Effects of Dienogest, a Synthetic Steroid, on Experimental Endometriosis in Rats

European Journal of Endocrinology (1998) 138 216–226 ISSN 0804-4643

Effects of dienogest, a synthetic steroid, on experimental endometriosis in rats

Yukio Katsuki, Yukiko Takano, Yoshihiro Futamura, Yasunori Shibutani, Daisuke Aoki1, Yasuhiro Udagawa1 and Shiro Nozawa1 Toxicology Laboratory, Mochida Pharmaceutical Co. Ltd, Fujieda, Shizuoka 426, Japan and 1Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo 160, Japan (Correspondence should be addressed to Y Katsuki, Toxicology Laboratory, Mochida Pharmaceutical Co. Ltd, 342 Gensuke Fujieda, Shizuoka 426, Japan)

Abstract Objective: Dienogest, a synthetic steroid with progestational activity, is used as a component of oral contraceptives and is currently being evaluated clinically for the treatment of endometriosis. The present study was conducted to confirm the effects of dienogest on experimental endometriosis in rats and to elucidate its mechanism of action. Design: Experimental endometriosis induced by autotransplantation of endometrium in rats. Methods: Endometrial implants, immune system, and bone mineral were investigated after 3 weeks of medication. Results: Dienogest (0.1–1 mg/kg per day, p.o.) reduced the endometrial implant volume to the same extent as danazol (100 mg/kg per day, p.o.). Simultaneously, dienogest ameliorated the endometrial implant-induced alterations of the immune system; i.e. it increased the natural killer activity of peritoneal fluid cells and splenic cells, decreased the number of peritoneal fluid cells, and decreased interleukin-1b production by peritoneal macrophages. In contrast, danazol (100 mg/kg per day, p.o.) and buserelin (30 mg/kg per day, s.c.) had none of these immunologic effects. Additionally, combined administration of dienogest (0.1 mg/kg per day) plus buserelin (0.3 mg/kg per day) suppressed the bone mineral loss induced by buserelin alone, with no reduction of the effect on endometrial implants. In vitro studies on dienogest revealed an antiproliferative effect on rat endometrial cells due to inhibition of protein kinase C activity plus a partial progestational effect. Conclusions: Dienogest appears to be a potent agent with mechanisms of action different from those of danazol and GnRH agonists currently available for the treatment of endometriosis.

European Journal of Endocrinology 138 216–226

Introduction Dienogest, 17a-cyanomethyl-17b-hydroxy-estra-4,9- dien-3-one, is a synthetic steroid that has prominent Endometriosis is an estrogen-dependent disease for progestational activity, and it has been clearly shown to which ovariectomy is the most effective treatment. lack androgenic, estrogenic, anti-estrogenic, and corti- However, many women with endometriosis wish to coid-like activities (8). With this hormonal proﬁle, become pregnant, so medication is the primary treat- dienogest appears to cause few side-effects in its clinical ment for this condition (1). The medications most use. This compound was initially assessed for its contra- commonly used include danazol (2), gonadotropin- ceptive value, and it is now available in Germany as a low- releasing hormone agonists (GnRH-a) (3), and proges- dose pill in combination with ethinylestradiol. Currently, tins such as medroxyprogesterone acetate (MPA) (4). it is being investigated as an agent for the treatment of Although these agents are relatively effective against endometriosis in Germany, France, and Japan. endometriosis, they produce a high incidence of adverse In the present study, the effects of dienogest on a rat reactions. Danazol causes weight gain due to its model of endometriosis were evaluated in comparison anabolic/androgenic activity, as well as abnormal lipid with those of danazol and buserelin, and the effective- metabolism, liver dysfunction, and cerebral thrombosis ness of combined therapy with dienogest plus buserelin (5). MPA causes weight gain and thrombosis (6), was also examined. Both alone and in combination with whereas GnRH-a causes menopause-like symptoms buserelin, dienogest was effective for the treatment of and a decrease in bone mineral density (BMD) experimental endometriosis in rats. The mechanism of attributable to estrogen lack (7). Therefore, the devel- its effect on endometriosis was also investigated by opment of a new agent that is safer than those currently assessing changes in immune function as well as those available for endometriosis would be highly desirable. in cAMP and protein kinase activity.

᭧ 1998 Society of the European Journal of Endocrinology

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Materials and methods vehicle alone. After 3 weeks of treatment, the dimensions of the endometrial implants were measured as Test drugs described above. Then each implant was excised and ﬁxed in 10% phosphate-buffered formalin for histologic Dienogest (Jenapharm GmbH, Jena, Germany), danazol evaluation. The uterus was also removed from each rat (Sigma Chemical Co., St Louis, MO, USA), progesterone and weighed. Moreover, the left femur was removed and (Sigma Chemical Co.), and buserelin (Hoechst Japan, ﬁxed in 10% phosphate-buffered formalin for examina- Tokyo, Japan) were obtained from the indicated sources. tion of BMD. The volumetric BMD of the distal femur RU-486 was kindly provided by Roussel-Uclaf Co. Ltd was determined by peripheral quantitative computed (Romainville, France). tomography (XCT-960A, Norland-Stratec, Fort Atkin- son, WI, USA) (10), and the density was calculated Animals separately for the trabecular, cortical, and total bone.

Eight-week-old female Sprague–Dawley rats were pur- Effect of buserelin or dienogest/buserelin on experi- chased from Charles River Japan Inc. (Yokohama, mental endometriosis Other rats with experimental Japan). The animals were maintained at room tempera- endometriosis were randomly divided into three groups: ture (23Ϯ2 ЊC) and 55Ϯ15% relative humidity in a vehicle alone (control; n = 10), buserelin (0.3 mg/kg per room with a 12 h light/12 h darkness cycle and were day, s.c.; n = 10), and dienogest (0.1 mg/kg per day, p.o.) given a sterilized solid diet (gamma ray-irradiated plus buserelin (0.3 mg/kg per day, s.c.) (n =10).After3 CRF-1, Oriental Yeast, Tokyo, Japan) and free access weeks of treatment, measurement of the endometrial to water during the experimental period. A vaginal implants, histologic examination of the implants, smear test was performed to check the estrous cycle in and assessment of BMD of the left femur were all rats before use in the experiment. At the end of the performed as described above. Additionally, peritoneal study period, the animals were killed by exsanguination fluid was recovered from the peritoneal cavity and the under anesthesia with sodium pentobarbital (50 mg/kg spleen was excised for the assay of natural killer (NK) i.p.). The guidelines of Mochida Pharmaceutical Co. Ltd activity. were followed for the care and use of the animals in this study. Measurement of NK activity Mononuclear cell suspen- sions from the peritoneal fluid and spleen were obtained In vivo studies by centrifugation at 450 g for 30 min at 4 ЊCona Percoll–Conray gradient (Dai-ichi Chemical Co., Tokyo, Preparation of a rat model of endometriosis At the Japan), and then the number of cells was adjusted to age of 9 weeks, experimental endometriosis was induced 1×106/ml. The NK assay was performed according to in rats at the proestrous stage of the estrous cycle by the method of Blomberg et al. (11). In brief, mono- autotransplantation of endometrium to a renal sub- nuclear cells were washed in RPMI 1640 supplemented capsular site (9). After laparotomy under anesthesia with 10% fetal calf (FCS) (Dainippon Pharmaceutical with sodium pentobarbital (50 mg/kg i.p.), the left Co., Osaka, Japan) prior to being combined with the uterine horn was resected, and the endometrium was × target cells, which were K562 cells (Dainippon Phar- dissected from the myometrium. Then a 5 5mm maceutical Co.) prelabeled with europium-DTPA(Sigma fragment of endometrial tissue was transplanted Chemical Co.). One hundred microliters (102 cells) of beneath the left renal capsule of the same animal. the labeled target cell suspension and 100 ml (104 cells) Two weeks later, a second laparotomy was done to of the effector cell suspension (effector to target ratio determine the viability of the endometrial implant from 100:1) were added to each well of a microplate (Nunc, the accumulation of fluid, and the dimensions (length, Roskilde, Denmark), and the cells were incubated at width, and height) of the implant were measured 37 ЊC for 4 h under a humidified atmosphere of 5% to calculate its volume. Animals with implants having 3 CO2–95% air. Then the supernatant was collected and a volume between 20 and 60 mm were used in the europium fluorescence was determined with a time- study. resolved fluorometer (DELFIA, Pharmacia Biotech Co., Uppsala, Sweden). NK activity was expressed as the Effect of various treatments on experimental percentage of lysis of target cells, taking the value for endometriosis Rats with experimental endometriosis cells treated with 2% sodium dodecyl sulfate as 100% were randomly divided into eight groups: vehicle alone and that for cells treated with medium alone as 0%. (control; n = 9), dienogest (0.03 mg/kg per day, p.o.; n = 10), dienogest (0.1 mg/kg per day, p.o.; n = 9), Effect of various treatments on NK activity More dienogest (0.3 mg/kg per day, p.o.; n = 10), dienogest rats with experimental endometriosis were randomly (1 mg/kg per day, p.o.; n = 9), danazol (100 mg/kg per divided into five groups (n = 7/group): vehicle alone day, p.o.; n = 10), buserelin (30 mg/kg per day, s.c.; (control), dienogest (0.1 mg/kg per day, p.o.), danazol n = 9), and an ovariectomized group (n = 9) treated with (100 mg/kg per day, p.o.), buserelin (30 mg/kg per day,

Downloaded from Bioscientifica.com at 09/25/2021 06:11:43PM via free access 218 Y Katsuki and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 138 s.c.), and an ovariectomized group treated with Tokyo, Japan) supplemented with 2% FCS pretreated vehicle alone. Additional age-matched animals with- with charcoal, 0.5% type I collagenase (Nitta Gelatin out endometriosis were used as an intact group Co., Tokyo, Japan), and 0.02% deoxyribonuclease I (n = 5). After 3 weeks of treatment, measurement of (Sigma Chemical Co.) (15). The uterus was incubated the endometrial implants, recovery of peritoneal repeatedly in the same medium, and the endometrial fluid, and splenectomy were carried out as described cells were collected after each 15 min incubation by above. Additionally, the volume of the peritoneal fluid filtering them through a nylon mesh filter (Nippon was determined according to a modification of the Becton Dickinson Co., Tokyo, Japan). The combined method of Carter et al. (12), and the number of cells in filtrate was then centrifuged at 300 g for 5 min at room the fluid was counted under a light microscope. temperature. The cell pellet was washed in culture medium (Dulbecco’s MEM supplemented with 2% FCS Effect of dienogest on interleukin-1b (IL-1b) pretreated with charcoal) and suspended in the same production by peritoneal macrophages A final set medium after adjustment of the cells to 5 × 104/ml. of rats with experimental endometriosis was randomly divided into two groups (n = 10/group): vehicle alone Effect of drugs on the proliferation of rat endo- (control) and dienogest (0.1 mg/kg per day, p.o.). metrial cells treated with b-estradiol One milliliter of Additional age-matched animals without endometriosis endometrial cell suspension (5 × 104 cells) was added to were used as an intact group (n = 5). After 3 weeks of each well of a 6-well plate and incubated at 37 ЊC for treatment, peritoneal macrophages were recovered for 17 h under a humidified atmosphere of 5% CO2–95% the assay of IL-1b production. air. Then 10 mlof17b-estradiol (Sigma Chemical Co.) Isolation of peritoneal macrophages and measurement of solution (4 ng/ml) were added to each well, and IL-1b Peritoneal macrophages were collected from the incubation was continued for an additional 8 h. Next, peritoneal fluid by the adhesion method (13) and were the medium was replaced with medium containing × ¹11 suspended in medium after adjustment to 105 cells/ml. 10 ml of a test agent solution, i.e. dienogest (3.2 10 , × ¹10 × ¹9 × ¹8 An aliquot of the cells (104 in 0.1 ml) was added to each 3.2 10 , 3.2 10 , and 3.2 10 M as final con- × ¹11 × ¹10 × well of a 24-well plate and incubated at 37 C for 24 h centrations), danazol (3.0 10 , 3.0 10 , 3.0 Њ ¹9 × ¹8 × ¹11 under a humidified atmosphere of 5% CO –95% air. 10 , and 3.0 10 M), buserelin (8.1 10 and 2 × ¹9 × ¹10 × Then the medium was centrifuged at 300 g for 5 min 8.1 10 M), or progesterone (3.2 10 , 3.2 ¹9 × ¹8 at 15 ЊC, the supernatant was concentrated with a 10 , and 3.2 10 M). Then the cells were incubated SMART system (Pharmacia Biotech Co.), and the IL-1b for 48 h in the absence or presence of RU-486 (1 ng/ml), level in the concentrate was measured by a time 100 mlof10mM 5-bromodeoxyuridine (BrdU, Sigma resolved-fluoroimmunoassay using the DELFIA system Chemical Co.) solution was added to each well, and (14). Briefly, rabbit anti-murine IL-1b antibody (Gen- incubation was continued at 37 ЊC for an additional 2 h. zyme Co., Cambridge, MA, USA) was added to each The supernatant was collected for the determination of well of a 96-well microplate precoated with anti-rabbit lactate dehydrogenase (LDH) activity, and the cells were IgG antibody and was incubated at room temperature washed with PBS (pH 7.4) prior to the measurement of for 1 h with shaking. Then each well was washed BrdU uptake. with DELFIA washing solution and blocked with Block Ace (Dainippon Pharmaceutical Co.). Next, the Measurement of BrdU uptake Cells were fixed with methyl concentrated supernatant and europium-labeled alcohol at room temperature for 30 min and then washed murine IL-1b (Genzyme Co.) were added to each well with PBS containing 0.1% Tween 20 (washing buffer). and incubated at room temperature for 1 h. After the Subsequently, a blocking agent solution (Block Ace, wells had been washed with DELFIA washing solution, Dainippon Pharmaceutical Co.) was added to each well. DELFIA enhancement solution was added; and incu- After preincubation for 30 min at room temperature, the bation was continued at room temperature for 5 min cells were incubated with an anti-BrdU antibody with shaking. Finally, europium fluorescence was and deoxyribonuclease (Amersham International plc., measured with a DELFIA fluorometer, and the IL-1b Buckinghamshire, UK) solution at room temperature for content was calculated from the standard curve 1 h. After washing of the cells with the washing buffer, the prepared previously. europium-labeled anti-mouse IgG antibody (Pharmacia Biotech Co.) was applied as described above (16). Europium fluorescence was measured with a DELFIA In vitro studies fluorometer, and the BrdU content was calculated from Isolation of rat endometrial cells Under anesthesia the standard curve prepared previously. with sodium pentobarbital (50 mg/kg, i.p.), the uterus was removed from 24 rats and immediately placed into Measurement of LDH leakage The incubation medium PBS. Each uterus was turned inside out and then of cells treated with various agents was centrifuged at incubated for 15 min at 37 ЊC with shaking in 300 g for 5 min at 15 ЊC, and the LDH activity in the Dulbecco’s medium (Nissui Pharmaceutical Co., supernatant was determined with an MTX LDH-kit

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had been added to each well of a 96-well microplate, the solution was decanted; and the microplate was washed with PBS (pH 6.8) containing 0.05% Tween 80 (washing buffer). Then the wells were blocked with Block Ace and washed again with the washing buffer. Next, 10 ml of endometrial cell homogenate (104 cells) and 100 ml of murine anti-cAMP antibody (Cayman Chemical Co.) were added to each well and incubated at 20 ЊC for 2 h with stirring. After discarding the solution and washing the wells with the washing buffer, 100 ml of europium-labeled anti-mouse IgG antibody was added to each well (18). Europium fluorescence was measured with a DELFIA fluorometer, and the cAMP content was calculated from the standard curve prepared previously. Measurement of PKA and PKC activities The activities of PKA and PKC were measured with a non-radioisotopic protein kinase assay kit (MBL Co. Ltd, Nagoya, Japan) modified for a time-resolved fluoroimmunoassay using the DELFIA system (19). Microplate wells coated with a synthetic peptide (PS-peptide: R-F-A-R-K-G-A-L-R-Q-K- N-V) were loaded with 12 ml of endometrial cell homogenate (104 cells) along with 108 ml of a reaction mixture containing 25 mM Tris–HCl (pH 7.0), 3 mM MgCl2, 0.1 mM ATP, 0.5 mM EDTA, 1 mM EGTA, and 5 mM 2-mercaptoethanol. For the PKA assay, 2 mM cAMP solution was added to the mixture, whereas 50 mg/ml phosphatidylserine and 2 mM CaCl2 were added for the PKC assay. After incubation at 25 ЊC for 5 min, the reaction was stopped by the addition of Figure 1 Effect of dienogest, danazol, buserelin, ovariectomy, or 100 ml of 20% H3PO4 solution. Then the wells were dienogest/buserelin on experimental endometriosis in rats. Upper washed, 100 ml of mouse anti-phosphorylated PS- panel: effect of dienogest (0.03, 0.1, 0.3 or 1 mg/kg per day, p.o.), peptide antibody (clone 2B9) were added to each well, danazol (100 mg/kg per day, p.o.), buserelin (30 mg/kg per day, s.c.), or ovariectomy (OVX). Lower panel: effect of buserelin (0.3 mg/ and incubation was carried out at 25 ЊC for 30 min with kg per day, s.c.) or dienogest (0.1 mg/kg per day, p.o.) plus shaking. After further washing, 100 ml of europium- buserelin (0.3 mg/kg per day, s.c.). Closed circles represent the labeled anti-mouse IgG antibody were added to each volume of the endometrial implants (mm3) in individual rats, and well. Europium fluorescence was measured with a open circles show the meanϮS.D. for each group. Data were DELFIA fluorometer, and the activities of PKA and analyzed by Dunnett’s test. Significant differences from the control group are marked: *P < 0:05, **P < 0:01. PKC were calculated from the standard curves prepared previously. One unit of enzyme activity was defined as (Kyokuto Kagaku Co., Tokyo, Japan) (17). LDH leakage the amount of enzyme producing 1 mmol of phosphory- was assessed from the absorbance at 560 nm. lated peptide per minute.

Effect of drugs on the cAMP content, protein kinase Statistical analysis A (PKA) activity, and protein kinase C (PKC) activity The results of each experiment were expressed as the of rat endometrial cells treated with b-estradiol The meanϮS.D. Dunnett’s test was used to assess the cAMP content and PKA and PKC activities were measured signiﬁcance of differences between groups in multiple after treatment of endometrial cells with dienogest comparisons. For comparison between two groups, × ¹11 × ¹10 × ¹9 × ¹8 (3.2 10 ,3.2 10 ,3.2 10 , and 3.2 10 M Student’s t-test was used. as ﬁnal concentrations), danazol (3.0 × 10¹11,3.0× ¹10 × ¹9 × ¹8 10 ,3.0 10 , and 3.0 10 M), or progesterone Results (3.2 × 10¹11,3.2×10¹10,3.2×10¹9, and 3.2 × 10¹8 M) in the absence or presence of RU-486 (1 ng/ml) as In vivo studies described above. Effect of various treatments on experimental Measurement of cAMP After 100 ml of cAMP tracer endometriosis At a dose of 0.03 mg/kg, dienogest did solution (Cayman Chemical Co., Ann Arbor, MI, USA) not decrease the volume of endometrial implants,

Downloaded from Bioscientifica.com at 09/25/2021 06:11:43PM via free access 220 Y Katsuki and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 138 whereas all the other doses tested (0.1, 0.3, and 1 mg/ implant cyst were composed of epithelium and stroma. kg) caused a significant decrease. The effect of dienogest In the control group, the implants showed histologic was comparable to that of danazol, but less marked than features reflecting the estrous cycle-dependent nature of that of buserelin or ovariectomy, both of which also the uterine epithelium. For example, the implants from significantly decreased the implant volume compared control rats at the proestrous stage were composed of with that of the control (Fig. 1, upper panel). tall columnar epithelial cells with large, round nuclei On histologic examination (Fig. 2), cysts protruding and slightly hypertrophic stromal cells (Fig. 2A and B). from the renal surface and covered by the renal capsule In the dienogest-treated groups, the implants displayed were found at the sites of all implants. The size of the epithelial cells similar to those of control rats at the cyst observed microscopically reflected the volume of proestrous stage (Fig. 2C and D). The implants of the implant at final laparotomy. The walls of each danazol-treated rats resembled those of dienogest- treated rats (Fig. 2E and F). In the buserelin-treated rats, the implants were composed of a thin, atrophic epithelium with pyknotic nuclei (Fig. 2G and H). In the ovariectomized rats, the epithelium was even more atrophic than in the buserelin-treated rats, and a sparse nuclear arrangement was noted (Fig. 2I and J), which was similar to that of the uterus during diestrus. The uterine weight in the dienogest-treated rats (192.8Ϯ48.9 mg) was comparable to that of the control rats (195.4Ϯ44.1 mg), whereas buserelin significantly decreased the uterine weight (74.0Ϯ12.5 mg, P<0.01). None of the doses of dienogest tested had any effect on the BMD of the distal femoral trabecular and cortical bone. On the other hand, danazol, buserelin, and ovariectomy caused a significant decrease in the BMD of trabecular bone in the distal femur. Bone mineral loss increased in the order of danazol

Effect of buserelin or dienogest/buserelin on experimental endometriosis A dose of 0.3 mg/kg of buserelin signiﬁcantly reduced the volume of the endometrial implants. Treatment with dienogest (0.1 mg/kg) plus buserelin (0.3 mg/kg) in combination also signiﬁcantly

Figure 2 Representative histologic appearance of experimental endometriosis and high-power views of the cyst walls of endometrial implants in a control rat at the proestrous stage (A, B) and in rats given dienogest (0.1 mg/kg per day, p.o.) (C, D), danazol (100 mg/ kg per day, p.o.) (E, F), buserelin (30 mg/kg per day, s.c.) (G, H), and ovariectomy (I, J). The left column of photomicrographs shows cysts protruding from the renal surface and encapsulated by the renal capsule. All the endometrial implants formed cysts. A decrease in the size of the cyst cavity is noticeable in the dienogest (C), danazol (E), buserelin (G), and ovariectomized (I) rats. The right column shows high-power views of the cyst walls composed of epithelium and stroma, indicating that they are endometrial tissues. In the control rat (B), tall columnar epithelial cells with large, round nuclei and slightly hypertrophic stromal cells are seen, and the histologic appearance coincides with that in the uterus at the proestrous stage. The dienogest (D) and danazol (F) rats show similar ﬁndings, i.e. the epithelium resembles that of the control rat at the proestrous stage. In the buserelin rat (H), the epithelium is atrophic. In the ovariectomized rat (J), the epithelium is more atrophic than in the buserelin-treated rat, and a sparse nuclear arrangement is seen. This is similar to that of the uterus at the diestrous stage. Hematoxylin and eosin stain; × 15 in A, C, E, G, and I; × 250 in B, D, F, H, and J.

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Table 1 Effect of treatment with dienogest, danazol, buserelin, or ovariectomy on BMD of the distal left femur in rats with experimental endometriosis.

BMD (mg/cm3) Dose Treatment (mg/kg per day) Total Trabecular Cortical

Control 0 702.3 Ϯ 31.6 428.1 Ϯ 44.7 940.3 Ϯ 38.8 Dienogest 0.03 693.8 Ϯ 48.5 403.7 Ϯ 84.2 949.8 Ϯ 31.9 0.1 739.1 Ϯ 65.4 471.7 Ϯ 64.8 943.2 Ϯ 32.7 0.3 729.9 Ϯ 84.6 456.3 Ϯ 109.2 944.0 Ϯ 15.9 1 709.3 Ϯ 49.0 434.3 Ϯ 69.3 936.8 Ϯ 45.8 Danazol 100 678.1 Ϯ 38.5 381.9 Ϯ 30.5** 947.3 Ϯ 40.8 Buserelin 0.03 639.5 Ϯ 79.3* 377.2 Ϯ 99.2** 911.4 Ϯ 38.2 Ovariectomy — 608.3 Ϯ 61.3** 329.3 Ϯ 84.1** 914.8 Ϯ 26.4

All values are expressed as the mean Ϯ S.D. Data were analyzed by Dunnett’s test. *P < 0:05, **P < 0:01 vs control group. decreased the implant volume and tended to reduce it more than buserelin alone, although there was no significant difference between the buserelin and dienogest/buserelin groups (Fig. 1, lower panel). On histologic examination of control rats at the diestrous stage, the epithelium of the implant was atrophic and showed a sparse nuclear arrangement (Fig. 3A and B). In the buserelin-treated rats, the columnar epithelium was low and displayed a dense nuclear arrangement (Fig. 3C and D). In the dienogest/buserelin-treated rats, the epithelium was also low, but it had a sparse nuclear arrangement (Fig. 3E and F). These findings were more similar to those for the control rats at the diestrous stage than to those for the buserelin-treated rats. On bone mineral examination, the buserelin-treated rats showed a significant decrease in the BMD of distal femoral trabecular bone. In contrast, combination therapy with dienogest and buserelin had no perceptible influence on BMD, which remained at the control level (Table 2).

Effect of various treatments on NK activity The NK activity of peritoneal fluid cells and splenic cells was significantly decreased by renal subcapsular endometrial tissue autotransplantation (Table 3). A dose of 0.1 mg/kg of dienogest stimulated a significant increase in the NK activity of both peritoneal fluid cells and splenic cells, and the NK activity of these animals practically reached that of the intact group. Danazol (100 mg/kg) slightly increased the NK activity of peritoneal fluid cells, but had no positive effect on NK activity in the spleen. Treatment with buserelin or Figure 3 Representative histologic appearance of experimental ovariectomy had no effect on the NK activity of cells endometriosis in a control rat at the diestrous stage (A, B), and in from the peritoneal fluid or spleen. Thus, NK activity in rats given buserelin (0.3 mg/kg per day, s.c.) (C, D) and dienogest the buserelin-treated or ovariectomized rats remained at (0.1 mg/kg per day, p.o.) plus buserelin (0.3 mg/kg per day, s.c.) (E, F). The control rat at the diestrous stage has epithelium similar to the control level, which was dramatically lower than that of the ovariectomized rat (Fig. 2J). In the buserelin rat, the that in the intact group. epithelium is low and has a dense nuclear arrangement. It is similar Dienogest also significantly decreased the number of to that in the buserelin (30 mg/kg per day, s.c.) rat (Fig. 2H). In the cells in the peritoneal fluid, whereas treatment with dienogest plus buserelin rat, the epithelium is also low, but it danazol, buserelin, or ovariectomy had no such effect. displays a sparse nuclear arrangement. It is not similar to that seen with buserelin or dienogest (Fig. 2D) monotherapy, but resembles The peritoneal fluid volume remained unchanged in all that of the uterus at the diestrous stage. Hematoxylin and eosin groups, including the intact one (data not shown). stain; × 15 in A, C, and E; × 250 in B, D, and F.

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Table 2 Effect of treatment with buserelin alone or dienogest plus buserelin on BMD of the distal left femur in rats with experimental endometriosis.

BMD (mg/cm3) Dose Treatment (mg/kg per day) Total Trabecular Cortical

Control 0 729.2 Ϯ 43.9 463.1 Ϯ 52.7 936.4 Ϯ 39.9 Buserelin 0.0003 654.4 Ϯ 41.6** 386.4 Ϯ 62.6** 924.4 Ϯ 20.8 Dienogest + buserelin 0.1 plus 0.0003 699.0 Ϯ 33.3## 433.1 Ϯ 34.6# 936.6 Ϯ 22.6

All values are expressed as the mean Ϯ S.D. Data were analyzed by Student’s test. **P < 0:01 vs control group. #P < 0:05, ##P <0:01 vs buserelin group.

The volume of the endometrial implants was sig- inhibited BrdU uptake by rat endometrial cells in a nificantly decreased by treatment with dienogest concentration-dependent manner (Table 4). Danazol (13.0Ϯ9.1, P < 0.01 vs control; 35.6 Ϯ 18.9 mm3), (3.0 × 10¹8 M) and progesterone (3.2 × 10¹8 M) also danazol (13.7 Ϯ 18.2 mm3, P < 0.01), buserelin (3.7 Ϯ significantly suppressed BrdU uptake. However, 4.5 mm3, P < 0.01), or ovariectomy (1.8 Ϯ 3.3 mm3, buserelin (8.1 × 10¹9 M) had no inhibitory effect on the P < 0.01), just as occurred in the experiment shown in uptake. In the presence of RU-486 (1 ng/ml), dienogest Fig. 1 (upper panel), indicating that data obtained with (Ն3.2 × 10¹10 M) also significantly inhibited BrdU this model were reproducible. uptake by rat endometrial cells in a concentration- Additionally, combination therapy with dienogest dependent manner. However, the inhibitory effect of and buserelin significantly enhanced the NK activity of dienogest was slightly reduced by RU-486. On the other both peritoneal fluid cells and splenic cells. hand, the suppressive effects of danazol and progesterone were abolished in the presence of RU-486. Additionally, Effect of dienogest on IL-1b production by perito- dienogest, danazol, and progesterone had no positive neal macrophages A significant increase in IL-1b effect on LDH leakage from the cells under these production by peritoneal macrophages from rats with conditions. endometriosis was observed compared with the production in the intact group (Fig. 4). Treatment with Effect of drugs on cAMP content, PKA activity, dienogest (0.1 mg/kg) significantly decreased IL-1b and PKC activity of rat endometrial cells treated production by peritoneal macrophages. with b-estradiol In the absence or presence of RU-486, dienogest (Ն3.2 × 10¹10 M) significantly reduced the In vitro PKC activity of rat endometrial cells (Table 5). This studies inhibitory action was specific to dienogest alone and Effect of drugs on the proliferation of rat endo- was not observed with danazol or progesterone. In the metrial cells treated with b-estradiol In the absence absence of RU-486, dienogest, danazol, and progester- of RU-486, dienogest (Ն3.2 × 10¹10 M) significantly one at their maximal concentrations significantly

Table 3 Effect of treatment with dienogest, danazol, buserelin, or ovariectomy alone, or with dienogest plus buserelin on the NK activity of peritoneal ﬂuid and splenic mononuclear cells and the number of peritoneal ﬂuid cells in rats with experimental endometriosis.

NK activity (% lysis) Number of peritoneal DosePeritoneal Splenic ﬂuid cells Treatment (mg/kg per day) ﬂuid cells cells ( × 106 cells/ml)

Control 0 6.8 Ϯ 0.3 8.4 Ϯ 0.6 5.1 Ϯ 0.5 Dienogest 0.1 17.1 Ϯ 0.6** 14.5 Ϯ 1.1** 3.1 Ϯ 0.7** Danazol 100 9.6 Ϯ 3.4** 9.5 Ϯ 1.8 4.7 Ϯ 0.6 Buserelin 0.03 7.1 Ϯ 0.3 8.5 Ϯ 0.7 5.5 Ϯ 1.0 Ovariectomy – 6.9 Ϯ 0.4 8.6 Ϯ 0.4 5.4 Ϯ 0.7 Intact – 19.5 Ϯ 0.6## 16.7 Ϯ 0.2## 3.2 Ϯ 0.5##

Control 0 6.9 Ϯ 0.3 8.6 Ϯ 0.5 NT Buserelin 0.0003 6.9 Ϯ 0.3 9.0 Ϯ 0.3 NT Dienogest + buserelin 0.1 plus 0.0003 16.5 Ϯ 1.4** 12.8 Ϯ 1.0** NT

All values are expressed as the mean Ϯ S.D. Data were analyzed by Dunnett’s test, except that for the intact group. **P < 0:01 vs control. ##P <0:01 for control vs intact group (Student’s t-test). NT, not tested.

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models of endometriosis (9, 21, 22). In the present study, we used a rat model of renal subcapsular endometrial tissue autotransplantation, because the good blood supply there ensures a high take rate for the implants. In this model, both danazol and buserelin significantly decreased the volume of endometrial implants, and the histologic findings corresponded to those described previously (22). In addition, this model showed a response to pregnancy (data not shown), which has been demonstrated to reduce endometriosis in humans. Accordingly, this seemed to be a valid model to assess the effect of dienogest compared with those effects of danazol and buserelin, and we were able to demon- strate that dienogest has some promising characteristics Figure 4 Effect of dienogest (0.1 mg/kg per day, p.o.) on interleukin- 1b production by peritoneal macrophages from rats with experi- as a possible therapeutic agent for endometriosis. mental endometriosis. Data are expressed as the meanϮS.D. and Dienogest significantly decreased the volume of were analyzed by Student’s t-test. A significant difference between endometrial implants, suggesting its effect on endo- the control and the intact groups is shown as ##P < 0:01; and that metriosis. However, the drug did not reduce the uterine from the control group, as **P < 0:01. weight in this model and had no effect on the release of luteinizing hormone and follicle-stimulating hormone decreased the cAMP content and PKA activity. In the from the pituitary gland at doses effective for implant presence of RU-486, however, this inhibitory action was volume reduction in this model (8), indicating a abolished. difference between this drug and GnRH-a in the mechanism of action. It has been shown clinically Discussion that surgical ovariectomy or medical ovariectomy with GnRH-a induces bone mineral loss. We therefore For investigation of potential endometriosis due to evaluated the effect of dienogest, danazol, buserelin, proliferation of the endometrial glands and stroma and ovariectomy on BMD. Our results showed that both outside the uterine cavity, experimental rat models have ovariectomy and buserelin treatment caused a decrease been created by autotransplantation of endometrial in the BMD of distal femoral trabecular bone. Unexpect- tissue into the abdominal wall (20), intestinal mesen- edly, danazol resembled buserelin in its action on bone. tery (21), or renal subcapsular space (9). Therapeutic Although clinical effects of danazol on bone are agents such as buserelin, danazol, and leuprolide controversial (23, 24), our finding on BMD is supported acetate are reported to be effective in these animal further by our observation that danazol increased the

Table 4 Direct effect of dienogest, danazol, buserelin, or progesterone on the proliferation of rat endometrial cells treated with b-estradiol.

Without RU-486 With RU-486 (1 ng/ml)

BrdU uptake LDH leakage BrdU uptake LDH leakage 4 4 Treatment Concentration (M) (pmol/10 cells) (absorbance at 560 nm) (pmol/10 cells) (absorbance at 560 nm)

Control (DMSO) 0 67.1 Ϯ 0.6 0.037 Ϯ 0.001 67.5 Ϯ 0.2 0.037 Ϯ 0.001 Dienogest 3:2 × 10¹11 66.8 Ϯ 0.6 0.035 Ϯ 0.002 67.2 Ϯ 0.2 0.038 Ϯ 0.002 3:2 × 10¹10 63.2 Ϯ 0.2** 0.036 Ϯ 0.002 62.2 Ϯ 0.8** 0.037 Ϯ 0.001 3:2 × 10¹9 52.2 Ϯ 0.1** 0.032 Ϯ 0.001** 59.3 Ϯ 1.4** 0.036 Ϯ 0.001 3:2 × 10¹8 35.5 Ϯ 0.4** 0.026 Ϯ 0.001** 56.4 Ϯ 2.2** 0.033 Ϯ 0.001** Danazol 3:0 × 10¹11 67.0 Ϯ 1.2 0.038 Ϯ 0.001 66.7 Ϯ 1.2 0.037 Ϯ 0.001 3:0 × 10¹10 66.9 Ϯ 1.2 0.038 Ϯ 0.002 67.6 Ϯ 1.5 0.037 Ϯ 0.001 3:0 × 10¹9 65.8 Ϯ 0.2 0.037 Ϯ 0.001 66.8 Ϯ 0.5 0.036 Ϯ 0.001 3:0 × 10¹8 63.5 Ϯ 0.3** 0.033 Ϯ 0.001** 64.9 Ϯ 2.2 0.036 Ϯ 0.001 Buserelin 8:1 × 10¹11 66.6 Ϯ 0.3 0.035 Ϯ 0.001 NT NT 8:1 × 10¹9 66.1 Ϯ 0.4 0.036 Ϯ 0.003 NT NT Progesterone 3:2 × 10¹10 67.5 Ϯ 0.4 0.038 Ϯ 0.001 67.1 Ϯ 0.3 0.037 Ϯ 0.002 3:2 × 10¹9 65.7 Ϯ 1.8 0.037 Ϯ 0.001 67.0 Ϯ 0.7 0.037 Ϯ 0.001 3:2 × 10¹8 53.5 Ϯ 2.6** 0.036 Ϯ 0.001** 67.3 Ϯ 0.4 0.037 Ϯ 0.001

All values are expressed as the mean Ϯ S.D. of triplicate determinations. Data were analyzed by Dunnett’s test. **P < 0:01 vs control. DMSO, dimethylsulfoxide; NT, not tested.

Downloaded from Bioscientifica.com at 09/25/2021 06:11:43PM via free access 224 Y Katsuki and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 138 cells) 0.01 0.00 0.02** 0.01** 0.01** 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 4 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ (1 ng/ml) cells) (mU/10 -estradiol. 0.02 0.20 0.050.060.160.02 0.19 0.05 0.11 0.04 0.12 0.01 0.12 0.04 0.19 0.04 0.19 0.02 0.19 0.03 0.19 0.02 0.20 0.20 0.20 0.20 4 b Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ cells) (mU/10 4 0.02 0.40 0.020.000.010.010.01 0.39 0.00 0.35 0.01 0.41 0.01 0.35 0.01 0.38 0.02 0.36 0.03 0.35 0.02 0.38 0.36 0.36 0.38 0.39 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 01 vs control. DMSO, dimethylsulfoxide. : 0 <

P cells) (pmol/10 0.01 0.54 0.000.01**0.00**0.01** 0.55 0.55 0.01 0.55 0.01 0.55 0.010.01 0.55 0.01 0.55 0.01 0.54 0.00 0.55 0.00 0.56 0.56 0.55 0.56 4 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ cells) (mU/10 0.05 0.19 0.030.020.010.02** 0.19 0.08 0.13 0.05 0.12 0.12 0.040.01** 0.19 0.05 0.19 0.02 0.19 0.19 0.030.03** 0.20 0.20 0.20 0.20 4 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Without RU-486 With RU-486 cells) (mU/10 4 0.01 0.45 0.010.010.010.01**0.01 0.39 0.00 0.41 0.01 0.39 0.23 0.02**0.01 0.40 0.01 0.37 0.01 0.40 0.27 0.02** 0.42 0.38 0.38 0.23 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ cAMP content PKA activity PKC activity cAMP content PKA activity PKC activity 0.55 0.55 0.55 0.33 0.55 0.55 0.54 0.44 0.55 0.56 0.55 0.42 of triplicate determinations. Data were analyzed by Dunnett’s test. ** S.D. (M) (pmol/10 Ϯ 11 10 9 8 11 10 9 8 11 10 9 8 ¹ ¹ ¹ ¹ ¹ ¹ ¹ ¹ ¹ ¹ ¹ ¹ 10 10 10 10 10 10 10 10 10 10 10 10 × × × × × × × × × × × × 2 2 2 2 0 0 0 2 2 2 0 2 : : : : : : : : : : : : 3 3 3 3 3 3 3 3 3 Direct effect of dienogest, danazol, or progesterone on the cAMP content, PKA activity, and PKC activity of rat endometrial cells treated with Dienogest 3 DanazolProgesterone 3 3 All values are expressed as the mean Control (DMSO) 0 0.55 Treatment Concentration Table 5

Downloaded from Bioscientifica.com at 09/25/2021 06:11:43PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 138 Effects of dienogest on experimental endometriosis 225 production of bone-resorptive cytokines by mouse RU-486, showing that their inhibitory action was osteoblasts in vitro (data not shown). These findings dependent on their progestational effect alone. suggest that danazol stimulates bone resorption in rats In order to investigate the antiproliferative action of with endometriosis. In contrast, dienogest had no effect dienogest, we then assessed its effect on intracellular on BMD in this model, which would make this drug signaling systems. Dienogest inhibited PKC activity, and preferable to avoid this unwanted side-effect associated this action was not influenced by RU-486. Therefore, with the treatment of endometriosis. For the control of this inhibitory action was clearly different from a adverse effects due to hypoestrogenism induced by progestational effect and may be specific to dienogest. GnRH-a, combination therapy with GnRH-a and a On the contrary, danazol and progesterone had no effect gestagen (e.g. MPA) has been proposed for the treatment on PKC activity. Moreover, the inhibitory actions of of endometriosis (25). Because dienogest has progesta- dienogest, danazol, and progesterone on intracellular tional activity and no effect on BMD, we also evaluated cAMP content and PKA activity were dependent on the effect of a combination of dienogest with buserelin their progestational activity. on experimental endometriosis. Dienogest clearly demon- Because MPA, having progestational activity, is clini- strated a striking protective effect against buserelin- cally as effective as danazol and has fewer side-effects induced bone mineral loss without reducing the potency than danazol (25), we consider that further study is of the effect on endometrial implants, indicating that needed to clarify the difference between dienogest and dienogest is a promising agent in combination with MPA in the treatment of experimental endometriosis. buserelin for the treatment of endometriosis. In conclusion, dienogest may be a potent agent for Recently, some investigators have reported an endometriosis with a direct inhibitory action (due to increase in the number of activated macrophages (26) inhibition of PKC activity and an additive progestational and an increase in the levels of IL-1b and tumor effect) on the proliferation of ectopic endometrial tissue. necrosis factor-a (27) in the peritoneal fluid of patients In addition, it normalizes the peritoneal environment, with endometriosis, as well as a decrease in these restores NK activity, and suppresses bone mineral loss, cytokine levels after medical treatment (27, 28). all actions which are clearly lacking in the case of Peritoneal macrophages and macrophage-induced IL-1 danazol and buserelin. Finally, because dienogest clearly may contribute to infertility caused by endometriosis differs in its mechanism of action from GnRH-a and (29). In addition, depressed NK activity has been danazol, it may be useful in combination with GnRH-a reported in patients with endometriosis (30) and in a for the treatment of endometriosis. rat model of experimental endometriosis (31). A significant correlation between reduced peritoneal NK activity and the severity of endometriosis was also Acknowledgements reported (32). Hence, we examined the NK activity of We wish to thank Mr Chihaya Kakinuma, Mr Noritsugu mononuclear cells from the peritoneal fluid and spleen Goda, and Ms Kyoko Motohashi for their technical as well as IL-1b production by peritoneal macrophages assistance. in our rat model. We found that dienogest normalized not only the NK activity but also the number of the cells in the peritoneal fluid and IL-1b production by References peritoneal macrophages, all of which showed abnorm- 1 Shaw RW. Treatment of endometriosis. Lancet 1992 340 1267– alities in the rats with endometriosis compared with 1270. intact rats. These effects of dienogest may combine to 2 Forbes KL & Thomas FJ. Tissue and endocrine responses to improve the peritoneal environment in endometriosis gestrinone and danazol in the treatment of endometriosis. Reproduction, Fertility and Development 1993 5 103–109. and contribute to diminish pain and infertility due 3 Fedele L, Bianchi S, Arcaini L, Vercellini P & Candiani GB. GnRH to endometriosis. In contrast, the NK activity in agonists in the treatment of endometriosis. Acta Europaea the danazol, buserelin, and ovariectomized groups Fertilitatis 1988 19 5–12. remained lower than in the intact group. 4 Metzger DA & Luciano AA. Hormonal therapy of endometriosis. Obstetrics and Gynecology Clinics of North America 1989 16 105– Finally, using rat endometrial cells in vitro, we 122. investigated the mechanism responsible for the effect 5 Barbieri RL & Ryan KJ. Danazol: Endocrine pharmacology and of dienogest. Dienogest directly inhibited the prolifera- therapeutic application. 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Drugs under Experimental and Clinical danazol and progesterone was completely abolished by Research 1997 23 45–62.

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