Elucidation of metabolic pathways of 25-hydroxyvitamin D3 mediated by CYP24A1 and CYP3A using Cyp24a1 knockout rats generated by CRISPR/Cas9 system

Abstract

Edited by F. Peter Guengerich CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher T max value than others. When 23S,25(OH) 2 D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH) 3 D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH) 3 D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH) 3 D3 via 23,25(OH) 2 D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH) 3 D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH) 3 D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 μg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.

Stereoselective Synthesis of 24-Fluoro-25-Hydroxyvitamin D3 Analogues and Their Stability to hCYP24A1-Dependent Catabolism

“…In contrast, we demonstrated that (23 S )-23-F-25(OH)D 3 ( 1 ) showed stronger resistance to CYP24A1 metabolism than (23 R )-23-F-25(OH)D 3 ( 2 ), as described in our previous study [ 9 ]. These results can be explained by the direction of hydroxylation at the C23 and C24 positions by CYP24A1 [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ].…”

“…Vitamin D 3 is a lipophilic vitamin, and hydroxylation steps promoted by the cytochrome P450 family are essential for both activation and deactivation pathways. In the deactivation step, human cytochrome P450 24A1 (hCYP24A1) is one of the main enzymes catalyzing hydroxylation at the C23 or C24 positions of the 25-hydroxyvitamin D 3 [25(OH)D 3 ] side-chain, and several subsequent hydroxylation steps lead to vitamin D 3 -23,26-lactone or calcitroic acid ( Scheme 1 ) [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ].…”

Design and Synthesis of Fluoro Analogues of Vitamin D

“…C23 hydroxylation and subsequent three-step oxidation lead to vitamin D 3 -26,23-lactone (3). On the other hand, C24 hydroxylation and subsequent five-step oxidation lead to calcitroic acid (4) (Scheme 1) [19,20].…”

Development of In Vitro and In Vivo Evaluation Systems for Vitamin D Derivatives and Their Application to Drug Discovery

“…A number of vitamin D derivatives have been approved by the FDA for clinical use in a variety of disorders. These derivatives include calcipotriol (Dovonex; Leo Pharmaceuticals) and 22-oxacalcitriol (Maxacalcitol; Chugai Pharmaceuticals) for treatment of psoriasis; 19-nor-1α,25(OH) 2 D 2 (Zemplar; Abbot Laboratories; Chicago, IL, USA), 26,26,26,27,27,27hexafluoro-(Falecalcitriol; Sumitomo Pharmaceuticals and Taisho Pharmaceuticals), and doxercalciferol (Hectorol; Bone Care Int. ; Middleton, WI, USA) for secondary hyperparathyroidism; and 1α(OH)D 3 (alfacalcidol; Chugai Pharmaceuticals Co., Ltd.; Tokyo, Japan) and eldecalcitol (Chugai Pharmaceuticals Co., Ltd.; Tokyo, Japan) for osteoporosis.…”

“…We have also generated genetically modified rats using genome editing as follows: Cyp27b1-gene-deficient rats (a type 1 rickets model animal), vitamin D receptor-gene-deficient rats, and rats harboring a mutant vitamin D receptor (R270L) gene (type II rickets model animals) [25]. We have also generated Cyp24a1gene-deficient rats to elucidate enzymes and metabolic pathways responsible for vitamin D derivative metabolism [26]. In this review, we describe the in vitro and in vivo systems we have developed for evaluation of vitamin D derivatives, and discuss the derivatives we have synthesized to date.…”