Validation of an HPLC Method for the Simultaneous Quantification of Metabolic Reaction Products Catalysed by CYP2E1 Enzyme Activity: Inhibitory Effect of Cytochrome P450 Enzyme CYP2E1 by Salicylic Acid in Rat Liver Microsomes.

Inhibition of cytochrome P450 (CYP) alters the pharmacokinetic parameters of the drug and causes drug-drug interactions. Salicylic acid been used for the treatment of colorectal cancer (CRC) and chemoprevention in recent decades. Thus, the aim of this study was to examine the in vitro inhibitory effect of salicylic acid on CYP2E1 activity in rat liver microsomes (RLMs) using high-performance liquid chromatography (HPLC). High-performance liquid chromatography analysis of a CYP2E1 assay was developed on a reversed phase C-18 column (SUPELCO 25 cm x 4.6 mm x 5 mu m) at 282 nm using 60% H2O, 25% acetonitrile, and 15% methanol as mobile phase. The CYP2E1 assay showed a good linearity (R-2 > 0.999), good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80-120%), and low detection (4.972 mu M and 1.997 mu M) and quantitation limit values (15.068 mu M and 6.052 mu M), for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. Salicylic acid acts as a mixed inhibitor (competitive and non-competitive inhibition), with K-i (inhibition constant) = 83.56 +/- 2.730 mu M and concentration of inhibitor causing 50% inhibition of original enzyme activity (IC50) exceeding 100 mu M (IC50 = 167.12 +/- 5.460 mu M) for CYP2E1 enzyme activity. Salicylic acid in rats would have both low and high potential to cause toxicity and drug interactions with other drugs that are substrates for CYP2E1.