Sumie Yoshitomi

Establishment and characterization of the transformants stably-expressing MDR1 derived from various animal species in LLC-PK1

PurposeStable transformants expressing human multidrug resistance 1 (MDR1), monkey MDR1, canine MDR1, rat MDR1a, rat MDR1b, mouse mdr1a, and mouse mdr1b in LLC-PK1 were established to investigate species differences in P-glycoprotein (P-gp, ABCB1) mediated efflux activity.MethodsThe seven cDNAs of MDR1 from five animals were cloned, and their transformants stably expressing the series of MDR1 in LLC-PK1 were established. Transport studies of clarithromycin, daunorubicin, digoxin, erythromycin, etoposide, paclitaxel, propranolol, quinidine, ritonavir, saquinavir, verapamil, and vinblastine were performed by using these cells, and efflux activity was compared among the species.ResultsExcept for propranolol, all compounds showed efflux activity in all transformants, and were judged to be substrates of P-gp. There were slight interspecies and interisoforms differences in the substrate recognition. However, the efflux ratio among the series of the MDR1 stably expressing cells showed good correlation as represented between human and monkey MDR1, and poor correlation as represented between human MDR1 and mouse mdr1a, and human and canine MDR1.ConclusionsResults in the present study indicate that all MDR1 stably expressing cells have efflux activity for various P-gp substrates, and that interspecies differences and similarities of the P-gp substrate efflux activity may exist.

Advantages of Human Hepatocyte-Derived Transformants Expressing a Series of Human Cytochrome P450 Isoforms for Genotoxicity Examination

Metabolites of chemicals can often be ultimate genotoxic species; thus, in vitro routine testing requires the use of rat liver S9. However, there is a question as to whether this represents an appropriate surrogate for human metabolism. We have previously demonstrated the usefulness of HepG2 transformants expressing major human cytochrome P450 (CYP) isoforms to assess the genotoxicity of metabolites. We further assessed the advantages of these transformants from the following three aspects. First, the sensitivity of these transformants was confirmed with micronucleus (MN) induction by 7,12-dimethylbenz[a]anthracene or ifosfamide in transformants expressing the corresponding CYP1A1 or CYP2B6 and CYP2C9, respectively. Second, by using these transformants, beta-endosulfan, a chemical for which the CYP isoforms contributing to its genotoxicity are unknown, was found to induce MN through the CYP3A4-mediated pathway. This result was confirmed by the facts that the decreased CYP3A4 activity using a inhibitor or short interfering RNA (siRNA) repressed MN induction by beta-endosulfan and that endosulfan sulfate, one of the metabolites produced by CYP3A4, induced MN in the transformants harboring an empty vector. Third, the interaction between phase I and II drug-metabolizing enzymes was demonstrated by MN induction with inhibitors of uridine diphosphate (UDP)-glucuronosyltransferases in tamoxifen-treated transformants harboring the corresponding CYP3A4 or with inhibitors of glutathione S-transferase in safrole-treated transformants harboring the corresponding CYP2D6, whereas neither tamoxifen nor safrole alone induced MN in any transformant. These advantages provide the benefits of newly established transformants for in vitro genotoxicity testing that reflects comprehensive metabolic pathways including not only human CYP isoforms but also the phase II enzymes.

Marked impact of P-glycoprotein on the absorption of TAK-427 in rats.

The role of P‐glycoprotein (P‐gp, ABCB1) on the absorption process was investigated by drug–drug interaction studies of TAK‐427 with P‐gp inhibitors (erythromycin, ketoconazole or quinidine) in rats and by transport studies using rat multidrug resistance (MDR1) stably expressing cells and rat small intestine mounted in a Ussing‐type chamber. TAK‐427 showed high efflux activity with low permeability in rat MDR1a and MDR1b stably expressing cells and was revealed to be a typical substrate for P‐gps. Although TAK‐427 was mainly absorbed from the small intestine in rats, a large part of the dosed compound remained in the gastrointestinal tract. Orally co‐administered P‐gp inhibitors (50 mg/kg) increased the AUC of TAK‐427 after a 5 mg/kg oral dose 5.4‐ to 18.3‐fold, whereas orally administered P‐gp inhibitors had a minor effect on the increase in the AUC of TAK‐427 (1.3‐ to 2.2‐fold) after a 0.5 mg/kg intravenous dose. Thus, the bioavailability of TAK‐427 after oral administration in rats (7.3%) markedly increased when co‐administered with P‐gp inhibitors (28.6–57.6%). Moreover, the transport of TAK‐427 was predominantly secretory throughout the rat small intestine and was inhibited by P‐gp inhibitors. In conclusion, P‐gp can markedly reduce the absorption of a typical P‐gp substrate by its efflux activity throughout the absorption site. Copyright