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The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25D3), plays an important role in the maintenance of calcium (Ca) homeostasis, bone formation, and cell proliferation and differentiation via nuclear vitamin D receptor (VDR). It is formed by the hydroxylation of vitamin D at the 1α position by 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1) in the kidney. However, Cyp27b1-/- mice, deficient in CYP27B1, and VDR-deficient mice (Vdr-/-) have not been extensively examined, particularly in a comparative framework. To clarify the physiological significance of 1α,25D3 and VDR, we produced Cyp27b1-/- mice and compared their phenotypes with those of Vdr-/- mice. Cyp27b1-/- mice exhibited hypocalcemia, growth defects, and skeletogenesis dysfunction, similar to Vdr-/- mice. However, unlike Cyp27b1-/- mice, Vdr-/- mice developed alopecia. Cyp27b1-/- mice exhibited cartilage mass formation and had difficulty walking on hindlimbs. Furthermore, a phenotypic analysis was performed on Cyp27b1-/- mice provided a high Ca diet to correct for the Ca metabolic abnormality. In addition, the effects of 1α,25D3 that are not mediated by Ca metabolic regulatory activity were investigated. Even when the blood Ca concentration was corrected, abnormalities in growth and cartilage tissue formation did not improve in Cyp27b1-/- mice. These results suggested that 1α,25D3 directly controls chondrocyte proliferation and differentiation. Using Cyp27b1-/- mice produced in this study, we can analyze the physiological effects of novel vitamin D derivatives in the absence of endogenous 1α,25D3. Accordingly, this study provides a useful animal model for the development of novel vitamin D formulations that are effective for the treatment and prevention of osteoporosis.

Calcium (Ca) absorption from the intestinal tract is promoted by active vitamin D (1α,25D3). Vitamin D not only promotes Ca homeostasis, but it also inhibits bone resorption and promotes osteogenesis, thus playing a role in the maintenance of normal bone metabolism. Because 1α,25D3 plays an important role in osteogenesis, vitamin D formulations, such as alfacalcidol (ALF) and eldecalcitol (ELD), are used for treating osteoporosis. While it is known that, in contrast to ALF, ELD is an active ligand that directly acts on bone, the reason for its superior osteogenesis effects is unknown. Cyp27b1-knockout mice (Cyp27b1−/− mice) are congenitally deficient in 1α,25D3 and exhibit marked hypocalcemia and high parathyroid hormone levels, resulting in osteodystrophy involving bone hypocalcification and growth plate cartilage hypertrophy. However, because the vitamin D receptor is expressed normally in Cyp27b1−/− mice, they respond normally to 1α,25D3. Accordingly, in Cyp27b1−/− mice, the pharmacological effects of exogenously administered active vitamin D derivatives can be analyzed without being affected by 1α,25D3. We used Cyp27b1−/− mice to characterize and clarify the superior osteogenic effects of ELD on the bone in comparison with ALF. The results indicated that compared to ALF, ELD strongly induces ECaC2, calbindin-D9k, and CYP24A1 in the duodenum, promoting Ca absorption and decreasing the plasma concentration of 1α,25D3, resulting in improved osteogenesis. Because bone morphological measurements demonstrated that ELD has stronger effects on bone calcification, trabecular formation, and cancellous bone density than ALF, ELD appears to be a more effective therapeutic agent for treating postmenopausal osteoporosis, in which cancellous bone density decreases markedly. By using Cyp27b1−/− mice, this study was the first to succeed in clarifying the osteogenic effect of ELD without any influence of endogenous 1α,25D3. Furthermore, ELD more strongly enhanced bone mineralization, trabecular proliferation, and cancellous bone density than did ALF. Thus, ELD is expected to show an effect on postmenopausal osteoporosis, in which cancellous bone mineral density decreases markedly.

The active form of vitamin D, 1α,25-dihydroxyvitamin D

Eldecalcitol [1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3, developing code: ED-71] is an analog of active vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] that possesses a hydroxypropoxy substituent at the 2beta-position of 1,25(OH)2D3. Eldecalcitol has potent biological effects on bone and is now in preparation for approval as a promising medicine for the treatment of osteoporosis in Japan. To explore chemical structure-biological activity relationships between eldecalcitol and related analogs, we have already synthesized 1-epi-eldecalcitol, 3-epi-eldecalcitol, and 1,3-diepi-eldecalcitol with inherent biological interests of each targeted analog and evaluated their biological responses. It has been reported that 20-epi-1,25(OH)2D3, a diastereomer of 1,25(OH)2D3 that possesses an inverted methyl substituent at the 20-position of the side chain, shows remarkably enhanced biological activities compared to parental compound, 1,25(OH)2D3. As a continuation of our modification studies on eldecalcitol, we took great interest in 20-epi-eldecalcitol and its biological responses. In this paper, the synthesis of 20-epi-eldecalcitol by the Trost coupling reaction between the A-ring fragment and the C/D-ring fragment as well as in vitro preliminary biological evaluation of 20-epi-eldecalcitol are described. In the induction of human myeloid leukemia cell (HL-60) differentiation, inhibition of the human histiocytic lymphoma cell (U937) proliferation, and increase in osteocalcin concentration in the human osteosarcoma cell (MG-63), 20-epi-eldecalcitol showed significantly enhanced activity compared to eldecalcitol.

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.

In vitamin D research, there have been three milestones from the perspective of pharmaceutical studies. The first was the early stages of vitamin D research and involved the discovery of vitamin D and its physiological significance as an anti-rickets factor. The second was the elucidation of the vitamin D activation pathway in the body and the discovery of biologically active metabolites. The third has been characterized by the discovery of the differentiation-inducing properties of active vitamin D. This indicated the diversity of the physiological actions of active vitamin D, which initially had been thought only to contribute to bone and calcium metabolism. Because these physiological actions were clinically useful, the third discovery revealed opportunities for the development of drugs appropriate to specific purposes by synthesizing derivatives to separate biological effects. Six active vitamin D derivatives (maxacalcitol, calcipotriol, tacalcitol, paricalcitol, doxercalciferol, and falecalcitriol) based on the findings during the third milestone are now commercially available and contributing to current clinical practice. This article discusses the various milestones outlining vitamin D and derivatives in terms of pharmaceutical studies and practical syntheses of the six derivatives developed during the third milestone.

Many readers may have only a vague idea about vitamin D. This is made complicated, in part, because it is also expressed with suffixes such as vitamin D(2) or vitamin D(3). Otherwise the prefix of "active" is also occasionally used. Vitamin D is often referred to as an important nutrient for calcium intake, especially for growing children and the elderly. On the other hand, it serves as a therapeutic drug for osteoporosis and psoriasis. Recent studies have suggested an association with a number of diseases such as cancer, diabetes and Alzheimer's disease. The author has been involved in the research and development of vitamin D applications for over 25 years, and has often witnessed even world-class experts confuse the two roles - vitamin and hormone - of "substance" D. Assuming some readers are not familiar with vitamin D, D hormone or osteoporosis, an outline of vitamin D and D hormone is delineated with a particular focus on the treatment of osteoporosis. Furthermore, development of alfacalcidol as the first prodrug of D hormone (calcitriol) and eldecalcitol as a characteristic new D hormone derivative and basic relationship between calcemic activity and effect on bone in vitamin D (cholecalciferol), D hormone (calcitriol/alfacalcidol), and a new D hormone derivative (eldecalcitol) are introduced.

Alfacalcidol (1alpha-hydroxyvitamin D(3)) has been widely used since 1981 as a prodrug for calcitriol (1alpha,25-dihydroxyvitamin D(3)) in the treatment of hypocalcemia, chronic renal failure, hypoparathyroidism and osteoporosis. More recently, doxercalciferol (1alpha-hydroxyvitamin D(2)) has been used since 1999 as a prodrug for 1alpha,25-dihydroxyvitamin D(2) for the treatment of secondary hyperparathyroidism. Currently, six forms of vitamin D are known. They range from vitamin D(2) to vitamin D(7 )and are distinguished by their differing side chains. Only vitamin D(2) and vitamin D(3) have been found to be biologically active based on the elucidation of activation pathways. Alfacalcidol and osteoporosis/doxercalciferol and secondary hyperparathyroidism are discussed, with a new look at old compounds including their practical syntheses.

1α,25-Dihydroxyvitamin D 3 (1α,25-D 3 ) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, the major limitation to its clinical use is that it causes hypercalcemia. Therefore, vitamin D analogs with potent cell regulatory effects but with weaker calcemic effects than 1α,25-D 3 are required. Among them, 22-oxa-1α,25-D 3 and 19-nor-1α,25-D 3 have anti-cancer effects with relatively low calcemic effects. Modifications at the C-2α position of the A-ring also produced analogs with a unique biological profile. Not only the side-chain but also the A-ring modification thus generates a unique analog with potent cell regulatory effects and low calcemic activity as well. We report here that the hybrid 1α,25-D 3 analog, synthesized via the highly regio- and stereo-selective ring opening 2α-fluorination and catalytic asymmetric carbonyl-ene cyclization, with 2α-fluoro, 19-nor, and 22-oxa modification exhibits unique cell regulatory activities against the development of metastatic lung carcinoma.

A 3-position diastereomer of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71, 2), 3-epi-ED-71 (4), was synthesized by the convergent method coupling the A-ring fragment (5) with the C/D-ring fragment (6). As the results of preliminary in vitro biological evaluation of 3-epi-ED-71 (4), the inhibition of parathyroid hormone secretion in bovine parathyroid cells and binding affinity to human recombinant vitamin D receptor and to human vitamin D binding protein in comparison with ED-71 (2), 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3, 1), and 3-epi-1,25(OH)2D3 (3) are described.

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.

Maxacalcitol, the 22-oxa-derivative of 1α,25-dihydroxyvitamin D3 and used currently as an antihyperparathyroidism and antipsoriatic drug, has been synthesized in seven chemical steps from 1α-hydroxydehydroepiandrosterone on the basis of our previously developed route. The present synthesis allows the production of the protected form of the penultimate intermediate in 26% overall yield in a kilogram scale reaction employing neither difficult reaction conditions nor chromatographic purification, having overcome all the difficulties involved in the previous route.

Recently, epimerization of the hydroxyl group at C-3 has been identified as a unique metabolic pathway of vitamin D compounds. We measured C-3 epimerization activity in subcellular fractions prepared from cultured cells and investigated the basic properties of the enzyme responsible for the epimerization. C-3 epimerization activity was detected using a NADPH-generating system containing glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and Mg(2+). The highest level of activity was observed in a microsomal fraction prepared from rat osteoblastic UMR-106 cells but activity was also observed in microsomal fractions prepared from MG-63, Caco-2, Hep G2, and HUH-7 cells. In terms of maximum velocity (V(max)) and the Michaelis constant (K(m)), 25-hydroxyvitamin D(3) [25(OH)D(3)] exhibited the highest specificity for the epimerization at C-3 among 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], 25(OH)D(3), 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], and 22-oxacalcitriol (OCT). The epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha-->beta)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D(3) at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.

Recently, it was revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and 24R,25-dihydroxyvitamin D3 (24,25(OH)2D3) were metabolized to their respective epimers of the hydroxyl group at C-3 of the A-ring. We now report the isolation and structural assignment of 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3 as a major metabolite of 25-hydroxyvitamin D3 (25(OH)D3) and the further metabolism of C-3 epimers of vitamin D3 metabolites. When 25(OH)D3 was incubated with various cultured cells including osteosarcoma, colon adenocarcinoma, and hepatoblastoma cell lines, 3-epi-25(OH)D3 and 24,25 (OH)2D3 were commonly observed as a major and minor metabolite of 25(OH)D3, respectively. 25(OH)D3 was at least as sensitive to C-3 epimerization as 1α, 25(OH)2D3 which has been reported as a substrate for the C-3 epimerization reaction. Unlike these cultured cells, LLC-PK1 cells, a porcine kidney cell line, preferentially produced 24,25(OH)2D3 rather than 3-epi-25(OH)D3. We also confirmed the existence of 3-epi-25(OH)D3 in the serum of rats intravenously given pharmacological doses of 25(OH)D3. The cultured cells metabolized 3-epi-25(OH)D3 and 3-epi-1α,25(OH)2D3 to 3-epi-24,25(OH)2D3 and 3-epi-1α,24,25(OH)3D3, respectively. In addition, we demonstrated that 3-epi-25(OH)D3 was metabolized to 3-epi-1α,25(OH)2D3 by CYP27B1 and to 3-epi-24,25(OH)2D3 by CYP24 using recombinant Escherichia coli cell systems. 3-Epi-25(OH)D3, 3-epi-1α,25(OH)2D3, and 3-epi-24,25(OH)2D3 were biologically less active than 25(OH)D3, 1α,25(OH)2D3, and 24,25(OH)2D3, but 3-epi-1α,25(OH)2D3 showed to some extent transcriptional activity toward target genes and anti-proliferative/differentiation-inducing activity against human myeloid leukemia cells (HL-60). These results indicate that C-3 epimerization may be a common metabolic pathway for the major metabolites of vitamin D3.

A one-pot modification method of steroid 20S-hydroxy functionality involving sequential alkylation and reductive cleavage of epoxy bond has been established for the industrial preparation of a medicinally important 1α,25-dihydroxy-22-oxa-vitamin D32 (22-oxa-calcitriol), Maxacalcitol.

22-Oxacalcitriol (OCT) is an analog of calcitriol, characterized by potent differentiation-inducing activity and low calcemic liability. The metabolism of OCT has been studied and its polar metabolites, such as 24/26-hydroxylated-OCT and hexanor-1 alpha,20-dihydroxyvitamin D(3) (1 alpha,20(OH)(2)D(3)), have been identified. In contrast, little is known about the less polar metabolites of OCT, which have been found in relatively large amounts. In this study, the in vitro metabolism of OCT was studied in UMR 106, Caco-2, and LLC-PK(1) cells to identify the less polar metabolites and to assess their biological activity. OCT was initially metabolized to three less polar metabolites, 3-epi-OCT and two dehydrates, 25-dehydroxy- 25-ene-22-oxa-1 alpha(OH)D(3) (25-ene-22-oxa-1 alpha(OH)D(3)) and 25-dehydroxy-24-ene-22-oxa-1 alpha(OH)D(3) (24-ene-22-oxa-1 alpha(OH)D(3)). We also observed further metabolites, the two C-3 epimers of the C-25 dehydrates, 25-ene-3-epi-22-oxa-1 alpha(OH)D(3) and 24-ene-3-epi-22-oxa-1 alpha(OH)D(3). The structures of these metabolites were successfully assigned by (1)H NMR and LC-MS analyses. The three cell lines differ in their ability to metabolize OCT through the C-3 epimerization or the C-25 dehydration pathway. The biological activity of the OCT metabolites assessed by a luciferase reporter gene transcriptional activation system, binding assays for the vitamin D receptor (VDR) and vitamin D-binding protein (DBP), and assays for regulatory activities of cell differentiation and proliferation was found to be lower than that of OCT. Thus, both the C-3 epimerization and C-25 dehydration may work to reduce the biological activity of OCT.

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.

Background. Since Slatopolsky et al. (J Clin Invest 1984; 74: 2136-2143) reported the effect of active vitamin D, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), on secondary hyperparathyroidism (2HPT) which accompanies chronic renal failure, there have been several studies of the therapeutic effects of 1,25(OH) 2 D 3 in this disease. Although parathyroid hormone (PTH) is suppressed by treatment with 1,25(OH) 2 D 3 , long-term treatment with 1,25(OH) 2 D 3 tends to induce hypercalcaemia. Therefore, an analogue of 1,25(OH) 2 D 3 , 1,25-dihydroxy-22-oxavitamin D 3 (22-oxacalcitriol, OCT) with less calcaemic activity, was developed for the treatment of 2HPT. Methods. In order to clarify the differences between the effects of 1,25(OH) 2 D 3 and OCT on 2HPT associated with chronic renal failure, these compounds were administered by intermittent i.v. injection for 2 weeks in rats with mild to moderate uraemia. Results. 1,25(OH) 2 D 3 markedly suppressed PTH levels, but increased serum calcium (Ca). OCT also markedly suppressed PTH levels, but induced only a slight increase in serum Ca. 1,25(OH) 2 D 3 caused a dose-dependent decrease in body weight, whereas OCT had no effect on body weight in uraemic rats. Based on those doses of OCT and 1,25(OH) 2 D 3 , which resulted in a 60% suppression of PTH, and induced hypercalcaemia, we consider the relative ratios for efficacy and Ca-elevating activity between OCT and 1,25(OH) 2 D 3 to be 1: 8 and 1: 48, respectively. Conclusions. OCT suppressed PTH levels with a slight increase in serum Ca without changing the body weight in uraemic rats. This observation suggests that OCT might be a useful vitamin D analogue for 2HPT management in long-term clinical treatment.

Background. 1,25-Dihydroxyvitamin D 3 (1,25(OH) 2 D 3 , calcitriol) has been used for the treatment of secondary hyperparathyroidism (2HPT) associated with chronic renal failure (CRF). However, hypercalcaemia frequently precludes the administration of ideal doses of 1,25(OH) 2 D 3 . 1,25-Dihydroxy-22-oxavitamin D 3 (22-oxacalcitriol, OCT) is an analogue of 1,25(OH) 2 D 3 with less calcaemic activity. Several investigators have reported the effect of this analogue on suppressing parathyroid hormone (PTH) in vitro and in vivo in rats and dogs. Methods and results. The first experiments were designed to compare the relative potency of an i.v. injection of OCT and 1,25(OH) 2 D 3 (i.v. OCT vs i.v. 1,25(OH) 2 D 3 ) on serum PTH and ionized calcium (ICa). A single dose of OCT (5 μg/kg) to uraemic dogs suppressed PTH by 81% without a statistical significant change in serum ICa. On the other hand, any of the effective doses of 1,25(OH) 2 D 3 on PTH suppression were hypercalcaemic. The intermittent administration of OCT (0.1 μg/kg) or 1,25(OH) 2 D 3 (0.025 μg/kg), three times per week i.v. suppressed serum PTH by 89 or 77%, respectively without hypercalcaemia. To evaluate OCT as an oral drug, it was given intermittently (three times per week) to a group of six dogs for a period of 4 weeks. Subsequently, it was changed to a daily administration (0.05 μg/kg) for a period of 2 weeks. Finally the dose was reduced to 0.025 μg/kg. Daily OCT 0.05 μg/kg suppressed serum PTH by 67%. Subsequently, 0.025 μg/kg maintained serum PTH within the normal range without hypercalcaemia for 4 weeks. The time course of serum OCT concentrations following a single i.v. or oral OCT dose to uraemic dogs showed that oral OCT was rapidly absorbed and reached maximum plasma concentration and its disappearance from blood was similar to that of i.v. injection. Conclusions. In conclusion, our results suggest that OCT is a useful vitamin D 3 analogue, with a potentially larger therapeutic window than that of i.v. 1,25(OH) 2 D 3 and which is available for i.v./oral, in the management of 2HPT.

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.

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.

A-ring diastereomers of 1α,25-dihydroxy-22-oxavitamin D3 (OCT) (2), 3-epi-1α,25-dihydroxy-22-oxavitamin D3 (3-epiOCT) (3) and 1,3-diepi-1α,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-1α,25-dihydroxy-22-oxavitamin D3 (1-epiOCT) (5) and 1α,25-dihydroxyvitamin D3 (1).

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.

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.

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.

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).

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.

The synthesis of tritiated 1α-25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71), [2β-(3-3H)]ED-71 ((3) and [26,27-3H6]ED-71 (4) is described. The former was prepared by reduction of a precausor containing a formyl group in the A-ring part with sodium borotritiide while the latter was labeled in the side chain using tritiated methylmagnesium iodide. The specific activity of 3 was 13.2 Ci/mmol and that of 4 138.0 Ci/mmol. Copyright © 1999 John Wiley & Sons, Ltd.

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.

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 (

SUMMARY: 22-Oxa-1,25-dihydroxyvitamin D3 (OCT) is an analogue of vitamin D with less calcemic action than 1,25-dihydroxyvitamin D3 (1,25D3), and thus may be advantageous in the treatment of secondary hyperparathyroidism in dialysis patients. to further elucidate the mechanisms of less-calcemic action of OCT in chronic renal failure, we examined the effects of OCT and 1,25D3 on mRNA levels for vitamin D-dependent 9-KDa calcium binding protein (CaBP-D9K) in the intestinal mucosa and 28-KDa (CaBP-D28K) in the kidney. In Sprague-Dawley rats made uremic by 5/6 nephrectomy for three months, OCT at doses of 0.25, 1.25 and 6.25 μg/kg, or 1,25D3 at 0.025,0.125 and 0.625 μg/kg were administered intravenously three times per week for two weeks. At 24 h after the final injection, enhanced serum PTH and PTH mRNA levels were successfully suppressed both by OCT and 1,25D3 in a dose dependent manner. However, OCT induced less hypercalcemia than 1,25D3. 1,25D3 markedly upregulated the expression of CaBP-D9K and CaBP-D28K genes, while they were not affected by OCT at all. In conclusion, such attenuated effects of OCT on calcium-binding proteins may play a role in the noncalcemic action, because number of CaBP-D9K has been suggested to correlate with calcium absorption in the intestine.

Hypercalcemia represents one of the important paraneoplastic syndromes affecting morbidity and mortality of cancer patients. We and others have demonstrated that vitamin D analogs with little calcemic activities suppress the transcription of the parathyroid hormone-related peptide (PTHrP) gene, a major humor responsible for cancer hypercalcemia, and thereby prevent the development of hypercalcemic syndrome. The present study was undertaken: to compare the therapeutic efficacy of a vitamin D analog, 22-oxa-1,25-dihydroxyvitamin D3 (OCT), and a bisphosphonate (disodium 3-amino-1-hydroxypropylidene-1,1-bisphosphonate pentahydrate [AHPrBP]), an inhibitor of osteoclastic bone resorption, on cancer-induced hypercalcemia; and to see if the effect could be enhanced by combination treatment, using a nude mouse model implanted with a human pancreas carcinoma (FA-6). After a single intravenous administration, OCT (5 microg/kg of body weight [BW]) was as effective as AHPrBP (10 mg/kg of BW) in lowering blood ionized calcium levels in tumor-bearing nude mice, and their combination further enhanced the therapeutic effect. Although AHPrBP lost its efficacy after repeated injections, OCT was still effective after the third administration. The therapeutic effect of OCT in cancer hypercalcemia was observed in four other human tumors, including another pancreas carcinoma (PAN-7), two squamous cell carcinomas of the lung (KCC-C1 and LC-6), and a squamous carcinoma of the pharynx (PHA-1), all of which elaborated PTHrP into the circulation. Treatment with OCT resulted in a decrease in circulating PTHrP levels by approximately 50% in two representative models. However, the mechanism underlying the antihypercalcemic effect of OCT seemed complex, involving inhibition of PTHrP production, suppression of excessive bone resorption, and an antitumor activity. OCT also markedly inhibited the body weight loss with tumor growth, while AHPrBP, which exhibited a similar antihypercalcemic effect, was less effective than OCT in preventing cachexia. The anticachectic activity of their combination did not exceed that of OCT alone, suggesting a hypercalcemia-dependent as well as an independent mechanism of cancer cachexia. It is concluded that OCT may be useful, either as a single agent or in combination with bisphosphonates, for the treatment of cancer-associated hypercalcemia and cachexia.

SUMMARY: 22-oxa-calcitriol (OCT), a vitamin D analogue, suppresses parathyroid hormone (PTH) secretion and has less calcaemic activity than 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in vivo. In this study, we evaluated the effect of OCT on PTH secretion in vitro using human hyperplastic parathyroid tissue obtained during surgery for advanced renal hyperparathyroidism and normal bovine parathyroid glands to compare the effects of 1,25(OH)2D3. 22-oxa-calcitriol suppressed PTH secretion by nodular hyperplastic parathyroid tissue and normal bovine tissue in a dose dependent manner, the same as 1,25(OH)2D3. We showed the additive effect of extracellular calcium level on suppression of PTH secretion by OCT and 1,25(OH)2D3. These results suggest that OCT suppresses PTH secretion, the same as 1,25(OH)2D3 not only in normal parathyroid cells but also on hyperplastic parathyroid cells.

The combination of the regioselective propiolate-ene reaction, catalytic enantioselective epoxidation and catalytic enantioselective carbonyl-ene cyclization completes the synthesis of the A ring of the hybrid 19-nor-22-oxa D3 analogue (1), which shows the significant activity in transactivation.

1Alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] mediates its biological activities through specific binding to the nuclear vitamin D receptor (VDR). The VDR, bound to 1alpha,25(OH)2D3, forms a heterodimer with a nuclear accessory factor, retinoid X receptor (RXR), and the complex subsequently binds to specific nucleotide sequences or a vitamin D-responsive element (VDRE) to induce gene transcriptions. Thus, an ideal analogue of 1alpha,25(OH)2D3 for therapeutic applications has been considered to be one which has a high binding affinity for VDR, thus forming a stable VDR/RXR complex, and binding strongly to VDRE. By contrast, we report here evidence contrary to this hypothesis. Several singly dehydroxylated A-ring analogues of 19-nor-1alpha,25-dihydroxyvitamin D3 and 19-nor-22-oxa-1alpha,25-dihydroxyvitamin D3, all of which have an extremely low binding affinity for VDR, and some of which lack the 1alpha-hydroxyl group that is considered to be essential for VDR-mediated gene expression, have greater or equivalent potencies to 1alpha,25(OH)2D3 for inhibiting the proliferation and inducing the differentiation of HL-60 cells, as well as inducing the transactivation of a luciferase reporter gene combining a rat 25-hydroxyvitamin D3 24-hydroxylase gene promoter containing two VDREs. The present findings open interesting possibilities as to the role of the VDR in the genomic action of 1alpha,25(OH)2D3 and the development of new 19-nor-analogues of 1alpha,25(OH)2D3.

A convergent and versatile synthesis of the potent vitamin D analog, 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 [1] (ED-71) is described.

A practically useful formation of γ-keto or γ-hydroxy ether linkage from alcohol, for the construction of the side chain of 1α, 25-dihydroxy-22-oxavitamin D3 (OCT), is described.

Interleukin-6 (IL-6) induces osteoclast-like cell (osteoclast) formation in a dose-dependent fashion in cocultures of mouse bone marrow cells and osteoblastic cells when soluble IL-6 receptor (sIL-6R) is present. Simultaneous treatment with submaximal doses of IL-1alpha and IL-6 with sIL-6R caused marked induction of osteoclast formation and PGE2 synthesis. These effects were suppressed by adding neutralizing antibodies against IL-1alpha or IL-6R and were totally abolished by adding nonsteroidal antiinflammatory drugs, such as indomethacin and a selective cyclooxygenase-2 (COX-2) inhibitor (NS398). In mouse osteoblastic cells, both IL-1alpha and IL-6 with sIL-6R markedly induced messenger RNA expression of COX-2, but not COX-1, as determined by Northern blot analysis, and luciferase activity in cells stably transfected with a COX-2 promoter-luciferase fusion construct. IL-6 and sIL-6R, when added separately, did not stimulate COX-2 messenger RNA expression. Simultaneous addition of IL-1alpha and IL-6 with sIL-6R to osteoblast cultures cooperatively induced transcription of COX-2, which was associated with a marked increase in COX activity measured by the conversion of arachidonic acid into PGE2. The increased PGE2 synthesis by osteoblasts may play an important role in osteoclastogenesis induced by submaximal doses of IL-1 and IL-6.

1,25-Dihydroxyvitamin D3 and 22-oxacalcitriol prevent the decrease in vitamin D receptor content in the parathyroid glands of uremic rats. Decreased content of the 1,25-dihydroxyvitamin D3 receptor (VDR) in parathyroid glands from patients and animals with chronic renal failure has been implicated in the pathogenesis of secondary hyperparathyroidism. In these studies, we examined the regulation of VDR by 1,25-dihydroxyvitamin D3 (1,25-D3) and 22-oxacalcitriol (OCT) in parathyroid glands of uremic rats. After eight weeks of renal failure, VDR content in parathyroid glands of uremic rats was decreased (400 ± 42 vs. 729 ± 47 fmol/mg protein in normal control rats, P < 0.05) and strongly correlated with serum 1,25-D3 levels (r = 0.829, P = 0.0001). Treatment with either 1,25-D3 or OCT prevented the decrease in VDR. We conclude that low serum 1,25-D3 levels, at least in part, account for the decrease in VDR content in parathyroid glands of uremic rats and that treatment with 1,25-D3 or OCT prevents this decrease ameliorating the development of secondary hyperparathyroidism.

In chronic renal failure, hyperphosphatemia, hypocalcemia, hyperparathyroidism, reduced activation of vitamin D, decreased level of calcium-sensing receptor, osteitis fibrosa, and osteomalacia are features related to calcium abnormalities. Hyperparathyroidism is a risk factor for survival of hemodialysis patients as well as hypoparathyroidism, which is another feature in hemodialysis patients. Treatment of these abnormalities includes control of parathyroid hormone (PTH) secretion, counteracting hyperphosphatemia, correction of hypocalcemia, and others. Various kinds of vitamin D analogs have been introduced recently in addition to calcitriol and alfacalcidol, which have a rather long history (eg, maxacalcitol and falecalcitriol). Sevelamer is a newly developed phosphate binder to treat soft-tissue calcification.

Synthesis of two tritiated 1α,25-dihydroxy-22-oxavitamin D 3 (OCT), [26- 3 H 3 ]OCT (3) and [2β- 3 H]OCT (4), is described. [26- 3 H 3 ]OCT (3) was prepared by tritiation at the side chain with tritiated methylmagnesium iodide and [2β- 3 H]OCT (4) was labeled at the A-ring by tritiation with sodium borotritiide.

The 22-thiolation reaction of the steroidal side chain and the synthesis of 1α,25-dihydroxy-22-thiavitamin D3 and related analogs bearing side chains of different sizes in combination with 20-epi configuration are described.

Eldecalcitol [1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D(3)], an analog of calcitriol (1α,25-dihydroxyvitamin D(3)), possesses a hydroxypropoxy substituent at the 2β-position of calcitriol. Eldecalcitol has potent biological effects on bone disease such as osteoporosis. The marketing of eldecalcitol has very recently started in Japan. In consideration of this, we have been investigating practical synthesis of eldecalcitol for industrial-scale production. Eldecalcitol was initially synthesized in a linear manner. The 27-step linear sequence was, however, suboptimal due to its lengthiness and low overall yield (ca. 0.03%). Next, we developed a convergent approach based on the Trost coupling reaction, in which the A-ring fragment (ene-yne part obtained in 10.4% overall yield) and the C/D-ring fragment (bromomethylene part obtained in 27.1% overall yield) are coupled to produce the triene system of eldecalcitol (15.6%). Although the overall yield of the convergent synthesis was better than that of the linear synthesis, significant improvements were still necessary. Therefore, additional biomimetic studies were investigated. Process development for the practical production of eldecalcitol is described herein.

As the postulated metabolites of 1α,25-dihydroxy-22-oxacitamin D 3 (OCT) (1), 24-hydroxylated OCT (3 and 4), 26-hydroxylated OCT (5 and 6), and pentanorOCT (7) were syntheiszed from the 20(S)-alcohol (9).