Inhibitory Effects of Calf Thymus DNA on Metabolism Activity of CYP450 Enzyme in Human Liver Microsomes

Abstract

The present study investigated the effect of calf thymus DNA (ctDNA) on human hepatic cytochrome P450s (CYP450s) in vitro. Specific substrate probes for each isoform, CYP1A2, 2C9, 2C19, 2D6 and 3A4, were incubated using pooled human liver microsomes with or without ctDNA, and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of probe metabolites. Enzyme kinetics parameters Ki and IC50 values were estimated to determine the types and strength of inhibition. ctDNA could specifically inhibit the metabolism of CYP2C9 probe substrates, with the IC50 = 0.9955 µg/ml, while it was not able to inhibit CYP1A2, CYP2C19, CYP2D6 or CYP3A4 (IC50 > 100 µg/ml). The results showed that ctDNA was a potent inhibitor of CYP2C9 enzyme, and has the metabolic interaction potential with the model drugs which are metabolism substrates of CYP2C9. The inhibition mechanism study suggested ctDNA may inhibit CYP2C9 by decreasing the activity of CYP450 reductase. These findings indicated that when the medical agents catalyzed mainly by CYP2C9 were co-administered in vivo with adsorptive material in vitro, the potential inhibitory effect of ctDNA on enzyme activity and the following metabolism character changes of the former should be highly focused on.

In Vitro Comparative Study of the Inhibitory Effects of Mangiferin and Its Aglycone Norathyriol towards UDP-Glucuronosyl Transferase (UGT) Isoforms

“…In practice, the [I]/ Ki ratio is used to predict the likelihood inhibitory drug-drug interactions. When assessing in vivo interaction potential, [I] represents the mean steady-state C max value following the administration of the highest proposed clinical dose [ 49 ]. Extensive research into the pharmacokinetics of MGF in rat had been investigated.…”

Evaluation and Comparison of the Inhibition Effect of Astragaloside IV and Aglycone Cycloastragenol on Various UDP-Glucuronosyltransferase (UGT) Isoforms

“…When assessing in vivo interaction potential, [I] represents the mean steady-state Cmax value following the administration of the highest proposed clinical dose [28]. When CAG was co-administrated with drugs mainly undergoing UGT1A8-or UGT2B7-catalyzed metabolism, in vivo herb-drug interaction could have occurred when the mean steady-state Cmax of CAG was above 0.034 µM and 20.98 µM, respectively.…”

Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes

“…However, the application of these methods was hindered by a number of defects. Methods involving the quantification of several metabolic reactions in human and rat plasma and urine, human liver microsomes, or hepatocytes were only used to evaluate the activity of a limited number of CYP isoforms . Additionally, a method that involved the quantification of eight metabolic reactions and one that quantified nine metabolic reactions in human liver microsomes were unable to comprehensively assess CYP3A4 activity because only one probe was used for CYP3A4.…”

“…Hepatic cytochrome P450 enzymes (CYPs) play a central role in the biotransformation of a wide variety of structurally unrelated compounds, including xenobiotics such as drugs and toxins, and various endogenous substrates . CYPs are encoded by genes belonging to a superfamily comprising a number of isozymes described by the US Food and Drug Administration (FDA) (2012), with CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 considered the major isoforms responsible for the metabolism of the majority of marketed drugs in humans.…”

“…Therefore, it is necessary to establish a fast and accurate method for simultaneous quantification of the above ten metabolic reactions to rapidly, accurately, and comprehensively evaluate the impact of drug candidates and other factors on the activity of CYP enzymes to guide rational drug use in clinical settings. Although thus far, several studies have reported analytical methods to detect the formation of multiple metabolites from probe substrates using LC–MS in biological samples, many disadvantages were associated with the proposed methods, including the facts that only a small number of metabolic reactions was detected and a single CYP 3A4‐specific probe was used to evaluate the activity of this isozyme in some of the methods [, ]. No rapid and accurate method currently exists that would be capable of simultaneous determination of ten metabolites to quantify ten different metabolic reactions in biological samples, which could thereby be used to evaluate the activities of multiple CYP isoforms.…”