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Comparison of the inhibitory effects of azole antifungals on cytochrome P450 3A4 genetic variants

https://doi.org/10.1016/j.dmpk.2021.100384Get rights and content

Abstract

Cytochrome P450 (CYP) 3A4 is one of the major drug-metabolizing enzymes. Genetic variants of CYP3A4 with altered activity are one of the factors responsible for interindividual differences in drug metabolism. Azole antifungals inhibit CYP3A4 to cause clinically significant drug-drug interactions. In the present quantitative study, we investigated the inhibitory effects of three azole antifungals (ketoconazole, voriconazole, and fluconazole) on testosterone metabolism by recombinant CYP3A4 genetic variants (CYP3A4.1 (WT), CYP3A4.2, CYP3A4.7, CYP3A4.16, and CYP3A4.18) and compared them with those previously reported for itraconazole. The inhibition constants (Ki) of ketoconazole, voriconazole, and fluconazole for rCYP3A4.1 were 3.6 nM, 3.2 μM, and 16.1 μM, respectively. The Ki values of these azoles for rCYP3A4.16 were 13.9-, 13.6-, and 6.2-fold higher than those for rCYP3A4.1, respectively, whereas the Ki value of itraconazole for rCYP3A4.16 was 0.54-fold of that for rCYP3A4.1. The other genetic variants had similar effects on the Ki values of the three azoles, whereas a very different pattern was seen for itraconazole. In conclusion, itraconazole has unique characteristics that are distinct from those shared by the other azole anti-fungal drugs ketoconazole, voriconazole, and fluconazole with regard to the influence of genetic variations on the inhibition of CYP3A4.

Introduction

Cytochrome P450 (CYP) 3A4 is a clinically significant oxidative enzyme that is involved in the metabolism of >50% of drugs used in the clinical setting, as well as certain endogenous substances (e.g., steroids) [1]. More than thirty non-synonymous genetic variants of CYP3A4 have been identified to date [2]. Among them, the allelic frequencies of CYP3A4∗2, CYP3A4∗7, CYP3A4∗16, CYP3A4∗18 are 2.7% in Caucasian [3], 1.4–3% in Caucasian [4,5], 1.4–5% in Japanese [5,6], and 1.3–2.8% in Japanese [6], respectively. In vitro and in vivo studies have shown that the metabolic activity of these genetic variants differs, and such variation is one of the factors responsible for interindividual variability in CYP3A4-mediated drug metabolism [7,8].

On the other hand, the inhibition of CYP3A4 is one of the major causes of drug-drug interactions (DDI). For example, a variety of azole antifungals are known to be potent inhibitors of CYP3A4 [9]. However, no reports have shown the extent of DDI with azole antifungals in subjects with CYP3A4 genetic variation. Using recombinant human CYP3A4 variants (rCYP3A4.1 (WT), rCYP3A4.2, rCYP3A4.7, rCYP3A4.16, and rCYP3A4.18) expressed in Escherichia coli, we previously found that the inhibitory kinetics of two typical CYP3A4 inhibitors, itraconazole (ITCZ, Fig. 1A) and cimetidine, were different among genetic variations in CYP3A4 [10]. ITCZ exhibited lower and higher inhibitory potency against CYP3A4.7 and CYP3A4.16, respectively, compared with its potency against CYP3A4.1, while cimetidine displayed the opposite trend. An in silico docking simulation demonstrated that these differences in the effects of genetic variations could be explained based on the ability of the two inhibitors to access the active site of each CYP3A4 variant, which was affected by conformational changes caused by amino acid substitution(s).

Among azole antifungals, the order of inhibitory potency towards CYP3A4.1 follows the pattern ketoconazole (KCZ, Fig. 1C) > ITCZ > voriconazole (VCZ, Fig. 1D) > fluconazole (FCZ, Fig. 1B) [9]. However, it remains to be investigated whether this is also the case for other CYP3A4 variants. Although azole nitrogen atoms are considered to play an important role in the interactions between antifungals and the catalytic site of CYP3A4 [11], the roles the non-azole parts of these inhibitors play in such interactions remain unclear.

The aim of this study was to quantitatively evaluate the inhibitory properties of three azole antifungals, KCZ, VCZ, and FCZ, on testosterone (TST) metabolism by CYP3A4 variants (rCYP3A4.1, rCYP3A4.2, rCYP3A4.7, rCYP3A4.16, and rCYP3A4.18) and compare them with those of ITCZ, which were reported previously.

Section snippets

Chemicals and materials

TST, FCZ, and KCZ were purchased from Nacalai Tesque Inc. (Kyoto, Japan), Wako Pure Chemical Industries Ltd (Osaka, Japan), and Sigma-Aldrich (St Louis, MO), respectively. VFEND (200 mg, a VCZ injection solution) was purchased from Pfizer (New York, NY, USA). Hydrocortisone and 6β-hydroxytestosterone (6β-OH TST) were obtained from Sigma-Aldrich and SPI-Bio Bertin Pharma (Bretonneux, France), respectively. The E. coli membrane fractions of CYP3A4.1, CYP3A4.2, CYP3A4.7, CYP3A4.16, and CYP3A4.18

Comparison of metabolic kinetics among CYP3A4 variants

The kinetic parameters of the CYP3A4 variants are shown in Table 1. The Vmax values of CYP3A4.16 and CYP3A4.18 were significantly lower and higher than that of the WT, respectively. The Km values of all variants were higher than that of the WT. In particular, the Km value of CYP3A4.16 was almost 7-fold higher than that of the WT. The “intrinsic clearance” (Vmax/Km) values of CYP3A4.7 and CYP3A4.16 were significantly lower than that of the WT enzyme.

Comparison of the inhibitory potency of azole antifungals among CYP3A4 variants

All inhibitors tested (FCZ, KCZ, and VCZ)

Discussion

In order to assess the risk of an individual to drug interactions with azole antifungals involving CYP3A4 inhibition, which can lead to severe clinical outcomes [[13], [14], [15], [16]], it is crucial to quantitatively investigate the inhibitory kinetics of such interactions for both the WT enzyme and the variants. The present study provides clinically useful information about the inhibitory potency of azole antifungals against major CYP3A4 genetic variants.

In this study, the inhibition

Conclusion

In this study, KCZ, VCZ, and FCZ inhibited the enzymatic activity of five CYP3A4 variants with different potencies. The order of inhibitory potency against the CYP3A4 variants was similar among these three azoles but different from that of ITCZ. As genetic variations affect the inhibitory kinetics of inhibitors differently, even within the same structural group, inhibitory kinetics should be determined experimentally, at least for major variants, in order to enable the personalized prediction

Authors’ contributions

Participated in research design: Akiyoshi and Ohtani.

Preparation of CYP enzyme: Yamaguchi, Kawamura, Miyazaki, Guengerich, Nakamura, Yamamoto.

Conducted experiments: Yamaguchi, Kawamura, Akiyoshi, Imaoka and Ohtani.

Performed data analysis: Yamaguchi, Kawamura and Akiyoshi.

Wrote or contributed to the writing of the manuscript: Yamaguchi, Akiyoshi, Imaoka, Guengerich, Nakamura, Yamamoto and Ohtani.

Declaration of competing interest

None.

Acknowledgements

This study was supported in part by JSPS Kakenhi Grant Numbers 15K08596 and 18K06758 [to H.O.] and United States National Institutes of Health grant R01 GM118122 [to F.P.G.].

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