Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

Figures & data

Figure 1 Structures, UPLC chromatogram, and the LC/MS profile of OMT and MT.

Notes: The UPLC chromatogram of OMT and MT standard substances (A); the UPLC chromatogram of OMT after metabolism in Phase I reduction reaction systems (B); the UPLC chromatogram of OMT after metabolism in Phase I reduction reaction systems without HLMs (C). The LC-MS profile of OMT and its metabolite after incubation with HLMs and NADPH at 37°C for 1.5 hours (D, E). (D) shows the original profile of OMT (m/z 265.1908); (E) shows the original profile of MT (m/z 249.1970).
Abbreviations: OMT, oxymatrine; MT, matrine; UPLC, ultra-performance liquid chromatography; LC/MS, liquid chromatography/mass spectroscopy; HLM, human liver microsomes.

Figure 2 Formation of MT by cDNA-expressed human cytochromes P450 enzymes.

Notes: OMT (50 μM) was incubated with cDNA-expressed P450 enzymes and an NADPH-regenerating system at 37°C for 1.5 hours. All incubations were done in triplicate. Data are expressed as mean ± SD. The asterisk (*) indicates a statistically significant difference (P<0.05) when compared to others, according to a one-way ANOVA with a post hoc test.
Abbreviations: OMT, oxymatrine; MT, matrine; ANOVA, analysis of variance.

Figure 3 Effects of various chemical inhibitors on the rate of MT formation.

Notes: OMT (50 μM) was incubated with HLMs and an NADPH-regenerating system in the presence of seven chemical inhibitors at 37°C for 1.5 hours (A). OMT control experiments were incubated without chemical inhibitor. These chemical inhibitors were fluvoxamine maleate (CYP1A2), gemfibrozil (CYP2C8), amiodarone hydrochloride (CYP2C9), omeprazole (CYP2C19), quinidine (CYP2D6), diethyldithiocarbamic acid (CYP2E1), and ketoconazole (CYP3A). The chemical inhibition test of TES was used as a positive control of CYP3A4 inhibitor (B). TES control experiments were incubated without CYP3A4 inhibitor. Each column represents mean percent control of formation rates of metabolite and the error bars are standard deviations of the mean (n=3). The asterisk (*) indicates a statistically significant difference (P<0.05) when compared to the control, according to a one-way ANOVA with a post hoc test.
Abbreviations: OMT, oxymatrine; MT, matrine; HLM, human liver microsomes; TES, testosterone; UPLC, ultra-performance liquid chromatography; ANOVA, analysis of variance.

Table 1 Apparent kinetic parameters of metabolism of oxymatrine by pooled HLMs, HIMs, and CYP3A4

Kinetic parameters	Pooled HLM	Pooled HIM	CYP3A4
Km (μM)	220.78±97.58	150.22±48.44	341.35±34.99
Vmax (nmol/min/mg)	1.49±0.24	0.46±0.05	1.29±0.05
CL (Vmax/Km, mL/min/mg)	0.0067	0.0031	0.0038

Notes: Calculated based on curve fitting using biphasic metabolism equation: V = (V_max1×C)/(K_m1+C)+(V_max2×C)/(K_m2+C).

Abbreviations: HLM, human liver microsomes; HIMs, human intestinal microsomes; K_m, Michaelis constant; V_max, maximum reaction rate; CL, intrinsic clearance.

Figure 4 Kinetics profiles of OMT metabolism by HLMs (A), HIMs (B), and CYP3A4 (C), respectively.

Notes: The formation rate of MT expressed as nmol/min/mg (mean ± SD). The biphasic metabolism equation was fit to the data from all three incubation system. The embedded figures are Eadie–Hofstee plots for the same data. Rhombuses and smooth lines denote the observed and predicted rates of OMT metabolism, respectively. Microsomal protein concentration was 0.1 mg/mL and CYP3A4 protein concentration was 0.08 mg/mL; incubation time was 1.5 hours. Each data point represented the average of three replicates.
Abbreviations: OMT, oxymatrine; MT, matrine; HLM, human liver microsomes; HIMs, human intestinal microsomes; V/C, velocity/concentration.

Figure 5 The amount of MT formed in different oxygen contents, the effects of nitrogen on OMT metabolism (A), the effects of helium on OMT metabolism (B), the effects of inactivated enzyme on OMT (50 μM) metabolism (C).

Notes: OMT (50, 100, and 200 μM) was incubated with HLMs and an NADPH-regenerating system at 37°C for 1.5 hours. All the experiments were carried out in triplicate. Data were expressed as mean ± SD. Differences were considered significant when the P-values were less than 0.05 (or P<0.05).
Abbreviations: OMT, oxymatrine; MT, matrine; HLM, human liver microsomes; SD, standard deviation.

Figure S1 The amount of 6-β-OH-testosterone formed in different oxygen contents, the effects of nitrogen on TES metabolism (A), and the effects of helium on TES metabolism (B).

Notes: TES (50, 100, and 200 μM) was incubated with HLMs and an NADPH-regenerating system at 37°C for 1.5 hours. All the experiments were carried out in triplicate. Data were expressed as mean ± SD. Differences were considered significant when the P-values were less than 0.05 (or *P<0.05).
Abbreviations: HLM, human liver microsomes; TES, testosterone.

Table S1 Intra- and interday precision and accuracy for MT in KPI solution (n=6)

Analyte	Concentration (μM)	Intraday		Interday
		Precision (RSD, %)	Accuracy (bias, %)	Precision (RSD, %)	Accuracy (bias, %)
MT	0.4	2.8	102.7	2.8	103.7
	2	4.3	100.7	3.6	100.8
	10	1.0	98.3	1.5	97.7

Abbreviations: MT, matrine; RSD, relative standard deviation; KPI, potassium phosphate buffer.

Table S2 Stability of MT in KPI solution at 37°C

Analyte	Concentration (μM)	30 min		60 min		90 min		120 min
		Average (%)	RSD (%)	Average (%)	RSD (%)	Average (%)	RSD (%)	Average (%)	RSD (%)
MT	0.4	107.0	1.3	106.7	2.5	107.1	2.8	105.5	3.5
	2	103.6	2.0	107.8	0.9	105.4	0.9	104.9	1.4
	10	100.9	2.5	102.1	1.2	102.7	2.1	102.5	2.6

Abbreviations: MT, matrine; RSD, relative standard deviation; KPI, potassium phosphate buffer.