Metabolism of 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 by CYP24A1 and biological activity of its 24R-hydroxylated metabolite

Our previous study revealed that the 2α-substituted vitamin D analog 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exhibited a higher osteocalcin promoter transactivation activity in human osteosarcoma cells and a greater effect on bone mineral density in a rat model of osteoporosis than 1α,25(OH)2D3　without increasing blood calcium concentration. Thus, we hypothesized that AH-1 could be a promising therapeutic agent for osteoporosis without any serious side effects. In this study, we compared the CYP24A1-dependent metabolism of AH-1 with that of 1α,25(OH)2D3. The resistance to CYP24A1-dependent metabolism could be an important property of vitamin D analogs in prolonging their biological effects. A kinetic analysis was performed using a membrane fraction prepared from recombinant E. coli expressing human CYP24A1. The kcat/Km (μM−1 min−1) value for AH-1 was 31% of that for 1α,25(OH)2D3, suggesting that AH-1 is not as resistant to CYP24A1-dependent metabolism as the other C2-substituted vitamin D analogs such as eldecalcitol [2β-hydroxypropoxy-1α,25(OH)2D3]. The major metabolite of AH-1 was the 24R-hydroxylated metabolite, which had high vitamin D receptor (VDR) binding affinity and high HL-60 cell differentiation activity similar to AH-1 itself. In contrast, 1α,25(OH)2D3 was metabolized by multistep monooxygenation reactions, which led to the loss of affinity for VDR. Thus, the greater therapeutic effects of AH-1 than those of 1α,25(OH)2D3 in in vivo studies using osteoporosis rat models may be due to 24R-hydroxy-AH-1 whose VDR affinity was 91% of that of AH-1.