1alpha,25-Dihydroxyvitamin D(3) (1alpha,25-D(3)) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, its calcemic effect in vivo limits its therapeutic applications. Here, we report the efficacy of 22-oxa-1alpha,25-dihydroxyvitamin D(3) (22-oxa-1alpha,25-D(3)), a low calcemic analog of vitamin D, against the development of metastatic lung carcinoma after an intravenous injection of green fluorescent protein-transfected Lewis lung carcinoma (LLC-GFP) cells in C57BL/6 mice. The mice injected with tumor cells were implanted simultaneously with osmotic minipumps containing either 1alpha,25-D(3), 22-oxa-1alpha,25-D(3) or vehicle. The 1alpha,25-D(3) treatment group had been hypercalcemic, but the 22-oxa-1alpha,25-D(3) and vehicle treatment groups remained normocalcemic for the duration of the experiment. The total number of lung metastases, lung weight and the expression of GFP mRNA in the lung were markedly decreased in 1alpha,25-D(3) and 22-oxa-1alpha,25-D(3)-treated mice. In the in vitro experiment, 1alpha,25-D(3) and 22-oxa-1alpha,25-D(3) reduced the expression of matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor and parathyroid hormone-related protein in LLC-GFP cells. Furthermore, in the angiogenesis assay, the number of tumor cells or basic fibroblast growth factor-induced angiogenesis was reduced in 1alpha,25-D(3) and 22-oxa-1alpha,25-D(3)-treated mice. Moreover, using a new experimental model of vitamin D receptor (VDR) null mutant (VDR(-/-)) mice with corrected hypocalcemia and hypervitaminosis D, we examine the anti-cancer effect of 22-oxa-1alpha,25-D(3) without other functions induced by 22-oxa-1alpha,25-D(3) in the host. In the VDR(-/-) mice, 22-oxa-1alpha,25-D(3) directly inhibited the metastatic activity of LLC-GFP cells in a dose-dependent manner without exerting a direct influence on the calcemic activity or other actions regulated by 22-oxa-1alpha,25-D(3) in the host. These results indicate that the inhibition of metastasis and angiogenesis-inducing activity in cancer cells seemed to be a major mechanism responsible for the anti-cancer effects of 22-oxa-1alpha,25-D(3). Our findings show that 22-oxa-1alpha,25-D(3) is beneficial for the prevention of metastasis in lung carcinoma.

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1, 2). 1␣,25(OH) 2 D 3 has potent anti-proliferative and cell differentiation-inducing activities in addition to its role in calcium homeostasis. After the expression of various biological activities, 1␣,25(OH) 2 D 3 is further metabolized through the C-24 (3-6)/C-23 (7-10) oxidation pathways and the C-3 epimerization pathway (11-14). The C-24 oxidation pathway, initiated by C-24 hydroxylation, leads to the conversion of 1␣,25(OH) 2 D 3 into a side chain cleavage product, calcitroic acid (4, 5). The C-23 oxidation pathway, initiated by C-23 hydroxylation, leads to the formation of 1␣,25(OH) 2 D 3 -26,23-lactone (7-10). The newly discovered C-3 epimerization pathway leads to the conversion of the configuration of the hydroxyl group at C-3 of the A-ring and produces 3-epi-1␣,25(OH) 2 D 3 from 1␣,25(OH) 2 D 3 . In view of this modification at the A-ring, the C-3 epimerization pathway is quite different from side chain oxidation pathways.The C-3 epimerization of 1␣,25(OH) 2 D 3 was observed in human colon carcinoma-derived Caco-2 cells (11), bovine parathyroid cells (12), rat osteoblastic UMR 106 and Ros17/2.8 cells (13), and various cultured cell lines (14). From these studies, the C-3 epimerization pathway is assumed to be cell-selective. It was considered that the C-3 epimerization pathway is cell differentiation-related in Caco-2 cells, because 3-epi-1␣,25 (OH) 2 D 3 was only observed in confluent, quiescent Caco-2 cells, not proliferating Caco-2 cells (11). 3-Epi-1␣,25(OH) 2 D 3 was also isolated as a circulating metabolite of 1␣,25(OH) 2 D 3 in rats treated with pharmacological doses of 1␣,25(OH) 2 D 3 (15). In addition, synthetic analogs of 1␣,25(OH) 2 D 3 , e.g. 22-oxacalcitriol (16), 20-epi-1␣,25(OH) 2 D 3 (17), and 1␣,25(OH) 2 -16-ene-23-yne-D 3 (18), have been reported to be metabolized to their * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, a grant for Cooperative Research administered by the Japan Private School Promotion Foundation, and a grant-in-aid from the Ministry of Health and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ § To whom correspondence should be addressed: Dept.

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 microg/kg BW) vis-à-vis vitamin D(3) (50-400 microg/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 D(3) at given urinary and serum Ca levels: larger doses of vitamin D(3) 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 D(3); 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 D(3), 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 D(3) in the treatment of osteoporosis.

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 17␤-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. 17␤-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 17␤-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. (J Bone Miner Res 2000;15:770 -779)