TABLE 5

Kinetic parameters of hydrolysis and uptake of ALS3 and Exp3174 in vitro incubation systems

Data are shown as the mean ± S.D. (n = 2 for hydrolysis assays and n = 3 for uptake assays).

Type	Enzyme/Transporter	Kinetic Model	K_m	V_max	CL_int	h
			µM	pmol/min/mg	µl/min/mg
ALS3 hydrolysis	HIM	Michaelis–Menten^{^a}	6.92 ± 0.39	895 ± 14	129 ± 6	NA
	CES1	Michaelis–Menten^{^a}	14.5 ± 1.3	3559 ± 108	245 ± 17	NA
	CES2	Allosteric sigmoidal	6.77 ± 0.15	7207 ± 65	1065 ± 19	1.77 ± 0.06
ALS3 uptake	OATP2B1	Allosteric sigmoidal	0.75 ± 0.08	182 ± 9	215 ± 14	1.72 ± 0.23
Exp3174 uptake	OATP2B1	Allosteric sigmoidal	7.85 ± 0.33	126 ± 3	16.1 ± 0.5	2.02 ± 0.14

NA, not available.
↵^a According to the Eadie–Hofstee plots in Fig. 4, the types of hydrolysis kinetics of ALS3 in HIM, rCES1, and rCES2 are all allosteric sigmoidal. However, the h values for ALS3 in HIM and rCES1 were 1.18 and 1.28, respectively, which showed weak allosteric sigmoidal curves. By comparison of the two fits using GraphPad Prism software based on the P value, Michaelis–Menten fit was chosen for calculating the kinetic parameters for HIM and rCES1. The h value for ALS3 in rCES2 was 1.77, and allosteric sigmoidal fit was thereby chosen based on the comparison of the two fits.