A novel synthesis of a radioactive compound of Icr-hydroxyvitamin D-3 (1 alpha OHD3) (1) and its pharmacokinetics are described. Radioactive 1 alpha OHD3 tritiated at 22 and 23 positions ([22,23-H-3(4)] 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-H-3(4)]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 D-3, 1 alpha,25-dihydroxy-vitamin D-3 [1 alpha,25(OH)(2)D-3)], after oral or intravenous administration of [22,23-H-3(4)] 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)(2)D-3 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)(2)D-3 (2).

. The present study was undertaken to clarify the receptor distribution and the pharmacokinetics of 3 H-1α(OH)D 3 , and 3 H-1α,25(OH) 2 D 3 for comparison. Receptor autoradiography was used after intravenous injection to 3-day-old neonatal rats and radioassay-HPLC after oral application to young adult rats. Corresponding results were obtained from both receptor autoradiography and radioassay. After 3 H-1α(OH)D 3 administration, uptake was delayed but sustained over a long period of time and the concentration of silver grains (autoradiography) or recovered 3 H-1α,25(OH) 2 D 3 (radioassay) peaked at a lower level. After 3 H-1α,25(OH) 2 D 3 administration, osteoblast nuclear, whole bone uptake and retention of radiolabeled compound were relatively rapid and short in duration. Nuclear uptake in osteoblasts after administration of 3 H-1α(OH)D 3 was abolished in competition studies with 10-fold unlabeled 1α,25(OH) 2 D 3 . These results indicate that 1α(OH)D 3 continuously supplies osteoblasts with converted 1α,25(OH) 2 D 3 and would not spread to the cells because of the low binding affinity of the receptor. Accordingly, 1α(OH)D 3 appears to have some therapeutic properties different from 1α,25(OH) 2 D 3 because of a relatively slow and sustained accumulation of the receptor and less C max (pharmacokinetics) compared with 1α,25(OH) 2 D 3 .

We examined whether 1α-hydroxyvitamin D3 (1α(OH)D3) is metabolized into 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in bone. Northern blot analysis indicated that the expression of vitamin D3 25-hydroxylase mRNA was highest in the liver, followed by the duodenum, calvaria, lung, kidney, skin and long bone, and lowest in the spleen. Of the bone cell fractions isolated from fetal mouse calvaria by a sequential enzymatic digestion, fraction 3, which consisted of mostly osteoblastic cells, showed the highest expression of vitamin D3 25-hydroxylase mRNA. When either cultured bone cells of fraction 3 or mouse calvaria were incubated with [3H]-1α(OH)D3, a radioactive peak which comigrated at the same position as authentic 1α,25(OH)2D3 was found on an HPLC chromatogram. The radioactive fraction obtained from the conditioned media of fetal mouse calvaria was tentatively identified as 1α,25(OH)2D3 by cochromatography with authentic 1α,25(OH)2D3 on three different HPLC systems and a thermal isomerization analysis. These results indicate that 1α,(OH)D3 is hydroxylated at the 25-position in bones, resulting in the local synthesis of 1α,25(OH)2D3 from 1α(OH)D3 in the skeletal tissues.