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METABOLISM OF 2-PROPOXY-1,25-DIHYDROXYVITAMIN D₃ AND 2-(3-HYDROXYPROPOXY)-1,25-DIHYDROXYVITAMIN D₃ BY HUMAN CYP27A1 AND CYP24A1

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan (D.A., T.K., H.H., K.I.); Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, Toyama, Japan (T.S., M.K.);Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan (A.K., N.S., Y.S., T.F., H.T.); and Laboratory of Nutrition, Koshien College, Nishinomiya, Japan (M.O.)

(Received November 16, 2004; accepted March 9, 2005)

	Abstract

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Recently, we demonstrated that some A-ring-modified vitamin D₃ analogs had unique biological activity. Of these analogs, 2-propoxy-1,25(OH)₂D₃ (C3O1) and 2-(3-hydroxypropoxy)-1,25(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 K_m and k_cat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1,25(OH)₂D₃. Thus, the catalytic efficiency, k_cat/K_m, of human CYP24A1 for O2C3 is only 2% of 1,25(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.

Analogs of 1,25(OH)₂D₃ are potentially useful for clinical treatment of type I rickets, osteoporosis, leukemia, psoriasis, renal osteodystrophy, and breast cancer (Binderup et al., 1991; Bishop et al., 1994; Bouillon et al., 1995; Yamada et al., 2003). For vitamin D analogs, the metabolism in such target tissues as kidneys, small intestine, and bones is pharmacologically essential, as reported by Komuro et al. (1998). Recently, we revealed that some A-ring-modified vitamin D₃ analogs had unique biological activity (Konno et al., 2000; Suhara et al., 2001; Takayama et al., 2001), and that ligands with a modification in the A-ring can alter the VDR-coactivator interaction, resulting in selective potentiation of the transcription function (Kittaka et al., 2000; Konno et al., 2000; Suhara et al., 2001; Takayama et al., 2001; Fujishima et al., 2003; Saito et al., 2004). This study examined two promising analogs for clinical use, 2-propoxy-1,25(OH)₂D₃ (C3O1) and 2-(3-hydroxypropoxy)-1,25(OH)₂D₃ (O2C3). O2C3 binds better than natural hormone to the mutant vitamin D receptor (R274A), which lost the hydrogen bond to the 1-hydroxyl group of 1,25(OH)₂D₃ (Kittaka et al., 2003). In addition, in our recent study, the high calcium effects of C3O1 and O2C3 were observed in animal experiments using rats. Note that O2C3 is the C₂-epimer of ED-71 (Fig. 1), which is being developed by Chugai Pharmaceutical Co. (Tokyo, Japan) as a potential therapeutic agent for osteoporosis (Okano et al., 1989).

View larger version (10K): [in this window] [in a new window]   FIG. 1. Structures of C3O1, O2C3, and ED-71.

In this paper, the metabolism of C3O1 and O2C3 by CYP27A1 and CYP24A1 is demonstrated. In addition, human CYP24A1 and rat CYP24A1 are compared in the metabolism of these compounds.

	Materials and Methods

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Materials. DNA-modifying enzymes, restriction enzymes, and DNA sequencing kit were purchased from Takara (Kyoto, Japan). E. coli JM109 (Takara) was used as a host strain. 1,25(OH)₂D₃ was purchased from Wako Pure Chemicals (Osaka, Japan). [26,27-Methyl-³H]1,25(OH)₂D₃ (specific activity 180 Ci/mmol) was purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). NADPH was purchased from Oriental Yeast Co (Tokyo, Japan). Terrific broth was purchased from Invitrogen (Carlsbad, CA). 2-Propyl-1,25(OH)₂D₃ (C3O1) and 2-(3-hydroxypropoxy)-1,25(OH)₂D₃ (O2C3) were synthesized as described previously (Kittaka et al., 2000; Saito et al., 2004). Other chemicals used were of the highest quality commercially available.

Construction of Expression Plasmids. The expression plasmids pKH27A1 (Sawada et al., 2000) for human CYP27A1, pKMath1 (Sawada et al., 1999) for human CYP27B1, pKSN24R2 (Sakaki et al., 1999a) for rat CYP24A1, and pKH24 (Sakaki et al., 2000) for human CYP24A1 were constructed as described previously. The coexpression plasmid pKARX for mature forms of human CYP27B1, bovine adrenodoxin (ADX), and bovine NADPH-adrenodoxin reductase (ADR) was constructed as described previously (Sawada et al., 1999).

Cultivation of the Recombinant E. coli Cells. Recombinant E. coli cells were grown in Terrific broth containing 50 µg ml^-1 ampicillin at 26°C under good aeration produced by bubbling. The induction of transcription of cDNAs for CYP27A1, CYP27B1, and CYP24A1 under tac promoter was initiated by the addition of isopropyl-thio-ß-D-galactopyranoside (IPTG) at a final concentration of 1 mM when the cell density (OD₆₆₀) reached 0.5. -Aminolevulinic acid was also added at a final concentration of 0.5 mM simultaneously. The recombinant cells were generally shaken at 26°C under good aeration by bubbling.

Measurement of Reduced CO-Difference Spectra. The reduced CO-difference spectra were measured with a Shimadzu UV-2200. The concentration of CYP27A1 was determined from the reduced CO-difference spectrum using a difference of the extinction coefficients at 446 nm and 490 nm of 91 mM^-1 cm^-1 (Omura and Sato, 1964). The absorption coefficient difference between 445 and 490 nm (105 mM^-1 cm^-1) was used for the calculation of the CYP24A1 hemoprotein concentration as described previously (Akiyoshi-Shibata et al., 1994)

Measurement of Enzyme Activity of CYP27A1, CYP27B1 and CYP24A1. The activity of both CYP27A1 and CYP24A1 toward O2C3 and C3O1 was measured in the reconstituted system containing the membrane fraction, 1.0 µM ADX, 0.1 µM ADR, 0.5 µM CYP27A1, or 0.01 to 0.02 µM CYP24A1, 0.5 mM NADPH, 100 mM Tris-HCl (pH 7.4), and 1 mM EDTA at 37°C. The reaction was initiated by addition of NADPH. Aliquots of the reaction mixture were collected after varying time intervals and extracted with 4 volumes of chloroform/methanol (3:1). The organic phase was recovered and dried up in vacuo. The resultant residue was dissolved in acetonitrile and applied to HPLC under the following conditions: column, YMC-Pack ODS-AM (5 µm) (4.6 mm x 300 mm) (YMC Co., Kyoto, Japan); UV detection, 265 nm; flow rate, 1.0 ml min^-1; column temperature, 40°C; mobile phase, acetonitrile: a linear gradient of 20 to 100% acetonitrile aqueous solution per 25 min and 100% acetonitrile for 12 min. The activity of CYP27B1 toward these substrates was measured with a highly sensitive in vivo system using E. coli cells expressing CYP27B1, ADX, and ADR (Sawada et al., 1999). Each of the substrates in the ethanol solution was added into the cell culture at a final concentration of 5.0 µM. After 30 min, IPTG was added at a final concentration of 1 mM. Aliquots of the cell culture were collected after varying time intervals and extracted with 4 volumes of chloroform/methanol (3:1).

LC-MS Analysis of the Metabolites. Isolated metabolites from HPLC effluents were subjected to mass spectrometric analysis using a Thermo Electron TSQ-70 (Thermo Electron, Waltham, MA) with atmospheric pressure chemical ionization, positive mode. The conditions of liquid chromatography are described as follows: column, reverse phase ODS column (µBondapak C18, 5 µm; Waters) (6 mm x 150 mm); mobile phase, 80% methanol aqueous solution per 25 min; flow rate, 1.0 ml min^-1; UV detection, 265 nm.

Binding Assay for Calf-Thymus Vitamin D Receptor. Displacement of [³H]1,25(OH)₂D₃ from calf-thymus cytosol receptor (Yamasa Shoyu, Chiba, Japan) by 1,25(OH)₂D₃ or metabolites of O2C3 and C3O1 was determined as described by Nakagawa et al. (2001). The increasing amounts of 1,25(OH)₂D₃ (0.0156-128 pg) or the metabolites (0.25-16,384 pg) in 20 µl of ethanol were added to 500 µl of the calf-thymus cytosol diluted with 50 mM potassium phosphate buffer (pH 7.4) containing 0.3 M KCl and incubated for 1 h at 20°C. Next, 34 fmol of [³H]1,25(OH)₂D₃ in 25 µl of ethanol was added and incubated for 1 h at 20°C. The 200 µl of dextran-charcoal (0.05% dextran T-150, 0.5% Charcoal Decolorizing Neutral) in 50 mM sodium phosphate buffer (pH 7.5), which was freshly prepared and stirred well before addition, was added to separate bound and free [³H]1,25(OH)₂D₃. The assay tube was shaken with a vortex mixer and centrifuged at 1000g for 10 min at 4°C. The radioactivity in the supernatant was measured with a liquid scintillation counter.

Other Methods. The concentration of vitamin D₃ derivatives was estimated by their molar extinction coefficient of 1.80 x 10⁴ M^-1 cm^-1 at 264 nm (Hiwatashi et al., 1982). Protein concentration was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.

	Results

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Expression of P450s in E. coli. Membrane fractions were prepared from the recombinant E. coli JM109/pKH27A1 cells for human CYP27A1, JM109/pKH24 cells for human CYP24A1, and JM109/pKSN24R2 cells for rat CYP24A1. The human CYP27A1, human CYP24A1, and rat CYP24A1 contents in the membrane fractions were calculated to be 1.10, 0.102, and 0.074 nmol/mg protein, respectively.

Metabolism by CYP27A1. The reconstituted system containing ADR, ADX, and the membrane fraction prepared from the recombinant E. coli JM109/pKH27A1 cells was examined for the metabolism of O2C3 and C3O1. Figure 2 shows the HPLC profiles of the substrates and their metabolites by CYP27A1. Although no metabolites of O2C3 were detected, a single peak was detected in the metabolism of C3O1. The metabolites were separated to two peaks under the HPLC conditions used in LC-MS analysis as shown in Fig. 2C. The retention time of the metabolite P2 was the same as that of O2C3 (data not shown). Figure 3 shows the mass spectra of O2C3, P1, and P2. The mass spectrum of O2C3 showed a molecular ion at m/z 491 (M + H), and fragment ions at 473 (M + H - H₂O), 455 (M + H - 2H₂O), and 437 (M + H - 3H₂O). The fragment ions at 397 (M + H - 76 - H₂O), 379 (M+H - 76 - 2H₂O), and 361 (M + H - 76 - 3H₂O) showing a loss of a 3-hydroxypropoxy group were observed. P2 showed the fragmentation pattern similar to O2C3, suggesting that P2 is O2C3. On the other hand, P1 showed a molecular ion at m/z 491(M + H), fragment ions at m/z 473 (M + H - H₂O), 455 (M + H - 2H₂O), 437 (M + H - 3H₂O), 413 (M + H - 60 - H₂O), 395 (M + H - 60 - 2H₂O), and 377 (M + H - 60 - 3H₂O). The fragment ion at m/z 413 indicated losses of a propoxy group and a water molecule. Thus, P1 appears to be produced by hydroxylation at a carbon atom except for the propoxy group.

View larger version (16K): [in this window] [in a new window]   FIG. 2. HPLC profiles of O2C3 (A), and C3O1 and its metabolites (B) by human CYP27A1. After incubation with 4.0 µM O2C3 (A) or 4.0 µM C3O1 (B) for 30 min, the reaction mixture was extracted and analyzed by HPLC as described under Materials and Methods. The metabolites designated as P1 and P2 were also analyzed by another HPLC condition (C) as described under Materials and Methods. O2C3 was not metabolized by CYP27A1 (A).

View larger version (19K): [in this window] [in a new window]   FIG. 3. Mass spectra of O2C3 (A), P2 (B), and P1 (C). The metabolites were extracted from the reconstituted system and isolated by HPLC, and then analyzed by mass spectrometry as described under Materials and Methods.

Affinity of the Metabolite P2 for VDR. The calf-thymus VDR binding assay demonstrated that C3O1 had slightly lower affinity than 1,25(OH)₂D₃, whereas O2C3 had higher affinity than 1,25(OH)₂D₃. Note that P2 had nearly the same affinity as O2C3 (Fig. 4). The concentrations of 1,25(OH)₂D₃, C3O1, O2C3, and P2 in the aqueous solution for 50% B/B₀ were 45, 70, 22, and 25 pM, respectively. Thus, the affinity of C3O1, O2C3, and P2 for VDR was estimated to be 64, 204, and 180% as compared with the affinity of 1,25(OH)₂D₃. These results confirm the assumption that the metabolite P2 is O2C3.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Binding assay of the control 1,25(OH)2D3 (), C3O1 () and its metabolite P2 (), and O2C3 () and its metabolite M3 () for calf-thymus vitamin D receptor. The designated concentrations are the final concentrations in the reaction mixture. B and B0 denote the concentration of [3H]1,25(OH)2D3 bound to VDR and the concentration of [3H]1,25(OH)2D3 added in the reaction mixture, respectively.

Metabolism of O2C3 by CYP24A1. The reconstituted system containing ADR, ADX, and the membrane fraction prepared from the recombinant E. coli JM109/pKH24 or JM109/pKSN24R2 cells was examined for the metabolism of O2C3. Figure 5 shows HPLC profiles of the substrate O2C3 and its metabolites by human CYP24A1 and rat CYP24A1. Five metabolites were observed in the metabolism by human CYP24A1. On the other hand, three metabolites, M2, M3, and M4, were observed in the metabolism by rat CYP24A1. The metabolites M1, M2, M3, M4, and M5 were considered to be 23,26(OH)₂-O2C3, 24-oxo-23(OH)-O2C3, a mixture of 23(OH)-O2C3 and 24(OH)-O2C3, 24-oxo-O2C3, and 25,26,27-trinor-24-ene-O2C3, respectively, based on HPLC pattern when compared with the corresponding metabolites of 1,25(OH)₂D₃ (Sakaki et al., 2000; Sawada et al., 2004).

View larger version (21K): [in this window] [in a new window]   FIG. 5. HPLC profiles of O2C3 and its metabolites by human CYP24A1 (A) and rat CYP24A1 (B). After incubation with 4.0 µM O2C3 for 60 min, the reaction mixture was extracted and analyzed by HPLC as described under Materials and Methods. M1 and M5 were the metabolites in the C-23 oxidation pathway observed in human CYP24A1-dependent metabolism but were not observed in rat CYP24A1-dependent metabolism.

To confirm the chemical structures of the metabolites, we collected the metabolites in the effluents from HPLC and subjected them to mass spectrometric analysis. As shown in Fig. 6, the mass spectrum of M3 showed a molecular ion at m/z 507 (M + H) and fragment ions at 489 (M + H - H₂O), 471 (M + H - 2H₂O), 453 (M + H - 3H₂O), 413 (M + H - 76 - H₂O), 395 (M + H - 76 - 2H₂O), and 377 (M + H - 76 - 3H₂O). The fragment ion at m/z 413 indicated losses of a 3-hydroxypropoxy group and a water molecule. In addition, fragment ions at 339 (M + H - 76 - 74), 321 (M + H - 76 - 74 - H₂O), and 303 (M + H - 76 - 74 - 2H₂O), which result after cleavage between C-23 and C-24, are characteristic of the 23-hydroxylated compound as described previously (Kusudo et al., 2004). These results indicate that M3 contains 23(OH)-O2C3. However, the intensity of these fragments is considerably small, and the putative products of the C-24 oxidation pathway, M4 and M2, are detected, suggesting that M3 contains both 23(OH)-O2C3 and 24(OH)-O2C3. The metabolite M4 showed a molecular ion at m/z 505 (M + H) and fragment ions at 487 (M + H - H₂O), 469 (M + H - 2H₂O), 411 (M + H - 76 - H₂O), 393 (M + H - 76 - 2H₂O), and 375 (M + H - 76 - 3H₂O). The fragment ion at m/z 411 indicated losses of a 3-hydroxypropoxy group and a water molecule. These results strongly suggest that M4 is 24-oxo-O2C3.

View larger version (24K): [in this window] [in a new window]   FIG. 6. Mass spectra of the metabolites M3 (A), M4 (B), and M5 (C). The metabolites were extracted from the reconstituted system and isolated by HPLC, and then analyzed by mass spectrometry as described under Materials and Methods.

The metabolite M5 showed a molecular ion at m/z 431 (M + H) and fragment ions at 413 (M + H - H₂O), and 337 (M + H - 76 - H₂O), which indicated losses of a 3-hydroxypropoxy group and a water molecule. The ion at 460 (M + H + 29), which has been characteristically observed under the LC-MS conditions (Sawada et al., 2000), was also observed. These results, together with our previous study (Sawada et al., 2004) on the metabolism of 1,25(OH)₂D₃, strongly suggest that M5 was 25,26,27-trinor-24-ene-O2C3 produced by side chain cleavage at the C24-C25 bond of O2C3.

The metabolite M1 showed a molecular ion at m/z 523 (M + H) and fragment ions at 505 (M + H - H₂O), 487 (M + H - 2H₂O), 429 (M + H - 76 - H₂O), and 411 (M + H - 76 - 2H₂O) (data not shown). The fragment ion m/z 429 indicated losses of a 3-hydroxypropoxy group and a water molecule. On the other hand, the metabolite M2 showed a molecular ion at m/z 521 (M + H) and fragment ions at 503 (M + H - H₂O), 485 (M + H - 2H₂O), 427 (M + H - 76 - H₂O), and 409 (M + H - 76 - 2H₂O) (data not shown). The fragment ion m/z 427 indicated losses of a 3-hydroxypropoxy group and a water molecule. These results confirmed the assumptions mentioned above that M1 and M2 are 23,26(OH)₂-O2C3 and 24-oxo-23(OH)-O2C3, respectively.

Affinity of the Metabolite M3 for VDR. The calf-thymus VDR binding assay demonstrated that O2C3 had higher affinity, whereas M3 had lower affinity than 1,25(OH)₂D₃ (Fig. 4). The concentrations of 1,25(OH)₂D₃, O2C3, and M3 in the aqueous solution for 50% B/B₀ were 45, 22, and 100 pM, respectively. Thus, the affinity of O2C3 and M3 for VDR was estimated to be 204 and 45% as compared with the affinity of 1,25(OH)₂D₃. Due to the low affinity of M5 for VDR assay, we could not evaluate the affinity of M5 precisely. Since our previous studies indicated that the metabolite with side chain cleavage at the C₂₄-C₂₅ bond of 1,25(OH)₂D₃ had much lower affinity for VDR than for 1,25(OH)₂D₃, it is reasonable that M5 has much lower affinity for VDR than does O2C3. On the metabolism of C3O1 by human CYP24A1 and rat CYP24A1, quite similar results were obtained (data not shown).

Kinetic Analysis of the Metabolism of 1,25(OH)₂D₃, O2C3, and C3O1 by CYP24A1. When the activity toward 1,25(OH)₂D₃, O2C3, and C3O1 was measured at the substrate concentrations of 0 to 5.0 µM, the reaction followed Michaelis-Menten-type kinetics. The kinetic parameters were calculated by nonlinear regression analysis using KaleidaGraph software (Abelbeck/Synergy, Reading, PA) (Table 1). The K_m and k_cat values of human CYP24A1 were calculated to be 0.06 µM and 6.3 (min^-1) for 1,25(OH)₂D₃; 0.94 µM and 1.9 (min^-1) for O2C3; and 1.1 µM and 1.0 (min^-1) for C3O1, respectively. Thus, the k_cat/K_m values of human CYP24A1 for O2C3 and C3O1 were approximately 2% and 1% as compared with that for 1,25(OH)₂D₃. In this study, the concentrations of ADX and ADR were significantly higher than those in our previous studies (Sakaki et al., 2000; Kusudo et al., 2004). Because the activities toward O2C3 and C3O1 were much lower than the activity toward 1,25(OH)₂D₃, in particular, at low substrate concentration, the concentrations of ADX and ADR were increased to enhance the activity. Under these conditions, multiple metabolites of 1,25(OH)₂D₃ were observed, although 1,23,25(OH)₂D₃ and 1,24,25(OH)₂ were major products. Thus, the actual k_cat value appears significantly higher than 6.3 (min^-1). On the other hand, the amounts of such metabolites as M1, M2, M4, and M5 were much less than that of M3, and the values for O2C3 and C3O1 are more accurate than that for 1,25(OH)₂D₃. These facts indicate that the actual k_cat/K_m values for O2C3 and C3O1 are significantly less than 2% and 1% of the k_cat/K_m value for 1,25(OH)₂D₃. Similar results were obtained in rat CYP24A1-dependent metabolism of 1,25(OH)₂D₃, O2C3, and C3O1. The k_cat/K_m values of rat CYP24A1 for O2C3 and C3O1 were much lower than that for 1,25(OH)₂D₃. However, a significant difference was observed in both K_m and k_cat values for C3O1 metabolism between rat CYP24A1 and human CYP24A1 (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Kinetic parameters of CYP24A1 and CYP27A1 for 1,25(OH)2D3, O2C3, and C3O1

	Discussion

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A large number of vitamin D analogs have been synthesized for clinical use in type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia, and breast cancer (Binderup et al., 1991; Bishop et al., 1994; Bouillon et al., 1995; Yamada et al., 2003). Of these, A-ring analogs have great potential for clinical use. For example, ED-71, which bears a 3-hydroxypropoxy group at the 2ß-position of 1,25(OH)₂D₃, was developed by Chugai Pharmaceutical Co. (Tokyo, Japan) as a potential therapeutic agent for osteoporosis because ED-71 significantly increases plasma calcium level for a long time (Okano et al., 1989). In addition, it was also reported that diastereomers of 2-methyl-1,25(OH)₂D₃ had unique activity profiles depending on their stereochemistry. In this study, we found the metabolism of O2C3 and C3O1 by three mitochondrial P450s, CYP27A1, CYP27B1, and CYP24A1, which are involved in the metabolism of vitamin D.

Note that two metabolites with a hydroxyl group were detected in the metabolism of C3O1 by CYP27A1. The metabolite designated as P2 was considered hydroxylated at a propoxy group based on LC-MS analysis. Both HPLC analysis and VDR assay strongly suggested that CYP27A1 catalyzes the conversion from C3O1 to O2C3. It is commonly known that CYP27A1 catalyzes the 25-hydroxylation of vitamin D₃. Thus, it was surprising that the enzyme catalyzed the introduction of a hydroxyl group to the propoxy group on the A-ring. However, we demonstrated that CYP27A1 can catalyze 1-hydroxylation on the A-ring of vitamin D₃ in addition to 25-, 26-, and 27-hydroxylation as described previously (Sawada et al., 2000). Thus, it might be possible to assume that C-3 of the propoxy group of C3O1 is close to C-1 in the substrate-binding pocket of CYP27A1. Note that O2C3 has 3 times greater affinity for VDR than does C3O1. Thus, conversion from C3O1 to O2C3 by CYP27A1 is considered a metabolic activation process. Although CYP27B1 shows higher 1-hydroxylation activity toward 25(OH)D₃ than does CYP27A1, CYP27B1 did not metabolize C3O1. Based on these results, it was assumed that a part of C3O1 administered to humans would be converted to O2C3 by CYP27A1 in the liver. The resultant O2C3 would be metabolized by CYP24A1 in the kidneys. Although other metabolism by liver microsomal P450s and UDP-glucuronosyltransferases cannot be ignored, metabolism by CYP27A1 and CYP24A1 appears quite important. The K_m and k_cat values of CYP27A1 for the conversion from C3O1 to O2C3 were 5.2 µM and 0.0074 min^-1, respectively (Table 1). Thus, this activity is much lower than the activity of CYP24A1 toward C3O1. Since the CYP27A1 content in the liver appears greater than CYP24A1 in the kidneys and small intestine, we cannot ignore the CYP27A1-dependent conversion of C3O1.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Putative metabolic pathways of C3O1 and O2C3 by human CYP27A1 and human CYP24A1. The putative structures of M1 and M2 were shown based on the LC-MS analysis and the metabolic pathways of 1,25(OH)2D3 and its A-ring diastereomers (Kusudo et al., 2004).

	Footnotes

 
This work was partly supported by Health Science Research Grants from the Ministry of Health Labour and Welfare of Japan, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.104.003038.

ABBREVIATIONS: VDR, vitamin D receptor; P450, cytochrome P450; ADX, adrenodoxin; ADR, NADPH-adrenodoxin reductase; C3O1, 2-propoxy-1,25-dihydroxyvitamin D₃; O2C3, 2-(3-hydroxypropoxy)-1,25-dihydroxyvitamin D₃; ED-71, 2ß-(3-hydroxypropoxy)-1,25-dihydroxyvitamin D3; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; IPTG, isopropyl-thio-ß-D-galactopyranoside; HPLC, high-performance liquid chromatography; B/B₀, ratio of the concentration of [³H]1,25(OH)₂D₃ bound to VDR to the concentration of [³H]1,25(OH)₂D₃ added in the reaction mixture; LC-MS, liquid chromatography-mass spectrometry.

Address correspondence to: Toshiyuki Sakaki, Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan. tsakaki{at}pu-toyama.ac.jp

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