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Drug Metabolism and Disposition Fast Forward
First published on June 6, 2007; DOI: 10.1124/dmd.107.015602

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Received for publication March 28, 2007.
Revised May 31, 2007.
Accepted for publication June 5, 2007.

Kinetic Studies of 25-Hydroxy-19-Nor-Vitamin D3 and 1{alpha},25-Dihydroxy-19-Nor-Vitamin D3 Hydroxylation by CYP27B1 and CYP24A1

Naoko Urushino 1, Sachie Nakabayashi 2, Midori A. Arai 3, Atsushi Kittaka 3, Tai C. Chen 4, Keiko Yamamoto 5, Keiko Hayashi 2, Shigeaki Kato 6, Miho Ohta 7, Masaki Kamakura 2, Shinichi Ikushiro 2, Toshiyuki Sakaki 2*

1 Kyoto University 2 Toyama Prefectural University 3 Teikyo University 4 Boston University School of Medicine 5 Tokyo Medical and Dental University 6 Tokyo University 7 Soai University

* Address correspondence to: E-mail: tsakaki{at}pu-toyama.ac.jp

Abstract

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D3 (25(OH)-19-nor-D3) inhibited the proliferation of immortalized non-cancerous PZ-HPV-7 prostate cells similar to 1{alpha},25-dihydroxyvitamin D3 (1{alpha},25(OH)2D3), suggesting that 25(OH)-19-nor-D3 might be converted to 1{alpha},25-dihydroxy-19-nor-vitamin D3 (1{alpha},25(OH)2-19-nor-D3) 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-D3 and 25-hydroxyvitamin D3 (25(OH)D3) and CYP24A1-dependent hydroxylation of 1{alpha},25(OH)-19-nor-D3 and 1{alpha},25(OH)2D3, we found that kcat/Km for 1{alpha}-hydroxylation of 25(OH)-19-nor-D3 was less than 0.1 % of that for 25(OH)D3 and the kcat/Km value for 24-hydroxylation was not significantly different between 1{alpha},25(OH)2-19-nor-D3 and 1{alpha},25(OH)2D3. The data suggest a much slower formation and a similar rate of degradation of 1{alpha},25(OH)2-19-nor-D3 as compared to 1{alpha},25(OH)2D3. We then analyzed the metabolites of 25(OH)D3 and 25(OH)-19-nor-D3 in PZ-HPV-7 cells by high performance liquid chromatography. We found that a peak that co-migrated with 1{alpha},25(OH)2D3 was detected in cells incubated with 25(OH)D3, whereas no 1{alpha},25(OH))2-19-nor-D3 was detected in cells incubated with 25(OH)-19-nor-D3. Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D3 is converted to 1{alpha},25(OH)2-19-nor-D3 by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D3 by itself may have a novel mechanism to activate VDR 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-D3 has potential as an attractive agent for prostate cancer therapy.


Key words: CYP expression, cytochrome P450 function, enzyme kinetics, vitamin D




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