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Serum Hormone Concentrations During Treatment with Multiple Rising Doses of Recombinant Follicle-Stimulating Hormone (Puregon) in Men with Hypogonadotropic Hypogonadism
FERTILITY AND STERILITYt VOL. 69, NO. 2 (SUPPL. 1), FEBRUARY 1998 Copyright ©1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Serum hormone concentrations during treatment with multiple rising doses of recombinant follicle-stimulating hormone (Puregon*) in men with hypogonadotropic hypogonadism† Bernadette Mannaerts, M.Sc.,‡ Bart Fauser, Ph.D.,§ Najiba Lahlou, M.D.,\ Jonas Harlin, M.D.,¶ Zeev Shoham, M.D.,** Herjan J. T. Coelingh Bennink, M.D., Ph.D.,‡ and Philippe Bouchard, Ph.D.†† Received April 7, 1995; revised and accepted August 2, 1995. Reprint requests: Bernadette Mannaerts, M.Sc., Medical Research and Development Unit, NV Organon, P.O. Box 20, 5340 BH Oss, the Netherlands (FAX: 31-412662555). * Puregon, NV Organon, Oss, the Netherlands. † Supported by NV Organon, Oss, the Netherlands. ‡ Medical Research and Development Unit, NV Organon. § Department of Obstetrics and Gynaecology, Dijkzigt Academic Hospital and Erasmus University, Rotterdam, the Netherlands. \ Present address: Institut National de la Sante´ et de la Recherche Me´dicale (INSERM U) 342, Hoˆpital Saint-Vincent-de-Paul, Paris, France. ¶ Department of Obstetrics and Gynaecology, Karolinska Hospital. ** Department of Obstetrics and Gynaecology, Kaplan Hospital. †† Present address: Service d’Endocrinologie et de Maladies de la Reproduction, Hoˆpital Saint-Antoine, Paris, France. 0015-0282/98/$19.00 PII S0015-0282(97)00513–X
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NV Organon, Oss; Dijkzigt University Hospital and Erasmus University, Rotterdam, the Netherlands; Karolinska Hospital, Stockholm, Sweden; Kaplan Hospital, Rehovot, Israel; Hoˆpital Bice`tre, Le Kremlin Bice`tre; and Fondation de Recherche en Hormonology, Fresnes, France
Objective: To study increases of serum FSH and gonadal response in gonadotropin-deficient men treated with recombinant FSH (Puregon; NV Organon, Oss, the Netherlands). Design: An open, prospective, multiple rising dose study in which volunteers received single daily IM doses of recombinant FSH for 3 weeks. The dose administered was increased at weekly intervals: the first 7 days, 75 IU/d; the subsequent 7 days, 150 IU/d; and the last 7 days, 225 IU/d. Participant(s): Nine men suffering from isolated gonadotropin deficiency or panhypopituitarism. Main Outcome Measurement(s): Immunoreactive FSH, LH, inhibin, T, and androstenedione. Result(s): Serum immunoreactive FSH (median) rose in accordance with the recombinant FSH doses administered from 0.5 mIU/mL (range ,0.05 to 1.9 mIU/mL) at baseline to 4.3 mIU/mL (range 2.0 to 8.5 mIU/mL), 8.4 mIU/mL (range 4.9 to 17.8 mIU/mL), and 13.6 mIU/mL (5.6 to 28.4 mIU/mL) after 1, 2, and 3 weeks of medication, respectively. The elimination half-life of recombinant FSH was 48 6 5 hours (mean 6 SD), which was slightly longer than that reported after single dose administration of recombinant FSH (32 6 12 hours). The bioactivity of recombinant FSH was reflected by serum inhibin levels, which rose from 116 U/L (range 34 to 356 U/L) at baseline to 350 U/L (range 63 to 1,109 U/L) at day 22. However, serum FSH and inhibin levels did not correlate when compared after 1, 2, and 3 weeks of recombinant FSH administration. Serum immunoreactive LH, T, androstenedione, and E2 were 0.2 mIU/mL (range ,0.05 to 0.7 mIU/mL [conversion factor to SI unit, 1.0]), 58 ng/dL (range ,12 to 222 ng/dL [conversion factor to SI unit, 0.0347]), 14 ng/dL (range 6 to 115 ng/dL [conversion factor to SI unit, 0.0349]), and 14 pg/mL (range ,14 to 16 pg/mL [conversion factor to SI unit, 3.67]), respectively, at baseline and remained unchanged during the entire treatment period. Conclusion(s): These data indicate that recombinant FSH treatment increases serum FSH in a dose-proportional fashion, increases inhibin secretion, and lacks intrinsic LH activity. (Fertil Sterilt 1996;65:406 –10. ©1996 by American Society for Reproductive Medicine.) Key Words: Hypogonadotropic men, recombinant human FSH, inhibin, androgens
In men, FSH secreted by the anterior pituitary gland interacts exclusively with the Sertoli cells in the testis. The mechanism of FSH action includes specific binding to plasma membrane receptors and subsequent activation of the adenylyl cyclase system, resulting in de novo synthesis of various regulatory factors (androgen-binding protein, inhibin, transferrin, plasminogen activator, prostaglandins, and aro-
matase). In general, FSH exerts a stimulatory effect on spermatogenesis, testicular size, and serum inhibin levels. The initiation and maintenance of complete spermatogenesis during puberty or in men with congenital hypogonadotropic hypogonadism requires both FSH and LH (1). Accordingly, the most commonly applied therapy to initiate sperm production is combined treatment with
hMG (containing FSH and LH activity) and a relatively high dose of hCG. Whereas the FSH in hMG is essential to stimulate the Sertoli cells, the LH activity contained by hMG is very low in comparison with the high amount of LH activity provided by hCG. Therefore, the replacement of hMG by pure FSH, i.e., recombinant FSH (Puregon; NV Organon, Oss, the Netherlands) for the treatment of hypopituitary men is likely to be equally effective. So far, this new biosynthetic hormone has shown to be safe and effective in female patients undergoing IVF-ET. Moreover, in a group-comparative study (including 981 treated patients) recombinant FSH (Puregon) appeared to be more efficacious than urinary FSH (Metrodin; Ares-Serono, Geneva, Switzerland) for controlled ovarian hyperstimulation (2). Recently, the pharmacokinetic properties of recombinant FSH in hypopituitary men after single administration of recombinant FSH have been reported (3). The current study describes the pharmacokinetic and pharmacodynamic properties of recombinant FSH in gonadotropin-deficient male volunteers treated daily for 21 days using weekly rising doses. Serum FSH, LH, inhibin, androstenedione (A), T, and E2 were measured at regular intervals during and after treatment.
MATERIALS AND METHODS Volunteers and Hormone Treatment A total of nine men suffering from hypogonadotropic hypogonadism volunteered in this four-center, multiple-dose study, which was part of a clinical phase I program. The study protocol was approved by the local ethics review committees, and written informed consent was obtained from all volunteers. Subjects receiving hormonal replacement refrained from this therapy during the study period, from $3 weeks in advance up to 1 week thereafter. Six of nine subjects had a history of normal gonadal function in that their testis had proven previously to respond to gonadotropins. Volunteers were injected IM, once daily, with recombinant FSH (Org 32489, Puregon, CP 090073), i.e., 75 IU/d from treatment days 1 to 7, 150 IU/d from days 8 to 14, and 225 IU/d from days 15 to 21. Blood samples were drawn before each recombinant FSH administration (thus 24 hours after the previous injection) at days 1, 3, 5, 8, 10, 12, 15, 17, and 19 and, after the last recombinant FSH injection (day 21), at days 22, 24, 26, 29, 31, 33, 36, and 40.
Hormone Assays Immunoreactive FSH and LH levels were measured by time-resolved immunofluorometric sandwich assays (Delfia; Wallac Oy, Turku, Finland) as described previously (7). Follicle-stimulating hormone and LH immunoreactivity was expressed in terms of the Second International Reference Preparation of pituitary FSH (code no. 78/549) and the Second International Standard for pituitary LH (code no. FERTILITY & STERILITYt
80/552). The sensitivity of immunofluorometric assay was 0.05 mIU/mL for both gonadotropins, and the intra-assay and interassay coefficients of variation (CV) were ,4.8% and 4.3% for FSH and 4.7% and 7.5% for LH, respectively. The cross-reactivity of LH in the FSH assay was ,0.08% and of FSH in the LH assay was ,0.01%. Serum T and A were assessed by RIA using a coat-acount RIA (Diagnostic Products Corporation, Los Angeles, CA). The detection limits of these assays were 0.38 and 0.07 nmol/L, respectively. The intra-assay and interassay CVs were 9% and 13% for the T assay and 5% and 9% for the A assay. Serum E2 was measured by time-resolved immunofluorometric sandwich assay (Delfia). The detection limit of this assay was 0.05 nmol/L and its intra-assay and interassay CV were 4.3% and 5.0%, respectively. Serum inhibin levels were measured by RIA using an antiserum (no. 1989) raised against purified bovine 31-kd inhibin. Purified bovine 31-kd inhibin iodinated by the lactoperoxidase method was used as a tracer. The standard was a pool of human follicular fluid (FF; 280 U/mL), which was calibrated against a rete testis standard preparation of defined bioactivity. The immunoactivity of 1 mU FF was equipotent to 8 pg recombinant human inhibin (Biotech Australia, East Roseville, Australia, specific in vitro bioactivity 51.060 U/mg protein using World Health Organization [WHO] standard 86/690 as the standard). The recombinant a-subunit of human inhibin exhibited complete cross-reactivity in this assay system. The standard pool, which was diluted in plasma from castrated subjects, provided dose responses parallel to the plasma dilution curves. The sensitivity of the assay was 28 U/L and the intra-assay and interassay CVs at the level 420 U/L were 2.1% and 6.5%, respectively. Normal mean values of healthy men indicated for the various assays described above were 3 mIU/mL (range 1 to 10.5 mIU/mL [conversion factor to SI unit, 1.00]) for FSH, 3.3 mIU/mL (range 1.0 to 8.4 mIU/mL [conversion factor to SI unit, 1.00]) for LH, 510 U/L (range 330 to 710 U/L) for inhibin, 123 ng/dL (range 52 to 307 ng/dL [conversion factor to SI unit, 0.0349]) for A, 605 ng/dL (range 271 to 1,066 ng/dL [conversion factor to SI unit, 0.0347]) for T, and 25 pg/mL (range ,14 to 35 pg/mL [conversion factor to SI unit, 3.67]) for E2.
Data Analysis Hormone values are presented as median values with ranges. One subject (M7) did not receive recombinant FSH injection at days 16 and 21 but was included in summary statistics of all hormone values. One subject (M5) who received replacement therapy of hCG (3,000 IU) and FSH (150 IU) up to 4 weeks before the study period had relatively high T (55.7 nmol/L) and A (10.8 nmol/L) levels at the first day of recombinant FSH treatment. These levels rapidly declined to undetectable, i.e., 25S
,0.4 and 0.62 nmol/L, respectively, at day 22 of the study period. This subject was included in the analysis of serum FSH and LH but was excluded from the summary statistics of other hormones. The elimination half-life (t 5 4) was calculated from serum immunoreactive FSH concentrations measured from 72 hours onward after the last recombinant FSH dose. A baseline correction was made by subtracting the lowest level in the wash-out phase from the concentrations measured.
FIGURE 1 Individual graphs of serum FSH and inhibin measured in gonadotropin-deficient men treated with single daily IM doses of recombinant FSH for 3 weeks. Doses were increased at weekly intervals from 75 to 225 IU/d; 1, M1 42 kg; ‚, M2 58 kg; E, M3 62 kg; 1, M4 71 kg; h, M5 92 kg; Œ, M6 54 kg; F, M7 61 kg; ƒ, M8 65 kg; {, M9 80 kg.
RESULTS Volunteers Nine gonadotropin-deficient, but otherwise healthy, male volunteers participated in this study. Four subjects were hypophysectomized because of a nonmalignant pituitary tumor, two subjects suffered from congenital isolated gonadotropin deficiency, two subjects were diagnosed as primary panhypopituitarism, and one was diagnosed as secondary panhypopituitarism due to pituitary stalk section. The age of the volunteers ranged between 29 and 50 years (mean 6 SD; 37.2 6 6.0 years) and their body mass index was between 18.7 and 26.0 kg/m2 (22.8 6 3.6 kg/m2).
Follicle-Stimulating Hormone Individual immunoreactive FSH levels measured in gonadotropin-deficient men during daily medication for 3 consecutive weeks are presented in Figure 1 (top). Serum FSH levels increased in a dose-proportional manner (Fig. 1, top). Steady-state levels of each dose level of recombinant FSH was reached after 3 to 5 days of treatment. Follicle-stimulating hormone immunoreactivity measured before the first recombinant FSH injection on day 1 and 1 day after completion of each treatment week are presented in Table 1. Increments of serum FSH were relatively high in one subject (M1) weighing only 42 kg (see Fig. 1), but the correlation between serum FSH measured at days 8, 15, and 22 and body weight was low (r 5 0.6 at all 3 days). The elimination half-life of recombinant FSH calculated from FSH concentrations measured after the last recombinant FSH injection was 48.4 6 5.4 hours.
Inhibin Individual graphs of serum inhibin are presented in Figure 1 (bottom). Baseline levels of serum inhibin measured before the first recombinant FSH administration varied to a great extent between subjects. During the treatment period, individual rises of inhibin were very gradual, and maximum inhibin concentrations represented a twofold to fourfold increase over baseline. Overall serum inhibin increased from 116 U/L (range 34 to 356 U/L) at day 1 to 350 U/L (range 63 to 1,109 U/L) at day 22 (Table 1). Comparison of serum FSH and inhibin at days 8, 15, and 22 did not reveal any correlation. 26S
A recombinant FSH
Luteinizing Hormone and Steroids Median values of serum LH, A, T, and E2 measured before the first recombinant FSH injection (day 1) and after 1 week (day 8), 2 weeks (day 15), and 3 weeks (day 22) of recombinant FSH treatment are presented in Table 1. In all subjects, serum LH was ,0.8 mIU/mL before the first injection, and none of the gonadotropin-deficient men showed changes of serum LH during the treatment period. Serum T was ,230 ng/dL (,8 nmol/L) at baseline but tended to decrease in time in four of eight subjects. In three subjects, serum T was undetectable (,12 ng/mL [,0.4 nmol/L]) at all time points. Serum A was ,43 ng/dL [,1.5 nmol/L] in six subjects, whereas two men (M3 and M4) with isolated gonadotropin deficiency had levels between 43 and 143 ng/dL (1.5 and 5 nmol/L), which were within the normal range of healthy men. In contrast, serum T of these two subjects was low (,72 ng/dL [,2.5 nmol/L]) and only one (M3) of these two Vol. 69, No. 2 (Suppl. 1), February 1998
TABLE 1 Concentrations of hormones measured on day 1 and 1 day after completion of each treatment week in hypogonadotropic men treated daily for 21 days with weekly rising doses of recombinant FSH.* Day
1 8 15 22
FSH
LH
Inhibin
A
T
E2
mIU/mL
(mIU/mL)
U/L
ng/dL
ng/dL
pg/mL
116 (34 to 356) 241 (49 to 579) 337 (97 to 965) 350 (63 to 1,109)
14 (6 to 115) 14 (6 to 95) 12 (3 to 106) 12 (6 to 146)
0.5 (,0.05 to 1.9) 4.3 (2.0 to 8.5) 8.4 (4.9 to 17.8) 13.6 (5.6 to 28.4)
0.2 (,0.05 to 0.2 (,0.05 to 0.1 (,0.05 to 0.1 (,0.05 to
0.7) 0.6) 0.8) 0.7)
58 (,12 to 26 (,12 to 20 (,12 to 17 (,12 to
222) 202) 195) 144)
14 (,14 to 16) ,14 (,14 to 17) 14 (,14 to 16) ,14 (,14 to 17)
* Values are medians with ranges in parentheses. Conversion factors to SI units are as follows: LH and FSH, 1.0; A, 0.0349; T, 0.0347; E2, 3.67.
subjects had relatively high inhibin concentrations (see Fig. 1). Serum E2 was either undetectable or extremely low and did not change during treatment.
DISCUSSION In the current study, treatment of hypogonadotropic men with weekly rising doses of recombinant FSH induced increases of serum FSH in a dose-proportional manner in the dose range tested (75 to 225 IU). At each dose, steady-state levels were reached within 5 days of treatment, which also was reported after daily IM administration of 75 and 150 IU in hypogonadotropic women (4). The elimination half-life of recombinant FSH was 48 6 5 hours and only slightly longer than the reported half-life (32 6 12 hours) after single-dose administration of recombinant FSH (3). The elimination half-life indicates the maximum dosing interval to reach steady-state levels. However, because both in the single- and multiple-dose study the elimination half-lives were calculated from serum FSH concentrations measured from 72 hours after (the last) injection, and basic FSH isohormones are thought to be cleared faster from the circulation than the more acidic isoforms, these half-lives probably reflect the more acidic fraction rather than that of the whole bioactive ingredient. In response to recombinant FSH, serum inhibin levels increased gradually, reflecting the bioactivity of the recombinant FSH doses injected. However, in several men, treatment with recombinant FSH treatment for 21 days did not increase serum inhibin up to the normal range (510 U/L [range 330 to 710 U/L]), most likely because of the relatively short treatment period.
FERTILITY & STERILITYt
In the current study, endogenous LH remained extremely low. In addition, serum steroids also were low, indicating that treatment with recombinant FSH did not affect androgen biosynthesis and thus lacks intrinsic LH activity. Accordingly, estrogens remained unchanged. In conclusion, our data indicate that recombinant FSH treatment of hypogonadotropic men increases serum FSH in a dose-proportional fashion, increases inhibin secretion, and lacks intrinsic LH activity.
Acknowledgments: The authors gratefully acknowledge Renato de Leeuw, Ph.D., NV Organon, Oss, the Netherlands, for the gonadotropin determinations and Ferdy Rombout, M.Sc., NV Organon, for calculating the elimination half-life of recombinant FSH.
References 1. Schaison G, Young J, Pholsena M, Nahoul K, Couzinet B. Failure of combined follicle-stimulating hormone-testosterone administration to initiate and/or maintain spermatogenesis in men with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 1993;77:1545–9. 2. Out HJ, Mannaerts BMJL, Driessen SGAJ, Coelingh Bennink HJT for The European Puregont Collaborative IVF Study Group. A prospective, randomized, assessor-blind, multicentre study comparing recombinant and urinary follicle-stimulating hormone (Puregon vs Metrodin) in invitro fertilization. Hum Reprod. 1995;10:534 – 40. 3. Mannaerts B, Shoham Z, Schoot D, Bouchard P, Harlin J, Fauser B, et al. Single-dose pharmacokinetics and pharmacodynamics of recombinant human follicle-stimulating hormone (Org 32489) in gonadotropin-deficient volunteers. Fertil Steril 1993;59:108 –14. 4. Schoot DC, Harlin J, Shoham Z, Mannaerts B, Lahlou N, Bouchard P, et al. Recombinant human follicle-stimulating hormone and ovarian response in gonadotropin deficient women. Hum Reprod 1994;9:1237– 42.
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Serum hormone concentrations during treatment with multiple rising doses of recombinant follicle-stimulating hormone (Puregon*) in men with hypogonadotropic hypogonadism† Bernadette Mannaerts, M.Sc.,‡ Bart Fauser, Ph.D.,§ Najiba Lahlou, M.D.,\ Jonas Harlin, M.D.,¶ Zeev Shoham, M.D.,** Herjan J. T. Coelingh Bennink, M.D., Ph.D.,‡ and Philippe Bouchard, Ph.D.†† Received April 7, 1995; revised and accepted August 2, 1995. Reprint requests: Bernadette Mannaerts, M.Sc., Medical Research and Development Unit, NV Organon, P.O. Box 20, 5340 BH Oss, the Netherlands (FAX: 31-412662555). * Puregon, NV Organon, Oss, the Netherlands. † Supported by NV Organon, Oss, the Netherlands. ‡ Medical Research and Development Unit, NV Organon. § Department of Obstetrics and Gynaecology, Dijkzigt Academic Hospital and Erasmus University, Rotterdam, the Netherlands. \ Present address: Institut National de la Sante´ et de la Recherche Me´dicale (INSERM U) 342, Hoˆpital Saint-Vincent-de-Paul, Paris, France. ¶ Department of Obstetrics and Gynaecology, Karolinska Hospital. ** Department of Obstetrics and Gynaecology, Kaplan Hospital. †† Present address: Service d’Endocrinologie et de Maladies de la Reproduction, Hoˆpital Saint-Antoine, Paris, France. 0015-0282/98/$19.00 PII S0015-0282(97)00513–X
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NV Organon, Oss; Dijkzigt University Hospital and Erasmus University, Rotterdam, the Netherlands; Karolinska Hospital, Stockholm, Sweden; Kaplan Hospital, Rehovot, Israel; Hoˆpital Bice`tre, Le Kremlin Bice`tre; and Fondation de Recherche en Hormonology, Fresnes, France
Objective: To study increases of serum FSH and gonadal response in gonadotropin-deficient men treated with recombinant FSH (Puregon; NV Organon, Oss, the Netherlands). Design: An open, prospective, multiple rising dose study in which volunteers received single daily IM doses of recombinant FSH for 3 weeks. The dose administered was increased at weekly intervals: the first 7 days, 75 IU/d; the subsequent 7 days, 150 IU/d; and the last 7 days, 225 IU/d. Participant(s): Nine men suffering from isolated gonadotropin deficiency or panhypopituitarism. Main Outcome Measurement(s): Immunoreactive FSH, LH, inhibin, T, and androstenedione. Result(s): Serum immunoreactive FSH (median) rose in accordance with the recombinant FSH doses administered from 0.5 mIU/mL (range ,0.05 to 1.9 mIU/mL) at baseline to 4.3 mIU/mL (range 2.0 to 8.5 mIU/mL), 8.4 mIU/mL (range 4.9 to 17.8 mIU/mL), and 13.6 mIU/mL (5.6 to 28.4 mIU/mL) after 1, 2, and 3 weeks of medication, respectively. The elimination half-life of recombinant FSH was 48 6 5 hours (mean 6 SD), which was slightly longer than that reported after single dose administration of recombinant FSH (32 6 12 hours). The bioactivity of recombinant FSH was reflected by serum inhibin levels, which rose from 116 U/L (range 34 to 356 U/L) at baseline to 350 U/L (range 63 to 1,109 U/L) at day 22. However, serum FSH and inhibin levels did not correlate when compared after 1, 2, and 3 weeks of recombinant FSH administration. Serum immunoreactive LH, T, androstenedione, and E2 were 0.2 mIU/mL (range ,0.05 to 0.7 mIU/mL [conversion factor to SI unit, 1.0]), 58 ng/dL (range ,12 to 222 ng/dL [conversion factor to SI unit, 0.0347]), 14 ng/dL (range 6 to 115 ng/dL [conversion factor to SI unit, 0.0349]), and 14 pg/mL (range ,14 to 16 pg/mL [conversion factor to SI unit, 3.67]), respectively, at baseline and remained unchanged during the entire treatment period. Conclusion(s): These data indicate that recombinant FSH treatment increases serum FSH in a dose-proportional fashion, increases inhibin secretion, and lacks intrinsic LH activity. (Fertil Sterilt 1996;65:406 –10. ©1996 by American Society for Reproductive Medicine.) Key Words: Hypogonadotropic men, recombinant human FSH, inhibin, androgens
In men, FSH secreted by the anterior pituitary gland interacts exclusively with the Sertoli cells in the testis. The mechanism of FSH action includes specific binding to plasma membrane receptors and subsequent activation of the adenylyl cyclase system, resulting in de novo synthesis of various regulatory factors (androgen-binding protein, inhibin, transferrin, plasminogen activator, prostaglandins, and aro-
matase). In general, FSH exerts a stimulatory effect on spermatogenesis, testicular size, and serum inhibin levels. The initiation and maintenance of complete spermatogenesis during puberty or in men with congenital hypogonadotropic hypogonadism requires both FSH and LH (1). Accordingly, the most commonly applied therapy to initiate sperm production is combined treatment with
hMG (containing FSH and LH activity) and a relatively high dose of hCG. Whereas the FSH in hMG is essential to stimulate the Sertoli cells, the LH activity contained by hMG is very low in comparison with the high amount of LH activity provided by hCG. Therefore, the replacement of hMG by pure FSH, i.e., recombinant FSH (Puregon; NV Organon, Oss, the Netherlands) for the treatment of hypopituitary men is likely to be equally effective. So far, this new biosynthetic hormone has shown to be safe and effective in female patients undergoing IVF-ET. Moreover, in a group-comparative study (including 981 treated patients) recombinant FSH (Puregon) appeared to be more efficacious than urinary FSH (Metrodin; Ares-Serono, Geneva, Switzerland) for controlled ovarian hyperstimulation (2). Recently, the pharmacokinetic properties of recombinant FSH in hypopituitary men after single administration of recombinant FSH have been reported (3). The current study describes the pharmacokinetic and pharmacodynamic properties of recombinant FSH in gonadotropin-deficient male volunteers treated daily for 21 days using weekly rising doses. Serum FSH, LH, inhibin, androstenedione (A), T, and E2 were measured at regular intervals during and after treatment.
MATERIALS AND METHODS Volunteers and Hormone Treatment A total of nine men suffering from hypogonadotropic hypogonadism volunteered in this four-center, multiple-dose study, which was part of a clinical phase I program. The study protocol was approved by the local ethics review committees, and written informed consent was obtained from all volunteers. Subjects receiving hormonal replacement refrained from this therapy during the study period, from $3 weeks in advance up to 1 week thereafter. Six of nine subjects had a history of normal gonadal function in that their testis had proven previously to respond to gonadotropins. Volunteers were injected IM, once daily, with recombinant FSH (Org 32489, Puregon, CP 090073), i.e., 75 IU/d from treatment days 1 to 7, 150 IU/d from days 8 to 14, and 225 IU/d from days 15 to 21. Blood samples were drawn before each recombinant FSH administration (thus 24 hours after the previous injection) at days 1, 3, 5, 8, 10, 12, 15, 17, and 19 and, after the last recombinant FSH injection (day 21), at days 22, 24, 26, 29, 31, 33, 36, and 40.
Hormone Assays Immunoreactive FSH and LH levels were measured by time-resolved immunofluorometric sandwich assays (Delfia; Wallac Oy, Turku, Finland) as described previously (7). Follicle-stimulating hormone and LH immunoreactivity was expressed in terms of the Second International Reference Preparation of pituitary FSH (code no. 78/549) and the Second International Standard for pituitary LH (code no. FERTILITY & STERILITYt
80/552). The sensitivity of immunofluorometric assay was 0.05 mIU/mL for both gonadotropins, and the intra-assay and interassay coefficients of variation (CV) were ,4.8% and 4.3% for FSH and 4.7% and 7.5% for LH, respectively. The cross-reactivity of LH in the FSH assay was ,0.08% and of FSH in the LH assay was ,0.01%. Serum T and A were assessed by RIA using a coat-acount RIA (Diagnostic Products Corporation, Los Angeles, CA). The detection limits of these assays were 0.38 and 0.07 nmol/L, respectively. The intra-assay and interassay CVs were 9% and 13% for the T assay and 5% and 9% for the A assay. Serum E2 was measured by time-resolved immunofluorometric sandwich assay (Delfia). The detection limit of this assay was 0.05 nmol/L and its intra-assay and interassay CV were 4.3% and 5.0%, respectively. Serum inhibin levels were measured by RIA using an antiserum (no. 1989) raised against purified bovine 31-kd inhibin. Purified bovine 31-kd inhibin iodinated by the lactoperoxidase method was used as a tracer. The standard was a pool of human follicular fluid (FF; 280 U/mL), which was calibrated against a rete testis standard preparation of defined bioactivity. The immunoactivity of 1 mU FF was equipotent to 8 pg recombinant human inhibin (Biotech Australia, East Roseville, Australia, specific in vitro bioactivity 51.060 U/mg protein using World Health Organization [WHO] standard 86/690 as the standard). The recombinant a-subunit of human inhibin exhibited complete cross-reactivity in this assay system. The standard pool, which was diluted in plasma from castrated subjects, provided dose responses parallel to the plasma dilution curves. The sensitivity of the assay was 28 U/L and the intra-assay and interassay CVs at the level 420 U/L were 2.1% and 6.5%, respectively. Normal mean values of healthy men indicated for the various assays described above were 3 mIU/mL (range 1 to 10.5 mIU/mL [conversion factor to SI unit, 1.00]) for FSH, 3.3 mIU/mL (range 1.0 to 8.4 mIU/mL [conversion factor to SI unit, 1.00]) for LH, 510 U/L (range 330 to 710 U/L) for inhibin, 123 ng/dL (range 52 to 307 ng/dL [conversion factor to SI unit, 0.0349]) for A, 605 ng/dL (range 271 to 1,066 ng/dL [conversion factor to SI unit, 0.0347]) for T, and 25 pg/mL (range ,14 to 35 pg/mL [conversion factor to SI unit, 3.67]) for E2.
Data Analysis Hormone values are presented as median values with ranges. One subject (M7) did not receive recombinant FSH injection at days 16 and 21 but was included in summary statistics of all hormone values. One subject (M5) who received replacement therapy of hCG (3,000 IU) and FSH (150 IU) up to 4 weeks before the study period had relatively high T (55.7 nmol/L) and A (10.8 nmol/L) levels at the first day of recombinant FSH treatment. These levels rapidly declined to undetectable, i.e., 25S
,0.4 and 0.62 nmol/L, respectively, at day 22 of the study period. This subject was included in the analysis of serum FSH and LH but was excluded from the summary statistics of other hormones. The elimination half-life (t 5 4) was calculated from serum immunoreactive FSH concentrations measured from 72 hours onward after the last recombinant FSH dose. A baseline correction was made by subtracting the lowest level in the wash-out phase from the concentrations measured.
FIGURE 1 Individual graphs of serum FSH and inhibin measured in gonadotropin-deficient men treated with single daily IM doses of recombinant FSH for 3 weeks. Doses were increased at weekly intervals from 75 to 225 IU/d; 1, M1 42 kg; ‚, M2 58 kg; E, M3 62 kg; 1, M4 71 kg; h, M5 92 kg; Œ, M6 54 kg; F, M7 61 kg; ƒ, M8 65 kg; {, M9 80 kg.
RESULTS Volunteers Nine gonadotropin-deficient, but otherwise healthy, male volunteers participated in this study. Four subjects were hypophysectomized because of a nonmalignant pituitary tumor, two subjects suffered from congenital isolated gonadotropin deficiency, two subjects were diagnosed as primary panhypopituitarism, and one was diagnosed as secondary panhypopituitarism due to pituitary stalk section. The age of the volunteers ranged between 29 and 50 years (mean 6 SD; 37.2 6 6.0 years) and their body mass index was between 18.7 and 26.0 kg/m2 (22.8 6 3.6 kg/m2).
Follicle-Stimulating Hormone Individual immunoreactive FSH levels measured in gonadotropin-deficient men during daily medication for 3 consecutive weeks are presented in Figure 1 (top). Serum FSH levels increased in a dose-proportional manner (Fig. 1, top). Steady-state levels of each dose level of recombinant FSH was reached after 3 to 5 days of treatment. Follicle-stimulating hormone immunoreactivity measured before the first recombinant FSH injection on day 1 and 1 day after completion of each treatment week are presented in Table 1. Increments of serum FSH were relatively high in one subject (M1) weighing only 42 kg (see Fig. 1), but the correlation between serum FSH measured at days 8, 15, and 22 and body weight was low (r 5 0.6 at all 3 days). The elimination half-life of recombinant FSH calculated from FSH concentrations measured after the last recombinant FSH injection was 48.4 6 5.4 hours.
Inhibin Individual graphs of serum inhibin are presented in Figure 1 (bottom). Baseline levels of serum inhibin measured before the first recombinant FSH administration varied to a great extent between subjects. During the treatment period, individual rises of inhibin were very gradual, and maximum inhibin concentrations represented a twofold to fourfold increase over baseline. Overall serum inhibin increased from 116 U/L (range 34 to 356 U/L) at day 1 to 350 U/L (range 63 to 1,109 U/L) at day 22 (Table 1). Comparison of serum FSH and inhibin at days 8, 15, and 22 did not reveal any correlation. 26S
A recombinant FSH
Luteinizing Hormone and Steroids Median values of serum LH, A, T, and E2 measured before the first recombinant FSH injection (day 1) and after 1 week (day 8), 2 weeks (day 15), and 3 weeks (day 22) of recombinant FSH treatment are presented in Table 1. In all subjects, serum LH was ,0.8 mIU/mL before the first injection, and none of the gonadotropin-deficient men showed changes of serum LH during the treatment period. Serum T was ,230 ng/dL (,8 nmol/L) at baseline but tended to decrease in time in four of eight subjects. In three subjects, serum T was undetectable (,12 ng/mL [,0.4 nmol/L]) at all time points. Serum A was ,43 ng/dL [,1.5 nmol/L] in six subjects, whereas two men (M3 and M4) with isolated gonadotropin deficiency had levels between 43 and 143 ng/dL (1.5 and 5 nmol/L), which were within the normal range of healthy men. In contrast, serum T of these two subjects was low (,72 ng/dL [,2.5 nmol/L]) and only one (M3) of these two Vol. 69, No. 2 (Suppl. 1), February 1998
TABLE 1 Concentrations of hormones measured on day 1 and 1 day after completion of each treatment week in hypogonadotropic men treated daily for 21 days with weekly rising doses of recombinant FSH.* Day
1 8 15 22
FSH
LH
Inhibin
A
T
E2
mIU/mL
(mIU/mL)
U/L
ng/dL
ng/dL
pg/mL
116 (34 to 356) 241 (49 to 579) 337 (97 to 965) 350 (63 to 1,109)
14 (6 to 115) 14 (6 to 95) 12 (3 to 106) 12 (6 to 146)
0.5 (,0.05 to 1.9) 4.3 (2.0 to 8.5) 8.4 (4.9 to 17.8) 13.6 (5.6 to 28.4)
0.2 (,0.05 to 0.2 (,0.05 to 0.1 (,0.05 to 0.1 (,0.05 to
0.7) 0.6) 0.8) 0.7)
58 (,12 to 26 (,12 to 20 (,12 to 17 (,12 to
222) 202) 195) 144)
14 (,14 to 16) ,14 (,14 to 17) 14 (,14 to 16) ,14 (,14 to 17)
* Values are medians with ranges in parentheses. Conversion factors to SI units are as follows: LH and FSH, 1.0; A, 0.0349; T, 0.0347; E2, 3.67.
subjects had relatively high inhibin concentrations (see Fig. 1). Serum E2 was either undetectable or extremely low and did not change during treatment.
DISCUSSION In the current study, treatment of hypogonadotropic men with weekly rising doses of recombinant FSH induced increases of serum FSH in a dose-proportional manner in the dose range tested (75 to 225 IU). At each dose, steady-state levels were reached within 5 days of treatment, which also was reported after daily IM administration of 75 and 150 IU in hypogonadotropic women (4). The elimination half-life of recombinant FSH was 48 6 5 hours and only slightly longer than the reported half-life (32 6 12 hours) after single-dose administration of recombinant FSH (3). The elimination half-life indicates the maximum dosing interval to reach steady-state levels. However, because both in the single- and multiple-dose study the elimination half-lives were calculated from serum FSH concentrations measured from 72 hours after (the last) injection, and basic FSH isohormones are thought to be cleared faster from the circulation than the more acidic isoforms, these half-lives probably reflect the more acidic fraction rather than that of the whole bioactive ingredient. In response to recombinant FSH, serum inhibin levels increased gradually, reflecting the bioactivity of the recombinant FSH doses injected. However, in several men, treatment with recombinant FSH treatment for 21 days did not increase serum inhibin up to the normal range (510 U/L [range 330 to 710 U/L]), most likely because of the relatively short treatment period.
FERTILITY & STERILITYt
In the current study, endogenous LH remained extremely low. In addition, serum steroids also were low, indicating that treatment with recombinant FSH did not affect androgen biosynthesis and thus lacks intrinsic LH activity. Accordingly, estrogens remained unchanged. In conclusion, our data indicate that recombinant FSH treatment of hypogonadotropic men increases serum FSH in a dose-proportional fashion, increases inhibin secretion, and lacks intrinsic LH activity.
Acknowledgments: The authors gratefully acknowledge Renato de Leeuw, Ph.D., NV Organon, Oss, the Netherlands, for the gonadotropin determinations and Ferdy Rombout, M.Sc., NV Organon, for calculating the elimination half-life of recombinant FSH.
References 1. Schaison G, Young J, Pholsena M, Nahoul K, Couzinet B. Failure of combined follicle-stimulating hormone-testosterone administration to initiate and/or maintain spermatogenesis in men with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 1993;77:1545–9. 2. Out HJ, Mannaerts BMJL, Driessen SGAJ, Coelingh Bennink HJT for The European Puregont Collaborative IVF Study Group. A prospective, randomized, assessor-blind, multicentre study comparing recombinant and urinary follicle-stimulating hormone (Puregon vs Metrodin) in invitro fertilization. Hum Reprod. 1995;10:534 – 40. 3. Mannaerts B, Shoham Z, Schoot D, Bouchard P, Harlin J, Fauser B, et al. Single-dose pharmacokinetics and pharmacodynamics of recombinant human follicle-stimulating hormone (Org 32489) in gonadotropin-deficient volunteers. Fertil Steril 1993;59:108 –14. 4. Schoot DC, Harlin J, Shoham Z, Mannaerts B, Lahlou N, Bouchard P, et al. Recombinant human follicle-stimulating hormone and ovarian response in gonadotropin deficient women. Hum Reprod 1994;9:1237– 42.
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