title: Functional ATP-binding cassette drug efflux transporters in isolated human and rat hepatocytes significantly affect assessment of drug disposition.
abstract: Freshly isolated hepatocytes are considered the gold standard for in vitro studies of hepatic drug disposition. To ensure a reliable supply of cells, cryopreserved human hepatocytes are often used. ABC-superfamily drug efflux transporters are key elements in hepatic drug disposition. These transporters are often considered lost after isolation of hepatocytes. In the present study, the expression and activity of ABC transporters BCRP, BSEP, P-gp, MRP2, MRP3, and MRP4 in human and rat cryopreserved hepatocytes were investigated. In commercially available human cryopreserved hepatocytes, all drug efflux transporters except human BCRP (hBCRP) exhibited similar expression levels as in fresh liver biopsies. Expression levels of hBCRP were 60% lower in cryopreserved human hepatocytes than in liver tissue, which could lead to, at most, a 2.5-fold reduction in hBCRP-mediated efflux. Fresh rat hepatocytes showed significantly lower levels of rat BCRP compared with liver expression levels; expression levels of other ABC transporters were unchanged. ABC transporters in human cryopreserved cells were localized to the plasma membrane. Functional studies could demonstrate P-gp and BCRP activity in both human cryopreserved and fresh rat hepatocytes. Inhibiting P-gp-mediated efflux by elacridar in in vitro experiments significantly decreased fexofenadine efflux from hepatocytes, resulting in an increase in apparent fexofenadine uptake. The results from the present study clearly indicate that ABC transporter-mediated efflux in freshly isolated as well as cryopreserved rat and human hepatocytes should be taken into account in in vitro experiments used for modeling of drug metabolism and disposition.
authors.affiliation: CNS and Pain Innovative Medicines DMPK, AstraZeneca R&D, Södertälje, (P.L., G.E., C.S., J.L., J.J., J.H., L.A.); Cardiovascular and Gastrointestinal Innovative Medicines DMPK, AstraZeneca R&D, Mölndal, (P.L., T.B.A.); Department of Pharmacy, Uppsala University, Uppsala, (P.L.); and Section of Pharmacogenetics, Departments of Physiology and Pharmacology, Karolinska Institutet, Stockholm, (T.B.A.), Sweden.
authors.forename: Patrik
authors.initials: P
authors.lastname: Lundquist
authors.forename: Gunilla
authors.initials: G
authors.lastname: Englund
authors.forename: Cristine
authors.initials: C
authors.lastname: Skogastierna
authors.forename: Johan
authors.initials: J
authors.lastname: Lööf
authors.forename: Jenny
authors.initials: J
authors.lastname: Johansson
authors.forename: Janet
authors.initials: J
authors.lastname: Hoogstraate
authors.forename: Lovisa
authors.initials: L
authors.lastname: Afzelius
authors.forename: Tommy B
authors.initials: TB
authors.lastname: Andersson
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D015925:Cryopreservation; D005260:Female; D005455:Fluorescent Antibody Technique; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008297:Male; D008657:Metabolic Clearance Rate; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013045:Species Specificity; D014018:Tissue Distribution
chemical_list: D018528:ATP-Binding Cassette Transporters; D004364:Pharmaceutical Preparations