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Kinetic Studies of 25-Hydroxy-19-nor-vitamin D₃ and 1,25-Dihydroxy-19-nor-vitamin D₃ Hydroxylation by CYP27B1 and CYP24A1

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto, Japan (N.U.); Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Kurokawa, Imizu, Toyama, Japan (S.N., K.H., S.I., M.K., T.S.); Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan (M.A.A., A.K.); Boston University School of Medicine, Boston, Massachusetts (T.C.C.); Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Kanda-Surugadai, Chiyoda-ku, Tokyo, Japan (K.Y.); Institute of Molecular and Cellular Biosciences, Tokyo University, Yayoi, Bunkyo, Tokyo, Japan (S.K.); and Development Nourishment Department, Soai University, Nankonaka, Suminoe, Osaka, Japan (M.O.)

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D₃ [25(OH)-19-nor-D₃] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], suggesting that 25(OH)-19-nor-D₃ might be converted to 1,25-dihydroxy-19-nor-vitamin D₃ [1,25(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-hydroxylation of 25(OH)-19-nor-D₃ and 25-hydroxyvitamin D₃ [25(OH)D₃] and CYP24A1-dependent hydroxylation of 1,25(OH)-19-nor-D₃ and 1,25(OH)₂D₃, we found that k_cat/K_m for 1-hydroxylation of 25(OH)-19-nor-D₃ was less than 0.1% of that for 25(OH)D₃, and the k_cat/K_m value for 24-hydroxylation was not significantly different between 1,25(OH)₂-19-nor-D₃ and 1,25(OH)₂D₃. The data suggest a much slower formation and a similar rate of degradation of 1,25(OH)₂-19-nor-D₃ compared with 1,25(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 comigrated with 1,25(OH)₂D₃ was detected in cells incubated with 25(OH)D₃, whereas no 1,25(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 1,25(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 vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D₃ has potential as an attractive agent for prostate cancer therapy.