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Danazol: Prolonged suppression of gonadotropins after subcutaneous administration in the castrate male rat
Danazol: Prolonged suppression of gonadotropins after subcutaneous administration in the castrate male rat v. Daniel Castracane, PhD," George M. Butterstein, PhD,b and Bruce R. Gauvin, MAC Amarillo, Texas, and Schenectady and Rensselaer, New York OBJECTIVE: Our purpose was to compare oral versus subcutaneous administration of danazol for its effect on elevated serum luteinizing hormone and follicle-stimulating hormone levels in castrated male rats. STUDY DESIGN: A single dose of danazol, either 100 or 400 mg/kg, was administered by gastric intubation or injected subcutaneously. Jugular venipuncture blood samples were taken at 0, 3, 24, and 48 hours and at 7, 10, 15, and 25 days, and serum levels of luteinizing hormone and follicle-stimulating hormone were determined by radioimmunoassay. RESULTS: Gonadotropin levels returned to control values 96 hours after oral administration, whereas 400 mg/kg of danazol administered SUbcutaneously resulted in suppression of gonadotropins for 25 days. CONCLUSIONS: Subcutaneous administration of danazol results in an unexpectedly prolonged suppression of serum gonadotropins compared with the same dose administered orally. A change from oral administration to a prolonged-release subcutaneous preparation of danazol may enhance the use of this drug in clincial situations and may lessen undesirable side effects. (AM J OeSTET GYNECOL 1994;171 :1597-600.)
Key words: Danazol, luteinizing hormone, follicle-stimulating hormone, rats
Danazol has been widely used in the treatment of endometriosis,l premenstrual syndrome,2 and fibrocystic disease, hereditary angioedema, and gynecomastia. 3 The effectiveness of danazol in the treatment of these disorders is based primarily on its anti gonadotropic effects'" 5 although other actions, including direct ovarian suppression and inhibition of the actions of steroid hormones at receptor sites, are also considered important. Although danazol possesses androgenic activity, it has also been shown that the suppressed levels of luteinizing hormone (LH) after danazol treatment results from activation of both estrogenic and androgenic receptors. 6 In the treatment of endometriosis women typically take an oral daily dose of danazol of up to 800 mg. The side eff~cts of the drug may include hirsutism, acne, seborrhea, myalgia, edema, deepening of the voice, clitorimegaly, and menometrorrhagia.?' 8 Clinical use of danazol has been exclusively by the oral route, and the same route predominates in experimental studies in From the Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center: the Department of Biology, Union College/ and the Sterling Drug Group.' Received for publication March 24, 1994; revised June 17, 1994; accepted June 27, 1994. Reprint requests: V. Daniel Castracane, PhD, Texas Tech University Health Sciences Center, Department of Obstetrics and Gynecology, 1400 Wallace Blvd., Amarillo, TX 79106. Copyright © 1994 by Mosby-Year Book, Inc. 0002-9378/94 $3.00 + 0 6/1/58786
laboratory animals. In this study we compared the effectiveness of oral with subcutaneous administration of danazol for its ability to suppress the elevated serum LH and follicle-stimulating hormone (FSH) levels in castrated male rats. We report the first and unexpected finding of a 25-day suppression of gonadotropins after a single subcutaneous treatment compared with nothing more than a transient suppression by the same dose administered by the oral route.
Material and methods Sexually mature male rats (200 gm) were obtained from Taconic Farms, Inc., Germantown, New York. Rats were castrated, and all treatments w,ere initiated 7 to 9 days after castration. Danazol was dissolved or suspended in lO% alcohol and oil (ethanol and cottonseed oil). A single dose of danazol, 100 or 400 mg/kg, was administered by gastric intubation or injected subcutaneously. Blood was collected by direct jugular venipuncture with the animal under light ether anesthesia immediately before danazol or vehicle administration and at 3, 24, and 48 hours and 7, 10, IS, and 25 days after administration. Controls were treated with 10% alcohol and oil vehicle, either by gastric intubation or subcutaneously, and blood was collected on the same schedule as for danazol-treated rats. This protocol was approved by the Animcal Care Committee of the SterlingWinthrop Research Institute, Rensselaer, New York. The serum was separated and frozen until LH and
1597
1598
Castracane, Butterstein, and Gauvin
December 1994
Am] Obstet Gynecol
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Fig. 1. Mean LH and FSH levels for first 96 hours after oral (PO) (A) or subcutaneous (SC) (B) administration of either 100 or 400 mglkg danazol. Each data point represents mean ± SEM of 10 animals for controls and eight for experiments. Oral administration of 100 mg/kg produced rapid decline in LH (p < 0.0001) within 3 hours, whereas only higher dose reduced FSH. Gonadotropins returned to control values after 4 days. Subcutaneous treatment at 100 mg reduced LH at 24 hours (p < 0.01) and FSH at 48 hours (p < 0.05), whereas 400 mg dose produced continued highly significant decline over 96 hours.
FSH levels were determined by radioimmunoassay after termination of the study. All rats were killed 25 days after treatment. Serum LH and FSH levels were measured by double antibody radioimmunoassay. Rat LH and FSH reference standards, highly purified hormones for iodination, and first antibodies were generously provided by Dr. Salvatore Raiti at the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases. Serum LH levels are expressed as nanograms of rLHRP-2 per milliliter. Intrassay and interassay coefficients of variance were 5.8% and 5.2%, respectively. Serum FSH levels are expressed as nanograms of rFSH-RP-l per milliliter. Intrassay and interassay coefficients of variance were 6.3% and 5.6%. Statistical comparisons were made with analysis of variance and Dunnett's method. The results are expressed as mean ± SE. The calculations were performed with JMPIN, a statistical visualization software
program (SAS Institute Inc., Duxbury Press, Belmont, Calif.). Results
Oral administration. The effect of oral administration of two doses of danazol (l00 and 400 mg/kg) on gonadotropin serum levels over the first 4 days is shown in Fig. 1. Serum LH was rapidly lowered by 50% by both doses within 3 hours of treatment (jJ < 0.0001). LH remained suppressed for 96 hours in animals receiving the 400 mg dose but returned to control levels by 48 hours after the lower dose. FSH was less responsive to danazol treatment. Oral administration of 100 mg had no significant effect on FSH levels, whereas 400 mg of danazol reduced FSH levels at 24 (jJ < 0.05) and 48 (jJ < 0.0001) hours. All animals had FSH values equal to control animals after 4 days. Both gonadotropins remained at control levels
Castracane, Butterstein, and Gauvin
Volume 171, Number 6 Am J Obstet Gynecol
A. Oral Administration
1599
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Fig. 2. Mean LH and FSH levels for 25 days after oral (PO) (A) or subcutaneous (SC) (B) administration of either 100 or 400 mglkg danazol. These are the same animals depicted in Fig. 1. There were no differences in gonadotropin levels after 4 days in orally treated danazol animals, whereas ani,mals receiving 400 mg/kg subcutaneously had suppressed levels of LH and FSH for entire 25 days of study.
throughout the remaining 25-day period, except for a significant rise (p < 0.05) ofLH at day 10 (Fig. 2). Two of the experimental animals receiving the 400 mg dose had very high LH levels at this time. Subcutaneous administration. One single sucutaneous administration of either 100 or 400 mglkg danazol significantly (p < 0.01) reduced LH values 24 hours after treatment (Fig. 1, B). Serum LH levels remained suppressed in animals receiving the higher dose, but hormone levels returned to control levels after 96 hours in the animals receiving lower doses. Subcutaneous treatment with 100 mg/kg danazol reduced FSH levels only after 48 hours (p < 0.05) (Fig. 1, B), whereas in animals receiving the higher dose there was a precipitous decline of FSH levels beginning 24 hours after treatment (p < 0.01). Both gonadotropins were highly significantly suppressed (p < 0.0001) by the 400 mg dose for up to 25 days of the study (Fig. 2, B). Postcastration rise of gonadotropins. All male rats used in this study were castrated 7 days before treatment
began. Throughout the 25 days of the study both gonadotropins continued to rise, following a typical postcastration event (Fig. 2, B). Treated animals were expected to follow this same pattern. The prolonged suppression of gonadotropin levels after subcutaneous treatment with danazol is therefore even more impressive. Comment Danazol has been frequently used for the treatment of endometriosis, and numerous reports exist to demonstrate a complex endocrinologic and pharmacologic profile, although suppression of pituitary gonadotropins remains its most important action. The suppression of LH was partially blocked or absent in ftutamidetreated males or in androgen receptor-deficient pseudohermaphrodite rats, indicating an androgen receptor-mediated event. 9 The circulating half-life of danazol is approximately 15 hours,1O so it is normally orally administered twice daily. It is well absorbed by the oral route and is metabolized to approximately 60
1600 Castracane, Butterstein, and Gauvin
different products. II The undesirable side effects of danazol treatment, in particular its androgenicity, may be accentuated by high-dose oral administration. In this study we have demonstrated for the first time that a single subcutaneous administration to the castrated male rat results in a prolonged suppression of circulating LH and FSH levels. In two other studies LH was shown to be reduced with low daily subcutaneous doses of 10 to 50 mg/kg for only 9 days in the male rat9 and 14 days in the female. 12 Neither of these studies was designed to evaluate the long-term effectiveness of this route of administration. Our unexpected finding suggests that danazol, probably because of its insolubility, !pay represent a natural sustained-release preparation by this route of administration. The studies were terminated 25 days before gonadotropin assay, because it seemed unlikely that any effect would be seen past this time. We were surprised to find that gonadotropin levels were still suppressed at the termination of the study. In a preliminary study we also found that a single subcutaneous administration of danazol can suppress the estrous cycle of rats for > 30 days (Butterstein and Castracane, unpublished observation). Future studies in the male will examine the involvement of danazol in other areas of physiologic regulation and will extend beyond the point of recovery of any observed response. There would be obvious advantages to a prolongedrelease preparation of danazol. Presumably this would decrease the total administered dose and would therefore be expected to decrease undesirable side effects. Both the reduced total dose and the elimination of any first-pass effect would reduce any deleterious actions of administered drug. Further studies are required to confirm this unexpectedly prolonged duration of effectiveness, but clearly the possible clinical results from
December 1994
Am] Obstet Gynecol
such a simple change in the route of administration may enhance the use of this drug in clinical situations. We thank Dr. Joseph Schmee for this assistance in statistical analysis. REFERENCES 1. Madanes AE, Farber M. Danazol. Ann Intern Med 1982; 96:625-30. 2. Halbreich U, Rojansky N, Palter S. Elimination of ovulation and menstrual cyclicity (with danazol) improves dysphoric premenstrual syndromes. Fertil Steril 1991;56: 1066-9. 3. American Hospital Formulary Service. Danazol. AHFS drug information. Hyattsville, Maryland: Taylor Printing, 1987:1616. 4. Shane JM, Kates R, Barbieri RL, Todd RB, Davies IJ. Pituitary gonadotropin responsiveness with danazol. Fertil Steril 1978;29:637-39. 5. Eldridge Cj, Dmowski PW, Mahesh VB. Effects of castration of immature rats on serum FSH and LH, and of ovarian steroid treatments after castration. Bioi Reprod 1974; 10:438-46. 6. Snyder BW, Beecham GD, Winneker RC. Danazol suppression of luteinizing hormone in the rat: evidence for mediation by both androgen and estrogen receptors. Soc Exp Bioi Med 1990;194:54-7. 7. Barbieri RL. New therapy for endometriosis. New Engl J Med 1988;318:512-4. 8. Henzl MR, Kwei L. Efficacy and safety of nafarelin in the treatment of endometriosis. AM J OBSTET GYNECOL 1990; 162:570-4. 9. Krey LC, Robbins R, McEwen BS. Danazol suppression of luteinizing hormone secretion: a role for danazol-androgen receptor interaction within the brain-pituitary complex. Fertil Steril 1981 ;35:467 -72. 10. Peterson JE, King ME, Banks WF, et al. Radioimmunoassay for danazol in human and monkey plasma. J Pharm Sci 1978;67: 1425-8. 11. Davison C, Banks W, Fritz A. The absorption, distribution and metabolic fate of danazol in rats, monkeys, and human volunteers. Arch Int Pharmacodyn 1976;221:294310. 12. Suzuki S, Sakamoto S, Kudo H, et al. Effects of danazol on endometrial DNA synthesis in rats. Steroids 1993;58: 551-3.
Key words: Danazol, luteinizing hormone, follicle-stimulating hormone, rats
Danazol has been widely used in the treatment of endometriosis,l premenstrual syndrome,2 and fibrocystic disease, hereditary angioedema, and gynecomastia. 3 The effectiveness of danazol in the treatment of these disorders is based primarily on its anti gonadotropic effects'" 5 although other actions, including direct ovarian suppression and inhibition of the actions of steroid hormones at receptor sites, are also considered important. Although danazol possesses androgenic activity, it has also been shown that the suppressed levels of luteinizing hormone (LH) after danazol treatment results from activation of both estrogenic and androgenic receptors. 6 In the treatment of endometriosis women typically take an oral daily dose of danazol of up to 800 mg. The side eff~cts of the drug may include hirsutism, acne, seborrhea, myalgia, edema, deepening of the voice, clitorimegaly, and menometrorrhagia.?' 8 Clinical use of danazol has been exclusively by the oral route, and the same route predominates in experimental studies in From the Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center: the Department of Biology, Union College/ and the Sterling Drug Group.' Received for publication March 24, 1994; revised June 17, 1994; accepted June 27, 1994. Reprint requests: V. Daniel Castracane, PhD, Texas Tech University Health Sciences Center, Department of Obstetrics and Gynecology, 1400 Wallace Blvd., Amarillo, TX 79106. Copyright © 1994 by Mosby-Year Book, Inc. 0002-9378/94 $3.00 + 0 6/1/58786
laboratory animals. In this study we compared the effectiveness of oral with subcutaneous administration of danazol for its ability to suppress the elevated serum LH and follicle-stimulating hormone (FSH) levels in castrated male rats. We report the first and unexpected finding of a 25-day suppression of gonadotropins after a single subcutaneous treatment compared with nothing more than a transient suppression by the same dose administered by the oral route.
Material and methods Sexually mature male rats (200 gm) were obtained from Taconic Farms, Inc., Germantown, New York. Rats were castrated, and all treatments w,ere initiated 7 to 9 days after castration. Danazol was dissolved or suspended in lO% alcohol and oil (ethanol and cottonseed oil). A single dose of danazol, 100 or 400 mg/kg, was administered by gastric intubation or injected subcutaneously. Blood was collected by direct jugular venipuncture with the animal under light ether anesthesia immediately before danazol or vehicle administration and at 3, 24, and 48 hours and 7, 10, IS, and 25 days after administration. Controls were treated with 10% alcohol and oil vehicle, either by gastric intubation or subcutaneously, and blood was collected on the same schedule as for danazol-treated rats. This protocol was approved by the Animcal Care Committee of the SterlingWinthrop Research Institute, Rensselaer, New York. The serum was separated and frozen until LH and
1597
1598
Castracane, Butterstein, and Gauvin
December 1994
Am] Obstet Gynecol
B. Sub-cutaneous Administration
A. Oral Administration 20
25
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Fig. 1. Mean LH and FSH levels for first 96 hours after oral (PO) (A) or subcutaneous (SC) (B) administration of either 100 or 400 mglkg danazol. Each data point represents mean ± SEM of 10 animals for controls and eight for experiments. Oral administration of 100 mg/kg produced rapid decline in LH (p < 0.0001) within 3 hours, whereas only higher dose reduced FSH. Gonadotropins returned to control values after 4 days. Subcutaneous treatment at 100 mg reduced LH at 24 hours (p < 0.01) and FSH at 48 hours (p < 0.05), whereas 400 mg dose produced continued highly significant decline over 96 hours.
FSH levels were determined by radioimmunoassay after termination of the study. All rats were killed 25 days after treatment. Serum LH and FSH levels were measured by double antibody radioimmunoassay. Rat LH and FSH reference standards, highly purified hormones for iodination, and first antibodies were generously provided by Dr. Salvatore Raiti at the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases. Serum LH levels are expressed as nanograms of rLHRP-2 per milliliter. Intrassay and interassay coefficients of variance were 5.8% and 5.2%, respectively. Serum FSH levels are expressed as nanograms of rFSH-RP-l per milliliter. Intrassay and interassay coefficients of variance were 6.3% and 5.6%. Statistical comparisons were made with analysis of variance and Dunnett's method. The results are expressed as mean ± SE. The calculations were performed with JMPIN, a statistical visualization software
program (SAS Institute Inc., Duxbury Press, Belmont, Calif.). Results
Oral administration. The effect of oral administration of two doses of danazol (l00 and 400 mg/kg) on gonadotropin serum levels over the first 4 days is shown in Fig. 1. Serum LH was rapidly lowered by 50% by both doses within 3 hours of treatment (jJ < 0.0001). LH remained suppressed for 96 hours in animals receiving the 400 mg dose but returned to control levels by 48 hours after the lower dose. FSH was less responsive to danazol treatment. Oral administration of 100 mg had no significant effect on FSH levels, whereas 400 mg of danazol reduced FSH levels at 24 (jJ < 0.05) and 48 (jJ < 0.0001) hours. All animals had FSH values equal to control animals after 4 days. Both gonadotropins remained at control levels
Castracane, Butterstein, and Gauvin
Volume 171, Number 6 Am J Obstet Gynecol
A. Oral Administration
1599
B. Sub-cutaneous Administration
30
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Fig. 2. Mean LH and FSH levels for 25 days after oral (PO) (A) or subcutaneous (SC) (B) administration of either 100 or 400 mglkg danazol. These are the same animals depicted in Fig. 1. There were no differences in gonadotropin levels after 4 days in orally treated danazol animals, whereas ani,mals receiving 400 mg/kg subcutaneously had suppressed levels of LH and FSH for entire 25 days of study.
throughout the remaining 25-day period, except for a significant rise (p < 0.05) ofLH at day 10 (Fig. 2). Two of the experimental animals receiving the 400 mg dose had very high LH levels at this time. Subcutaneous administration. One single sucutaneous administration of either 100 or 400 mglkg danazol significantly (p < 0.01) reduced LH values 24 hours after treatment (Fig. 1, B). Serum LH levels remained suppressed in animals receiving the higher dose, but hormone levels returned to control levels after 96 hours in the animals receiving lower doses. Subcutaneous treatment with 100 mg/kg danazol reduced FSH levels only after 48 hours (p < 0.05) (Fig. 1, B), whereas in animals receiving the higher dose there was a precipitous decline of FSH levels beginning 24 hours after treatment (p < 0.01). Both gonadotropins were highly significantly suppressed (p < 0.0001) by the 400 mg dose for up to 25 days of the study (Fig. 2, B). Postcastration rise of gonadotropins. All male rats used in this study were castrated 7 days before treatment
began. Throughout the 25 days of the study both gonadotropins continued to rise, following a typical postcastration event (Fig. 2, B). Treated animals were expected to follow this same pattern. The prolonged suppression of gonadotropin levels after subcutaneous treatment with danazol is therefore even more impressive. Comment Danazol has been frequently used for the treatment of endometriosis, and numerous reports exist to demonstrate a complex endocrinologic and pharmacologic profile, although suppression of pituitary gonadotropins remains its most important action. The suppression of LH was partially blocked or absent in ftutamidetreated males or in androgen receptor-deficient pseudohermaphrodite rats, indicating an androgen receptor-mediated event. 9 The circulating half-life of danazol is approximately 15 hours,1O so it is normally orally administered twice daily. It is well absorbed by the oral route and is metabolized to approximately 60
1600 Castracane, Butterstein, and Gauvin
different products. II The undesirable side effects of danazol treatment, in particular its androgenicity, may be accentuated by high-dose oral administration. In this study we have demonstrated for the first time that a single subcutaneous administration to the castrated male rat results in a prolonged suppression of circulating LH and FSH levels. In two other studies LH was shown to be reduced with low daily subcutaneous doses of 10 to 50 mg/kg for only 9 days in the male rat9 and 14 days in the female. 12 Neither of these studies was designed to evaluate the long-term effectiveness of this route of administration. Our unexpected finding suggests that danazol, probably because of its insolubility, !pay represent a natural sustained-release preparation by this route of administration. The studies were terminated 25 days before gonadotropin assay, because it seemed unlikely that any effect would be seen past this time. We were surprised to find that gonadotropin levels were still suppressed at the termination of the study. In a preliminary study we also found that a single subcutaneous administration of danazol can suppress the estrous cycle of rats for > 30 days (Butterstein and Castracane, unpublished observation). Future studies in the male will examine the involvement of danazol in other areas of physiologic regulation and will extend beyond the point of recovery of any observed response. There would be obvious advantages to a prolongedrelease preparation of danazol. Presumably this would decrease the total administered dose and would therefore be expected to decrease undesirable side effects. Both the reduced total dose and the elimination of any first-pass effect would reduce any deleterious actions of administered drug. Further studies are required to confirm this unexpectedly prolonged duration of effectiveness, but clearly the possible clinical results from
December 1994
Am] Obstet Gynecol
such a simple change in the route of administration may enhance the use of this drug in clinical situations. We thank Dr. Joseph Schmee for this assistance in statistical analysis. REFERENCES 1. Madanes AE, Farber M. Danazol. Ann Intern Med 1982; 96:625-30. 2. Halbreich U, Rojansky N, Palter S. Elimination of ovulation and menstrual cyclicity (with danazol) improves dysphoric premenstrual syndromes. Fertil Steril 1991;56: 1066-9. 3. American Hospital Formulary Service. Danazol. AHFS drug information. Hyattsville, Maryland: Taylor Printing, 1987:1616. 4. Shane JM, Kates R, Barbieri RL, Todd RB, Davies IJ. Pituitary gonadotropin responsiveness with danazol. Fertil Steril 1978;29:637-39. 5. Eldridge Cj, Dmowski PW, Mahesh VB. Effects of castration of immature rats on serum FSH and LH, and of ovarian steroid treatments after castration. Bioi Reprod 1974; 10:438-46. 6. Snyder BW, Beecham GD, Winneker RC. Danazol suppression of luteinizing hormone in the rat: evidence for mediation by both androgen and estrogen receptors. Soc Exp Bioi Med 1990;194:54-7. 7. Barbieri RL. New therapy for endometriosis. New Engl J Med 1988;318:512-4. 8. Henzl MR, Kwei L. Efficacy and safety of nafarelin in the treatment of endometriosis. AM J OBSTET GYNECOL 1990; 162:570-4. 9. Krey LC, Robbins R, McEwen BS. Danazol suppression of luteinizing hormone secretion: a role for danazol-androgen receptor interaction within the brain-pituitary complex. Fertil Steril 1981 ;35:467 -72. 10. Peterson JE, King ME, Banks WF, et al. Radioimmunoassay for danazol in human and monkey plasma. J Pharm Sci 1978;67: 1425-8. 11. Davison C, Banks W, Fritz A. The absorption, distribution and metabolic fate of danazol in rats, monkeys, and human volunteers. Arch Int Pharmacodyn 1976;221:294310. 12. Suzuki S, Sakamoto S, Kudo H, et al. Effects of danazol on endometrial DNA synthesis in rats. Steroids 1993;58: 551-3.