Microsome biocolloids for rapid drug metabolism and inhibition assessment by LC-MS

Abstract

Rat liver microsomes attached to nanoparticles were used for LC-MS studies of CYP3A and 2E1 enzymes in metabolism of N-nitroso compounds. Using these biocolloids, turnover rates were measured within 2 min. Inhibitor IC(50) values for ketoconazole (KET) and 4-methylpyrazole (4-MEP) were estimated.

Figure 1

capLC-photodiode array (PDA) chromatograms showing the generation of metabolite peaks after 2 min. exposure of RLM biocolloids at pH 7.0 and 37° C to 1 mM (a) NNK or (b) NPYR, and NAPDH generating system c) NNK exposed to RLM biocolloids in absence of NADPH. Insets show MS spectra for metabolites 4-HPB and 2-OH-THF (Scheme 1) from NNK and NPYR respectively; (d) Influence of reaction time on amount of HPB (dashed line, closed circle) or 2-OH-THF (full line, open circle) formed in the presence of RLM-biocolloids and HPB (dashed line, diamonds) formed with RLMs in solution.

Figure 2

a) The effects of the specific inhibitors (KET and 4-MEP) on the catalytic activity of RLM-biocolloids during the metabolism of NNK and NPYR. Data collected after 10 min. incubation of RLM-biocolloids with respective N-nitrosamines and inhibitors, in the presence of NADPH generating system. b) CapLC chromatograms illustrating the metabolites formed with exposure of NNK to RLM films on silica nanospheres in the presence of NAPDH generating system with and without KET, 10 minute incubation.

Figure 3

Enzyme activity remaining with respect to control (no inhibitor) after 10 min incubation of RLM biocolloids, NADPH generating system with: a) different concentrations of KET in the presence of 0.1 mM NNK(open circle, dashed line) and 1.0mM NNK (closed squares, full line) b) different concentrations of 4-MEP in the presence of 0.1 mM NPYR (half closed squares, dashed line) and 1.0mM NPYR(closed circle, full line)

Scheme 1

Conceptual illustration of RLM film fabrication on silica nanospheres. Layer-by-layer electrostatic assembly was used to first immobilize positively charged poly(ethylenimine) PEI on the negative silica, then rat liver microsomes (RLM).

Scheme 2

NNK and NPYR metabolism featuring α-hydroxylation catalyzed by CYPs [16].