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

**Kaori Yasuda <sup>1</sup> , Miyu Nishikawa <sup>2</sup> , Hiroki Mano <sup>1</sup> , Masashi Takano <sup>3</sup> , Atsushi Kittaka <sup>3</sup> , Shinichi Ikushiro <sup>2</sup> and Toshiyuki Sakaki 1,\***


**Abstract:** We have developed an in vitro system to easily examine the affinity for vitamin D receptor (VDR) and CYP24A1-mediated metabolism as two methods of assessing vitamin D derivatives. Vitamin D derivatives with high VDR affinity and resistance to CYP24A1-mediated metabolism could be good therapeutic agents. This system can effectively select vitamin D derivatives with these useful properties. We have also developed an in vivo system including a *Cyp27b1-*gene-deficient rat (a type I rickets model), a *Vdr*-gene-deficient rat (a type II rickets model), and a rat with a mutant *Vdr* (R270L) (another type II rickets model) using a genome editing method. For *Cyp27b1*-gene-deficient and *Vdr* mutant (R270L) rats, amelioration of rickets symptoms can be used as an index of the efficacy of vitamin D derivatives. *Vdr-*gene-deficient rats can be used to assess the activities of vitamin D derivatives specialized for actions not mediated by VDR. One of our original vitamin D derivatives, which displays high affinity VDR binding and resistance to CYP24A1-dependent metabolism, has shown good therapeutic effects in *Vdr* (R270L) rats, although further analysis is needed.

**Keywords:** vitamin D; vitamin D receptor; split luciferase-based biosensor; CYP24A1-dependent metabolism; CYP27B1; rickets; genome editing
