Suppressive actions of a gonadotropin-releasing hormone antagonist on luteinizing hormone, follicle-stimulating hormone, and prolactin release in estrogen-deficient postmenopausal women Randall J. Urban, MD: Spyros N. Pavlou, MD: Jean E. Rivier, MD,c Wylie W. Vale, MD,c Maria L. Dufau, MD/ and Johannes D. Veldhuis, MD"

Charlottesville, Virginia, Nashville, Tennessee, San Diego, California, and Bethesda, Maryland We investigated time- and dose-dependent actions of a gonadotropin-releasing hormone antagonist, the "Nal-Glu" peptide [Ac-D2Nal" 4CIDPhe2 , D3PaP, Arg 5 , DGlu 6(AA), DAla'O], in nine healthy estrogen-withdrawn postmenopausal women. Gonadotropin-releasing hormone antagonist was administered subcutaneously at doses of 10, 30, 100, and 300 j.Lg/kg. Suppression of immunoactive luteinizing hormone concentrations was achieved with a 30 j.Lg/kg dose of antagonist. Suppression of immunoactive follicle-stimulating hormone levels was less (40%) even at the highest antagonist dose (300 j.Lg/kg). Bioactive luteinizing hormone concentrations also significantly decreased (>60%) at the two antagonist doses tested (30 and 300 j.Lg/kg). However, the lower antagonist dose showed an "escape" of bioactive luteinizing hormone values after 18 hours. No suppressive effects of the antagonist on prolactin secretion occurred at any dose tested. We conclude that this gonadotropin-releasing hormone antagonist can achieve effective, potent, and long-lasting suppression of pituitary secretion of biologically active luteinizing hormone at higher doses, but secretion of biologically active luteinizing hormone may "escape" at lower doses. (AM J OSSTET GVNECOL 1990;162:1255-60.)

Key words: Gonadotropin-releasing hormone antagonist, gonadotropin secretion, bioactive luteinizing hormone, gonadotropin-releasing hormone, prolactin secretion A recently developed class of compounds termed gonadotropin-releasing hormone (GnRH) antagonists has potential for clinical application based on an ability to produce immediate suppression of serum gonadotropins by binding competitively with endogenous GnRH to pituitary gonadotroph receptors. l • 2 The early GnRH antagonists had limited potency and produced From the Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia School of Medicine," the Department of Internal Medicine, Vanderbilt Universi(v: The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, and the Section of Molecular Endocrinology, Endocrine Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health. Supported in part by National Institutes of Health (NIH) Grant no. RR 00847 to the Clinical Research Center of the University of Virginia, Research Career Development Award No. 1 K04 HD 00634 (j. D. V.), National Institutes of Health Clinical Associate Physician Award no. 3 MOl RR00847-1491 (R.]. U.), BiomedicalResearch Support Grant No. 5-S07-RR 05431-26 (R..f. U.), Diabetes and Research Training Center Grant no. 5 P60 AM 22125-05, National Institutes of Health-supported Clinfo Data Reduction Systems, and National Institutes of Health Grant no. HD 13527 (j. R., W. V.). Received for publication October 17, 1989; revised December 21, 1989; accepted January 18,1990. Reprint requests: Johannes D. Veldhuis, MD, Box 202, University of Virginia School of Medicine, Charlottesville, VA 22908. 611119449

significant local histamine release at the site of subcutaneous injection in human subjects. 36 Later GnRH antagonists have shown an improvement in potency while producing only minimal local histamine release with subcutaneous injection. 7 . 'o Recently, another GnRH antagonist that causes prolonged suppression of gonadotropins after a single injection while producing no apparent local histamine release at the site of injection has been tested in monkeys. II Whether this action will be similar in humans is yet to be determined. Suppression of serum gonadotropin levels in hypergonadotropic postmenopausal women has been achieved with a GnRH agonist ' " and GnRH antagonists. 3 • 13 The latter resulted in measurable local edematous responses.' In this study we tested the dose-dependent suppression of gonadotropins and prolactin in estrogen-deficient hypergonadotropic postmenopausal women with the new investigative GnRH antagonist [Ac-D2NAL ' , 4CIDPhe", D3Pai', ArgO, DGlu 6 (AA), DAla 'O ] GnRH, the "Nal-Glu" peptide. In this primary hypogonadal setting with increased serum gonadotropin concentrations (from presumed increased GnRH secretion), the true suppressive actions of this GnRH antagonist can be determined in the absence of feedback changes otherwise introduced 1255

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Am J Obstet Gynecol

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sence of the antagonist. The lower dose affected minimally the apparent potency of the standard preparation (standard, 22 IVlmg [range, 21 to 25 IVlmg], whereas the maximal dose caused a slight overestimation of potency (l IJ-g Nal-Glu, 30 IV Img [range, 27 to 37 IV/mg]). Immunoactive FSH. The Nal-Glu antagonist produced much less marked suppression of immunoactive FSH than immunoactive or bioactive LH concentrations. The maximal percentage suppression (40%) occurred at the 300 IJ-g/kg antagonist dose (Fig. 2). Only the 300 IJ-g I kg antagonist dose resulted in a significant increase in the duration of suppression. Prolactin. There was no significant suppression of serum prolactin concentrations demonstrable with any antagonist dose. Comment We have studied the time-course and dose-response effects of a new potent GnRH antagonist (Nal-Glu) in estrogen-deficient postmenopausal women with im-

1258 Urban at al.

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munoactive LH, FSH, and prolactin and bioactive LH release as end points of effective blockade of endogenous GnRH. Our findings are consistent with those of a recent similar study.13 However, by testing additional doses (30 and 300 fJ-g/kg) of antagonist in a more detailed time course of bioactive LH, we report several novel and clinially relevant findings. We observed that the immediate suppression of bioactive LH release parallels that of immunoactive LH, but "escape" of bioactive LH secretion from the suppressive effects of antagonist occurs at the lower antagonist dose (30 fJ-g/kg) while the immunoactive LH values are still suppressed. Similar "escape" has been described in gonadally intact men,8 in whom antagonistinduced withdrawal of testosterone negative feedback may be considered a contributing factor. However, since a similar degree of gonadal-steroid dependence on endogenous LH secretion is not expected in the postmenopausal setting,' withdrawal of steroidal negative feedback cannot be so readily invoked in this setting. Although our LH immunoassay measures uncombined n-subunit only sparingly « 15%), the rat interstitial cell testosterone bioassay is devoid of responsivity to free glycoprotein subunits." 15. 16 Thus "escape" of LH bioactivity from suppression also cannot be explained by a secondary rise in n-subunit secretion. At the high dose of GnRH antagonist (300 fJ-g/kg) our findings are consistent with the previous study": bioactive LH is suppressed to a greater degree than immunoactive LH, thus lowering the bioactive/immunoactive ratio. Although the exact biochemical basis for the reduced LH bioimmuno ratio is not known, this finding could indicate changes in the LH molecule,

possibly altered glycosylation, the appearance of an antagonist molecule, or both, as has been proposed for FSH.17 Practically, this disparity between immunoactive and bioactive LH release may be of clinical importance, and suggests the use of higher doses of GnRH antagonist if more sustained suppression of LH bioactivity is desired. Moreover, inadequate dosing of the GnRH antagonist will result in suppression of immunoactive LH values while continued release of bioactive LH can still be producing unwanted clinical effects. Second, inspection of the time series showed apparent continued LH pulsations, although at markedly reduced amplitude. Because these studies were conducted with 20-minute sampling intervals, detailed LH secretion and clearance dynamics cannot be accurately discerned from this low-amplitude signal. 18,19 To further validate these findings of marked suppression of LH bioactivity in vivo, we investigated possible direct effects of the Nal-Glu antagonist in the rat interstitial cell testosterone assay. Because maximal concentrations of the Nal-Glu GnRH antagonist in plasma are initially approximately 300 ng/ml after a 300 fJ-g/kg dose (Fig. 5) and decline the rafter, it can be inferred that circulating levels of the antagonist do not interfere with rat interstitial cell testosterone assay measurements. Thus direct effects of the antagonist in vitro on the rat interstitial cell testosterone bioassay cannot account for in vivo suppression of LH bioactivity. There was relatively minimal (-40%) suppression of immunoactive FSH concentrations at the 300 fJ-g/kg dose. This observation is consistent with previous reports in men. 7 • 1O, 13 However, the apparently lesser de-

GnRH release-Suppression of gonadotropin and prolactin release

Volume 162 Number 5

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gree of suppression of FSH release may not fully reflect the action of bioactive FSH secretion, since Nal-Glu antagonist administration has produced a significant suppression of bioactive FSH release while only minimally suppressing immunoactive FSH levels. I'. 20 Moreover, GnRH antagonist administration has been postulated to stimulate the secretion of FSH antagonists (FHS isoforms devoid of bioactivity), which could be measured by FSH immunoassay but not bioassayY The inability of the Nal-Glu GnRH antagonist to sup-

press prolactin secretion is distinct from but consistent with a previous study that showed no suppression of serum prolactin concentrations in postmenopausal women receiving a GnRH agonist, goserelin. 12 Such observations imply that the presumptive temporal coupling between LH and prolactin secretion that has been reported in some circumstances 21 -21 is not substantially GnRH-dependent, at least in the estrogen-deficient postmenopausal setting. In summary, we have tested the dose-response and

1260 Urban et al.

time-course of action of a potent GnRH antagonist, Nal-Glu, in estrogen-deficient postmenopausal women and found that the antagonist (1) achieves suppression (