Functional characteristics of CYP3A4 allelic variants on the metabolism of loperamide in vitro

Figures & data

Figure 1 The transformation of loperamide to its main metabolite N-demethylated loperamide (DLOP).

Figure 2 UPLC-MS/MS chromatographs of N-demethylated loperamide and midazolam (10 μg/mL midazolam) in the 200-μL incubation system: (A) without loperamide and midazolam; (B) with activity-abolished microsomes and spiked with 0.25 μM N-demethylated loperamide; (C) incubating with 20 μM loperamide and 1 pmol CYP3A4.1. Abbreviations: CYP3A4, cytochrome P450 3A4; MRM, multiple reaction monitoring.

Table 1 Kinetic parameters for N-demethylated loperamide activity of CYP3A4.1 and other CYP3A4 variants on loperamide metabolism

Variants	Vmax (pmol/min/pmol P450)	Km (μM)	Clint (Vmax/Km) (μL/min/pmol P450)
3A4.1	2.079±0.064	2.196±0.218	0.951±0.066
3A4.2	0.568±0.010**	8.023±0.492**	0.071±0.003**
3A4.3	1.777±0.052	5.530±0.290**	0.322±0.007**
3A4.4	1.467±0.018*	3.293±0.019*	0.446±0.003**
3A4.5	2.153±0.131	4.804±0.246**	0.448±0.004**
3A4.6	ND	ND	ND
3A4.7	3.259±0.078**	8.849±0.318**	0.368±0.004**
3A4.8	5.302±0.367**	7.828±1.111**	0.682±0.048**
3A4.9	0.371±0.004**	2.884±0.247	0.129±0.010**
3A4.10	1.180±0.028**	2.500±0.076	0.472±0.003**
3A4.11	2.854±0.021**	17.163±0.229**	0.166±0.003**
3A4.12	0.969±0.042**	4.016±0.346**	0.242±0.010**
3A4.13	0.651±0.014**	5.812±0.249**	0.112±0.002**
3A4.14	4.862±0.226**	10.494±0.711**	0.464±0.016**
3A4.15	1.074±0.054**	3.423±0.313*	0.315±0.013**
3A4.16	10.703±0.430**	15.550±1.370**	0.690±0.033**
3A4.17	ND	ND	ND
3A4.18	9.223±0.116**	11.567±0.319**	0.798±0.012**
3A4.19	0.914±0.042**	2.234±0.394	0.416±0.056**
3A4.20	ND	ND	ND
3A4.23	0.434±0.034**	5.331±0.922**	0.082±0.008**
3A4.24	0.209±0.001**	1.329±0.040	0.157±0.004**
3A4.28	1.230±0.056*	2.356±0.223	0.524±0.026**
3A4.29	1.327±0.009*	3.356±0.045*	0.395±0.003**
3A4.30	ND	ND	ND
3A4.31	0.834±0.004**	2.900±0.167	0.288±0.016**
3A4.32	0.553±0.012**	3.568±0.199*	0.155±0.005**
3A4.33	0.851±0.016**	4.849±0.338**	0.176±0.009**
3A4.34	0.626±0.002**	1.902±0.040	0.329±0.007**

Notes: Significantly different from wild-type CYP3A4, *P<0.05, **P<0.01.

Abbreviations: Cl_int, intrinsic clearance; ND, not determined; CYP3A4, cytochrome P450 3A4.

Figure 3 Michaelis–Menten curve of the enzymatic activities of the wild-type CYP3A4 and other CYP3A4 variants on loperamide metabolism. Data are presented as mean ± SD of 3 parallel experiments. The variants with designated allele names have been arranged into 6 groups (A–F).

Abbreviation: CYP3A4, cytochrome P450 3A4.

Figure 4 Relative clearance of CYP3A4 variants toward loperamide metabolism compared with the wild type, arranged in the order.

Notes: Significant differences between the wild-type CYP3A4 and CYP3A4 variants analyzed by the mean of one-way ANOVA with Dunnett’s test, *P<0.05, **P<0.01. Abbreviation: CYP3A4, cytochrome P450 3A4.