Establishment Of A Automated Enzyme Reaction System And Applied To Research Of The Inhibition Of CYPs By Drug Candidates

Cytochrome P450(Cytochrome P450 proteins, CYP) is a diverse class of mixed-function oxidase and heme enzyme systems, widely found in animals, plants and microorganisms, which biotransform a wide variety of chemicals, including drugs, pesticides, carcinogens, herbicides, food additives and other types of drug candidates. Cytochrome P450 is involved in 90% of drug metabolism in humans. However, when compounds inhibit its activity, metabolism of other compounds may be obstructed, so that drug-drug interactions(DDI) may occur. 96% metabolic interaction is mediated by CYPs, and inhibition of enzyme leading to DDI accounts for 70%. In the process of drug development, it is important to identify the specific CYP isoforms responsible for the metabolism of a drug candidatess and assess the inhibition/induction potential of the drug candidates to the CYP isoforms.One of the most common strategie in vitro to evaluate the inhibitory effect of a drug candidates on CYPs is to monitor its effect on the biotransformation of CYPs probe substrates in human liver microsomes(HLM). Manual incubation using glass tubes for a long time. Restricted by operator’s experience in scientific research and experimental practices, skilled level, and even emotional fluctouation, manual incubation has some defects in terms of accuracy. And it is also difficult to achieve high throughput for a great number of drug candidatess research. The actual operation of the probe substrates approach mostly based on using automation platform instead of manual incubation to study the inhibition of compounds to CYP has been published rarely in the literature.In this article, A Tecan RSP 100/8 Genesis series liquid handling workstation and IKA KS 130 small Orbital Shaker have been customized and integrated to provide an automated miniaturized cytochrome P450 inhibition assay platform. Fully automated incubations were conducted in a 96-well format under optimized enzyme kinetic conditions. All reagent additions to the 96-well microplates were performed exclusively by the dispenser of Tecan liquid handling workstation. It was used to perform larger volume bulk reagent and compounds dilution operations along with constant temperature and shake for enzyme reaction using the customized orbital shaker. Metabolites of probe substrates were analyzed with rapid LC-MS/MS methods.Two incubation programs have been established, including one preliminary study program which was set up for the determination of inhibition to several CYPs by single concentration of test drug candidates and another was for the determination of inhibition to single CYP by Different concentrations of test drug candidates, namely, this could be used to calculate concentration of test drug that will cause 50% inhibition(IC50). Setting a preliminary study program will help to determine whether it is necessary to calculate the IC50, or set concentration levels of test drug candidates if necessary.A positive control CYP inhibitor were included in each assay. The IC50 values determined for typical CYPs inhibitors were reproducible and consistent with those reported in the literature. This proved the correctness of the system to a certain extent.Finally, we used this automated system to study the inhibition of four main active ingredient of Rheum palmatum L. to CYPs. The results showed that Chrysophanol had a relatively inhibition to 2E1, Rhein had a relatively inhibition to 2C19 and 2E1; Emodin had a relatively inhibition to 2C8 and 2C19; and Aloe-Emodin had a relatively inhibition to 2B6 and 2C19. We calculated the IC50 of the three CYP isoforms of greatest significance in the metabolism(3A4/5, 2D6 and 2C9), and the results showed that the IC50 of Emodin to 3A4/5 and 2D6 was 15.82 mol/L and 12.68 mol/L;IC50 of Chrysophanol to 3A4/5 was 18.40mol/L;IC50 of Rhein to 2C9 was 24.17 mol/L;Others were more than 25.00 mol/L. This provided a reference for the possibility of DDI while giving further development of the medicinal value of Rheum palmatum L.