Sadaoki Sakai

Sphingosine-1-phosphate-mediated osteoclast precursor monocyte migration is a critical point of control in antibone-resorptive action of active vitamin D.

The migration and positioning of osteoclast precursor monocytes are controlled by the blood-enriched lipid mediator sphingosine-1-phosphate (S1P) and have recently been shown to be critical points of control in osteoclastogenesis and bone homeostasis. Here, we show that calcitriol, which is the hormonally active form of vitamin D, and its therapeutically used analog, eldecalcitol, inhibit bone resorption by modulating this mechanism. Vitamin D analogs have been used clinically for treating osteoporosis, although the mode of its pharmacologic action remains to be fully elucidated. In this study, we found that active vitamin D reduced the expression of S1PR2, a chemorepulsive receptor for blood S1P, on circulating osteoclast precursor monocytes both in vitro and in vivo. Calcitriol- or eldecalcitol-treated monocytoid RAW264.7 cells, which display osteoclast precursor-like properties, migrated readily to S1P. Concordantly, the mobility of circulating CX3CR1+ osteoclast precursor monocytes was significantly increased on systemic administration of active vitamin D. These results show a mechanism for active vitamin D in controlling the migratory behavior of circulating osteoclast precursors, and this action should be conducive to limiting osteoclastic bone resorption in vivo.

Daily administration of eldecalcitol (ED-71), an active vitamin D analog, increases bone mineral density by suppressing RANKL expression in mouse trabecular bone.

Eldecalcitol (ED‐71) is a new vitamin D3 derivative recently approved for the treatment of osteoporosis in Japan. Previous studies have shown that the daily administration of ED‐71 increases bone mineral density (BMD) by suppressing bone resorption in various animal models. In this study, we examined how ED‐71 suppresses bone resorption in vivo, by analyzing bone histomorphometry and ex vivo osteoclastogenesis assays. Daily administration of ED‐71 (50 ng/kg body weight) to 8‐week‐old male mice for 2 and 4 weeks increased BMD in the femoral metaphysis without causing hypercalcemia. Bone and serum analyses revealed that ED‐71 inhibited bone resorption and formation, indicating that the increase in BMD is the result of the suppression of bone resorption. This suppression was associated with a decrease in the number of osteoclasts in trabecular bone. We previously identified cell cycle‐arrested receptor activator of NF‐κB (RANK)‐positive bone marrow cells as quiescent osteoclast precursors (QOPs) in vivo. Daily administration of ED‐71 affected neither the number of RANK‐positive cells in vivo nor the number of osteoclasts formed from QOPs in ex vivo cultures. In contrast, ED‐71 suppressed the expression of RANK ligand (RANKL) mRNA in femurs. Immunohistochemical experiments also showed that the perimeter of the RANKL‐positive cell surface around the trabecular bone was significantly reduced in ED‐71‐treated mice than in the control mice. ED‐71 administration also increased BMD in 12‐week‐old ovariectomized mice, through the suppression of RANKL expression in the trabecular bone. These results suggest that the daily administration of ED‐71 increases BMD by suppressing RANKL expression in trabecular bone in vivo.

Comparison of effects of alfacalcidol and alendronate on mechanical properties and bone collagen cross-links of callus in the fracture repair rat model.

Both bone density and quality are important determinants of bone strength. Bone quality is prescribed by matrix characteristic including collagen cross-linking and bone structural characteristics and is important in reinforcement of bone strength. We investigated the effects of alfacalcidol (ALF), a prodrug of calcitriol, and alendronate (ALN), a bisphosphanate, on the mechanical properties and content of enzymatic cross-links in femoral bone using a fracture repair rat model. Forty 3-month-old female Wistar-Imamichi rats were randomized into 4 groups: SHAM (sham-operated+vehicle), OVX (ovariectomy+vehicle), ALF (ovariectomy+ALF, 0.1 microg/kg/d, p.o.) and ALN (ovariectomy+ALN, 10 microg/kg/d, s.c.). Treatment began immediately after SHAM or OVX surgery. Three weeks later, all animals underwent transverse osteotomies at the midshaft of the left femur. Treatment was continued and rats were sacrificed at 12 weeks post-fracture for evaluation by X-ray radiography, micro-CT, pQCT, biomechanical testing and bone histomorphometry. In the ALN group, no new cortical shell appeared and the callus diameter was significantly larger than in the OVX group (p<0.05). Stiffness of fractured callus in the ALF group, but not in the ALN group, was significantly higher than in the OVX group. Youngs modulus in the ALN group was significantly decreased compared to the OVX group. Moreover, micro-CT analysis showed that ALN treatment increased the lowly mineralized bone in the callus by, resulting in the highest content of woven bone area and lowest content of lamellar bone. The total amount of enzymatic cross-links in both the ALF and ALN groups was significantly higher than in the OVX control group. Of particular interest, the Pyr-to-Dpyr ratio was significantly decreased by ALF administration, suggesting that ALF but not ALN normalized the enzymatic cross-link patterns in fractured bone to the control level. In conclusion, ALN and ALF treatment increased bone strength via the distinctive effect on bone mass and quality. ALN formed larger calluses and increased enzymatic cross-links despite delayed woven bone remodeling into lamellar bone, whereas ALF treatment induced lamellar bone formation coincided with increasing in the enzymatic cross-linking and normalizing the cross-link pattern in callus to native bone pattern.

Combination therapy with eldecalcitol and alendronate has therapeutic advantages over monotherapy by improving bone strength

Eldecalcitol (ED-71), a 2β-hydroxypropyloxy derivative of 1α,25(OH)(2)D(3), inhibits bone resorption more potently than does alfacalcidol while maintaining osteoblastic function in an estrogen-deficient, high-turnover osteoporosis rat model. Alendronate (ALN) has been reported to increase bone mass by suppressing bone resorption mainly by inducing apoptosis of osteoclasts. The aim of this study was to clarify the combination effect of ED-71 and ALN on bone loss in ovariectomized rats. Wistar-Imamichi rats (32weeks old) were ovariectomized and randomly assigned to 10 groups (n=9-11); 11 rats were sham-operated. Rats were orally administered either vehicle alone, ALN (0.05, 0.2mg/kg), ED-71 (0.015, 0.03μg/kg), or a combination of ALN and ED-71. The treatment started 2weeks after surgery and continued for 12weeks. ED-71 significantly increased calcium and phosphorus in serum and urine; however, the mean values were within the normal range. Bone mineral density (BMD) and maximum load in both the lumbar spine and femur significantly increased with ED-71 monotherapy, and showed a tendency to increase with ALN monotherapy. Compared with ALN monotherapy, the combination of ALN and ED-71 significantly increased BMD and maximum load in both the lumbar spine and femur, suggesting that the combination therapy is more beneficial than ALN monotherapy in this protocol. The combination treatment had an additive suppressive effect on eroded surface and osteoclast number, with the suppressive effect more potent than either ALN or ED-71 monotherapy. Moreover, the combination therapy partially counteracted the suppressive effects of ALN on bone formation and on the histomorphometric indices of osteoblast number and activity. Interestingly, ALN had no effect on the anabolic action of ED-71. In conclusion, the combination therapy of ALN and ED-71 has therapeutic advantages over ALN monotherapy in terms of improving bone mechanical strength without excessive suppression of bone turnover.

Vitamin D Receptor Signaling Enhances Locomotive Ability in Mice

Bone fractures markedly reduce quality of life and life expectancy in elderly people. Although osteoporosis increases bone fragility, fractures frequently occur in patients with normal bone mineral density. Because most fractures occur on falling, preventing falls is another focus for reducing bone fractures. In this study, we investigated the role of vitamin D receptor (VDR) signaling in locomotive ability. In the rotarod test, physical exercise enhanced locomotive ability of wild‐type (WT) mice by 1.6‐fold, whereas exercise did not enhance locomotive ability of VDR knockout (KO) mice. Compared with WT mice, VDR KO mice had smaller peripheral nerve axonal diameter and disordered AChR morphology on the extensor digitorum longus muscle. Eldecalcitol (ED‐71, ELD), an analog of 1,25(OH)2D3, administered to rotarod‐trained C57BL/6 mice enhanced locomotor performance compared with vehicle‐treated nontrained mice. The area of AChR cluster on the extensor digitorum longus was greater in ELD‐treated mice than in vehicle‐treated mice. ELD and 1,25(OH)2D3 enhanced expression of IGF‐1, myelin basic protein, and VDR in rat primary Schwann cells. VDR signaling regulates neuromuscular maintenance and enhances locomotive ability after physical exercise. Further investigation is required, but Schwann cells and the neuromuscular junction are targets of vitamin D3 signaling in locomotive ability.

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

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 (0.3mg/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.

Histological Effects of the Combined Administration of Eldecalcitol and a Parathyroid Hormone in the Metaphyseal Trabeculae of Ovariectomized Rats

Summary Intermittent administration of human parathyroid hormone (1-34) (hPTH(1-34)) promotes anabolic action in bone by stimulating bone remodeling, while eldecalcitol, an analog of active vitamin D3, suppresses osteoclastic bone resorption, and forms new bone by minimodeling. We have examined the biological effects of combined administration of eldecalcitol and hPTH(1-34) on 9-week-old Wistar rats that underwent an ovariectomy (OVX) or Sham operation. They were divided into a Sham group, OVX with vehicle (OVX group), OVX with 10 µg/kg/day of hPTH(1-34) (PTH group), OVX with 20 ng/kg/day of eldecalcitol (eldecalcitol group) or OVX with 10 μg/kg/day of hPTH(1-34), and 20 ng/kg/day of eldecalcitol (combined group) for 4 or 8 weeks. As a consequence, the combined group showed a marked increase in bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) than OVX and had the highest bone mineral density (BMD) compared with other groups. OVX and PTH groups exhibited a high osteoblastic surface/bone surface (Ob.S/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS) indices and many TRAP-reactive osteoclasts. Contrastingly, eldecalcitol and combined groups tended to attenuate the indices of osteoclastic surface/bone surface (Oc.S/BS) and Ob.S/BS than that the other groups. The combined group revealed histological profiles of minimodeling- and remodeling-based bone formation. Thus, the combined administration of eldecalcitol and hPTH(1-34) augments their anabolic effects by means of minimodeling and remodeling.