Receptor activator of NF-kappa B ligand inhibition suppresses bone resorption and hypercalcemia but does not affect host immune responses to influenza infection.

Receptor activator of NF-kappaB (RANK) and its ligand (RANKL) are essential for osteoclast formation, function, and survival. Osteoprotegerin (OPG) inhibits RANK signaling by sequestering RANKL. This study evaluated the antiosteoclast and immunoregulatory effects of mouse rRANK-Fc, which, similar to OPG, can bind RANKL. The effect of RANKL inhibition by RANK-Fc on osteoclast function was determined by inhibition of vitamin D(3) (1,25(OH)(2)D(3))-induced hypercalcemia. Mice were injected with a single dose of 0, 10, 100, 500, or 1000 microg of RANK-Fc; 100 microg of OPG-Fc; or 5 microg of zoledronate 2 h before 1,25(OH)(2)D(3) challenge on day 0, and sacrificed on days 1, 2, 4, 6, 8, 12, 16, and 20. RANK-Fc doses of 100 or 500 microg were tested in a mouse respiratory influenza virus host-resistance model. A single dose of RANK-Fc > or =100 microg suppressed elevation of serum calcium levels and suppressed the bone turnover marker serum pyridinoline at day 4 and later time points, similar to those observed with OPG-Fc and zoledronate (p < or = 0.01 vs controls). By day 6, both immature and mature osteoclasts were depleted by high doses of RANK-Fc (500 and 1000 microg) or 100 microg of OPG-Fc. RANK-Fc doses of 100 or 500 microg had no detectable effect on immune responses to influenza infection, as measured by activation of cytotoxic T cell activity, influenza-specific IgG response, and virus clearance. RANK-Fc inhibition of RANKL has antiosteoclast activity at doses that have no detectable immunoregulatory activity, suggesting that RANKL inhibitors be further studied for their potential to treat excess bone loss.

1alpha-hydroxyvitamin D2 is less toxic but not bone selective relative to 1alpha-hydroxyvitamin D3 in ovariectomized rats.

Identification of bone selective vitamin D analogues would provide an interesting substance class for the treatment of osteoporosis. The synthetic prodrug 1alpha-hydroxyvitamin D2 [1alpha(OH)D2] has been shown to combine equal bone-preserving activity with distinctly reduced calcemic effects relative to 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] in 3-month-old ovariectomized (OVX) rats. Therefore, 1alpha(OH)D2 may be a bone-selective compound. The aim of this study was to compare the bone protective and the calcemic activities of chronically administered 1alpha(OH)D2 and 1alpha(OH)D3 in 6-month-old OVX rats over a broad dose range from ineffective to toxic doses. Ninety-six female 6-month-old Fischer-344 rats were used for this experiment. Eighty rats were bilaterally OVX, 8 rats were sham-operated (SHAM), and 8 rats were killed at the time of surgery as a baseline control. Groups of OVX rats received vehicle alone (n = 16) or daily doses in the diet of 0.025, 0.05, 0.1, and 0.2 microg of 1alpha(OH)D2 or 1alpha(OH)D3 per kg body weight (BW) per day (n = 8 each). After calcein double-labeling, all animals were killed 3 months post-OVX. Orally administered 1alpha(OH)D2 was significantly less toxic compared with 1alpha(OH)D3 in terms of BW gain and kidney calcium content. The effects of 1alpha(OH)D2 and 1alpha(OH)D3 on serum calcium and urinary calcium excretion were generally similar at all doses in this study. Both 1alpha(OH)D2 and 1alpha(OH)D3 prevented the estrogen deficiency-induced bone loss in OVX rats, and induced profound bone anabolic effects at high dosages. 1alpha(OH)D3 and 1alpha(OH)D2 also dose-dependently increased total bone mineral density (BMD), cortical area, and cortical thickness in the tibial diaphysis of OVX rats. Bone resorption as assessed by osteoclast numbers (Oc.Ns) in vertebral cancellous bone and urinary excretion of deoxypyridinoline (DPD) was dose-dependently suppressed by 1alpha(OH)D2 and 1alpha(OH)D3. These data show that although 1alpha(OH)D2 was slightly but significantly less toxic compared with 1alpha(OH)D3, it did not have increased skeletal effects at any dose. Taken together, our findings argue against selective metabolic activation of 1alpha(OH)D2 in bone.

[Experimental and clinical studies on calcium urolithiasis: (I) Animal model for calcium oxalate urolithiasis using ethylene glycol and 1-alpha (OH) D3].

As calcium oxalate stones are the most important component in urolithiasis, an experimental model has to be designed to clarify the pathogenesis and aid in their prevention. Hyperoxaluria as well as hypercalciuria were produced in rats by administering ethylene glycol (0.5%, in drinking water administered ad libitum) and 1-alpha (OH) D3 (0.5 micrograms/rat given every other day), respectively, for three to four weeks. Neither drug alone produced stones efficiently as did the combination regimen of these two compounds. The occurrence of stones was 77.3%, and with only a moderate degree of renal functional impairment. Biochemical and histological data were obtained using this model.

Parathyroid morphology in rats after administration of active vitamin D3.

The morphology of the parathyroid gland was examined in rats treated for one month with an active vitamin D3, 1 alpha-hydroxyvitamin D3. On continuous administration of 12.5 micrograms/kg/day of 1 alpha-hydroxyvitamin D3, the first histological change of the parathyroid gland, seen on day 10, was atrophy of the chief cells with marked accumulation of prosecretory granules. Replacement of the parenchyma by small or large cysts was evident on days 20 and 30. The remaining portion of the parathyroid parenchyma showed various histological changes: widened intercellular spaces intermingled with many cytoplasmic processes, shrinkage of the cytoplasm of the chief cells, and the presence of a few ghost cells in cysts. The appearance of cysts may be caused by suppression of parathyroid hormone secretion and is a characteristic lesion in hypervitaminosis in rats induced by treatment with active vitamin D3.

An investigation of the toxicity of 1alpha-hydroxycholecalciferol to calves.

Two calves were treated with 15 micrograms/kg body weight of 1alpha-hydroxycholecalciferol by intramuscular injection on four occasions at seven-day intervals. Anorexia and reduced water consumption persisted for 48 h after each treatment. No clinical signs of iridocyclitis or any other lesions of the eyes were present at any time either macroscopically or microscopically. After the first treatment serum GOT and GD activities increased, serum AP activity fell, serum concentrations of calcium and inorganic phosphate increased, and magnesium concentrations decreased. The reduced serum magnesium concentrations and increased calcium and inorganic phosphate concentrations were maintained for the duration of the experiment, but there was no evidence of a cumulative effect of successive treatments. Blood urea concentrations increased after the third treatment. The gross pathology at post mortem examination was similar to that reported after vitamin D3 supplementation.

[Comparative study of the biological activity and toxic effect of 1alpha-hydroxycholecalciferol and ergocalciferol in rats]. / Sravnitel'noe izuchenie biologicheskoi aktivnosti i toksicheskogo deistviia 1alpha-oksikholekal'tsiferola i érgokal'tsiferola na krysakh.

Single administration of 0.25 microgram of sunthetic Ialpha-hydroxycholecalciferol (IalphaOHD3) into nephrectomized rats, maintained at D-avitaminous diet, improved the active transport of calcium ions against the concentration gradient in small intestine of these animals, whereas ergocalciferol was biologically inactive under the same conditions. Administration of IalphaOHD3 during 5 days at a dose 0.025 microgram normalized calcium content in blood serum of rats with D-avitaminosis, Increased doses of IalphaOHD3, administered into intact animals, caused transient hyperphosphatemia, hypercalcemia, calcinosis of internal tissues (kidney heart, aorta) as well as death of some animals. IalphaOHD3 exceeded 400-fold the hypercalcemic and calcinose effects of ergocalciferol. LD50 for IalphaOHD3 was equal to 100 microgram/kg, if it was administered during 5 days per os. Tissue calcinosis was developed after administration of a daily dose 10 microgram/kg, moderate hypercalcemia was caused by a daily dose 1 microgram/kg or 0.25 microgram per an animal; this amount is only 10-fold higher as compared with the physiologic requirement. Ergocalciferol caused hypercalcemia and metastatic calcification only at a dose 4000 microgram/kg. Clinical use of IalphaOHD3 at doses, exceeding the physiologic requirements, has to be prohibited due to high activity of the preparation and to toxicity of its increased doses.

Relative toxicity and metabolic effects of cholecalciferol and 25-hydroxycholecalciferol in chicks.

The relative toxicity and metabolic effectiveness of cholecalciferol (CC) and 25-hydroxycholecalciferol (25-HCC) in chicks were evaluated by feeding six graded levels of each and observing gross and microscopic pathology as well as several metabolic parameters of calcium metabolism. Renal tubular calcification was observed when CC was fed at the rate of 10.0 mg/kg of diet and when 25-HCC was fed at the rate of 0.1 mg/kg diet. Thus, 100-fold increase in toxicity results when the hydroxylated form of CC is fed. Both microscopic renal lesions and increased renal calcium and inorganic phosphate concentrations occurred in chicks with normal serum calcium concentrations.