Marked reduction of bone turnover by alendronate attenuates the acute response of bone resorption marker to endogenous parathyroid hormone

Bone 44 (2009) 634–638

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Bone j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b o n e

Marked reduction of bone turnover by alendronate attenuates the acute response of bone resorption marker to endogenous parathyroid hormone Vit Zikan a, Jan J. Stepan b,⁎ a b

Department of Internal Medicine 3, Charles University, Faculty of Medicine, Prague, Czech Republic Institute of Rheumatology, Charles University, Faculty of Medicine, Na Slupi 4, CZ 12850, Prague, Czech Republic

a r t i c l e

i n f o

Article history: Received 30 June 2008 Revised 3 December 2008 Accepted 4 December 2008 Available online 24 December 2008 Edited by: T. Jack Martin Keywords: Aminobisphosphonates Bone resorption Hypocalcemia Parathyroid hormone Raloxifene

a b s t r a c t The aim of this study was to assess the effects of the antiresorptive treatments of alendronate (ALN), risedronate (RIS) and raloxifene (RLX) on the response of bone to endogenous parathyroid hormone (PTH) induced by acute hypocalcemia. Forty women (age, 55–80 years) with postmenopausal osteoporosis (treated with ALN, RIS and RLX or untreated-control group) were given infusions of sodium ethylenediaminetetraacetic acid (EDTA; 10 mg/kg of body weight). Serum ionized calcium (iCa), plasma intact PTH and marker of bone resorption, serum β C-terminal telopeptide of type I collagen (β-CTX; β CrossLaps) were followed for 180 min. In all women, decrease in serum iCa following the EDTA load resulted in an acute increase in serum PTH. Between 60 and 180 min, plasma PTH in the ALN and RIS treated women remained significantly higher than in the control group. The integrated β-CTX responses (area under curves, AUCs) to peaks of PTH were significantly lower in the ALN treated women than in those treated with RIS, RLX or control group. There was no significant difference in β-CTX AUC response to PTH between RIS, RLX and control women. Taken together, these findings suggest that in women with postmenopausal osteoporosis treated with ALN, a substantial reduction of bone turnover blunts the acute bone resorbing effect of endogenous PTH. © 2008 Elsevier Inc. All rights reserved.

Introduction Antiresorptive therapies, which include bisphosphonates, raloxifene, calcitonin and estrogen, decrease the rate of bone turnover by initially reducing the level of bone resorption and subsequently by reducing the level of bone formation [1].These treatments reduce the risk of fractures by preventing osteoclasts from further degrading the bone structure, particularly in cancellous bone. In contrast, intermittent administration of teriparatide (human recombinant parathyroid hormone, PTH (1–34)) or PTH (1–84) stimulates bone turnover. Osteoclasts are involved in the initial phase of PTH anabolic action on the bone [2–5]. Accordingly, pre-treatment or concomitant treatment with alendronate (ALN) may attenuate the initial bone mineral density (BMD) and biochemical marker's response to treatment with teriparatide or PTH (1–84) [6–10]. Moreover, the effects of ALN may differ from those of risedronate (RIS) or raloxifene (RLX) [8,11,12], suggesting that the degree of inhibition of bone turnover might influence the bone's responsiveness to anabolic stimuli. However, twenty-four months of teriparatide therapy increases matrix and bone formation, regardless of whether or not the patients had received prior ALN antiresorptive

⁎ Corresponding author. Fax: +42 0 224914451 E-mail address: [email protected] (J.J. Stepan). 8756-3282/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.12.013

therapy [13]. These findings suggest that the early treatment response to teriparatide is attenuated by previous use of potent nitrogen-containing bisphosphonates but that these effects are overcome with longer treatment. Experimentally, intermittent PTH administration stimulated a transient increase in RANKL [2,14] which may result in a transient increase in osteoclastic resorption [15]. However in humans, to the best of our knowledge, the acute effect (minute to hours) of PTH on osteoclastic resorption has not been systematically studied. The degradation products of resorbed bone such as hydroxyproline, deoxypyridinoline or telopeptides of mature collagen type I are released into the circulation during osteoclastic bone resorption. The acute responses of biochemical markers of bone resorption to a single dose of PTH were not observed by using urinary hydroxyproline and deoxypyridinoline [16,17]. However, type I collagen crosslinked C telopeptide (β-CTX) is significantly increased in the circulation of healthy young men in response to acute stimulation of endogenous PTH as well as after a single teriparatide injection [4]. However, it is not known whether the long-term treatment of antiresorptive agents, such as aminobisphosphonates or RLX, affects the acute bone resorbing effect of PTH. The aim of this study was to test our hypothesis that the response of the bone resorption marker β-CTX to acute hypocalcaemia is influenced by long-term antiresorptive treatment with RIS, ALN, or RLX in women with postmenopausal osteoporosis.

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Materials and methods

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to avoid the venous irritation and followed by 20 ml 0.9% NaCl alone over 1 min after the end of EDTA infusion.

Subjects Biochemical analysis Patients for the study were recruited from the Center for Metabolic Bone Diseases of the University Hospital, Prague from May to October 2005. Ambulatory postmenopausal women 50 to 80 years of age, who were at least 5 years postmenopausal, were eligible to enter the study if they had a BMD T-score below −2.5 at the lumbar spine and/or the femoral neck, and had been on an anticatabolic treatment for at least 24 months or untreated. Women were excluded from the study if they had diseases other than osteoporosis which affected bone metabolism, if they were vitamin D insufficient (serum 25-hydroxyvitamin D concentration b50 nmol/l), or had abnormal values for serum or urinary calcium. Also excluded were patients who had received therapy with other drugs affecting bone metabolism such as corticosteroids, cyclosporine, fluoride, or thiazide diuretics in the past. Out of 90 patients who received the invitation, 44 women were eligible for the study. Thirty women with postmenopausal osteoporosis who were treated with ALN (n = 10), RIS (n = 10) or RLX (n = 10) and 10 untreated osteoporotic women (control group) were included. The 4 patients excluded from the EDTA infusions had either peripheral veins unsuitable for intravenous cannulation (n = 3) or serious hypersensitiveness and drug allergy in medical history (n = 1). All patients had a total calcium intake of at least 1000 mg/day, either through diet or diet plus calcium supplements and were on vitamin D supplementation (600–800 IU daily). The study was undertaken with the understanding and written consent of each subject, with the approval of the Ethics committee of the Faculty of Medicine, Charles University, Prague, and in compliance with national legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (Declaration of Helsinki). Study design Patients were treated with weekly ALN (70 mg per tablet), weekly RIS (35 mg per tablet) or daily RLX (60 mg per tablet). Patients with weekly ALN or RIS have been instructed to use the last tablet 7 days before the start of the EDTA test. Patients on RLX treatment were instructed to take the last tablet 24 h before the testing. All women continued their calcium and vitamin D supplements on the day before the study. On the day of the study, however, no supplements or medication were given in the morning. All women fasted overnight, and for safety reasons and to standardize the baseline concentrations of the variables, all tests were performed 3 h after a standard calcium load (250 mg of elemental calcium in 200 ml of plain water and 30 g of white bread) [4]. The endogenous PTH secretion was stimulated by hypocalcaemia induced by a disodium ethylenediaminetetraacetic acid (EDTA) infusion. Blood samples were obtained immediately before the test at 09:00 (baseline) and at 33, 60, 90, 120 and 180 min after the beginning of the test. The subjects were fasted until the end of each study session with only a steady intake of plain water (about 500 ml). The plasma and serum specimens were stored at −70 °C for the later measurement of the bone markers and PTH. Measurement of the serum ionized calcium (iCa) was performed on the same day after anaerobic collection of all specimens, which were stored anaerobically at 4 °C to avoid pH shifts. EDTA infusions An intravenous cannula was placed in the forearm and was used for both the EDTA infusion and blood sampling. EDTA at a dose of 10 mg/kg body weight in 100 ml 0.9% NaCl solution was administered for 30 min, along with 10–15 ml of 10% trimecaine in 100 ml 0.9% NaCl

All samples, except the serum ionized calcium, were assayed simultaneously. Serum ionized calcium (iCa) was measured using an ion selective electrode with an AVL 9180 (Roche Diagnostics GmbH, Germany). The within run imprecision was below 2% and between run imprecision was below 4%. The plasma concentrations of the immunoreactive intact PTH were determined using an electrochemiluminescence-based immunoanalysis (the Elecsys 1010 Analyzer, Roche Diagnostics GmbH, Germany). The within run imprecision was below 6%. The serum concentrations of β-CTX were measured using the electrochemiluminescence-based immunoanalysis (the Elecsys 1010 Analyzer, Roche Diagnostics, Germany). The within run imprecision for the β-CTX was below 7%. The serum concentrations of intact Nterminal propeptide of type I procollagen (PINP) were measured by radioimmunoassay (Procollagen Intact PINP, Orion Diagnostica, Finland). The within run imprecision was below 5%. Serum concentrations of 25-hydroxyvitamin D (25-OHD) were determined by using enzymoimmunoassay (OCTEIA-25-Hydroxy Vitamin-D, Immunodiagnostic Systems Limited, UK). Concentrations of 25-OHD above 50 nmol/l were considered normal. Statistical analysis The primary end-point of the study was acute β-CTX change. The study was designed to enroll 40 patients, with approximately 9 patients from each pretreatment group. Calculation of the sample size to detect differences in β-CTX after PTH administration between the treatment naive and ALN pretreated groups was based on data from this laboratory indicating that assuming a common standard deviation of 0.090, at least 80% power existed to detect a difference of 0.150 in βCTX between the long-term ALN-treated and treatment-naive patients with 9 patients in each treatment group. To assess changes, the values were calculated for each person and expressed as the percentage of the initial baseline value. Data were expressed as mean ± SD if not otherwise stated. The time differences were assessed by oneway ANOVA for repeated measurements. If the treatment effects were not normally distributed, the Friedman repeated measures ANOVA on

Table 1 Baseline patients' characteristics for the control group and anti-resorption treatment groups (mean ± SD) Baseline characteristics

Control (n = 10)

ALN treated (n = 10)

RIS treated (n = 10)

RLX treated (n = 10)

Age (years) Years since menopause Duration of treatment Height (cm) Weight (kg) BMI (kg/m2) Spine L1–L4 (T-score) Total hip (T-score) Serum iCa (mmol/l) Serum 25-OHD (nmol/l) Plasma intact PTH (ng/l) Serum β-CTX (ng/l) Serum P1NP (μg/l)

68.3 ± 6.1 18.9 ± 7.7 – 162 ± 6.0 60.5 ± 8.9 23.1 ± 3.0 −2.7 ± 0.9 −1.7 ± 0.7 1.30 ± 0.02 104.1 ± 24.8 36.2 ± 11.7 408.1 ± 132 49.5 ± 11.5

67.4 ± 8.1 20.6 ± 6.0 4.6 ± 1.9 164 ± 4.0 65.3 ± 7.2 24.3 ± 2.1 −2.2 ± 1.4 − 1.2 ± 0.9 1.30 ± 0.02 80.8 ± 19.1 26.5 ± 7.5 104.1 ± 52.2⁎,† 19.2 ± 9.9⁎

69.4 ± 8.9 19.7 ± 8.5 3.9 ± 2 160.1 ± 4.4 62.1 ± 5.3 24.2 ± 1.7 − 2.9 ± 0.9 −1.5 ± 0.7 1.33 ± 0.05 87.9 ± 14.6 37.02 ± 8.6 209.8 ± 68.5⁎ 31.4 ± 9.8

67.6 ± 7.0 21.1 ± 6.9 3.4 ± 1.4 166 ± 3.8 69.2 ± 9.9 24.9 ± 3.1 −2.5 ± 1.3 −1.5 ± 0.6 1.29 ± 0.03 79.4 ± 23.3 27.4 ± 7.3 247.8 ± 88 33.2 ± 13.9

Results were compared by ANOVA, Tukey test was used for all pairwise multiple comparison procedures. Abbreviations: iCa: ionized calcium; 25-OHD: 25-hydroxy-vitamin D3; intact PTH: intact parathyroid hormone; β-CTX: β C-terminal telopeptide of type I collagen; P1NP: N-terminal propeptide of procollagen type I. ⁎ p b 0.05 compared with control group. † p b 0.05 ALN treated compared with RLX treated group.

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ranks was used. If a significant difference was found with ANOVA, subsequent multiple comparison procedures (Tukey test) were used to determine the difference between the basal values and other time points. The trapezoidal method was applied for calculation of the timeaveraged mean change from baseline (the area under the curve, AUC). The differences between the study groups were evaluated using the analysis of variance (ANOVA) with subsequent multiple comparison procedures (Tukey test). Analyses were made with SigmaStat statistical software, version 3.1 (Jandel Corporation, San Rafael, USA). For all tests p b 0.05 was considered significant. Results At baseline, the characteristics of the study groups and biochemical variables with the exception of the markers of bone turnover were not significantly different (Table 1). Indices of bone resorption were significantly lower in the women on ALN and RIS treatment. Indices of bone formation were significantly lower only in ALN treated women. All subjects completed the full study protocol with no adverse event. The expected decline in mean serum ionized calcium (iCa) during EDTA infusions was very similar in all groups of women, with the nadir at the end of the infusion. Calcium levels remained suppressed after the infusion was discontinued with subsequent slow increases,

Fig. 2. The areas under the curve (AUC, %⁎min) for the serum β-CTX concentrations between 0 and 180 min (upper panel) and for the plasma PTH concentrations between 60 and 180 min (lower panel) after EDTA-infusions in treated women (ALN, RIS and RLX) and in control group (CRL). Significance of difference between the study groups (ANOVA, Tukey test for multiple comparisons). Results shown are mean ± SE.

which did not return to baseline values within 180 min of the study period (Fig. 1). In all women, a decrease in serum iCa concentrations resulted in a peak of plasma PTH after the end of the 30 min infusion (at 33 min), with a subsequent rapid decrease to near baseline values (Fig. 1). Although there was no difference in the PTH peak responses among the treatment groups of women, the AUCs for PTH between 60 and 180 min in both the ALN and RIS treated women remained significantly higher than in the control group (ANOVA, p = 0.014; Tukey test, p b 0.05; Fig. 2). Increase in PTH concentration was followed by a significant increase in serum β-CTX as early as at 60 min in the control group (RM ANOVA, p b 0.001; Tukey test, p = 0.002) and at 90 min in RLX or RIS treated patients (RM ANOVA, p b 0.001; Tukey test, p b 0.001). In ALN treated patients we documented a significant decrease in serum β-CTX at 33 min (RM ANOVA p b 0.001; Tukey test, p = 0.036) with subsequent increase which reached statistical significance at 120 min as compared with baseline (Tukey test, p = 0.005) (Fig. 1). The AUCs for serum β-CTX were significantly lower in the ALN treated women than in those treated with RIS, RLX or in control group (ANOVA, p b 0.001; Tukey test, p = 0.048 for RIS group, p b 0.001 for RLX and control group; Fig. 2). There was no significant difference in β-CTX AUC response between RIS or RLX treated women and control group. Discussion

Fig. 1. Serum ionized calcium (S-iCa; upper panel), plasma intact parathyroid hormone (PTH; mid panel) and serum β C-terminal telopeptide of type I collagen (β-CTX; lower panel) responses to EDTA-infusions in ALN (◊), RIS (▴) and RLX (●) treated women and in control group (○). The values are mean ± SE. Significances are shown in Fig. 2.

This study of postmenopausal women with osteoporosis demonstrates that acute stimulation of the parathyroid gland by EDTAinduced hypocalcemia results in an acute increase of the serum β-CTX, the degradation product of mature collagen type I, which is released into the circulation during osteoclastic bone resorption. These results are in accordance with our previous investigation in healthy young men [4] and clearly show that significant acute effects on the bone resorption marker can result from transient elevation in the plasma levels of PTH. Although we did not measure directly the osteoclast

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activity, the initial involvement of osteoclasts on the PTH effect is supported by previously published experimental studies [2,14,18]. The present results indicate that reduction of bone turnover by ALN significantly blunts the acute bone resorbing effects of PTH. Interestingly, there appears to be an initial decrease in serum β-CTX after the hypocalcaemic stimulus in ALN-treated women. In contrast to ALN, long-term treatment with RIS or RLX, which caused a lesser reduction of bone turnover, did not significantly alter the bone's responsiveness to acute hypocalcemia. These results are in agreement with previous long-term studies which showed that the effects of ALN may differ from those of RIS or RLX [8,11,12]. However, our present study was not designed or powered to study the mechanisms by which aminobisphosphonates and RLX interfere with the acute bone response to endogenous PTH and therefore, we only speculate on the explanation for these differences. It could be related to the reduced bone remodeling units available for PTH effect in ALN treated patients. This view is consistent with previous histomorphometric data, indicating that during the early hPTH(1–34) treatment period, the majority (70%) of new bone formation occurs in remodeling units in which bone resorption was already ongoing or completed at the start of treatment, while 30% of new bone formation is both spatially and temporally unrelated to prior resorption (modeling based formation) [5]. Alternatively, the lesser response to PTH effect in ALN treated subjects could be directly attributed to diminished bone cells responsiveness to PTH action. It has been suggested in vitro that ALN may exert a suppressive effect on cells of the osteoblast lineage [19]. Additional studies are needed to elucidate the mechanisms for these differences. Also, a longer period of observation would be needed to better assess differences between groups treated with aminobisphosphonates where the initial decrease in β-CTX concentrations affects AUC calculations. Changes of serum calcium concentrations after EDTA infusion in treated patients did not differ significantly from that of the control group, indicating that the correction of acutely induced hypocalcemia is not dependent exclusively on bone turnover. Urinary calcium excretion was not measured in this study. Also, we cannot exclude any difference from a later recovery of serum calcium concentrations (after 180 min). Concerning acute changes of PTH secretion in response to EDTAinduced hypocalcemia, a similar peak PTH response was observed in all studied groups of women. However, during the period between 60 and 180 min after EDTA administration, PTH remained significantly higher in the aminobisphosphonate-treated women than in the control group. This observation, which is supported by the previous study of patients with osteoporosis on long-term oral therapy with pamidronate [20], may suggest that for maintenance of normal serum calcium concentrations under conditions of calcium stress, more PTH is needed. In contrast to aminobisphosphonates, PTH responses in the RLX group did not significantly differ from the control group, indicating either a lesser suppression of bone turnover by RLX or a decrease in sensitivity caused by RLX of the parathyroids to hypocalcemia, analogous to estrogen action [21]. This latter possibility was not supported by the study results of Oleksik et al. [22]. In addition, RLX may also exert estrogen effect on renal calcium reabsorption and therefore can decrease a need on PTH secretion. Limitations of our study should be noted. Firstly, there was a short time of observation with a lack of bone formation marker measurement. In our previous study in healthy men [4] bone formation markers, plasma N-MID osteocalcin and serum propeptide of procollagen type I (PINP) remained stable after both teriparatide and EDTA administrations over a study period of 3 h. Recently, Eastell et al. have shown in postmenopausal women that treatment with teriparatide (20 μg/day) significantly increased PINP after 3 days but serum βCTX did not change [23]. Secondly, given the small sample size, the power of the test to observe a smaller difference in AUCs between RIS or RLX treated women and the control group is limited. Thirdly, we did

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not measure magnesium concentrations in this study, and therefore we cannot exclude a possibility that magnesium deficiency could decrease PTH action in some patients [24]. In conclusion, our data in postmenopausal osteoporotic women indicate that significant effect on bone resorption marker serum βCTX results from transient elevation in the plasma concentration of PTH. This marker will probably be useful for testing the bone's response to other drugs which stimulate PTH secretion, such as calcium receptor antagonists (calcilytics). More importantly, we observed that in women with postmenopausal osteoporosis treated with ALN, a substantial reduction of bone turnover blunts the acute bone resorbing effect of endogenous PTH. Our data suggest a larger response to the acute hypocalcemia in RLX- and RIS-treated patients than in ALN-treated patients. Whether bone resistance to the acute effect of PTH in ALN treated patients contributes to the delayed response in bone formation markers and BMD shown in previous studies after long-term PTH treatment will require further study. If so, our findings would be important to optimize the use of antiresorptive therapy with anabolics. Acknowledgments We thank Oldriska Lukaskova for excellent technical assistance. The work was supported by MZ ČR 000 237280. References [1] Delmas PD. Treatment of postmenopausal osteoporosis. Lancet 2002;359:2018–26. [2] Ma YL, Cain RL, Halladay DL, Yang X, Zeng Q, Miles RR, et al. Catabolic effects of continuous human PTH (1–38) in vivo is associated with sustained stimulation of RANKL and inhibition of osteoprotegerin and gene-associated bone formation. Endocrinology 2001;142:4047–54. [3] Martin TJ, Sims NA. Osteoclast-derived activity in the coupling of bone formation to resorption. Trends Mol Med 2005;11:76–81. [4] Zikán V, Štěpán JJ. Marker of bone resorption in acute response to exogenous or endogenous parathyroid hormone. Biomark Insights 2008;3:19–24. [5] Lindsay R, Cosman F, Zhou H, Bostrom MP, Shen VW, Cruz JD, et al. A novel tetracycline labeling schedule for longitudinal evaluation of the short-term effects of anabolic therapy with a single iliac crest bone biopsy: early actions of teriparatide. J Bone Miner Res 2006;21:366–73. [6] Cosman F, Nieves J, Woelfert L, Shen V, Lindsay R. Alendronate does not block the anabolic effect of PTH in postmenopausal osteoporotic women. J Bone Miner Res 1998;13:1051–5. [7] Finkelstein JS, Leder BZ, Burnett SA, Wyland JJ, Lee H, de la Paz AV, et al. Effects of teriparatide, alendronate, or both on bone turnover in osteoporotic men. J Clin Endocrinol Metab 2006;91:2882–7. [8] Miller P, Lindsay R, Watts N, Meeves S, Lang T, Delmas P, et al. Patients previously treated with risedronate demonstrate greater responsiveness to teriparatide than those previously treated with alendronate: the OPTAMISE study. J Bone Miner Res 2007;22:S26. [9] Boonen S, Marin F, Obermayer-Pietsch B, Simoes ME, Barker C, Glass EV, et al. Effects of previous antiresorptive therapy on the bone mineral density response to two years of teriparatide treatment in postmenopausal women with osteoporosis. J Clin Endocrinol Metab 2008;93:852–60. [10] Black DM, Greenspan SL, Ensrud KE, Palermo L, McGowan JA, Lang TF, et al. The effects of parathyroid hormone and alendronate alone or in combination in postmenopausal osteoporosis. N Engl J Med 2003;349:1207–15. [11] Ettinger B, San Martin J, Crans G, Pavo I. Differential effects of teriparatide on BMD after treatment with raloxifene or alendronate. J Bone Miner Res 2004;19: 745–51. [12] Delmas P, Watts N, Miller P, Cahall D, Bilezikian J, Lindsay R. Bone turnover markers demonstrate greater earlier responsiveness to teriparatide following treatment with risedronate compared with alendronate: the OPTAMISE study. J Bone Miner Res 2007;22:S27. [13] Stepan JJ, Dobnig H, Burr DB, Li J, Ma YL, Sipos A, et al. Histomorphometric changes by teriparatide in alendronate pre-treated women with osteoporosis. J Bone Miner Res 2008;23:S6–7. [14] Onyia JE, Miles RR, Yang X, Halladay DL, Hale J, Glasebrook A, et al. In vivo demonstration that human parathyroid hormone 1–38 inhibits the expression of osteoprotegerin in bone with the kinetics of an immediate early gene. J Bone Miner Res 2000;15:863–71. [15] Holtrop ME, King GJ, Cox KA, Reit B. Time-related changes in the ultrastructure of osteoclasts after injection of parathyroid hormone in young rats. Calcif Tissue Int 1979;27:129–35. [16] Lindsay R, Nieves J, Henneman E, Shen V, Cosman F. Subcutaneous administration of the amino-terminal fragment of human parathyroid hormone-(1–34): kinetics and biochemical response in estrogenized osteoporotic patients. J Clin Endocrinol Metab 1993;77:1535–9.

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