title: An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics.
abstract: Many adverse drug reactions are caused by the cytochrome P450 (CYP) dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-mediated toxicity and to improve safety of drug candidates, we developed two in vitro cell-based assays by combining an activating system (human CYP3A4) with target cells (HepG2 cells): in the first method we incubated microsomes containing cDNA-expressed CYP3A4 together with HepG2 cells; in the second approach HepG2 cells were transiently transfected with CYP3A4. In both assay systems, CYP3A4 catalyzed metabolism was found to be comparable to the high levels reported in hepatocytes. Both assay systems were used to study ten CYP3A4 substrates known for their potential to form metabolites that exhibit higher toxicity than the parent compounds. Several endpoints of toxicity were evaluated, and the measurement of MTT reduction and intracellular ATP levels were selected to assess cell viability. Results demonstrated that both assay systems are capable to metabolize the test compounds leading to increased toxicity, compared to their respective control systems. The co-incubation with the CYP3A4 inhibitor ketoconazole confirmed that the formation of reactive metabolites was CYP3A4 dependent. To further validate the functionality of the two assay systems, they were also used as a "detoxification system" using selected compounds that can be metabolized by CYP3A4 to metabolites less toxic than their parent compounds. These results show that both assay systems can be used to screen for metabolic activation, or de-activation, which may be useful as a rapid and relatively inexpensive in vitro assay for the prediction of CYP3A4 metabolism-mediated toxicity.
authors.affiliation: Department of Pre-Clinical Development, Nerviano Medical Sciences S.r.l., V.le Pasteur, 10, 20014, Nerviano, MI, Italy. luisella.vignati@nervianoms.com
authors.forename: Luisella
authors.initials: L
authors.lastname: Vignati
authors.forename: Elisa
authors.initials: E
authors.lastname: Turlizzi
authors.forename: Sonia
authors.initials: S
authors.lastname: Monaci
authors.forename: Pietro
authors.initials: P
authors.lastname: Grossi
authors.forename: Ruben de
authors.initials: Rd
authors.lastname: Kanter
authors.forename: Mario
authors.initials: M
authors.lastname: Monshouwer
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D015766:Albendazole / Q000378:metabolism / Q000633:toxicity; D000639:Amitriptyline / Q000378:metabolism / Q000633:toxicity; D000818:Animals; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002220:Carbamazepine / Q000378:metabolism / Q000633:toxicity; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002839:Chromans / Q000378:metabolism / Q000633:toxicity; D018920:Coculture Techniques; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003622:Dapsone / Q000378:metabolism / Q000633:toxicity; D004789:Enzyme Activation / Q000187:drug effects; D005485:Flutamide / Q000378:metabolism / Q000633:toxicity; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007538:Isoniazid / Q000378:metabolism / Q000633:toxicity; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009793:Ochratoxins / Q000378:metabolism / Q000633:toxicity; D010879:Piperazines / Q000378:metabolism / Q000633:toxicity; D011802:Quinidine / Q000378:metabolism / Q000633:toxicity; D013379:Substrate Specificity; D013629:Tamoxifen / Q000378:metabolism / Q000633:toxicity; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism / Q000633:toxicity; D045162:Thiazolidinediones / Q000378:metabolism / Q000633:toxicity; D018675:Toxicity Tests / Q000379:methods*; D014229:Triazolam / Q000378:metabolism / Q000633:toxicity; D000077288:Troglitazone; D015262:Xenobiotics / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*
chemical_list: D002839:Chromans; D065607:Cytochrome P-450 Enzyme Inhibitors; D009793:Ochratoxins; D010879:Piperazines; D013778:Tetrazolium Salts; D013844:Thiazoles; D045162:Thiazolidinediones; D015262:Xenobiotics; D013629:Tamoxifen; C025589:ochratoxin A; D000639:Amitriptyline; D014229:Triazolam; D002220:Carbamazepine; D019328:Buthionine Sulfoximine; C092292:ziprasidone; D005485:Flutamide; D000255:Adenosine Triphosphate; D003622:Dapsone; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C022616:thiazolyl blue; D015766:Albendazole; D005978:Glutathione; D000077288:Troglitazone; D011802:Quinidine; D007538:Isoniazid