ZK 98.299; onapristone; endometrium; hormone levels; ovulation; bonnet monkeys.

Introduction

RU 486, ZK 98.734 and ZK 98.299 are structurally related progesterone antag-onists. These antiprogestins, particularly RU 486, have undergone extensive evaluation for termination of early pregnancy (for review see 1). Oral treatment with these antiprogestins produces incomplete abortion in about 30 to 40 per cent of women with an amenorrhoea of less than 63 days. However, supplementation of treatment with a prostaglandin analogue leads to the rapid onset of expulsive behaviour, with expulsion of conceptus within a few hours thereafter. In this regard, ZK 98.299 has been found to be more synergistic as compared to the other antiprogestins in the guinea pig ‘model’ (2).

The endometrium and the gonadotropic cells of the pituitary are the preferential target cells of these antiprogestins (3). Treatment with ZK 98.299 during the follicular phase usually terminates folliculogenesis and consequently ovulation is blocked in bonnet monkeys (4,5). The rise in the serum levels of estradiol and the preovulatory surge of LH are either completely blocked or attenuated and these effects appear to be due to impaired gonadotropin release. Similar effects on folliculogenesis have

58 Contraception 1993:48, July

ZK 98.299 delays endometrial development: Ishwad et al.

also been observed with ZK 98.734 in bonnet monkeys (6) and RU 486 in both non-human primates (7) and women (8,9). In addition, administration of these antiprogestins also has inhibitory effects on the endometrial development. Treatment during the luteal phase induces premature menstruation which could be due to an effect on the endometrium, or corpus luteum function and/or on the hypothalamo-pituitary system. The observations that in the bonnet (10) and rhesus monkeys (11) and in women (12,13) these antiprogestins can provoke early menstruation in spite of high progesterone levels during pseudopregnancy, induced by hCG administration, further suggest that the local effect on the endometrium is the most important. These observations suggest that these antiprogestins can possibly be developed as contraceptive agents where the drug could be administered at regular intervals to block ovulation or other gonadotropin- dependent events, or alternatively to delay the endometrial development and disturb luteal-endometrial integrity such that it is not compatible for implantation.

Studies were, therefore, undertaken to investigate the effects of different doses of ZK 98.299, administered at weekly intervals, on hormonal dynamics, uterine bleeding and endometrial morphology of bonnet monkeys. ZK 98.299 was preferred because of its less antiglucocorticoid activity as compared to RU 486 (14). Moreover, in our pilot studies, using bonnet monkey as an animal ‘model’, daily treatment with ZK 98.299 during the different phases of folliculogenesis has been shown to arrest folliculogenesis and ovulation (4,5).

Materials and methods

Animals

Female bonnet monkeys (Macaca radiata) weighing between 3.5 to 4.5 kg and exhibiting at least two consecutive menstrual cycles of normal duration, immediately prior to induction in this study, were used. In the Primate Facility of the Institute, maintained at 26” + 1°C and photoperiod of 14h light : 10h dark schedule, the monkeys are kept in individual cages with built-in restraining device. A diet comprising of fresh vegetables, fruit, peanuts, soaked grams and water is fed to them. The menstrual cycle length and the duration of menstrual bleeding are recorded by daily examination of vaginal swabs. This study was carried out during Septem- ber to April when the menstrual cycles of most of the animals are consis- tently of normal duration and ovulatory.

Drug

ZK 98.299 (onapristone, 11@(4 dimethyl aminophenyl)- 17ol-hydroxy- 17p- (3-hydroxypropyl)-13a-methyl-4,9-gonadien-3-one) was dissolved in ben-

Contraception 1993:48, July 59

Clinical Article

zyl benzoate at 90°C and then further diluted with castor oil ( 1: 10) to get the desired concentration.

Treatment

Twelve animals were randomly distributed into four equal groups. The animals in Group 1 were treated with the vehicle alone, and in Groups 2,3 and 4 with 5 mg, 10 mg and 20 mg ZK 98.299 at weekly intervals. Treatment was initiated on Day 1 (Dl) of the menstrual cycle and it was administered for two consecutive cycles in Groups 1, 2 and 3. Since the treatment cycle length of animals in Group 4 was considerably prolonged, they were treated for one menstrual cycle only.

Collection of blood samples

Blood samples were collected, without sedating the animals, during the pretreatment and treatment cycles of all the groups of animals. In addition, from the animals treated with 20 mg dose, a few samples were also collected during the post-treatment cycles. Blood samples were collected once daily during the first ten days of each menstrual cycle and thereafter every alternate day throughout the menstrual cycle. Serum was separated and kept frozen at -20°C until further processed.

Endometrial histology

Uterine biopsy was taken from two representative animals in each group. The biopsies were taken around day 20 of the second treatment cycle of the first three groups, and around day 50 of the fourth group cycle following abdominal surgery from animals which had been sedated with Ketamin hydrochloride (10 mg/kg body wt). The tissue was fixed in Bouin’s fixative and processed for routine histology.

Estimation of hormone levels

Serum levels of estradiol and progesterone were measured by radioimmunoassay method as described previously (15). The reagents including the monoclonal antibodies raised against estradiol-G-CMO-BSA and progesterone-3-CMO-BSA were provided by the Human Reproduction Pro- gramme of the WHO under the matched assay reagent programme (16).

Statistical analysis

Changes in the duration of menstrual cycle length, duration of menses and the levels of gonadal hormones during the pretreatment and treatment cycles were statistically analysed by the Student’s t-test.



Results

Effect on menstrual cycle

Treatment with vehicle, and 5 mg or 10 mg ZK 98.299 at weekly intervals had no significant effect on the menstrual cycle length of both treatment cycles (Table 1). In one animal (# 208), which had been treated with 10 mg dose (Group 31, the cycle length of the second treatment cycle was increased to 53 days. Analysis of the hormonal data showed that this menstrual cycle had two distinct ovarian cycles. The menstruation during the second treatment cycle was probably very scanty and missed on the vaginal swabs. This animal was, therefore, treated for three consecutive ovarian cycles.

Increase in the dose of ZK 98.299 to 20 mg/week, significantly prolonged the menstrual cycle length. However, analysis of the hormonal data of these animals was also suggestive of two ovarian cycles. However, each ovarian cycle was considerably longer than the pretreatment cycle of the same animal.

Menstrual bleeding was generally scanty during the treatment cycles as compared to the vehicle-treated cycles or post-treatment cycles.

Effects on gonadal hormones

Treatment with the different doses of ZK 98.299 had no effect on ovulation in both treatment cycles. Preovulatory rise in the levels of estradiol, and rise in the levels of progesterone (above 1 ng/ml) after the mid cycle peak of estradiol levels was suggestive of ovulation having occurred in all the animals treated with ZK 98.299.

In animals treated with 5 mg dose, serum levels of progesterone during the luteal phase increased above 3 ng/ml and were suggestive of adequate luteal function. Serum levels of estradiol and progesterone during the pretreatment and two treatment cycles of one representative animal (E 250) of this group are shown in Figure 1.

Treatment with 10 mg dose also had no effect on gonadal hormones, as shown in one representative animal (# 180) in Figure 1. Animal #208, which had been treated for three consecutive ovarian cycles, also had normal luteal function in all the three cycles.

Treatment with 20 mg dose caused prolongation of the follicular phase and menstrual cycle length. The mid cycle rise in the levels of estradiol was delayed. An increase in the levels of progesterone (above 1 ng/ml) was observed after the estradiol peak but the levels did not rise above 3 ng/ml, thereby indicating luteal insufficiency (Figure 2).

Effect on endometrial histology

The vehicle-treated animals showed secretory endometrium with tortu- ous glands and secretion in the lumen (Figure 3a). Treatment with ZK


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FIGURE 1. Serum levels of estradiol and progesterone during the pretreatment and two treatment cycles of two representative bonnet monkeys treated with 5 mg (f 250) or 10 mg (f 180) ZK 98.299 at weekly intervals. The profile of gonadal hormones in the treatment cycles is similar to that of the pretreatment cycle, and also to the vehicle-treated control group (data not shown).

98.299 caused atrophic changes in the glands as well as stroma. The epithelial cell height of the glands was markedly decreased (Figure 3b). The effect on the glands was more marked with 10 mg dose (Figure 3c) as compared to 5 mg dose. The glands became small and inactive with apparent increase in the stroma. Similar effects were also seen in the animals treated with 20 mg dose (Figure 3d).

Discussion

This study demonstrates that the endometrium is the primary target of onapristone and it is possible to impair endometrial development without affecting ovulation, and possibly menstrual cycle length. Treatment with this antiprogestin (5 to 20 mg) at weekly intervals caused atrophic changes


Clinical Article

-

I TREATMENT 1 1 1 I 1 1 1 1 I

FIGURE 2. Serum levels of estradiol and progesterone during the pretreatment and treatment cycles of one

representative animal (f 230) treated with 20 mg ZK 98.299. The menstrual cycle length of the treatment cycle,

as monitored by vaginal swabs, was increased to 82 days. However, distinct peaks in the levels of estradiol

on days 16 and 48 followed by rising levels of progesterone are suggestive of two ovarian cycles. Recording

of menstruation was probably missed as a result of scanty bleeding. Serum progesterone levels during both treatment ovarian cycles did not rise above 2 ng/ml and are suggestive of luteal insufficiency.

in the endometrium in a dose-dependent manner without inhibiting ovulation. This study, therefore, opens up the possibility of developing onapristone and possibly other related antiprogestins, as contraceptive agents where the treatment may disturb the luteal-endometrial integrity such that it is not compatible for implantation.

We have also observed that when the frequency of treatment with the highest dose (20 mg) of onapristone was increased to every third day, an amenorrheic state was induced in the animals (unpublished observations). Treatment blocked the normal follicular development and ovulation was arrested during the treatment period. The endometrium had a few small glands which were distinctly atrophic. These effects are similar to our earlier studies in which high doses of onapristone were administered daily on days 5 to 10 or 5 to 15 of the menstrual cycle and the treatment either completely blocked or attenuated the rise in estradiol and bioactive LH levels (4,5).

These observations suggest that the endometrium, the gonadotropic cells of the pituitary and possibly the ovary are the target organs of onapristone. Endometrium seems to be the primary target as it is sensitive to even low doses of the drug. At high doses, both the endometrium and the gonadal function were affected. The observations that in both monkeys and women the induction of uterine bleeding with structurally related antiprogestins ZK 98.734 (10) and RU 486 (11,12) during the luteal phase does not require a decrease in ovarian steroid production, and bleeding can be induced even when high progesterone levels are maintained by exogenous administration of hCG, support the concept of direct impact of antiprogestins on the endometrium.



FIGURE 3. Endometrial morphology of bonnet monkeys treated with the vehicle (a), 5 mg (b),

10 mg (c), and 20 mg (d) of ZK 98.299. The animals in the vehicle-treated group had glandular and stromal cells exhibiting normal secretory activity. The endometrial structure seems to be

affected in ZK 98.299~treated animals. The epithelial cell height of the glands is reduced, the amount of stromal tissue is increased in comparison with the glandular tissue, and the glands are devoid of secretions.


Clinical Article

The local effect of antiprogesterone on the endometrium seemingly is most important. These drugs have high affinity for progesterone receptors (3,17,18) and antagonize the effects of progesterone on the endometrium. When onapristone is administered only during the proliferative phase, treatment does not induce premature bleeding and light microscopic examination does not reveal any significant effect on the endometrium. Similarly, oral administration of RU 486 on days 7 to 10 of the menstrual cycle has no effect on the morphology of the endometrium or other mani- festations of a progesterone effect (19).

Administration of onapristone during the post-ovulatory phase results in the shedding of the mucosa and subsequent bleeding (15). Similarly, treatment with a single dose, as low as 5 mg, of RU 486 administered during the early luteal phase has been shown to inhibit endometrial glandular secretory activity, increase the degenerative changes in gland cells and induce several changes in the vessels of the stroma of women (20).

The mechanisms through which these antiprogestins influence endometrial development are not precisely known. The observed inhibitory effects of onapristone on endometrial growth and development may not be solely due to the inhibition of progesterone action. The proliferation of endometrium is a well established function of estrogen action during the proliferative phase of the cycle. Antiprogestins generally do not bind to the estrogen receptors. However, there is evidence that these com- pounds may also inhibit estrogen’s effects on the endometrium in ovariectomized, estradiol-substituted cynomolgus monkeys (21,22) and rabbits (23). Interestingly, in ovariectomized rabbits, this functional antiestro- genie effect of onapristone was seen within the endometrial glands but not in the endometrial stroma and in the myometrium which were rather stimulated by this compound. Furthermore, the degenerative changes in the vascular structure could be the result of alterations in the metabolism of prostaglandins. RU 486 and ZK 98.734 have been shown to stimu-late the synthesis of prostaglandins by glandular cells of the early human decidua through an effect on the cycle-oxygenase activity (24,251. Graham et al. (26) have shown that at least during the early luteal phase, which is a critical period of progesterone-dependent gene expression, treatment with RU 486 blocks the production of endometrial secretory glycan in women.

Treatment with high doses of onapristone, administered once daily during the follicular phase, not only affects the endometrium but also impairs folliculogenesis and consequently ovulation (5). These inhibitory effects of onapristone on follicular development could be due to impaired release of gonadotropins. Once-weekly administration of RU 486 (27) and ZK 98.734 (6) in a 25 mg dose has been shown to block mid-cycle gonadotropin surges in non-human primates. RU 486 has also been reported to reduce LH secretion and amplitude of LH pulses (8,28,29)



and blunt the pituitary LH response to gonadotropin-releasing hormone (30). The inhibitory effects of antiprogestins on gonadotropin release are also substantiated by the observation that ZK 98.734-induced blockade of folliculogenesis in bonnet monkeys is restored by treatment with LH and FSH (hMG) or human FSH alone (1). Similarly, the estradiol-induced positive feed-back release of LH is blocked by ZK 98.734 in male common marmosets (31) and RU 486 in the cynomolgus monkeys (21). The blockade of folliculogenesis with high doses of antiprogestins is likely to be due to their direct effect on gonadotropin release. Under in vitro conditions also, RU 486 has been shown to inhibit in a dose-dependent manner GnRH-induced LH and FSH secretion by rat pituitary cells which had been primed with estradiol to maintain progesterone receptors (32). The antigonadotropic effects of onapristone appear to be specific to its progesterone antagonistic activity rather than due to its progestomimetic properties. Treatment of ovariectomized estrogen-primed rabbits with onapristone (5 mg/kg/day) for seven consecutive days had no progestational effects on the uterus (unpublished observations).

In brief, this study suggests that onapristone has the ability to interrupt endometrial development regardless of whether ovulation is blocked or not. At low doses, onapristone induces functional state of progesterone deficiency without blocking ovulation or corpus luteum function. Since the undisturbed function of endometrial glands is essential for uterine receptivity, the inhibition of gland formation by an antiprogesterone compound should result in the prevention of implantation. This study, therefore, opens up a possibility of using antiprogestin as a contraceptive where the drug could be administered in low doses to block endometrial development and possibly implantation.

Acknowledgement/Footnote

The authors are grateful to the Special Programme of Research, Develop- ment and Research Training in Human Reproduction of the World Health Organization for providing reagents for the estimation of steroid hormones by radioimmunoassay methods.

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