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The affinity of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] and analogs with side-chain modifications [MC 903 or calcipotriol, MC 1147 or 24,24-dihomo-1 alpha,25-(OH)2D3 and 1,25-(OH)2-16ene-23yne-D3] for the vitamin D receptor and the serum vitamin D binding protein (DBP) were compared. The affinity of MC 903 for the receptor from chick and rat duodenum or from human peripheral blood mononuclear cells or HL-60 cells varied between 60 and 100% relative to the affinity of 1,25-(OH)2D3. The relative affinity of 1,25-(OH)2-16ene-23yne-D3 and MC 1147 varied for the same receptors between 45-70 and 3.5-25%, respectively. The relative affinity of MC 903 for human DBP was 30-fold decreased, whereas the two other analogs did not bind to DBP at all even in more than 1000-fold excess. The in vitro biologic activity of 1 alpha,25-(OH)2D3 on phytohemagglutinin-stimulated normal human lymphocyte proliferation was markedly inhibited by the addition of physiologic amounts of DBP to the cell culture medium. No such inhibition was observed when MC 903 or 1147 was evaluated similarly. DBP therefore reversed the rank order of the in vitro potency of these analogs. Intramuscular injections for 10 consecutive days to vitamin D-deficient chicks demonstrated a greater than or equal to 100-fold lower biologic activity of MC 903, MC 1147, and 1,25-(OH)2-16ene-23yne-D3 compared to that of 1 alpha,25-(OH)2D3 as evaluated by serum calcium and osteocalcin concentrations, as well as by duodenal calbindin D28K and bone calcium content.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin D3 and its hydroxylated metabolites are normally in thermal equilibrium with their previtamin D isomers. To evaluate the biologic activity of 1 alpha, 25-dihydroxyprevitamin D3, we synthesized 19-nor analogs of 1 alpha, 25-dihydroxy(pre)vitamin D3 because the absence of a C19 methylene group prevents the isomerization of these analogs. The affinity of 1 alpha, 25-(OH)2D3-19-nor-D3 for the intestinal vitamin D receptor and plasma vitamin D binding protein was mildly decreased [30 and 20% of the affinity of 1 alpha, 25-(OH)2D3, respectively], but the affinity of 1 alpha, 25-(OH)2-19-nor-previtamin D3 was only 1 and 6% of that of 1 alpha, 25-(OH)2D3 for the receptor and DBP, respectively. The in vitro effects on human promyeloid leukemia (HL-60 cell) differentiation and osteocalcin secretion by human osteosarcoma (MG-63) cells by 1 alpha, 25-(OH)2-19-nor-D3 were nearly identical to those of 1 alpha-25-(OH)2D3, whereas 19-nor-previtamin D3 showed poor activity (2%). The in vivo calcemic effects of both analogs, studied in vitamin D-deficient chicks treated for 10 consecutive days with the analogs, showed no activity of the previtamin D3 analog and reduced calcemic effects (< or = 10%) of 1 alpha, 25-(OH)2-19-nor-D3. We conclude that the previtamin D form of 1 alpha, 25-(OH)2D3 has lost most of its biologic activity in vitro and in vivo.

The biological activity of 16-epoxy side-chain analogs of 1 alpha,25-dihydroxyvitamin D3, (1 alpha,25(OH)2D3) was evaluated in vitro and in vivo. Compared to 1 alpha,25(0H)2D3, all analogs had lower affinities for the pig duodenal vitamin D receptor and also for the human serum vitamin D binding protein. The in vitro effects on cell proliferation or differentiation of human promyeloid leukemia (induction of superoxide production in HL-60 cells), human osteosarcoma MG-63 cells (osteocalcin secretion), or human breast cancer cells (incorporation of thymidine in MCF-7 cells), was markedly inhibited by several epoxy analogs, compared to 1 alpha,25(OH)2D3, but the rank order of their activity widely varied among different cancer cells. The most potent analogs (24S,25S-24-hydroxy-25,26-epoxy-22-ene-1 alpha-OHD3, 25,26-epoxy-23-yne-1 alpha-OHD3, and 25,26-epoxy-23-yne-20-epi-1 alpha-OHD3 or compounds, 16, 5, and 7, respectively) were equipotent (16 and 5) or 30-fold (compound 7 on MG-63 cells) to 40-fold (compound 7 on MCF-7 cells) more active than 1 alpha,25-(OH)2D3. These analogs were nevertheless poorly antirachitic (3%) when tested in vitamin D-deficient chicks (using serum and bone calcium, serum osteocalcin and duodenal calbindin D-28K, as end points). Compound 7 was also 100-fold more active than 1 alpha,25-(OH)2D3 in inhibition of proliferation of human foreskin keratinocytes. Some epoxy analogs of 1 alpha,25-(OH)2D3 thus display interesting dissociations between their receptor affinity and their potency to induce cell differentiation, whereas their effect on cell proliferation/differentiation exceed their calcemic effects more than 100- to 1000-fold.

Maternal and fetal 1,25-dihydroxyvitamin D 3 (l,25(OH) 2 D 3 ) and osteocalcin were measured in guinea pigs, to examine their potential use as animal models for fetal bone development and calcium homeostasis. Measurements were performed on days 42, 57 and 63 of gestation. Maternal serum total l,25(OH) 2 D 3 concentrations were increased only at the end of gestation (day 63). However, because the vitamin D binding protein (DBP) and albumin levels were decreased by 35–50% from day 42 onwards, the unbound l,25(OH) 2 D 3 , calculated as the l,25(OH) 2 D 3 /DBP molar ratio, was increased before day 63. Osteocalcin concentrations during gestation were 50–54% of levels found in nongravid animals. Fetal serum total l,25(OH) 2 D 3 concentrations were 20% of those in maternal guinea pigs. Since DBP levels were only 9–15% of maternal levels, the unbound l,25(OH) 2 D 3 was consistently higher in fetuses, from day 42 onwards. There was a rise in total and unbound l,25(OH) 2 D 3 between days 57 and 63 of fetal life. Osteocalcin concentrations were higher in fetal than in adult guinea pigs, and reached peak values on day 57 (1023 μ g/l, i.e. 4.2 times higher than in adult female guinea pigs). Fetuses of guinea pigs that had received a restricted food supply for 14 days (days 49–63) had normal l,25(OH) 2 D 3 concentrations, but decreased osteocalcin concentrations compared with normal fetuses. The data obtained in fetal guinea pigs are comparable with those found in human fetuses, and suggest that the guinea pig may be a suitable model for studies on fetal bone and mineral development.

The kinetics of 1,25-dihydroxyvitamin D3 [1,25(OH)2-D3] and the in vivo response to 1,25(OH)2D3 (7.5, 15, and 30 ng/100 g body wt), infused or injected subcutaneously for 12-14 days, were studied in male spontaneously diabetic and control BB rats. In control rats, increasing doses of 1,25(OH)2D3 produced parallel increases in plasma 1,25(OH)2D3 and calcium, urinary calcium, duodenal CaBP9K, and renal CaBP28K. 1,25-(OH)2D3 at 30 ng/100 g markedly raised plasma osteocalcin and osteoblast/osteoid surfaces in the tibial metaphysis, but inhibited bone mineralization rate. In diabetic rats, plasma 1,25-(OH)2D3 concentrations were decreased, and the rise of plasma 1,25(OH)2D3 during 1,25(OH)2D3 infusion was blunted, but the free 1,25(OH)2D3 index remained normal or above normal. Diabetic rats had an increased metabolic clearance rate of 1,25-(OH)2D3 (0.38 +/- 0.015 vs. 0.24 +/- 0.007 ml.min-1.kg-1), with no further increase in 1,25(OH)2D3-infused diabetic rats; their relative production rate of 1,25(OH)2D3 was unchanged. The responses of plasma and urinary calcium, duodenal CaBP9K, and renal CaBP28K to infused 1,25(OH)2D3 were normal, as was duodenal calcium absorption in 1,25(OH)2D3-injected diabetic rats. However, the virtual absence of osteoblasts/osteoid in trabecular bone was unaltered in diabetic rats infused with 30 ng/100 g 1,25(OH)2D3, with only minimal increase of their low plasma osteocalcin levels. 1,25(OH)2D3 treatment therefore cannot be expected to reverse diabetic osteopenia.

24-Oxa-vitamin D 3 (24-oxa-D 3 ) and 24-oxa-1α-hydroxyvitamin D 3 were designed as possible inhibitors of the vitamin D metabolic activation pathway. Their affinity for the vitamin D receptor (from pig intestine) and human vitamin binding protein were reduced, and their potency to induce cell differentiation of human leukemia cells (HL 60) or osteosarcoma cells (MG 63) was markedly reduced (19% and 3%, respectively), in comparison with calcitriol. A single or chronic injection of 24-oxa-D 3 had no biological activity, whereas chronic administration of 24-oxa-1α-hydroxy-D 3 showed weak agonist activity in rachitic chicks. When the 24-oxa-D 3 was given prior to a single injection of vitamin D 3 , lower values of serum calcium (64% of the value obtained in vitamin D—treated animals), osteocalcin (52%), 25-(OH)D 3 (45%) and duodenal calbindin-D 28K (9.4%) were found. When given chronically in a 100-fold more excess no clear antagonistic effects were observed. 24-Oxa-D 3 is thus a new metabolic weak antagonist of vitamin D 3 , but adding a hydroxyl group at C-1 creates a weak agonist.