**4. Role of the CYP4 Family in the Metabolism of Drugs**

The roles of most CYP4 family proteins in the metabolism of drugs and xenobiotic compounds appear to be minor compared to those of CYP1, 2, and 3. However, CYP4F2 metabolizes the ester prodrug of gemcitabine and the antiparasitic pafuramidine [57]. In addition, CYP4A11 exhibited metabolism of the immune suppressant tacrolimus to an inactive form [58]. Although the turnover rates were low compared to those of CYP3A4, CYP4F11 exhibited catalytic activity towards commonly used drugs such as erythromycin, benzphetamine, and chlorpromazine [36,37,59]. CYP4F12 has been reported to slowly metabolize the antihistamine ebastine [60] and the antifungal terfenadine [61]. CYP4 enzymes are indirectly involved in drug metabolism and drug responses. For example, CYP4F2 and CYP4F11 are involved in the metabolism of vitamin K, facilitating vitamin K inactivation and elimination [29,62]. The amount of active vitamin K is important for maintenance of warfarin dosing, as it is metabolized strongly by CYP2C9 [63,64], indicating that CYP4 enzymes are indirectly involved in warfarin dose maintenance. CYP4 enzymes show catalytic activity toward various fatty acids and their metabolites have the potential to act as ligands or activators of nuclear receptors, such as peroxisome proliferator-activated receptors (PPARs) [65,66]. Therefore, drugs targeting the activation or inactivation of PPARs may show altered pharmacokinetics or toxic responses [67,68]. Such indirect involvement may affect the drug response to conditions such as fatty liver diseases, diabetic diseases, and inflammatory diseases.

#### **5. The CYP4 Family and Inflammation**

CYP4 enzymes are involved in inflammation through the metabolism of inflammatory molecules. They metabolize inflammatory mediators such as leukotrienes (LTs) and also produce 20-HETE [53]. While CYP4F11 possesses lower affinity toward leukotriene B4 (LTB4), neutrophilic CYP4F3A has the highest affinity for LTB4 ω-hydroxylation [36]. CYP4F3A metabolizes LTB4 into the inactive form 20-hydroxy leukotriene B4, mediating a critical step in regulation of the inflammatory response. However, CYP4A11 has shown low activity toward LTB4 using in vitro methods [69]. CYP4F3B ω-hydroxylates omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to their 20-hydroxy and 22-hydroxy metabolites, respectively [32], which are lipid mediators that can activate inflammatory PPARs [70]. Studies have shown that hepatic and renal rat *CYP4F* genes were upregulated under inflammatory conditions following treatment with barium sulfate [71]. On the other hand, rat hepatic CYP4A mRNAs were downregulated in response to lipopolysaccharides used as a model of inflammation [72]. Human CYP4V2 was first identified in inflammatory cell macrophages, and its gene expression was reduced following selective treatment with a PPARγ agonist [41]. Depending on the clinical situation, ω-hydroxylase activity associated with the CYP4 family could be considered as a potential drug target for reducing the inflammatory response, providing a novel mechanism for future anti-inflammatory drugs.

#### **6. The CYP4 Family and Cancers**

Induction of CYP4 family members, including CYP4F2, CYP4F3, CYP4A11, and CYP4Z1, has been reported in various types of cancer [73,74]. Upregulation of CYPF2 and CYP4A11 was confirmed through Western blot assays in human thyroid, ovarian, breast, pancreatic, and colon cancer tissues [75]. CYP4Z1 is expressed in mammary tissue and upregulated in breast cancer tissue [74]. These findings suggest that ω-hydroxylase activity may be a biomarker of cancer prognosis. Evaluation of the CYP4 expression profile in hepatocellular carcinoma (HCC) showed that CYP4F2, CYP4F12, and CYP4V2 mRNA levels were negatively correlated with cell-cycle-associated genes, suggesting that these *CYP4* genes are favorable prognostic factors in HCC [76]. In addition, expression of CYP4 has been reported to be associated with angiogenesis through production of 20-HETE, which activates vascular endothelial receptors in arteries and thus increases blood supply to cancer cells [77]. Among CYP4 enzymes, CYP4F3B, CYP4A11, and CYP4F2 are major enzymes involved in the generation of 20-HETE, which plays an important role in tumor progression and angiogenesis. Therefore, their tissue expression and omega-hydroxylase activity levels play roles in cancer progression. CYP4B1 metabolizes several protoxic xenobiotics, including 2-aminofluorine, 2-naphthylamine, 4-ipomeanol, and benzidine [78–81]. Therefore, CYP4B1 involvement in cancers has been suggested based on its expression levels and metabolism of pro-carcinogens in the bladder and lung [78,82]. CYP4B1 may play a role in detoxification or activation in tissues. Sasaki et al. reported that the individuals carrying the *CYP4B1\*2* allele have an increased risk of bladder cancer [27]. However, it has also been reported that there is no association between the *CYP4B1* genotype and the risk of lung cancer in the Japanese population [83]. Downregulation of CYP4B1 proteins represented an unfavorable indicator in patients with urothelial carcinomas of the upper urinary tract and bladder, indicating a protective role of CYP4B1 in patients with urotherial carcinomas [84]. Involvement of CYP4Z1 in breast cancer has been suggested, as it was identified in breast tissue and upregulated in breast carcinoma [74,76]. Therefore, CYP4Z1 was proposed as a biomarker for malignancy and/or progression of ovarian and prostate cancer [85]. It was reported that breast cancer cells exhibited the abnormal translocation of CYP4Z1 protein to the plasma membrane instead of targeting to the intracellular membrane of the endoplasmic reticulum, which caused the CYP4Z1 autoantibody production that might serve as a biomarker for the diagnosis [86]. Expression of CYP4Z1 has been reported to promote angiogenesis and tumor growth by increasing 20-HETE synthesis [74]. However, a recent functional study of CYP4Z1 in a recombinant enzyme system indicated that 20-HETE was not detected in the CYP4Z1 reaction with arachidonic acid, and suggested that CYP4Z1 may modulate breast cancer without direct 20-HETE synthesis [87]. Further studies are needed to clarify the roles of CYP4Z1 in carcinogenesis in various tissues.

#### **7. The CYP4 Family and Cardiovascular Diseases**

Several studies have shown that *CYP4* family genes are associated with cardiovascular diseases, including hypertension and myocardial infarction, through the production of 20-HETE or perturbation of fatty acid metabolism [88,89]. Multiple aspects of the mechanism underlying the effect of 20-HETE

on the cardiovascular complex have been reported. In a metabolomics study in mice, increased 20-HETE levels in the blood (>120-fold) with chronic rofecoxib treatment were associated with reduced bleeding time and increased platelet aggregation [47]. Additionally, 20-HETE has been suggested to mediate androgen-induced hypertension through increasing the level of Cyp4a12 in a mouse study [90], wherein the increased level of Cyp4a12 produced more eicosanoids, which were predicted to mediate androgen-induced hypertension. In the kidney, however, 20-HETE exerts anti-hypertensive effects through inhibition of sodium reabsorption in the proximal tubule and thick ascending limb of Henle [91]. Furthermore, 20-HETE was found to act as a vasoconstrictor of vascular smooth muscle cells by allowing increased calcium entry into cells and enhanced phosphorylation of contractile elements [92–94]. Several studies have suggested interplay between 20-HETE and the renin–angiotensin aldosterone system (RAAS) in hypertension. Briefly, angiotensinogen II has been reported to increase renal production of 20-HETE [95], and 20-HETE can activate the RAAS by inducing angiotensin-converting enzyme [96,97]. Further investigations are needed to fully elucidate the mechanistic link between 20-HETE and the RAAS in humans. Rat CYP4A was downregulated in the kidney of hypertensive rats, which was associated with reduced formation of 20-HETE in the kidney and reduction of the diuretic effect [98]. CYP4A was upregulated in studies of doxorubicin-induced cardiotoxicity, where it was associated with myocardial infarction and increased 20-HETE synthesis [51]. Furthermore, Jarrar et al. found that heart cyp4a12 was highly upregulated in mice after cardiac toxicity induced by non-steroidal anti-inflammatory drugs [44]. Thus, targeting of 20-HETE synthesis or modulation of eicosanoid levels through manipulation of CYP4 enzymes can decrease the cardiotoxicity of such drugs. This application should be considered in future development of the drug for cardiovascular health care.

#### **8. Role of the CYP4 Family in Other Diseases**

Bietti's crystalline dystrophy (BCD) is an autosomal recessive disease characterized by the presence of numerous small, yellow or white crystal-like deposits of fatty compounds in the light-sensitive retina tissue [52,99,100]. These deposits damage the retina, resulting in progressive atrophy of the retinal pigment epithelium and progressive vision loss at approximately 40 or 50 years of age [101,102]. The occurrence of BCD is more common in East Asian populations than other ethnic groups [103,104]. BCD is caused by mutations in the *CYP4V2* gene, which is comprised of 11 exons encoding a 525 amino acid protein on chromosome 4 [99,105,106]. CYP4V2 is known to metabolize fatty acids, and thus CYP4V2 in the retina is most likely involved in the breakdown and elimination of fatty acids from the retina [52]. Impaired CYP4V2 function due to genetic mutations may affect lipid metabolism and elimination from the retina. The severity and progression of BCD symptoms varies widely among patients. These variations may be influenced by differing levels of defectiveness in CYP4V2 function caused by mutations of different severities. Various mutations in *CYP4V2* have been found, including stop codon creation, an amino acid change in an important region, destruction of a splice site, and a frameshift in the CYP4V2 protein-coding cDNA. More than 60 mutations of the *CYP4V2* gene have been reported in BCD patients [99,103,105,107–112]. A number of mutations of *CYP4V2* have significant impacts on CYP4V2 activity. The most common mutation in BCD is an insertion–deletion mutation at the end of intron 6 and the beginning of exon 7 (IVS6-8del17insGC, c.802-8del17/insGC) [103,105,106,108,109,111–125]. This mutation causes the deletion of exon 7 in the CYP4V2 protein, resulting in a major structural change and the complete loss of CYP4V2 activity.

Type 3 lamellar ichthyosis, a skin keratinization disease, was found to be caused by genetic mutation of *CYP4F22* [126]. Since the discovery that *CYP4F22* is one of the causative genes for ichthyosis, the molecular mechanisms underlying the role of CYP4F22 in the etiology of ichthyosis have remained largely unknown until recently. Acylceramide is an important lipid of the skin permeability barrier, and patients with ichthyosis show strongly repressed acylceramide production [127–130]. Ohno et al. (2015) reported that CYP4F22 is responsible for the generation of acylceramide through ω-hydroxylation of long-chain fatty acids [18]. Recently, a *CYP4F22* genetic variant associated with lamellar ichthyosis was reported in a Tunisian family [131]. A missense mutation in exon 8, CYP4F22

Arg243Leu, was suggested to be linked to lamellar ichthyosis and predicted to be a functionally defective variant based on in silico analysis. Genetic screening for *CYP4F22* mutations associated with lamellar ichthyosis should be extended in future works.
