Induction of hypothalamic opioid activity with transdermal estradiol* administration in postmenopausal woment*

Transcription

1 FERTILITY AND STERILITY Copyright e 1991 The American Fertility Society Printed on acid-free paper in U.S.A. Induction of hypothalamic opioid activity with transdermal estradiol* administration in postmenopausal woment* Joseph F. D'Amico, M.D. II Gail A. Greendale, M.D.~ John K. H. Lu, Ph.D. Howard L. Judd, M.D. # Cedars-Sinai Medical Center and the University of California-Los Angeles, (UCLA) School of Medicine, Los Angeles, California The effects of estradiol (E 2) administered by a transdermal system on the induction of hypothalamic opioid activity was examined in 18 postmenopausal women. Women were given 25, 50, 100, or 200!Lg/d dosages of E 2 for 28 days each. Naloxone at a dosage of 2 mg/h was infused intravenously for 4 hours, and serum was obtained every 15 minutes for 6 hours. With increasing doses, rises of serum E 2 and estrone were elicited across the range seen in premenopausal women. A stepwise reduction of luteinizing hormone (LH) levels was observed with increasing dosages of E 2 The naloxone infusions resulted in significant increases of LH when E 2 was being given but not during the pretreatment studies. These data indicate E 2, in physiological concentrations, induces hypothalamic opioid activity. Fertil Steril 55:754, 1991 In primates the secretion of luteinizing hormone (LH) is pulsatile in character 1 and is consequent to the episodic secretion of gonadotropin-releasing hormone (GnRH) from the medial basal hypothalamus,2 the region from where the pulsatile signal originates. 3 Proopiomelanocortin -derived opioid peptides are found in high concentrations in this Received August 6, 1990; revised and accepted November 28, * Estraderm, Ciba Geigy, Summit, New Jersey. t Supported by grants from Ciba-Geigy Corporation, Summit, New Jersey, and by grant RR 865 from the United States Public Health Service, Bethesda, Maryland. :j: Presented at the 37th Annual Meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21 to 24, Department of Obstetrics and Gynecology. 1\ Present address: Fertility and Reproductive Health Institute of North California, San Jose, California. 1f Department of General Internal Medicine. # Reprint requests: Howard L. Judd, M.D., Department of Obstetrics and Gynecology, UCLA School of Medicine, LeConte Avenue, Los Angeles, California D'Amico et al. E 2 and opioids region. The suppression of LH release by endogenous opioid peptides has been well documented in humans. 4 That the hypothalamic opioidergic system exerts a tonic inhibition on gonadotropin secretion is supported by the ability of naloxone, an opioid receptor antagonist, to elicit rises of LH levels and to modulate pulsatile LH release in normally menstruating women. 5 Regulation of gonadotropin secretion by endogenous opioids is not apparent in hypoestrogenic, postmenopausal women. 6 However, administration of conjugated equine estrogens at a dosage of 1.25 mg to postmenopausal women increased opioidergic regulation of gonadotropin secretion, whereas the addition of a progestin further enhanced this activity.7 To date, the effects on hypothalamic opioidergic activity of exogenous estradiol (E2) at doses that raise the concentration of the hormone in the circulation to values seen in premenopausal women has not been reported. The present dose-response study examined the effects of E2 given by a transdermal system (Estraderm; Ciba-Geigy Corp, Summit, NJ) on circu-

2 lating LH levels. To determine indirectly hypothalamic opioidergic activity, a standard naloxone infusion experiment was performed. MATERIALS AND METHODS Eighteen women were studied who were postmenopausal for at least 1 year and had received no hormonal medication for at least 60 days before the study. They were randomly divided into two groups. No statistically significant difference was identified between the two groups for age, years since menopause, height, and weight. In group 1 (n = 8), 25 and 100 JLg/d dosages of E 2 were administered daily via a transdermal system. Each system was worn on the lower abdominal or buttock area and changed every 3! days. Each dosage was administered for 28 days with a 7 -day washout period between doses. In group 2 (n = 10), 50 and 200 JLg/d dosages were given in a similar manner for 28 days each. The opioid antagonist, naloxone, was utilized as a probe to test for hypothalamic opioid activity. Naloxone was infused in normal saline at a rate of 2 mg/h for 4 hours. Infusions were performed before estrogen administration (pretreatment study) and on the 27th day of each dosage of the E 2 systems. Thus, all patients were studied on the 2nd day of application of the final E 2 system for each dosage. Blood samples were obtained every 15 minutes for 6 hours beginning 1 hour before the naloxone infusion. All serum samples were separated immediately and stored at -20 C until analyzed for LH, E 2, and estrone (E 1). Luteinizing hormone levels were measured every 15 minutes by radioimmunoassay (RIA) as previously described. 8 Estradiol find E 1 levels were assessed hourly by RIA after ether extraction of the serum sample and partial purification ofthe extract over a microcelite column. 9 The intra-assay and interassay variations of all assays did not exceed 9% and 11%, respectively. All measurements of a given hormone were assessed in the same assay for each subject. For comparison, blood samples were also obtained from 15 premenopausal women early and late in the follicular phase of their menstrual cycles. Estradiol and E 1 levels were measured on these samples. Data Analysis Analysis of variance was used to test for overall differences among mean serum E 2 and E 1 levels by dose of transdermal E 2 given. Pair-wise differences between E 2 by all doses and E 1 levels by all doses were tested using Tukey's test for unequal sample sizes. Differences among mean E 2 levels before, during, and after the naloxone infusion were tested by repeated measures ANOV A. To reduce in part the impact of large pulsatile excursions of LH seen in postmenopausal women, four time zones were constructed for the analysis of the LH response during the naloxone infusions. Luteinizing hormone measurements during each time zone were averaged to provide one LH value for each subject for each time period. The zone I LH value is the average of four measurements obtained at 15- minute intervals before naloxone infusion. Zone II LH is the average of eight LH measures obtained every 15 minutes for the 1st 2 hours of naloxone administration. Zone III LH is the same amount of data for the 2nd 2 hours of the infusion. Zone IV is the average of the four LH measurements during the 1-hour postinfusion period. Data are reported as a percent change of the LH level from the values measured before the naloxone infusion during the pretreatment study (before estrogen administration). Repeated measures ANOV A was used to test differences in mean percent change in LH by the time of naloxone infusion and by dose of estrogen administered. RESULTS Figure 1 shows the mean ± SE levels of E 2 and E 1 measured in the 18 postmenopausal women before estrogen treatment and with each dosage of the E 2 systems. On the right are shown the levels of these two estrogens in the premenopausal women early and late in the follicular phase of their menstrual cycles. Before E 2 therapy, estrogen levels were in the range reported for other postmenopausal women. 10 Clear dose-response effects were seen with the circulating levels of these two estrogens rising across the range seen in young women as the dosages were increased. Overall dosage effects were significant at P = At each dosage, the increase of E 2 was greater than E 1 Figure 2 shows the mean± SE levels of E 2 measured hourly before, during, and after the naloxone infusion. The before serum sample was obtained just before commencement of the naloxone infusion, and the postspecimen was drawn 1 hour after cessation of the infusion. Again the mean values observed in the young women when E 2 levels were at the lowest and highest concentrations seen during their menstrual cycles are shown for comparison. During the pretreatment study (before estrogen administration) and with each dose of the E 2 system, the E 2 levels D'Amico et al. E 2 and opioids 755

3 pmoi/l I pmoi/l 400 Estradiol d zoo rt21 F!9 Estrone 0 ll9ljl IT7J r i 1 r.il Bose Early Late Line Estradiol Dose Follicular (,ug /24 hr) Figure 1 The mean ± SE levels of E 2 and E 1 observed in the postmenopausal women before the naloxone infusion during the pretreatment study (before estrogen administration) and on 25, 50, 100, and 200 Jtg/d of transdermal E 2. Serum samples were obtained on day 27 of 28-day treatment of each E 2 dosage. For reference are shown the levels of these two estrogens in premenopausal women during the early and late follicular phases of their menstrual cycles. showed minimal variation before, during, and after the naloxone infusions. The effects of naloxone infusion on LH levels at the four doses of estrogen tested are shown in Figure 3. Again, data are reported as a percent change of the LH level from the values measured before the naloxone infusion during the pretreatment study (before estrogen administration). For zone I (before naloxone infusion), a clear dose-response suppression of LH was observed with increasing dosages of E 2 (P < 0.05). Naloxone infusions were associated with a statistically significant increase of LH over time (P = ). Luteinizing hormone increased significantly between zones I and III and this persisted in zone IV (P < 0.05). Five hours after initiation of the naloxone infusions, LH values approximated those observed in the women before estrogen was administered, with the exception of the 200-~g/d dose study. A similar increase with the naloxone infusion was not seen before E 2 was administered (pretreatment study). 756 D'Amico et al. E 2 and opioids DISCUSSION This study provides the first dose-response evaluation of the change in circulating estrogen levels elicited by the commercially available transdermal E 2 systems. Previously, Powers et aly reported reasonably stable E 2 levels during the 72-hour wearing of prototype systems, after a transient rise during the 1st day of application. Thus, to minimize withinand between-patient variations ofe2 levels, we only sampled the patients on the 2nd day of application of the last system or day 27 of a 28-day treatment period for each E 2 dosage. Powers et al. 11 also reported mean levels of E 2 of 92, 147, and 275 pmol/l on the 25, 50, and 100-~g/d systems, respectively. These values were similar to those reported by us in women also administered prototype systems. 10 In the present study, the mean levels of E 2 were 45% to 138% higher than were reported earlier with the prototype systems. The administration of E 2 to postmenopausal women provided a unique opportunity to assess the feedback effects of this estrogen on gonadotropin release in the absence of other ovarian factors known to exert central feedback actions, i.e., progesterone (P) and inhibin. In particular, attention was directed to the possible influence E 2 had on the induction of opioidergic activity. The majority of evidence indicates that opiates affect anterior pituitary function indirectly at the level of the hypothalamusp- 14 Studies of cultured pituitaries have failed to demonstrate effects of naloxone and opiate peptides on LH secretion. However, in vitro perfusion of the human fetal medioba ~ pmol/l 2l Baseline..., /D o-o 200pg/ D ct-<j 251Jg/D._...IOOpg/D NALOXONE INFUSION Pre Hours of Infusion Past Early Late Follicular Figure 2 The mean ± SE levels of E 2 before, during, and after the naloxone infusions in the postmenopausal women during the pretreatment study and on each dosage oftransdermal E 2 Naloxone infusions were performed on day 27 of 28-day treatment period for each E 2 dosage. The levels in premenopausal women are shown for reference.

4 sal hypothalamus elicited an acute increase in GnRH release after a pulse injection of naloxone with prompt inhibition by a pulse of beta-endorphin administered halfway through a naloxone infusion. 15 In pituitary stalk-sectioned monkeys, in vivo studies have shown that beta-endorphin had no effect on gonadotropin secretion and did not interfere with the stimulation of gonadotropin release by exogenously administered GnRH. 16 From these observations, it appears that opiates act on the hypothalamus to affect pituitary function by changing the synthesis or release of the hypothalamic-releasing hormones, thus altering the concentration of these hormones in portal bloodp During the present pretreatment studies, the naloxone infusions failed to elicit an increase in circulating LH levels. These findings were similar to observations made previously by us and others in postmenopausal women This lack of effect of opiate receptor blockade suggests a very low or absent input of hypothalamic opioid peptides on gonadotropin secretion in women lacking ovarian function and is consistent with the undetectable levels of beta-endorphin measured in hypophyseal portal blood in ovariectomized monkeys. 19 As expected, the administration of transdermal E 2 elicited a dose-dependent lowering of LH levels. This finding was similar to those we have reported earlier with prototype systems. 10 The reduction in circulating LH produced by changing concentrations of E 2 and E 1 closely paralleled the ability of naloxone to stimulate LH release. With the exception of the 200-~tg/d dosage of E 2, LH levels returned to concentrations similar to those seen in the pretreatment study (before estrogen administration) when naloxone was infused. These observations constitute evidence that in postmenopausal women the negative feedback effect of E 2 on LH release operates in a dose-dependent manner and that the inhibition of LH by this estrogen is, in large part, mediated through the induction of opioidergic activity. That ovarian steroids enhance endogenous opioid activity on GnRH release has been suggested by several lines of evidence. Increases in the frequency and amplitude of LH pulses during the luteal phase 5 and rises of LH levels in the late follicular and luteal phases of the menstrual cycles of young women given naloxone infusions 7 have been reported. Increases of hypophyseal portal vein levels of beta-endorphin in some ovariectomized monkeys given E 2 and E 2 plus P have also been observed. 2 Finally, increases I n o---o 25 JJ9/ JJQ/0-100~/0 C>----<) 200!J9/ 0 Figure 3 The mean ± SE percent change of LH levels from values observed before the naloxone infusion in the pretreatment study (before estrogen administration) in the study subjects. The data are divided into zone I (prenaloxone), zone II (1st 2 hours of naloxone), zone III (last 2 hours of naloxone) and zone IV (postnaloxone). Again, naloxone infusions were performed on day 27 of 28-day treatment period for each E 2 dosage. of LH with naloxone infusions in postmenopausal women given 1.25 mg of conjugated equine estrogens for 3 weeks have been found. 7 The present findings provide the first indication of induction of hypothalamic opioidergic activity with exogenous E 2 administered at doses that raised circulating E 2 to levels seen during the follicular phase of the menstrual cycle in younger women. This supports the potential physiological role of this induction in the feedback effects of ovarian estrogens on gonadotropin release. The induction of hypothalamic opioidergic activity by E 2 in postmenopausal women may be relevant to the ability of this hormone to relieve menopausal symptoms such as hot flushes. Currently, it is believed that menopausal hot flushes result from. a disturbance of thermoregulatory centers within the hypothalamus. Numerous studies have implicated opioids in the regulation of body temperature, with opioid administration raising and its withdrawal lowering central temperature. 21 Narcotic addicts frequently report the sensation of hot and cold flushes during withdrawal. 22 Three consistently observed events of the menopausal hot flush, i.e., skin temperature rise, LH surge, and acceleration of heart rate are similar in magnitude and temporal rela- m Ill D'Amico et al. E 2 and opioids 757

5 tionship with these alterations induced by morphine withdrawal in the narcotic-addicted rat Hot flushes also appear to be a type of withdrawal response. In patients with gonadal dysgenesis and low estrogen levels, hot flushes are not observed. Estrogen treatment and its subsequent withdrawal will initiate these disturbances. Finally, the thermoregulatory disturbances experienced with opioid withdrawal in addicts and hot flushes in postmenopausal women are each ameliorated by clonidine Based on the above observations, the following hypothesis is proposed. Loss of ovarian function with its accompanying reduction of hypothalamic opioid activity results in thermoregulatory instability and the triggering of hot flushes. Estradiol administration, through enhancement of hypothalamic opioid activity, lowers gonadotropin levels and relieves hot flushes. Experiments are underway in this laboratory to examine this hypothesis. REFERENCES 1. 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