0021-972X/83/5704-0732$02.00/0
`Journal of Clinical Endocrinology and Metabolism
`Copyright © 1983 by The Endocrine Society
`
`Vol. 57, No.4
`Printed in U.S.A.
`
`Site of Action of Low Dose Ketoconazole on Androgen
`Biosynthesis in Men*
`
`R. J. SANTEN, H. VAN DEN BOSSCHE, J. SYMOENS, J. BRUGMANS, AND
`R. DECOSTER
`Department of Medicine, Division of Endocrinology, The Milton S. Hershey Medical Center, The
`Pennsylvania State University (R.J.S.), Hershey, Pennsylvania 17033; and Janssen Pharmaceutica,
`Beerse, Belgium
`
`ABSTRACT. Ketoconazole inhibits testosterone biosynthesis
`in men, but the exact site of its action on the androgen pathway
`remains to be established. To examine this question, we meas(cid:173)
`ured several steroids in the androgen and glucocorticoid path(cid:173)
`ways in normal men before and after receiving either a single
`dose of 200 mg ketoconazole or placebo in a cross-over random(cid:173)
`ized trial. Total and free plasma testosterone fell to levels 60%
`below basal within 4-8 h (P < 0.02 in all) and then returned to
`control concentrations by 24 h after drug administration. The
`transient alterations of plasma testosterone correlated well with
`ketoconazole blood levels, which peaked at 2 h and fell exponen(cid:173)
`tially thereafter. A compensatory increase in plasma LH at 24 h
`in the drug but not placebo group was consistent with the
`decrease in plasma testosterone. The levels of plasma artdro(cid:173)
`stenedione paralleled those of testosterone in the ketoconazole(cid:173)
`treated subjects. In marked contrast, plasma 17a-hydroxypro(cid:173)
`gesterone increased at 4-8 h (all P < 0.02) before returning to
`basal values at 24 h. This rise in precursor with fall in product
`
`steroid implicated an effect of ketoconazole on the c17-20 lyase
`enzyme. 'Fhis conclusion was supported by the highly significant
`increase in the ratio of plasma 17 a-hydroxyprogesterone to
`androstened~one observed between 2 and 24 h after drug admin(cid:173)
`istration. T4e effect of ketoconazole at this dose level appeared
`relatively specific, since no decrements in plasma cortisol or 11-
`desoxycortisof were found.
`During chronic admin\stration of 200 mg ketoconazole daily,
`decrements of plasma testosterone 2-4 h after drug administra(cid:173)
`tion were minimal and documented only by paired comparisons
`within subjects but not by unpaired tests between normal men
`and men receiving drug. The lack of major effects on testosterone
`levels long term at this dosage probably explain why few andro(cid:173)
`gen-related side effects with this drug were previously reported.
`Ketoconazole, therefo:e, represents another compound with rel(cid:173)
`atively selective effects on a cytochrome P-450-mediated steroid
`hydroxylation step, namely that involved with c17-20 lyase. (J
`. Clin Endocrinol Metab 57: 732, 1983)
`
`K ETOCONAZOLE (Nizoral; Janssen Pharmaceu(cid:173)
`
`tica, Beerse, Belgium) is an orally active, imida(cid:173)
`zole derivative used clinically as an antifungal agent (1).
`This drug inhibits lanosterol conversion to ergosterol in
`yeast at concentrations equal to or greater than 10-9 M,
`but in mammalian tissue only at concentrations of 10-6
`M (2, 3). Observations of gynecomastia in a small per(cid:173)
`centage of men given this drug led to studies of androgen
`levels after acute administration of ketoconazole ( 4, 5).
`A dose-response inhibition of plasma testosterone with
`single doses from 200-600 mg was found (5). Plasma
`testosterone decreased maximally to 14% of basal values
`8 h after an oral dose of 600 mg ketoconazole. The onset
`of suppression at 2 h and complete recovery at 24 h
`paralleled the concentrations of orally administered drug.
`At the higher doses (i.e. 400-600 mg, orally), ketocona-
`
`Received March 21, 1983.
`Address requests for reprints to: Dr. Richard J. Santen, Department
`of Medicine, Division of Endocrinology, Pennsylvania State University,
`Hershey, Pennsylvania 17033.
`*Presented in part at the American Society for Clinical Investiga(cid:173)
`tion, Washington, D.C., May 8-10, 1982.
`
`zole also blunted the increments in plasma cortisol after
`ACTH injections (6).
`The finding of marked but transient decreases in
`plasma testosterone acutely and lack of other clinically
`apparent endocrine effects in men during chronic keto(cid:173)
`conazdle administration suggested a relatively selective
`block of steroidogenesis. It appeared pertinent, then, to
`determine the site of action of ketoconazole on the an(cid:173)
`drogen biosynthetic pathway. We measured the levels of
`various plasma steroids in normal men to examine pre(cid:173)
`cursor to product relationships. This report identifies an
`effect of ketoconazole in low doses on the cytochrome P-
`450-dependent c17-20 lyase, one of two enzymes that
`mediate the conversion of 17 a-hydroxyprogesterone to
`androstenedione.
`
`Materials and Methods
`
`Patient studies
`
`Experimental protocol: acute. Ten normal men between the ages
`of 27 and 55 yr volunteered to take a single dose of ketoconazole
`(200 mg) orally and placebo on another occasion. A randomized
`
`732
`
`WCK1039
`Page 1
`
`

`
`ANDROGEN INHIBITION WITH KETOCONAZOLE
`
`733
`
`Testosterone
`
`I
`
`I
`
`I
`
`AI
`
`I
`
`0
`
`I
`
`I
`
`I
`2
`4
`8
`Free Testosterone
`
`I
`
`I
`
`I
`
`I
`
`-
`
`-
`
`I
`
`24
`
`E -C)
`
`c:::
`
`8-
`
`6-
`4-
`
`2-
`
`2.0-
`
`1.5-
`
`1.0-
`
`.5
`
`I
`
`0
`
`I
`2
`
`I
`
`4
`Hours
`
`I
`
`8
`
`I
`
`24
`
`B
`
`cross-over design was used, with each subject serving as his
`own control. After a basal sample was drawn at 0900 h, the
`subjects took either drug or placebo. Subsequent blood samples
`were collected 2, 4, 8, and 24 h later. After an interval of 1
`week, the identical protocol was repeated, but the men were
`crossed over to receive either drug or placebo, respectively.
`
`Experimental protocol: chronic. Testosterone levels were meas(cid:173)
`ured between 2 and 4 h after daily doses of 200 mg ketoconazole.
`In 10 men (aged 38-70 yr) receiving this drug for onychomy(cid:173)
`cosis, blood samples were obtained basally and at the 3rd, 6th,
`9th, and 12th months of ketoconazole administration. Testos(cid:173)
`terone levels in these patients were compared to those in 10
`normal men in whom blood was also collected and processed
`simultaneously with those in men given ketoconazole.
`
`Assay methods. Plasma 17 a-hydroxyprogesterone, androstene(cid:173)
`dione, and LH concentrations were measured by standard RIA
`methods used in our laboratories and previously characterized
`as to specificity, sensitivity, and precision (7-9). The assay for
`plasma androstenedione used Celite column chromatography
`before assay, and that for 17a-hydroxyprogesterone used LH-
`20 chromatography. The remaining methods employed J)lasma
`extraction without chromatography (testosterone and 11-
`desoxycortisol) (10, 11) or direct assay for plasma LH (9) and
`cortisol (commercial kit assay, CORT-CTK-125 internatio~al
`Cis-Sorin). Free testosterone [free and weakly bound or non(cid:173)
`testosterone-estrogen-binding globulin (non-TeBG) bound]
`was measured with the method of Tremblay and Dube (12), as
`slightly modified in our laboratory. Ketoconazole levels were
`measured after purification on high pressure-liquid chromatog(cid:173)
`raphy, as previously described (13).
`
`Statistical methods. Student's paired t tests (two-tailed) were
`employed to compare values during placebo or drug adminis(cid:173)
`tration at each time point during acute studies, and unpaired t
`tests were used for chronic studies.
`\
`
`Results
`
`Acute studies
`
`Plasma testosterone fell from 4.92 ± 0.35 (±SEM) ng/
`ml to nadir levels of 2.08 ± 0.23 ng/ml 4 h after the
`administration of drug but not after placebo (P < 0.01
`us. placebo). Recovery from suppression began at 8 hand
`was complete at 24 h (Fig. 1A). Free testosterone (i.e.
`non-TeBG bound) fell to a similar extent. These decre(cid:173)
`ments correlated well with drug blood levels, which
`peaked at 2 h [2.65 ± 0.45 (±SEM) JLg/ml) and then fell
`exponentially at 4 h (1.47 ± 0.32 JLg/ml) and 8 h (0;25 ±
`0.09 JLg/ml). No drug was detectable in plasma at 24 h.
`In response to the testosterone decrement, plasma LH
`increased in the drug- but not in placebo-treated patients
`(Fig. lB). There was a high degree of variability in LH
`measurements, which is attributable to pulsatile LH
`secretion (9). Nonetheless, at 24 h, plasma LH levels
`were significantly higher after drug (8.2 ± 0.9 ng/ml)
`
`24
`
`8
`
`/
`
`4
`Hours
`FIG. 1. A, Plasma total and free testosterone levels (mean ± SEM)
`basally and at various time points after a single 200-mg dose of
`ketoconazole or placebo given to normal men in a· randomized cross(cid:173)
`over study (see Materials and Methods). *, P < 0.05; **, P < 0.02; ***,
`P < 0.01. 0-0, Placebo (n = 10); •- -•, 200 mg ketoconazole. B,
`Plasma LH levels. Study design was identical to that in A. 0-0,
`Placebo; •- -•, 200 mg ketoconazole.
`
`than after placebo (5.6 ± 0.7; P < 0.01) administration.
`The concentrations of plasma androstenedione paral(cid:173)
`leled those of testosterone (Fig. 2A), decreasing from 1.0
`
`WCK1039
`Page 2
`
`

`
`734
`
`A
`
`.6.4 Androstenedione
`
`17 -OH Progesterone
`
`E -Ol
`
`1::
`
`Hours
`
`Ratio 17 -OH Progesterone/ Androstendione
`
`SANTEN ET AL.
`
`. JCE & M • 1983
`Vol57•No4
`± 0.13 to 0.64 ± 0.04 (±SEM) ng/ml (P < 0.01) at 0800
`h, while recovering at 24 h after drug administration. In
`marked contrast, the levels of 17 a-hydroxyprogesterone
`increased after drug administration to 2.8 ± 0.4 ng/ml at
`8 h, while, after placebo, a diurnal decrement to 1.5 ±
`0.2 was found (Fig. 2A). Thus, ketoconazole caused a
`highly significant increase in 17 a-hydroxyprogesterone
`(P < 0.01) at 8 h, with complete return to basal concen(cid:173)
`trations at 24 h.
`The decline in plasma androstenedione and testoster(cid:173)
`one with an increase in 17 a-hydroxyprogesterone sug(cid:173)
`gested ablock of the C17_20 lyase with accumulation of
`precursor (17a-hydroxyprogesterone) and a fall in prod(cid:173)
`uct (androstenedione). To examine this more closely, the
`ratio of 17 a-hydroxyprogesterone to androstenedione
`was calculated (Fig. 2B). A significant increase in this
`ratio occurred at 2 h, with a plateau at 4-8 h (P < 0.001
`and P <: 0.01 at 4 and 8, respectively) and persistent but
`minimal i~crements at 24 h (Fig. 2B).
`As a m·easure of the selectivity of blockade after a
`single low dose of ketoconazole, plasma cortisol levels
`were not significantlf different at any time point in drug ..
`treated men from those in placebo-treated men. As a
`result of diurnal changes, cortisol declined by 30% in
`both groups at 4-8 h and returned to baseline the next
`morning [placebo: basal, 15 ± 1.3; 4 h, 9.4 ± 1.2; 8 h, 8.4
`± 1.4 Jtg/dl; ketoG,onazole: basal, 16.5 ± 1.3; 4 h, 8.0 ±
`1.2; 8 h, 9.9 ± 1.5 Jtg/dl (mean± SEM)]. 11-Desoxycortisol
`levels were also similar in both groups of men, except at
`8 h when drug-treated men had significantly (P < 0.01)
`hi~her levels (0. 73 ± 0.04 Jtg/dl) than after placebo treat(cid:173)
`ment (0.63 ± 0.05 Jtg/dl).
`
`Chron~c studies
`
`During chronic therapy with a single daily dose of 200
`mg ketoconazole, testosterone levels 2-4 h after the
`morning dose were slightly lower than basal levels in
`drug-tteated men (Fig. 3). However, nonpaired compar(cid:173)
`isons of values in normal subjects or in men receiving
`drug at each time point revealed no statistically signifi(cid:173)
`cant differences. Drug levels in plasma increased during
`chronic administration from 0.75 ± 0.15 Jtg/ml at 3
`months to 2.9 ± 0.59 at 12 months. This probably reflects
`the variable times after drug ingestion (i.e. 2-4 h) that
`the blood samples were collected. This conclusion is
`supported by an additional study (clinical files of Janssen
`Pharmaceutica), where plasma ketoconazole levels were
`constant in five men sampled similarly 2-4 h after morn(cid:173)
`ing doses (i.e. 3 months, 3. 7 ± 0. 76 Jtg/ml; 6 months, 2.9
`± 0.55 Jtg/ml; 9 months, 2.4 ± 0.53 Jtg/ml).
`
`Discussion
`
`A wide variety of clinically available drugs exert inhib(cid:173)
`itory effects on the hypothalamic-pituitary-targ¢t gland
`
`Hours
`FIG. 2. A, Plasma androstenedione (top) and 17a-hydroxyprogesterone
`(bottom) levels. Study design was identical to that in Fig. lA. 0-0,
`Placebo; •- -•, 200 mg ketoconazole. B, Ratios of 17a-hydroxyproges(cid:173)
`terone to androstenedione.
`
`WCK1039
`Page 3
`
`

`
`ANDROGEN INHIBITION WITH KETOCONAZOLE
`
`735
`
`Testosterone
`
`ri
`
`r*
`
`,.:t
`
`*
`
`*
`
`*
`
`6
`
`E 4 -C) c:
`
`2
`
`0
`
`0
`3
`6
`9
`12 months
`N
`9
`9
`10
`9
`10
`FIG. 3. Plasma testosterone levels during chronic administration of
`200 mg ketoconazole as a single oral dose daily in 10 patients (~) and
`in 10 normal men (o). All blood samples were collected 2-4 h after
`single doses of drug in the patients and at the same time in the normal
`subjects. *, P < 0.05, basal us. treatment. The differences between
`normal and drug-treated patients were not significantly different at
`any time point.
`f
`
`axes. Certain of these compounds inhibit the biosyn(cid:173)
`thesis of steroids involved in negative feedback control
`systems. Compensatory increments of the respective
`trophic hormones result,_ which can attenuate or com(cid:173)
`pletely mask the endocrine inhibitory properties of these
`drugs. For this reason, subtle endocrine actions of certajn
`drugs have not been detected in basic toxicological, pre(cid:173)
`clinical, and early clinical testing. Only after exposure of
`large numbers of patients have definite and often potent
`endocrinological effects of certain ~ompounds been
`noted. For example, the potent adrenal inhibitory and
`T4-blocking properties of aminoglutethimide were unde(cid:173)
`tected during early testing and clinical introduction (14).
`Because of compensatory ACTH and TSH increments
`o~curring during aminoglutethimide\administration, the
`majority of subjects initially receiving this ,compound
`experienced no signs or symptoms of cortisol qr T 4 defi-
`ciency (15, 16).
`\.
`Ketoconazole, similarly, produced no effects on the
`male reproductive tract or symptoms of androg~n defi(cid:173)
`ciency in preclinical and early clinical trials (17-20).
`Reports of gynecomastia in a limited number of patients
`receiving larger doses led to initial endocrinological stud(cid:173)
`ies (4, 5). The decrements in testosterone and compen(cid:173)
`satory increments in plasma LH observed in this and
`earlier studies ( 5) demonstrated a potent effect of keto(cid:173)
`conazole on androgen biosynthesis. Two factors mini(cid:173)
`mize the significance of this effect on a practical basis.
`First, the drug is given as a single daily dose, and the
`half-life is short (i.e. 4 h). Consequently, the acute re(cid:173)
`duction of testosterone is transient, and complete recov(cid:173)
`ery in androgen levels occurs by 24 h. Secondly, a com(cid:173)
`pensatory increase in LH results, which, ~nalogous to
`the ACTH increment after aminoglutethimide treat(cid:173)
`ment, may allow the effect of low doses of drug to be
`Partially or completely overcome during chronic admin(cid:173)
`istration (5). This latter effect may explain why plasma
`
`testosterone levels were minimally suppressed during
`daily administration of 200 mg, orally, for up to 12
`months. Unfortunately, additional plasma for LH, FSH,
`and estradiol assays is not currently available for further
`assessment of the drug effects during chronic (or acute)
`administration.
`Doses of ketoconazole higher than 200 mg daily, when
`administered chronically, may persistently suppress an(cid:173)
`drogen production in men (Dunn, J ., and J. Graybill,
`presented at a workshop meeting on ketoconazole at
`Janssen Pharmaceutica, Beersa, Belgium, January 22,
`1983). Pont et al. (5) found greater suppression of testos(cid:173)
`terone with a single 600-mg dose than with 200 mg. This
`could be due in part to additional blocking effects of
`ketoconazole. For example, enzymes in addition to the
`c17-20 lyase could be inhibited with higher ketoconazole
`doses. Cortisol responsiveness to ACTH is blunted dur(cid:173)
`ing the administration of 400-600 mg ketoconazole (6).
`Such pharmacological effects would be analogous to
`those observed with aminoglutethimide which blocks
`aromatase at low doses (i.e. 0.3 MM), and also cholesterol
`side-chain cleavage and 18-hydroxylation of corticoster(cid:173)
`one at higher concentrations (3 MM) (14, 21). These
`considerations indicate that further dose-response stud(cid:173)
`ies of ketoconazole on various steroid biosynthetic en-
`zymes are now required.
`.
`Inferences regarding the site of action of drugs such as
`ketoconazole with· measurements of circulating plasma
`levels of steroid are indirect. Alterations of steroid
`MCRs, for example, can perturb the ratios of precursor
`to product in plasma independent of an enzymatic block.
`However, a change in the androstenedione or 17-hydrox(cid:173)
`yprogesterone MCRs within 4-8 h after the administra(cid:173)
`tion of a single dose of ketoconazole would not be ex(cid:173)
`pected. The drug effect on plasma steroid ratios could
`also be explained on another basis. A greater release of
`steroid precursor from its site of synthesis and a lesser
`leak of product from tissue without an associated enzy(cid:173)
`matic blockade could explain altered precursor to product
`steroid ratios. This possibility is unlikely, since in vitro
`3H -labeled steroid metabolic studies also demonstrated
`an inhibitory effect of ketoconazole on the conversion of
`17 a-hydroxyprogesterone to androstenedione (H. Van
`den Bossche, H., presented at a workshop conference on
`ketoconazole at Janssen Pharmaceutica, Beersa, Bel(cid:173)
`gium, January 23, 1983).
`Androstenedione and 17a-hydroxyprogesterone origi(cid:173)
`nate from both adrenal and gonadal sources in men.
`Ketoconazole could block the testis or the adrenal exclu(cid:173)
`sively, or both glands simultaneously. The fall in plasma
`testosterone, a nearly exclusive testicular steroid in men,
`suggests a direct gonadal effect. An additional action on
`the adrenal is suggested by the blunting of ACTH-stim(cid:173)
`ulated cortisol during the administration of higher doses
`
`WCK1039
`Page 4
`
`

`
`736
`
`SANTEN ET AL.
`
`JCE&M•l983
`Vol57•No4
`
`ofketoconazole (6). Whether a single 200-mg dose blocks
`adrenal c17-20 lyase as well as its testicular counterpart
`remains to be established.
`It would provide additional support for our conclusions
`to demonstrate that steroid levels in pubertal boys with
`congenital c17-20 lyase deficiency are similar to the con(cid:173)
`centrations in our patients. Zachmann et al. (22) recently
`reported 3 patients with this congenital defect and re(cid:173)
`viewed the literature in 11 others. In all 3 patients, the
`17 a-hydroxyprogesterone levels were only slightly ele(cid:173)
`vated and increased further after hCG or ACTH admin(cid:173)
`istration. The degree of inhibition of androstenedione
`and testosterone biosynthesis varied from mild in two
`patients to profound in the third. Taken together, the
`degree of changes in 17a-hydroxyprogesterone, andro(cid:173)
`stenedione, and testosterone in Zackmann' s patients are
`similar to those 4-8 h after a single dose of ketoconazole.
`Zackmann et al. (22) speculated that two enzymes exist
`for the conversion of 17 a-hydroxyprogesterone to andro(cid:173)
`stenedione. The first enzyme is involved in the conver(cid:173)
`sion of both 17 a-hydroxypregnenolone to dehydroepian(cid:173)
`drosterone (~5 pathway) and 17a-hydroxyprogesterone
`to androstenedione (~4 pathway), and the second (the
`c17-20 desmolase, strictore sensu) exclusively mediates
`the ~4 pathway conversion. In vitro studies to be pub(cid:173)
`lished separately by Van den Bossche suggest that ke(cid:173)
`toconazole inhibits the. latter of these two enzymes,
`which mediates the conversion of 17 a,20a-dihydroxypro(cid:173)
`gesterone to androstenedione. Studies to determine the
`effects of ketoconazole on the ~ 5 pathway in vitro are
`ongoing. This methodological approach will allow eval(cid:173)
`uation of these effects without the confounding action of
`LH in producing a switch from the ~ 4 to the ~ 5 pathway
`(23).
`In summary, ketoconazole blocked C17_20 lyase and
`caused an acute rise in 17 a-hydroxyprogesterone, with
`decrements in androstenedione and testosterone. This
`effect induced a compensatory rise in LH secretion.
`During chronic administration of 200 mg ketoconazole
`as a single daily dose, testosterone levels were not sup(cid:173)
`pressed, perhaps as a result of persistent mild increments
`in LH secretion.
`
`Acknowledgments
`The authors are grateful to Mr. R. Woestenbroghs for determination
`of ketoconazole plasma levels. Mrs. Marlene Thompson provided ex(cid:173)
`cellent secretarial assistance. The authors acknowledge the support of
`the Core Endocrine Laboratory for assistance with the RIA measure(cid:173)
`ments.
`
`References
`1. @raybill JR, Drutz DJ 1980 Ketoconazole: a major innovation for
`treatment of fungal disease. Ann Intern Med 93:921
`
`2. Van den Bossche H, Willemsens G, Cools W, Cornelissen F,
`Lauwers W, Cutsem J 1980 In vitro and in vivo effects of the
`antimycotic drug ketoconazole on sterol synthesis. Antimicrob
`Agents Chemother 17:922
`3. Willemsens G, Cools W, Van den Bossche H 1980 Effects of
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`4. DeFelice R, Johnson DG, Galgiani JN 81 Gynecomastia with
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`7. Samojlik E, Santen RJ 1978 Adrenal suppression with aminoglu(cid:173)
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`8. Samojlik E, Santen RJ, Wells SA 1977 Adrenal suppression with
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`9. Santen RJ, Bardin CW 1973 Episodic luteinizing hormone secre(cid:173)
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`-
`10. De Coster R, Becke:il's JF, Wouters-Ballman P, Ectors F 1979
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`12. Tremblay RR, Dube JY 197 4 Plasma concentrations of free and
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`14. Santen RJ, Henderson IC 1981 Pharmanual: A Comprehensive
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`and New York
`15. Santen RJ, Wells SA, Cohn N, Demers LM, Misbin RI, Foltz EL
`1977 Compensatory increase in TSH secretion without effect on
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`16. Fishman LM, Liddle GW, Island DP, Fleischer N, Kuchel 0 1967
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`17. Restrepo A, Stevens DA, Utz JP 1980 First International Sympos(cid:173)
`ium on Ketoconazole. Rev Infect Dis 2:519
`18. Stevens DA, Stiller RL, Williams PL, Sugar AM 1982 Experience
`with ketoconazole in three major presentations of progressive
`coccidioidomycosis. Am J Med 7 4:58
`19. Levine HB 1982 Ketoconazole in the Management of Fungal
`Disease. ADIS Press, New York
`20. Graybill JR 1983 Introduction: an update on progress with keto(cid:173)
`conazole. Am J Med 7 4:1
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