Calcium supplementation does not reproduce the pharmacological efficacy of alfacalcidol for the treatment of osteoporosis in rats.

The purpose of this study was to assess whether a nutritional supply of calcium (Ca) could be substituted for alfacalcidol (ALF) administration in preventing bone loss due to estrogen deficiency. Female Wistar-Imamichi rats (8 months old) were ovariectomized (OVX) or sham-operated. OVX rats received ALF administration (0.025, 0.5, or 0.1 microg/kg, p.o., 5 times a week) with standard rodent chow [Ca 1.2%, phosphorus (P) 1.04%], a Ca-enriched diet containing 2%, 4%, or 6% Ca (Ca/P ratio of 2, 4, and 6, respectively), or a Ca/P-enriched diet (Ca/P ratio of 1.2). After 12 weeks of treatment, all rats were killed to harvest the spine, serum, and urine samples. Neither the ALF treatment nor the Ca supplement caused hypercalcemia. In the spine, ALF prevented decreases in bone mineral density (BMD) and compressive strength of lumbar spine induced by OVX. Micro-computed tomographic analysis confirmed that ALF significantly improved the trabecular bone pattern factor and the structure model index and suppressed bone destruction. In contrast, of particular interest, high-dose Ca administration did not have marked effects on bone fragility. Also, when both Ca and P were administered in high doses, BMD and mechanical strength decreased dose-dependently, urinary P excretion significantly increased, and serum parathyroid hormone level increased. Together, it is difficult to adjust the Ca supply through diet alone without disrupting the balance between serum Ca and P levels. Consequently, we conclude that ALF is beneficial for the treatment of osteoporosis, which is not achieved by the use of a Ca supplement.

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, 1alpha,25-dihydroxy-2beta-(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 1alpha-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 microgram) 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.

Anabolic effects of recombinant human parathyroid hormone (1 - 84) and synthetic human parathyroid hormone (1 - 34) on the mandibles of osteopenic ovariectomized rats with maxillary molar extraction.

In rodent osteoporosis models such as ovariectomized (OVX) rats, intermittently administered human parathyroid hormone (hPTH) has an anabolic effect in vertebrae and long bones. In the present experiments, subcutaneously injected hPTH(1 - 34) or hPTH(1 - 84) dose- and time-dependently increased bone mineral density (BMD) as measured by dual energy X-ray absorptiometry in mandibles, L2 to L4 vertebrae and femurs of such rats. The highest dose (15.9 nmol/kg, s. c.) of either peptide given four times weekly for 10 weeks completely reversed the effects of overiectomy on BMD. Significant elevation in lumbar BMD after 10 weeks was observed with hPTH(1 - 34) or hPTH(1 - 84) at 1.1 nmol/kg, whereas hPTH(1 - 34) at 1.1 and 4.2 nmol/kg significantly increased BMD of the whole bone and the metaphysis of the femur and the diaphysis of the bone, respectively. In contrast, significant effects of hPTH(1 - 84) administration on BMD increase in the femur were observed at 4.2 and 15.9 nmol/kg in the whole bone and the metaphysis, and in the diaphysis, respectively. Maxillary molar extraction left mandibular BMD in rats with intact ovaries unchanged, but significantly decreased mandibular BMD in OVX rats. Administration of hPTH(1 - 84) for 10 weeks in OVX rats without or with extraction significantly increased BMD in the mandibular molar region at doses of 15.9 and 4.2 nmol/kg, respectively, indicating that efficacy was increased by extraction. A significant BMD increase in the molar region in OVX rats with extraction occurred at only 1.1 nmol/kg of hPTH(1 - 34) and 4.2 nmol/kg of hPTH(1 - 84). Also, BMD of the ramus region was increased by administration of both peptides to a lesser extent than that of the molar region in these rats. Thus, intermittent administration of hPTH, especially hPTH(1 - 34), has an anabolic effect on bone, particularly alveolar bone. Such treatment may increase alveolar bone mass in postmenopausal women with osteoporosis.

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.

Isolation, identification and biological activity of 24R,25-dihydroxy-3-epi-vitamin D3: a novel metabolite of 24R,25-dihydroxyvitamin D3 produced in rat osteosarcoma cells (UMR 106).

We recently identified 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)2-3-epi-D3] as a metabolite of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] produced in rat osteosarcoma cells (UMR 106). We now report the isolation of 24R,25-dihydroxy-3-epi-vitamin D3 [24R,25(OH)2-3-epi-D3] as a metabolite of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] by high-performance liquid chromatography (HPLC) with chiral column and its structure assignment by proton nuclear magnetic resonance (1H-NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. We also demonstrated the production of 24R,25(OH)2-3-epi-D, in two other cell lines [human colon carcinoma cells (Caco-2) and porcine kidney cells (LLC-PK1)] which were previously shown to convert 1alpha,25(OH)2D3 into 1alpha,25(OH)2-3-epi-D3. It can be seen that the production of 24R,25(OH)2- 3-epi-D3 from 24R,25(OH)2D3 is lower than that of 1alpha,25(OH)2-3-epi-D3 from 1alpha,25(OH)2D3 in all the cells studied. 24R,25(OH)2-3-epi-D3 was found to be inactive in terms of its ability to bind to the vitamin D receptor (VDR), in inhibiting proliferation and in inducing differentiation of human promyelocytic leukemia cells (HL-60). Thus, our study indicates that the C-3 epimerization pathway is common to both 1alpha,25(OH)2D3 and 24R,25(OH)2D3 and may play an important role in modulating the concentration and the biological activity of these two major vitamin D3 metabolites in target tissues.

Asymmetric catalytic ene-cyclization approach to 2-fluoro-19-nor-1,25-dihydroxyvitamin D(3) A-ring analog with significant transactivation activity.

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)2D3) has been shown to modulate not only proliferation and differentiation, but also apoptosis in malignant cells, indicating that it could be useful for the treatment of cancer and psoriasis. However, little information has been available on the binding conformation of the 1alpha,25(OH)2D3 molecule and its analogs with the vitamin D receptor (VDR). Therefore, we synthesized 2alpha-fluorinated A-ring analogs of 19-nor-1alpha,25(OH)2D3 in order to investigate the VDR-binding conformation of the A-rings on the basis of the (19)F NMR analysis. The 2alpha-fluoro-19-nor-1alpha,25-dihydroxyvitamin D3 A-ring analog thus synthesized via a asymmetric catalytic carbonyl-ene cyclization, shows significant activity in transactivation.

Synthesis and evaluation of A-ring diastereomers of 1alpha,25-dihydroxy-22-oxavitamin D3 (OCT).

A-ring diastereomers of 1alpha,25-dihydroxy-22-oxavitamin D3 (OCT) (2), 3-epi-1alpha,25-dihydroxy-22-oxavitamin D3 (3-epiOCT) (3) and 1,3-diepi-1alpha,25-dihydroxy-22-oxavitamin D3 (1,3-diepiOCT) (4) were synthesized by the convergent method. In vitro binding affinity for rat vitamin D binding protein and calf-thymus vitamin D receptor, differentiation-inducing activity on HL-60 cells, and transcriptional activity of 3-epiOCT (3) and 1,3-diepiOCT (4) were evaluated in comparison with OCT (2), 1-epi-1alpha,25-dihydroxy-22-oxavitamin D3 (1-epiOCT) (5) and 1alpha,25-dihydroxyvitamin D3 (1).

Synthesis and biological characterization of 1alpha,24,25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (24-hydroxylated ED-71).

24-Hydroxylated derivatives were synthesized in 24(R) and 24(S) forms by the convergent method as analogs related to 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3). In the convergent synthesis, the A-ring fragment, synthesized from diethyl D-tartarate, and the C/D-ring fragments in 24(R) and 24(S) forms (vitamin D numbering), obtained from vitamin D(2) via the Inhoffen-Lythgoe diol, were coupled in moderate yields to give 1alpha,24(R),25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) and 1alpha,24(S),25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3). In preliminary biological evaluations, 24-hydroxylation of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) caused weakened affinity to vitamin D binding protein in vitro and less calcemic activity in vivo compared to the parent compound. While the affinity to vitamin D receptor in 24(R) epimer was sustained, the affinity in 24(S) epimer was less than that of the parent compound.

Biological activities of 19-nor-1alpha,25-dihydroxyvitamin D3 analogs singly dehydroxylated at the C-1 or C-3 position of the A-ring.

Growing interests have been focused on the development of hybrid-analogs with modifications of the A-ring and the side chain of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. An exocyclic methylene group at C-10, a hydroxy group at C-1 and a hydroxy group at C-3 play a crucial role in the expression of biological activities of 1alpha,25(OH)2D3. However, relationship between the functional groups and activities has not been fully understood. We have synthesized and evaluated biological activities of several singly dehydroxylated A-ring analogs of 19-nor-1alpha,25(OH)2D3 and 19-nor-22-oxa-1alpha,25(OH)2D3. All of them have an extremely low binding affinity for vitamin D receptor (VDR). Some of them lack the 1alpha-hydroxy group that is considered to be essential for VDR-mediated gene expression, have greater or equivalent potencies to 1alpha,25(OH)2D3 for inducing differentiation and cell cycle G0-G1 arrest of human promyelocytic leukemia cells as well as for the transactivation of target genes including a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter and a human osteocalcin gene promoter in transfected mammalian cells. The assessment of a ligand/VDR/Retinoid X receptor complex formation using a two-hybrid luciferase assay revealed that the liganded VDR has high potency to form a heterodimer, but this could not explain the high biological potency of the 19-nor analogs. Other reason(s) including an interaction with transcriptional cofactors should be considered to explain the mechanism of action of 19-nor analogs.

Characterization of new conjugated metabolites in bile of rats administered 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3).

The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25-dihydroxyvitamin D(3) to rats, 23, 25-dihydroxy-24-oxovitamin D(3) 23-glucuronide, 3-epi-24, 25-dihydroxyvitamin D(3) 24-glucuronide, and 24,25-dihydroxyvitamin D(3) 3-sulfate were obtained as new biliary metabolites together with 24,25-dihydroxyvitamin D(3) 3- and 24-glucuronides. The above metabolites, except 24,25-dihydroxyvitamin D(3) 3-glucuronide, were obtained from rats dosed with 25-hydroxyvitamin D(3). 23, 25-Dihydroxyvitamin D(3) 23-glucuronide was also obtained from the bile of rats administered 25-hydroxyvitamin D(3) in addition to its 3-glucuronide, 25-glucuronide, and 3-sulfate. Thus, it was found that 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were directly conjugated as glucuronide and sulfate, whereas at the C-23 position, they were hydroxylated and then conjugated. Furthermore, we found that the C-3 epimerization acts as one of the important pathways in vitamin D metabolism.

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 17beta-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. 17beta-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 17beta-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.

Synthesis and pharmacokinetics of 1 alpha-hydroxyvitamin D3 tritiated at 22 and 23 positions showing high specific radioactivity.

A novel synthesis of a radioactive compound of 1 alpha-hydroxyvitamin D3 (1 alpha OHD3) (1) and its pharmacokinetics are described. Radioactive 1 alpha OHD3 tritiated at 22 and 23 positions ([22,23-(3)H4]1 alpha OHD3) (5) was prepared via key reactions of the reduction of acetylenic side chain in the ketone (12) with tritium gas in the presence of palladium-charcoal and the subsequent Wittig reaction with the A-ring synthon (16). [22,23-(3)H4]1 alpha OHD3 (5) showed high specific radioactivity (111.5 Ci/mmol) and was used successfully in pharmacokinetics studies with rats. In the pharmacokinetics studies, the plasma concentration level of the active form of vitamin D3, 1 alpha,25-dihydroxy-vitamin D3 [1 alpha,25(OH)2D3], after oral or intravenous administration of [22,23-(3)H4]1 alpha OHD3 (5), showed longer half-life, lower maximum concentration, and lower area under the curve than those after treatment of 1 alpha,25(OH)2D3 tritiated at 26 and 27 positions (4). These results might suggest a beneficial therapeutic utility of 1 alpha OHD3 (1) over the treatment of 1 alpha,25(OH)2D3 (2).

In vitro biological activities of a series of 2 beta-substituted analogues of 1 alpha,25-dihydroxyvitamin D3.

Biological activities of a series of 2beta-substituted analogues of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] were evaluated in vitro in terms of their binding affinity with regard to calf thymus cytosolic vitamin D receptor (VDR) and rat plasma vitamin D-binding protein (DBP). Additionally, reporter gene luciferase activities using either a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), in transfected rat osteoblast-like ROS17/2.8 cells, or a human VDR-GAL4 modified two-hybrid system in transfected human epitheloid carcinoma, cervix HeLa cells were examined. Binding affinity for VDR, transactivation potency on the target gene and VDR-mediated gene regulation of the hydroxyalkyl and hydroxyalkoxy 2beta-substituted analogues were almost comparable to those of 1alpha,25(OH)2D3, while the alkyl and alkenyl analogues were much less active than 1alpha,25(OH)2D3. This study investigated the biological evaluation of a series of 2beta-substituted analogues at the molecular level, with regard to the structural differences of alkyl, alkenyl, hydroxyalkyl, hydroxyalkoxy, alkoxy, hydroxy and chloro substituents at the 2beta-position of 1alpha,25(OH)2D3.

Catalytic asymmetric syntheses and biological activities of singly dehydroxylated 19-nor-1alpha,25-dihydroxyvitamin D(3) A-ring analogs in cancer cell differentiation and apoptosis.

BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) has been shown to modulate not only proliferation and differentiation, but also apoptosis in malignant cells, indicating that it could be useful for treating cancer. Little information is available concerning the structural motifs of the 1alpha, 25(OH)(2)D(3) molecule responsible for modulation of differentiation and apoptosis, however. We set out to synthesize singly dehydroxylated A-ring analogs of 19-nor-1alpha,25(OH)(2)D(3) in a catalytic asymmetric fashion, and to investigate their biological activities in leukemia HL-60 cells. RESULTS: A series of singly dehydroxylated 19-nor-1alpha,25-dihydroxyvitamin D(3) A-ring analogs were synthesized using a combinatiorial sequence of regioselective propiolate-ene reaction and catalytic asymmetric carbonyl-ene cyclization. Surprisingly, the analogs could be clearly divided into two categories; one group, bearing 1alpha-hydroxy or 3beta-hydroxy groups in the A-ring, were potent differentiators and the second group, bearing 1beta-hydroxy or 3alpha-hydroxy groups, were potent stimulators of apoptosis. CONCLUSIONS: We have clearly identified the structural motifs of 19-nor-1alpha,25(OH)(2)D(3) analogs responsible for differentiation and apoptosis in HL-60 cells. These findings will provide useful information not only for development of therapeutic agents for treatment of leukemia and other cancers, but also for structure-function studies of 1alpha,25(OH)(2)D(3).

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.

Rapid control of transmembrane calcium influx by 1alpha,25-dihydroxyvitamin D3 and its analogues in rat osteoblast-like cells.

1alpha,25-Dihydroxyvitamin D3 [1alpha,25(OH)2D3] has been shown to exert both its nuclear vitamin D receptor (nVDR)-mediated genomic actions and membrane vitamin D receptor (mVDR)-mediated nongenomic actions. In this study, the effects of 1alpha,25(OH)2D3 and its analogues on transmembrane Ca2+ influx were examined in the growth phase of rat osteosarcoma ROS17/2.8 cells. Like BAYK8644 (2 x 10(-5)M), a well-known L-type Ca2+ channel agonist, 1alpha,25(OH)2D3 (10(-8)M) increased transmembrane influx of Ca2+ through voltage-dependent Ca2+ channels and increased intracellular Ca2+ concentration within 2 min of addition to the medium. The 1alpha,25(OH)2D3-induced Ca2+ influx was completely blocked by pre-treatment with nifedipine (2 x 10(-5)M), an L-type Ca2+ channel antagonist. Two vitamin D analogues, 22-oxa-1alpha,25(OH)2D3 (OCT, 10(-8) M) and 20-epi-22-oxa-24a, 26a,27a-trihomo-1alpha,25(OH)2D3 (KH1060, 10(-8)M), which were 3.8 and 3600-fold more active than 1alpha,25(OH)2D3 in stimulating differentiation on human promyelocytic leukemic HL-60 cells, respectively, also increased intracellular Ca2+ concentration, while their Ca2+ channeling activities were similar to or significantly weaker than that of 1alpha,25(OH)2D3. Furthermore, the enhanced transmembrane Ca2+ influx induced by 1alpha,25(OH)2D3 (10(-8)M) or OCT (10(-8)M) was completely blocked by pre-treatment with the respective 1beta epimer [1beta,25(OH)2D3 and 1beta-OCT] at equal concentration. These findings suggest that 1alpha,25(OH)2D3 and its analogues modulate transmembrane Ca2+ influx in osteoblast-like cells by opening L-type Ca2+ channels which can recognize 1alpha-hydroxy analogues as agonists and 1beta-hydroxy analogues as antagonists.

The advantage of alfacalcidol over vitamin D in the treatment of osteoporosis.

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.

New metabolic pathway of (24R)-24,25-dihydroxyvitamin D3: epimerization of the 3-hydroxy group.

A new metabolic pathway of (24R)-24,25-dihydroxyvitamin D3 [24,25(OH)2D3] was clarified in the in vivo experiments. After the administration of 24,25(OH)2D3 to rats, a new monoglucuronide of a vitamin D metabolite was obtained from the bile together with 24,25(OH)2D3 3- and 24-glucuronides. The genin of the metabolite was identified as 3-epi-24,25(OH)2D3 in comparison with the synthetic sample based on the data from 1H-NMR, GC/MS, and LC/atmospheric pressure chemical ionization-MS. The conjugation position was determined to be the 24-hydroxy group by the LC/electrospray ionization-MS and -MS/MS/MS combined with derivatization. To our knowledge, this is the first reported instance of the epimerization of the 3-hydroxy group of vitamin D compound with no hydroxy group at the 1alpha-position.

22-oxacalcitriol suppresses secondary hyperparathyroidism without inducing low bone turnover in dogs with renal failure.

BACKGROUND: Calcitriol therapy suppresses serum levels of parathyroid hormone (PTH) in patients with renal failure but has several drawbacks, including hypercalcemia and/or marked suppression of bone turnover, which may lead to adynamic bone disease. A new vitamin D analogue, 22-oxacalcitriol (OCT), has been shown to have promising characteristics. This study was undertaken to determine the effects of OCT on serum PTH levels and bone turnover in states of normal or impaired renal function. METHODS: Sixty dogs were either nephrectomized (Nx, N = 38) or sham-operated (Sham, N = 22). The animals received supplemental phosphate to enhance PTH secretion. Fourteen weeks after the start of phosphate supplementation, half of the Nx and Sham dogs received doses of OCT (three times per week); the other half were given vehicle for 60 weeks. Thereafter, the treatment modalities for a subset of animals were crossed over for an additional eight months. Biochemical and hormonal indices of calcium and bone metabolism were measured throughout the study, and bone biopsies were done at baseline, 60 weeks after OCT or vehicle treatment, and at the end of the crossover period. RESULTS: In Nx dogs, OCT significantly decreased serum PTH levels soon after the induction of renal insufficiency. In long-standing secondary hyperparathyroidism, OCT (0.03 microg/kg) stabilized serum PTH levels during the first months. Serum PTH levels rose thereafter, but the rise was less pronounced compared with baseline than the rise seen in Nx control. These effects were accompanied by episodes of hypercalcemia and hyperphosphatemia. In animals with normal renal function, OCT induced a transient decrease in serum PTH levels at a dose of 0.1 microg/kg, which was not sustained with lowering of the doses. In Nx dogs, OCT reversed abnormal bone formation, such as woven osteoid and fibrosis, but did not significantly alter the level of bone turnover. In addition, OCT improved mineralization lag time, (that is, the rate at which osteoid mineralizes) in both Nx and Sham dogs. CONCLUSIONS: These results indicate that even though OCT does not completely prevent the occurrence of hypercalcemia in experimental dogs with renal insufficiency, it may be of use in the management of secondary hyperparathyroidism because it does not induce low bone turnover and, therefore, does not increase the risk of adynamic bone disease.

In vivo nuclear uptake of a vitamin D analog (OCT) in different tumor cell populations of FA-6 cancer xenograft in nude mice by receptor autoradiography.

1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3] and its analogs have been shown to repress the production of parathyroid hormone-related peptide (PTHrP) in tumors, which is a major factor causing humoral hypercalcemia associated with various cancers. Since vitamin D analogs may be applicable to the treatment of cancer patients, the present study was undertaken to examine whether OCT, an analog with little calcemic activity, is incorporated into tumor tissues, and to identify cellular and subcellular sites of its specific uptake and retention. [26-3H]OCT was injected i.v. into nude mice inoculated with a human pancreatic carcinoma cell line (FA-6). At 1 hour after the injection, intracellular concentration of radioactivity was visualized by receptor (thaw-mount) autoradiography. The results indicate a heterogeneous distribution of radioactivity in nuclei of certain large cancer cells as well as in single or clustered small and elongated cells within the tumor, while connective tissue cells outside of the tumor remained free of nuclear labeling. The data suggest that OCT acts selectively at the genome of cancer cells during a certain maturational stage and also of a second population of small fibroblast-like cells that may have been transplanted with the tumor or are host-derived.