* Presented in part at the 37th Japanese Congress of Fertility and Sterility, Tokyo, Japan, November 6 to 7, 1992.

Transcription

1 FERTILITY AND STERILITY Copyright 1995 Amerian Soiety for Reprodutive Mediine Vol. 63. No.6, June 1995 Printed on aid-free paper in U. S. A. Danazol onentrations in ovary, uterus, and serum and their effet on the hypothalami-pituitary-ovarian axis during vaginal administration of a danazol suppository* Takahiro Mizutani, M.D.t:J: Shigeru Nishiyama, M.D.t Ihiro Amakawa, M.D.t Akitoshi Watanabe, M.D.t Karo Nakamuro, M.D.t Nobuyuki Terada, M.D. Osaka Prefetural Hospital, and The Center for Adult Diseases, Osaka, Japan Objetive: To determine danazol onentrations in the ovary, uterus, and serum during daily vaginal administration of a danazol suppository and to examine its effet on the hypothalamipituitary-ovarian axis. Design: Sampling of tissues after vaginal or oral administration of danazol and sampling of blood during ontrol and danazol-administration menstrual yles. Setting: Outpatient volunteers and inpatients at a publi hospital. Partiipants: Thirty patients who were to undergo hysteretomy and oophoretomy beause of uterine leiomyoma and eight regularly menstruating volunteers. Interventions: Danazol was administered as a vaginal suppository (1 mg) or orally (4 mg). Main Outome Measure: Danazol onentrations in the ovary, uterus, and serum, and serum E2 and P levels. Results: Danazol onentrations in the ovary and uterus after daily vaginal administration of a suppository ontaining 1 mg danazol were omparable to those after daily oral administration of 4 mg danazol, but the serum danazol onentration was muh lower. Menstrual yle patterns of serum E2 and P levels were normal during daily vaginal administration of a danazol suppository. Conlusion: Daily administration of a suppository ontaining 1 mg danazol produes high ovarian and uterine onentrations but low serum onentrations, and no effet was deteted on the hypothalami-pituitary-ovarian axis. Fertil Steril 1995;63: Key Words: Danazol, suppository, vaginal administration, danazol onentration, serum estradiol, serum progesterone, ovary, uterus Reeived July 25, 1994; revised and aepted January 1, * Presented in part at the 37th Japanese Congress of Fertility and Sterility, Tokyo, Japan, November 6 to 7, t Department of Obstetris and Gyneology, Osaka Prefetural Hospital. :j: Reprint requests: Takahiro Mizutani, M.D., Department of Obstetris and Gyneology, Osaka Prefetural Hospital, Bandai-Higashi, Sumiyoshi-ku, Osaka 558, Japan (FAX: ). Department of Pathology, The Center for Adult Diseases Mizutani et al. Vaginal administration of danazol Danazol, a syntheti derivative of 17 a-ethinyltestosterone, has been used widely in the treatment of endometriosis (1, 2). Oral administration of 4 to 6 mg/d results in high serum onentrations of danazol, whih may produe androgeni side effets, suh as ane, hirsutism, and weight gain (3, 4). Danazol has androgeni ativity (5) and inreases serum free T by dereasing serum sex hormone-binding globulin (SHBG) (6, 7). Furthermore, beause orally administered danazol is absorbed from the gastrointestinal trat and enters the liver via the portal vein, liver dysfuntion may result (3). The severity of these side effets sometimes ompels patients to disontinue treatment with oral danazol. Reently, Igarashi (8) reported that intravaginal administration of a danazol ring ontaining 2 to 3.5 g of danazol was effetive in treatment of endometriosis and that intravaginal administration resulted in muh lower serum levels of danazol than oral administration of 4 mg of danazol one per day. These results suggest that vaginal administration an serve as an alternative route for danazol therapy Fertility and Sterility

2 that might produe fewer and less severe side effets. Therefore, we prepared a suppository ontaining 1 mg of danazol to treat patients with endometriosis by intravaginal administration. In the present study, we measured danazol onentrations in the ovary, uterus, and serum after daily vaginal administration of a danazol suppository and investigated its effets on the hypothalami-pituitary-ovarian axis. Subjets MATERIALS AND METHODS Subjets in this study were women with regular menstrual yles who were between 23 and 43 years of age. Thirty patients (47.5 ± 2.3 years; mean ± SEM) who were to undergo hysteretomy and unilateral oophoretomy beause of uterine leiomyoma underwent assay of danazol onentrations in the ovary, uterus, and serum. Two volunteers (31 and 33 years) and six volunteers (31.2 ± 5.1 years) underwent assay of serum E2 and P levels throughout menstrual yles and at the midluteal phase, respetively. Informed onsent was obtained from all patients. Preparation of a Danazol Suppository One hundred milligrams of danazol was suspended in 1 ml ofvoso-h 15 (Maruishi Pharmaeutial Co., Ltd., Osaka, Japan) at 6 C. The suspension was transferred into a ylindrial ontainer, where it turned solid at room temperature. Measurement of Danazol Conentrations in Ovary, Uterus, and Serum Thirty patients who were to undergo hysteretomy and unilateral oophoretomy were divided into three groups. Patients of group I reeived a danazol suppository in the posterior vaginal fornix 4, 12, or 36 hours before hysteretomy and oophoretomy. Patients of group II were instruted to insert a danazol suppository into the posterior vaginal fornix by themselves every day for 3 days until surgery. Patients in group III took a apsule ontaining 2 mg of danazol twie per day (4 mg/d) for 3 days until surgery. The uterus and ovary were removed at surgery 12 hours after the last administration of danazol in groups II and III. Blood was drawn at hysteretomy and oophoretomy in eah group, and serum was separated by entrifugation. Serum, endometrium and myometrium of the uterine ervix and orpus, and the ovary were stored at -2 C until the assay for danazol. Endometrium, myometrium, and ovaries were homogenized with four volumes of saline in a Polytron PT1 homogenizer (Brinkman In- Vol. 63, No.6, June 1995 struments, Westbury, NY). Myometrium and ovaries were mined with sissors before homogenization. Danazol in the tissue homogenate and in serum was extrated with a solution of n-hexane-hloroform (7:3, vol/vol), and then applied to a Sep-Pak Silia artridge absorption olumn (Millipore Co., Bedford, MA). The absorbed danazol was eluted with hloroform. Mter evaporation of hloroform, danazol was resolved in a small amount of the n-hexane-hloroform solution and measured by high-pressure liquid hromatography with a Lihrosorb Si 65 olumn (25 X 4.6 m; Merk & Co., In. Rahway, NJ). Serum E2 and P Levels During Daily Vaginal Eight volunteers with regular menstrual yles reeived a danazol suppository in the posterior vaginal fornix one per day, from the 4th day of eah menstrual yle to the beginning of menstruation, for two to six suessive menstrual yles. The menstrual yle immediately before the menstrual yle in whih danazol was administered was regarded as a ontrol yle. Two volunteers had blood drawn to measure serum E2 and P levels every 2nd day during the ontrol menstrual yle and the seond menstrual yle of danazol administration; BBT was reorded during both yles. Six volunteers had blood drawn to measure serum E2 and P levels at the midluteal phase (7 to 9 days after LH surge) of both the ontrol menstrual yle and the seond menstrual yle of danazol administration. The LH surge was determined by measurement of urine LH levels and by observation of folliular development by transvaginal ultrasonography. Endometrial Histology During Daily Vaginal Endometrial biopsy was performed on the 5th day after ovulation of the seond menstrual yle of danazol administration in eah of the volunteers. Time of ovulation was onfirmed by LH surge and disappearane of Graffian folliles on ultrasonography. Assay of Serum E2 and P levels Serum E2 and P levels were determined by RIA kits (Diagnosti Produts Co., Los Angeles, CA). The intra-assay and interassay oeffiients of variation were 6.% and 6.8% for E2 and 5.8% and 6.5% for P. Detetion of Urine LH Urine LH onentration was measured with an L CHECK enzymeimmunoassay kit (Nipro Co., Osaka, Japan). An LH onentration> 4 miu/ml (onversion fator to SI unit, 1.) was onsidered positive. Mizutani et al. Vaginal administration of danazol 1185

3 9r--r , Ovary o Endometrium o Myometrium (ervix) A Myometrium (orpus) Serum 1L;:=~~~~ Time after administration of a danazol suppository (hours) Figure 1 Time ourse of danazol onentrations in the ovary, endometrium and myometrium of the uterine ervix and orpus, and serum after single vaginal administration of a danazol suppository. Eah point represents a mean ± SEM of three to five samples. Statistis Values are presented as means ± SEM. Statistial signifiane (P <.5) was evaluated by Student's t-test and the paired t-test. RESULTS Danazol Conentrations in Ovary, Uterus, and Serum After a Single Vaginal Administration of Danazol Suppository Danazol onentrations in the ovary, uterus, and serum after a single vaginal administration of danazol suppository are shown in Figure 1. Danazol administered via the vagina is distributed rapidly to the ovaries and uterus, and danazol onentrations in the ovary and uterus reahed high levels 4 hours after administration, in order of dereasing onentration, in the ovary, endometrium, myometrium of the uterine ervix, and myometrium of the uterine orpus. Danazol onentrations in these tissues 12 hours after administration dereased to one third to one fourth the levels after 4 hours, and those 36 hours after the danazol administration were <5 ng/ mg tissue. In ontrast, serum danazol onentrations in all patients were always < 1 ng/ml. Danazol Conentrations in the Ovary, Uterus, and Serum Mter Daily Vaginal Administration of a Danazol Suppository Figure 2 shows danazol onentrations in the ovary, uterus, and serum 12 hours after the last administration of danazol in patients who reeived either a single vaginal suppository ontaining 1 mg of danazol, a daily vaginal danazol suppository for 3 days, or daily oral administration of 4 mg of danazol for 3 days. Danazol onentrations in the ovary and uterus after daily vaginal administration. of 1 mg of danazol were two to three times higher than those after a single administration and were omparable to those after daily oral administration of a fourfold higher dose (4 mg). However, the serum danazollevel after daily vaginal administration of danazol was <-do of that after daily oral administration. Serum E2 and P Levels During Daily Vaginal Eight volunteers with regular menstrual yles reeived a vaginal danazol suppository one per day for two to six suessive menstrual yles. Figure 3 shows BBT and serum levels of E2 and P during the ontrol menstrual yle and the seond menstrual yle of danazol administration of one volunteer. During the ontrol menstrual yle, BBT showed a biphasi pattern, and LH surge ourred, as shown by urinary LH levels. Serum E2 and P levels also showed normal patterns (Fig. 3A). During the seond menstrual yle of danazol administration, BBT and serum levels of E2 and P showed patterns similar to those during the ontrol menstrual yle, and LH surge was onfirmed (Fig. 3B). Folliular development in the ovaries and ovulation were onfirmed Q) :::l CI) CI).;::l 26 -Single vaginal administration (1 mg) *t 25 D Daily vaginal administration (1 mg) 24 ~ Daily oral administration (4 mg) 23 1 E * * C '-' 6.;::l... ~ C Q) 4 U 5 * * * (5 3 N I1J * * I1J 2 1 Ovary Endometrium Myometrium Myometrium Serum (ervix) (orpus) Figure 2 Danazol onentrations in the ovary, endometrium and myometrium of the uterine ervix and orpus, and serum after single vaginal administration of 1 mg danazol, daily vaginal administration of 1 mg danazol for 3 days, and daily oral administration of 4 mg danazol for 3 days. Danazol onentrations were measured 12 hours after the last administration. The height of a bar represents a mean + SEM ofthree to five samples. *p <.5, signifiant differene from the values after single vaginal administration of danazol by Student's t-test. tp <.5, signifiant differene from the values after daily vaginal administration of danazol by Student's t-test. * 1186 Mizutani et ai. Vaginal administration of danazol Fertility and Sterility

4 A B IlIys From LH PIlI< Urine LH IlIys From LH PIlI< Urine LH ++ 4 :J E Figure 3 The BBT and se rum E2 and P levels during 5 the ontrol menstrual yle 2 (A) and the seond menstrual yle (B) of daily vaginal ad- 1 1 ministration of danazol (on- version fators to 81 unit for E2 and Pare and 3.18, IlIys From LH PIlI< respetively). 4 :J :J E :J E... E to to..s 5..s.,., ' 1 2 ~ l! J!!.,., In 5.fi 1 5 ~ e "- "- +1 IlIys From LH Peak by transvaginal ultrasonography during both the ontrol menstrual yle and the seond menstrual yle of danazol administration. Results in a seond volunteer were similar (data not shown). Serum E2 and P levels of the other six volunteers were measured at the midluteal phase in the ontrol menstrual yle and the seond menstrual yle of danazol administration. Serum E 2 levels ofthese volunteers in the ontrol yle versus those in the seond yle of danazol administration were 158 versus 18, 119 versus 15, 121 versus 12, 111 versus 12, and 95 versus 124 pg/ml (onversion fator to Sl unit, 3.617), respetively; the means ± SEM of the former and the latter were 12.8 ± 1.4 and 12.8 ± 8.3, respetively. Their serum P levels in the ontrol yle versus those in the seond yle of danazol administration were 11.5 versus 1.5, 11. versus 11.2, 1.8 versus 9.3, 8.2 versus 7.9, 8. versus 12.8, and 6. versus 8.1 ng/ml (onversion fator to Sl unit, 3.18), respetively; the means ± SEM of the former and the latter were 9.3 ±.9 and 1. ±.8, respetively. There were no signifiant differenes in serum E2 and P levels between the yles when examined by the paired t-test. Of the eight volunteers, six reeived a danazol suppository for three to six suessive menstrual yles (total, 24 yles). The BBT of eah menstrual yle showed the normal biphasi pattern, and no menstrual disorders were found. Furthermore, no ane, hirsutism, or weight gain was found in five volunteers. Only one volunteer omplained of very mild ane. Serum glutami oxaloaeti transaminase (GOT) and glutami pyruvi transaminase (GPT) levels of these six volunteers Vol. 63, No.6, June 1995 after the last administration of danazol suppository were 9, 13, 15, 7, 13, and 1 lull, and 6, 1, 8, 6, 11, and 8 lull, respetively, and were within normal ranges (normal ranges of GOT and GPT were 7 to 34 and 4 to 32 lull, respetively). Endometrial Histology During Vaginal To investigate the diret effet of vaginally administered danazol on endometrium, endometrial biopsy was performed in the eight volunteers on the 5th day after the ovulation ofthe seond menstrual yle of vaginal danazol administration. A representative endometrial histologi pattern is shown in Figure 4. The endometrium was abundant in tortuous glands, and the aumulation of seretion was found often in their lumen. In some glands the nulei of epithelial ells displaed toward the free surfae, forming subnulear vauoles, but in the rest of glands their nulei were found in the basal ytoplasm. The stroma of the endometrium was partly edematous. These histologi developments orrespond to the early seretory phase. DISCUSSION Orally administered danazol is absorbed from the gastrointestinal trat, enters the systemi irulation via the portal vein, and then is distributed to various tissues, inluding the ovaries and uterus. Therefore, oral administration of danazol results in high serum onentrations, as shown in the present study. In ontrast, a single vaginal administration Mizutani et a!. Vaginal administration of danazol 1187

5 r Figure 4 Endometrium on the 5th day after the ovulation during the seond menstrual yle of daily vaginal administration of danazol. Hematoxylin and eosin, Magnifiation, x71. of danazol produed high danazol onentrations in the ovary and uterus while keeping the serum danazol onentration at muh lower levels. Furthermore, danazol onentrations in the ovary and uterus after daily vaginal administration (1 mg) were omparable to those after daily oral administration at a fourfold higher dose (4 mg), whereas the serum danazol onentration after daily vaginal administration was <~ of that after daily oral administration. Therefore, it is likely that vaginally and orally administered danazol reah the ovary and uterus through different routes. A venous plexus or the lymphati system is the probable route from the vagina to these organs. Danazol has antigonadotropi ativities, and daily oral administration of 4 mg of danazol inhibits both ovulation and LH surge (9-11). However, neither was inhibited by daily vaginal administration. This result is attributable to the lower serum onentration of danazol produed by vaginal administration. Daily vaginal administration of 1 mg danazol had no effet on either serum levels of E2 and P or menstrual yles, whereas the danazol onentration in the ovary was omparable with that from daily oral administration of 4 mg danazol. These results suggest that danazol at onentrations produed by daily oral administration of 4 mg danazol does not inhibit estrogen prodution by the ovaries. Therefore, the dereased serum levels of E2 after daily administration of 4 mg danazol reported by several investigators (9,12,13) apparently were due to the ation of danazol at high serum onentrations on the hypothalami-pituitary axis (13, 14). On the other hand, there are many reports that danazol in1188 Mizutani et a1. Vaginal administration of danazol hibited steroidogenesis in the ovary in vitro (15-17). In these studies, danazol was used at onentrations greater than 1 j.lm. However, aording to the present and previous studies (18), danazol onentrations in the ovary after oral administration of 4 mg danazol are <.3 j.lm. Therefore, the extrapolation of in vivo effets of danazol from in vitro studies requires great are. Endometrium during daily vaginal administration of 1 mg danazol was not atrophi. Beause daily vaginal administration of danazol did not affet serum levels of E2 and P, this finding suggests that danazol at a onentration reahed in the endometrium during daily vaginal administration (1 mg) does not inhibit the ations of estrogen and P there. Danazol at high serum onentrations during daily oral administration sometimes produes side effets, suh as ane, hirsutism, and weight gain (3, 4). In ontrast, daily vaginal administration of danazol resulted in muh lower serum onentrations, and only mild ane was found in one of six volunteers. Moreover, serum GOT and GPT levels of volunteers were within normal ranges. Thus, daily vaginal administration of danazol would be aompanied by fewer and less severe side effets than daily oral administration. In the present study, the effetiveness of daily vaginal administration of danazol in the treatment of endometriosis was not examined. However, Igarashi (8) reported that a single vaginal administration of a danazol ring ontaining 2 to 3.5 g of danazol redued dysmenorrhea and the extent of pelvi endometriosis in eah of 35 women treated. Therefore, vaginal administration ould be an alternative therapeuti route with fewer and less severe side effets. Furthermore, if the effetiveness of vaginal administration of danazol for treatment of endometriosis is onfirmed, it would indiate that danazol has a diret effet on endometrioti tissue beause vaginal administration of danazol does not affet the hypothalami-pituitary-ovarian axis, although danazol may not have a diret effet on the endometrium as suggested in this study. The effetiveness of oral administration of danazol for treatment of endometriosis may be asribed to a diret effet of danazol on endometrioti tissue and to its effet on the hypothalami-pituitary axis. Aknowledgement. Danazol was kindly supplied by Tokyo Tanabe Co., Tokyo, Japan. REFERENCES 1. Biberoglu KO, Behrman SJ. Dosage aspets of danazol therapy in endometriosis: short-term and long-term effetiveness. Am J Obstet Gyneol 1981; 139: Ward GD. Dosage aspets ofdanazol therapy in the treatment of endometriosis. Postgrad Med J 1979; 55:7-9. Fertility and Sterility

6 3. Spooner JB. Classifiation of side-effets to danazol therapy. J Int Med Res 1977;5: Henzl MR, Kwei L. Effiay and safety of nafarelin in the treatment of endometriosis. Am J Obstet Gyneol 199; 162: Barbieri RL, Lee H, Ryan KJ. Danazol binding to rat androgen, gluoortioid, progesterone and estrogen reeptors: orrelation with biologi ativity. Fertil Steril 1979;31: Dowsett M, Forbes KL, Rose GL, Mudge JE, Jeffoate SL. A omparison of the effets of danazol and gestrinone on testosterone binding to sex hormone binding globulin in vitro and in vivo. Clin Endorinol (Ox ) 1986;24: Wynn V. Metaboli effets of danazol. J Int Med Res 1977;5: Igarashi M. A new therapy for pelvi endometriosis and uterine adenomyosis: loal effet of vaginal and intrauterine danazol appliation. Asia Oeania J Obstet Gynaeol 199; 16: Wood GP, Wu C-H, Flikinger GL, Mikhail G. Hormonal hanges assoiated with danazol therapy. Obstet Gyneol 1975;45: Lauersen N, Wilson KH. Evaluation of danazol as an oral ontraeptive. Obstet Gyneol 1977;5: Barbieri RL, Ryan KJ. Danazol: endorine pharmaology and therapeuti appliations. Am J Obstet Gyneol 1981; 141: Dikey RP, Taylor SN, Curole DN. Serum estradiol and danazol. I. Endometriosis response, side effets, administration interval, onurrent spironolatone and dexamethasone. Fertil Steril1984;42: Dmowski WP, Headley S, Radwanska E. Effets of danazol on pulsatile gonadotropin patterns and on serum estradiol levels in normally yling women. Fertil Steril 1983;39: Maouris P, Dowsett M, Edmonds DK, Sullivan D. The effet of danazol on pulsatile gonadotropin seretion in women with endometriosis. Fertil Steril 1991;55: Olsson J-H, Hillensjo T, Nilsson L. Inhibitory effets of danazol on steroidogenesis in ultured human granulosa ells. Fertil SteriI1986;46: Barbieri RL, Canik JA, Makris A, Todd RB, Davies IJ, Ryan KJ. Danazol inhibits steroidogenesis. Fertil Steril 1977;28: Adashi EY, Resnik CEo Diret biphasi effets of danazol on gonadotropin-dependent differentiation of ultured rat granulosa ells. Fertil SteriI1986;45: Mizutani T, Sakata M, Miyake A, Tanizawa, Terada N, Terakawa N. No inhibitory effets of danazol on estrogen prodution by ovaries of hypophysetomized rats stimulated by gonadotropins. In Vivo 1993; 7: Vol. 63, No.6, June 1995 Mizutani et al. Vaginal administration of danazol 1189