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.     

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.     

Disposition and metabolism of F6-1alpha,25(OH)2 vitamin D3 and 1alpha,25(OH)2 vitamin D3 in the parathyroid glands of rats dosed with tritium-labeled compounds.

26,26,26,27,27,27-Hexafluoro-1alpha,25(OH)2 vitamin D3, a hexafluorinated analog of 1alpha,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound with respect to some vitamin D actions. The reason for enhanced biological activity in the bones, kidneys, and small intestine appears to be related to F6-1alpha,25(OH)2 vitamin D3 metabolism to ST-232 (26,26,26,27,27,27-hexafluoro-1alpha,23S,25-trihydroxyvitamin D3), a bioactive 23S-hydroxylated form that is resistant to further metabolism. We compared the disposition and metabolism of [1beta-3H]F6-1alpha,25(OH)2 vitamin D3 and [1beta-3H]1alpha,25(OH)2 vitamin D3 in parathyroid glands of rats intravenously administered with labeled compounds at a dose of 10 microg/kg. In the [1beta-3H]F6-1alpha,25(OH)2 vitamin D3-dosed group, radioactivity was highly detected in the kidneys, parathyroid glands, and the small intestine. The radioactivity in the parathyroid glands remained high until 48 h postdosing, with values of 2.5, 8.4, and 14.6 times higher at 6, 24, and 48 h postdosing than after dosing with [1beta-3H] 1alpha,25(OH)2 vitamin D3. In the group given [1beta-3H]F6-1alpha,25(OH)2 vitamin D3, the unchanged compound was mainly detected with a small amount of ST-232 at 6 h postdosing. At the 24- and 48-h time points, over half of the radioactivity was observed as ST-232, and additionally, ST-233, the 23-oxo form, accounted for a small amount at the 48-h time point. The present study demonstrated local retention of [1beta-3H]F6-1alpha,25(OH)2 vitamin D3 and the bioactive metabolite ST-232 in parathyroid glands after intravenous administration. The findings may indicate one of the reasons for the higher potency of F6-1alpha,25(OH)2 vitamin D3 than 1alpha,25(OH)2 vitamin D3 in parathyroid.     

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.     

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.     

In vivo and in vitro pharmacokinetics and metabolism studies of 26,26,26,27,27,27-F6-1,25(OH)2 vitamin D3 (Falecalcitriol) in rat: induction of vitamin D3-24-hydroxylase (CYP24) responsible for 23S-hydroxylation in target tissues and the drop in serum levels.

1. 26,26,26,27,27,27-F6,-1,25(OH)2 vitamin D3, Falecalcitriol, the hexafluorinated analogue of 1,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound regarding some vitamin D actions. The reason for enhanced biological activity appears related to F6-1,25(OH)2 vitamin D3 metabolism to F6-1,23S,25(OH)3 vitamin D3, a bioactive 23S-hydroxylated form which is resistant to further metabolism. 2. In the present in vivo studies, the repeated oral administration of [3H]F6-1,25(OH)2 vitamin D3 to rat resulted in a significant reduction of the radioactivity and the F6-1,25(OH)2 vitamin D3 concentrations in serum, especially at the 2 h maximum point after each dosing. Additionally, F6-1,23S,25(OH)3 vitamin D3 in the serum and small intestine was increased by the prior administration of F6-1,25(OH)2 vitamin D3. 3. Further in vitro investigation showed [3H]F6-1,25(OH)2 vitamin D3 to be metabolized to F6-1,23S,25(OH)3 vitamin D3 by kidney and small intestine homogenates of rat, the reaction being increased by the prior administration of F6-1,25(OH)2 vitamin D3. Moreover, this latter treatment was associated with a marked increase of CYP24 mRNA in the small intestine within 4 h after dosing. 4. The results indicate that in vivo metabolism of F6-1,25(OH)2 vitamin D3 to F6-1,23S,25(OH)3 vitamin D3 is catalysed by CYP24, the enzyme being induced by prior substrate exposure.     

Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1alpha,25-dihydroxyvitamin D3 conjugation

The biological effects of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) are terminated primarily by P450-dependent hydroxylation reactions. However, the hormone is also conjugated in the liver and a metabolite, presumably a glucuronide, undergoes enterohepatic cycling. In this study, the identity of human enzymes capable of catalyzing the 1,25(OH)2D3 glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D3. Among 12 different UGT isozymes tested, only UGT1A4 > 2B4 and 2B7 supported the reaction. Two different 1,25(OH)2D3 monoglucuronide metabolites were generated by recombinant UGT1A4 and human liver microsomes. The most abundant product was identified by mass spectral and NMR analyses as the 25-O-glucuronide isomer. The formation of 25-O-glucuronide by UGT1A4 Supersomes and human liver microsomes followed simple hyperbolic kinetics, yielding respective Km and Vmax values of 7.3 and 11.2 microM and 33.7 +/- 1.4 and 32.9 +/- 1.9 pmol/min/mg protein. The calculated intrinsic 25-O-glucuronide M1 formation clearance for UGT1A4 was 14-fold higher than the next best isozyme, UGT2B7. There was only limited (four-fold) inter-liver variability in the 25-O-glucuronidation rate, but it was highly correlated with the relative rate of formation of the second, minor metabolite. In addition, formation of both metabolites was inhibited >80% by the selective UGT1A4 inhibitor, hecogenin. If enterohepatic recycling of 1,25(OH)2D3 represents a significant component of intestinal and systemic 1,25(OH)2D3 disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step.     

Glucuronidation of DRF-6574, hydroxy metabolite of DRF-4367 (a novel COX-2 inhibitor) by pooled human liver, intestinal microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT1A1, 1A3 and 1A8.

DRF-4367 is a novel COX-2 inhibitor, which showed good efficacy in several animal models of inflammation. In a comparative in vitro metabolism in various liver microsomes, DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by CYP2D6 and 2C19 isoenzymes. DRF-6574 readily undergoes Phase-II metabolism and forms glucuronide and sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for glucuronidation of DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e., UGT1A8 showed the catalytic activity. The enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg protein for UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by phenylbutazone.     

Metabolism of a 20-methyl substituted series of vitamin D analogs by cultured human cells: apparent reduction of 23-hydroxylation of the side chain by the 20-methyl group

We describe here for the first time the effect of introducing a 20-methyl group on the side-chain metabolism of the vitamin D molecule. Using a series of 20-methyl-derivatives of 1alpha,25-(OH)2D3 incubated with two different cultured human cell lines, HPK1A-ras and HepG2, previously shown to metabolize vitamin D compounds, we obtained a series of metabolic products that were identified by comparison to chemically synthesized standards on HPLC and GC-MS. 24-Hydroxylated-, 24-oxo-hydroxylated-, and 24-oxo-23-hydroxylated products of 20-methyl-1alpha,25-(OH)2D3 were observed, but the efficiency of 23-hydroxylation was low as compared with that of the natural hormone and, in contrast to 1alpha,25-(OH)2D3, no truncated 23-alcohol was formed from the 20-methyl analog. These data, taken together with results from other analogs with changes in the vicinity of the C17-C20 positions, lead us to speculate that such changes must alter the accessibility of the C-23 position to the cytochrome P450 involved. Using the HepG2 cell line, we found evidence that the 24S-hydroxylated product of 20-methyl-1alpha,25-(OH)2D3 predominates, implying that the liver cytochrome involved in metabolism is a different isoform. Studies with a more metabolically resistant analog of the series, 20-methyl-Delta(23)-1alpha,25-(OH)2D3, gave the expected block in 23- and 24-hydroxylation, and evidence of an alternative pathway, namely 26-hydroxylation. 20-Methyl-Delta(23)-1alpha,25-(OH)2D3 was also more potent in biological assays, and the metabolic studies reported here help us to suggest explanations for this increased potency. We conclude that the 20-methyl series of vitamin D analogs offers new perspectives into vitamin D analog action, as well as insights into the substrate preferences of the cytochrome(s) P450 involved in vitamin D catabolism.     

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.     

Kinetic studies of 25-hydroxy-19-nor-vitamin D3 and 1 alpha,25-dihydroxy-19-nor-vitamin D3 hydroxylation by CYP27B1 and CYP24A1.

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D(3) [25(OH)-19-nor-D(3)] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)], suggesting that 25(OH)-19-nor-D(3) might be converted to 1 alpha,25-dihydroxy-19-nor-vitamin D(3) [1 alpha,25(OH)(2)-19-nor-D(3)] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) and 25-hydroxyvitamin D(3) [25(OH)D(3)] and CYP24A1-dependent hydroxylation of 1 alpha,25(OH)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3), we found that k(cat)/K(m) for 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) was less than 0.1% of that for 25(OH)D(3), and the k(cat)/K(m) value for 24-hydroxylation was not significantly different between 1 alpha,25(OH)(2)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3). The data suggest a much slower formation and a similar rate of degradation of 1 alpha,25(OH)(2)-19-nor-D(3) compared with 1 alpha,25(OH)(2)D(3). We then analyzed the metabolites of 25(OH)D(3) and 25(OH)-19-nor-D(3) in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1 alpha,25(OH)(2)D(3) was detected in cells incubated with 25(OH)D(3), whereas no 1 alpha,25(OH)(2)-19-nor-D(3) was detected in cells incubated with 25(OH)-19-nor-D(3). Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D(3) is converted to 1 alpha,25(OH)(2)-19-nor-D(3) by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D(3) by itself may have a novel mechanism to activate vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1 alpha-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D(3) has potential as an attractive agent for prostate cancer therapy.     

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.     

Stereoselective metabolism of racemic carvedilol by UGT1A1 and UGT2B7, and effects of mutation of these enzymes on glucuronidation activity

Carvedilol, an alpha- and beta-adrenergic blocking drug, is mainly metabolized by CYP2D6, UGT1A1, UGT2B4 and UGT2B7. This drug is administered orally as a racemic mixture of R(+)- and S(-)-enantiomers. It has been reported that CYP2D6 prefers metabolizing S-carvedilol to R-carvedilol stereoselectively. On the other hand, stereoselective metabolism of carvedilol by UGTs is still unclear. Moreover, we have reported that patients with chronic heart failure who had polymorphism in CYP2D6, UGT1A1 and/or UGT2B7 had lower metabolic activity and oral clearance than did patients with no polymorphism. The aim of this study was to clarify stereoselective metabolism of carvedilol by UGT1A1 and UGT2B7 and to determine by using a recombinant enzyme-introduced mutation whether genetic mutation in UGT1A1 and UGT2B7 causes reduction in metabolic activity for carvedilol. A glucuronidation assay using human liver microsomes and recombinant UGT1A1 and UGT2B7 expressed in HeLa cells demonstrated that UGT1A1 prefers metabolizing R-carvedilol to S-carvedilol. On the other hand, UGT2B7 prefers metabolizing S-carvedilol to R-carvedilol. Moreover, G71R mutation of UGT1A1 reduced both affinity and capacity but did not affect stereoselective metabolism. On the other hand, both A71S and H268Y mutations of UGT2B7 reduced capacity but did not affect affinity and, as a result, the efficiency of metabolism was remarkably reduced. However, as in the case of UGT1A1, neither of the mutations affected stereoselective metabolism.     

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.     

New 2-alkylidene 1alpha,25-dihydroxy-19-norvitamin D3 analogues of high intestinal activity: synthesis and biological evaluation of 2-(3'-alkoxypropylidene) and 2-(3'-hydroxypropylidene) derivatives

In a search for novel vitamin D compounds of potential therapeutic value, E- and Z-isomers of 1alpha,25-dihydroxy-2-(3'-hydroxypropylidene)-19-norvitamin D(3), as well as a derivative of the former compound possessing a 3'-(methoxymethoxy)propylidene substituent at C-2, were efficiently prepared. All vitamins were obtained in convergent syntheses, starting with (-)-quinic acid and the protected 25-hydroxy Grundmann ketones. Quinic acid was converted into keto lactone 11, and a substituted hydroxypropylidene group was attached by Wittig reaction yielding pairs of isomeric compounds 12, 13 and 14, 15. These olefinic products were then transformed into phosphine oxides 32-34 which were subjected to Lythgoe type Wittig-Horner coupling with C,D-fragments 35a and 35b. An alternative route was also elaborated that comprised Julia coupling of sulfones 39a and 39b with the cyclohexanone derivative 23. The binding of all synthesized vitamins to the full-length rat recombinant vitamin D receptor (VDR) is either similar to or within one log of 1alpha,25(OH)(2)D(3). The in vivo tests have revealed that the calcemic activity of all analogues in the E-series (5a, 6a, 6b) is considerably higher than that of the native hormone.     

Structure-function analysis of vitamin D 24-hydroxylase (CYP24A1) by site-directed mutagenesis: amino acid residues responsible for species-based difference of CYP24A1 between humans and rats

Our previous studies revealed the species-based difference of CYP24A1-dependent vitamin D metabolism. Although human CYP24A1 catalyzes both C-23 and C-24 oxidation pathways, rat CYP24A1 shows almost no C-23 oxidation pathway. We tried to identify amino acid residues that cause the species-based difference by site-directed mutagenesis. In the putative substrate-binding regions, amino acid residue of rat CYP24A1 was converted to the corresponding residue of human CYP24A1. Among eight mutants examined, T416M and I500T showed C-23 oxidation pathway. In addition, the mutant I500F showed quite a different metabolism of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] from both human and rat CYP24A1. These results strongly suggest that the amino acid residues at positions 416 and 500 play a crucial role in substrate binding and greatly affect substrate orientation. A three-dimensional model of CYP24A1 indicated that the A-ring and triene part of 1alpha,25(OH)2D3 could be located close to amino acid residues at positions 416 and 500, respectively. Our findings provide useful information for the development of new vitamin D analogs for clinical use.