Danazol and gestagen displacement of testosterone and influence on sex-hormone-binding globulin capacity*

Transcription

1 FERTILITY AND STERILITY Copyright c 1982 The American Fertility Society Printed in U.8A. Danazol and gestagen displacement of testosterone and influence on sex-hormone-binding globulin capacity* Bo Nilsson, M.D.t:J: Ragnar SOdergiird, Ph.D. Mats-Goran Damber, M.D.t Bo von Schoultz, M.D.t University of Umed, Umed, Sweden The mechanism of action of danazol is poorly understood, but this testosterone (T) derivate is frequently used in the clinical treatment of endometriosis, and its tendency to androgenic/anabolic side effects is well known. The interaction of danazol with T binding to sex-hormone-binding globulin (SHBG) was studied with the use of an aqueous two-phase system with polyethylene glycol (PEG) and dextran for equilibrium partition. Competitive binding studies were also performed with norethisterone (NET), d-norgestrel (d-n g), medroxyprogesterone acetate (MP A), and tamoxifen (TMX). Danazol, d-ng, and NET were found. to exert a marked T displacing activity, while MPA and TMX had no significant effect. The low values for SHBG binding capacity that were found during danazol therapy mainly reflect occupation of binding sites by danazol and to a lesser degree a real decrease in protein concentration. It was calculated that during treatment the total SHBG capacity in serum is approximately 20 times exceeded. Therapeutic danazol serum levels are 1000 times those of normal female total T levels; and since the affinity to SHBG for danazol was found to be 1/20 that of T one should conclude an almost total occupation of binding sites. The endocrine effects of danazol might be interpreted in terms of T displacement and as a consequence of increased levels of free T during therapy. Fertil Steril 38:48, 1982 Although frequently used in the clinical treatment of endometriosis, the mechanism of action of danazol remains poorly understood, and controversy exists about its effects on the human endocrine system. An inhibition of gonadotropin secretion was previously thought to be the major Received September 25, 1981; revised and accepted March 4,1982. *Supported by the Swedish Research Council (5982); the Medical Faculty, University ofumea, Umea, Sweden; Fertility Research Institute, Malmo, Sweden; Sterling-Winthrop AB, Stockholm, Sweden; and Lovens Liikemedel AB, Malmo, Sweden. tdepartment of Obstetrics and Gynecology. :j:reprint requests: Bo Nilsson, M.D., Department ofobstetrics and Gynecology, University of Umell., S Umell., Sweden. Department of Physiology..48 Nilsson et al. Danazol displacement of testosterone action of this 17~-ethinyl testosterone (T) derivate, but this effect has been difficult to confirm in clinical studies. I, 2 Alternatively, direct effects on steroid metabolism and receptor interaction have been suggested. 3-5 The most obvious effects of danazol therapy are a rapid and marked reduction of circulating estrogens and androgenic/ anabolic side effects. I, 2, 6-9 Acne, hirsutism, and weight gain are sometimes also observed in women taking other T derivates, such as the 19-norsteroids. Recently, d norgestrel (d-ng) and norethisterone (NET) were shown to bind to sex-hormone-binding globulin (SHBG), thereby displacing T.IO, 11 A similar T displacing activity of danazol might provide an important explanation for some of its endocrine effects. Therefore, competitive studies of the binding to SHBG for T, danazol, and some gestagens Fertility and Sterility

2 were performed, and the influence of danazolon serumshbg binding capacity was 'studied. MATERIALS AND METHODS THE TWO-PHASE SYSTEM An-aqueous two~phase system composed by two water-soluble polymers polyethylene glycol (PEG) (upper phase 7%wtlwt) and dextran (lower phase 12% wt/wt),was used for equilibrium partition. 12 A M K-phosphate buffer containing 0.1 M K-thiocyanate with a ph of 7.4 was used. All experiments were performed at room temperature (20 0 C). In this system serum proteins are almost exclusively confined to the lower dextrancontaining phase, while steroids distribute more equally between the two phases in a well-defined manner, different for each steroid. The partition coefficient is determined with the use of labeled steroids in serum-free separation experiments. Then, with serum in the system, it is possible from the specifi-c radioactivity in the upper phase for us to calculate the amounts of free and protein-bound steroid in the lower phase. REAGENTS [1,2-3 H]-5a-Dihydrotestosterone and [1, 2-3 H]-testosterone (New England Nuclear Corporation, Boston, MA)were purified by, paper chromatography with the Bush B3 system. 13 Unlabeled dihydrotestosterone (DHT), testosterone (T), cortisol (F), NET, bovine serum albumin (BSA), activated charcoal Norit A, dextran (Mw 17500), and PEG (Mw6000) were purchased from Sigma Chemical Company (St. Louis, MO). Danazol was obtained from Sterling-Winthrop AB (Stockholm, Sweden), dong from KABI AB (Stockholm), tamoxifen (TMX) from Imperial Chemical Industries Ltd. (Macclesfield, England), and medroxyprogesterone acetate (MPA) from L~vens KemiskeFabrik (Ballerup, Denmark). (Systematic names: see footnote*). All salts were of analytic grade, and' the water was twice distilled in quartz. *Trivial and systematic names of substances used: Testosterone (17~-hydroxy-4-androsten-3-one); d-norgestrel (d-13~-ethyl-17a-ethinyl-17~-hydroxyg()n-4-en-3-one); 'Norethisterone (17~-hydroxy-17a-ethinyl-4-estren-3-one); Medroxyprogesterone acetate (17a-acetoxy-6-methyl-pregna- 4-en-3;20-dione); Danazol (17a-pregn-4-en-20-yno-(2,3-d) isoxazol-17-ol); Tamoxifen ([trans 1-(4~-dimetylaminoetoxyphenyl) 1,2 diphenylbut-1-ene]). SERUM Venous blood samples were drawn from healthy women in the third trimester of pregnancy taking no drugs; five women with endometriosis before and after 3 to 6 months of treatment with danazol, 600 mg daily (200 mg x 3);,and one apparently healthy nonpregnant woman taking no drugs. After coagulation all sera were decanted and stored at C until used. DISPLACEMENT STUDIES Pooled pregnancy serum with a high SHBG concentration was used. One hundred microliters of serum was incubated for 15 minutes in glass tubes with 10,000 dpm [1,2-3 H]-T, 10 ng unlabeled T, 0.2 J.1g F (in order to avoid any possible disturbance due tothe presence of corticosteroidbindiiig globulin (CBG), and various amounts of the other steroids to be tested. Thereafter, 2.4 gm of the phase system was added. After equilibration for 30, minutes and centrifugation at 1000 x g for 10 minutes, 800 J.11 of the upper phase was analyzed in a liquid scintillation counter. T, dong, NET, MPA, danazol, and TMX in amounts of 10-3, 10-1, 1, 10, 10 2, and 10 3 ng were used as test substances. T BINDING TO SHBG Pooled pregnancy serum was used. One hundred microliters of serum, 10,000 dpm [1, 2-3 H]-T, 0.2 J.1g F, and various amounts (5,10,15,20, and 30 ng) of unlabeled T were incubated with 2.4 gm of the phase system. Experiments were also performed with fixed amounts of test substance (25 ng dong, 250 ng NET, and 250 ng danazod added to the system. Scatchard analyses were performed for the ratio of bound and free T plotted as a function of bound T.14 SHBG binding was calculated by subtraction of albumin binding according to Rosenthal. 15 Albumin binding was calculated from experiments using heat-treated (60 0 C, 30 minutes) serum. 12 SHBG BINDING CAPACITY AND DANAZOL THERAPY Individual serum samples from five women with endometriosis were analyzed before and after 3 to 6 months of treatment with danazol 200 mg x 3. One hundred microliters of serum, 10,000 dpm [1,2-3 H]-DHT, and 3.5 ng of unlabeled DHT were incubated with 2.4' gm of the phase system. Single-point assays for SHBG binding capacity were performed. With knowledge of Nilsson et ai. Danazol displacement of testosterone 49

3 Tes tos terone Bound % of total 100' 0-0 Testosterone _Danazol... d-norgestrel I!r----t. Medroxyprogesterone acetate Norethisterone T amoxifen o I, I, " : I ' I I', I I, I I II I I' I ', I, ' ' I' ", ' amount ~ I ', i i i i i. steroid a 0,01 0, added (ng) Figure 1 Displacement of3h-testosterone from SHBO by different syntetic steroids. The amount of steroid necessary to reduce bound T to 50% of total is indicated by the association constants for the DHT -SHBG and DHT-albumin complexes, albumin concentration,16 and values for bound and free fractions of DHT, the SHBG binding capacity was calculated.12 In order to study whether it was possible to remove danazol from serum by treatment with activated charcoal, at first a control serum sample from a normal healthy women was used. Danazol, to a final concentration of 2.5 jj.g/ml serum, was added, and with the use of the phase system, the SHBG binding capacity for DHT was calculated by the Scatchard plot method. The presence of danazol decreased SHBG binding capacity for DHT, while DHT-albumin binding was not affected. After treatment with activated charcoal,17 the SHBG binding capacity was restored to the same value as it had before the addition of danazol. Thereafter, the pooled sera from the five danazoltreated women were investigated. Part of the serum was heat-treated (60 0 C, 30 minutes). One hundred microliters of serum, 10,000 dpm [1, 2-3 H]-DHT, and varying amounts (2, 3.5, 5, 9, and 15 ng) of unlabeled DHT was incubated with 2.4 gm of the phase system. SHBG binding capacity was calculated in samples treated and not treated with activated charcoal. 150 Nilsson. et ai. Danazol displacement of testosterone DISPLACEMENT STUDIES RESULTS The T displacing activity of the substances tested are shown in Figure 1. d-ng, NET, and danazol had fa marked effect, while MPA and TMX showed no significant displacement at the concentrations used. The r-elative amounts of steroid needed to reduce binding of radioactive T to 50% were, in comparison with unlabeled T, 1:2.5, 1:13.5, and 1:21 for d-ng, NET and danazol, respectively. T BINDING TO SHBG As shown in Figure 2, all three added steroids (25 ng d-ng, 250 ng NET, and 250 ng danazol) markedly depressed the Scatchard curve for T binding. Precise calculations of SHBG binding capacity from these curves were not possible; but, roughly estimated, a 50% reduction in binding capacity occurred after adding 25 ng d-ng and 250 ng danazol. The effect of 250 ng NET was even more pronounced. T es tos terone Bound/Free Testosterone only (5,10,15, 20 and 3Ongl.... Testosterone+d-Norgestrel 25ng.... Testosterone + Danazol 250 ng. o-a Testosterone+Norethisterone 250ng.,... Heat-treated serum (albumin binding) + Testosterone 10, 20, and 30ng. Bound '------L-----L-..l L..testosterone ng/l00}j1 serum Figure 2 Scatchard analysis of T binding to SHBG, with and without the presence of 25 ng dong; 250 ng NET, and 250 ng danazol. The straight line represents SHBG steroid binding after subtraction of nonspecific albumin binding, and the intercept of this line on the abscissa represents total SHBG binding capacity. Fertility and Sterility

4 Table 1. SHBG Binding Capacity (nm) Before and During Danazol Treatment Patient No. Before danazol 3 months 6 months SHBG BINDING CAPACITY AND DANAZOL THERAPY Values for SHBG binding capacity in the five women before and during danazol treatment are given in Table 1. In all five women, after 3 months a pronounced decrease was found, which remained stable also after 6 months. In two of the women values were not measurable. Scatchard curves of DHT binding in pooled sera before and during danazol therapy are shown in Figure 3. The depression of the curve that occurred during danazol therapy was partly restored when serum was pretreated with activated charcoal. The values for SHBG binding capacity before danazol therapy were 42 ng/ml or 145 nm. Precise calculation of SHBG binding capacity after treatment was not possible; but, roughly estimated, a 5-fold reduction occurred. After removal of danazol, the corresponding figures were 25 ng/ml or 86nM. DISCUSSION The present study shows that danazol has a marked T-displacing activity with respect to SHBG binding. Previous data10, 11 for d-ng and NET were also confirmed. Danazol binds to SHBG with approximately 1120 of the affinity with which T is bound. This is on the same order as for NET and around 1110 of d-ng binding. T displaceme~t represents a potential hazard for androgenic/anabolic side effects during therapy. Therapeutic doses and the resultant serum levels of the respective steroids are vitally important in this respect. For example, d-ng serum levels following oral contraception in regular doses range between 1 and 4 ng/ml,18, 19 while NET levels reach 5 to 10 ng/ml.i8, 20 Average T levels in normal women are around 0.2 to 0.8 ng/ml.21 In comparison, the serum concentrations during danazol therapy are much higher. A dose of 400 mg daily was found to give serum levels between 300 and 700 ng/ml. 22 Thus, therapeutic levels of danazol are more than 50 times those of NET, around 500 times those of d-ng, and approximately 1000 times normal T levels. In clinical practice, furthermore, daily danazol doses of 600 and even 800 mg are frequently used, which means that T displacement from SHBG during danazol treatment is about 50 times that during oral contraception with d-ng and NET. Average values of SHBG binding capacity in normal women are around 30 ng DHT or Tlml, or 105 nm,12 corresponding to 35 ng danazollml. The interaction ofdanazol, d-ng, and NET with T binding is further illustrated in Figure 2. SHBG binding capacity in the system was 11.2 ng T or approximately 13 ng danazol. The addition of 250 ng danazol means that the binding capacity is exceeded around 20 times, which roughly corresponds to the clinical situation during treatment with 400 ng daily. Twenty-five nanograms d-ng and 250 ng NET had quite similar effects on the Scatchard curve, but these amounts, in terms of a resultant serum concentration, are much higher than those used for hormonal contraception. So far, most assays for SHBG have been based on determinations of binding capacity and not on the concentration of the protein. Thus, re- DHT Bound/Free Before Danazol!DHT: 2, and 15ng)... Danazol treatment 600 mg doily 3-6 months IDHT: and 15 ng)... Danazol treatment- serum pretreated with activated charcoal (oht: and 15ng) ><--->< Heat-treated serum (albumin binding) (OHT: 15, 20 and 3Ong) 5 ~'----~ ~x----~ ,x' Bound DHT L "----..L-, r----_-.-ng /100 J.l serum Figure 3 Scatchard analysis of DHT binding to SHBG. Sera from patients before and during danazol therapy, with and without pretreatment with activated charcoal. The straight lines represent SHBG binding of DHT before (-) and during C...) danazol treatment (serum pretreated with activated charcoal). The intercepts on the abscissa represent SHBG binding capacity. Nilsson et ai. Danazol displacement of testosterone 51

5 r ported alterations in SHBG levels may reflect either real changes in the amount of protein or occupation of binding sites on the protein molecules by competitive substances. This latter effect is certainly caused by danazol and is further illustrated in Figure 3. Depression of SHBG levels during danazol therapy has previously been described by Laurell and Rannevik23 and was further confirmed in this study (Table 1). Accordingly, the Scatchard curve for pooled treatment sera (Fig. 3) was markedly depressed. Pretreatment of serum with activated charcoal partly restored the binding capacity. Thus, the low values for SHBG binding capacity during danazol therapy mainly reflect occupation of binding sites by danazol and, to a lesser degree, a real decrease in protein concentration. The decrease in SHBG protein concentration, in tum, might reflect increased levels of free T. 24 It is tempting from the present data to explain the endocrine effects of danazol mainly in terms of its T displacing ability and as a consequence increased levels of free T during treatment. Data in the literature on the levels of total plasma T following danazol treatment show considerable disagreement. Cross-reactivity of danazol and its metabolites in T assays and species variations complicates the intepretation.2, 3, 5, 25 Human studies using specific assays report significant dose-related increases in total serum T.25 It should be recalled that the biologic effect of a steroid is mediated by its free fraction. Our data strongly suggest a marked increase in free T during danazol treatment. This interpretation is also supported by previous data.26, 27 In view of the fact that therapeutic danazol levels are 1000 times those of normal female total serum T levels, and that the affinity to SHBG for danazol is 1120 of that of T, one should conclude an almost total occupation of SHBG binding sites. However, SHBG is not the only steroid-binding protein in human serum, and the possible modulating effect of albumin and the influence offeedback mechanisms remains to be studied. Acknowledgments. Skillful technical assistance was provided by Mrs. M. Isaksson and Mrs. M. Wallen. REFERENCES 1. Potts GO: Pharmacology of danazol. J Int Med Res 5 (Suppl 3):1, ROnnberg L, Ylostalo P, Jiirvinen PA: Effects of danazol in the treatment of severe endometriosis. Postgrad Med J 55 (Suppl 5):21, Barbieri RL, Canick JA, Makris A, Todd RB, Davies IJ, Ryan KJ: Danazol inhibits steroidogenesis. Fertil Steril 28:809, Barbieri RL, Lee H, Ryan KJ: Danazol binding to rat androgen, glycocorticoid, progesterone, and estrogen receptors: correlation with biologic activity. Fertil Steril 31:182, Sherins RJ, Gandy HM, Thorslund TW, Paulsen CA: Pituitary and testicular function studies. I. Experience with a new gonadal inhibitor, 17a-pregn-4-en-20-yno(2,3-d) isoxazol-17-ol(danazol). J Clin Endocrinol Metab 32:522, Dmowski WP, Sholer HFL, Mahesh VB, Greenblatt RB: Danazol-a syntetic steroid derivate with interesting physiologic properties. Fertil Steril 22:9, Malkonen M, Mauninen V, HirvonenE: Effects ofdanazol and lynestrenol on serum lipoproteins in endometriosis. Clin Pharmacol Ther 28:602, Wynn V: Metabolic effects of danazol. J Int Med Res 5 (Suppl 3):25, Goebel R, Rjosk HK: Ein neues syntetisches Gestagen Behandlungsergebnisse bei Endometriose, Mastopathie und langjahriger primarer Ehesterilitat. 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6 23. Laurell CB, Rannevik G: A comparison of plasma protein changes induced by danazol, pregnancy and estrogens. J Clin Endocrinol Metab 49:719, Anderson DC: Sex-hormone-binding globulin. Clin Endocrinol 3:69, Floyd WS: Danazol: endocrine and endometrial effects. Int J Fertil 25:75, Schwarz S, Hintner H, Tappeiner G: Endocrinological study in 4 patients with hereditary angioneurotic edema (HAE) before and during danazol therapy: effects on sex hormone binding globulin (SHBG). Acta Endocrinol [Suppl 91] (Copenh)225:114, Schwarz S, Tappeiner G, Hintner H: Hormone binding globulin levels in patients with hereditary angioedema during treatment with danazol. Clin Endocrinol 14:563, 1981 Nilsson et al. Danazol displacement of testosterone 63