*3.3. CYP24A1-Dependent Multi-Step Reaction toward the Active form of Vitamin D<sup>3</sup>*

CYP24A1 plays central roles in vitamin D metabolism and produces a wide variety of metabolites. We revealed that rat or human CYP24A1 catalyzes a six-step reaction, starting with hydroxylation at the 24*R* position of 1α,25(OH)2D<sup>3</sup> to produce the final metabolite, calcitroic acid (Figure 3). In addition, human CYP24A1 catalyzes a four-step reaction, starting with hydroxylation at the 23*S* position to produce the 26,23-lactone form (Figure 3). In the reaction of P450, it is often seen that the reaction product is not released from the substrate binding pocket and the reaction proceeds further. Thus, the two- or three-step reaction is not special in the P450 reaction; however, there is no other P450 that catalyzes

such a multi-step reaction for one substrate. Moreover, it is noted that the reaction by human CYP24A1 proceeds in a dual pathway, the C-24 pathway and the C-23 pathway. Interestingly, the ratio of the C-24 to C-23 pathways varies among animal species. In human CYP24A1, it is about 4:1, but, in rat CYP24A1, about 25:1; however, in animal species such as guinea pig and opossum, the C-23 pathway is major. In rat and human CYP24A1, the 326th amino acid residue from the N-terminus is Ala, whereas it is Gly in guinea pigs and opossum, and, when the Ala326 in rat and human CYP24A1 is replaced by Gly, it changes to the guinea pig type [59]. Given that inactivating the active form of vitamin D is the physiological role of CYP24A1, it may be less important whether the C-24 or C-23 pathway is predominant. *NADPH-Adrenodoxin Reductase (ADR)*  The mitochondrial P450 system consists of three components: CYP, ADX, and ADR. Electrons are sequentially transferred from NADPH through ADR and ADX to CYP24A1 (Figure 2). Thus, CYP24A1-dependent activity was measured in an in vitro reconstituted system containing purified ADX and ADR proteins. On the other hand, in a whole-cell system, co-expression of mature forms of CYP24A1, ADX, and ADR in *E. coli* is required. We have demonstrated that the *E. coli* expression system is quite useful to investigate enzymatic properties of CYP24A1. Using this *E. coli* expression system, we have determined kinetic parameters of CYP24A1 in the metabolism of the native vitamin D and various vitamin D derivatives, and revealed their metabolic pathways [45–58]. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 7 of 16 *3.3. CYP24A1-Dependent Multi-Step Reaction toward the Active form of Vitamin D3* CYP24A1 plays central roles in vitamin D metabolism and produces a wide variety of metabolites. We revealed that rat or human CYP24A1 catalyzes a six-step reaction, starting with hydroxylation at the 24*R* position of 1α,25(OH)2D3 to produce the final metabolite, calcitroic acid (Figure 3). In addition, human CYP24A1 catalyzes a four-step reaction,

starting with hydroxylation at the 23*S* position to produce the 26,23-lactone form (Figure

*3.2. Construction of a CYP24A1 Enzyme System Containing Adrenodoxin (ADX) and* 

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 6 of 16

*3.1. Expression of Rat or Human CYP24A1 in E. coli Cells* 

the level of the active form of vitamin D.

zymatic properties [20–24].

**3. In Vitro Evaluation of CYP24A1-mediated Metabolism of Vitamin D Derivatives**

The rat *Cyp24a1* cDNA was cloned from the rat kidney cDNA library [39], and the isolated cDNA clone contained the open reading frame consisting of 514 amino acids. Since the amino acid sequence showed less than 40 % homology with already known CYPs, the new CYP family name, CYP24, was given to this vitamin-D-24-hydroxylase.

The molecular mechanism of *CYP24A1* gene regulation is quite complicated, and many factors are tissue-specifically involved in the expression of *CYP24A1* [40–44]. These facts strongly suggest that CYP24A1 is a physiologically essential enzyme that regulates

When the deduced amino acid sequence from its cDNA was compared to that aminoterminal amino acid sequence of the CYP24A1 purified from rat kidney, it was found that the mature form of rat CYP24A1 lacks amino-terminal 32 amino acids. These results suggest that amino-terminal 32 amino acids function as a mitochondrial targeting signal, which is removed after translocation of CYP24A1 to mitochondria. We have successfully expressed the mature forms of rat and human CYP24A1 in *E. coli* cells to reveal their en-

**Figure 2.** Mitochondrial electron transport chain of CYP24A1. CYP24A1-dependent mono-oxygenase activity re-**Figure 2.** Mitochondrial electron transport chain of CYP24A1. CYP24A1-dependent mono-oxygenase activity requires the electron transfer from NADPH via NADPH-adrenodoxin oxidoreductase (ADR) and adrenodoxin (ADX) to the heme iron of CYP24A1 situated on the inner membrane of mitochondria. RH represents substrate of CYP24A1.

**Figure 3.** C-23 and C-24 oxidative pathways of 1α,25(OH)2D3 catalyzed by human CYP24A1. Human CYP24A1 catalyzes 6-step mono-oxygenation from C-24 hydroxylation to produce calcitroic acid, and 4-step mono-oxygenation **Figure 3.** C-23 and C-24 oxidative pathways of 1α,25(OH)2D<sup>3</sup> catalyzed by human CYP24A1. Human CYP24A1 catalyzes 6-step mono-oxygenation from C-24 hydroxylation to produce calcitroic acid, and 4-step mono-oxygenation from C-23 hydroxylation to the lactone formation.

#### *3.4. Metabolism of Vitamin D Derivatives by CYP24A1*

from C-23 hydroxylation to the lactone formation.

*3.4. Metabolism of Vitamin D Derivatives by CYP24A1*  The CYP24A1 gene has two VDREs in the promoter region [40,41,60] and, when the active form of vitamin D binds to VDR, remarkable transcriptional induction of CYP24A1 occurs. When a large amount of CYP24A1 protein is expressed in the cell, the active form of vitamin D is inactivated via the metabolic pathways described above. This mechanism appears to be crucial for keeping the level of the active form of vitamin D. However, when a vitamin D derivative with a high affinity for VDR is developed as a drug, the drug binds to VDR to induce CYP24A1. Therefore, vitamin D derivatives that are not easily metabolized by CYP24A1 could be excellent drugs with long-lasting efficacy. Eldecalcitol, an osteoporosis treatment drug developed by Chugai Pharmaceutical Co., Ltd., has a 3-hydroxy-propyloxy group at the 2β position of 1α,25(OH)2D3 (Figure 4). We revealed that CYP24A1 hardly metabolizes Eldecalcitol [53] and suggest that the resistance to CYP24A1-dependent metabolism may be a key factor that keeps its efficacy for a long time The CYP24A1 gene has two VDREs in the promoter region [40,41,60] and, when the active form of vitamin D binds to VDR, remarkable transcriptional induction of CYP24A1 occurs. When a large amount of CYP24A1 protein is expressed in the cell, the active form of vitamin D is inactivated via the metabolic pathways described above. This mechanism appears to be crucial for keeping the level of the active form of vitamin D. However, when a vitamin D derivative with a high affinity for VDR is developed as a drug, the drug binds to VDR to induce CYP24A1. Therefore, vitamin D derivatives that are not easily metabolized by CYP24A1 could be excellent drugs with long-lasting efficacy. Eldecalcitol, an osteoporosis treatment drug developed by Chugai Pharmaceutical Co., Ltd., has a 3-hydroxy-propyloxy group at the 2β position of 1α,25(OH)2D<sup>3</sup> (Figure 4). We revealed that CYP24A1 hardly metabolizes Eldecalcitol [53] and suggest that the resistance to CYP24A1-dependent metabolism may be a key factor that keeps its efficacy for a long time [53,61–63]. We have investigated the metabolism of many vitamin D deriva-

[53,61–63]. We have investigated the metabolism of many vitamin D derivatives by

in the metabolic mode of 1α,25(OH)2D3 by CYP24A1 suggests that there are also animal

tives by CYP24A1 and have clearly demonstrated the importance of CYP24A1-dependent metabolism. In addition, as mentioned above, the fact that there are animal species differences in the metabolic mode of 1α,25(OH)2D<sup>3</sup> by CYP24A1 suggests that there are also animal species differences in the metabolism of vitamin D derivatives. Therefore, the development of vitamin D derivatives requires not only animal studies, but also metabolic studies using human CYP24A1 enzyme. species differences in the metabolism of vitamin D derivatives. Therefore, the development of vitamin D derivatives requires not only animal studies, but also metabolic studies using human CYP24A1 enzyme.

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 8 of 16

**Figure 4.** Structures of three CYP24A1-resistant VDR agonists: Eldecalcitol (ED-71), O2C3, MART-10, and AH-1. **Figure 4.** Structures of three CYP24A1-resistant VDR agonists: Eldecalcitol (ED-71), O2C3, MART-10, and AH-1.

#### *3.5. CYP24A1-Resistant Vitamin D Derivatives with a Substituent at C2*α *Position 3.5. CYP24A1-Resistant Vitamin D Derivatives with a Substituent at C2α Position*

We have synthesized many of A-ring-modified derivatives with a substituent at the C2α position, which have unique biological activities [64–68]. Of these derivatives, 2α-(3 hydroxypropoxy)-1α,25(OH)2D3 (O2C3), which is a C2-epimer of Eldecalcitol, was examined for the metabolism by CYP24A1. Five metabolites were detected in its metabolism by human CYP24A1, including both C-23 and C-24 oxidation pathways [48]. The *K*m and *k*cat values of human CYP24A1 for O2C3 were estimated to be approximately 16 times greater and 3 times lower than those for 1α,25(OH)2D3, respectively [48]. Accordingly, the catalytic efficiency (*k*cat/*K*m) of human CYP24A1 for O2C3 is only about 3% of 1α,25(OH)2D3. These results strongly suggest that O2C3 is much more resistant to CYP24A1-dependent metabolism than 1α,25(OH)2D3. It is noted that another C-2-substituted derivative, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3 (MART-10) (Figure 4), was more resistant to CYP24A1-dependent degradation than O2C3 [69–74]. The *k*cat/*K*m values We have synthesized many of A-ring-modified derivatives with a substituent at the C2α position, which have unique biological activities [64–68]. Of these derivatives, 2α- (3-hydroxypropoxy)-1α,25(OH)2D<sup>3</sup> (O2C3), which is a C2-epimer of Eldecalcitol, was examined for the metabolism by CYP24A1. Five metabolites were detected in its metabolism by human CYP24A1, including both C-23 and C-24 oxidation pathways [48]. The *K*<sup>m</sup> and *k*cat values of human CYP24A1 for O2C3 were estimated to be approximately 16 times greater and 3 times lower than those for 1α,25(OH)2D3, respectively [48]. Accordingly, the catalytic efficiency (*k*cat/*K*m) of human CYP24A1 for O2C3 is only about 3% of 1α,25(OH)2D3. These results strongly suggest that O2C3 is much more resistant to CYP24A1-dependent metabolism than 1α,25(OH)2D3. It is noted that another C-2-substituted derivative, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D<sup>3</sup> (MART-10) (Figure 4), was more resistant to CYP24A1 dependent degradation than O2C3 [69–74]. The *k*cat/*K*<sup>m</sup> values of human CYP24A1 for MART-10 were about 0.3 % of those for 1α,25(OH)2D3.

of human CYP24A1 for MART-10 were about 0.3 % of those for 1α,25(OH)2D3. Our in vivo studies using rats revealed that MART-10 had a potent anticancer effect, with a low calcemic effect, which is a suitable property as an anticancer drug. The re-Our in vivo studies using rats revealed that MART-10 had a potent anticancer effect, with a low calcemic effect, which is a suitable property as an anticancer drug. The resistance to CYP24A1 is also a suitable property of MART-10 as an anticancer drug.

#### **4. In Vivo Evaluation System for Vitamin D Derivatives Using Genetically Modified 4. In Vivo Evaluation System for Vitamin D Derivatives Using Genetically Modified Rats Generated by Genome Editing**

sistance to CYP24A1 is also a suitable property of MART-10 as an anticancer drug.

#### **Rats Generated by Genome Editing**  *4.1. Appearance and Growth of Genetically Modified (GM) Rats*

*4.1. Appearance and Growth of Genetically Modified (GM) Rats*  Figure 5a shows WT, *Vdr* (R270L), and *Vdr-*KO rats fed an F-2 diet containing 0.75% Ca, and *Cyp27b1*-KO rats fed a diet containing 1.15% Ca at 15 weeks after birth. Although *Cyp27b1-*KO rats were much smaller than WT rats, body sizes of *Vdr* (R270L) and *Vdr-*KO rats were not so different from that of the WT rats. Figure 5a shows the *Vdr-* abnormal skin and alopecia of KO rats. Elasticity and softness of the skin of *Vdr*-KO rats were substantially lowered and the wavy skin was formed [25]. Keratinization was elevated and Figure 5A shows WT, *Vdr* (R270L), and *Vdr-*KO rats fed an F-2 diet containing 0.75% Ca, and *Cyp27b1*-KO rats fed a diet containing 1.15% Ca at 15 weeks after birth. Although *Cyp27b1-*KO rats were much smaller than WT rats, body sizes of *Vdr* (R270L) and *Vdr-*KO rats were not so different from that of the WT rats. Figure 5A shows the *Vdr-* abnormal skin and alopecia of KO rats. Elasticity and softness of the skin of *Vdr*-KO rats were substantially lowered and the wavy skin was formed [25]. Keratinization was elevated and follicles decreased, and formation of cysts appeared in the dorsal skin of *Vdr-*KO rats [25].

follicles decreased, and formation of cysts appeared in the dorsal skin of *Vdr-*KO rats [25]. Figure 5a shows that growth was substantially diminished in *Cyp27b1*-KO rats compared to WT rats. However, only a slight decrease was observed in *Vdr* (R270L) and *Vdr-*KO rats. It was noted that approximately a half of male *Cyp27b1*-KO rats fed with the diet containing 0.75 % Ca died prior to 9 weeks of age, and none survived to 10 weeks of age (data not shown), whereas no animals had died at 15 weeks of age in the *Cyp27b1*-

KO rats fed with the diet containing 1.15% Ca. Thus, the diet that contained 0.75% Ca was used for mutant *Vdr* (R270L) and *Vdr-*KO rats, while the diet containing 1.15 % Ca

**Figure 5.** The appearance of GM rats and their abnormal bone formation [25]. (A) Comparison of body size and skin phenotype at 15 weeks of age. (B) First panels, 2D μ-CT images of horizontal section at distal femur; second panels, von Kossa staining of distal femur; bottom panels, toluidine blue staining of epiphyseal cartilage. **Figure 5.** The appearance of GM rats and their abnormal bone formation [25]. (A) Comparison of body size and skin phenotype at 15 weeks of age. (B) First panels, 2D µ-CT images of horizontal section at distal femur; second panels, von Kossa staining of distal femur; bottom panels, toluidine blue staining of epiphyseal cartilage.

*4.2. Osteogenesis and Plasma Ca, PTH, and 1α,25(OH)2D3 Levels in the GM Rats*  It is noted that *Cyp27b1*-KO rats are remarkably smaller than other rats. Figure 5b shows the middle region of the femur in 2D μCT scan images. The femur lengths of *Cyp27b1-*KO, *Vdr* (R270L), and *Vdr-*KO rats were found to be remarkably shorter than those of WT rats. The μCT scanning and von Kossa staining of femurs showed hyperplasia of calcified trabecular bones with a narrow medullary cavity in all the *Vdr* (R270L), *Cyp27b1*-KO, and *Vdr-*KO rats (Figure 5b). The *Vdr* (R270L) and *Vdr-*KO rats expressed no clear differences in total bone mineral density (BMD). In contrast, the BMD of cortical bone Figure 5A shows that growth was substantially diminished in *Cyp27b1*-KO rats compared to WT rats. However, only a slight decrease was observed in *Vdr* (R270L) and *Vdr-*KO rats. It was noted that approximately a half of male *Cyp27b1*-KO rats fed with the diet containing 0.75 % Ca died prior to 9 weeks of age, and none survived to 10 weeks of age (data not shown), whereas no animals had died at 15 weeks of age in the *Cyp27b1*-KO rats fed with the diet containing 1.15% Ca. Thus, the diet that contained 0.75% Ca was used for mutant *Vdr* (R270L) and *Vdr-*KO rats, while the diet containing 1.15 % Ca was used for *Cyp27b1*-KO rats.

#### in *Cyp27b1*-KO rats was substantially diminished [25]. *4.2. Osteogenesis and Plasma Ca, PTH, and 1α,25(OH)2D<sup>3</sup> Levels in the GM Rats*

Histological analysis of the epiphyseal cartilage demonstrated structural disorder of the growth plate in all the *Vdr* (R270L), *Cyp27b1*-KO, and *Vdr-*KO rats. Whereas WT growth plates contained aligned cartilage cells in the layered structure, growth plates in all three GM rats lost the sequential plate structure and cartilage cell alignment (Figure 5). It is noted that *Cyp27b1*-KO rats are remarkably smaller than other rats. Figure 5B shows the middle region of the femur in 2D µCT scan images. The femur lengths of *Cyp27b1-* KO, *Vdr* (R270L), and *Vdr-*KO rats were found to be remarkably shorter than those of WT rats. The µCT scanning and von Kossa staining of femurs showed hyperplasia of calcified trabecular bones with a narrow medullary cavity in all the *Vdr* (R270L), *Cyp27b1*-KO, and *Vdr-*KO rats (Figure 5B). The *Vdr* (R270L) and *Vdr-*KO rats expressed no clear differences

in total bone mineral density (BMD). In contrast, the BMD of cortical bone in *Cyp27b1*-KO rats was substantially diminished [25].

Histological analysis of the epiphyseal cartilage demonstrated structural disorder of the growth plate in all the *Vdr* (R270L), *Cyp27b1*-KO, and *Vdr-*KO rats. Whereas WT growth plates contained aligned cartilage cells in the layered structure, growth plates in all three GM rats lost the sequential plate structure and cartilage cell alignment (Figure 5). Thus, the morphology of bone was abnormal in all three GM rats, and, in *Cyp27b1*-KO rats, the most significant disorders of bone were observed.

It is well known that rickets type I model *Cyp27b1-*KO mice, and rickets type II model *Vdr-*KO mice, have significantly lower plasma Ca levels than WT mice [75,76]. Expectedly, the plasma Ca level was substantially reduced, and the level of parathyroid hormone (PTH) in plasma was greatly increased in *Vdr* (R270L) rats and *Cyp27b1*-KO rats [25]. Unexpectedly, the plasma Ca level in *Vdr*-KO rats was normal at 15 weeks. In *Vdr*-KO rats, until 10 weeks, the plasma level of Ca was significantly lower than that in WT rats, and PTH level was substantially higher than that in WT rats [25]. Plasma PTH level in *Vdr*-KO rats was remarkably higher than that in WT rats; although, at 15 weeks, the level of Ca in plasma in *Vdr*-KO rats returns to normal. These findings might indicate that hyperparathyroidism occurred in *Vdr*-KO rats [25]. In addition, the putative incomplete formation of intercellular barriers in epithelial tissues, including the small intestine, in VDR-KO rats might cause the increased calcium permeability to result in the normal level of plasma Ca concentration [77].

Although plasma 1α,25(OH)2D<sup>3</sup> level was significantly increased in *Vdr* (R270L) and *Vdr-*KO rats, it was significantly decreased in *Cyp27b1*-KO rats (8.0 ± 3.2 pg/mL (mean ± SEM, n = 7)) compared to WT rats (24.8 ± 5.2 pg/mL, (mean ± SEM, n = 7)) [25].
