ABSTRACT
Among many of the validated methods for testing skin sensitization, direct peptide reactivity assay (DPRA) employs no cells or animals. Although no immune cells are involved in this assay, it reliably predicts the skin sensitization potential of a chemical in chemico. Herein, a new method was developed using endogenous small-molecular-weight compounds, cysteamine and glutathione, rather than synthetic peptides, to differentiate skin sensitizers from non-sensitizers with an accuracy as high as DPRA. The percent depletion of cysteamine and glutathione by test chemicals was measured by an HPLC equipped with a PDA detector. To detect small-size molecules, such as cysteamine and glutathione, a derivatization by 4-(4-dimethylaminophenylazo) benzenesulfonyl chloride (DABS-Cl) was employed prior to the HPLC analysis. Following test method optimization, a cut-off criterion of 7.14% depletion was applied to differentiate skin sensitizers from non-sensitizers in combination of the ratio of 1:25 for cysteamine:test chemical with 1:50 for glutathione:test chemical for the best predictivity among various single or combination conditions. Although overlapping HPLC peaks could not be fully resolved for some test chemicals, high levels of sensitivity (100.0%), specificity (81.8%), and accuracy (93.3%) were obtained for 30 chemicals tested, which were comparable or better than those achieved with DPRA.
Subject(s)
Cysteamine/antagonists & inhibitors , Drug Eruptions/prevention & control , Drugs, Investigational/adverse effects , Glutathione/antagonists & inhibitors , Models, Molecular , Skin/drug effects , Analytic Sample Preparation Methods , Chromatography, High Pressure Liquid , Cysteamine/chemistry , Drug Evaluation, Preclinical/methods , Drugs, Investigational/chemistry , Glutathione/chemistry , Humans , Indicators and Reagents/chemistry , Kinetics , Photometry , ROC Curve , Reproducibility of Results , Small Molecule Libraries , Spectrophotometry, Ultraviolet , p-Dimethylaminoazobenzene/analogs & derivatives , p-Dimethylaminoazobenzene/chemistryABSTRACT
Deoxypodophyllotoxin (DPT) is a bioactive compound of Anthriscus sylvestris (Apiaceae). In the present study, the inhibition of cytochrome P450 (CYP) by DPT was evaluated in human liver microsomes (HLM) and the baculovirus-insect cell-expressed human CYPs using a cocktail probe assay. When a mixture of specific CYP substrates was incubated with DPT in HLM, CYP2C9-catalyzed diclofenac 4-hydroxylation and CYP3A4-catalyzed midazolam 1-hydroxylation were strongly inhibited by DPT, with IC (50) values of 6.3 and 9.2 microM, respectively. The Lineweaver-Burke plots for the inhibition of CYP2C9 and CYP3A4 in HLM and baculovirus-insect cell-expressed human CYPs were consistent with a competitive type of inhibition. From these results, DPT was characterized to be a competitive inhibitor of CYP2C9 and CYP3A4, with K(i) values of 3.5 and 10.8 microM in HLM and 24.9 and 3.5 microM in baculovirus-insect cell-expressed human CYPs, respectively.
Subject(s)
Apiaceae , Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Cytochrome P-450 CYP3A Inhibitors , Podophyllotoxin/analogs & derivatives , Cytochrome P-450 CYP2C9 , Cytochrome P-450 CYP3A , Drug Evaluation, Preclinical , Drugs, Chinese Herbal , Humans , Microsomes, Liver , Podophyllotoxin/pharmacologyABSTRACT
The in vitro metabolism of deoxypodophyllotoxin (DPT), a medicinal herbal product isolated from Anthriscus sylvestris (Apiaceae), was investigated in rats and human microsomes and human recombinant cDNA-expressed CYPs. The incubation of DPT with pooled human microsomes in the presence of NADPH generated five metabolites while its incubation with dexamethasone (Dex)-induced rat liver resulted in seven metabolites (M1-M7) with major metabolic reactions including mono-hydroxylation, O-demethylation and demethylenation. Reasonable structures of the seven metabolites of DPT could be proposed, based on the electrospray tandem mass spectra. Chemical inhibition by ketoconazole and metabolism studies with human recombinant cDNA-expressed CYPs indicated that CYP 3A4 and 2C19 are the major CYP isozymes in the metabolism of DPT in human liver microsomes.