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Reproductive Endocrinology |
Institute of Reproductive Medicine of the University (WHO Collaborating Center for Research in Human Reproduction) (H.M.B., S.K., G.P., E.N.), Münster; and Asta Medica (T.R.), Frankfurt am Main, Germany
Address all correspondence and requests for reprints to: Prof. Dr. E. Nieschlag, FRCP, Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Münster, Germany.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Today, effective GnRH agonist depot preparations are available for clinical therapy, e.g. of prostate carcinoma. However, we could show that GnRH agonists are not suited for male contraception because they do not cause a prolonged and effective FSH decrease and thus fail to suppress spermatogenesis in male volunteers (2). In contrast, the GnRH antagonist Nal-Glu given at relatively high doses of 7.5–20 mg/day caused a significant suppression of both gonadotropins and spermatogenesis (3, 4, 5). However, high daily doses of the GnRH antagonist Nal-Glu, which can result in local adverse side-effects, are impractical and too expensive for long term use. Therefore, we were interested to determine whether a suppression of LH, FSH, and sex hormones achieved by initial high dose GnRH antagonist administration can be maintained by continued low dose injections. To test this concept, we performed a controlled clinical trial with cetrorelix, an effective modern GnRH antagonist (6, 7), given at high loading doses followed by low maintenance doses in normal male volunteers.
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Subjects and Methods |
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The study was performed as a single blind, randomized, placebo-controlled examination. The protocol was approved by the ethics committee of the University of Münster and the State Medical Board. Male volunteers, aged 20–40 yr, were recruited for the study. Detailed information about the experiment was provided, and written informed consent was obtained before commencement of the study. A thorough medical history was taken, followed by a physical examination and routine clinical chemistry and hematology. Criteria for participation included an uneventful medical history and normal results of physical examination, serum hormones, blood chemistry, and hematology at two baseline control examinations. Additional medication was not allowed during the study.
After this screening procedure, 16 normal healthy male volunteers were
randomly allocated to 1 of 4 study groups (Table 1
). The
GnRH antagonist or placebo was injected sc to all volunteers into
adipose tissue at the abdominal wall laterally to the rectus abdominis
muscle. The volunteers of cetrorelix verum groups I, II, and III
received 10 mg cetrorelix, given as 2 injections of 5 mg at 2 sites,
from study days 0–4. This initial loading dose period was followed by
maintenance injections of 2 mg cetrorelix/day in group I, 2 injections
of 1 mg cetrorelix/day in group II (1 mg every 12 h), and 1 mg
cetrorelix/day in group III up to study day 20. Volunteers of group IV
received 2 placebo mannitol injections/day of 5 mL for 5 days (study
days 0–4) followed by placebo mannitol injections of 2 mL up to study
day 20. All injections were given at 0800 h and in group II
additionally at 2000 h.
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The GnRH antagonist cetrorelix ([Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]GnRH; SB-75) (8) was synthesized and provided by Asta Medica (Frankfurt am Main, Germany). Lyophilized cetrorelix was stored at -20 C. Before injection, cetrorelix was dissolved in bacteriostatic water containing 5.2% (wt/vol) mannitol to a final concentration of 1.0 g/L. Injections of bacteriostatic water containing 5.2% (wt/vol) mannitol served as the placebo control.
Blood samples
Blood samples for LH, FSH, and T were collected at 0800 and 2000 h at the three baseline control examinations (study days -6, -4, and -2), then daily on study days 0–28, every other day from study days 30–42, and on study days 49, 63, and 91. Morning levels of estradiol and sex hormone-binding globulin (SHBG) were determined at two control examinations (study days -6 and -4), on day 0 and then weekly up to study day 49, and on study days 63 and 91. Serum samples for cetrorelix determinations were collected at 0800 and 2000 h on study day -6, on study days 0–20, then only at 0800 h daily from study days 21–28, every other day from study days 30–42, and on study days 49, 63, and 91. When blood samples were collected twice on any study day for hormone and cetrorelix measurements, the results are displayed as the respective mean for each volunteer; on day 0, however, values at 0800 and 2000 h are displayed separately. Blood samples for determinations of serum levels of hormones and cetrorelix were separated at 800 x g and stored at -20 C until assayed.
Immunoassays
Serum LH, FSH, and SHBG were determined by specific fluoroimmunoassays (Delfia hLH Spec, Delfia hFSH, Delfia SHBG, Pharmacia, Freiburg, Germany). The lower detection limits for FSH, LH, and SHBG were 0.25 IU/L, 0.12 IU/L, and 6.3 nmol/L, respectively. The intra- and interassay coefficients of variation for LH were 4.5% and 6.2%, respectively; those for FSH were 3.9% and 5.9%, respectively; and those for SHBG were 4.9% and 7.2%, respectively. In our laboratory, the normal range for LH is 2–10 IU/L, that for FSH is 1–7 IU/L, and that for SHBG is 11–71 nmol/L, respectively.
T was measured by RIA in extracted serum samples. The detection limit for T was 0.7 nmol/L. The intra- and interassay coefficients of variation for T were 6.3% and 9.8%, respectively. The lower normal limit for T is 12 nmol/L. Estradiol was measured by RIA (Sorin Biomedica, Saluggia, Italy). The detection limit for estradiol was 37 pmol/L. The intra- and interassay coefficients of variation for estradiol were 6.6% and 8.1%, respectively. The upper normal limit for estradiol is 250 pmol/L. Cetrorelix was measured by RIA, as described recently (6). The detection limit for cetrorelix was 0.28 µg/L, and the intra- and interassay coefficients of variation were 9.0% and 14.0%, respectively.
Local side-effects
Local side-effects after sc cetrorelix administration were documented daily on transparency paper. The erythema area was determined in a blinded manner by applying a digital planimeter (Haff, Pfronten, Germany).
Statistics
Significant variations over time and differences between study groups in any parameter were evaluated by multifactor ANOVA for repeated measures. When differences between study groups were significant, variations over time were tested separately for each group. In case of a general effect over time, values at single time points were analyzed in more detail by comparison with the baseline control value using Duncan’s multiple comparison test for repeated measures. The respective values of the second prestudy control examination were considered baseline values. When necessary, analysis was performed on logarithmically transformed data. P < 0.05 was considered significant. Computations were performed using the statistical software package SPSS, version 6.1.3 (SPSS, Chicago, IL), and Statgraphics Plus, version 7.1 (STSC, Rockville, MD). Unless otherwise stated, results are given as the mean ± SE.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Cetrorelix
Daily injections of the loading dose of 10 mg/day cetrorelix
resulted in a rapid increase in mean serum concentrations of
cetrorelix, with maximal levels on day 4 (Fig. 1).
Statistical analysis revealed no significant difference in the serum
levels of cetrorelix among groups I–III during the loading dose
period. During the following maintenance dose phase, cetrorelix levels
decreased and stabilized during the last week of maintenance dose
injections. For the three verum groups, multifactor ANOVA revealed a
plateau of stable cetrorelix concentrations for this time period. This
plateau was statistically not different between groups I and II (daily
maintenance dose of 2 mg) and ranged between 7.5 ± 0.7 µg/L
(group I, day 21) and 11.2 ± 1.2 µg/L (group II, day 16), with
an average level of 9.5 µg/L. The plateau in group III with daily
maintenance doses of 1 mg was, on the average, 4.8 µg/L and ranged
between 4.4 ± 0.4 µg/L at day 21 and 5.1 ± 0.4 µg/L at
day 15. During the maintenance dose phase, no significant differences
were detected between morning and evening levels of cetrorelix. After
the last injection, serum concentrations of cetrorelix declined and
were consistently lower than 2 µg/L in groups I and II and lower than
1 µg/L in group III after study day 30.
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No significant change in LH was seen in group IV during the study
period. In the three cetrorelix groups, injections of 10 mg/day for the
first 5 days resulted in suppression of LH levels to the subnormal
range (Fig. 2). During the maintenance dose period, LH
levels further declined in groups I–III. From study day 8 to the end
of the injection phase, individual LH levels remained near the assay
detection limit. No significant difference could be detected among the
three cetrorelix groups; however, LH levels at 2000 h were lower
than those at 0800 h. After the last cetrorelix injection, LH
levels returned to the normal range. Mean serum concentrations greater
than 2 IU/L were measured in groups I and II first on study day 30. In
group III, which was given the lowest maintenance dose, LH levels
returned to the normal range on day 26. A significant rebound increase
in LH after the last injection was seen in all verum groups. In group
I, a maximal LH level of 11.3 ± 2.6 IU/L, exceeding the normal
range, was measured on day 49; in group III, a maximal level of
11.3 ± 1.8 IU/L was measured on day 42. In group II, a maximal
level of 7.3 ± 3.0 IU/L measured on day 40 remained in the normal
range. All four study groups showed values in the prestudy control
range on day 91.
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No significant change in FSH was detected in placebo group IV. In
groups I–III, injections of 10 mg/day cetrorelix for the first 5 days
resulted in significant suppression of FSH levels compared to baseline;
however, levels still remained in the normal range (Fig. 3). During the maintenance dose injection phase, FSH
levels further declined in all cetrorelix groups to levels in the
subnormal range. Minimal serum concentrations were measured from study
day 15 to the end of the injection phase. During this period, FSH
levels were near the assay detection limit. No significant difference
could be detected among the three cetrorelix groups or between morning
and evening levels. After the last cetrorelix injection, FSH levels
increased, returning to the normal range. Mean serum concentrations
greater than 1 IU/L were measured in groups II and III first on study
day 26. In group I, FSH levels failed to return to the normal range
before day 34. A small rebound increase in FSH after the last injection
achieved significance only in group III. In group I, a maximal FSH
level of 4.9 ± 1.5 IU/L was measured on day 49; in group II, a
maximal level of 4.7 ± 1.2 IU/L was measured on day 63; and in
group III, a maximal level of 4.9 ± 0.7 IU/L was measured on day
49. All groups showed FSH levels in the prestudy control range (mean,
<4 IU/L) at the follow-up examination on day 91.
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Whereas no significant change was observed in the placebo group, T
was significantly suppressed in all three cetrorelix groups during the
loading dose period (Fig. 4). A further decline in T was
seen during the maintenance dose period, with minimal levels in all
three verum groups from study days 8–25, i.e. 5 days after
the last cetrorelix injection. During this period, individual T levels
were near the assay detection limit in all volunteers; at no time point
did they exceed 2 nmol/L. No significant differences in T
concentrations were detected among the three cetrorelix groups;
however, T levels at 2000 h tended to be lower than levels at
0800 h. Mean serum concentrations of T in the normal range higher
than 12 nmol/L were first recorded in study groups I and II on days 36
and 38, respectively. In group III, with the lowest maintenance dose of
cetrorelix, T levels in the normal range were seen as early as day 32.
A small, but significant, rebound increase in T compared to the
baseline control levels was seen in groups II and III. At the follow-up
examination, all groups had regained T concentrations comparable to the
baseline levels.
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Serum concentrations of estradiol mirrored the respective T levels
in the different study groups. Whereas no change was seen in the
placebo group, estradiol levels were suppressed close to the assay
detection limit by cetrorelix during the entire injection period (Fig. 5). Values in the prestudy range were regained on day 35
in groups II and III; in group I, this did not occur before day 42. In
groups II and III, a small, but significant, rebound increase in
estradiol concentrations was noticed. Mean serum levels were in the
prestudy control range in all study groups during the follow-up
examination on day 91.
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During the study course, mean SHBG levels remained unchanged in
the placebo group (Fig. 6). In the three verum groups, a
small, but uniform, increase in serum concentrations of SHBG was noted.
In group I, a maximal level of 33.1 ± 7.3 nmol/L was measured on
day 14 compared to a baseline control level of 26.1 ± 4.8 nmol/L.
Group II had a maximal level of 41.5 ± 10.8 nmol/L on day 21
compared to the baseline level of 32.7 ± 6.9 nmol/L; group III
had a maximal level of 39.7 ± 6.7 nmol/L on study day 21 compared
to the baseline value of 27.6 ± 6.9 nmol/L. The increase in SHBG
was significant in groups II and III; it did not reach statistical
significance in group I. Mean serum levels of SHBG returned to the
baseline control range in verum groups at the follow-up examination on
day 91.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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These data prompted us to investigate whether after initial effective suppression of gonadotropins and sex hormones by loading doses of 10 mg/day cetrorelix, lower maintenance doses might be sufficient to sustain the suppressive effect. In the present study we could demonstrate that much lower doses of 2 or even 1 mg/day cetrorelix can uniformly maintain suppression, once achieved, in all 12 volunteers. The same doses, when injected for 8 days without an initial loading dose, were unable to achieve any prolonged suppression of gonadotropins or sex hormones (9).
The finding that low GnRH antagonist doses, which are ineffective during the first week, suppress gonadotropins effectively during long term treatment argues against the concept that GnRH antagonists act through a purely competitive mechanism. Similar evidence is provided by preclinical studies. When adult male cynomolgus monkeys received high daily doses of 1400–1600 µg/kg of the GnRH antagonist ORG 30276 for 8 weeks and repeated GnRH stimulation tests were performed, a dose of 500 µg GnRH, which caused a clear increase in LH serum levels at study week 3 did not result in a significant increase at week 8 (12). As the magnitude of the LH response to GnRH is correlated with the concentration of GnRH receptors at the pituitary (13, 14), this preclinical study suggests that chronic administration of GnRH antagonists not only acts competitively at the GnRH receptor, but also might lead to receptor down-regulation in the pituitary gland.
Recently, in vitro ligand competition assays showed that single dose administration of 100 µg cetrorelix to rats resulted in down-regulation of pituitary GnRH receptors for at least 72 h. The receptor concentration was lowest 3–6 h after cetrorelix administration, and recovery of receptor numbers began within 24 h (15). Another experiment demonstrated that chronic sc administration of cetrorelix to male rats at a dose of 100 µg/day for 4 weeks resulted in a decrease in the pituitary receptors for GnRH by 77% (14). As a control, the injection of the GnRH agonist [D-Trp6]GnRH at the same dose resulted in a decrease in GnRH receptors by 69% after 4 weeks. In addition, it was demonstrated that GnRH antagonist administration caused a 83% reduction of the messenger ribonucleic acid for GnRH receptors within 2 weeks. Therefore, it is likely that the loss of GnRH receptors can be attributed to a decreased expression of the GnRH receptor gene or an increased degradation of the messenger ribonucleic acid for the receptor (14). These studies provide initial preclinical evidence that in addition to the well described competitive receptor blockage, GnRH antagonists, similar to GnRH agonists, cause a decrease in the number of GnRH receptors in the pituitary gland.
This mechanism of action could well explain the effective and sustained suppression of gonadotropins and sex hormones by very low daily maintenance doses of cetrorelix in the present study. It should be noted that in our previous studies, much higher doses, i.e. 5 mg/day cetrorelix, resulting in significantly higher serum levels of the GnRH antagonist with nadir levels above 7 µg/L before the next injection, were not able to suppress LH and FSH to levels lower than the normal range (9). However, if gonadotropins are significantly suppressed by loading dose injections, probably resulting in GnRH receptor down-regulation, much lower serum concentrations of cetrorelix with mean levels lower than 5 µg/L can maintain the suppression of gonadotropins. Serum levels of LH, FSH, and T decreased even further during the maintenance dose phase compared to those during the initial loading dose period to levels consistently near assay detection limits. The validity of this loading dose/maintenance dose concept was recently demonstrated in a clinical trial of male contraception, in which effective suppression of gonadotropins and endogenous T could be sustained by maintenance dose injections of 2 mg/day cetrorelix for up to 12 weeks (16).
Previously, it was believed that the development of the GnRH antagonist depot preparation is impracticable, as only GnRH antagonist doses of 7.5–20 mg/day effectively suppressed gonadotropins as well as sex hormones, whereas lower doses were ineffective (3, 4, 5, 9). However, this study demonstrates that after an effective loading dose period, much smaller doses of the GnRH antagonist are sufficient. Thus, GnRH antagonist depot preparations, which release relatively small amounts of the GnRH antagonist per day, might be effective for sustained suppression during the maintenance dose phase. Recently, we demonstrated in a first phase I, placebo-controlled, clinical study that a GnRH antagonist depot preparation, cetrorelix pamoate, which was relatively ineffective when injected into normal men at a single dose (17), was able to suppress gonadotropins and sex hormones effectively and uniformly when given after a loading dose phase of 5 days (18).
In conclusion, the results of this study show that compared to previous long term studies, much lower daily doses of the GnRH antagonist are sufficient for effective suppression of LH, FSH, and T after initial high loading dose injections. This loading dose/maintenance dose scheme might be important for future clinical applications of GnRH antagonists. With respect to dose-dependent local side-effects, treatment costs, and the development of GnRH antagonist depot preparations, this concept could be of significant relevance for the future clinical development of GnRH antagonists for treatment of sex hormone-dependent diseases as well as for male contraception.
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Acknowledgments |
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Footnotes |
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Received December 17, 1996.
Revised February 5, 1997.
Accepted February 12, 1997.
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; 10
mg/day cetrorelix for 5 days, followed by 2 x 1 mg/day up to
study day 20), and group III (
: 10 mg/day cetrorelix for 5 days,
followed by 1 mg/day up to study day 20).
; placebo
injections). The medium dashed line indicates the normal
range of LH; the short dashed line indicates the lower
detection limit of the assay.
