Noboru Kubodera

Alfacalcidol Inhibits Bone Resorption and Stimulates Formation in an Ovariectomized Rat Model of Osteoporosis: Distinct Actions from Estrogen

Although alfacalcidol has been widely used for the treatment of osteoporosis in certain countries, its mechanism of action in bone, especially in the vitamin D–replete state, remains unclear. Here we provide histomorphometric as well as biochemical evidence that alfacalcidol suppresses osteoclastic bone resorption in an ovariectomized rat model of osteoporosis. Furthermore, when compared with 17β‐estradiol, a representative antiresorptive drug, it is evident that alfacalcidol causes a dose‐dependent suppression of bone resorption, and yet maintains or even stimulates bone formation, as reflected in increases in serum osteocalcin levels and bone formation rate at both trabecular and cortical sites. 17β‐Estradiol, which suppresses bone resorption to the same extent as alfacalcidol, causes a parallel reduction in the biochemical and histomorphometric markers of bone formation. As a final outcome, treatment with alfacalcidol increases bone mineral density and improves mechanical strength more effectively than 17β‐estradiol, with a more pronounced difference in cortical bone. We conclude that estrogens depress bone turnover primarily by suppressing bone resorption and, as a consequence, bone formation as well, whereas alfacalcidol “supercouples” these processes, in that it suppresses bone resorption while maintaining or stimulating bone formation.

ED-71, a vitamin D analog, is a more potent inhibitor of bone resorption than alfacalcidol in an estrogen-deficient rat model of osteoporosis

Although active vitamin D is used in certain countries for the treatment of osteoporosis, the risk of causing hypercalcemia/hypercalciuria means that there is only a narrow therapeutic window, and this has precluded worldwide approval. The results of our previous animal studies have suggested that the therapeutic effect of active vitamin D on bone loss after estrogen deficiency can be dissociated at least partly from its effect of enhancing intestinal calcium absorption and suppressing parathyroid hormone (PTH) secretion. To test this, we compared the effects of ED-71, a hydroxypropoxy derivative of 1alpha,25-dihydroxyvitamin D3, with orally administered alfacalcidol, on bone mineral density (BMD) and the bone remodeling process as a function of their effects on calcium metabolism and PTH, in a rat ovariectomy (ovx) model of osteoporosis. ED-71 increased bone mass at the lumbar vertebra to a greater extent than alfacalcidol, while enhancing calcium absorption (indicated by urinary calcium excretion) and decreasing serum PTH levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers. These results suggest that active vitamin D exerts an antiosteoporotic effect by inhibiting osteoclastic bone resorption while maintaining osteoblastic function, and that these anticatabolic/anabolic effects of active vitamin D take place independently of its effects on calcium absorption and PTH. The demonstration that ED-71 is more potent in these properties than alfacalcidol makes it an attractive candidate as an antiosteoporotic drug.

The Advantage of Alfacalcidol Over Vitamin D in the Treatment of Osteoporosis

Abstract. Although alfacalcidol is widely used in the treatment of osteoporosis, its mechanism of action in bone is not fully understood. Alfacalcidol stimulates intestinal calcium (Ca) absorption, increases urinary Ca excretion and serum Ca levels, and suppresses parathyroid hormone (PTH) secretion. It remains to be clarified, especially under vitamin D-replete conditions, whether alfacalcidol exerts skeletal effects solely via these Ca-related effects, whether the resultant suppression of PTH is a prerequisite for the skeletal actions of alfacalcidol, and, by inference, whether alfacalcidol has an advantage over vitamin D in the treatment of osteoporosis. To address these issues, we (1) compared the effects of alfacalcidol p.o. (0.025–0.1 μg/kg BW) vis-à-vis vitamin D3 (50–400 μg/kg BW) on bone loss in 8-month-old, ovariectomized (OVX) rats as a function of their Ca-related effects, and (2) examined whether the skeletal effects of alfacalcidol occur independently of suppression of PTH, using parathyroidectomized (PTX) rats continuously infused with hPTH(1–34). The results indicate that (1) in OVX rats, alfacalcidol increases BMD and bone strength more effectively than vitamin D3 at given urinary and serum Ca levels: larger doses of vitamin D3 are required to produce a similar BMD-increasing effect, in the face of hypercalcemia and compromised bone quality; (2) at doses that maintain serum Ca below 10 mg/dl, alfacalcidol suppresses urinary deoxypyridinoline excretion more effectively than vitamin D3; and (3) alfacalcidol is capable of increasing bone mass in PTX rats with continuous infusion of PTH, and therefore acts independently of PTH levels. It is suggested that alfacalcidol exerts bone-protective effects independently of its Ca-related effects, and is in this respect superior to vitamin D3, and that the skeletal actions of alfacalcidol take place, at least in part, independently of suppression of PTH. Together, these results provide a rationale for the clinical utility of alfacalcidol and its advantage over vitamin D3 in the treatment of osteoporosis.

Enhancement of PGI2 formation by a new vasodilator, 2-nicotinamidoethyl nitrate in the coupled system of platelets and aortic microsomes

Exogenous arachidonate addition to the coupled system of platelets and aortic microsomes resulted in production of TXA2 and PGI2 (detected as the stable degradation products, TXB2 and 6-keto PGF1 alpha, respectively). Imidazole, papaverine and dipyridamole increased PGI2 and decreased TXA2 in the coupled system. All of these agents inhibited TXA2 formation by platelets from arachidonate. Nitroglycerin did not show any effect on PGI2 and TXA2 formation in the coupled system and on TXA2 formation by platelets. In contrast with these compounds, in spite of showing no inhibitory effect on TXA2 formation by platelets alone, 2-nicotinamidoethyl nitrate (SG-75) increased PGI2 and decreased TXA2 in the coupled system. It is suggested that SG-75 accelerated the conversion of PGH2 to PGI2 so that smaller amounts of TXA2 was produced in the coupled system.

1,25-dihydroxyvitamin D3 as well as its analogue OCT lower blood calcium through inhibition of bone resorption in hypercalcemic rats with continuous parathyroid hormone-related peptide infusion.

The effects of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] and its analogue 22‐oxa‐1,25(OH)2D3 (22‐oxacalcitriol) (OCT) on calcium and bone metabolism were examined in an animal model of hypercalcemia with continuous infusion of parathyroid hormone–related peptide (PTHrP), to determine whether active vitamin D could counteract the skeletal action of PTHrP in addition to its reported effect in suppressing the production of PTHrP in cancer cells. Parathyroid glands were removed from 8‐week‐old Sprague–Dawley rats to eliminate the confounding effects of endogenous PTH. Animals were then continuously infused with human PTHrP(1–34) at a constant rate via osmotic minipumps for 2 weeks, and at the same time treated orally or intravenously with OCT or 1,25(OH)2D3 four to nine times during the 2‐week period. Under these conditions, OCT and, surprisingly, 1,25(OH)2D3 alleviated hypercalcemia in a dose‐dependent manner. 1,25(OH)2D3 and OCT suppressed the urinary excretion of deoxypyridinoline, although they did not affect renal calcium handling, suggesting that the antihypercalcemic effect is attributable to the inhibition of bone resorption. These active vitamin D compounds also counteracted the effects of PTHrP at the proximal renal tubules, as reflected by a decrease in phosphate excretion. Histomorphometric analysis of bone revealed a dose‐related decrease in parameters of bone resorption. These results suggest that 1,25(OH)2D3 as well as OCT has the potential to alleviate hypercalcemia, at least in part, through the inhibition of bone resorption in hypercalcemic rats with constant PTHrP levels. We propose that the main function of active vitamin D in high bone‐turnover states is to inhibit bone resorption, and this may have important implications for the understanding of the role of active vitamin D in the treatment of metabolic bone diseases, such as osteoporosis.

ED-71, a new active vitamin D3, increases bone mineral density regardless of serum 25(OH)D levels in osteoporotic subjects.

A previous randomized placebo-controlled double-blinded clinical trial revealed that treatment of osteoporotic subjects supplemented with 200 or 400IU/day vitamin D3 with 0.75 microg/day ED-71 for 12 months increased lumbar and hip bone mineral density (BMD) by 3.4 and 1.5%, respectively, compared to placebo group (JCE&M 90:5031,2005). These effects on BMD were stronger than any previous results using 1alpha(OH)D3 or 1,25(OH)2D3. However, there still was a concern that the effect of ED-71 could be observed because serum 25(OH)D in many of these subjects were below its optimal level. In order to address this issue, we performed post hoc analysis to compare the effect of ED-71 on lumbar and hip BMD between subjects with upper (>29 ng/mL) and lower tertiles (<25 ng/mL) of serum 25(OH)D. Lumbar BMD after 12-month treatment with 0.5, 0.75 and 1.0 microg/day ED-71 increased similarly in both lower and upper tertile groups of serum 25(OH)D. In addition, hip BMD also showed a tendency to increase when 0.75 and 1.0 microg/day ED-71 groups were combined together in both upper and lower serum 25(OH)D tertile groups, although the increase was not statistically significant. These results demonstrate that the effect of ED-71 on bone is independent of supplementary effect for nutritional vitamin D insufficiency, and suggest that ED-71 may exert its effect as a unique VDR ligand with stronger effect on bone compared to the natural ligand, 1,25(OH)2D3.

A new active vitamin D analog, ED-71, causes increase in bone mass with preferential effects on bone in osteoporotic patients

As a candidate for active vitamin D analogs that have selective effects on bone, 1α,25‐dihydroxy‐2β‐(3‐hydroxypropoxy)vitamin D3 (ED‐71) has been synthesized and is currently under clinical trials. In ovariectomized rat model for osteoporosis, ED‐71 caused an increase bone mass at the lumbar vertebra to a greater extent than 1α‐hydroxyvitamin D3 (alfacalcidol), while enhancing calcium absorption and decreasing serum parathyroid hormone levels to the same degree as alfacalcidol. ED‐71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers.An early phase II clinical trial was conducted with 109 primary osteoporotic patients. The results indicate that oral daily administration of ED‐71 (0.25, 0.5, 0.75, and 1.0 μg) for 6 months increased lumbar bone mineral density in a dose‐dependent manner without causing hypercalcemia and hypercalciuria. ED‐71 also exhibited a dose‐dependent suppression of urinary deoxypyridinoline with no significant reduction in serum osteocalcin. These results demonstrate that ED‐71 has preferential effects on bone with diminished effects on intestinal calcium absorption. ED‐71 offers potentially a new modality of therapy for osteoporosis with selective effects on bone. J. Cell. Biochem. 88: 286–289, 2003.

In Vitro Metabolism of the Vitamin D Analog, 22-Oxacalcitriol, Using Cultured Osteosarcoma, Hepatoma, and Keratinocyte Cell Lines

Using four cultured cell models representing liver, keratinocyte, and osteoblast, we have demonstrated that the vitamin D analog, 22-oxacalcitriol is degraded into a variety of hydroxylated and side chain truncated metabolites. Four of these metabolic products have been rigorously identified by high pressure liquid chromatography, diode array spectrophotometry, and gas chromatography-mass spectrometry analysis as 24-hydroxylated and 26-hydroxylated derivatives as well as the cleaved molecules, hexanor-1α,20-dihydroxyvitamin D and hexanor-20-oxo-1α-hydroxyvitamin D. Comparison with chemically synthesized standards has revealed the stereochemistry of the biological products. Although differences exist in the amounts of products formed with the different cell types, it is apparent that 22-oxacalcitriol is subject to metabolism by both vitamin D-inducible and noninducible enzymes. Time course studies suggest that the truncated 20-alcohol is derived from a side chain hydroxylated molecule via a hemiacetal intermediate and the 20-oxo derivative is likely formed from the 20-alcohol. Biological activity measurements of the metabolites identified in our studies are consistent with the view that these are catabolites and that the biological activity of 22-oxacalcitriol is due to the parent compound. These results are also consistent with recent findings of others that the biliary excretory form of 22-oxacalcitriol is a glucuronide ester of the truncated 20-alcohol.

Syntheses and preventive effects of analogues related to 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71) on bone mineral loss in ovariectomized rats

Analogues related to 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71) (2), 26,27-dimethyl ED-71 (3) and 26,27-diethyl ED-71 (4), were synthesized from lithocholic acid (5). In the study of the preventive effects of these analogues and ED-71 (2) on bone mineral loss in ovariectomized rats, 26,27-dimethyl ED-71 (3) showed the most potent activity.

Synthesis and biological evaluation of a 3-positon epimer of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71)

1alpha,25-Dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71), an analog of active vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], possesses a hydroxypropoxy substituent at the 2beta-position of 1,25(OH)(2)D(3). ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)(2)D(3). Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)(2)D(3), an epimer of 1,25(OH)(2)D(3) at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)(2)D(3) at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D(3) analogs were found to be 1,25(OH)(2)D(3)>ED-71> or =3-epi-1,25(OH)(2)D(3)>>3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.