OF 2-PROPOXY-1 , 25-DIHYDROXYVITAMIN D 3 AND 2-( 3-HYDROXYPROPOXY )-1 , 25-DIHYDROXYVITAMIN D 3 BY HUMAN CYP 27 A 1 AND CYP 24 A 1

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 Km and kcat 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, kcat/Km, 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. Vitamin D3 is initially converted to 25(OH)D3 by CYP27A1 in the liver, and then 25(OH)D3 is converted to 1 ,25(OH)2D3 by CYP27B1 in the kidneys. On the other hand, CYP24A1 is recognized as the key enzyme in the biological inactivation of 1 ,25(OH)2D3. The 1 ,25(OH)2D3 level is precisely regulated via gene regulation of CYP27B1 and CYP24A1. 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 -propoxy1 ,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). Recently, we revealed the enzymatic properties of CYP27A1, CYP27B1, and CYP24A1 expressed in Escherichia coli cells (Sakaki et al., 1999a,b, 2000; Sawada et al., 1999, 2000; Uchida et al., 2004). A remarkable species-based difference was observed in the CYP24A1-dependent metabolism of 1 ,25(OH)2D3 (Beckman et al., 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, 2 -(3-hydroxypropoxy)-1 ,25-dihydroxyvitamin D3; 1 ,25(OH)2D3, 1 ,25-dihydroxyvitamin D3; IPTG, isopropyl-thio-D-galactopyranoside; HPLC, high-performance liquid chromatography; B/B0, ratio of the concentration of [ H]1 ,25(OH)2D3 bound to VDR to the concentration of [ H]1 ,25(OH)2D3 added in the reaction mixture; LC-MS, liquid chromatography-mass spectrometry. 0090-9556/05/3306-778–784$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 6 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 3038/3033571 DMD 33:778–784, 2005 Printed in U.S.A. 778 at A PE T Jornals on N ovem er 6, 2017 dm d.aspurnals. org D ow nladed from 1996; Sakaki et al., 2000) and its analogs (Sakaki et al., 2000; Kusudo et al., 2003; Kusudo et al., 2004) in humans and rats. These facts mean that preclinical tests using animals cannot correctly predict the metabolism of vitamin D analogs in humans. 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 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-H]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.,