0021-972X/91/7306-1360$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 73, No. 6 Printed in U.S.A.
Combined Administration of a Gonadotropin-Releasing Hormone Antagonist and Testosterone in Men Induces Reversible Azoospermia without Loss of Libido* SPYROS N. PAVLOU, KIM BREWER, M. GINNIE FARLEY, JILL LINDNER, MARIA-CRISTINA BASTIAS, B. JANE ROGERS, LARRY L. SWIFT, JEAN E. RIVIER, WYLIE W. VALEf, P. MICHAEL CONN, AND CARL M. HERBERT Departments of Medicine (S.N.P., M.G.F., J.L.), Obstetrics and Gynecology (KB., M.-C.B., J.R., C.M.H.), and Pathology (L.L.S.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232; the Department of Pharmacology, University of Iowa College of Medicine (P.M.CJ, Iowa City, Iowa 52242; and The Salk Institute (J.E.R., W. W. V.), La Jolla, California 92037
ABSTRACT. GnRH antagonists suppress pituitary and gonadal function by competing with endogenous GnRH for binding to receptors on pituitary gonadotrophs. We studied the effects of GnRH antagonist administration to men in a protocol simulating a likely male contraceptive regimen combined with a low dose of testosterone. The GnRH antagonist Nal-Glu was given daily (10 mg, sc) for 20 weeks to eight normal men, and a low dose of testosterone enanthate (25 mg, sc) was given every week. Sperm counts started declining during week 4, and complete azoospermia was reached within 6-12 weeks in six of the eight subjects. Subjects 7 and 8, whose sperm counts and serum gonadotropin levels were not suppressed after 10 weeks, were given 20 mg Nal-Glu starting at week 10. One became azoospermic at week 16, while the other's total sperm counts continued declining and reached a nadir of 1.4 million by week 20. Sperm motility and viability in this subject were completely suppressed
D
URING the last 20 yr, investigators have pursued several approaches to develop a safe, reversible, practical, reliable, and effective contraceptive for men Received January 14,1991. Address all correspondence and requests for reprints to: Spyros N. Pavlou, M.D., Division of Endocrinology, AA-4206 MCN, Vanderbilt University Medical Center, Nashville, Tennessee 37232. * This work was supported by the Contraceptive Research and Development Program (CONRAD, CSA-87-013), Eastern Virginia Medical School, under a Cooperative Agreement with the United States Agency for International Development (AID, DPE-3044-A-00-606300). The views expressed by the authors do not necessarily reflect the view of AID or CONRAD. Supported also by the following grants and grants-in-aid: Clinical Research Center Grant-in-Aid 5-M01-RR-95, Population Center Grant-in-Aid HD-05797, DK-26657, NIH Grant HD-13527 (to W.W.V. and J.E.R.), and HD-19899 (to P.M.C.). The Nal-Glu was synthesized at The Salk Institute under Contract N01HD-7-2907 and made available by the Contraceptive Development Branch, Center for Population Research, NICHHD. Research was conducted in part by the Clayton Foundation for Research, California division. t Clayton Foundation Investigator.
after week 14. Sperm counts returned to baseline levels 12-14 weeks after the end of Nal-Glu administration. The mean serum LH level of the first six subjects decreased from 3 ± 03. U/L at baseline to less than 0.1 U/L until week 20, and then levels returned to baseline. FSH levels similarly decreased from a combined mean of 3.6 ± 0.9 U/L at baseline to below 0.3 U/L after 4 weeks of Nal-Glu administration. Serum mean testosterone levels between weekly injections of testosterone enanthate ranged from 27.4 ± 5.9 to 4.8 ± 1.4 nmol/L, but remained in the hypogonadal range ( 0.05).
5
10
15
20
25
30
35
40
TIME (weeks)
FIG. 2. Serum FSH, LH, and testosterone levels (mean ± SEM) in the six men who received 10 mg Nal-Glu and TE for 20 weeks. • , LH values below the 0.1 U/L assay detectability limits. Samples during antagonist administration were drawn immediately before the injection ofTE.
During week 10, frequent samples drawn every 10 min for 24 h showed that LH levels were suppressed below 0.1 U/L (assay sensitivity), and no pulses could be identified in the first six subjects. FSH and testosterone
None of the subjects complained of changes in libido, potency, frequency of ejaculations, or quality of erections during the biweekly clinical interviews, with the exception of the first 2 weeks of Nal-Glu administration alone, when no TE was given. Libido score, derived from the questionnaire is shown in Fig. 8. In three men libido remained unchanged while in the other five subjects some fluctuations from baseline were noted. Overall, libido, quality of sexual activity and quality of erections scores did not change significantly during any phase of the study. Toxicology
No changes were seen throughout the study in serum chemistry (sodium, potassium, chloride, CO2, glucose, blood urea nitrogen, creatinine, serum glutamic oxalacetic transaminase, lactate dehydrogenase, alkaline phosphatase, bilirubin, calcium, phosphorus, protein, and albumin) or urinalysis. No significant changes were
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 April 2015. at 07:41 For personal use only. No other uses without permission. . All rights reserved.
GnRH ANTAGONIST AS MALE CONTRACEPTIVE
1365
7 -i
300 6 -
250 5 -
E 200 -
150 3 -
2 100 cc
2 -
LU
50
0
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-5
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J
40
5
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Nal-Glu 10 mg Nal-Glu 20 mg 7 -
TE 25 mg q week
6 5 -
o cc LU H CO
O H CO
3 2 -
111
1 -
0
1 -
J
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10
15 20 25 TIME (weeks)
J
-5
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FiG. 3. Sperm density profile and serum levels of FSH, LH, and testosterone of the two men who received 10 mg Nal-Glu for the first 10 weeks and 20 mg for the next 10 weeks. • , Either total absence of sperm in the ejaculate or LH values below the 0.1 U/L assay detectability limits. Samples during antagonist administration were drawn immediately before the injection of TE.
noted in body weight. Uric acid levels decreased (P < 0.01) from 6.6 ± 0.5 at baseline to 5.4 ± 0.4 at 10 weeks and 5.5 ± 0.5 at 20 weeks. Levels were still lower (P < 0.02) than baseline 5.8 ± 0.3 10 weeks after discontinuation of Nal-Glu administration. Hemoglobin also decreased (P < 0.05) from 15.3 ± 0.3 at baseline to 14.3 ± 0.4, 13.9 ± 0.3, and 14.8 ± 0.2 at weeks 10, 20, and 10 of follow-up, respectively. Similarly, the hematocrit decreased (P < 0.05) from 44.1 ± 0.6 at baseline to 41.7 ± 1.2 and 40.9 ± 0.8 at 10 and 20 weeks, and returned to 45 ± 0.3 at 10 weeks of follow-up. Occasional local reactions were seen at the injection sites in the form of erythema (1-5 cm diameter) and mild induration. The occurrence of these local reactions seemed to subside after the first weeks of Nal-Glu administration.
Discussion It has been over 25 yr since women assumed the major responsibility for fertility control with the introduction
of the first oral contraceptive pill (32). Male contraception has not advanced substantially during the last 65 yr (33) and still relies on the condom, the oldest reversible method (34). Despite renewed interest in the condom as a means of protection against human immunodeficiency virus infection, it remains by and large for casual use. The results of this study demonstrate that combined GnRH antagonist and testosterone can completely suppress spermatogenesis without loss of libido. Seven of the eight subjects became azoospermic during the study, while in the eighth, total sperm counts were still declining during the last week of Nal-Glu administration. It is conceivable that if Nal-Glu administration had continued he might have become azoospermic. Sperm motility and viability in this subject were completely suppressed after week 14. Full recovery of sperm density was achieved in all subjects within 14 weeks. These data show for the first time that consistent and reversible azoospermia is a pragmatic goal in male contraceptive research. Azoospermia is critical in the development of a male
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 April 2015. at 07:41 For personal use only. No other uses without permission. . All rights reserved.
PAVLOU ET AL.
1366 10 -n
JCE & M • 1991 Vol 73 • No 6
TE 25 mg
8 -
LLJ
FIG. 4. Mean (±SEM) serum testosterone levels measured daily between two injections of 25 mg TE.
6 -
O cc 4 -
O
h(/) LJJ
2 -
0
J
3
4
TIME (days) contraceptive, because it is consistent with infertility. Until now hormonal approaches to suppress spermatogenesis had, in general, failed to induce azoospermia (5). In Chinese and Indonesian men, however, higher rates of azoospermia (up to 90%) have been reported (6). Combined administration of GnRH agonists and testosterone had also failed to induce azoospermia. Testosterone can restore spermatogenesis in the hypophysectomized rat (35) and may maintain a low grade spermatogenesis in primates (36) or humans (37). In our study the low dose of testosterone perhaps contributed to the high rate of azoospermia. Future studies should show whether higher doses of testosterone will attenuate the antagonist effects on spermatogenesis. The role of FSH has been implicated in some other studies, because hypophysectomy predictably results in azoospermia (38). When men were given a GnRH agonist and TE, FSH levels decreased by only 50%, while no significant decrease in serum FSH levels was seen in the nonresponders (17). Bioassayable FSH levels in these studies changed in parallel to immunoreactive levels (19). Moreover, during long term GnRH agonist administration, serum FSH levels escape and tend to return toward baseline (10, 20). In contrast, GnRH antagonists suppress FSH and further decrease the biological potency of the FSH molecule (23). Administration of a GnRH antagonist to postmenopausal women produces circulation of FSH species that bind to the FSH receptor without any intrinsic FSH activity (39). Recent studies provide evidence that the Nal-Glu antagonist can even suppress FSH secretion from gonadotroph pituitary adenomas
(40). Therefore, effective suppression of FSH seems to be a key factor in the success of the GnRH antagonist to induce azoospermia in men. The use of the highly specific and sensitive assays in the present study shows that gonadotropins were suppressed to levels below assay detectability and that the gonadotropin response to natural GnRH was totally blunted. Furthermore, this suppression persisted for a few weeks after discontinuation of antagonist administration. It is of interest that the pituitary recovered faster than the gonads, as LH increased past baseline levels before testosterone levels returned to normal and reestablished negative feedback. However, higher doses of Nal-Glu were required for the heavier subjects. This observation is similar to hypogonadal men given authentic GnRH (41). Pharmacokinetics of Nal-Glu, measured by RIA, remained the same throughout the study, similar to those we reported earlier using a radioreceptor assay (25). In the only man who did not reach azoospermia, Nal-Glu levels followed a different pattern, with no postinjection peak phase. When the serum of this man was analyzed for the presence of antibodies to Nal-Glu or material interfering with the assay, none could be found (data not shown). However, high binding of Nal-Glu to his serum proteins seems likely, because GnRH analogs bind to serum proteins (42). Sperm concentration declined within 4 weeks in the first six volunteers, and the first man became azoospermic at 6 weeks. This fall occurred faster than in previous studies that used high doses of steroids or GnRH agonists (4, 5,14). Recovery was achieved within 14 weeks, which
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 April 2015. at 07:41 For personal use only. No other uses without permission. . All rights reserved.
GnRH ANTAGONIST AS MALE CONTRACEPTIVE 1.0
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18
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TIME (hours)
FIG. 6. Upper panel, Mean (±SEM) serum immunoreactive Nal-Glu levels at weeks 10 (solid line) and 20 {broken line) in the six men who received 10 mg Nal-Glu and TE for 20 weeks. Lower panel, Nal-Glu serum levels on weeks 10 (solid line) and 20 (broken line) in the two men who received 10 mg Nal-Glu for the first 10 weeks and 20 mg for the next 10 weeks.
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Vol. 73, No. 6 Printed in U.S.A.
Combined Administration of a Gonadotropin-Releasing Hormone Antagonist and Testosterone in Men Induces Reversible Azoospermia without Loss of Libido* SPYROS N. PAVLOU, KIM BREWER, M. GINNIE FARLEY, JILL LINDNER, MARIA-CRISTINA BASTIAS, B. JANE ROGERS, LARRY L. SWIFT, JEAN E. RIVIER, WYLIE W. VALEf, P. MICHAEL CONN, AND CARL M. HERBERT Departments of Medicine (S.N.P., M.G.F., J.L.), Obstetrics and Gynecology (KB., M.-C.B., J.R., C.M.H.), and Pathology (L.L.S.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232; the Department of Pharmacology, University of Iowa College of Medicine (P.M.CJ, Iowa City, Iowa 52242; and The Salk Institute (J.E.R., W. W. V.), La Jolla, California 92037
ABSTRACT. GnRH antagonists suppress pituitary and gonadal function by competing with endogenous GnRH for binding to receptors on pituitary gonadotrophs. We studied the effects of GnRH antagonist administration to men in a protocol simulating a likely male contraceptive regimen combined with a low dose of testosterone. The GnRH antagonist Nal-Glu was given daily (10 mg, sc) for 20 weeks to eight normal men, and a low dose of testosterone enanthate (25 mg, sc) was given every week. Sperm counts started declining during week 4, and complete azoospermia was reached within 6-12 weeks in six of the eight subjects. Subjects 7 and 8, whose sperm counts and serum gonadotropin levels were not suppressed after 10 weeks, were given 20 mg Nal-Glu starting at week 10. One became azoospermic at week 16, while the other's total sperm counts continued declining and reached a nadir of 1.4 million by week 20. Sperm motility and viability in this subject were completely suppressed
D
URING the last 20 yr, investigators have pursued several approaches to develop a safe, reversible, practical, reliable, and effective contraceptive for men Received January 14,1991. Address all correspondence and requests for reprints to: Spyros N. Pavlou, M.D., Division of Endocrinology, AA-4206 MCN, Vanderbilt University Medical Center, Nashville, Tennessee 37232. * This work was supported by the Contraceptive Research and Development Program (CONRAD, CSA-87-013), Eastern Virginia Medical School, under a Cooperative Agreement with the United States Agency for International Development (AID, DPE-3044-A-00-606300). The views expressed by the authors do not necessarily reflect the view of AID or CONRAD. Supported also by the following grants and grants-in-aid: Clinical Research Center Grant-in-Aid 5-M01-RR-95, Population Center Grant-in-Aid HD-05797, DK-26657, NIH Grant HD-13527 (to W.W.V. and J.E.R.), and HD-19899 (to P.M.C.). The Nal-Glu was synthesized at The Salk Institute under Contract N01HD-7-2907 and made available by the Contraceptive Development Branch, Center for Population Research, NICHHD. Research was conducted in part by the Clayton Foundation for Research, California division. t Clayton Foundation Investigator.
after week 14. Sperm counts returned to baseline levels 12-14 weeks after the end of Nal-Glu administration. The mean serum LH level of the first six subjects decreased from 3 ± 03. U/L at baseline to less than 0.1 U/L until week 20, and then levels returned to baseline. FSH levels similarly decreased from a combined mean of 3.6 ± 0.9 U/L at baseline to below 0.3 U/L after 4 weeks of Nal-Glu administration. Serum mean testosterone levels between weekly injections of testosterone enanthate ranged from 27.4 ± 5.9 to 4.8 ± 1.4 nmol/L, but remained in the hypogonadal range ( 0.05).
5
10
15
20
25
30
35
40
TIME (weeks)
FIG. 2. Serum FSH, LH, and testosterone levels (mean ± SEM) in the six men who received 10 mg Nal-Glu and TE for 20 weeks. • , LH values below the 0.1 U/L assay detectability limits. Samples during antagonist administration were drawn immediately before the injection ofTE.
During week 10, frequent samples drawn every 10 min for 24 h showed that LH levels were suppressed below 0.1 U/L (assay sensitivity), and no pulses could be identified in the first six subjects. FSH and testosterone
None of the subjects complained of changes in libido, potency, frequency of ejaculations, or quality of erections during the biweekly clinical interviews, with the exception of the first 2 weeks of Nal-Glu administration alone, when no TE was given. Libido score, derived from the questionnaire is shown in Fig. 8. In three men libido remained unchanged while in the other five subjects some fluctuations from baseline were noted. Overall, libido, quality of sexual activity and quality of erections scores did not change significantly during any phase of the study. Toxicology
No changes were seen throughout the study in serum chemistry (sodium, potassium, chloride, CO2, glucose, blood urea nitrogen, creatinine, serum glutamic oxalacetic transaminase, lactate dehydrogenase, alkaline phosphatase, bilirubin, calcium, phosphorus, protein, and albumin) or urinalysis. No significant changes were
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 April 2015. at 07:41 For personal use only. No other uses without permission. . All rights reserved.
GnRH ANTAGONIST AS MALE CONTRACEPTIVE
1365
7 -i
300 6 -
250 5 -
E 200 -
150 3 -
2 100 cc
2 -
LU
50
0
J
0
-5
10
15
20
25
30
35
J
40
5
0
5
10
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Nal-Glu 10 mg Nal-Glu 20 mg 7 -
TE 25 mg q week
6 5 -
o cc LU H CO
O H CO
3 2 -
111
1 -
0
1 -
J
0
10
15 20 25 TIME (weeks)
J
-5
10
15
20
TIME (weeks)
FiG. 3. Sperm density profile and serum levels of FSH, LH, and testosterone of the two men who received 10 mg Nal-Glu for the first 10 weeks and 20 mg for the next 10 weeks. • , Either total absence of sperm in the ejaculate or LH values below the 0.1 U/L assay detectability limits. Samples during antagonist administration were drawn immediately before the injection of TE.
noted in body weight. Uric acid levels decreased (P < 0.01) from 6.6 ± 0.5 at baseline to 5.4 ± 0.4 at 10 weeks and 5.5 ± 0.5 at 20 weeks. Levels were still lower (P < 0.02) than baseline 5.8 ± 0.3 10 weeks after discontinuation of Nal-Glu administration. Hemoglobin also decreased (P < 0.05) from 15.3 ± 0.3 at baseline to 14.3 ± 0.4, 13.9 ± 0.3, and 14.8 ± 0.2 at weeks 10, 20, and 10 of follow-up, respectively. Similarly, the hematocrit decreased (P < 0.05) from 44.1 ± 0.6 at baseline to 41.7 ± 1.2 and 40.9 ± 0.8 at 10 and 20 weeks, and returned to 45 ± 0.3 at 10 weeks of follow-up. Occasional local reactions were seen at the injection sites in the form of erythema (1-5 cm diameter) and mild induration. The occurrence of these local reactions seemed to subside after the first weeks of Nal-Glu administration.
Discussion It has been over 25 yr since women assumed the major responsibility for fertility control with the introduction
of the first oral contraceptive pill (32). Male contraception has not advanced substantially during the last 65 yr (33) and still relies on the condom, the oldest reversible method (34). Despite renewed interest in the condom as a means of protection against human immunodeficiency virus infection, it remains by and large for casual use. The results of this study demonstrate that combined GnRH antagonist and testosterone can completely suppress spermatogenesis without loss of libido. Seven of the eight subjects became azoospermic during the study, while in the eighth, total sperm counts were still declining during the last week of Nal-Glu administration. It is conceivable that if Nal-Glu administration had continued he might have become azoospermic. Sperm motility and viability in this subject were completely suppressed after week 14. Full recovery of sperm density was achieved in all subjects within 14 weeks. These data show for the first time that consistent and reversible azoospermia is a pragmatic goal in male contraceptive research. Azoospermia is critical in the development of a male
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 April 2015. at 07:41 For personal use only. No other uses without permission. . All rights reserved.
PAVLOU ET AL.
1366 10 -n
JCE & M • 1991 Vol 73 • No 6
TE 25 mg
8 -
LLJ
FIG. 4. Mean (±SEM) serum testosterone levels measured daily between two injections of 25 mg TE.
6 -
O cc 4 -
O
h(/) LJJ
2 -
0
J
3
4
TIME (days) contraceptive, because it is consistent with infertility. Until now hormonal approaches to suppress spermatogenesis had, in general, failed to induce azoospermia (5). In Chinese and Indonesian men, however, higher rates of azoospermia (up to 90%) have been reported (6). Combined administration of GnRH agonists and testosterone had also failed to induce azoospermia. Testosterone can restore spermatogenesis in the hypophysectomized rat (35) and may maintain a low grade spermatogenesis in primates (36) or humans (37). In our study the low dose of testosterone perhaps contributed to the high rate of azoospermia. Future studies should show whether higher doses of testosterone will attenuate the antagonist effects on spermatogenesis. The role of FSH has been implicated in some other studies, because hypophysectomy predictably results in azoospermia (38). When men were given a GnRH agonist and TE, FSH levels decreased by only 50%, while no significant decrease in serum FSH levels was seen in the nonresponders (17). Bioassayable FSH levels in these studies changed in parallel to immunoreactive levels (19). Moreover, during long term GnRH agonist administration, serum FSH levels escape and tend to return toward baseline (10, 20). In contrast, GnRH antagonists suppress FSH and further decrease the biological potency of the FSH molecule (23). Administration of a GnRH antagonist to postmenopausal women produces circulation of FSH species that bind to the FSH receptor without any intrinsic FSH activity (39). Recent studies provide evidence that the Nal-Glu antagonist can even suppress FSH secretion from gonadotroph pituitary adenomas
(40). Therefore, effective suppression of FSH seems to be a key factor in the success of the GnRH antagonist to induce azoospermia in men. The use of the highly specific and sensitive assays in the present study shows that gonadotropins were suppressed to levels below assay detectability and that the gonadotropin response to natural GnRH was totally blunted. Furthermore, this suppression persisted for a few weeks after discontinuation of antagonist administration. It is of interest that the pituitary recovered faster than the gonads, as LH increased past baseline levels before testosterone levels returned to normal and reestablished negative feedback. However, higher doses of Nal-Glu were required for the heavier subjects. This observation is similar to hypogonadal men given authentic GnRH (41). Pharmacokinetics of Nal-Glu, measured by RIA, remained the same throughout the study, similar to those we reported earlier using a radioreceptor assay (25). In the only man who did not reach azoospermia, Nal-Glu levels followed a different pattern, with no postinjection peak phase. When the serum of this man was analyzed for the presence of antibodies to Nal-Glu or material interfering with the assay, none could be found (data not shown). However, high binding of Nal-Glu to his serum proteins seems likely, because GnRH analogs bind to serum proteins (42). Sperm concentration declined within 4 weeks in the first six volunteers, and the first man became azoospermic at 6 weeks. This fall occurred faster than in previous studies that used high doses of steroids or GnRH agonists (4, 5,14). Recovery was achieved within 14 weeks, which
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 April 2015. at 07:41 For personal use only. No other uses without permission. . All rights reserved.
GnRH ANTAGONIST AS MALE CONTRACEPTIVE 1.0
|Nal-Glu 10 mg
Nal-Glu 10 mg
to Mg '0 MO 100 (jg
0.8 -
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1367
•
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8
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10 mg
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20
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10
12
14
16
18
20
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6
8
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20
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TIME (hours)
FIG. 6. Upper panel, Mean (±SEM) serum immunoreactive Nal-Glu levels at weeks 10 (solid line) and 20 {broken line) in the six men who received 10 mg Nal-Glu and TE for 20 weeks. Lower panel, Nal-Glu serum levels on weeks 10 (solid line) and 20 (broken line) in the two men who received 10 mg Nal-Glu for the first 10 weeks and 20 mg for the next 10 weeks.
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