Biomaterial Database

Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes: assessment of human CYP inhibition potential. 1999

G C Moody, and S J Griffin, and A N Mather, and D F McGinnity, and R J Riley

Department of Physical & Metabolic Sciences, Astra Charnwood, Loughborough, UK.

1. Fully automated inhibition screens for the major human hepatic cytochrome P450s have been developed and validated. Probe assays were the fluorometric-based ethoxyresorufin O-deethylation for CYP1A2 and radiometric analysis of erythromycin N-demethylation for CYP3A4, dextromethorphan O-demethylation for CYP2D6, naproxen O-demethylation for CYP2C9 and diazepam N-demethylation for CYP2C19. For the radiometric assays > 99.7% of 14C-labelled substrate was routinely extracted from incubations by solid-phase extraction. 2. Furafylline, sulphaphenazole, omeprazole, quinidine and ketoconazole were identified as specific markers for the respective CYP1A2 (IC50 = 6 microM), CYP2C9 (0.7 microM), CYP2C19 (6 microM), CYP2D6 (0.02 microM) and CYP3A4 (0.2 microM) inhibition screens. 3. For the radiometric methods, a two-point IC50 estimate was validated by correlating the IC50 obtained with a full (seven-point) assay (r2 = 0.98, p < 0.001). The two-point IC50 estimate is useful for initial screening, while the full IC50 method provides more definitive quantitation, where required. 4. IC50 determined for a series of test compounds in human liver microsomes and cytochrome P450 cDNA-expressed enzymes were similar (r2 = 0.89, p < 0.001). In particular, the CYP1A2, CYP2D6 and CYP3A4 screens demonstrated the flexibility to accept either enzyme source. As a result of incomplete substrate selectivity, expressed enzymes were utilized for analysis of CYP2C9 and CYP2C19 inhibition. Good agreement was demonstrated between IC50 determined in these assays to IC50 published by other laboratories using a wide range of analytical techniques, which provided confidence in the universality of these inhibition screens. 5. These automated screens for initial assessment of P450 inhibition potential allow rapid determination of IC50. The radiometric assays are flexible, sensitive, robust and free from analytical interference, and they should permit the identification and eradication of inhibitory structural motifs within a series of potential drug candidates.

UI	MeSH Term	Description	Entries
D008745	Methylation	Addition of methyl groups. In histo-chemistry methylation is used to esterify carboxyl groups and remove sulfate groups by treating tissue sections with hot methanol in the presence of hydrochloric acid. (From Stedman, 25th ed)	Methylations
D008862	Microsomes, Liver	Closed vesicles of fragmented endoplasmic reticulum created when liver cells or tissue are disrupted by homogenization. They may be smooth or rough.	Liver Microsomes,Liver Microsome,Microsome, Liver
D011874	Radiometry	The measurement of radiation by photography, as in x-ray film and film badge, by Geiger-Mueller tube, and by SCINTILLATION COUNTING.	Geiger-Mueller Counters,Nuclear Track Detection,Radiation Dosimetry,Dosimetry, Radiation,Geiger Counter,Geiger-Mueller Counter Tube,Geiger-Mueller Probe,Geiger-Mueller Tube,Radiation Counter,Counter Tube, Geiger-Mueller,Counter Tubes, Geiger-Mueller,Counter, Geiger,Counter, Radiation,Counters, Geiger,Counters, Geiger-Mueller,Counters, Radiation,Detection, Nuclear Track,Dosimetries, Radiation,Geiger Counters,Geiger Mueller Counter Tube,Geiger Mueller Counters,Geiger Mueller Probe,Geiger Mueller Tube,Geiger-Mueller Counter Tubes,Geiger-Mueller Probes,Geiger-Mueller Tubes,Probe, Geiger-Mueller,Probes, Geiger-Mueller,Radiation Counters,Radiation Dosimetries,Tube, Geiger-Mueller,Tube, Geiger-Mueller Counter,Tubes, Geiger-Mueller,Tubes, Geiger-Mueller Counter
D002250	Carbon Radioisotopes	Unstable isotopes of carbon that decay or disintegrate emitting radiation. C atoms with atomic weights 10, 11, and 14-16 are radioactive carbon isotopes.	Radioisotopes, Carbon
D002624	Chemistry, Clinical	The specialty of ANALYTIC CHEMISTRY applied to assays of physiologically important substances found in blood, urine, tissues, and other biological fluids for the purpose of aiding the physician in making a diagnosis or following therapy.	Clinical Chemistry
D003577	Cytochrome P-450 Enzyme System	A superfamily of hundreds of closely related HEMEPROTEINS found throughout the phylogenetic spectrum, from animals, plants, fungi, to bacteria. They include numerous complex monooxygenases (MIXED FUNCTION OXYGENASES). In animals, these P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs (BIOTRANSFORMATION). They are classified, according to their sequence similarities rather than functions, into CYP gene families (>40% homology) and subfamilies (>59% homology). For example, enzymes from the CYP1, CYP2, and CYP3 gene families are responsible for most drug metabolism.	Cytochrome P-450,Cytochrome P-450 Enzyme,Cytochrome P-450-Dependent Monooxygenase,P-450 Enzyme,P450 Enzyme,CYP450 Family,CYP450 Superfamily,Cytochrome P-450 Enzymes,Cytochrome P-450 Families,Cytochrome P-450 Monooxygenase,Cytochrome P-450 Oxygenase,Cytochrome P-450 Superfamily,Cytochrome P450,Cytochrome P450 Superfamily,Cytochrome p450 Families,P-450 Enzymes,P450 Enzymes,Cytochrome P 450,Cytochrome P 450 Dependent Monooxygenase,Cytochrome P 450 Enzyme,Cytochrome P 450 Enzyme System,Cytochrome P 450 Enzymes,Cytochrome P 450 Families,Cytochrome P 450 Monooxygenase,Cytochrome P 450 Oxygenase,Cytochrome P 450 Superfamily,Enzyme, Cytochrome P-450,Enzyme, P-450,Enzyme, P450,Enzymes, Cytochrome P-450,Enzymes, P-450,Enzymes, P450,Monooxygenase, Cytochrome P-450,Monooxygenase, Cytochrome P-450-Dependent,P 450 Enzyme,P 450 Enzymes,P-450 Enzyme, Cytochrome,P-450 Enzymes, Cytochrome,Superfamily, CYP450,Superfamily, Cytochrome P-450,Superfamily, Cytochrome P450
D004791	Enzyme Inhibitors	Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction.	Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
D005456	Fluorescent Dyes	Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags.	Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D005561	Formates	Derivatives of formic acids. Included under this heading are a broad variety of acid forms, salts, esters, and amides that are formed with a single carbon carboxy group.	Formic Acids,Acids, Formic
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man