0021-972x/92/7403-0623$03.00/0 Journal of Clinical Endocrinology Copyright 0 1992 by The Endocrine
and Metabolism Society
Vol.
Printed
74, No. 3
in U.S.A.
Enhanced Transdermal Delivery of Testosterone across Nonscrotal Skin Produces Physiological Concentrations of Testosterone and Its Metabolites in Hypogonadal Men* A. WAYNE MEIKLE, JOHN D. STRINGHAM, WILLIAM D. ODELL
NORMAN KEITH
A. MAZER, JOHN F. MOELLMER, G. TOLMAN, STEPHEN W. SANDERS,
AND
Departmentsof Medicine (A. W.M., J.D.S., K.G.T., S.W.S., W.D.O.) and Pharmaceutics(N.A.M.), University of Utah, and TheraTech, Inc. (N.A.M., J.F.M.), Salt Lake City, Utah 84132
ABSTRACT. None of the current or experimental androgen treatment modalities for male hypogonadism has been reported to produce physiological concentrations or circadian variations in testosterone (T) and its metabolites, dihydrotestosterone (DHT) and estradiol (Ez), This investigation describes a novel transdermal dosage form designed to enhance the delivery of native T across nonscrotal skin. The main objective was to determine whether the nightly application of two experimental transdermal patches to different sites on the body (e.g. back, chest, arms, etc.) would result in normal plasma levels of T, DHT, and Ez for men and mimic the normal circadian variation. Six hypogonadal males (aged 24-66 yr) were studied 4 weeks after stopping T ester treatment. After single application of two patches, T levels increased from a pretreatment baseline of 5.8 f 0.94 nmol/L (mean + SE; 167 f 27 ng/dL) to an average peak concentration of 44.1 & 4.8 nmol/L (1273 f 138 ng/dL) 5.7 f 0.6 h after application and reached a 24-h level of 16.9 f 2.9 nmol/L (488 + 85 ng/dL). DHT and E, levels exhibited parallel variations within the normal reference ranges. During 4 weeks of daily evening application to various sites on the torso, the mean delivery of T from two patches was 5.2 + 0.1 mg/day (-20% of the patch content), and morning T levels were within
the normal limits. On day 28 of treatment, the 24-h plasma profiles of T, DHT, and E, (obtained with two patches on the back) approximately mimicked the normal circadian variations reported in healthy young men. The time-averaged T level was 21.8 + 2.9 nmol/L (629 + 84 ng/dL), and the plasma concentration ratios of DHT/T (0.07 + 0.01) and Ez/T (0.005 + 0.001) were within the normal range. SHBG concentrations were not significantly altered over the 4 weeks of treatment. The patches were well tolerated, except for one patient who developed a local reaction to an excipient during the third week of treatment. Two of the patients (one with Klinefelter’s syndrome) completed several months of continuous therapy. T, DHT, and Es have remained in the range of normal, and plasma LH levels in the patient with Klinefelter’s syndrome became normal. Subjective improvement in symptoms has continued, and tolerability has been good in both patients. These results indicate that the enhanced transdermal delivery of T across nonscrotal skin is a patient-friendly androgen replacement modality and produces physiological concentrations of T and its metabolites, which are unattainable with other treatment modalities. (J Clin Endocrirwl Metab 74: 623-628, 1992)
T
(T) secretion and plasma concenESTOSTERONE trations are reduced below normal in many pathophysiological conditions. These include diseases directly affecting Leydig cells of the testes and disorders of the pituitary and hypothalamus that reduce LH secretion and secondarily decrease T synthesis by Leydig cells. In such patients, replacement androgen therapy is usually prescribed as treatment. However, none of the current replacement therapies [including oral administration (l5), sublingual treatment (6), depot injections (7-ll), SC Received April 5, 1991. Address all correspondence and requests for reprints to: A. Wayne Meikle, M.D., Division of Endocrinology, 50 North Medical Drive, Salt Lake City, Utah 84132. * This work was supported by TheraTech, Inc. and USPHS Grant MOl-RR00064. A.W.M., J.D.S., K.G.T., S.W.S., and W.D.O. were independent from TheraTech and contributed to the experimental design and data analysis of the investigation.
implants (12, 13), and experimental “transscrotal” patches (14-19)] has been reported to produce physiological concentrations of T and its metabolites or to mimic the normal circadian variation of T (20-23). Therapy with oral preparations of synthetic androgens is associated with hepatotoxicity, including cholestatic jaundice, hepatic tumors, and vascular lesions (5, 24, 25). We have investigated a novel enhanced transdermal system, designed to deliver native T across nonscrotal skin, to determine if it produces physiological plasma concentrations of T and its metabolites in six hypogonadal men. Materials
and Methods
Study participants
The six men participating in the study ranged in age from had primary hypogonadism [Klinefelter’s
24-66 yr. One patient 623
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624
MEIKLE
syndrome(KS)], and five had hypogonadotropichypogonadism of various causes.All patients had been treated with a depot parenteral T ester until 1 month before the study began.None of the menwasreceiving drugsthat are known to interfere with androgen metabolism, although one was receiving a tricyclic antidepressantthat may causesexualdysfunction. The protocol was approved by the Institutional Review Board for research with human subjects,University of Utah, and written consent was obtained from each subject before enrollment into the study. Each participant underwent a prestudy screeningexamination, which included a medical history, physical examination (including the prostate), electrocardiogram, complete blood count, chemistry profile, lipid profile, urinalysis, thyroid function studies, and serum T, dihydrotestosterone (DHT), 17/3-estradiol(E2),LH, FSH, and sex hormone-bindingglobulin (SHBG) measurements. Transdermal
ET AL.
JCE & M .1992 Vol74.No3
Patch T assay
The residualcontent of T in all usedpatcheswasdetermined by reverse phase high pressure liquid chromatography after methanol extraction. The amount of T released from the patches was calculated as the difference between their initial content (12.8 mg) and the measuredresiduals. Blood hormone
assays
T, DHT, EP,gonadotropins(LH and FSH), and SHBG were assayedby previously describedprocedures(22, 23). DHT was determinedby RIA after solvent extraction and isolation using the Bush A solvent system,which separatesDHT from steroids having any cross-reactionwith the antisera. Internal recovery standardswere usedto correct for procedural losses.The interassaycoefficients of variation were about 10% or lessfor all of the assays.
T patches
Experimental T transdermal patches (code 6275-O) were suppliedby the sponsor,TheraTech, Inc. (Salt Lake City, UT). Each patch contained 12.8 mg T in a proprietary permeationenhancing vehicle composedof polar, nonpolar, and amphiphilic excipients (26) and glycerine, an antiirritant (27). The delivery area of each patch was 7.5 cm’, and the total area (including the adhesive)was25 cm’. Basedon in uitro permeation studies (TheraTech, Inc.), the 24-h application of two patcheswasexpected to deliver approximately 7 mg T. Patch applications
On day 1 of the single application steady-stateprofile (SlB) study, two patches were applied to the midback region of each patient at 2200 h and securedwith additional adhesivetape (Elastikon, Johnson and Johnson, New Brunswick, NJ). The patcheswere removed 24 h later, and the subjectsremainedin the clinic for an additional 24 h (without patches) for measurementsof the elimination kinetics. The 4-weekS2B study began immediately thereafter. In this phase,subjectsself-appliedtwo patchesnightly (at -2200 h) to various siteson the torso (e.g. back, upper arms,chest, abdomen,legs,etc.) for 26 consecutive days. On day 27, the patcheswere again applied in the clinic to the midback region (with additional tape) for measurementsof 24-h steady state hormoneprofiles and subsequentelimination kinetics. Blood collection for hormone
assays
Plasmaconcentrations of T, DHT, and E2 were performed before treatment and on days l-3 (SlB study) and days 27-29 (S2B study) 1, 2, 4, 6, 8, 12, 16, 20, and 24 h after patch application and 1, 2, 4,6, 12, 18, and 24 h after patch removal. Hormone levels were also determined at weekly intervals (on days 7, 14, and 21 of the S2B study) approximately 12 h after self-application of the patches.Blood sampleswere collectedin tubescontaining heparin andcentrifuged at 4 C, and the plasma was stored at -20 C until assayedfor sex hormones. The concentrations of LH, FSH, and SHBG were also measured during eachin-patient study phase.
Data analysis
The initial (prestudy) baselinehormoneconcentrationswere computed as the average of the concentrations measuredat 0 and 24 h after removal of the first patches. This was done to compensatefor inherent fluctuations in the endogenoussecretion of T and improve the accuracy of the baseline adjusted profiles. The poststudy baselineconcentrations were taken as the values measured24 h after removing the day 27 patches. The baselineadjusted hormone profiles (for day 1) were calculated for each subjectby subtracting the baselineconcentration from the measuredlevel, with negative values set at zero. The tl,z values were estimated for each subject by visually fitting a straight line to a log linear plot of their baseline adjustedT levels measured0, 1, 2, 4, 6, and 12 h after patch removal. The results are essentially equivalent to a weighted least squaresfit of the data, but are lesssensitive to spurious fluctuations. The maximum plasmaconcentration (Cpmsx) and time of the maximum concentration (T,,,) were determinedby inspection. The area under the curve was computed using the trapezoidal rule. The kinetics of T absorption from the transdermal patches were calculated from the baseline adjusted T profile from day 1, using the method of Wagner and Nelson (28) and assuminga normal plasma T clearance rate of 1100 L/day (29-31). The resultsare expressedas the meanf SE.
Results Sex steroid study)
profiles
after single patch
application
(SIB
T, DHT, and E2 concentrations were determined over an interval of 48 h after the application of two transderma1 patches to the back for 24 h at the beginning of the study (Fig. 1). Plasma T concentrations increased from a baseline of 5.8 f 0.94 nmol/L (mean + SE; 167 f 27 ng/dL) to an average peak concentration of 44.1 +- 4.8 nmol/L (1273 f 138 ng/dL) at 5.7 f 0.6 h and decreased thereafter to a plateau of about 17 nmol/L (500 ng/dL) for the remaining 12 h. The tI,z of the elimination phase
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ENHANCED
TRANSDERMAL
625
T DELIVERY
10.0 E 8.0 Patch nalysis c---
‘r E -
6.0
0.0
4.0
8.0
12.0
16.0
20.0
1
I
Time (h) FIG. 2. Cumulative T input kinetics based on in uiuo plasma profiles by the method of Wagner and Nelson (28). The arrow indicates the total T input for 24 h based on residual content of the patches determined by high pressure liquid chromatography.
Daily patch release data and weekly plasma concentrations of T
I
I 0
u
24 Time
56
4s
(h)
FIG. 1. The plasma concentrations of T, DHT, and E2 before and after application of two patches to the back of six hypogonadal men for 24 h and after removal of the patches (mean + SE). The shaded area represents the range of hormone concentrations (95% confidence interval) in normal men (22, 23).
after removal of the patches was 116 f 17 min. DHT and EP (Fig. 1) reached peak concentrations at about 612 h, and these two metabolites of T paralleled the values observed for T and were both in the range observed in eugonadal men. Transdermal absorption kinetics
Based on analysis of the baseline adjusted T profiles, the cumulative amount of T absorbed from the patches during 24 h is shown in Fig. 2. During the first 8 h, T was absorbed at a faster rate than during the last 12 h. This change in absorption rate accounts for the peak in the T profile and the subsequent plateau phase. The total amount absorbed at 24 h was in excellent agreement with the calculated amount of T released from the patches (i.e. 6.4 f 0.5 mg/two patches).
Based on patch analysis, the total amount of T delivered per two patches varied on a daily basis from 4.1-6.8 mg, with a mean of 5.2 f 0.1 mg over the entire 28-day study (see Fig. 3, lower panel). In all cases the values were within the normal physiological range of endogenous T production (i.e. 3-10 mg/day) (29-32). The mean T concentrations measured about 12 h postpatch application on days 1, 7, 14, 21, and 28 of the study are shown in Fig. 3 (upper panel). Compared to the pre- and poststudy baselines (also shown), the weekly concentrations remained within the normal range and correlated (r = 0.82; n = 5 time points) with the patch release data (Fig. 3, lower panel). The mean baseline T concentration was lower after day 28 than on day 1 (n = 5; P < 0.05). This suggests that endogenous secretion of T, which was low in these men, was further reduced by the treatment. During the 4 weeks of continuous treatment in which the participants applied the patches, one patient developed a skin reaction to the patches and was removed from the study on day 23. Steady state sex steroid profiles after the last patch application (S2B study)
The average peak T concentration after the second 24h study was 33.5 f 4.3 nmol/L (966 f 122 ng/dL; n = 5; Fig. 4) and was also achieved about 6 h after application of the patch. The time-averaged T level of 21.8 + 4.3 nmol/L (629 f 84 ng/dL; n = 5; Fig. 4) was also achieved about 6 h after application of the patch. The patterns of T concentrations were similar during both the first and second 24-h study periods (Figs. 1 and 4). Further, the
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626
MEIKLE
’
I
SlB -
Outpatient-
528
I I
I
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JCE & M. 1992 Vol74.No3
AL.
’
B. 212.0111 ;: > g.” L D
6.0
E f
3.0
0
7
Day
U
21
26
of Application
FIG. 3. The daily total T release (lower panel) and the weekly plasma T concentration (upper panel) before and after wearing two patches daily; the patches were applied at 2200 h, and the plasma T values were measured in samples collected at 1000 h. The normal ranges of T production (29-32) and morning T levels (22, 23) are shown by the shaded area. T levels denoted BL represent baseline levels before and after SlB and S2B studies. Values are the mean + SE (0, n = 6; 0, n = 5).
steady state concentration profiles of T and DHT (Fig. 4) mimicked the circadian variation that has been reported in various studies of T (20, 21, 33-36) and DHT (21,34,35) in normal young men. No circadian variation has been reported in the literature thus far for Ez (32, 35, 37). Ratio of DHT/T
and EJT
0 0.0
4.0
2200 h
8.0
12.0
Time (h)
15.0
20.0
24.0
2200 h
FIG. 4. Plasma concentrations of T (lower panel), DHT (middle panel), and El (upper panel) after application of two patches to the back in five subjects on day 28. Values are the mean f SE. Shaded areaa are normal ranges, as described in Fig. 1.
values were 39 + 5 nmol/L at the beginning of the study and 41.33 k 4.8 nmol/L at the end of 4 weeks. The LH decreased toward normal in the patient with KS, and the FSH level declined somewhat. Local tolerability
Based on the areas under the plasma concentration profiles measured in the steady state study (S2B), the ratios of DHT/T and EJT were 0.07 f 0.01, and that of EJT was 0.005 f 0.001, respectively. These ratios were not significantly different between the two observation periods (SlB us. S2B; P > 0.4) and are comparable to the DHT/T and EJT ratios observed for normal men in our laboratory (22, 23, 29).
The patches were well tolerated, except for one patient who developed a local reaction to the patches on day 23, requiring discontinuation. This reaction was demonstrated to be an allergic sensitivity to one of the excipients in the patch formulation. In general, there was only a mild and transient redness (without edema) at the application site, but otherwise no unpleasant reactions were observed.
SHBG, LH and FSH
Continuation study
The SHBG DHT-binding capacity did not change significantly during the course of the study. The mean
Two subjects were studied for 6 additional months. The patches were well tolerated by them, and they re-
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ENHANCED
TRANSDERMAL
ported excellent subjective responses in sexual function, libido, energy level, and mood. The plasma concentrations of T, DHT, and Ez measured about 12 h after application of the patches are shown in Fig. 5. With rare exception, these values have remained within the normal reference ranges. SHBG concentrations (data not shown) have also remained in the normal range (30-110 nmol/ L). The subject with primary hypogonadism had baseline elevated concentrations of LH and FSH (44 and 35 IU/ L, respectively). The LH level became normal after 4 months of therapy, whereas FSH declined, but was still elevated above the normal range. Gonadotropin levels in the patient with secondary hypogonadism also decreased. Plasma concentrations of cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, and triglycerides remained relatively constant in both patients over the course of therapy.
12.0
20.0
26.0
T DELIVERY
627
Discussion
This study demonstrates that a novel enhanced transdermal delivery system is capable of producing normal plasma concentrations (23, 23), including circadian variation (20, 21, 33-37), of T and its potent metabolites, DHT and Ez, over 24 h in hypogonadal men. Transderma1 delivery of T across nonscrotal skin produces normal ratios of DHT/T and EJT. This contrasts with reports of transdermal delivery across scrotal skin, in which DHT levels are initially normal relative to T and then become elevated, resulting in a ratio of DHT/T of 1:2.5, rather than the normal ratio of about 1:lO (14-19, 22, 23). The scrotal skin contains higher activity of 5areductase than nonsexual skin such as the trunk, back, and legs (38,39). Apparently, the 5a-reductase of scrotal skin, which converts T to DHT, is inducible by androgen and results in increased delivery of DHT relative to T during extended use of the scrotal T patch. In contrast, enhanced delivery of T across nonscrotal skin did not induce the already low activity of 5a-reductase during the time of the studies reported herein and resulted in a pattern of sex steroid concentrations that more closely resembles secretion of T by the Leydig cells of the testes and peripheral conversion of T to its active metabolites. Sex steroid concentrations after administration of depot T esters (11, 40) or oral T undecanoate (l-5) do not mimic normal T concentrations as provided by the current patch system. Depot T esters result in supraphysiological concentrations of both T and Ez, followed over several days by subnormal plasma T concentrations (11, 40). T undecanoate produces multiple daily peaks and valleys of the androgen and must be administered several times daily (l-5). The patches were well tolerated, except by one individual who developed an allergic reaction to an excipient of the patches. Based on recent dermatologic studies in 52 normal men, where no such allergic reactions were observed (Mazer, N. A., unpublished data), the frequency of such reactions among patients is expected to be low. We conclude that transdermal delivery of T across nonscrotal skin produces physiological plasma concentrations and circadian variations of T and its potent metabolites, DHT and El, in hypogonadal men. This patient-friendly treatment system should provide new insights into the potential benefits and safety issues associated with the correction of androgen deficiency in a variety of clinical disorders (24, 25, 41).
36.0
Weeks
FIG. 5. Continuation study; plasma concentrations of T (lower panel), DHT (middle paneel), and E2 (upper panel) in two subjects. Samples were obtained at 2- to 4-week intervals, approximately 12 h after application of two patches to various sites on the torso. Shaded areas are normal ranges, as described in Fig. 1.
References 1. Johnsen SG, Bennett EP, Jensen VG. Therapeutic effectiveness of oral testosterone. Lancet. 1974;2:1473-5. 2. Cantrill J, Dewis P, Large DM, Newman M, Anderson DC. Which testosterone replacement therapy? Clin Endocrinol (Oxf). 1984;21:97-107.
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MEIKLE 3. Franchi F, Luisi M, Kicovic PM. Long term study of oral testosterone undecanoate in hypogonadal males. Int J Androl. 1978;1:270-8. 4. Franchimont P, Kicovic PM, Mattei A, Roulier R. Effects of oral testosterone undecanoate in males. Clin Endocrinol (Oxf). 1978;9:313-20. 5. Werner SC, Hanger FM, Kritzler RA. Jaundice due to methyltestosterone therapy. Am J Med. 1950;8:325. 6. Stuendel CS, Dudley RE. Sublingual (SL) testosterone (T) simulates episodic androgen release. Proc of the 73rd Annual Meet of The Endocrine Sot. 1990;426. 7. Sokol RZ, Palacios A, Camptiedl LA, Saul CC, Swerdloff RS. Comparison of the kinetics of injectable testosterone in eugonadal and hypogonadal men. Fertil Steril. 1982;37:425-30. 8. Aakvaag A, Vogt JH. Plasma testosterone values in different forms of testosterone replacement. Acta Endocrinol (Copenh). 1969:60:537-42. 9. Fujioka M, Shinohara Y, Baba S, et al. Pharmacokinetic properties of testosterone propionate in normal men. J Clin Endocrinol Metab. 1986;63:1361-4. 10. Schulte-Beerbuhl M, Nieschlag E. Comparison of testosterone, dihydrotestosterone, luteinizing hormone, and follicle-stimulating hormone in serum after injection of testosterone enanthate or testosterone cypionate. Fertil Steril. 1980;33:201-3. 11. Snyder PJ, Lawrence DA. Treatment of male hypogonadism with testosterone enanthate. J Clin Endocrinol Metab. 1980:51:1335-g. 12. Burris AS, Ewing LL, Sherins RJ. Initial trial of slow-release testosterone microspheres in hypogonadal men. Fertil Steril. 1988;50:493-7. 13. Handelsman DJ, Conway J, Boylan LN. Pharmacokinetics and pharmacodynamics of testosterone pellets in man. J Clin Endocrino1 Metab. 1990;71:216-22. 14. Korenman SG, Viosca S, Garza D, et al. Androgen therapy of hypogonadal men with transscrotal testosterone systems. Am J Med. 1987;83:471-8. 15. Findlay JC, Place VA, Snyder PJ. Transdermal delivery of testosterone. J Clin Endocrinol Metab. 1987;64:266-8. 16. Bals-Pratsch M, Yoon Y-D, Knuth UA, Nieschlag E. Transdermal testosterone substitution therapy Lancet. __ for male hypogonadism. __ 1986;2:943-6. 17. Ahmed SR, Boucher AE, Manni A, et al. Transdermal testosterone theranv in the treatment of male hvnoeonadism. J Clin Endocrinol I. I Metab: 1988;66:546-51. 18. Findlay JC, Place V, Snyder PJ. Treatment of primary hypogonadism in men by the transdermal administration of testosterone. J Clin Endocrinol Metab. 1989;69:369-73. 19. Cunningham GR, Corder0 E, Thornby JI. Testosterone replacetherapeutic ment with transdermal systems. JAMA. 1989;261:2525-30. 20. Bremner WJ, Vitiello MV, Prinz PN. Loss circadian of rhythmicity in blood testosterone levels with aging in normal men. J Clin Endocrinol Metab. 1983;56:1278-81. 21. Sjoberg B, de la Torre B, Hedman G, et al. Circadian variation in system hormone levels in healthy men. A study by frequent sampling. J Endocrinol Invest. 1979;2:131-7. 22. Meikle AW, Stringham JD, Wilson DE, et al. Plasma 5cY-reduced androgens in men and hirsute women: role of adrenal and gonads. J Clin Endocrinol Metab. 1979;48:969-75.
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23. Meikle AW, Bishop DT, Stringham JD, West DW. Quantitating genetic and non-genetic factors that determine plasma sex steroid variation in normal male twins. Metabolism. 1987:35:1090-5. 24. Snyder PJ. Clinical use of androgens. Annu Rev Med. 1984;35: 207-17. 25. Matsumoto AM, Bremner WJ. Hypogonadism: androgen therapy. In: Krieger DT, Bardin CW, eds. Current therapy in endocrinolo-6. Philadeuhia: Decker/Morlev: 1985:145-g. 26. Pate1 DC, Chang Y. ‘Penetration enhancement with binary system of oleic acid, oleins, and oleyl alcohol with lower alcohols. U.S. patent no. 4,863,970. 1989. 27. Pate1 DC, Ebert CD. Method for reducing skin irritation associated with drug/penetration enhancer compositions. U.S. patent no. 4,855,294. 1989. 28. Wagner JG, Nelson E. Percent absorbed time plots derived from blood level and/or urinary excretion data. J Pharm Sci. 1963;52:610-1. 29. Meikle AW, Stringham JD, Bishop DT, West DE. Quantitating genetic and nongenetic factors influencing androgen production and clearance rates in men. J Clin Endocrinol Metab. 1988;67: 104-g. 30. Southern AL, Gordon GC, Tochimoto S. Further study of factors affecting the metabolic clearance rate of testosterone in man. J Clin Endocrinol Metab. 1968;28:1105-12. 31. Vermeulen A, Rubens R, Verdonck L. Testosterone secretion and metabolism in male senescence. J Clin Endocrinol Metab. 1972;34:730-5. 32. Ishimaru T, Pages L, Horton R. Altered metabolism of androgens in elderly men with benign hvnernlasia. J Clin Endocrinol - prostatic _ -_ Metab. i977;45:695-701. 33. Leymarie P, Roger M, Castanier M, Scholler R. Circadian variations of plasma testosterone and estrogens in normal men. A study by frequent sampling. J Steroid Biochem. 1974;5:161-71. 34. Guignard MM, Pesuies PC, Serrurier BD, et al. Circadian rhythms in plasma levels of cortisol, dehydroepiandrosterone, androstenedione, testosterone, and dihydrotestosterone of healthy young men. Acta Endocrinol (Copenh). 1980;94:536-45. 35. Cortes-Gallegos V, Alonso R, Castaneda G, et al. Corticosteroid potency us hypothalamic-pituitary-gonadal axis. J Steroid Biochem. 1984;20:353-6. 36. Shiang-Mian Y, Rong W, Yi-Xue Z, et al. Circadian variations of serum sex hormone binding globulin binding capacity in normal adult men and women. J Steroid Biochem. 1990;36:111-5. 37. Montanini V, Simoni M, Chiossi G, et al. Age-related changes in plasma dehydroepiandrosterone sulfate, cortisol, and testosterone and free testosterone circadian rhythms in adult men. Horm Res. 1988;29:1-6. 38. Wilson JD, Walker JD. The conversion of testosterone to 5aandrostane-17&ol-3-one (dihydrotestosterone) by skin slices of man. J Clin Invest. 1969:48:371-g. 39. Imperato-McGinley J, Guerrero L, Gautier T, et al. Steroid 5areductase deficiency in man: an inherited form of male pseudohermaphroditism. Science. 1974:186:1213-5. 40. Plymate SR, Leonard JM, Paulsen CA, et al. Sex hormone-binding globulin changes with androgen replacement. J Clin Endocrinol Metab. 1983;57:645-8. 41. Korenman SG, Morley JE, Mooradian AD, et al. Secondary hypogonadism in older men: its relation to impotence. J Clin Endocrinol Metab. 1990;71:963-9.
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and Metabolism Society
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74, No. 3
in U.S.A.
Enhanced Transdermal Delivery of Testosterone across Nonscrotal Skin Produces Physiological Concentrations of Testosterone and Its Metabolites in Hypogonadal Men* A. WAYNE MEIKLE, JOHN D. STRINGHAM, WILLIAM D. ODELL
NORMAN KEITH
A. MAZER, JOHN F. MOELLMER, G. TOLMAN, STEPHEN W. SANDERS,
AND
Departmentsof Medicine (A. W.M., J.D.S., K.G.T., S.W.S., W.D.O.) and Pharmaceutics(N.A.M.), University of Utah, and TheraTech, Inc. (N.A.M., J.F.M.), Salt Lake City, Utah 84132
ABSTRACT. None of the current or experimental androgen treatment modalities for male hypogonadism has been reported to produce physiological concentrations or circadian variations in testosterone (T) and its metabolites, dihydrotestosterone (DHT) and estradiol (Ez), This investigation describes a novel transdermal dosage form designed to enhance the delivery of native T across nonscrotal skin. The main objective was to determine whether the nightly application of two experimental transdermal patches to different sites on the body (e.g. back, chest, arms, etc.) would result in normal plasma levels of T, DHT, and Ez for men and mimic the normal circadian variation. Six hypogonadal males (aged 24-66 yr) were studied 4 weeks after stopping T ester treatment. After single application of two patches, T levels increased from a pretreatment baseline of 5.8 f 0.94 nmol/L (mean + SE; 167 f 27 ng/dL) to an average peak concentration of 44.1 & 4.8 nmol/L (1273 f 138 ng/dL) 5.7 f 0.6 h after application and reached a 24-h level of 16.9 f 2.9 nmol/L (488 + 85 ng/dL). DHT and E, levels exhibited parallel variations within the normal reference ranges. During 4 weeks of daily evening application to various sites on the torso, the mean delivery of T from two patches was 5.2 + 0.1 mg/day (-20% of the patch content), and morning T levels were within
the normal limits. On day 28 of treatment, the 24-h plasma profiles of T, DHT, and E, (obtained with two patches on the back) approximately mimicked the normal circadian variations reported in healthy young men. The time-averaged T level was 21.8 + 2.9 nmol/L (629 + 84 ng/dL), and the plasma concentration ratios of DHT/T (0.07 + 0.01) and Ez/T (0.005 + 0.001) were within the normal range. SHBG concentrations were not significantly altered over the 4 weeks of treatment. The patches were well tolerated, except for one patient who developed a local reaction to an excipient during the third week of treatment. Two of the patients (one with Klinefelter’s syndrome) completed several months of continuous therapy. T, DHT, and Es have remained in the range of normal, and plasma LH levels in the patient with Klinefelter’s syndrome became normal. Subjective improvement in symptoms has continued, and tolerability has been good in both patients. These results indicate that the enhanced transdermal delivery of T across nonscrotal skin is a patient-friendly androgen replacement modality and produces physiological concentrations of T and its metabolites, which are unattainable with other treatment modalities. (J Clin Endocrirwl Metab 74: 623-628, 1992)
T
(T) secretion and plasma concenESTOSTERONE trations are reduced below normal in many pathophysiological conditions. These include diseases directly affecting Leydig cells of the testes and disorders of the pituitary and hypothalamus that reduce LH secretion and secondarily decrease T synthesis by Leydig cells. In such patients, replacement androgen therapy is usually prescribed as treatment. However, none of the current replacement therapies [including oral administration (l5), sublingual treatment (6), depot injections (7-ll), SC Received April 5, 1991. Address all correspondence and requests for reprints to: A. Wayne Meikle, M.D., Division of Endocrinology, 50 North Medical Drive, Salt Lake City, Utah 84132. * This work was supported by TheraTech, Inc. and USPHS Grant MOl-RR00064. A.W.M., J.D.S., K.G.T., S.W.S., and W.D.O. were independent from TheraTech and contributed to the experimental design and data analysis of the investigation.
implants (12, 13), and experimental “transscrotal” patches (14-19)] has been reported to produce physiological concentrations of T and its metabolites or to mimic the normal circadian variation of T (20-23). Therapy with oral preparations of synthetic androgens is associated with hepatotoxicity, including cholestatic jaundice, hepatic tumors, and vascular lesions (5, 24, 25). We have investigated a novel enhanced transdermal system, designed to deliver native T across nonscrotal skin, to determine if it produces physiological plasma concentrations of T and its metabolites in six hypogonadal men. Materials
and Methods
Study participants
The six men participating in the study ranged in age from had primary hypogonadism [Klinefelter’s
24-66 yr. One patient 623
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624
MEIKLE
syndrome(KS)], and five had hypogonadotropichypogonadism of various causes.All patients had been treated with a depot parenteral T ester until 1 month before the study began.None of the menwasreceiving drugsthat are known to interfere with androgen metabolism, although one was receiving a tricyclic antidepressantthat may causesexualdysfunction. The protocol was approved by the Institutional Review Board for research with human subjects,University of Utah, and written consent was obtained from each subject before enrollment into the study. Each participant underwent a prestudy screeningexamination, which included a medical history, physical examination (including the prostate), electrocardiogram, complete blood count, chemistry profile, lipid profile, urinalysis, thyroid function studies, and serum T, dihydrotestosterone (DHT), 17/3-estradiol(E2),LH, FSH, and sex hormone-bindingglobulin (SHBG) measurements. Transdermal
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Patch T assay
The residualcontent of T in all usedpatcheswasdetermined by reverse phase high pressure liquid chromatography after methanol extraction. The amount of T released from the patches was calculated as the difference between their initial content (12.8 mg) and the measuredresiduals. Blood hormone
assays
T, DHT, EP,gonadotropins(LH and FSH), and SHBG were assayedby previously describedprocedures(22, 23). DHT was determinedby RIA after solvent extraction and isolation using the Bush A solvent system,which separatesDHT from steroids having any cross-reactionwith the antisera. Internal recovery standardswere usedto correct for procedural losses.The interassaycoefficients of variation were about 10% or lessfor all of the assays.
T patches
Experimental T transdermal patches (code 6275-O) were suppliedby the sponsor,TheraTech, Inc. (Salt Lake City, UT). Each patch contained 12.8 mg T in a proprietary permeationenhancing vehicle composedof polar, nonpolar, and amphiphilic excipients (26) and glycerine, an antiirritant (27). The delivery area of each patch was 7.5 cm’, and the total area (including the adhesive)was25 cm’. Basedon in uitro permeation studies (TheraTech, Inc.), the 24-h application of two patcheswasexpected to deliver approximately 7 mg T. Patch applications
On day 1 of the single application steady-stateprofile (SlB) study, two patches were applied to the midback region of each patient at 2200 h and securedwith additional adhesivetape (Elastikon, Johnson and Johnson, New Brunswick, NJ). The patcheswere removed 24 h later, and the subjectsremainedin the clinic for an additional 24 h (without patches) for measurementsof the elimination kinetics. The 4-weekS2B study began immediately thereafter. In this phase,subjectsself-appliedtwo patchesnightly (at -2200 h) to various siteson the torso (e.g. back, upper arms,chest, abdomen,legs,etc.) for 26 consecutive days. On day 27, the patcheswere again applied in the clinic to the midback region (with additional tape) for measurementsof 24-h steady state hormoneprofiles and subsequentelimination kinetics. Blood collection for hormone
assays
Plasmaconcentrations of T, DHT, and E2 were performed before treatment and on days l-3 (SlB study) and days 27-29 (S2B study) 1, 2, 4, 6, 8, 12, 16, 20, and 24 h after patch application and 1, 2, 4,6, 12, 18, and 24 h after patch removal. Hormone levels were also determined at weekly intervals (on days 7, 14, and 21 of the S2B study) approximately 12 h after self-application of the patches.Blood sampleswere collectedin tubescontaining heparin andcentrifuged at 4 C, and the plasma was stored at -20 C until assayedfor sex hormones. The concentrations of LH, FSH, and SHBG were also measured during eachin-patient study phase.
Data analysis
The initial (prestudy) baselinehormoneconcentrationswere computed as the average of the concentrations measuredat 0 and 24 h after removal of the first patches. This was done to compensatefor inherent fluctuations in the endogenoussecretion of T and improve the accuracy of the baseline adjusted profiles. The poststudy baselineconcentrations were taken as the values measured24 h after removing the day 27 patches. The baselineadjusted hormone profiles (for day 1) were calculated for each subjectby subtracting the baselineconcentration from the measuredlevel, with negative values set at zero. The tl,z values were estimated for each subject by visually fitting a straight line to a log linear plot of their baseline adjustedT levels measured0, 1, 2, 4, 6, and 12 h after patch removal. The results are essentially equivalent to a weighted least squaresfit of the data, but are lesssensitive to spurious fluctuations. The maximum plasmaconcentration (Cpmsx) and time of the maximum concentration (T,,,) were determinedby inspection. The area under the curve was computed using the trapezoidal rule. The kinetics of T absorption from the transdermal patches were calculated from the baseline adjusted T profile from day 1, using the method of Wagner and Nelson (28) and assuminga normal plasma T clearance rate of 1100 L/day (29-31). The resultsare expressedas the meanf SE.
Results Sex steroid study)
profiles
after single patch
application
(SIB
T, DHT, and E2 concentrations were determined over an interval of 48 h after the application of two transderma1 patches to the back for 24 h at the beginning of the study (Fig. 1). Plasma T concentrations increased from a baseline of 5.8 f 0.94 nmol/L (mean + SE; 167 f 27 ng/dL) to an average peak concentration of 44.1 +- 4.8 nmol/L (1273 f 138 ng/dL) at 5.7 f 0.6 h and decreased thereafter to a plateau of about 17 nmol/L (500 ng/dL) for the remaining 12 h. The tI,z of the elimination phase
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ENHANCED
TRANSDERMAL
625
T DELIVERY
10.0 E 8.0 Patch nalysis c---
‘r E -
6.0
0.0
4.0
8.0
12.0
16.0
20.0
1
I
Time (h) FIG. 2. Cumulative T input kinetics based on in uiuo plasma profiles by the method of Wagner and Nelson (28). The arrow indicates the total T input for 24 h based on residual content of the patches determined by high pressure liquid chromatography.
Daily patch release data and weekly plasma concentrations of T
I
I 0
u
24 Time
56
4s
(h)
FIG. 1. The plasma concentrations of T, DHT, and E2 before and after application of two patches to the back of six hypogonadal men for 24 h and after removal of the patches (mean + SE). The shaded area represents the range of hormone concentrations (95% confidence interval) in normal men (22, 23).
after removal of the patches was 116 f 17 min. DHT and EP (Fig. 1) reached peak concentrations at about 612 h, and these two metabolites of T paralleled the values observed for T and were both in the range observed in eugonadal men. Transdermal absorption kinetics
Based on analysis of the baseline adjusted T profiles, the cumulative amount of T absorbed from the patches during 24 h is shown in Fig. 2. During the first 8 h, T was absorbed at a faster rate than during the last 12 h. This change in absorption rate accounts for the peak in the T profile and the subsequent plateau phase. The total amount absorbed at 24 h was in excellent agreement with the calculated amount of T released from the patches (i.e. 6.4 f 0.5 mg/two patches).
Based on patch analysis, the total amount of T delivered per two patches varied on a daily basis from 4.1-6.8 mg, with a mean of 5.2 f 0.1 mg over the entire 28-day study (see Fig. 3, lower panel). In all cases the values were within the normal physiological range of endogenous T production (i.e. 3-10 mg/day) (29-32). The mean T concentrations measured about 12 h postpatch application on days 1, 7, 14, 21, and 28 of the study are shown in Fig. 3 (upper panel). Compared to the pre- and poststudy baselines (also shown), the weekly concentrations remained within the normal range and correlated (r = 0.82; n = 5 time points) with the patch release data (Fig. 3, lower panel). The mean baseline T concentration was lower after day 28 than on day 1 (n = 5; P < 0.05). This suggests that endogenous secretion of T, which was low in these men, was further reduced by the treatment. During the 4 weeks of continuous treatment in which the participants applied the patches, one patient developed a skin reaction to the patches and was removed from the study on day 23. Steady state sex steroid profiles after the last patch application (S2B study)
The average peak T concentration after the second 24h study was 33.5 f 4.3 nmol/L (966 f 122 ng/dL; n = 5; Fig. 4) and was also achieved about 6 h after application of the patch. The time-averaged T level of 21.8 + 4.3 nmol/L (629 f 84 ng/dL; n = 5; Fig. 4) was also achieved about 6 h after application of the patch. The patterns of T concentrations were similar during both the first and second 24-h study periods (Figs. 1 and 4). Further, the
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MEIKLE
’
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Outpatient-
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JCE & M. 1992 Vol74.No3
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’
B. 212.0111 ;: > g.” L D
6.0
E f
3.0
0
7
Day
U
21
26
of Application
FIG. 3. The daily total T release (lower panel) and the weekly plasma T concentration (upper panel) before and after wearing two patches daily; the patches were applied at 2200 h, and the plasma T values were measured in samples collected at 1000 h. The normal ranges of T production (29-32) and morning T levels (22, 23) are shown by the shaded area. T levels denoted BL represent baseline levels before and after SlB and S2B studies. Values are the mean + SE (0, n = 6; 0, n = 5).
steady state concentration profiles of T and DHT (Fig. 4) mimicked the circadian variation that has been reported in various studies of T (20, 21, 33-36) and DHT (21,34,35) in normal young men. No circadian variation has been reported in the literature thus far for Ez (32, 35, 37). Ratio of DHT/T
and EJT
0 0.0
4.0
2200 h
8.0
12.0
Time (h)
15.0
20.0
24.0
2200 h
FIG. 4. Plasma concentrations of T (lower panel), DHT (middle panel), and El (upper panel) after application of two patches to the back in five subjects on day 28. Values are the mean f SE. Shaded areaa are normal ranges, as described in Fig. 1.
values were 39 + 5 nmol/L at the beginning of the study and 41.33 k 4.8 nmol/L at the end of 4 weeks. The LH decreased toward normal in the patient with KS, and the FSH level declined somewhat. Local tolerability
Based on the areas under the plasma concentration profiles measured in the steady state study (S2B), the ratios of DHT/T and EJT were 0.07 f 0.01, and that of EJT was 0.005 f 0.001, respectively. These ratios were not significantly different between the two observation periods (SlB us. S2B; P > 0.4) and are comparable to the DHT/T and EJT ratios observed for normal men in our laboratory (22, 23, 29).
The patches were well tolerated, except for one patient who developed a local reaction to the patches on day 23, requiring discontinuation. This reaction was demonstrated to be an allergic sensitivity to one of the excipients in the patch formulation. In general, there was only a mild and transient redness (without edema) at the application site, but otherwise no unpleasant reactions were observed.
SHBG, LH and FSH
Continuation study
The SHBG DHT-binding capacity did not change significantly during the course of the study. The mean
Two subjects were studied for 6 additional months. The patches were well tolerated by them, and they re-
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ENHANCED
TRANSDERMAL
ported excellent subjective responses in sexual function, libido, energy level, and mood. The plasma concentrations of T, DHT, and Ez measured about 12 h after application of the patches are shown in Fig. 5. With rare exception, these values have remained within the normal reference ranges. SHBG concentrations (data not shown) have also remained in the normal range (30-110 nmol/ L). The subject with primary hypogonadism had baseline elevated concentrations of LH and FSH (44 and 35 IU/ L, respectively). The LH level became normal after 4 months of therapy, whereas FSH declined, but was still elevated above the normal range. Gonadotropin levels in the patient with secondary hypogonadism also decreased. Plasma concentrations of cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, and triglycerides remained relatively constant in both patients over the course of therapy.
12.0
20.0
26.0
T DELIVERY
627
Discussion
This study demonstrates that a novel enhanced transdermal delivery system is capable of producing normal plasma concentrations (23, 23), including circadian variation (20, 21, 33-37), of T and its potent metabolites, DHT and Ez, over 24 h in hypogonadal men. Transderma1 delivery of T across nonscrotal skin produces normal ratios of DHT/T and EJT. This contrasts with reports of transdermal delivery across scrotal skin, in which DHT levels are initially normal relative to T and then become elevated, resulting in a ratio of DHT/T of 1:2.5, rather than the normal ratio of about 1:lO (14-19, 22, 23). The scrotal skin contains higher activity of 5areductase than nonsexual skin such as the trunk, back, and legs (38,39). Apparently, the 5a-reductase of scrotal skin, which converts T to DHT, is inducible by androgen and results in increased delivery of DHT relative to T during extended use of the scrotal T patch. In contrast, enhanced delivery of T across nonscrotal skin did not induce the already low activity of 5a-reductase during the time of the studies reported herein and resulted in a pattern of sex steroid concentrations that more closely resembles secretion of T by the Leydig cells of the testes and peripheral conversion of T to its active metabolites. Sex steroid concentrations after administration of depot T esters (11, 40) or oral T undecanoate (l-5) do not mimic normal T concentrations as provided by the current patch system. Depot T esters result in supraphysiological concentrations of both T and Ez, followed over several days by subnormal plasma T concentrations (11, 40). T undecanoate produces multiple daily peaks and valleys of the androgen and must be administered several times daily (l-5). The patches were well tolerated, except by one individual who developed an allergic reaction to an excipient of the patches. Based on recent dermatologic studies in 52 normal men, where no such allergic reactions were observed (Mazer, N. A., unpublished data), the frequency of such reactions among patients is expected to be low. We conclude that transdermal delivery of T across nonscrotal skin produces physiological plasma concentrations and circadian variations of T and its potent metabolites, DHT and El, in hypogonadal men. This patient-friendly treatment system should provide new insights into the potential benefits and safety issues associated with the correction of androgen deficiency in a variety of clinical disorders (24, 25, 41).
36.0
Weeks
FIG. 5. Continuation study; plasma concentrations of T (lower panel), DHT (middle paneel), and E2 (upper panel) in two subjects. Samples were obtained at 2- to 4-week intervals, approximately 12 h after application of two patches to various sites on the torso. Shaded areas are normal ranges, as described in Fig. 1.
References 1. Johnsen SG, Bennett EP, Jensen VG. Therapeutic effectiveness of oral testosterone. Lancet. 1974;2:1473-5. 2. Cantrill J, Dewis P, Large DM, Newman M, Anderson DC. Which testosterone replacement therapy? Clin Endocrinol (Oxf). 1984;21:97-107.
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MEIKLE 3. Franchi F, Luisi M, Kicovic PM. Long term study of oral testosterone undecanoate in hypogonadal males. Int J Androl. 1978;1:270-8. 4. Franchimont P, Kicovic PM, Mattei A, Roulier R. Effects of oral testosterone undecanoate in males. Clin Endocrinol (Oxf). 1978;9:313-20. 5. Werner SC, Hanger FM, Kritzler RA. Jaundice due to methyltestosterone therapy. Am J Med. 1950;8:325. 6. Stuendel CS, Dudley RE. Sublingual (SL) testosterone (T) simulates episodic androgen release. Proc of the 73rd Annual Meet of The Endocrine Sot. 1990;426. 7. Sokol RZ, Palacios A, Camptiedl LA, Saul CC, Swerdloff RS. Comparison of the kinetics of injectable testosterone in eugonadal and hypogonadal men. Fertil Steril. 1982;37:425-30. 8. Aakvaag A, Vogt JH. Plasma testosterone values in different forms of testosterone replacement. Acta Endocrinol (Copenh). 1969:60:537-42. 9. Fujioka M, Shinohara Y, Baba S, et al. Pharmacokinetic properties of testosterone propionate in normal men. J Clin Endocrinol Metab. 1986;63:1361-4. 10. Schulte-Beerbuhl M, Nieschlag E. Comparison of testosterone, dihydrotestosterone, luteinizing hormone, and follicle-stimulating hormone in serum after injection of testosterone enanthate or testosterone cypionate. Fertil Steril. 1980;33:201-3. 11. Snyder PJ, Lawrence DA. Treatment of male hypogonadism with testosterone enanthate. J Clin Endocrinol Metab. 1980:51:1335-g. 12. Burris AS, Ewing LL, Sherins RJ. Initial trial of slow-release testosterone microspheres in hypogonadal men. Fertil Steril. 1988;50:493-7. 13. Handelsman DJ, Conway J, Boylan LN. Pharmacokinetics and pharmacodynamics of testosterone pellets in man. J Clin Endocrino1 Metab. 1990;71:216-22. 14. Korenman SG, Viosca S, Garza D, et al. Androgen therapy of hypogonadal men with transscrotal testosterone systems. Am J Med. 1987;83:471-8. 15. Findlay JC, Place VA, Snyder PJ. Transdermal delivery of testosterone. J Clin Endocrinol Metab. 1987;64:266-8. 16. Bals-Pratsch M, Yoon Y-D, Knuth UA, Nieschlag E. Transdermal testosterone substitution therapy Lancet. __ for male hypogonadism. __ 1986;2:943-6. 17. Ahmed SR, Boucher AE, Manni A, et al. Transdermal testosterone theranv in the treatment of male hvnoeonadism. J Clin Endocrinol I. I Metab: 1988;66:546-51. 18. Findlay JC, Place V, Snyder PJ. Treatment of primary hypogonadism in men by the transdermal administration of testosterone. J Clin Endocrinol Metab. 1989;69:369-73. 19. Cunningham GR, Corder0 E, Thornby JI. Testosterone replacetherapeutic ment with transdermal systems. JAMA. 1989;261:2525-30. 20. Bremner WJ, Vitiello MV, Prinz PN. Loss circadian of rhythmicity in blood testosterone levels with aging in normal men. J Clin Endocrinol Metab. 1983;56:1278-81. 21. Sjoberg B, de la Torre B, Hedman G, et al. Circadian variation in system hormone levels in healthy men. A study by frequent sampling. J Endocrinol Invest. 1979;2:131-7. 22. Meikle AW, Stringham JD, Wilson DE, et al. Plasma 5cY-reduced androgens in men and hirsute women: role of adrenal and gonads. J Clin Endocrinol Metab. 1979;48:969-75.
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23. Meikle AW, Bishop DT, Stringham JD, West DW. Quantitating genetic and non-genetic factors that determine plasma sex steroid variation in normal male twins. Metabolism. 1987:35:1090-5. 24. Snyder PJ. Clinical use of androgens. Annu Rev Med. 1984;35: 207-17. 25. Matsumoto AM, Bremner WJ. Hypogonadism: androgen therapy. In: Krieger DT, Bardin CW, eds. Current therapy in endocrinolo-6. Philadeuhia: Decker/Morlev: 1985:145-g. 26. Pate1 DC, Chang Y. ‘Penetration enhancement with binary system of oleic acid, oleins, and oleyl alcohol with lower alcohols. U.S. patent no. 4,863,970. 1989. 27. Pate1 DC, Ebert CD. Method for reducing skin irritation associated with drug/penetration enhancer compositions. U.S. patent no. 4,855,294. 1989. 28. Wagner JG, Nelson E. Percent absorbed time plots derived from blood level and/or urinary excretion data. J Pharm Sci. 1963;52:610-1. 29. Meikle AW, Stringham JD, Bishop DT, West DE. Quantitating genetic and nongenetic factors influencing androgen production and clearance rates in men. J Clin Endocrinol Metab. 1988;67: 104-g. 30. Southern AL, Gordon GC, Tochimoto S. Further study of factors affecting the metabolic clearance rate of testosterone in man. J Clin Endocrinol Metab. 1968;28:1105-12. 31. Vermeulen A, Rubens R, Verdonck L. Testosterone secretion and metabolism in male senescence. J Clin Endocrinol Metab. 1972;34:730-5. 32. Ishimaru T, Pages L, Horton R. Altered metabolism of androgens in elderly men with benign hvnernlasia. J Clin Endocrinol - prostatic _ -_ Metab. i977;45:695-701. 33. Leymarie P, Roger M, Castanier M, Scholler R. Circadian variations of plasma testosterone and estrogens in normal men. A study by frequent sampling. J Steroid Biochem. 1974;5:161-71. 34. Guignard MM, Pesuies PC, Serrurier BD, et al. Circadian rhythms in plasma levels of cortisol, dehydroepiandrosterone, androstenedione, testosterone, and dihydrotestosterone of healthy young men. Acta Endocrinol (Copenh). 1980;94:536-45. 35. Cortes-Gallegos V, Alonso R, Castaneda G, et al. Corticosteroid potency us hypothalamic-pituitary-gonadal axis. J Steroid Biochem. 1984;20:353-6. 36. Shiang-Mian Y, Rong W, Yi-Xue Z, et al. Circadian variations of serum sex hormone binding globulin binding capacity in normal adult men and women. J Steroid Biochem. 1990;36:111-5. 37. Montanini V, Simoni M, Chiossi G, et al. Age-related changes in plasma dehydroepiandrosterone sulfate, cortisol, and testosterone and free testosterone circadian rhythms in adult men. Horm Res. 1988;29:1-6. 38. Wilson JD, Walker JD. The conversion of testosterone to 5aandrostane-17&ol-3-one (dihydrotestosterone) by skin slices of man. J Clin Invest. 1969:48:371-g. 39. Imperato-McGinley J, Guerrero L, Gautier T, et al. Steroid 5areductase deficiency in man: an inherited form of male pseudohermaphroditism. Science. 1974:186:1213-5. 40. Plymate SR, Leonard JM, Paulsen CA, et al. Sex hormone-binding globulin changes with androgen replacement. J Clin Endocrinol Metab. 1983;57:645-8. 41. Korenman SG, Morley JE, Mooradian AD, et al. Secondary hypogonadism in older men: its relation to impotence. J Clin Endocrinol Metab. 1990;71:963-9.
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