Changes in bone turnover during tibolone treatment

Maturitas 47 (2004) 83–90

Changes in bone turnover during tibolone treatment Gregorio Riera-Espinoza∗ , Jenny Ramos, Rosal´ıa Carvajal, Emma Belzares, Guadalupe Stanbury, Rosa Far´ıas, Inés Valderrama, Kelia Alvarez, Gregorio Riera-González UNILIME, Universidad de Carabobo, Hospital Universitario Dr. Angel Larralde, IVSS, Valencia, Venezuela Received 8 April 2002; received in revised form 5 May 2003; accepted 2 June 2003

Abstract Objectives: An open study was carried out to evaluate changes in bone remodeling markers such as N-telopeptide (NTx), tartrate-resistant acid phosphatase (TRAP), total alkaline phosphatase (TAP), and bone alkaline phosphatase (BAP) during a 1-year continuous tibolone treatment in postmenopausal women. Material and methods: Thirty-six postmenopausal women were recruited for receiving tibolone 2.5 mg per day for 1 year. Densitometry and determination of biochemical markers of bone metabolism in serum and urine were performed at 1, 3, 6, and 12 months. Results: Comparing baseline with 12 month’s values, BAP and all resorption markers decreased significantly. NTx began to decrease since the initiation of the treatment (baseline: 74.4 ± 5.3; 1 month: 57.5±4.2; 12 months: 36.6 ± 2.8). BAP increased at the first month (baseline: 37.3 ± 2.1; 1 month: 42.6 ± 3.0) but diminished in the following months (12 months: 23.1 ± 1.5). TAP started to decrease significantly only after 6 months of treatment (baseline: 37.3 ± 2.1; 12 months: 31.4 ± 2.3) and TRAP after 3 months (baseline: 9.8 ± 0.4; 6 months: 9.1 ± 0.5; 12 months: 8.2 ± 0.4). Normal bone mineral density at distal and ultradistal forearm was maintained during the 1-year treatment (baseline: 0.42 ± 0.01; 12 months: 0.42 ± 0.01 and baseline: 0.33 ± 0.01; 12 months: 0.33 ± 0.01, respectively). Conclusion: The use of tibolone 2.5 mg per day diminished progressively and significantly bone resorption and formation markers during 1-year treatment period. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Bone markers; N-telopeptide; Bone alkaline phosphatase; Tibolone; Postmenopausal

1. Introduction It has been well established that during the menopause, the rate of bone loss is increased with the subsequent osteopenia and osteoporosis condition that ∗ Corresponding author. Present address: PAKMAIL 19053, P.O. Box No. 02 5304, Miami, FL 33102-5304, USA. Tel.: +58-416-6412110; fax: +58-241-8687776. E-mail address: [email protected] (G. Riera-Espinoza).

could be present in this period or even after. For years, bone mineral density (BMD) has been used to assess the efficacy of bone loss prevention treatments. BMD has been measured using dual-energy X-ray absorptiometry, dual-photo absorptiometry and quantitative computed tomography. Bone remodeling markers are also useful in evaluating therapies that are used for preventing bone loss [1–16,35]. However, in our review of the available literature, we did not find any published reports where simultaneously N-telopeptide

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(NTx) as a resorption marker and BAP as formation marker during tibolone therapy were assessed. Tibolone (Livial®, Organon, Oss, The Netherlands) is a synthetic steroid with tissue-specific activity: it controls climacteric symptoms and prevents postmenopausal bone loss with less concern for women, as it does not cause a return of withdrawal bleeding. Tibolone has estrogenic, progestagenic, and androgenic properties [17]. Reported effects of tibolone on bone include inhibition of cortical bone loss [2,18,19] and evidences of beneficial effects on lumbar spine and femoral neck [2]. Even in postmenopausal women with vertebral fractures, tibolone increases the mineral density of trabecular bone [3]. Its effects on climacteric symptoms are well known [4]; tibolone does not induce endometrial stimulation [20,21] and genital bleeding is mild [8,22,23]. Bone resorption markers’, including tartrate-resistant acid phosphatase (TRAP), pyrilinkns, C-terminal and/or N-terminal telopeptides, determinations call worldwide attention in the assessments of the degree of bone remodeling in osteoporotic patients, enhancing the choice of treatment schedules, monitoring the therapeutic response, and even predicting the degree of the expected bone mass increase from any treatment [6,24,25], mainly when antiresorptive drugs—such as alendronate, pamidronate or etidronate, as well as calcitonin—are used. Therefore, an evaluation of the effect of tibolone on these markers is of great interest. We also determined alkaline phosphatase bone isoenzyme, currently one of the most widely accepted bone formation markers. The main objective of this study was to evaluate changes in bone remodeling markers (NTx, TRAP, total alkaline phosphatase (TAP), and bone alkaline phosphatase (BAP)) during 1-year continuous tibolone treatment of 2.5 mg per day in postmenopausal women.

Centre and the “Dr. Amaury Rengel” clinic in Valencia, Venezuela, were recruited for an open noncomparative study and treated with tibolone 2.5 mg per day on a continuous basis for 1 year. A written informed consent was obtained from each participant as well as the approval from the Ethics Committee of the University Hospital and the Regional Medical Association. Patients with history of fractures resulting from minor trauma were excluded. Also patients exceeding 30% of their ideal body weight were excluded. Other exclusion criteria for enrolment were: • patients who had been treated during the last year with anabolic steroids, PTH, glucocorticoids, calcitonin, biphosphonates, vitamin D, thyroid hormones, antiepileptic drugs, and hormone replacement therapy; • patients with the following diseases: diabetes mellitus, hyperparathyroidism, abnormalities of skeletal development, hyperthyroidism, renal or hepatic failure, and osteomalacia; • patients smoking more than 20 cigarettes per day; • patients with an ethanol ingestion >2 drinks per day; and • patient asking to start hormone replacement treatment (HRT) with estrogen/progestogens. 2.2. Vital signs, weight, and height parameters Weight, height, blood pressure (systolic and diastolic), and heart rate were measured at baseline, 1, 3, 6, and 12 months. Blood pressure was taken in sit position. For each of the vital sign parameters, calculations were performed for the absolute change from baseline. 2.3. Bone markers

2. Material and methods 2.1. Study population Thirty-six women, at least 1 year after menopause, attending to the osteoporotic clinic at UNILIME-UC, “Angel Larralde” University Hospital, Universidad de Carabobo, the “Guerra Mendez” Medical

Second morning urine sample was collected without any prior diet restriction at baseline, 1, 3, 6, and 12 months. At the same time, a venous blood sample was taken while fasting. All samples were store frozen at −70 ◦ C until their measurements were done. TRAP was processed within 24 h of sampling. Laboratory and bone marker assessments were performed at baseline, 1, 3, 6, and 12 months.

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The following laboratory parameters were measured: • Ca (serum and urine) SI (mg/dl): atomic absorptiometry (Perkin–Elmer 3100). • Phosphorus (serum and urine) SI (mg/dl): Eagle diagnostics based on phosphomolibdene reduction (UV) (Daly and Ertinghausen 1972). • Creatinine (serum and urine) SI (mg/dl): Modified Jaffe (Labtest). • Urinary calcium/creatinine ratio. For biochemical estimation of bone metabolism, the following bone markers were used: • TAP (IU/l): Labtest, Roy Modified (VC intra-assay 2.0%, inter-assay 2.4%). • BAP (IU/l): enzyme immunoassay, Alkphase B (Metra Biosystems, USA) (VC intra-assay 4.9%, inter-assay 5.8%). • TRAP (IU/l): hydrolysis of paranitrophenyl phosphate at pH 4.8 [33,34] (VC intra-assay 4.2%). • NTx (mmol BCE/mmol creatinine): enzyme immunoassay (Osteomark, Ostex, USA) (VC intra-assay 7.6%, inter-assay 4.0%). • Bone content equivalent (BCE). 2.4. Densitometry Bone mineral density from distal and ultradistal non-dominant forearm at baseline, 6, and 12 months was measured using the Osteometer DTX-200 (variation coefficient: 1.5%).

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Table 1 Clinical characteristics and laboratory values of postmenopausal women treated with tibolone 2.5 mg per day Baseline (mean ± S.D.) Weight 64.3 ± Height 158.5 ± Blood pressure 122.8 ± (systolic, mmHg) Blood pressure 76.1 ± (diastolic, mmHg) Heart rate 78.4 ± Calcium (serum, mmol/l) 2.35 ± Phosphorous (serum, mmol/l) 1.29 ± Creatinine (serum, mmol/l) 61.9 ± Ca/Cr ratio 0.2 ± Calcium (urine, mmol/l) 0.33 ± Phosphorous (urine, mmol/l) 2137.6 ± Creatinine (urine, mmol/l) 823.0 ±

12 months (mean ± S.D.)

6.8 6.1 13.9

63.6 ± 6.9 158.4 ± 6.2 121.5 ± 13.2

9.9

75.0 ± 9.0

4.6 76.4 0.125 2.35 0.2 1.13 17.6 61.9 0.1 0.1 0.18 0.27 3003 1359 469.4 1176.6

± ± ± ± ± ± ± ±

4.6 0.1 0.35 8.8 0.1 0.15 839.54 612.6

No statistical significance between baseline and 12 months in any parameter.

3. Results Mean age was 52.56 ± 8.35 and years since menopause was 6.06 ± 4.82. Table 1 shows clinical characteristics and laboratory values at baseline and 12 months. Blood pressure and heart rate measurements at baseline and throughout the study were normal. Serum or urine calcium, phosphate, and creatinine were maintained within normal limits. Although weight comparison between baseline (64.3 ± 6.8 kg) and 12 months (63.6 ± 6.9 kg) was

2.5. Statistical analysis The baseline and 1, 3, 6, and 12 months clinical characteristics, laboratory values, bone markers, and percentage changes were compared using a paired Student’s t-test. Data for clinical characteristics and laboratory values are given as mean ± S.D. Data for bone markers, percentage changes of bone markers, and BMD are given as mean ± S.E.M. Parameters with a (two-sided) P-value ≤ 0.05 are discussed. All statistical analyses were performed on complete data between periods, meaning that dropout subjects data were included in the analysis at all dates.

Fig. 1. Percentage change in bone markers in postmenopausal women during 1-year tibolone 2.5 mg per day treatment. Alkphase B (BAP), TAP, TRAP, and NTx.

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Fig. 2. Individual data for NTx in postmenopausal women during 1-year tibolone 2.5 mg per day treatment.

not statistically significant (Table 1), it must be said that at the first month there was a statistical significant increase that disappeared in the following months. After 12 months of treatment, BAP, NTx, TRAP, and TAP decreased significantly (Fig. 1). Forearm BMD did not change during the study. These patients had, as a group, normal BMD at study entry. Fig. 1 shows the percentage changes of each marker during the study. NTx was significantly lower since the

initiation of the treatment. BAP did so after 3 months of tibolone use. TRAP diminished after 3 months. TAP did not change significantly during the first 6 months but after this period, the values decreased and reached statistical significance. Specifically, NTx diminished progressively: −15% at month 1, −32% at month 3, −35% at month 6, and −51% at month 12. BAP increased 29% in comparison to baseline after 1 month of treatment, but decreased

Fig. 3. Individual data for BAP in postmenopausal women during 1-year tibolone 2.5 mg per day treatment.

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Fig. 4. Bone mineral density by DEXA at forearm (distal and ultradistal) in postmenopausal women during 1-year tibolone 2.5 mg per day treatment.

significantly thereafter: −25, −23, and −38% at 3, 6, and 12 months, respectively. Mean NTx value was 74.4 nmol BCE/mmol creatinine at baseline and decreased to 57.7, 47.6, 41.8, and 36.6 at 1, 3, 6, and 12 months, respectively (Fig. 1). Individual data about NTx and BAP changes throughout the study are presented in Figs. 2 and 3. Bone mineral density at distal and ultradistal forearm measured by DEXA was maintained within normal limits during the 1-year treatment period (Fig. 4). Of the 36 women involved in the study, 27 (75%) finished and nine (25%) discontinued the therapy, three because of unacceptable adverse experiences. Adverse experiences were undesirable weight gain in one patient, although weight changes were not significant at the end of the treatment, mammary tenderness in other, and a finding of a breast nodule in the third one. The other dropout reasons were unwillingness to co-operate or insufficient compliance with the treatment. Seven women (19.4%) reported weight gain as an adverse experience, four (11.1%) suffered from genital mycosis, and three (8.3%) had vaginal bleeding. Other adverse experiences seen in two patients were pruritus and breast tenderness (5.5%).

4. Discussion Tibolone is a steroid hormone with tissue-specific activity which alleviates climacteric symptoms and prevents osteoporosis. In the present study, in early postmenopausal women, our main objective was to

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show changes in bone turnover after 1-year treatment. Tibolone showed effective antiresorptive action by decreasing urinary excretion of NTx since the first month of treatment and serum BAP from the third month onwards. As far as we know, there are no publications evaluating simultaneously NTx and BAP during tibolone treatment. Women loose bone mass after estrogen deprivation, secondary to an increase in bone remodeling due to osteoclastic activity [7,26–28]. Elevated bone markers have been well correlated with rapid deterioration of bone mass [25] and with an increase in fracture rate [11]. The use of other antiresorptive therapies, estrogens [7,29], raloxifene [30], or biphosphonates [8], inhibits bone remodeling and improves bone density proportionally to its antiresorptive efficacy measured by bone markers. Adequate evaluation of bone markers is important to estimate and control the effect of treatment of any antiresorptive drug. Both resorption and formation markers will diminish after antiresorptive treatment because the remodeling cycle is inhibited as a whole: resorption and formation phases. We assessed specific markers for bone resorption and formation: NTx, TRAP, and BAP (Alkphase B). In a recent animal study performed by Ederveen and Kloosterboer [9], an efficacy of tibolone in suppression bone turnover after ovariectomy as same as estrogens was observed. Previous studies with tibolone have shown subtle decrease of hydroxyproline (OHP) [1,2,10,11]. In most of these studies, there was considerable overlap of treated and non-treated patients, and the percentage changes of OHP during treatment was less than 30%, which is the minimum acceptable level of change for an urinary marker [12]. Also, the same studies reported a slight decrease in TAP with tibolone. Other markers of bone turnover also diminish after tibolone treatment. Bjarmason et al. [13] reported a decrease of 46% in C-telopeptide (CTx) and 8.9% in osteocalcin using tibolone 2.5 mg per day in late postmenopausal women. TRAP diminished significantly 16.7% after a year of treatment but there was no change at 1, 3, or 6 months of therapy. This may be explained by its variability (short-term coefficient of variation: 12.7% [36]) and because this study does not include a high volume of subjects. TAP diminished only after 1-year treatment and there was a very significant reduction

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(−37.8%) in BAP after the third month, which is the most important bone marker of the alkaline phosphatase family. Interestingly, this marker increased after 1 month of treatment. Even though the explanation for this is not clear, it would be probably due to the primary antiresorptive effect of the drug and to some anabolic action of the
4 isomer metabolite or by the delay in synchronization between bone resorption and formation as it occurs with other HRT regimens [59]. In this last study, the response was delayed specially for BAP when oral route of administration of estrogens was used. Similar observation has been made for osteocalcin and another formation marker: PICP (procollagen type I carboxyterminal propeptide) [60] or BAP and PINP (procollagen type I aminoterminal propeptide) [61]. In this study, NTx, a very specific marker of bone resorption, decreased significantly since the first month of tibolone treatment reaching −51.5% after 12 months. NTxs have been used in the control and prediction of bone response to treatment with biphosphonates [8] or conventional HRT [14]. Other resorption markers like pyridinium cross-links also decrease with tibolone [15]. During the treatment period not only absolute values of NTx and BAP decreased significantly, but also the variability of both markers diminished and the standard error of the means decreased from 5.3 to 2.8 for NTx and from 2.7 to 1.5 for BAP (S.E.M. at baseline and 12 months). This may be because tibolone could progressively favor the coupling mechanisms of the bone remodeling cycle. This fact is probably implicated in the so-called “bone quality effect” of different antiresorptive drugs by which resorption cavities are rebuilt by new bone and the net effect of bone strength is achieved. There are no many comparison studies between tibolone, estrogens, and raloxifene regarding bone actions, however tibolone has shown a more profound effect in suppressing bone turnover markers than estrogens [37] and bone remodeling suppression is greater with estrogens than with raloxifene [38]. Even though there are no head to head studies with these three drugs, comparison of three independent studies with similar designs and methodology [39–41] have shown after 2 years of treatment that markers of bone turnover were decreased less with raloxifene than with tibolone or estrogens.

Bone markers have shown seasonal variations that account for up to 20–30% lower values in summer than in winter [47–51]. These variations include both marker’s formation and resorption. However, in some studies these changes have not been found [52–54]. The difference between these studies may be due to some of them being longitudinal and the others being cross-sectional. Observed high turnover in winter time may be due to vitamin D deficiency and may be less marked at low latitudes [55]. In Venezuela, we do not have significant seasonal changes (dry and rainy periods) and our latitude is very low (8◦ N). Because of this, we believe that seasonal changes in bone markers do not play a role in our results. Osteoporosis is a systemic disease and bone loss usually occurs at all sites, and low bone density at a variety of measurement sites is associated with the risk of osteoporotic fractures [42–44]. The predictive values of available BMD measurement sites are similar when considering overall fracture risk [45,46]. Also, there has been found a consistent association between bone loss rate at the forearm and bone markers [56,57]. In a large population-based prospective cohort [58], 51 untreated women who had the highest rate of bone loss, the predictive value of osteocalcin and serum CTx were very significant. Moreover, for women with bone markers at baseline 2 S.D. above the premenopausal range, the rate of forearm bone loss was two- to sixfold higher than women with low turnover. These data show that forearm BMD and its correlation with bone marker measurements are adequate to evaluate the effect of any antiresorptive therapy. Our patients had normal BMD at the forearm and this condition was preserved during the study. Other reports have shown increase in BMD in subject taking tibolone with previous low BMD in early [2,11,16] and late postmenopausal women [3,13] or inclusive subjects with fracture history [10]. Also, positive effects on bone density have been reported similar to estrogens [31,32]. In our study, the objective was not bone mass but bone turnover, however bone mass peak was preserved by the use of tibolone. In summary, tibolone probably diminishes bone remodeling as shown by decreasing bone resorption and formation markers as NTx and BAP during 1-year use of 2.5 mg per day in postmenopausal women. Both markers diminished significantly and progressively during 1-year treatment period.

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Acknowledgements To TSU Elio Padilla, Computer Science, and Dr. Jesus Urbina, Statistics, for their technical assistance in the preparation of this manuscript. To Organon Venezolana for all its support during the performance of this study.

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