Calcium regulating activity of 26,27-dialkyl analogs of 1α,25-dihydroxyvitamin D3

Summary A series of analogs of 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] with alkyl substitutions in 26- and 27-positions were tested for calcium (Ca) regulating activity. The potencies of dialkyl analogs in stimulating bone resorption in neonatal mouse calvaria cultures were the highest in 1,25-dihydroxy-26,27-dimethylvitamin D₃[1,25(OH)₂-(Me)₂D₃], followed by 1,25(OH)₂D₃, 1,25-dihydroxy-26,27-diethylvitamin D₃[1,25(OH)₂(Et)₂D₃], and 1,25-dihydroxy-26,27-dipropylvitamin D₃[1,25(OH)₂(Pr)₂D₃] in that order. A similar order of potential regarding formation of osteoclast-like cells in mouse bone marrow cell cultures and on bone Ca mobilization with long-term vitamin D-deficient rats was observed in the same series. The relative potencies of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 1,25(OH)₂(Et)₂D₃, and 1,25(OH)₂(Pr)₂D₃ in competing with 1,25(OH)₂D₃ for binding to chick intestinal cytosol receptors were 1:1:0.16:0.036. A similar order of potential in case of intestinal Ca transport in situ was observed in the same series. The potencies of dialkyl analogs in competing with 25-hydroxy-vitamin D₃ for binding to rat serum vitamin D binding protein were much lower than that of 1,25(OH)₂D₃. Effect of 1,25(OH)₂(Me)₂D₃ on osteopenia in rats induced by ovariectomy and right sciatic neurotomy was higher than that of 1,25(OH)₂D₃. From these results, the lengthening by one carbon at 26- and 27-positions was shown to maintain the Ca regulatory activity of 1,25(OH)₂D₃.     

Bone-resorbing activities of 24-epi-1α-hydroxyvitamin D2 and 24-epi-1α,25-dihydroxyvitamin D2

Bone-resorbing activities of 24-epi-1-hydroxyvitamin D₂ [24-epi-1(OH)D₂], 24-epi-1,25-dihydroxyvitamin D₂ [24-epi-1,25(OH)₂D₂], and 1,24S,25-trihydroxyvitamin D₂ [1,24S,25(OH)₃D₂], which might be a metabolite of 24-epi-1,25(OH)₂D₂, were investigated. In an in vitro bone resorption test, the activity of 24-epi-1(OH)D₂ was similar to that of 1-hydroxyvitamin D₃ [1(OH)D₃] at 10^-9 M-10^-6 M. The activity of 24-epi-1,25(OH)₂D₂ was weaker than that of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] at 10^-11 M-10^-8 M. On the other hand, the activity of 1,24S,25(OH)₃D₂ was similar to that of 24-epi-1,25(OH)₂D₂ at 10^-11 M-10^-9 M. In the formation assay of osteoclast-like cells, the activity of 24-epi-1(OH)D₂ was weaker than that of 1(OH)D₃ at 10^-7 M. The activity of 24-epi-1,25(OH)₂D₂ was almost similar to that of 1,25(OH)₂D₃ at 10^-11 M-10^-7 M. The activity of 1,24S,25(OH)₃D₂ was significantly weaker than that of 24-epi-1,25(OH)₂D₂ at 10^-11 M-10^-9 M. In the two experiments, the potencies of 24-epi-1,25(OH)₂D₂ were about 100 times higher than those of 24-epi-1(OH)D₂. In an in vivo/in vitro bone resorption test, the activity of 24-epi-1(OH)D₂ was almost similar to those of 1(OH)D₃ and 1,25(OH)₂D₃ and higher than those of 24-epi-1,25(OH)₂D₂ and 1,24S,25(OH)₃D₂. 24-epi-1-(OH)D₂ and 1(OH)D₃ were longer lasting than 24-epi-1,25(OH)₂D₂ and 1,25(OH)₂D₃ in this experiment. These results suggested that 24-epi-1(OH)D₂ as well as 1(OH)D₃ was converted into dihydroxy form in vivo.     

Elongation of the side chain of analogs of 1α,25-dihydroxyvitamin D3 prevents osteopenia in a rat model

Five analogs of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] [1], 26,27-dimethyl-1,25-dihydroxyvitamin D₃ [1,25(OH)₂(Me)₂D₃] [2], 26,27-dimethyl-1,25-dihydroxyvitamin D₃ [1,25(OH)₂(Et)₂D₃] [3], 26,27-dipropyl-1, 25-dihydroxyvitamin D₃ [1,25(OH)₂(Pr)₂D₃] [4], 26,27-dimethyl-24, 24-difluoro-1,25-dihydroxyvitamin D₃ [24F₂-1,25(OH)₂(Me)₂D₃, and [5] 24a-homo-24,24-difluoro-1,25-dihydroxyvitamin D₃ [24aF₂-homo-1,25(OH)₂D₃] were investigated to clarify the possibility that prevents osteopenia induced in rats by ovariectomy and sciatic neurotomy. The objective of our studies was to determine whether these analogs may be effective for treatment of subjects with osteoporosis. 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 24aF₂-homo-1,25(OH)₂D₃ prevented decreases in bone mineral density (BMD) of the femur, as measured by dual energy X-ray absorptiometry (DXA). The potency of 1,25(OH)₂(Me)₂D₃ in this test was higher than that of 1,25(OH)₂D₃. The potencies of 24F₂-1,25(OH)₂(Me)₂D₃ and 24aF₂-homo-1,25(OH)₂D₃ were similar to that of 1,25(OH)₂D₃. On the other hand, though 1,25(OH)₂(Et)₂D₃ and 1,25(OH)₂(Pr)₂D₃ had a preventive effect on the decease in BMD, the potency of two analogs was lower than that of 1,25(OH)₂D₃. Decreases in cortical and trabecular bone areas of the femur were prevented by three analogs of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 24aF₂-homo-1,25(OH)₂D₃. Serum calcium (Ca) concentration was elevated at the last administration of three analogs of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃ and 24aF₂-homo-1,25(OH)₂D₃. Decreases in the Ca concentration in untreated rats were noted a few days after the last administration. In light of these positive effects, we are continuing research on 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 24aF₂-homo-1,25(OH)₂D₃ as putative treatment for osteoporosis.     

Low utility of serum 25–hydroxyvitamin D3 and 1, 25-dihydroxyvitamin D3 in predicting peripheral Treg and Th17 cell counts in ESRD and renal transplant patients

BackgroundVitamin D has shown an immune-modulatory effect in different studies. Vitamin D stimulates Tregs and inhibits Th17 cells. The immune-modulatory role of vitamin D in chronic kidney disease (CKD) and renal transplant patients is unclear. We measured whether different serum levels of vitamin D were associated with an increased or decreased presence of lymphocyte subsets including Treg and Th17 cells in end-stage renal disease (ESRD) and renal transplant recipients.MethodsEighty-seven renal transplant recipients and 53 end-stage renal disease (ESRD) patients were enrolled in this study. The absolute counts of CD4 + and CD8 + T, CD16 + CD56 + NK, CD19 + B, CD4 + CD25 + CD127- Foxp3 + (Tregs), Helios + Tregs, CD38 + Tregs, and CD4 + CD17 + (Th17) cells were analyzed in peripheral blood in both patient groups. In addition, serum 25 (OH) D₃, 1, 25 (OH)₂ D₃, IL-6, IL-17, IL-23, and TGF-β₁ were measured. The association between lymphocyte subset counts and 1, 25 (OH)₂ D₃ or 25 (OH) D₃ was studied, as was the association between serum IL-6, IL-17, IL-23, or TGF-β₁ and 1,25 (OH)₂ D₃ or 25 (OH) D₃.ResultsSerum 25 (OH) D₃ and 1,25 (OH)₂ D₃ levels were not independently associated with peripheral CD4 + T, CD19 + B, CD16 + CD56 + NK, Treg, or Th17 cell counts. In contrast to serum 25 (OH) D₃, serum1, 25 (OH)₂ D₃ was positively associated with CD8 + T cells counts in renal transplant recipients.ConclusionOur findings indicate low utility of serum 25 (OH) D₃ and 1, 25 (OH)₂ D₃ levels in predicting a change in lymphocyte subset counts in ESRD and renal transplant patients.     

Effect of 25-hydroxyvitamin D3 and 1 α,25 dihydroxyvitamin D3 on differentiation and apoptosis of human osteosarcoma cell lines

Osteosarcoma (OS) is a malignant bone tumor predominantly affecting children and adolescents. OS has a 60% survival rate with current treatments; hence, there is a need to identify novel adjuncts to chemotherapeutic regimens. In this pilot study, we investigated the dose‐response to 1α,25‐dihdroxyvitamin D₃ (1,α 25(OH)₂D₃) and 25‐hydroxyvitamin D₃ (25(OH)D₃) by human OS cell lines, SaOS‐2, and 143B. We hypothesized that 1,α 25(OH)₂D₃ and 25(OH)D₃ would stimulate differentiation and induce apoptosis in OS cells in a dose‐dependent manner. Human OS cell lines, SaOS‐2, and 143B, were treated with 1,α 25(OH)₂D₃ or 25(OH)D₃ or an ethanol control, respectively, at concentrations ranging from 1 to 1,000 nM. Ki67 (a marker of cellular proliferation) immunocytochemistry revealed no significant changes in the expression of Ki‐67 or MIB‐1 in 1α,25(OH)₂D₃ or 25(OH)D₃ treated SaOS‐2 or 143B cells. Both control and 1α,25(OH)₂D₃ treated SaOS‐2 and 143B cells expressed vitamin D receptor (VDR). Markers of osteoblastic differentiation in 143B cells and SaOS‐2 cells were induced by both 25(OH)D₃ and 1α,25(OH)₂D, and evident by increases in alkaline phosphatase (ALP) activity, osteocalcin (OCN) mRNA expression, and mineralization of extra‐cellular matrix (ECM) by alizarin red staining. An increasing trend in apoptosis in response to 25(OH)D₃, in both SaOS‐2 and 143B cells was detected by terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end labeling (TUNEL) staining. With 1α,25(OH)₂D₃ treatment, apoptosis was evident at higher concentrations only. These preliminary findings suggest that OS cells express VDR and respond to 25(OH)D₃ and 1α,25(OH)₂D₃ by undergoing differentiation and apoptosis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:831–844, 2012     

A novel synthetic vitamin D analogue, 2β-(3-hydroxypropoxy)1α, 25-dihydroxyvitamin D3 (ED-71), increases bone mass by stimulating the bone formation in normal and ovariectomized rats

We performed dosing experiments to evaluate the bone mass increasing action of a novel, synthetic vitamin D derivative, 2-(3-hydroxypropoxy)-1, 25(OH)₂D₃ (ED-71), in normal and estrogen-deficient rats. The first experiment consisted of 31 Sprague-Dawley rats, 28 weeks of age. The second experiment consisted of 44 animals who were ovariectomized (OVX) or sham operated at the age of 12 weeks. ED-71 was given twice a week for the duration of 12 weeks. At the end of the experiments, serum chemistries were examined and lumbar vertebrae were assessed histomorphometrically. Serum alkaline-phosphatase levels tended to decrease by ED-71 administration in the first experiment and their elevated values after ovariectomy were also depressed by ED-71 in the second experiment. Serum osteocalcin levels, however, increased by the agent. In the first experiment, cancellous bone volume (BV/TV) increased dose dependently. Bone formation rates (BFR/BS) also increased. In the second experiment, BV/TV significantly decreased by ovariectomy and it increased in ED-71-treated groups, but not in 1-(OH)D₃-treated group. BFR/BS increased by ED-71. Activation frequency did not decreased by ED-71 in either experiment. These data clearly demonstrated that ED-71 administration was capable of increasing the bone mass by stimulating bone formation in normal and estrogen-deficient rats.     

The Vitamin D Analog 1��,25-Dihydroxy-2��-(3-Hydroxypropyloxy) Vitamin D3 (Eldecalcitol) is a Potent Regulator of Calcium and Phosphate Metabolism

The vitamin D analog 1α,25-dihydroxy-2β-(3-hydroxypropyloxy)vitamin D(3) (ED-71 or eldecalcitol) has been developed for treatment of osteoporosis, but its effects on mineral metabolism have not been investigated in detail. In the present study, we compared the effects of eldecalcitol and calcitriol on calcium (Ca) and phosphate (Pi) handling in rats. Oral administration of eldecalcitol (0, 7.5, 20, or 50?pmol) q.o.d. for 2?weeks dose-dependently increased ionized Ca, intestinal Ca absorption, and urinary Ca excretion, while these doses of calcitriol had no significant effects. The highest dose of eldecalcitol did not alter serum Pi but stimulated both intestinal Pi absorption and urinary Pi excretion; the latter was attributable, in part, to increased serum FGF-23. The effects of high-dose eldecalcitol on Ca and Pi absorption and urinary excretion and FGF-23 persisted for several days following cessation of treatment. The higher potency of eldecalcitol on Ca and Pi handling was also observed in parathyroidectomized rats infused with PTH, excluding a role for differential regulation of PTH. Direct measurement of duodenal Ca absorption by the in situ loop method confirmed the higher potency of eldecalcitol in this segment via induction of TRPV6. These studies indicated that with chronic administration eldecalcitol is more potent than calcitriol at stimulating intestinal absorption of Ca and Pi, as well as FGF-23. The mechanisms responsible for the higher potency of eldecalcitol are speculated to be its higher vitamin D-binding protein (DBP) affinity and resistance to metabolism.     

Kinetics of β-glycerophosphate-induced endochondral mineralization In vitro. Calcium accumulation,alkaline phosphatase activity,and effects of levamisole

Summary Isolated mesenchymal limb bud cells from day-12 mouse embryos grown at high density in organoid culture at the medium/air interphase differentiate into chondrocytes and form cartilage nodules. Upon addition of -glycerophosphate (-GP), cartilage undergoes endochondral mineralization. This -GP-induced mineralization was investigated by measuring the calcium content in the cultures and the activity of alkaline phosphatase (AP) in the cell mass and the medium. Calcium incorporation depended on the amount of -GP added. After continuous treatment, mineralization began on day 8 of the culture period and increased linearly until day 15. In long-term cultures, periodical treatment for 6 days caused an increase in mineralization the older the cultures were, but the slope of increase was proportionately less steep. Treatment at the latest period on days 19–24 resulted in a markedly reduced mineralization. After short-term treatment (48 hours), mineralization increased also the older the cultures were and proceeded during further cultivation in -GP-free medium. This kinetic behavior indicates a dependency of mineralization on cartilage maturation in this in vitro system. AP activity increased enormously and nearly logarithmically in the cell mass in -GP-free medium, whereas -GP treatment inhibited this drastic increase. In the medium, considerable activities of AP were also measurable from day 10 onward. It increased in -GP-free medium up to day 14, but was diminished after mineralization had been induced. Levamisole inhibited AP activity dose dependently when added directly to the enzyme-containing medium (100% inhibition at 10^-3 M). Added to the cultures from day 7 to 14, it partially inhibited AP activity and mineralization at 5×10^-5 M; mineralization was totally inhibited at 10^-3 M, but AP activity was still present. This high concentration was cytotoxic, as revealed ultrastructurally and by GAG estimation. This in vitro system comprises cartilage development and maturation, -GP-inducible endochondral mineralization, and final degenerative changes; it may be an appropriate model for investigations on endochondral mineralization.     

Early Postmenopausal Bone Loss is Prevented by Estrogen and Partially by 1α-OH-vitamin D3: Therapeutic Effects of Estrogen and/or 1α-OH-vitamin D3

A total of 79 Japanese women who were within 5 years of menopause were randomly assigned 1α-hydroxyvitamin D₃ [1α(OH)D₃] 1.0 μg/day, conjugated estrogens 0.625 mg/day, a combination of both, or control (no treatment). Lumbar spine and proximal femur bone mineral density (BMD) and biochemical indices were monitored over 2 years. In the 1α(OH)D₃-treated group, there was a nonsignificant decrease in lumbar spine BMD compared with controls, and no significant loss in the femoral neck compared with controls. In the estrogen-treated group, there was a nonsignificant increase in spine BMD (+2.17% in the first year and +1.71% in the second year), and no loss in femoral neck BMD. The combination of conjugated estrogens +1α(OH)D₃ was more effective in increasing BMD in the spine (+3.68% in the first year and +3.63% in the second year) and femur (+2.56% in the first year and +4.44% in the second year) BMD. There was a significant difference in lumbar spine BMD in both the first and second years between the combination-treated group and the 1α(OH)D₃-treated and control groups (P < 0.01). Serum osteocalcin (OC) significantly decreased in the combination-treated group (−23.8% in the first year) and the estrogen-treated group (−37.6% and −41.2% at 6 and 18 months, respectively), and serum alkaline phosphatase (Alp) decreased significantly in the first year in the combination-treated (−31.5%), estrogen-treated (−27.3%), and 1α(OH)D₃-treated (−7.9%) groups, whereas serum OC increased (+45.4% in the first year) in women without treatment. The results of this study indicate that early postmenopausal bone loss in the femoral neck is prevented by conjugated estrogens, 1α(OH)D₃, or both, whereas bone loss in the spine is not prevented by 1α(OH)D₃. Estrogen proves effective in preventing early postmenopausal bone loss by markedly inhibiting bone turnover. Moreover, a synergistic bone-sparing effect can be expected when estrogen is administered concomitantly with 1α(OH)D₃ rather than when used alone. Received: 28 April 1998 / Accepted: 23 December 1998     

The Effect of Recombinant PTH(1–34) and PTH(1–84) on Serum Ionized Calcium, 1,25-Dihydroxyvitamin D,and Urinary Calcium Excretion: A Pilot Study

We investigated the frequency of hypercalcemia and/or hypercalciuria following parathyroid hormone (PTH) 1–34 and 1–84 administration in a crossover trial. Ten postmenopausal osteoporotic women previously treated with bisphosphonates were subdivided into two groups of five patients each. A 24-h urine collection to determine baseline calcium (Ca) and creatinine (Cr) the day before administration of PTH was followed by determination of serum ionized Ca (Ca²⁺), Cr, 25(OH)D, and 1,25(OH)₂D at baseline. Thereafter, 100 mcg of PTH(1–84) or 20 mcg of PTH(1–34) was administered. A 24-h urinary collection and blood samples 2, 4, and 24-h after each PTH administration were again taken. One week after the first PTH administration patients were rechallenged with the second PTH. The PTH peptides did not differ with respect to changes in Ca²⁺ at 2, 4, and 24 h postinjection; at the last time point the values were virtually identical to the initial values. There was no difference in urinary Ca on the day following PTH injection compared to baseline, in terms both of Ca/Cr and of Ca excretion. The two PTH peptides did not differ with respect to changes in 1,25(OH)₂D at 2, 4, and 24 h considering both the absolute values and the percent changes with respect to baseline (24-h 1–84 = 125.6 ± 58.6 pg/ml, 153% increase; 1–34 = 124.1 ± 64.7, 130%). Our results indicate no difference in postinjection serum Ca²⁺, 1,25(OH)₂D, or urinary Ca excretion after a single dose of either PTH(1–84) or PTH(1–34) in patients previously treated with bisphosphonates.     

The effect of a new vitamin D analog, 22-oxa-1α,25(OH)2D3, on bone mineral metabolism in normal male rats

Summary Thein vivo effects of 22-oxa-1, 25 dihydroxyvitamin D₃ (OCT), on bone mineral metabolism were investigated in normal male Sprague-Dawley rats. The rats were administered either vehicle (control), low-dose OCT (25 ng/100 g body weight), or high-dose OCT (250 ng/100 g body wt). High-dose OCT increased serum ionized calcium (P<0.05) and decreased serum parathyroid hormone (PTH) (P<0.05) at all time points and increased serum bone Gla protein on days 7 and 28 (P<0.05) compared with controls. Lowdose OCT decreased serum PTH at all the time points (P<0.05) compared with controls. Tibial bone histomorphometry showed no significant differences between the two doses of OCT and controls. We found that OCT has minimal direct effects on bone metabolism in normal male rats in contrast to 1,25 dihydroxyvitamin D₃. This property may be advantageous in the treatment with OCT of cell-proliferative diseases.Michael Fallon is deceased     

Effect of 1α-vitamin D3 on bone strength and composition in growing rats with and without corticosteroid treatment

The effects of 1-vitamin D₃ were studied for 6 months in 2-month-old male and female rats on a moderately low calcium diet with or without low-dose prednisolone treatment. Both cortical bone mechanical and biochemical properties were examined. Femoral bone specimens were subjected to torsional loading tests. With age, bone strength and stiffness increased in both sexes, accompanied by an increased degree of mineralization (bone ash and calcium concentrations). During growth, strength and stiffness increased more in male than in female rats. When 1-vitamin D₃ (0.5 g/kg/day) was given alone, bone mechanical competence improved significantly whereas insulin-like growth factor-I (IGF-I) and calcium concentrations in the bone matrix were significantly reduced. Treatment with low-dose prednisolone (0.5 mg/kg/day) alone did not influence bone mechanical properties compared with intact control rats (without prednisolone) although a significant reduction in calcium concentration and an increased phosphorus concentration were measured. A combined therapy with prednisolone and 1-vitamin D₃ significantly increased bone strength, toughness, and stiffness compared with control bones. Both mineralization degree (ash and calcium concentration) and IGF-I concentration were decreased.We conclude that (1) mechanical properties of rat cortical bones improve relatively more in males compared with agematched females during growth which is related to increased bone mass and size, (2) low-dose prednisolone treatment does not change mechanical properties in males, and altered them only nonsignificantly in females despite a change in mineralization degree in both sexes; (3) treatment with 1-vitamin D₃ results in a consistent increase in mechanical competence of the bone accompanied by a significant decrease in IGF-I concentration in the bone matrix.     

Effect of 1α-hydroxyvitamin D3 and egg-shell calcium on bone metabolism in ovariectomized osteoporotic model rats

Egg-shell calcium (Ca) is one of the effective Ca sources for bone metabolism. In the present study, we investigated whether egg-shell Ca had similar effects compared with calcium carbonate (CaCO₃) when vitamin D₃ (1α(OH)D₃) treatment was given to an osteopo-rotic rat model. In both 1α(OH)D₃-supplemented and -unsupplemented rats, the bone mineral density (BMD) of the lumber spine in the vitamin-supplemented group increased significantly compared with the unsupplemented group. In a Ca balance study, there were also significant differences in intestinal Ca absorption, urinary Ca and fecal Ca between the vitamin-supplemented and -unsupplemented groups. These results show that egg-shell Ca could have similar effects to CaCO₃ on bone metabolism. In contrast with CaCO₃, vitamin D₃ supplementation did not significantly increase serum Ca levels in the egg-shell Ca group; however, the mechanism of Ca absorption is still unclear. Our results suggest that egg-shell Ca may be an effective nutrient in Ca metabolism for people treated with vitamin D₃. Received: January 6, 2000 / Accepted: July 31, 2000