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Suppression of a Pituitary-Ovarian Axis by Chronic Oral Administration of a Novel Nonpeptide Gonadotropin-Releasing Hormone Antagonist, TAK-013, in Cynomolgus Monkeys

Pharmaceutical Research Division (T.H., H.A., M.K., M.H., N.C., N.S., M.F.) and Strategic Product Planning Department (S.F., M.F.), Takeda Chemical Industries, Ltd., Osaka 532-8686, Japan

Address all correspondence and requests for reprints to: Takahito Hara, M.D., Pharmaceutical Research Laboratories I, Takeda Chemical Industries, Ltd., 17-85 Jusohonmachi 2-chome Yodogawa-ku, Osaka 532-8686, Japan. E-mail: Hara_Takahito{at}takeda.co.jp.

	Abstract

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TAK-013 is a novel nonpeptide and orally active GnRH antagonist. We first examined the effect of TAK-013 on GnRH-stimulated LH release using primary-cultured pituitary cells of cynomolgus monkeys. TAK-013 suppressed LH release to below basal levels at concentrations higher than 100 nM with the IC₅₀ value of 36 nM. Next, we examined the effect of chronic oral administration of TAK-013 on serum hormone levels in regularly cycling female cynomolgus monkeys. TAK-013 administered at 90 mg/kg·d (30 mg/kg 3 times daily) for approximately 80 d continued to suppress LH, estradiol, and progesterone, but not FSH. The suppressive effect was reversible, in that normal profiles of sex steroids were observed immediately after discontinuation of the TAK-013 treatment. Interestingly, the suppressive effect of TAK-013 was not observed in marmoset monkeys. In summary, TAK-013 by oral administration suppresses a pituitary-ovarian axis continuously and reversibly in cynomolgus monkeys. Considering that TAK-013 has more potent antagonistic properties for human GnRH receptor than for monkey receptor, our data suggest that TAK-013 would be effective for reproductive disorders such as endometriosis and uterine leiomyoma and useful for assisted reproductive technology procedures.

	Introduction

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GnRH AGONISTS HAVE therapeutic effects on hormone-dependent diseases, such as breast cancer, endometriosis, and uterine leiomyomata by suppression of estrogen production (1, 2). Because GnRH agonists cause flare-up initially, several peptide GnRH antagonists are currently being evaluated clinically (2, 3, 4, 5). However, these peptide antagonists must be administered sc by daily injection or sustained delivery system (2, 3, 4, 5), which can be a disadvantage in some patients. Thus, there is a need for the development of an orally active GnRH antagonist. We have reported that a thienopyridine derivative compound, T-98475, has high affinity for the human and monkey GnRH receptors and has the GnRH antagonistic properties in cynomolgus monkeys (6). Further studies have revealed that a novel thienopyrimidine derivative compound, TAK-013, has higher affinity for the human and monkey GnRH receptors and more potent antagonistic properties than T-98475 (7). We investigated the effect of TAK-013 by prolonged oral administration on a pituitary-ovarian axis in cynomolgus and marmoset monkeys.

	Materials and Methods

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Animal experiments

The protocols of animal experiments were approved by the Takeda experimental animal use and care committee in accordance with National Institutes of Health standards. Six adult female cynomolgus monkeys weighing 2.4–2.6 kg were divided into two treatment groups. One group served as controls and received vehicle only (n = 3; animal no. 1060, 1178, and 1144), and the other group received TAK-013 (n = 3; animal no., 776, 1065, and 1173). Blood samples (1 ml) were obtained from the cubital vein without anesthesia two times per week at approximately 1330 h to measure the serum LH, FSH, estradiol, and progesterone levels by which the menstrual cycles were monitored. The first day of a luteal phase was defined as the day on which serum progesterone levels rose above a value of 0.3 ng/ml, and the last day of the luteal phase was defined as the day on which serum progesterone levels from the previous cycle declined to 0.3 ng/ml or less. Cynomolgus monkeys in our colony (9 animals) have a mean ± SEM cycle length of 37.1 ± 2.3 d, with a luteal phase of 15.0 ± 0.7 d (n = 32). The protocol included nine consecutive menstrual cycles consisting of two pretreatment control cycles, four treatment cycles, and three posttreatment cycles. Within 10–17 d after the end of the second pretreatment cycle (animal no. 1060, 1178, 1144, 776, 1065, and 1173; 10, 10, 15, 14, 17, and 14 d, respectively), the animals started to receive vehicle or TAK-013 three times daily (30 mg/kg per dose orally) at approximately 0830 h, 1430 h, and 2030 h. The vehicle treatment was continued for 82–93 d (animal no. 1060, 1178, and 1144; 93, 82, and 85 d, respectively), and the TAK-013 treatment was continued for 79–83 d (animal no. 776, 1065, and 1173; 79, 83, and 79 d, respectively). The next three cycles were monitored to confirm the resumption of menstrual cycles. The study of female marmoset monkeys weighing 330–430 g was performed basically in the same way as that of cynomolgus monkeys. The marmoset monkeys received vehicle or TAK-013 three times daily (30 mg/kg per dose orally) at approximately 0800 h, 1400 h, and 2000 h. A blood sample (0.1 ml) was obtained from the femoral vein two times per week at approximately 1330 h for the measurement of progesterone levels. The first day of a luteal phase was defined as the day on which plasma progesterone levels rose above 25 ng/ml, and the last day of the luteal phase was defined as the day on which the plasma progesterone levels from the previous cycle declined to 25 ng/ml or less in marmoset monkeys. Marmoset monkeys in our colony (12 animals) have a mean ± SEM cycle length of 28.1 ± 0.8 d, with a luteal phase of 18.5 ± 0.6 d (n = 71). Within 3–27 d after the end of the second pretreatment cycle (animal no. N313, 11371, 11522, 11456, 11524, and 11517; 4, 5, 7, 3, 5, and 27 d, respectively), the animals started to receive vehicle or TAK-013 (30 mg/kg three times a day orally) at approximately 0840 h, 1440 h, and 2040 h. The vehicle treatment was continued for 77–85 d (animal no. N313, 11371, and 11522; 85, 77, and 79 d, respectively), and the TAK-013 treatment was continued for 70–79 d (animal no. 11456, 11524, and 11517; 79, 70, and 79 d, respectively).

For the study of primary-cultured pituitary cells, two male cynomolgus monkeys at the age of 10 yr and 10 female marmoset monkeys at the age of 1 yr were used.

Reagents

TAK-013 (N-{4-[5-{[benzyl(methyl)amino]methyl}-1-(2,6-difluorobenzyl)-2,4-dioxo-3-phenyl-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-N'-methoxyurea) was synthesized in our company. Methylcellulose (Metolose, Shin-Etsu Chemical Ltd., Tokyo, Japan) was dissolved in distilled water containing 6 mg/ml citric acid to make a 0.5% solution. This solution was used as the vehicle for TAK-013 in animal studies. GnRH was purchased from Peptide Institute, Inc. (Osaka, Japan).

Preparation of monkey pituitary cells

Monkey pituitary cells were prepared according to the method modified from that of Shiota et al. (8). Pituitaries from cynomolgus and marmoset monkeys were removed and washed with Krebs-Ringer bicarbonate solution containing 5 mg/ml BSA, 5 U/ml penicillin, and 5 µg/ml streptomycin (KRBGA). The tissues were then dispersed by shaking (1 h, 100 cycles/min, 37 C) in KRBGA containing 4 mg/ml collagenase and 10 µg/ml deoxyribonuclease I, followed by incubation at 37 C for 8 min in KRBGA containing 2.5 mg/ml pancreatin. The digestion was stopped by adding 10% fetal bovine serum. The cells were resuspended in DMEM supplemented with 10% fetal bovine serum, 5 U/ml penicillin, and 5 µg/ml streptomycin, filtered through a cell strainer (70 µm, Becton Dickinson and Co., Franklin Lakes, NJ), and washed twice with the medium. The cells were counted with a particle counter (Beckman Coulter, Tokyo, Japan) and plated in a 48-well tissue culture plate at a density of 50,000 cells/0.5 ml.

LH release from pituitary cells

After 3 d in culture (37 C; 95% air-5% CO₂), the monkey pituitary cells were washed with DMEM, followed by preincubation at 37 C for 1 h in the medium. Then, the medium was exchanged with fresh DMEM, and TAK-013 (3, 10, 30, 100, 300, 1000, or 3000 nM) was added to the cells. After incubation for 1 h, GnRH at the concentration of 1 nM was added to the cells, and the mixture was incubated 5 h longer. The conditioned medium was stored at -80 C until assays for LH were performed. All culture media were supplemented with penicillin 5 U/ml and streptomycin 5 µg/ml.

Bioassays for LH

Concentrations of LH in the conditioned medium of monkey pituitary cells and in the serum obtained from cynomolgus monkeys were determined by bioassays using dispersed mouse testicular cells modified from the method of Van Damme et al. (9). An assay buffer, DMEM supplemented with 0.5% calf serum, 5 U/ml penicillin, and 5 µg/ml streptomycin, was used in all steps in this experiment. Testes from 9- to 11-wk-old BALB/C mice (Charles River Laboratories, Inc., Kanagawa, Japan) were decapsulated and cut into four pieces. The tissues were then dispersed by shaking (1 h, 125 cycles/min, 37 C) in the assay buffer, followed by filtration with a cell strainer (70 µm). The cells were placed in ice for 10 min, washed three times with the buffer, and then counted with a particle counter. The cells (4 x 10⁵ cells/0.4ml) were mixed with 44.4 µl of standards (Equine LH, Sigma, St. Louis, MO) or with the samples to be tested, and the mixture was incubated at 37 C for 2 h. After the cells were incubated at 60 C for 30 min, the conditioned medium was obtained. The testosterone concentrations in the conditioned media were determined using a commercially available RIA kit (DiaSorin, Inc., Saluggia, Italy), and the LH concentrations in the samples were calculated by linear regression analysis over the linear portion of the standard curves. All samples in each experiment were analyzed within the same assay. The least-detectable concentration of the assay was 0.037 mU/ml. Between- and within-assay coefficients of variation were 31.5% (n = 4 assays) and 14.9% (n = 10 assays), respectively.

RIAs

Estradiol and progesterone levels were determined with commercially available RIA kits (bioMérieux, Marcy L’Etoile, France). The detection limit for the estradiol RIA was 10 pg/ml. Between- and within-assay coefficients of variation were 10.6% and 9.1%, respectively (n = 10 assays). The detection limit for the progesterone RIA was 0.05 ng/ml. Between- and within-assay coefficients of variation were both 4.1% (n = 10 assays) in cynomolgus monkeys, and were 6.8% and 3.1%, respectively, in marmoset monkeys (n = 10 assays). FSH levels were determined by a heterologous RIA using commercially available rat FSH RIA kits (Amersham Pharmacia Biotech, Little Chalfont, UK) where rhesus monkey FSH (Scripps Laboratories, San Diego, CA) instead of the attached rat FSH was used as standard. The detection limit was 0.437 IU/ml. Between- and within-assay coefficients of variation were 1.5% (n = 4 assays) and 2.2% (n = 5 assays), respectively. Serial dilutions of the serum paralleled those of the standard preparation.

Statistics

In the experiments of the monkey pituitary cell culture, differences between the GnRH-stimulated control group and the TAK-013-treated groups were analyzed by the Shirley-Williams test. A value of P less than 0.025 in the one-tailed Shirley-Williams test was considered significant. In addition, the dose of TAK-013 estimated to engender 50% suppression of the LH production (IC₅₀ and 95% confidence interval) was calculated by linear regression analysis over the descending linear portion of the dose-response curve, where the mean of the GnRH-stimulated control group and that of the non-GnRH-stimulated group were set at 100 and 0, respectively. In the in vivo experiments, differences of the treatment-caused change of serum estradiol levels between the vehicle- and TAK-013-treated groups were analyzed by unpaired Student’s t test with Holm’s correction for repeated tests, following the calculation of the treatment-caused change between the pretreatment and treatment periods, between the treatment and posttreatment periods, and between the pretreatment and posttreatment periods using the formulas (a - b)/a x 100, (b - c)/b x 100, and (a - c)/a x 100, respectively, where a, b, and c represent the average of the total values during the pretreatment, treatment, and posttreatment periods, respectively. A value of P less than 0.05 in unpaired Student’s t test with Holm’s correction was considered significant.

All statistical analyses were performed with SAS statistical software (version 6.1, SAS Institute, Inc., Cary, NC).

	Results

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Suppressive effect of TAK-013 on GnRH-stimulated LH release from cynomolgus but not marmoset monkey pituitary cells

The effect of TAK-013 on GnRH-stimulated gonadotropin release was examined using primary-cultured pituitary cells of cynomolgus and marmoset monkeys. As shown in Fig. 1A, 100-1000 nM of TAK-013 significantly suppressed LH release induced by 1 nM GnRH (P < 0.025), and moreover, 300 and 1000 nM of TAK-013 suppressed LH release to below basal levels in pituitary cells of cynomolgus monkeys. The IC₅₀ value was 36 nM (95% confidence interval, 25–50 nM) in cynomolgus monkeys (Table 1). The FSH level in the conditioned medium was too low to study the effect of TAK-013 on FSH release (data not shown). As shown in Fig. 1B, TAK-013 did not suppress LH release significantly, even at 3000 nM, and the IC₅₀ value was higher than 3000 nM in marmoset monkeys (Table 1).

View larger version (15K): [in this window] [in a new window]   Figure 1. Effect of TAK-013 on GnRH-stimulated LH release in pituitary cells of cynomolgus (A) and marmoset (B) monkeys. Data are shown as mean ± SEM (n = 3). *, Significant difference (P < 0.025) between the GnRH-stimulated control vs. the TAK-013-treated groups by the one-tailed Shirley-Williams test.

Serum LH levels in cynomolgus monkeys treated with the vehicle or TAK-013 were determined. LH surges were observed in monkeys treated with the vehicle (Fig. 2) and were also observed during the pretreatment and posttreatment periods in monkeys treated with TAK-013 (Fig. 3). However, LH surges were abrogated, and the LH levels were generally low during the TAK-013 treatment period (Fig. 3). The LH levels recovered immediately after discontinuation of the TAK-013 administration (Fig. 3). In contrast to LH, serum FSH levels were not suppressed by TAK-013 (Figs. 2 and 3).

View larger version (40K): [in this window] [in a new window]   Figure 2. Serum LH and FSH levels in individual cynomolgus monkeys before, during, and after treatment with vehicle. Days represent time from the beginning of treatment, and the duration of treatment is indicated with a thick horizontal bar.

View larger version (40K): [in this window] [in a new window]   Figure 3. Serum LH and FSH levels in individual cynomolgus monkeys before, during, and after treatment with TAK-013 at 90 mg/kg·d. Days represent time from the beginning of treatment, and the duration of treatment is indicated with a thick horizontal bar.

As shown in Fig. 4 and Table 2, cynomolgus monkeys continued to display the normal menstrual cycles and steroid hormone profiles during and after treatment with vehicle. In monkey no. 1060, the maximal estradiol levels were 205, 454, and 611 pg/ml in the three consecutive cycles before treatment; 528, 543, 317, and 243 pg/ml during vehicle treatment; and 653, 285, and 199 pg/ml in the three consecutive cycles after vehicle treatment (Fig. 4A). The maximal progesterone levels were 12.2, 19.4, and 44.5 ng/ml in the three consecutive cycles before treatment; 16.3, 21.4, 26.3, and 7.6 ng/ml during vehicle treatment; and 9.7, 11.2, and 11.1 ng/ml in the three consecutive cycles after vehicle treatment (Fig. 4A). The other two monkeys treated with vehicle (no. 1178 and 1144) showed similar steroid hormone dynamics on the whole (Fig. 4, B and C). In contrast, treatment of cynomolgus monkeys, which had displayed normal cyclicity, with TAK-013 at a total daily dose of 90 mg/kg for approximately 80 d resulted in continuous suppression of serum estradiol and progesterone levels (Fig. 5) and abolishment of the cyclicity (Table 2). After the discontinuation of TAK-013 administration, the normal cyclicity and steroid hormone profiles resumed immediately (Fig. 5 and Table 2). In monkey no. 776, the maximal estradiol levels fell from 239, 657, and 342 pg/ml in the three consecutive cycles before treatment to 109 pg/ml during the TAK-013 treatment, and recovered to 665, 212, and 257 pg/ml in the three consecutive cycles after the TAK-013 treatment (Fig. 5A). The maximal progesterone levels in this monkey fell from 20.9, 13.6, and 22.3 ng/ml in the three consecutive cycles before treatment to 2.9 ng/ml during the TAK-013 treatment and recovered to 40.9, 37.8, and 15.3 ng/ml in the three consecutive cycles after the TAK-013 treatment (Fig. 5A). The other two monkeys treated with TAK-013 (no. 1065 and 1173) showed similar steroid hormone dynamics on the whole (Fig. 5, B and C). The decrease of estradiol levels by TAK-013 (P < 0.05) and the increase of estradiol levels after the cessation of TAK-013 (P < 0.01) were statistically significant, and the change of estradiol levels between the pretreatment and posttreatment periods was not statistically significant (P > 0.05).

View larger version (40K): [in this window] [in a new window]   Figure 4. Serum estradiol and progesterone levels in individual cynomolgus monkeys before, during, and after treatment with the vehicle. Days represent time from the beginning of treatment, and the duration of treatment is indicated with a thick horizontal bar.

View larger version (39K): [in this window] [in a new window]   Figure 5. Serum estradiol and progesterone levels in individual cynomolgus monkeys before, during, and after the treatment with TAK-013 at 90 mg/kg·d. Days represent time from the beginning of treatment, and the duration of treatment is indicated with a thick horizontal bar.

Marmoset monkeys treated with vehicle displayed normal progesterone profile (Fig. 6, left panels, and Table 3). In contrast to cynomolgus monkeys, treatment of marmoset monkeys with TAK-013 at a total daily dose of 90 mg/kg for 70–79 d did not suppress the plasma progesterone levels on the whole (Fig. 6, right panels) and did not abolish the cyclicity (Fig. 6, right panels, and Table 3).

View larger version (46K): [in this window] [in a new window]   Figure 6. Plasma progesterone levels in individual marmoset monkeys before, during, and after treatment with vehicle (left) or TAK-013 at 90 mg/kg·d (right). Days represent time from the beginning of treatment, and the duration of treatment is indicated with a thick horizontal bar.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Interestingly, nonpeptide GnRH antagonists have species specificity (6, 7, 18). TAK-013 has a 220-fold more potent antagonistic property for the human GnRH receptor than for the monkey receptor (7). In this study, TAK-013 did not suppress GnRH-stimulated LH release from marmoset pituitary cells and consistently did not affect the steroid hormone profiles in gonadally intact female marmoset monkeys either, demonstrating that TAK-013 could not possess GnRH antagonistic properties in marmoset monkeys. GnRH receptor cDNA sequence of marmoset monkeys has been published (19), and that of cynomolgus monkeys has been cloned and sequenced in our company (our unpublished data). They are different at 12 amino acids: residues at positions 15, 16, 19, 50, 113, 140, 152, 153, 169, 222, 264, and 273. Currently, it remains to be clarified which residues are involved in this species difference. It was stated that Phe³¹³ of the human GnRH receptor is critical for the species difference of the binding affinity of a nonpeptide quinolone-based GnRH antagonist between human and dog (18). However, because Phe³¹³ is common in both cynomolgus and marmoset monkeys, Phe³¹³ does not seem to be related to the species specificity observed in this study.

Our data demonstrate that TAK-013 suppresses the GnRH-stimulated LH release to the basal level in cynomolgus monkey pituitary cells. Consistently, our results show that TAK-013 abrogates the LH surges and has a tendency to suppress the basal serum LH levels in female cynomolgus monkeys. In addition, TAK-013 clearly suppressed the elevated serum LH levels in castrated male monkeys (7). Therefore, the inhibitory effect of TAK-013 on the menstrual cycles of sex steroid hormones in cynomolgus monkeys could be exerted at least at the pituitary level. Although the GnRH receptor gene is expressed in the human ovary (20) and GnRH agonists have been reported to possess direct inhibitory effects on steroidogenesis in the rat ovary, it is still controversial whether the receptor is functional in the primate ovary (21). Recently, it was stated that the GnRH agonist, triptorelin, and peptide GnRH antagonists, cetrorelix and ganirelix, exert no effect on steroidogenesis in the human ovary (22). Further study is required to clarify whether a nonpeptide antagonist, TAK-013, exerts an inhibitory or stimulatory effect at the ovarian level.

Our results demonstrate that the serum sex steroid levels and the duration of luteal phase in the posttreatment cycles were not different from those of pretreatment cycles, suggesting that the suppressive effect of TAK-013 on the pituitary-ovarian function should be reversible. Furthermore, the normal menstrual cycles resumed immediately after the discontinuation of chronic treatment with TAK-013. These characteristics of TAK-013 are favorable when used clinically in the gynecological field, because irreversible or prolonged suppression of steroid hormones will cause side effects such as osteoporosis and hot flush and might cause low pregnancy rate in case of assisted reproductive technology.

In summary, our results indicate that TAK-013 suppresses LH release from pituitary cells, and oral administration of TAK-013 continuously and reversibly suppresses a pituitary-ovarian axis in cynomolgus monkeys. Together with the findings that TAK-013 demonstrates a 220-fold more potent antagonistic property for the recombinant human GnRH receptor than for the monkey receptor (7), our data suggest that TAK-013 would be effective for the medical treatment of reproductive disorders, such as endometriosis and leiomyoma, and would be a useful tool for assisted reproductive technology.

Received July 10, 2002.

Accepted December 23, 2002.

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