title: (R)-[(11)C]verapamil is selectively transported by murine and human P-glycoprotein at the blood-brain barrier, and not by MRP1 and BCRP.
abstract: BACKGROUND
  Positron emission tomography (PET) with [(11)C]verapamil, either in racemic form or in form of the (R)-enantiomer, has been used to measure the functional activity of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) at the blood-brain barrier (BBB). There is some evidence in literature that verapamil inhibits two other ABC transporters expressed at the BBB, i.e. multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). However, previous data were obtained with micromolar concentrations of verapamil and do not necessarily reflect the transporter selectivity of verapamil at nanomolar concentrations, which are relevant for PET experiments. The aim of this study was to assess the selectivity of verapamil, in nanomolar concentrations, for Pgp over MRP1 and BCRP.
  
  
  METHODS
  Concentration equilibrium transport assays were performed with [(3)H]verapamil (5 nM) in cell lines expressing murine or human Pgp, human MRP1, and murine Bcrp1 or human BCRP. Paired PET scans were performed with (R)-[(11)C]verapamil in female FVB/N (wild-type), Mrp1((-/-)), Mdr1a/b((-/-)), Bcrp1((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice, before and after Pgp inhibition with 15 mg/kg tariquidar.
  
  
  RESULTS
  In vitro transport experiments exclusively showed directed transport of [(3)H]verapamil in Mdr1a- and MDR1-overexpressing cells which could be inhibited by tariquidar (0.5μM). In PET scans acquired before tariquidar administration, brain-to-blood ratio (Kb,brain) of (R)-[(11)C]verapamil was low in wild-type (1.3 ± 0.1), Mrp1((-/-)) (1.4 ± 0.1) and Bcrp1((-/-)) mice (1.8 ± 0.1) and high in Mdr1a/b((-/-)) (6.9 ± 0.8) and Mdr1a/b((-/-))Bcrp1((-/-)) mice (7.9 ± 0.5). In PET scans after tariquidar administration, Kb,brain was significantly increased in Pgp-expressing mice (wild-type: 5.0 ± 0.3-fold, Mrp1((-/-)): 3.2 ± 0.6-fold, Bcrp1((-/-)): 4.3 ± 0.1-fold) but not in Pgp knockout mice (Mdr1a/b((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-))).
  
  
  CONCLUSIONS
  Our combined in vitro and in vivo data demonstrate that verapamil, in nanomolar concentrations, is selectively transported by Pgp and not by MRP1 and BCRP at the BBB, which supports the use of (R)-[(11)C]verapamil or racemic [(11)C]verapamil as PET tracers of cerebral Pgp function.
authors.affiliation: Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, and Center for Systems Neuroscience, Hannover, Germany; Department of Clinical Pharmacology, Medical University of Vienna, Austria.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Römermann
authors.forename: Thomas
authors.initials: T
authors.lastname: Wanek
authors.forename: Marion
authors.initials: M
authors.lastname: Bankstahl
authors.forename: Jens P
authors.initials: JP
authors.lastname: Bankstahl
authors.forename: Maren
authors.initials: M
authors.lastname: Fedrowitz
authors.forename: Markus
authors.initials: M
authors.lastname: Müller
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Löscher
authors.forename: Claudia
authors.initials: C
authors.lastname: Kuntner
authors.forename: Oliver
authors.initials: O
authors.lastname: Langer
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000000981:diagnostic imaging / Q000378:metabolism*; D002250:Carbon Radioisotopes; D004285:Dogs; D005260:Female; D055786:Gene Knockout Techniques; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D049268:Positron-Emission Tomography; D014700:Verapamil / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D002250:Carbon Radioisotopes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D014700:Verapamil; C116614:ABCG2 protein, human; C421109:multidrug resistance-associated protein 1
keywords: (R)-[(11)C]verapamil; Blood–brain barrier; Breast cancer resistance protein; Multidrug resistance protein 1; P-glycoprotein; Positron emission tomography