Synthesis and biological activity of (22E,25R)- and (22,25S)-22-dehydro- 1 alpha,25-dihydroxy-26-methylvitamin D3

Both 25-epimers of (22E)-22-dehydro-1 alpha,25-dihydroxy-26-methylvitamin D3 [22-dehydro-26-methyl-1,25-(OH)2D3] were synthesized. The biological activity of these compounds was tested in binding affinity to chick intestinal receptor protein of 1 alpha,25-dihydroxy-vitamin D3 [1,25-(OH)2D3] and in stimulating for intestinal calcium transport and bone calcium mobilization with vitamin D-deficient rats. The relative potency of (25R)- and (25S)-22-dehydro-26-homo-1,25-(OH)2D3 and 1,25-(OH)2D3 in competing for the intestinal cytosolic binding was 1.7:1.5:1. A similar order of activity was observed on intestinal calcium transport and bone calcium mobilization. In the ability for stimulation of intestinal calcium transport, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were about 3.6 and 2.1 times as active as 1,25-(OH)2D3, respectively. In bone calcium mobilization tests, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were estimated to be 2.2 and 1.6 times as potent as 1,25-(OH)2D3, respectively.     

Intestinal synthesis of 24-keto-1,25-dihydroxyvitamin D3. A metabolite formed in vivo with high affinity for the vitamin D cytosolic receptor

24-Keto-1,25-dihydroxyvitamin D3 has been identified as an intestinal metabolite of 1,25-dihydroxyvitamin D3 by ultraviolet absorbance, mass spectroscopy, and chemical reactivity. The metabolite was produced from 1,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3 in rat intestinal mucosa homogenates. 24-Keto-1,25-dihydroxyvitamin D3 is present in vivo in the plasma and small intestinal mucosa of rats fed a stock diet, receiving no exogenous 1,25-dihydroxyvitamin D3, and in the plasma and small intestinal mucosa of rats dosed chronically with 1,25-dihydroxyvitamin D3. 24-Keto-1,25-dihydroxyvitamin D3 has affinity equivalent to 1,24R,25-trihydroxyvitamin D3 for the 3.7 S cytosolic receptor specific for 1,25-dihydroxyvitamin D3 in the intestine and thymus. In cytosolic preparations contaminated with the 5 S vitamin D-binding protein, both metabolites are about 7-fold less potent than 1,25-dihydroxyvitamin D3. In contrast, in cytosolic preparations largely free of the 5 S binding protein, both metabolites are equipotent with the parent compound. No evidence was obtained supporting a substantial presence of 23-keto-1,25-dihydroxyvitamin D3 in vivo; nor was the latter compound generated in detectable amounts from 1,25-dihydroxyvitamin D3 by intestinal homogenates. Thus, C-24 oxidation is a significant pathway of intestinal 1,25-dihydroxyvitamin D3 metabolism that produces metabolites with high affinity for the cytosolic receptor which mediates vitamin D action.     

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.     

Synthesis of 25-hydroxy-[26,27-3H]vitamin D2, 1,25-dihydroxy-[26,27-3H]vitamin D2 and their (24R)-epimers

Synthesis of a C-24-epimeric mixture of 25-hydroxy-[26,27-3H]vitamin D2 and a C-24-epimeric mixture of 1,25-dihydroxy-[26,27-3H]vitamin D2 by the Grignard reaction of the corresponding 25-keto-27-nor-vitamin D2 and 1 alpha-acetoxy-25-keto-27-nor-vitamin D3 with tritiated methyl magnesium bromide is described. Separation of epimers by high-performance liquid chromatography afforded pure radiolabeled vitamins of high specific activity (80 Ci/mmol). The identities and radiochemical purities of 25-hydroxy-[26,27-3H[vitamin D2 and 1,25-dihydroxy-[26,27-3H]vitamin D2 D2 were established by cochromatography with synthetic 25-hydroxyvitamin D2 or 1,25-dihydroxyvitamin D2. Biological activity of 25-hydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the rat plasma binding protein for vitamin D compounds, and by its in vitro conversion to 1,25-dihydroxy-[26,27-3H]vitamin D2 by kidney homogenate prepared from vitamin D-deficient chickens. The biological activity of 1,25-dihydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3.     

24-homologated 1,25-dihydroxyvitamin D3 compounds: separation of calcium and cell differentiation activities

A series of 24-homologated 1,25-dihydroxyvitamin D3 compounds have been chemically synthesized and studied with regard to their activity in inducing differentiation of human promyelocyte HL-60 cells to monocytes and in calcium mobilizing activity in vitamin D deficient rats. Homologation of 1,25-dihydroxyvitamin D3 or its delta 22 analogue by one or two carbons increases by 10-fold and three-carbon homologation reduces by half the activity in causing differentiation of HL-60. On the other hand, homologation causes a substantial decrease in in vivo calcium mobilization activity. The addition of each carbon at the 24-position decreases binding to the HL-60 receptor or rat intestinal receptor by 5-10-fold so that binding affinity of the trihomo compound for the receptors is 130 times less that of 1,25-dihydroxyvitamin D3. Thus, binding affinity for the receptor cannot account for the preferential activity of the 24-homologated compounds in inducing cell differentiation.     

Highly potent cell differentiation-inducing analogues of 1alpha,25-dihydroxyvitamin D3: synthesis and biological activity of 2-methyl-1,25-dihydroxyvitamin D3 with side-chain modifications

Eight 2-methyl substituted analogues of 20-epi-22R-methyl-1alpha,25-dihydroxyvitamin D3 (5) and 20-epi-24,26,27-trihomo-22-oxa-1alpha,25-dihydroxyvitamin D3 (6: KH-1060) were convergently synthesized. Preparation of the CD-ring portions with modified side chains of 5 and 6, followed by palladium-catalyzed cross-coupling with the A-ring enyne synthons (20a-d), (3S,4S,5R)-, (3S,4R,5R)-, (3S,4S,5S)- and (3R,4R,5S)-3,5-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded two sets of four A-ring stereoisomers of 20-epi-2,22-dimethyl-1,25-dihydroxyvitamin D3 (7a-d) and 20-epi-24,26,27-trihomo-2-methyl-22-oxa-1,25-dihydroxyvitamin D3 (8a-d). The biological profiles of the hybrid analogues were assessed in terms of affinity for vitamin D receptor (VDR) and HL-60 cell differentiation-inducing activity in comparison with the natural hormone. The combined modifications of the A-ring at the 2-position and the side chain yielded analogues with high potency.     

The synthesis and biological activity of 25-hydroxy-26,27-dimethylvitamin D3 and 1,25-dihydroxy-26,27-dimethylvitamin D3: highly potent novel analogs of vitamin D3

We synthesized 25-hydroxy-26,27-dimethylvitamin D3, 9, and 1,25-dihydroxy-26,27-dimethylvitamin D3, 14, from chol-5-enic acid-3 beta-ol and tested their biological activity in vivo and in vitro. 9 was found to be highly potent vitamin D analog with bioactivity similar to that of 25-hydroxyvitamin D3. 9 bound to rat plasma vitamin D binding protein with approximately one-third the affinity of 25-hydroxyvitamin D3. In a duodenal organ culture system and in a competitive binding assay with chick intestinal 1,25-dihydroxyvitamin D receptor, 9 was significantly more potent than 25-hydroxyvitamin D3. 1,25-Dihydroxy-26,27-dimethylvitamin D3, 14 was also highly active in vivo. At doses of 1000-5000 pmol/rat, its action was more sustained than that of 1,25-dihydroxyvitamin D3. 14 bound to vitamin D binding protein about 18 times less effectively than 1,25-dihydroxyvitamin D3. 14 bound to the chick intestinal cytosol receptor with an affinity one-half that of 1,25-dihydroxyvitamin D3. In a duodenal organ culture system, 14 was about half as active as 1,25-dihydroxyvitamin D3. Extension of the sterol side chain, at C-26 and C-27, by methylene groups, prolongs the bioactivity of a vitamin D sterol hydroxylated at C-1 and C-25; the corresponding sterol, hydroxylated only at C-25, does not show any alteration of its bioactivity in vivo. These newly synthesized analogs may potentially be of therapeutic use in various mineral disorders.     

1alpha,25-dihydroxyvitamin D3 stimulates phosphorylation of IkappaBalpha and synergizes with TPA to induce nuclear translocation of NFkappaB during monocytic differentiation of NB4 leukemia cells.

Treatment of NB4 acute promyelocytic leukemia cells with 1,25-dihydroxyvitamin D3 (1,25D3) or analogs 20-epi-22-oxa-24a,26a,27a-trihomo-1alpha,25-dihydroxyvitamin D3, 1,24-dihydroxy-22-ene-24-cyclopropylvitamin D3, 1alpha,25-dihydroxylumisterol3, or 1alpha,25(OH)2-d5-previtamin D3 in combination with TPA induces monocytic differentiation. The role of 1,25D3 in the induction of maturation has been shown to be a priming effect. Differentiation in response to these agents requires VDR-independent signaling of 1,25D3, PKC signaling, intracellular calcium, and calpain activity. In this study we identify the NFkappaB/IkappaB signaling pathway as a target of 1,25D3 and TPA action. One of the priming effects of 1,25D3 appears to be the rapid phosphorylation of serine residues on IkappaBalpha. On their own, 1,25D3, its analogs, and TPA do not alter IkappaBalpha expression; however, combinations of analogs with TPA result in a synergistic decrease in IkappaBalpha expression. Decreased expression of IkappaBalpha likely results from enhanced degradation, which allows the observed subsequent nuclear translocation of NFkappaB subunit p65. Since nuclear-localized NFkappaB was observed only in combination-treated cells, it is proposed that nuclear targets of NFkappaB are required for monocytic differentiation. Intracellular calcium and proteolytic activity are both necessary for the induction of IkappaB regulation and translocation of NFkappaB and are critical components of the nongenomic signaling cascades of the 1,25D3-induced differentiation pathway.     

Ligand-specific structural changes in the vitamin D receptor in solution

Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogues, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogues cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)?D?], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D? [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D? [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of coactivators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and -12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of coactivators.     

Synthesis and evaluation of a 3-position diastereomer of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71)

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.     

Characterization of a specific, high affinity binding macromolecule for 1 alpha, 25-dihydroxyvitamin D3 in cultured chick kidney cells

Cytosol prepared from vitamin D3-deficient kidney cells in culture contains a 3.7 S protein that specifically binds 1,25-dihydroxyvitamin D3 with high affinity and low capacity. Whole kidney homogenate cytosol preparations are shown to possess two 1,25-dihydroxyvitamin D3 binding macromolecules. One of the binding proteins sediments at 3.5 to 3.7 S while the second sediments at 6.0 S. The 6.0 S component has a greater affinity for 25-dihydroxyvitamin D3 than for 1,25-dihydroxyvitamin D3. Cultured cell cytosol was found to have little 6.0 S 25-hydroxyvitamin D3 binding protein. Scatchard analysis of the cultured cell cytosol reveals an equilibrium binding constant (KD) of 5.6 x 10 (-11) with 57 fmol of sites/mg of protein. The receptor-like protein has a Mr = 72,000 and as with other steroid receptors it aggregates in the presence of low potassium concentrations. Analog competition for receptor binding reveals the following potency order: 1,25-dihydroxyvitamin D3 > 25-hydroxyvitamin D3 > 1 alpha-hydroxyvitamin D3 > 24(R),25-dihydroxyvitamin D3; the receptor had no detectable affinity for vitamin D3. The kidney cells respond to 1,25-dihydroxyvitamin D3 by diminishing 25-hydroxyvitamin D3 1 alpha-hydroxylation and increasing 24R-hydroxylation. Cultured cells provide a preparation of cytosol which has allowed extensive characterization of the renal 1,25-dihydroxyvitamin D3 receptor and should facilitate investigations into the role this receptor plays in renal control of vitamin D3 metabolism.     

Isolation and identification of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, a new metabolite of 1,25-dihydroxyvitamin D3 produced in rat kidney

A new metabolite of vitamin D3 was produced in vitro by perfusing rat kidneys with 1,25-dihydroxyvitamin D3 (4 X 10(-6) M). It was isolated and purified from the lipid extract of the kidney perfusate by high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, chemical derivatization, and chemical synthesis, the new metabolite was identified as 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Along with the new metabolite, three other previously identified metabolites, namely, 1,24,25-trihydroxyvitamin D3, 1,25-dihydroxy-24-oxovitamin D3, and 1,23,25-trihydroxy-24-oxovitamin D3, were also isolated. The new metabolite was also formed when 1,23,25-trihydroxy-24-oxovitamin D3 was used as the substrate. Thus, the new metabolite fits into the following metabolic pathway: 1,25-dihydroxyvitamin D3----1,24(R),25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3----1,23,25-trihydroxy-24-oxovitamin D3----1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Further, we used 1 alpha,25-dihydroxy[1 beta-3H]vitamin D3 in the kidney perfusion system and demonstrated 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 as the major further metabolite of 1,25-dihydroxyvitamin D3, circulating in the final perfusate when kidneys were perfused with 1,25-dihydroxyvitamin D3 (6 X 10(-10) M) for 4 h. The biological activity of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-3 alcohol) and its metabolic relationship to 1-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D3 (calcitroic acid or C-23 acid), the other previously identified side-chain cleavage metabolite of 1,25-dihydroxyvitamin D3, are unknown and are presently undergoing investigation.     

Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway

About a decade ago calcitroic acid was isolated as a major side chain cleaved water-soluble metabolite of 1,25-dihydroxyvitamin D3 [Esvelt, R. P., Schnoes, H. K., & Decula, H. F. (1979) Biochemistry 18, 3977]. Presently, calcitroic acid is being considered as the major excretory form of 1,25-dihydroxyvitamin D3. However, the exact site or sites of calcitroic acid production and the possible side chain modified intermediary metabolites that may be formed during the conversion of 1,25-dihydroxyvitamin D3 into calcitroic acid are not fully understood. In the mean time there have been many advances in our understanding of the side-chain metabolism of 1,25-dihydroxyvitamin D3. It is now well established that both the kidney and the intestine metabolize 1,25-dihydroxyvitamin D3 through the C-24 oxidation pathway according to the following steps: 1,25-dihydroxyvitamin D3----1,24,25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3-----1,23,25-trihydroxy-24-oxovitamin D3. Recently, we identified 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-23 alcohol) as a major side chain cleaved lipid-soluble metabolite of 1,25-dihydroxyvitamin D3 and further extended the aforementioned C-24 oxidation pathway in the kidney by demonstrating 1,23,25-trihydroxy-24-oxovitamin D3 as the precursor of C-23 alcohol [Reddy, G. S., Tserng, K. Y., Thomas, B. R., Dayal, R., & Norman, A. W. (1987) Biochemistry 26, 324]. In this present study, we investigated the metabolic fate of 1,25-dihydroxyvitamin D3 (3 X 10(-10) M) in the perfused rat kidney and identified calcitroic acid as the major water-soluble metabolite of 1,25-dihydroxyvitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)     

A synthetic analogue of vitamin D3, 22-oxa-1,25-dihydroxy-vitamin D3, stimulates the production of prostacyclin by vascular tissues.

We investigated the effect of 22-oxa-1,25-dihydroxyvitamin D3, a synthetic analogue of vitamin D3, on the production of prostacyclin by vascular tissues using rat aortic rings and A7r5 cells derived from fetal rat aortic smooth muscle. Prostacyclin synthesis by aortic rings of rats treated with 22-oxa-1,25-dihydroxyvitamin D3 was much higher than that of non-treated controls, but did not cause any significant hypercalcemia. Treatment with 22-oxa-1,25-dihydroxyvitamin D3 significantly increased the production of prostacyclin by A7r5 cells for 48 hours in a dose-dependent manner. In time-course studies, cells incubated with 22-oxa-1,25-dihydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 produced prostacyclin progressively over a period of 48 hours. The shortest period of incubation that produced a significant amount of prostacyclin compared with control cultures was 24 hours. We observed that treatment with 22-oxa-1,25-dihydroxyvitamin D3 induced cyclooxygenase mRNA in A7r5 cells. Our data suggest that 22-oxa-1,25-dihydroxyvitamin D3 may possibly be a protective substance against the development of atherosclerosis by modulating prostaglandin metabolism.     

Isolation and identification of 1,24,25-trihydroxyvitamin D2, 1,24,25,28-tetrahydroxyvitamin D2, and 1,24,25,26-tetrahydroxyvitamin D2: new metabolites of 1,25-dihydroxyvitamin D2 produced in rat kidney

Three new metabolites of vitamin D2 were produced in vitro by perfusing isolated rat kidneys with 1,25-dihydroxyvitamin D2. They were isolated and purified from the kidney perfusate by the techniques of methanol-methylene chloride lipid extraction and high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, and specific chemical reactions, the metabolites were identified as 1,24,25-trihydroxyvitamin D2, 1,24,25,28-tetrahydroxyvitamin D2, and 1,24,25,26-tetrahydroxyvitamin D2. Both 1,24,25,28-tetrahydroxyvitamin D2 and 1,24,25,26-tetrahydroxyvitamin D2 were also produced when a kidney was perfused with 1,24,25-trihydroxyvitamin D2. Thus, it becomes clear that 1,25-dihydroxyvitamin D2 is first hydroxylated at C-24 to form 1,24,25-trihydroxyvitamin D2, which is then further hydroxylated at C-28 and C-26 to form 1,24,25,28-tetrahydroxyvitamin D2 and 1,24,25,26-tetrahydroxyvitamin D2, respectively. From several recent studies, it has been well established that 1,25-dihydroxyvitamin D3 is converted into various further metabolites in the kidney as a result of chemical reactions such as C-23, C-24, and C-26 hydroxylations, C-24 ketonization, and C-23:C-26 lactonization. From our study it is obvious that 1,25-dihydroxyvitamin D2 does not undergo all of the aforementioned chemical reactions except C-24 and C-26 hydroxylations. Also, our study indicates that C-28 hydroxylation plays a significant role in the further metabolism of 1,25-dihydroxyvitamin D2. Thus, for the first time, we describe a novel further metabolic pathway for 1,25-dihydroxyvitamin D2 in a mammalian kidney.     

Effect of age on duodenal 1,25-dihydroxyvitamin D-3 receptors in Wistar rats

We confirmed our previous observation that duodenal Ca2+ absorption and serum 1,25-dihydroxyvitamin D (1,25-(OH)2D) levels declined concurrently in old (24 months old) rats as compared to young (6 months old) rats. It is well known that 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) expresses its action after binding to specific receptor molecules. In this paper, we compared certain properties of rat duodenal 1,25-(OH)2D3 receptors from old and young animals. Receptor preparations were incubated with [3H]1,25-(OH)2D3 to quantitate the number of unoccupied and total receptor sites and showed that total and unoccupied receptor sites decreased by 22 and 16%, respectively in old rats. Endogenously occupied sites were reduced by 43% in duodenum of the old rat and, consequently, the percentage of receptor occupancy also declined. Age did not affect the dissociation constant (KD) of 1,25-(OH)2D3 from the receptor; the sedimentation coefficient (3.3 S) of the tritiated 1,25-(OH)2D3-receptor complex in sucrose density centrifugation; or its affinity for DNA. The data are consistent with the hypothesis that the age-related decline in Ca2+ absorption in the intestine may be due, in part, to the decrement in the circulating level of 1,25-(OH)2D and a reduction of intestinal 1,25-(OH)2D3 receptor occupancy status.     

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D3

To evaluate possible functional roles for 24,25-dihydroxyvitamin D₃, 24,24-difluoro-25-hydroxyvitamin D₃ has been synthesized and shown to be equally as active as 25-hydroxyvitamin D₃ in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D₃ was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D₃, while large amounts were found when 25-hydroxyvitamin D₃ was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D₃, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D₃ from 24,24-difluoro-25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D₃ is less active than 25-hydroxyvitamin D₃ and 24R,25-dihydroxyvitamin D₃. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D₃ is more active than 25-hydroxyvitamin D₃ which is itself more active than 24R,25-dihydroxyvitamin D₃. The 24,24-difluoro-1,25-dihydroxyvitamin D₃ is equal to 1,25-dihydroxyvitamin D₃, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D₃ in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D₃ cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds.     

Up-regulation of the intestinal 1,25-dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D2 and vitamin D3

Concentrations of intestinal 1,25-dihydroxyvitamin D receptor were measured in rats receiving pharmacological amounts (25,000 IU/rat daily for 6 days) of either vitamin D2 or vitamin D3. The data showed that both hypervitaminosis D2 and hypervitaminosis D3 resulted in significant up-regulation of intestinal 1,25-dihydroxyvitamin D receptor (fmol/mg protein) relative to controls (409 +/- 24, vitamin D2-treated; 525 +/- 41, vitamin D3-treated; and 249 +/- 19, control). The 1,25-dihydroxyvitamin D receptor enhancement also was accompanied by elevated plasma 25-hydroxyvitamin D and hypercalcemia. These data suggest that increased target-tissue 1,25-dihydroxyvitamin D receptor may play a role in enhancing target-tissue responsiveness and, thus, have a significant role in mediating the toxic effects of hypervitaminosis D.     

Synthesis, biologic activity, and protein binding characteristics of a new vitamin D analog, 22-hydroxyvitamin D3

We synthesized a novel vitamin D analog, 22-hydroxyvitamin D3 9 and tested its biologic activity (and antivitamin properties) in vivo in vitamin D-deficient rats, and in vitro in the chick embryonic duodenum. We examined its ability to bind to the sterol carrier protein, vitamin D binding protein and the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. The new vitamin 9 was synthesized from 3 beta-hydroxy-22,23-dinorcholenic acid 1 in 12 steps. The vitamin 9 displayed no vitamin D agonist activity in the intestine or in bone in vivo and did not block the activity of vitamin D3 or 25-hydroxyvitamin D3. It was a weak vitamin D3 agonist in the chick embryonal duodenum in vitro. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. Vitamin 9 bound to the chick intestinal cytosol receptor with low affinity. 22-Hydroxyvitamin D3 and various vitamin D sterols were bound to vitamin D binding protein in the following order: 25-hydroxyvitamin D3. (24R)-24,25-dihydroxyvitamin D3, and (25S)-25,26-dihydroxyvitamin D3 greater than 22-hydroxyvitamin D3 greater than 11 alpha-hydroxyvitamin D3 greater than 1,25-dihydroxyvitamin D3 greater than vitamin D3. We conclude that the introduction of a hydroxyl group at C-22 in the side chain of the vitamin D3 molecule decreases its biological activity.