title: Chemical inhibitors of CYP450 enzymes in liver microsomes: combining selectivity and unbound fractions to guide selection of appropriate concentration in phenotyping assays.
abstract: 1. Chemical inhibition is the widely used method in reaction phenotyping assays for estimation of specific enzyme contribution to a given metabolic pathway. The results from phenotyping assays depend on the selectivity of chemical inhibitor and the concentration of inhibitor used in the incubation. 2. The higher protein concentrations used in the in vitro phenotyping assays will impact the inhibitory potency of chemical inhibitors. The objective of the study is to evaluate comprehensively the selectivity of chemical inhibitors and to guide in selecting appropriate concentration of the chemical inhibitors to be used in the phenotyping assays based on unbound fractions. 3. Selectivity of chemical inhibitors against nine major CYP450 isoforms was determined in liver microsomes using standard probe substrates. The unbound fractions of the selective inhibitors were determined in human liver microsomes using high-throughput equilibrium dialysis. Combining unbound inhibitor concentrations that are required to inhibit the CYP450 activities by 90% and unbound fractions of the chemical inhibitors in liver microsomes appropriate total concentrations of the inhibitors to be used in the phenotyping assays were reported. 4. The findings suggest that non-specific binding of the chemical inhibitors need to be taken into account while selecting concentrations for phenotyping assays.
authors: Discovery Research, Suven Life Sciences Ltd, Serene Chambers , Banjara Hills, Hyderabad, Andhra Pradesh , India and.
authors: Ramakrishna
authors: R
authors: Nirogi
authors: Raghava Choudary
authors: RC
authors: Palacharla
authors: Venkatesham
authors: V
authors: Uthukam
authors: Arunkumar
authors: A
authors: Manoharan
authors: Surya Rao
authors: SR
authors: Srikakolapu
authors: Ilayaraja
authors: I
authors: Kalaikadhiban
authors: Rajesh Kumar
authors: RK
authors: Boggavarapu
authors: Ranjith Kumar
authors: RK
authors: Ponnamaneni
authors: Devender Reddy
authors: DR
authors: Ajjala
authors: Gopinadh
authors: G
authors: Bhyrapuneni
mesh_terms: D001681:Biological Assay; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D055808:Drug Discovery / Q000379:methods; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D008862:Microsomes, Liver / Q000201:enzymology*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System
keywords: Chemical inhibitors; cytochrome P450; reaction phenotyping; unbound fractions