Functional Measurement of CYP2C9 and CYP3A4 Allelic Polymorphism on Sildenafil Metabolism

Figures & data

Figure 1 Chemical structure of the analytes and metabolic pathway of sildenafil.

Table 1 Michaelis-Menten Kinetic Parameters for Demethylation Activities of Wild-Type CYP2C9*1 and 37 Mutant CYP2C9 Alleles Toward Sildenafil

Variants	Vmax (pmol/min/pmol)	Km (µM)	CLint (µL/min/µmol)	Relative Clearance (% of Wild Type)
CYP2C9*1	0.75±0.02	42.75±2.45	17.46±0.45	100.00
CYP2C9*2	0.88±0.05	64.75±6.99	13.71±0.79	78.10
CYP2C9*3	0.30±0.01**	89.98±3.22**	3.30±0.07**	18.89
CYP2C9*8	0.35±0.00*	153.73±0.06**	2.25±0.01**	12.87
CYP2C9*11	1.13±0.01**	60.71±0.60	18.55±0.23	106.20
CYP2C9*13	0.11±0.01	155.23±12.01*	0.74±0.02**	4.21
CYP2C9*14	0.96±0.13	57.48±22.69	17.72±3.96	100.72
CYP2C9*16	0.47±0.00*	45.96±8.98	10.58±0.12	60.64
CYP2C9*19	0.19±0.06*	280.63±114.29	0.70±0.08**	3.96
CYP2C9*23	0.54±0.01*	42.57±1.45	12.60±0.13*	72.09
CYP2C9*27	0.92±0.02*	59.77±1.89*	15.44±0.29	88.42
CYP2C9*29	0.96±0.20	85.60±48.25	12.49±3.53	71.05
CYP2C9*31	0.76±0.03	39.48±3.94	19.30±1.18	110.66
CYP2C9*34	0.39±0.03**	111.70±8.74*	3.51±0.16**	20.05
CYP2C9*36	0.07±0.00**	224.97±12.59*	0.32±0.00**	1.82
CYP2C9*37	0.51±0.09	73.53±33.54	7.69±2.54	41.00
CYP2C9*38	0.31±0.03**	20.47±5.50	15.59±3.01	87.04
CYP2C9*39	0.28±0.03**	46.46±14.72	6.24±1.44*	34.91
CYP2C9*40	1.17±0.12	140.93±26.79	8.36±0.69**	47.83
CYP2C9*41	1.13±0.12	178.47±52.69	6.63±1.40*	37.11
CYP2C9*42	0.07±0.00**	83.50±31.53	0.89±0.25***	5.00
CYP2C9*43	0.46±0.23	1,044.00±540.35	0.45±0.03**	2.38
CYP2C9*44	0.82±0.16	142.40±68.39	6.37±1.93	34.98
CYP2C9*45	0.18±0.02***	54.15±13.77	3.39±0.54***	19.17
CYP2C9*46	0.29±0.02**	66.54±11.44	4.48±0.51***	25.62
CYP2C9*47	0.25±0.04**	52.47±15.16	4.91±0.69**	27.95
CYP2C9*48	0.70±0.06	193.07±33.91	3.68±0.29***	20.97
CYP2C9*49	1.11±0.01**	63.31±2.64	17.51±0.53	100.15
CYP2C9*50	0.28±0.00**	70.54±3.24*	3.94±0.11**	22.56
CYP2C9*51	0.20±0.00**	51.84±2.83	3.89±0.15**	22.27
CYP2C9*52	0.19±0.01**	62.53±6.19	3.00±0.16**	17.13
CYP2C9*53	0.29±0.02**	47.46±1.69	6.09±0.18**	34.84
CYP2C9*54	0.22±0.01**	60.71±5.28	3.61±0.22**	20.69
CYP2C9*55	0.26±0.00**	49.92±3.98	5.18±0.32***	29.64
CYP2C9*56	0.37±0.02**	76.22±7.41	4.83±0.20**	27.64
CYP2C9*58	0.41±0.02**	129.13±12.93	3.18±0.15**	18.24
CYP2C9*59	0.10±0.00**	231.80±33.88	0.45±0.04**	2.54
CYP2C9*60	0.25±0.04*	156.97±58.23	1.64±0.27***	9.20

Notes: *Significantly different from wild-type CYP2C9, *p < 0.05, **p < 0.01, ***p < 0.001.

Abbreviation: N.D., not determined.

Figure 2 Michaelis–Menten curves of the enzymatic activity of the recombinant wild-type CYP2C9*1 and 37 variants toward sildenafil N-demethylation (each point represents the mean ±SD of three parallel experiments).

Table 2 Michaelis-Menten Kinetic Parameters for Demethylation Activities of Wild-Type CYP3A4*1 and 22 Mutant CYP3A4 Alleles Toward Sildenafil

Variants	Vmax (pmol/min/pmol)	Km (µM)	CLint (µL/min/µmol)	Relative Clearance (% of Wild Type)
CYP3A4*1	9.29±0.45	64.02±5.07	0.15±0.02	100.03
CYP3A4*2	13.39±0.43***	169.77±11.26***	0.08±0.00**	55.54
CYP3A4*3	10.68±0.17**	56.99±3.79	0.19±0.01*	132.00
CYP3A4*4	10.91±0.10**	71.13±1.20	0.15±0.00	107.79
CYP3A4*5	6.01±0.43**	147.87±26.30**	0.04±0.00**	28.91
CYP3A4*9	10.82±0.14**	85.07±6.63*	0.13±0.01	89.53
CYP3A4*10	12.88±0.15***	67.05±1.03	0.19±0.00*	134.97
CYP3A4*11	21.31±0.74***	167.87±16.17***	0.13±0.01	89.56
CYP3A4*14	9.87±0.20	53.13±3.69*	0.19±0.01*	130.70
CYP3A4*15	10.06±0.10*	62.29±3.60	0.16±0.01	113.64
CYP3A4*17	ND	ND	ND	ND
CYP3A4*19	10.65±0.29*	62.22±2.04	0.17±0.00	120.34
CYP3A4*23	11.33±0.30**	76.10±4.29*	0.15±0.00	104.76
CYP3A4*24	3.36±0.56***	1,317.67±298.59**	0.00±0.00***	1.80
CYP3A4*28	5.91±0.13***	68.89±5.69	0.09±0.01**	60.47
CYP3A4*29	9.18±0.03	86.90±2.89**	0.11±0.00*	74.25
CYP3A4*30	ND	ND	ND	ND
CYP3A4*31	7.49±0.02**	51.67±3.65*	0.15±0.01	102.20
CYP3A4*32	13.20±0.31***	69.34±2.67	0.19±0.00*	133.84
CYP3A4*33	10.45±0.33*	60.64±3.97	0.17±0.01	121.15
CYP3A4*34	11.78±0.40**	77.36±5.91*	0.15±0.01	107.11

Notes: *Significantly different from wild-type CYP3A4, *p < 0.05, **p < 0.01, ***p < 0.001.

Abbreviation: ND, not determined.

Figure 3 Michaelis–Menten curves of the enzymatic activity of the recombinant wild-type CYP3A4*1 and 22 variants toward sildenafil N-demethylation (each point represents the mean ±SD of three parallel experiments).