Combination treatment with eldecalcitol (ED-71) and raloxifene improves bone mechanical strength by suppressing bone turnover and increasing bone mineral density in ovariectomized rats

Bone 53 (2013) 167–173

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Original Full Length Article

Combination treatment with eldecalcitol (ED-71) and raloxifene improves bone mechanical strength by suppressing bone turnover and increasing bone mineral density in ovariectomized rats Satoshi Takeda a, Sadaoki Sakai a, Ayako Shiraishi b, Nobuo Koike a, Masahiko Mihara a, Koichi Endo a,⁎ a b

Product Research Department, Chugai Pharmaceutical Co., Ltd., Japan Medical Plan Management Department, Chugai Pharmaceutical Co., Ltd., Japan

a r t i c l e

i n f o

Article history: Received 7 September 2012 Revised 22 November 2012 Accepted 3 December 2012 Available online 8 December 2012 Edited by: Toshio Matsumoto Keywords: Eldecalcitol Raloxifene Bone turnover Bone mineral density Mechanical strength

a b s t r a c t The aim of this study was to investigate the effect of combination treatment with eldecalcitol (ELD) and raloxifene (RAL) on bone turnover, bone mineral density (BMD), and bone strength. Eight-month-old rats were ovariectomized (OVX) or sham operated, and divided into five groups (Sham, OVX+vehicle, OVX+RAL, OVX+ELD and OVX+ELD+RAL). ELD (7.5 ng/kg) and RAL (0.3 mg/kg) were orally administered alone or in combination daily. Urinary deoxypyridinoline (DPD) levels were measured after 4, 8, and 12 weeks of treatment. After 12 weeks of treatment, BMD and mechanical properties of the lumbar spine and femur were assessed, and bone histomorphometry was performed. Urinary DPD levels in all the treatment groups were significantly decreased compared with the OVX+vehicle group. At 4 weeks of treatment, urinary DPD level of the combination group was significantly lower than that of either monotherapy group. The reduction in the BMD of the lumbar spine and femur by OVX was significantly prevented in all the treatment groups, and the BMD in the combination group was significantly higher than that in either monotherapy group. The ultimate load and work to failure of the fifth lumbar vertebra were significantly improved only by the combination treatment. The femoral midshaft ultimate load was significantly increased in the OVX+ELD group and the combination group, and the femoral midshaft work to failure was increased only in the combination group. Bone histomorphometric analysis using the third lumbar vertebra revealed that osteoblast surface (Ob.S/BS), osteoclast surface (Oc.S/BS) and osteoclast number (N.Oc/BS) significantly decreased in all treatment groups, and osteoid surface (OS/BS) and bone formation rate (BFR/BS) significantly decreased in the ELD-treated and combination groups. The values of Ob.S/BS and OS/BS in the combination group were lower than those in either of the monotherapy groups. The bone formation parameters in the combination group were not reduced to below levels of the sham-operated control, suggesting that the combination therapy with ELD and RAL may not cause oversuppression of bone turnover. These results indicated that the combination treatment with ELD and RAL might be a beneficial therapy with respect to their combined effects of enhancing the mechanical properties of trabecular and cortical bone by suppressing bone turnover and increasing BMD more than either monotherapy. © 2012 Elsevier Inc. All rights reserved.

Introduction Eldecalcitol (1α, 25-dihydroxy-2β-(3-hydroxypropyloxy) vitamin D3; ELD), a new active vitamin D analog, has recently been approved for the treatment of osteoporosis in Japan. A 3-year clinical study comparing the effect of ELD with that of alfacalcidol in patients with osteoporosis showed that ELD significantly reduced the incidence of vertebral fractures as well as wrist fractures [1]. Compared to alfacalcidol, ELD also more potently increased bone mineral density (BMD) and reduced bone turnover markers [1]. It has also been shown that ELD promotes urinary calcium excretion similarly to ⁎ Corresponding author at: Product Research Department, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka, 412-8513, Japan. Fax: + 81 550 87 6782. E-mail address: [email protected] (K. Endo). 8756-3282/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2012.12.001

alfacalcidol, but has a lower potency to suppress blood parathyroid hormone (PTH) [2]. A longitudinal analysis of hip geometry revealed that ELD increased cortical cross-sectional area, volumetric BMD, and bone mass, and maintained cortical thickness, resulting in an improvement of biomechanical properties [3]. These clinical studies thus showed that ELD improves mechanical strength in both of trabecular and cortical bone by suppressing bone turnover and increasing BMD. The effects of ELD on bone metabolism were also observed in animal studies [4–6]. In ovariectomized (OVX) rats, ELD inhibited osteoclastic bone resorption and increased bone mass. These effects were greater than for alfacalcidol and were independent of effects on calcium absorption and blood PTH level [7]. Raloxifene (RAL), a selective estrogen receptor modulator, has been shown to increase BMD in the lumbar spine and femoral neck and reduce the incidence of vertebral fractures in clinical trials in

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patients with osteoporosis [8,9]. It is reported that RAL decreased bone turnover markers, and that this inhibitory effect on bone turnover contributed in part to prevention of vertebral fractures [10]. In a 1-year clinical trial in patients with osteoporosis, combination therapy with RAL and alendronate showed additive effects on biochemical markers of bone turnover as well as on BMD of the lumbar spine and femoral neck [11]. In OVX rats, treatment with both RAL and alendronate resulted in increased bone mass and improved biomechanical properties in the lumbar vertebrae [12]. These reports indicate that combined therapy with antiresorptive agents produces beneficial effects on bone mass and bone turnover. On the other hand, there is a possibility that combination therapy with antiresorptive agents can reduce bone formation to below normal levels, causing adverse effects on bone quality [13]. Indeed, severely suppressed bone turnover increases microdamage and results in increased susceptibility to atypical skeletal fragility [14,15]. Recently, Sakai et al. have shown that combination treatment with alendronate and ELD in OVX rats attenuates the reduction of bone formation parameters induced by treatment with alendronate alone [16], suggesting that combination therapy with an antiresorptive agent and active vitamin D3 may have a more favorable effect on bone formation than a combination of antiresorptive agents. Supplementation with alfacalcidol to RAL therapy showed a greater bone-sparing effect than RAL-alone therapy by suppressing the increment of serum PTH in postmenopausal Japanese women with osteoporosis or osteopenia [17]. However, there are as yet no reports describing the efficacy of combination treatment with ELD and RAL in osteoporosis patients or in animal models. The aim of this study was to investigate the efficacy of ELD and RAL, alone or in combination, on bone turnover, BMD, and bone mechanical strength in OVX rats.

body weight. Tetracycline and calcein were injected subcutaneously for bone labeling at 7 and 2 days, respectively, before necropsy. The dose of ELD was selected based on our preliminary observation that 7.5 ng/kg of ELD produced a significant increase in BMD without causing hypercalcemia in OVX rats. The dose of RAL employed was based on a previous study which showed that the effect of RAL on preventing bone loss induced by OVX in rats plateaued at a dose of 0.3 mg/kg [18]. After 4, 8, and 12 weeks of treatment, urine samples were collected over a 24-h period by using metabolic cages, and stored at −65 °C. Under isoflurane anesthesia, blood was collected from the abdominal aorta. The blood was then centrifuged, and the supernatant was collected and stored at −65 °C until measurement of parameters. The lumbar spine and bilateral femur were excised. The second through fourth lumbar vertebrae (L2–L4) and the right femur were preserved in 70% ethanol. The fifth lumbar vertebra (L5) and the left femur were wrapped in saline soaked gauze and stored at − 65 °C until biomechanical analysis. This study was performed according to the experimental protocol approved by the Institutional Animal Care and Use Committee at Chugai Pharmaceutical Co., Ltd. Biochemical analysis Serum calcium (Ca), serum total cholesterol (CHO), urinary Ca, and urinary creatinine (Cre) concentrations were measured with an automatic analyzer (7180, Hitachi, Ltd., Tokyo, Japan). Urinary deoxypyridinoline (DPD) levels were measured by enzyme-immunoassay using an Osteolinks-DPD kit (DS Pharma Biomedical Co., Ltd., Osaka, Japan), and the data were normalized with respect to urinary Cre concentration. Serum osteocalcin (OC) levels were measured by enzyme-linked immunosorbent assay using a Rat Osteocalcin ELISA System (GE Healthcare Bioscience Co., Ltd., Tokyo, Japan).

Materials and methods Measurement of bone mineral density Reagents ELD was synthesized by Chugai Pharmaceutical Co., Ltd (Tokyo, Japan). ELD was dissolved in, and diluted to the given concentration in, medium chain triglyceride (MCT, Nisshin Oillio Group, Ltd., Tokyo, Japan). RAL was provided by Eli Lilly and Co. (Indianapolis, IN, USA). RAL was dissolved in, and diluted to the given concentration in, 20% (w/v) hydroxypropyl-β-cyclodextrin (Wako Chemical, Ltd., Miyazaki, Japan).

The BMD of the L2–L4 vertebrae and of the right femur was measured by DXA using a DCS-600EX-ШR. During data analysis, the femur was divided into ten equal segments along its major axis. The mean values of BMD for the three most proximal scanned areas, those for the next four scanned areas, and those for three most distal areas were calculated as the densities of the proximal, middle, and distal parts of the femur, respectively. Biomechanical analysis

Animals Seven-month-old female Wistar-Imamichi rats were obtained from the Institute for Animal Reproduction (Ibaraki, Japan), and acclimatized for 1 month. Animals were housed individually in stainless-steel wire cages under standard laboratory conditions at 21–25 °C, 40–70% humidity, and a 12 h:12 h light/dark cycle. All animals had free access to tap water and a commercial standard rodent chow (CE-2, CLEA Japan, Inc., Tokyo, Japan). Experimental design Before ovariectomy or sham operation, lumbar vertebral BMD (L2–L5) was measured in vivo by dual-energy X-ray absorptiometry (DXA) using a DCS-600EX-IIIR bone densitometer (Aloka Co., Ltd., Tokyo, Japan). Rats were divided into 5 groups, each with a similar mean BMD (Sham, OVX +vehicle, OVX+ ELD, OVX + RAL, OVX + ELD +RAL; n =10/group). All rats except those in the Sham group were subjected to bilateral ovariectomy under anesthesia. From the day following surgery, OVX animals were orally administered ELD (7.5 ng/kg of body weight) and RAL (0.3 mg/kg of body weight), alone or in combination, daily for 12 weeks. In the Sham group and OVX +vehicle group, rats received vehicle at a dose of 1 mL/kg of

Biomechanical testing was performed using a mechanical testing machine (TK-252C; Muromachi Kikai Co., Ltd, Tokyo, Japan) [19]. For the three-point bending test of the mid femur, the left femur was placed on a holding device with supports located 12 mm apart. The upper loading device was aligned to the center of the femoral shaft on the anterior side. The load was applied at a rate of 20 mm/s until failure occurred. Prior to the compression test of the L5 vertebral body, the vertebral arch and end plates were removed to obtain a specimen with planoparallel ends. The loading rate of the compression test was set at 2.5 mm/min. Ultimate load (N), stiffness (N/mm), and work to failure (mJ) were calculated from the load–displacement curve. Bone histomorphometry Bone histomorphometry was performed on the L3 vertebral body and the femoral diaphysis. Specimens were fixed in 70% ethanol and stained in Villanueva bone stain. After dehydration with ethanol, the specimens were defatted and embedded in methyl methacrylate. Five-micrometer thick sections of the L3 vertebral body were prepared to evaluate trabecular bone. Static and dynamic parameter measurements were collected with a Histomorphometric System (System Supply Co. Ltd., Nagano, Japan) linked to a microscope

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equipped with bright-field and epifluorescence illumination. The following parameters were measured: bone volume (BV/TV, %), trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, /mm), trabecular separation (Tb.Sp, μm), osteoblast surface (Ob.S/BS, %), osteoid surface (OS/BS, %), osteoclast surface (Oc.S/BS, %), osteoclast number (N.Oc/BS, /mm), mineral apposition rate (MAR, μm/day), and bone formation rate (BFR/BS, mm3/mm2/year). For the femoral diaphysis, 20- to 30-μm-thick cross-cut ground sections were obtained using a microgrinding machine system, and the following parameters were measured: total core (Tt.C, mm 2), marrow volume (Ma.V, mm 2), cortical area (Ct.Ar, mm 2), cortical width (Ct.Wi, mm), periosteal surface (Ps.S, mm), endosteal surface (Es.S, mm), endosteal mineral apposition rate (Es.MAR, μm/day), endosteal bone formation rate (Es.BFR/BS, mm3/mm2/year), periosteal mineral apposition rate (Ps.MAR, μm/day), and periosteal bone formation rate (Ps.BFR/BS, mm 3/mm2/year). Nomenclature and units used in this study follow the report of the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee [20]. Statistical analysis Data are presented as mean+standard deviation (SD) or mean±SD. The differences among the groups were examined with one-way analysis of variance (ANOVA). When the ANOVA indicated a significant difference, statistical differences between individual groups were evaluated with Tukey's multiple comparison test. Statistical comparisons between the Sham group and the OVX+vehicle group were performed by unpaired t-test. For all tests, Pb 0.05 was considered statistically significant. Statistical Analysis System software package (SAS Institute Inc., Cary, NC, USA) was used for all statistical analyses.

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decreased significantly compared with the OVX + vehicle group at 4 weeks of treatment. Ovariectomy significantly decreased serum Ca concentration compared with the Sham group (Table 1). ELD significantly increased serum Ca concentration as well as urinary Ca excretion (Ca/Cre) compared with the OVX + vehicle group, while RAL did not alter these parameters. The combination treatment with ELD and RAL significantly suppressed ELD-induced increases in serum Ca concentration and urinary Ca excretion. RAL significantly decreased serum total CHO compared with the OVX + vehicle group, while ELD did not alter this parameter (Table 1). The combination treatment with ELD and RAL did not affect the decrease in serum total CHO achieved by the treatment with RAL alone.

Bone mineral density The BMD of the L2–L4 vertebrae and the femur in the OVX + vehicle group was significantly lower than that of Sham group at 12 weeks after OVX (Fig. 2). The treatment with ELD or RAL alone and the combination treatment with ELD and RAL prevented the OVX-induced reduction of the BMD of the vertebrae and the femur. The BMD in the combination group was significantly higher than that in either of the monotherapy groups (Figs. 2A, B). In the femur, the BMD of the proximal and distal femur in all of the agent-treated groups was significantly higher than that in the OVX + vehicle group (Figs. 2C, E). The BMD of the mid femur was significantly increased in the OVX + ELD and OVX + ELD+ RAL groups compared with the OVX + vehicle group (Fig. 2D). In the OVX + ELD + RAL group, the BMD of the distal femur was significantly higher than that in either of the monotherapy groups (Fig. 2E).

Results Biomechanical properties Serum and urinary biochemical parameters Urinary DPD excretion in the OVX+ vehicle group was significantly increased compared with the Sham group at 4, 8, and 12 weeks of treatment (Fig. 1). These increases in urinary DPD excretion by OVX were significantly suppressed in all the agent-treated groups. At 4 weeks of treatment, urinary DPD excretion in the OVX+ ELD+ RAL group was significantly lower than that in the OVX+ELD group or in the OVX+RAL group (Fig. 1A). Serum OC concentration in the OVX+vehicle group was significantly higher than that in the Sham group through the whole experimental period (Table 1). In the OVX + ELD and the OVX+ RAL groups, serum OC concentration did not change in comparison with the OVX + vehicle group. On the other hand, serum OC in the OVX + ELD+ RAL group

4 weeks

A DPD/Cre (nmol/mmol)

120

a 100

100

80

80

60

b

8 weeks

B

120

b

60

b,c,d

40 20

Ovariectomy significantly decreased the ultimate load and work to failure of the L5 vertebral body (Figs. 3A, C). The treatment with ELD or RAL alone showed a slight, but not significant, increase in those parameters. The combination treatment with ELD and RAL significantly increased the ultimate load and work to failure compared with the OVX + vehicle group. Ovariectomy and agent treatment did not alter the stiffness of the L5 vertebral body (Fig. 3B). Ovariectomy did not induce a significant reduction in the biomechanical properties of the femoral midshaft (Figs. 3D, E, F). The ultimate load of the femoral midshaft significantly increased in the OVX + ELD group and the OVX+ ELD+ RAL group (Fig. 3D). In the OVX + ELD+ RAL group, the work to failure also significantly increased compared with the OVX + vehicle group (Fig. 3F).

C 120

a

100 80

RAL ELD+RAL

OVX

b,c

60

b

40

b,d

40

b

b,d

20 0

0 Sham Vehicle ELD

a

b,c

20

0

12 weeks

Sham Vehicle ELD

RAL ELD+RAL

OVX

Sham Vehicle ELD

RAL ELD+RAL

OVX

Fig. 1. Urinary deoxypyridinoline excretion after 4 (A), 8 (B), and 12 (C) weeks of treatment. Data are presented as mean + SD of 10 animals. aP b 0.05 vs. Sham by unpaired t-test. b P b 0.05 vs. OVX + vehicle, cP b 0.05 vs. OVX + ELD, dP b 0.05 vs. OVX + RAL by Tukey's multiple comparison test.

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Table 1 Serum and urinary biochemical parameters after 4, 8, or 12 weeks of treatment. OVX Sham Serum osteocalcin (ng/mL)

4 weeks 8 weeks 12 weeks 12 weeks 12 weeks 12 weeks

Serum total cholesterol (mg/dL) Serum calcium (mg/dL) Urinary calcium/creatinine

Vehicle a

18.7 ± 4.8 20.7 ± 4.1 16.3 ± 3.3 178 ± 31 10.34 ± 0.31 0.037 ± 0.011

33.6 ± 6.6 29.5 ± 7.5a 22.1 ± 5.1a 178 ± 14 9.92 ± 0.23a 0.038 ± 0.011

ELD

RAL

ELD + RAL

30.5 ± 4.7 29.3 ± 4.4 22.7 ± 5.4 194 ± 22 10.72 ± 0.36b 0.346 ± 0.058b

29.4 ± 2.7 26.1 ± 4.3 18.2 ± 3.0 118 ± 21b,c 9.62 ± 0.24c 0.042 ± 0.054c

25.0 ± 3.6b 23.8 ± 3.9 17.6 ± 2.1c 131 ± 30b,c 10.25 ± 0.34c,d 0.198 ± 0.046b,c,d

Data are presented as mean ± SD (n = 10). a P b 0.05 vs. Sham by unpaired t-test. b P b 0.05 vs. OVX + vehicle, c P b 0.05 vs. OVX + ELD, d P b 0.05 vs. OVX + RAL by Tukey's multiple comparison test.

Bone histomorphometry Ovariectomy decreased BV/TV, Tb.Th and Tb.N, and increased Tb.Sp of the L3 vertebra, suggesting that trabecular bone architecture deteriorated following ovariectomy (Table 2). This ovariectomy-induced deterioration of the trabecular structure was prevented in the OVX + ELD group and the OVX + ELD + RAL group. In the OVX + RAL group, the decrease in BV/TV and Tb.N and the increase in Tb.Sp were significantly prevented. There were no significant differences in these structural parameters between the agent-treated groups. In the OVX + vehicle group, Ob.S/BS, OS/BS, Oc.S/BS, N.Oc/BS, MAR, and BFR/BS significantly increased compared with the Sham group. The treatment with ELD suppressed the increases in Ob.S/BS,

A

Lumbar spine

200

250

BMD (mg/cm2)

200

OS/BS, Oc.S/BS, N.Oc/BS, and BFR/BS, while RAL suppressed the increases in Ob.S/BS, Oc.S/BS, and N.Oc/BS. The combination treatment with ELD and RAL significantly inhibited the increases of all the dynamic parameters. In the combination group, Ob.S/BS and OS/BS were significantly lower than those in either of the monotherapy groups. In the femoral diaphysis, ovariectomy increased Ma.V and Es.S, and decreased Ct.Wi in comparison with the Sham group (Table 3). In the OVX + ELD group and the OVX + ELD + RAL group, Ma.V and Es.S decreased and Ct.Ar and Ct.Wi increased significantly compared with the OVX + vehicle group. On the other hand, RAL treatment did not affect these structural parameters of cortical bone. Es.MAR and Es.BFR/BS in the OVX + vehicle group increased significantly compared with the Sham group. In the combination group, Es.MAR

B

Whole femur

150

b

a

b,c

a

b,c,d

b

b,c,d

b

150 100 100 50 50 0

0 Sham Vehicle ELD

Sham Vehicle ELD

RAL ELD+RAL

OVX

OVX

C

D

Proximal femur

200

BMD (mg/cm2)

E

Mid femur

Distal femur

200

200

b,d

b 150

RAL ELD+RAL

b

a

150

a

b,d

b

150

100

100

100

50

50

50

Sham Vehicle ELD

RAL ELD+RAL

OVX

b

a

0

0

0

b,c,d

b

Sham Vehicle ELD

RAL ELD+RAL

OVX

Sham Vehicle ELD

RAL ELD+RAL

OVX

Fig. 2. Bone mineral density at 12 weeks after OVX measured by DXA in the lumbar spine (L2–L4) (A), the whole femur (B), the proximal femur (C), the mid femur (D), and the distal femur (E). Data are presented as mean + SD of 10 animals. aP b 0.05 vs. Sham by unpaired t-test. bP b 0.05 vs. OVX + vehicle, cP b 0.05 vs. OVX + ELD, dP b 0.05 vs. OVX + RAL by Tukey's multiple comparison test.

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A

B

600

a 400

200

C

2500

250

2000

200

Work to failure (mJ)

b

Stiffness (N/mm)

Ultimate Load (N)

800

1500

1000

500 0

Sham Vehicle ELD

RAL ELD+RAL

150

a

100

50

Sham Vehicle ELD

RAL ELD+RAL

D

E

300

RAL ELD+RAL

OVX

OVX

OVX

F

700

140

600

120

Stiffness (N/mm)

c 200 150 100 50

Work to failure (mJ)

b,d

b

250

b

0

0 Sham Vehicle ELD

Ultimate Load (N)

171

500 400 300 200 100

Sham Vehicle ELD

RAL ELD+RAL

100 80 60 40 20

0

0

b,d

0 Sham Vehicle ELD

RAL ELD+RAL

Sham Vehicle ELD

OVX

OVX

RAL ELD+RAL

OVX

Fig. 3. Biomechanical properties of the fifth lumbar vertebral body and femoral midshaft. The ultimate load (A), stiffness (B), and work to failure (C) of the vertebra were measured by compression test. The femoral midshaft ultimate load (D), stiffness (E), and work to failure (F) were measured by 3-point bending test. Data are presented as mean + SD of 10 animals. aP b 0.05 vs. Sham by unpaired t-test. bP b 0.05 vs. OVX + vehicle, cP b 0.05 vs. OVX + ELD, dP b 0.05 vs. OVX + RAL by Tukey's multiple comparison test.

and Es.BFR/BS decreased significantly compared with either of the monotherapy groups. No significant changes in Ps.MAR or Ps.BFR/BS were observed in the OVX + vehicle group or any of the agenttreated groups. Discussion ELD prevents vertebral and non-vertebral fractures and increases the BMD of the lumbar spine and total hip in osteoporotic patients [1]. A previous study has shown that ELD maintains cortical thickness

and cortical cross-sectional area and increases volumetric BMD and bone mass in femoral neck, resulting in improvement of the biomechanical properties [3]. These beneficial effects on BMD and bone geometry are thought to be attributed to ELD's action of suppressing bone turnover that is elevated by estrogen deficiency. Clinical [1,21] and pre-clinical [7,16] studies have shown that ELD reduces bone resorption markers, and that this action of ELD is stronger than that of alfacalcidol. In the present study, urinary DPD levels as well as N.Oc/BS values of lumbar vertebral trabecular bone decreased in the ELD-treated group, suggesting that ELD inhibits bone resorption by

Table 2 Bone histomorphometry of the trabecular bone of the third lumbar vertebra. OVX

BV/TV (%) Tb.Th (μm) Tb.N (/mm) Tb.Sp (μm) Ob.S/BS (%) OS/BS (%) Oc.S/BS (%) N.Oc/BS (/mm) MAR (μm/day) BFR/BS (mm3/mm2/year)

Sham

Vehicle

ELD

RAL

ELD + RAL

30.7 ± 5.6 93.1 ± 17.7 3.31 ± 0.24 210.6 ± 25.4 1.71 ± 1.46 2.79 ± 2.41 2.87 ± 0.85 1.28 ± 0.37 1.26 ± 0.22 0.017 ± 0.014

21.8 ± 3.8a 74.4 ± 11.0a 2.93 ± 0.22a 268.5 ± 31.5a 11.93 ± 3.94a 18.14 ± 4.96a 5.23 ± 1.34a 1.91 ± 0.53a 1.71 ± 0.14a 0.128 ± 0.030a

37.2 ± 6.4b 102.4 ± 15.4b 3.63 ± 0.31b 174.9 ± 28.3b 7.26 ± 4.39b 12.28 ± 6.82b 1.03 ± 0.74b 0.50 ± 0.33b 1.49 ± 0.27 0.056 ± 0.037b

32.6 ± 4.0b 90.8 ± 11.1 3.60 ± 0.28b 188.8 ± 22.7b 8.06 ± 1.88b 13.27 ± 2.63 3.63 ± 0.92b,c 1.36 ± 0.36b,c 1.73 ± 0.19 0.104 ± 0.029c

35.3 ± 6.0b 98.7 ± 20.0b 3.60 ± 0.24b 180.2 ± 18.3b 2.48 ± 1.09b,c,d 4.75 ± 1.61b,c,d 1.22 ± 0.66b,d 0.59 ± 0.31b,d 1.26 ± 0.29b,d 0.024 ± 0.009b,d

Data are presented as mean ± SD (n = 10). a P b 0.05 vs. Sham by unpaired t-test. b P b 0.05 vs. OVX + vehicle, c P b 0.05 vs. OVX + ELD, d P b 0.05 vs. OVX + RAL by Tukey's multiple comparison test.

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Table 3 Bone histomorphometry of the cortical bone of the femoral diaphysis. OVX

2

Tt.C (mm ) Ma.V (mm2) Ct.Ar (mm2) Ct.Wi (mm) Ps.S (mm) Es.S (mm) Es.MAR (μm/day) Es.BFR/BS (mm3/mm2/year) Ps.MAR (μm/day) Ps.BFR/BS (mm3/mm2/year)

Sham

Vehicle

ELD

RAL

ELD + RAL

9.60 ± 0.49 3.42 ± 0.24 6.17 ± 0.45 0.65 ± 0.04 11.60 ± 0.28 7.26 ± 0.35 1.02 ± 0.25 0.020 ± 0.014 1.04 ± 0.25 0.320 ± 0.135

10.00 ± 0.54 4.17 ± 0.31a 5.83 ± 0.40 0.59 ± 0.03a 11.77 ± 0.34 7.79 ± 0.26a 1.33 ± 0.22a 0.079 ± 0.035a 0.98 ± 0.11 0.278 ± 0.066

9.97 ± 0.71 3.56 ± 0.26b 6.41 ± 0.54b 0.67 ± 0.04b 11.81 ± 0.42 7.32 ± 0.42b 1.50 ± 0.22 0.137 ± 0.104 1.01 ± 0.14 0.335 ± 0.080

9.90 ± 0.36 3.90 ± 0.26 6.01 ± 0.19 0.62 ± 0.02c 11.71 ± 0.25 7.71 ± 0.43 1.56 ± 0.17 0.114 ± 0.030 0.92 ± 0.11 0.273 ± 0.061

10.06 ± 0.38 3.52 ± 0.30b,d 6.55 ± 0.18b,d 0.69 ± 0.02b,d 11.78 ± 0.20 7.13 ± 0.35b,d 1.10 ± 0.23c,d 0.022 ± 0.020c,d 0.98 ± 0.06 0.328 ± 0.030

Data are presented as mean ± SD (n = 8–10). a P b 0.05 vs. Sham by unpaired t-test. b P b 0.05 vs. OVX + vehicle, c P b 0.05 vs. OVX + ELD, d P b 0.05 vs. OVX + RAL by Tukey's multiple comparison test.

decreasing the number of osteoclasts at remodeling sites on the bone surface. The mechanism by which ELD inhibits bone resorption is not fully elucidated. Harada et al. reported that daily administration of ELD suppressed the expression of receptor activator of NF-κB ligand (RANKL) mRNA and reduced RANKL-positive cells in the trabecular bone of OVX mice [22]. Calcitriol, an active form of vitamin D3, inhibits osteoclast formation by suppressing c-Fos expression in osteoclast precursor cells [23]. Moreover, active vitamin D3 inhibits osteoclastogenesis by up-regulating IFN-β expression, which in turn suppresses the expression of nuclear factor of activated T cells c1 (NFATc1), an essential transcription factor for osteoclast formation [24]. Taken together, it is suggested that ELD suppresses bone resorption by acting on bone marrow cells including osteoclast precursor cells and osteoblastic cells, which in turn inhibit osteoclast formation in vivo. RAL reduces bone turnover markers in osteoporotic patients [8–10] and in OVX animal models [25,26]. An in vitro study showed that RAL lowers production of bone-resorptive cytokines such as IL-1α, IL-1β, and IL-6 by osteoblastic cells, and also inhibits the expression of RANKL protein [27]. In addition, RAL stimulates osteoblastic production of osteoprotegerin (OPG), which acts as a decoy receptor for RANKL and prevents RANKL from binding to RANK on the surface of osteoclastic lineage cells [28,29]. Krum et al. reported that RAL induces apoptosis in osteoclasts via up-regulation of Fas ligand in osteoblasts [30]. These reports suggest that RAL modulates osteoblastic production of various factors involved in osteoclastogenesis and osteoclast survival, resulting in suppression of bone resorption. Indeed, in the present study, osteoclast number in the trabecular bone and urinary DPD excretion decreased with RAL administration. In this study, the combination treatment with ELD and RAL significantly decreased urinary DPD excretion in comparison with either ELD or RAL treatment alone. In addition, bone histomorphometric analysis showed that the combination therapy significantly reduced the osteoblastic parameters Ob.S/BS and OS/BS compared with either monotherapy, and the BFR/BS level in the combination group was lower than that in the RAL-treated group. These results indicated that in states of high bone turnover such as in estrogen-deficient rats, combination therapy with ELD and RAL suppresses bone turnover more greatly than monotherapy with either agent. As described above, active vitamin D3 acts on osteoclast precursor cells and inhibits osteoclast formation, while RAL inhibits osteoclastic bone resorption by suppressing production of bone-resorbing factors from osteoblasts. The fact that suppression of bone turnover is enhanced by combination therapy with ELD and RAL might be attributed to the combined effects of their different antiresorptive mechanisms.

Importantly, the histomorphometric indices of bone formation did not fall to below the levels of the sham-oparated group; therefore, the suppressive effect on bone turnover observed in the combined treatment was not excessive. It has been shown that long-term administration of bisphosphonates can result in oversuppression of bone turnover with increased risk of atypical bone fractures [14,15]. A previous report has shown that 16-week combination treatment of RAL and alendronate in OVX rats suppressed histomorphometric bone formation parameters to below sham control levels [12]. Although the administration period in this study was short, it appears that use of combination therapy with ELD and RAL makes it possible to avoid severely suppressed bone turnover. ELD promotes bone minimodeling, which is focal bone formation that does not depend on bone resorption [6]. This bone minimodeling by ELD may contribute to the maintenance of bone formation in the combination treatment. The values of lumbar and femoral BMD in the OVX + ELD + RAL group were higher than those in either of the agent-treated groups. From this result, it is thought that the combination treatment of ELD and RAL leads to an additive effect on BMD. The inhibitory action on bone resorption, as mentioned above, may contribute to this BMDincreasing effect in the combination therapy. Boivin et al. reported that the degree of mineralization in iliac bone increases after 2-years of RAL treatment in osteoporotic patients who are supplemented with vitamin D and Ca [31]. It is also possible that this increase in mineralization may contribute to the increase in BMD observed in the combined treatment with ELD and RAL. Histomorphometric analysis of the trabecular bone of the L3 vertebra revealed that the effects of the combination treatment with ELD and RAL to increase BV/TV, Tb.Th, and Tb.N and to decrease Tb.Sp were not significantly different from those of each monotherapy. This observation indicates that the combination therapy with ELD and RAL offered no additive effect on trabecular bone structure in comparison with either monotherapy. On the other hand, the values of Ct.Ar and Ct.Wi for the cortical bone of the mid femur were higher in the combination group than in the RAL-treated group. Ma.V also decreased with the combination treatment more than with RAL administration alone. These data suggest that, compared with RAL monotherapy, the combination therapy has greater beneficial effect on the cortical bone structure. Biomechanical testing of the L5 vertebral body showed that the combined treatment with ELD and RAL significantly increased the ultimate load and the work to failure, while each monotherapy did not. In the 3-point bending test of the femoral diaphysis, only the combination treatment increased the work to failure, and the combination group and the ELD-treated group augmented the ultimate load. These results indicated that the combination therapy has beneficial effects on the

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mechanical properties in the lumbar spine and femoral midshaft. Bone strength is defined by both BMD and bone quality, which includes factors such as bone turnover, microarchitecture, mineralization, microdamage, and collagen cross-links [32]. The effect of the combination therapy with ELD and RAL on enhancing bone mechanical properties can be accounted for by at least the BMD-increasing action and the bone turnover suppressing action described above. Active vitamin D3 enhances intestinal Ca absorption and maintains a positive Ca balance. Indeed, ELD increased urinary Ca excretion as well as serum Ca concentration in this study. The combination treatment with ELD and RAL reduced ELD-induced increases in urinary Ca excretion and serum Ca concentration. This observation indicates that the combination therapy with ELD and RAL could decrease the incidence of adverse effects such as hypercalcemia or hypercalciuria induced by ELD monotherapy. In conclusion, the combination treatment with ELD and RAL improved bone mechanical strength in OVX rats more than either monotherapy by increasing BMD and suppressing bone turnover. This combination therapy may be useful in the treatment of osteoporotic patients.

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