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Original Article |
Chair of Obstetrics and Gynecology (S.P., M.M., T.R., F.Z.), University of Catanzaro, 88100 Catanzaro, Italy; Departments of Molecular and Clinical Endocrinology and Oncology (F.O., G.L., A.C.) and Gynecology, Obstetrics and Human Reproduction (M.P., C.N.), University of Naples "Federico II", 80131 Naples, Italy; and Department of Obstetrics and Gynecology (P.M.), University of Messina, 98166 Messina, Italy
Address all correspondence and requests for reprints to: Dr. Stefano Palomba, Via Nicolardi 188, Napoli 80131, Italy. E-mail: stefanopalomba{at}tin.it.
Abstract
This prospective randomized, single-blind, placebo-controlled clinical trial was performed to evaluate the efficacy of raloxifene in preventing the bone loss associated with GnRH agonist (GnRH-a) administration.
One hundred premenopausal women with uterine leiomyomas were treated with leuprolide acetate depot at a dosage of 3.75 mg/d for 28 d and then randomized into two groups to receive raloxifene hydrochloride at 60 mg/d (group A) or placebo (1 tablet/d; group B). Bone mineral density (BMD) and serum bone metabolism markers were evaluated at admission and after six treatment cycles.
Posttreatment BMD differed significantly from baseline BMD in group B but not in group A. BMD was significantly higher in group A than in group B. In group A, serum osteocalcin and bone alkaline phosphatase levels and urinary deoxypyridinoline and pyrilinks-D excretion were unchanged vs. baseline. Differently, posttreatment concentrations of these bone turnover markers were significantly lower in group B compared with baseline and group A values.
In conclusion, raloxifene prevents GnRH-a related bone loss in premenopausal women with uterine leiomyomas.
CONTINUOUS ADMINISTRATION of GnRH agonist (GnRH-a) inhibits the release of gonadotropins inducing a down-regulation of pituitary GnRH receptors and a state of hypogonadotropic hypogonadism (1, 2). The hypoestrogenic state induced by GnRH-a is effective in the treatment of various sex hormone-related diseases (3, 4, 5, 6, 7, 8, 9, 10). It has been shown that the GnRH-a administration is useful in breast and prostate cancer (3, 4) and in such benign diseases as uterine leiomyomatosis, endometriosis, and premenstrual syndrome (5, 6, 7, 8, 9, 10).
The GnRH-a related hypoestrogenism frequently causes climacteric-like symptoms, such as vasomotor symptoms and overall severe bone loss (1, 2). Several drugs have been associated with GnRH-a to reduce these side effects and to allow prolonged treatment (1, 2, 5, 6, 7, 8, 9, 10). However, very few treatment regimens are effective to alleviate the GnRH-a related symptoms in women with leiomyomas without compromising the effectiveness of the analog alone (11, 12, 13, 14). For instance, estroprogestins or estriol may be used only after a window period of treatment with GnRH-a alone (11, 12, 13, 14). At present, only tibolone, a steroid compound structurally related to 19-nortestosterone derivatives, which exhibits a concomitant weak estrogenic, progestinic, and androgenic activity (15), is an effective add-back therapy when administrated contemporarily with analog (5, 6, 7, 8, 9).
Raloxifene is a synthetic nonsteroidal drug derived from the benzothiophene and afferent to selective estrogen receptor modulators, which constitute a group of compounds that interact with estrogen receptors eliciting tissue-specific responses (16). Raloxifene exerts estrogenic effects on the metabolism, the central nervous system, the skeleton, and the cardiovascular system (16, 17, 18, 19, 20), whereas it shows a weak estrogenic antagonist effect on the breast and the uterus (21, 22, 23, 24, 25). These diverse effects could be due to the different distribution of the specific estrogen receptor subtypes that mediate gene transcription if activated by raloxifene (26).
Thus far, there are no data in literature about raloxifene administration for the prevention of the bone loss associated with GnRH-a. In addition, raloxifene reduces the size of the uterus and leiomyomas in postmenopausal women (25), but not in premenopausal women (27). Consequently, raloxifene appears to be a good add-back candidate to associate with GnRH-a.
On the basis of these considerations, the present study was designed to investigate the effect on bone metabolism of adding 60 mg daily of raloxifene hydrochloride in women treated with GnRH-a for symptomatic uterine leiomyomas.
Patients and Methods
The procedures used in this study were in accordance with the guidelines of the Helsinki Declaration on human experimentation. The protocol was approved by the Local Ethic Committees. The purpose of the protocol was explained to each woman attending the Department of Gynecology of the universities of Catanzaro, Messina, and Naples "Federico II" before she entered the study. Written informed consent was obtained from all subjects.
Patients
Between June 2000 and January 2001, 100 premenopausal women affected by symptomatic uterine leiomyomas were enrolled in the study. Exclusion criteria were: neoplastic, metabolic, endocrine, liver, hematological, and infectious diseases; active rheumatoid arthritis; history of acute or recurrent vascular thrombosis; bone mineral density (BMD) values less than 1.0 SD, the mean bone density of the peak value for sex-matched healthy young adults (-1.0 T-score) at posterior-anterior lumbar spine; a body mass index (BMI) less than 18 kg/m2 or more than 30 kg/m2; previous or current treatment with bisphosphonates, sodium fluoride, calcitonin, estroprogestins, or anabolic steroids, corticosteroids, calcium (Ca) or vitamin D, phosphate, thiazidic diuretics, or other drugs interfering with bone metabolism; and abnormal serum levels of creatinine (Cr; >133 µmol/liter), 25-OH-vitamin D (<20 µmol/liter), Ca (normal values, 2.2–2.6 mmol/liter), phosphorus (P; normal values, 1.0–1.4 mmol/liter), and PTH (normal values, 10–65 ng/liter). We also excluded women smoking more than 20 cigarettes per day and drinking more than three alcoholic beverages per day. The presence of a hypoechoic or calcified leiomyoma and endometrial abnormalities detected at transvaginal ultrasonography (TV-USG) was considered another exclusion criterion.
Treatment protocol
At the entry, all subjects were randomized in single blocks into a single-blind, placebo-controlled study design using a computer-generated randomization list. The subjects were assigned to one of two groups of 50 women each. All women received leuprolide acetate depot (Enantone, Takeda, Rome, Italy) at a dosage of 3.75 mg/d for 28 d in association with or raloxifene hydrochloride (Evista, Eli Lilly \|[amp ]\| Co., Sesto Fiorentino, Italy) at a dosage of 60 mg/d p.o. (group A) or placebo tablets (1 tablet/d; group B). The study lasted for 6 cycles of 28 d each, during which single blinding was maintained in both groups. After the 6 cycles, the women of group A continued the treatment for another 12 cycles.
Study protocol
At baseline and after every six cycles of treatment, BMD and bone metabolism were measured, and Ca intake, alcohol consumption, and physical activity were evaluated in both groups (28). Ca intake and alcohol consumption were assessed by a dietary history of patients using a semiquantitative diet questionnaire developed by our dieticians. Ca intake was expressed as a score ranging from 1–3 according to the following scale: 1, less than 500 mg/d; 2, 500-1000 mg/d; and 3, more than 1000 mg/d. Alcohol consumption was also expressed as a score ranging from 1–3 according the following scale: 1, less than 1000 mg/d; 2, 1000–2000 mg/d; and 3, more than 2000 mg/d. A semiquantitative questionnaire was used to evaluate patients’ daily physical activity, job, and daily activities. Physical activity was expressed as a score ranging from 1–3: score 3 was assigned to women who exercised regularly (high physical activity); score 2 was assigned to women who did not exercise regularly but participated daily in activities like cleaning house, climbing stairs, or walking to work, to the bus stop, or to a restaurant (moderate physical activity); score 1 was assigned to women who did not participate in any of above mentioned activities (low physical activity).
No dietary restrictions or changes were implemented during the study. To ensure adequate Ca intake, all patients with a Ca intake less than 1000 mg received daily supplements of elemental Ca in the form of an effervescent tablet composed of calcium carbonate (Cacit, Procter \|[amp ]\| Gamble, Rome, Italy). This supplement was taken at lunch.
At the beginning of the study and after six cycles of treatment, uterine and leiomyoma size, number of tumors, and endometrial thickness were evaluated by TV-USG.
All women agreed to use barrier contraception during the study.
The subjects were instructed to report in a daily diary the characteristics of their menstrual cycle (length and severity of uterine bleedings) as well as the onset of side effects.
Every three cycles, each subject underwent a standard clinical evaluation and laboratory analyses, including hematological, renal function, and liver function tests, and microscopic examination of sediment from midstream urine specimens.
BMD measurement
The BMD was determined by dual energy x-ray absorptiometry (Dexa QDR 1000, Hologic, Inc., Waltham, MA) at posterior-anterior lumbar spine (vertebrae L1 to L4) and at hip (trochanter and femoral neck). The precision of the measurements expressed as coefficient(s) of variation (CV) in vitro for repeated BMD determinations in two standard phantom in our laboratory was 0.41%. The CV in vivo had been evaluated comparing two measurements performed at 7-d intervals in 30 volunteers and was 1.1%, 1.8%, and 1.0% for lumbar spine, trochanter, and femoral neck, respectively. An instrument calibration with a standard phantom was obtained every day at all sites examined. We used the mean of three scans to calculate bone mineral content. The BMD values were calculated by the software of the bone densitometer dividing the bone mineral content (grams per centimeter) for the bone width (centimeter) and expressed directly as an index (grams per square centimeter).
The results of absorptiometry were examined by a single observer blind in respect to different treatment regimens. The primary end-point was lumbar spine BMD. Hip trochanter and femoral neck BMD were considered secondary end-points. Absorptiometric findings were also expressed as percentage change of baseline values.
Biochemical assays
At study entry, all women underwent blood analyses to verify a healthy state. Bone metabolism was evaluated at entry and after six cycles of treatment by determining the serum levels of Ca and P, PTH, osteocalcin (OC), and bone alkaline phosphatase (BAP) levels, as markers of bone formation, and urinary Cr-corrected free deoxypyridinoline (DPD) and pyrilinks-D (PYD), as markers of bone resorption. Blood and 24-h urine samples were collected after an overnight fasting between 0830 and 0930 h to avoid the interference of circadian changes. Patients were asked to refrain from eating foods containing fat or gelatin within 12 h of their clinic visit. Serum samples were separated within 1 h from collection and kept frozen at -80 C, and urine was stored at -20 C until biochemical analysis. All samples from each woman were analyzed in the same assay and were analyzed blind by a central laboratory.
Serum Ca, P, PTH, OC, and BAP, and urinary Cr, DPD, and PYD levels were measured with commercial kits. Serum Ca (reference range, 2.2–2.6 mmol/liter) and P (reference range, 1.0–1.4 mmol/liter) levels were assessed by spectrophotometry. Serum PTH levels (reference range, 10–65 ng/liter) were determined using an intact PTH immunoradiometric assay (Diagnostic Systems Laboratories Inc., Webster, TX) with a sensitivity of 1.0 ng/liter and intra-assay and interassay CV of 7.1% and 3.5%, respectively. Serum OC levels (reference range, 3.1–13.7 µg/liter) were assayed by an immunoradiometric assay (Diagnostic Products Corp., Los Angeles, CA) with a sensitivity of 0.1 µg/liter and intra-assay and interassay CV of 4.5% and 3.5%, respectively. Serum BAP levels (reference range for postmenopausal women, 14.8–43.4 IU/liter) were measured using an immunoenzymatical assay (EIA) (Metra Biosystems, Milan, Italy) with a sensitivity of 0.7 IU/liter and intra-assay and interassay CV of 5.2% and 5.0%, respectively. Urinary DPD concentrations (reference range normalized for Cr levels, 3.0–7.4 nmol/mmol) were assayed by an EIA (Metra Biosystems) with a sensitivity of 1.1 nmol/liter and intra-assay and interassay CV of 7.6% and 5.5%, respectively. Urinary PYD concentrations (reference range normalized for Cr levels, 16.0–37.0 nmol/mmol) were assayed by an EIA (Metra Biosystems) with a sensitivity of 7.5 nmol/liter and intra-assay and interassay CV of 8.5% and 6.8%, respectively. Urinary concentrations of Cr (reference range, 8.8–14.1 mmol/24 h) were measured with the use of an autoanalyzer (Monarch 1000, Instrumentation Laboratory, Milan, Italy). Cr-corrected values were calculated by dividing DPD and PYD by urinary Cr measured using a standard colorimetric assay (DPD/Cr and PYD/Cr). The biochemical data of bone turnover markers were also expressed as percentage change from the baseline values.
Safety evaluation
Standard clinical evaluations and laboratory analyses, including hematological, renal function, and liver function tests, serum Ca and P measurements, and microscopic examinations of sediment from midstream urine specimens were performed before treatment and after each three treatment cycles.
The subjects were instructed to report in a daily diary the appearance of adverse experiences. The adverse experience was defined as any undesirable clinical experience occurring to patients during the study, whether or not related to the drugs administrated. A serious adverse experience was defined as death, overdose, diagnosis of cancer, or any event that was life threatening, permanently disabling, or requiring hospitalization. From the time the patients received the first dose of the drugs, all subjects were seen every 3 months to check the personal diary. All patient data were carefully considered to establish the severity, duration, seriousness, and a possible cause-effect relationship.
Statistical analysis
On the basis of previous data (28), the required sample size was calculated to be 40 subjects per group to detect an effect (2% difference in the mean percentage change from baseline in lumbar spine BMD within- and between-group) on the size of 2 SD with an 
value of 0.05 (two-sided) and a power 1-
of 0.8. After evaluating our expected drop-out rate, we enrolled 50 subjects per group. The power analysis of the present study showed a value of ß = 0.903.
Repeated measures ANOVA, followed by the Newman-Keuls multiple range test, was used to compare multiple measures of age, BMI, BMD, and biochemical data. Wilcoxon’s signed-rank test was used to compare parity, cigarettes smoked, alcohol consumption, Ca intake, and physical activity. The proportion of women receiving Ca supplements in the two groups of treatment was compared using the 
2 test. The Fisher’s exact test was used to compare the incidence of adverse experiences between treatment groups. The statistical analysis was performed using the SPSS 9.0 (SPSS, Inc., Chicago, IL). Data were normally distributed and were expressed as mean ± SD.
Results
Demographic data
Ninety-one of the 100 enrolled patients completed the study. After randomization, the two groups were similar for age, parity, BMI, cigarettes smoked, Ca intake, alcohol consumption, and physical activity (Table 1
). The proportion of women receiving Ca supplements was similar at baseline between the groups (Table 1) and throughout the six cycles of treatment (data not shown).
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BMD measurements
After randomization, lumbar spine, trochanter, and femoral neck BMD were similar in the two groups (Table 1
).
At sixth cycle of treatment, lumbar spine, trochanter, and femoral neck BMD did not differ from baseline values in group A. In group B, these values were significantly (P < 0.05) lower as compared with baseline and group A values (Fig. 1).
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After randomization, the biochemical parameters of bone turnover were similar in the two groups (Table 1).
Serum Ca and P levels were unchanged in the two groups after six cycles of treatment. After treatment, serum PTH levels were unchanged in group A compared with baseline values, but significantly (P < 0.05) decreased in group B compared with baseline and group A (Fig. 2).
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). Side effects and drop-outs
Throughout the study, the two treatment schedules were generally well tolerated. The total incidence of all adverse experiences and of drug-related adverse experiences did not differ between the two groups. Raloxifene was well tolerated, and its safety profile was similar to that of placebo. No serious adverse experience was reported during the study. Similarly, the incidence of clinical effects and/or laboratory abnormalities did not differ between the two treatment groups.
The numbers of withdrawals were similar in the two groups (five women in group A and four in group B). Drop-outs were due to lack of compliance to the treatment in three patients (two women in group A and one in group B) and to missed BMD and TV-USG examinations in six (three in each treatment group). No drop-out was due to drug-related adverse experiences.
Discussion
The major side effect of long-term treatment with GnRH-a is the negative impact on bone metabolism. In fact, continuous administration of GnRH-a for 6 months or more induces a significant bone loss depending on the dose of the analog, the length of the treatment, and the degree of induced hypoestrogenism (2, 29). The wide range of BMD changes observed in the subjects treated with GnRH-a suggests that responses to GnRH-a may vary greatly from subject to subject (30). Moreover, there is some controversy as to how discontinuation of GnRH-a treatment affects bone metabolism. According to some reports (31, 32, 33), the bone loss is slowly recovered after treatment withdrawal, whereas other reports (34, 35, 36) indicate that it is not significantly recovered or is reduced after treatment withdrawal.
Our study confirms that the bone loss induced by GnRH-a occurs at the lumbar spine, trochanter, and proximal femur (31, 37, 38) at a rate of about 1% per month during the first 6 months of treatment (2). This loss of the trabecular component of bone may adversely affect the cancellous microstructures, causing damage that is unlikely to be reversed by cessation of therapy and may increase the risk of osteoporosis (39).
Raloxifene at the standard dosage of 60 mg daily prevents postmenopausal bone loss in women without osteoporosis and is used also to treat established postmenopausal osteoporosis (40, 41). The BMD gain induced by raloxifene, furthermore, is significantly lower than the gains observed with estrogen replacement therapy/hormone replacement therapy and/or alendronate (1, 28, 42). In addition, raloxifene reduces the risk of vertebral fractures in postmenopausal osteoporotic women with or without preexisting fractures by about 40% vs. placebo (42, 43, 44, 45), thereby improving quality of life (46). Interestingly, only 4% of the reduced risk of vertebral fracture obtained with raloxifene is due to BMD gain, and the remaining 96% of the risk reduction remains unexplained (47).
This study demonstrates that administration of raloxifene at standard doses prevents the GnRH-a related bone loss at the axial and appendicular bone sites. In women treated with GnRH-a, the positive effect of raloxifene on bone metabolism was also confirmed by the lack of significant change in biochemical parameters of bone formation and reabsorption. The percentage of change in lumbar BMD observed after six cycles of treatment with GnRH-a plus raloxifene was similar to that obtained with GnRH-a plus tibolone (5). In addition, tibolone administration was useful to prevent bone loss during a GnRH-a long-term treatment of 2 yr (6).
No single add-back regimen is appropriate for all gynecological indications during GnRH-a treatments. The ideal add-back therapy would preserve the efficacy of the analog, while preventing bone loss and hot flushes. Although the add-back therapy is well established in women with endometriosis (48), in patients affected by uterine leiomyomas the addition of progestins or estroprogestins at the start of the GnRH-a administration seemed to reduce the effect of the analog on the uterine and leiomyoma size (2, 11, 12, 13), and only tibolone addition may be used at the start of the analog treatment (5, 6).
A variety of anti-reabsorptive drugs have been used to preserve the bone tissue during GnRH-a treatment. Cyclic intermittent etidronate administration did not affect lumbar BMD in women treated with GnRH-a for 6 months (49). Somekawa et al. (50, 51) have shown that the superimposition of ipriflavone, vitamin K2, 1,25-dihydroxyvitamin D3, or vitamin K2 plus 1,25-dihydroxyvitamin D3 in women treated with GnRH-a did not change the bone turnover markers alleviating slightly the analog-related bone loss, i.e. -3.70%, -3.72%, -4.13%, and -3.59% vs. baseline values, respectively.
It is not know whether the add-back therapy preserves the bone tissue, or whether women treated with GnRH-a plus add-back therapy are at high risk of postmenopausal osteoporosis. A randomized trial (30) conducted to identify the spontaneous reversibility of BMD at the lumbar spine and hip up to 6 yr after long-term GnRH-a treatment, with and without add-back therapy, suggested that the bone loss is not completely recovered, notwithstanding the add-back therapy. Finkelstein and Arnold (52) demonstrated that the daily administration of PTH reduces the bone loss during GnRH-a treatment and exerts a beneficial effect on bone metabolism even when the analog administration is discontinued. We recently observed that postmenopausal women previously treated with long-term GnRH-a plus tibolone administration have a reduction in BMD and in bone turnover markers similar to that observed in surgically postmenopausal women (53). This suggests that besides adversely affecting bone tissue by inducing hypogonadism, GnRH-a also exerts a direct effect on the bone. It is not know whether the addition of raloxifene or tibolone reduces only the deleterious effects of GnRH-a related hypoestrogenism on bone metabolism, whereas it did not protect the bone from direct analog damage. This would suggest that great caution should be given to the use of GnRH-a, even with add-back therapy addition, in gynecological patients (54).
In conclusion, our study shows that raloxifene administration in women treated with GnRH-a prevents the analog-related bone loss during treatment. Long-term studies are needed to determine the mechanism of this protective effect and how discontinuation of treatment affects bone metabolism.
Acknowledgments
We are grateful to Jean Ann Gilder (Scientific Communication) for editing and revising the text.
Footnotes
Abbreviations: BAP, Bone alkaline phosphatase; BMD, bone mineral density; BMI, body mass index; Cr, creatinine; CV, coefficient(s) of variation; DPD, deoxypyridinoline; EIA, immunoenzymatical assay; GnRH-a, GnRH agonist; OC, osteocalcin; P, phosphorus; PYD, pyrilinks-D; TV-USG, transvaginal ultrasonography.
Received May 21, 2002.
Accepted June 27, 2002.
References
and ERb at AP1 sites. Science 277:1508–1510This article has been cited by other articles:
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  S. Palomba, F. Orio Jr, T. Russo, A. Falbo, T. Cascella, P. Doldo, C. Nappi, G. Lombardi, P. Mastrantonio, and F. Zullo Long-term effectiveness and safety of GnRH agonist plus raloxifene administration in women with uterine leiomyomas Hum. Reprod., June 1, 2004; 19(6): 1308 - 1314. [Abstract] [Full Text] [PDF]
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S. Palomba, T. Russo, F. Orio Jr, A. Sammartino, F. M. Sbano, C. Nappi, A. Colao, P. Mastrantonio, G. Lombardi, and F. Zullo Lipid, glucose and homocysteine metabolism in women treated with a GnRH agonist with or without raloxifene Hum. Reprod., February 1, 2004; 19(2): 415 - 421. [Abstract] [Full Text] [PDF]
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S. Palomba, T. Russo, F. Orio Jr, L. Tauchmanova, E. Zupi, P. L. B. Panici, C. Nappi, A. Colao, G. Lombardi, and F. Zullo Effectiveness of combined GnRH analogue plus raloxifene administration in the treatment of uterine leiomyomas: a prospective, randomized, single-blind, placebo-controlled clinical trial Hum. Reprod., December 1, 2002; 17(12): 3213 - 3219. [Abstract] [Full Text] [PDF]
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