Intestinal and hepatic CYP3A4 catalyze hydroxylation of 1alpha,25-dihydroxyvitamin D(3): implications for drug-induced osteomalacia

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D(3) metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23- and 24-hydroxylation of 1,25(OH)(2)D(3). No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH)(2)D(3) hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH)(3)D(3), 1,24S,25(OH)(3)D(3), and 1,23S,25(OH)(3)D(3). Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH)(2)D(3) 23- and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH)(2)D(3) metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.     

Metabolism of 26,26,26,27,27,27-F6-1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The compound 26,26,26,27,27,27-F(6)-1alpha,25(OH)(2)D(3) is a hexafluorinated analog of the active form of Vitamin D(3). The enhanced biological activity of F(6)-1alpha,25(OH)(2)D(3) is considered to be related to a decreased metabolic inactivation of the compound in target tissues such as the kidneys, small intestine, and bones. Our previous study demonstrated that CYP24 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues. In this study, we compared the human and rat CYP24-dependent metabolism of F(6)-1alpha,25(OH)(2)D(3) by using the Escherichia coli expression system. In the recombinant E. coli cells expressing human CYP24, bovine adrenodoxin and NADPH-adrenodoxin reductase, F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3), F(6)-23-oxo-1alpha,25(OH)(2)D(3), and the putative ether compound with the same molecular mass as F(6)-1alpha,25(OH)(2)D(3). The putative ether was not observed in the recombinant E. coli cells expressing rat CYP24. These results indicate species-based difference between human and rat CYP24 in the metabolism of F(6)-1alpha,25(OH)(2)D(3). In addition, the metabolite with a cleavage at the C(24)z.sbnd;C(25) bond of F(6)-1alpha,25(OH)(2)D(3) was detected as a minor metabolite in both human and rat CYP24. Although F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3) had a high affinity for Vitamin D receptor, the side-chain cleaved metabolite and the putative ether showed extremely low affinity for Vitamin D receptor. These findings indicate that human CYP24 has a dual pathway for metabolic inactivation of F(6)-1alpha,25(OH)(2)D(3) while rat CYP24 has only one pathway. Judging from the fact that metabolism of F(6)-1alpha,25(OH)(2)D(3) in rat CYP24-harboring E. coli cells is quite similar to that in the target tissues of rat, the metabolism seen in human CYP24-harboring E. coli cells appear to exhibit the same metabolism as in human target tissues. Thus, this recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of Vitamin D analogs in human target tissues before clinical trials.     

Isolation and identification of 1alpha-hydroxy-24-oxovitamin D3 and 1alpha,23-dihydroxy-24-oxovitamin D3: metabolites of 1alpha,24(R)-dihydroxyvitamin D3 produced in rat kidney

1alpha,24(R)-Dihydroxyvitamin D3 [1alpha,24(R)(OH)2D3], a synthetic vitamin D3 analog, has been developed as a drug for topical use in the treatment of psoriasis. At present, the target tissue metabolism of 1alpha,24(R)(OH)2D3 is not understood completely. In our present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in the isolated perfused rat kidney. The results indicated that 1alpha,24(R)(OH)2D3 is metabolized in rat kidney into several metabolites, of which 1alpha,24(R),25-trihydroxyvitamin D3, 1alpha,25-dihydroxy-24-oxovitamin D3, 1alpha,23(S),25-trihydroxy-24-oxovitamin D3, and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D3 are similar to the previously known metabolites of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. In addition to these aforementioned metabolites, we also identified two new metabolites, namely 1alpha-hydroxy-24-oxovitamin D3 and 1alpha,23-dihydroxy-24-oxovitamin D3. The two new metabolites do not possess the C-25 hydroxyl group. Thus, the metabolism of 1alpha,24(R)(OH)2D3 into both 25-hydroxylated and non-25-hydroxylated metabolites suggests that 1alpha,24(R)(OH)2D3 is metabolized in the rat kidney through two pathways. The first pathway is initiated by C-25 hydroxylation and proceeds further via the C-24 oxidation pathway. The second pathway directly proceeds via the C-24 oxidation pathway without prior hydroxylation at the C-25 position. Furthermore, we demonstrated that rat kidney did not convert 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] into 1alpha,25(OH)2D3. This finding indicates that the rat kidney does not possess the classical vitamin D3-25-hydroxylase (CYP27) activity. However, from our present study it is apparent that prior hydroxylation of 1alpha(OH)D3 at the C-24 position in the 'R' orientation allows 25-hydroxylation to occur. At present, the enzyme responsible for the C-25 hydroxylation of 1alpha,24(R)(OH)2D3 is unknown. Our observation that the side chain of 1alpha,24(R)(OH)2D3 underwent 24-ketonization and 23-hydroxylation even in the absence of the C-25 hydroxyl group suggests that 1alpha,25(OH)2D3-24-hydroxylase (CYP24) can perform some steps of the C-24 oxidation pathway without prior C-25 hydroxylation. Thus, we speculate that CYP24 may be playing a dual role in the metabolism of 1alpha,24(R)(OH)2D3.     

Metabolism of 26,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 and 26,27-hexafluoro-1 alpha,23(S)25-trihydroxyvitamin D3 in ROS17/2.8 cells transfected with a plasmid expressing CYP24

1. To clarify the possibility that the metabolism of 26,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 [F6-1,25(OH)2D3] to 26,27-hexafluoro-1 alpha,23(S),25-trihydroxyvitamin D3 [F6-1,23,25(OH)3D3 and that of F6-1,23,25(OH)3D3 to 26,27-hexafluoro-23-oxo-1 alpha,25-dihydroxyvitamin D3 [F6-23-oxo-1,25(OH)2D3] are catalysed by 25-hydroxyvitamin D3 24-hydroxylase (CYP24), ROS17/2.8 cells transfected with a plasmid expressing CYP24 [pSVL-CYP24(+)] and a corresponding blank plasmid [pSLV-CYP24R(-)] were used. 2. Incubation of [1 beta-3H]-F6-1,25(OH)2D3 for 2 and 5 days with ROS17/2.8 cells transfected with pSVL-CYP24(+) generated a metabolite that co-migrated with authentic F6-1,23,25(OH)3D3 in both normal phase and reversed-phase HPLC systems. 3. Incubation of [1 beta-3H]-F6-1,23,25(OH)3D3 for 5 days with pSVL-CYP24(+)- transfected ROS 17/2.8 cells generated a metabolite that co-migrated with authentic F6-23-oxo-1,25(OH)2D3. In contrast, the metabolites F6-1,23,25(OH)3D3 or F6-23-oxo-1,25(OH)2D3 were not generated in the cells transfected with pSVL-CYP24R(-). 4. The results indicate that CYP24 catalyses the conversion of F6-1,25(OH)2D3 to F6-1,23,25(OH)3D3 and that of F6-1,23,25(OH)3D3 to F6-23-oxo-1,25(OH)2D3.     

Metabolism of 2 alpha-propoxy-1 alpha,25-dihydroxyvitamin D3 and 2 alpha-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 by human CYP27A1 and CYP24A1.

Recently, we demonstrated that some A-ring-modified vitamin D3 analogs had unique biological activity. Of these analogs, 2alpha-propoxy-1alpha,25(OH)2D3 (C3O1) and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)2D3 (O2C3) were examined for metabolism by CYP27A1 and CYP24A1. Surprisingly, CYP27A1 catalyzed the conversion from C3O1 to O2C3, which has 3 times more affinity for vitamin D receptor than C3O1. Thus, the conversion from C3O1 to O2C3 by CYP27A1 is considered to be a metabolic activation process. Five metabolites were detected in the metabolism of C3O1 and O2C3 by human CYP24A1 including both C-23 and C-24 oxidation pathways. On the other hand, three metabolites of the C-24 oxidation pathway were detected in their metabolism by rat CYP24A1, indicating a species-based difference in the CYP24A1-dependent metabolism of C3O1 and O2C3 between humans and rats. Kinetic analysis revealed that the Km and kcat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1alpha,25(OH)2D3. Thus, the catalytic efficiency, kcat/Km, of human CYP24A1 for O2C3 is only 2% of 1alpha,25(OH)2D3. These results and a high calcium effect of C3O1 and O2C3 in animal experiments using rats suggest that C3O1 and O2C3 are promising for clinical treatment of osteoporosis.     

Metabolism of 26,26,26,27,27,27-F6-1 alpha,23S,25-trihydroxyvitamin D3 by human UDP-glucuronosyltransferase 1A3.

26,26,26,27,27,27-Hexafluoro-1alpha,25-dihydroxyvitamin D(3) [F(6)-1alpha, 25(OH)(2)D(3)], which is now clinically used as a drug for secondary hyperparathyroidism, is a hexafluorinated analog of the active form of vitamin D(3). Our previous studies demonstrated that CYP24A1 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues and that F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3). In this study, we examined the metabolism of F(6)-1alpha,25(OH)(2)D(3),F(6)-1alpha,23S,25(OH)(3)D(3), and F(6)-23-oxo-1alpha,25(OH)(2)D(3) by human UDP-glucuronosyltransferases (UGTs). Of these compounds, F(6)-1alpha,23S,25(OH)(3)D(3) was remarkably glucuronidated both in human liver microsomes and in the recombinant system expressing human UGT. No significant interindividual differences were observed among 10 human liver samples. The recombinant system for 12 species of human UGTs revealed that F(6)-1alpha,23S,25(OH)(3)D(3) glucuronidation was specifically catalyzed by UGT1A3. The information obtained in this study seems very useful to predict the metabolism and efficacy of vitamin D analogs in human bodies before clinical trials. In addition, note that for the first time a possible probe substrate for UGT1A3 has been found.     

An inducible cytochrome P450 3A4-dependent vitamin D catabolic pathway

Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4β,25-dihydroxyvitamin D(3) [4β,25(OH)(2)D(3)] dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4β,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4β,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4β,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.     

Rapid and drastic induction of CYP3A4 mRNA expression via vitamin D receptor in human intestinal LS180 cells

The aim of this study was to evaluate the usefulness of human intestinal LS180 cells for studying the induction of CYP3A4 mRNA expression via vitamin D receptor (VDR). CYP3A4 mRNA expression in LS180 cells treated with 100 nM 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) for 6 and 24 h was approximately 80- and 500-fold higher than the control, respectively. A protein kinase (PK) inhibitor (staurosporine), c-jun N-terminal kinase (JNK) pathway inhibitor (curcumin), and JNK inhibitor (SP600125) attenuated 1alpha,25(OH)(2)D(3)-induced CYP3A4 mRNA expression, suggesting that the PK-JNK pathway contributed to the rapid and drastic induction of CYP3A4 expression via VDR in LS180 cells. The ability of CYP3A4 mRNA induction in LS180 cells was highly dependent on the site and number of vitamin D(3) and D(2) hydroxylation. In addition, short-time (6 h) treatment of LS180 cells with cytotoxic secondary bile acids, lithocholic acid (LCA) and 3-keto-LCA also significantly induced the mRNA expression of CYP3A4. LS180 cells may be useful to quickly investigate the CYP3A4-inducing effect of drugs, xenobiotics, and/or endogenous substrates in the intestinal epithelia.     

Protective effects of 1 alpha,25-(OH)(2)D(3) against the neurotoxicity of glutamate and reactive oxygen species in mesencephalic culture

This study was undertaken to determine whether 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)(2)D(3)], an active metabolite of vitamin D, protects dopaminergic neurons against the neurotoxic effects of glutamate and dopaminergic toxins using rat mesecephalic culture. Brief glutamate exposure elicited cytotoxicity in both dopaminergic and non-dopaminergic neurons. Pretreatment, but not co-administration, of 1 alpha,25-(OH)(2)D(3) protected both types of neurons against the cytotoxicity of glutamate in a concentration- and time-dependent manner. The neuroprotective effect of 1 alpha,25-(OH)(2)D(3) was inhibited by the protein synthesis inhibitor, cycloheximide. To investigate the mechanisms of these neuroprotective effects, we examined the effects of 1 alpha,25-(OH)(2)D(3) on neurotoxicity induced by calcium ionophore and reactive oxygen species (ROS). Pretreatment with 1 alpha,25-(OH)(2)D(3) protected both types of neurons against the cytotoxicity induced by A23187 in a concentration-dependent manner. Furthermore, 24-h pretreatment with 1 alpha,25-(OH)(2)D(3) concentration-dependently protected both types of neurons from ROS-induced cytotoxicity. A 24-h incubation with 1 alpha,25-(OH)(2)D(3) inhibited the increase in intracellular ROS level following H(2)O(2) exposure. A 24-h exposure to 1-methyl-4-phenylpyridium ion (MPP(+)) or 6-hydroxydopamine (6-OHDA) exerted selective neurotoxicity on dopaminergic neurons, and these neurotoxic effects were ameliorated by 1 alpha,25-(OH)(2)D(3). These results suggest that 1 alpha,25-(OH)(2)D(3) provides protection of dopaminergic neurons against cytotoxicity induced by glutamate and dopaminergic toxins by facilitating cellular functions that reduce oxidative stress.     

The biological activities of 1alpha,25-dihydroxyvitamin D3 and its synthetic analog 1alpha,25-dihydroxy-16-ene-vitamin D3 in normal human osteoblastic cells and human osteosarcoma SaOS-2 cells are modulated by 17-beta estradiol and dependent on stage of differentiation

We compared the effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and its analog, 1alpha,25-dihydroxy-16-ene-vitamin D3 [1alpha,25(OH)2-16-ene-D3], as well as their interactions with 17-beta estradiol (E2) on osteoblastic function in our human normal (HOB) and osteosarcoma SaOS-2 cell models representing two different stages of differentiation, the more differentiated HOB+DEX cells and SaOS+DEX cells, and the corresponding less differentiated HOB-DEX and SaOS-DEX cells. The differential effects of 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 and the modulation by E2 on ALP activity in HOB-DEX and HOB+DEX cells were small but significant. The most significant effects were seen in SaOS+DEX cells, in which 1alpha,25(OH)2-16-ene-D3 was 100-fold more potent than 1alpha,25(OH)2D3, the maximal enhancement being exerted at 0.1 nM and 10 nM, respectively. E2 enhanced the stimulatory effects of both compounds, with ALP being increased 2-fold at 0.1 nM (p<0.001). Osteocalcin (OC) production in HOB-DEX cells was stimulated 1.3 to 1.4-fold by 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 at a concentration of 0.01 nM, with E2 inhibiting the effect of 1alpha,25(OH)2-16-ene-D3. In SaOS-DEX and SaOS+DEX cells, 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 stimulated OC production 1.6-fold at 0.1 nM with E2 slightly enhancing the effect of 1alpha,25(OH)2D3. Western blot analysis of 1alpha,25(OH)2D3 receptor (VDR) levels showed that in SaOS+DEX cells, the effect of 1alpha,25(OH)2D3 was larger than that of 1alpha,25(OH)2-16-ene-D3. These results show that 1alpha,25(OH)2-16-ene-D3 is biologically active in human osteoblasts.     

Induction of CYP3A4 by 1 alpha,25-dihydroxyvitamin D3 is human cell line-specific and is unlikely to involve pregnane X receptor.

Under certain culture conditions, exposure of the human colon adenocarcinoma cell line Caco-2 to 1,25-(OH)(2)-D(3) induces expression of CYP3A4 to levels comparable to that in human small intestinal epithelium. To determine whether 1,25-(OH)(2)-D(3) could be used to restore CYP3A expression in other culture models, we examined several cell lines derived from malignancies of human tissues known to express CYP3A enzymes: Hep G2 (liver), LS180 (colon), HPAC (pancreas), Hs746T (stomach). Primary cultures of human hepatocytes from two donors were also examined. 1,25-(OH)(2)-D(3) increased CYP3A catalytic activity in LS180 (15-fold), HPAC (6-fold), and hepatocytes (2- to 3-fold); this was accompanied by induction of CYP3A4 mRNA and CYP3A immunoreactive protein. However, 1,25-(OH)(2)-D(3) had no effect on CYP3A expression in Hs746T or Hep G2. Known ligands for pregnane X receptor (PXR) (rifampin, dexamethasone, and dexamethasone t-butyl acetate) markedly induced CYP3A4 expression in human hepatocytes. In contrast, these ligands had little or no effect on CYP3A4 expression in Caco-2 cells, even at concentrations 1 to 2 orders of magnitude greater than effective concentrations of 1,25-(OH)(2)-D(3) or two other vitamin D receptor (VDR) ligands (25-OH-D(3) and 1-OH-D(3)). The retinoic acid receptor ligand all-trans-retinoic acid augmented the 1,25-(OH)(2)-D(3)-mediated induction of CYP3A4 catalytic activity up to 2-fold in Caco-2 cells, while having no demonstrable effect on levels of CYP3A4 mRNA or protein. The retinoid X receptor ligand 9-cis-retinoic acid appeared to slightly reduce CYP3A4 catalytic activity. We conclude that 1,25-(OH)(2)-D(3) can be used to increase CYP3A4 expression in some, but not all, human cell lines derived from tissues known to express CYP3A enzymes. The mechanisms involved in this induction are unlikely to involve PXR and may involve VDR.     

Manipulation of thromboxane synthesis by novel 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 in human promyelocytic leukemia (HL-60) cells.

Using new steroidal side-chain-lengthened 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], we manipulated the synthesis of thromboxane and thromboxane-producing enzymes, cyclo-oxygenase and thromboxane synthase, in human promyelocytic leukemia (HL-60) cells in serum-free culture. The order of potency of the analogues for stimulating thromboxane B2 synthetic activity from arachidonic acid (reflecting combined cyclo-oxygenase activity and thromboxane synthase activity) and from prostaglandin H2 (thromboxane synthase activity only) as well as for cyclo-oxygenase induction was 1 alpha,25-(OH)2D3 > or = 1 alpha,25-(OH)2-26,27-CH3)2D3 > 1 alpha,25-(OH)2-26,27-(C2H5)2D3 > 1 alpha,25-(OH)2-26,27-(C3H7)2D3. These results suggest that there are functional and structural limits to the chain length of C-26 and C-27 dialkyl groups flanking the C-25-OH group in the 1 alpha,25-(OH)2D3 molecule for expressing thromboxane synthetic activity in HL-60 cells. Removal of the C-1 alpha-OH group from 1 alpha,25-(OH)2D3 led to markedly decreased thromboxane synthetic activity in HL-60 cells. These structure-activity relationships indicate that both the C-25-OH and C-1 alpha-OH groups in the 1 alpha,25-(OH)2D3 molecule are essential for expressing thromboxane synthesis in HL-60 cells. Also, the rank order for stimulating thromboxane synthesis correlated well with the binding affinity of these dialkyl analogues for the 1 alpha,25-(OH)2D3 receptor of HL-60 cells, suggesting a 1 alpha,25-(OH)2D3 receptor-mediated induction mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of 3-epi-1alpha,25-dihydroxyvitamin D3 involved in 1alpha,25-dihydroxyvitamin D3 metabolic pathway in cultured cell lines

Using six different cultured cell models representing osteoblast, intestine, kidney and keratinocyte, we have demonstrated that 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) is metabolized into 3-epi-1alpha,25(OH)2D3 in vitamin D-target cells. Although differences existed in the amount of 3-epi-1alpha,25(OH)2D3 formed with different cell types, it was apparent that 1alpha,25(OH)2D3 was subjected to metabolism both through the C24-oxidation and 3-epimerization pathways. Time course and dose response studies showed that the production of 3-epi-1alpha,25(OH)2D3 was enzymatic. It is interesting to note that this epimerization proceeded from 3beta towards 3alpha unidirectionally, and this conversion was not inhibited by ketoconazole. These data suggest that cytochrome P450 related enzymes including the 24-hydroxylase would not affect this reaction. The biological activity of 3-epi-1alpha,25(OH)2D3 was found to be lower than the native 1alpha,25(OH)2D3 in suppressing of proliferation of HL-60 cells, while the affinity of 3-epi-1alpha,25(OH)2D3 for vitamin D-binding protein was 2.5-fold higher than that of 1alpha,25(OH)2D3. The results indicate that 3-epimerization may change the pharmacokinetics and catabolism of 1alpha,25(OH)2D3 in vitamin D-target cells.