title: P-glycoprotein, CYP3A, and plasma carboxylesterase determine brain and blood disposition of the mTOR Inhibitor everolimus (Afinitor) in mice.
abstract: OBJECTIVE
  To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models.
  
  
  METHODS
  We used wild-type, Abcb1a/1b(-/-), Abcg2(-/-), Abcb1a/1b;Abcg2(-/-), and Cyp3a(-/-) mice to study everolimus oral bioavailability and brain accumulation.
  
  
  RESULTS
  Following everolimus administration, brain concentrations and brain-to-liver ratios were substantially increased in Abcb1a/1b(-/-)and Abcb1a/1b;Abcg2(-/-), but not Abcg2(-/-)mice. The fraction of everolimus located in the plasma compartment was highly increased in all knockout strains. In vitro, everolimus was rapidly degraded in wild-type but not knockout plasma. Carboxylesterase 1c (Ces1c), a plasma carboxylesterase gene, was highly upregulated (∼80-fold) in the liver of knockout mice relative to wild-type mice, and plasma Ces1c likely protected everolimus from degradation by binding and stabilizing it. This binding was prevented by preincubation with the carboxylesterase inhibitor BNPP. In vivo knockdown experiments confirmed the involvement of Ces1c in everolimus stabilization. Everolimus also markedly inhibited the hydrolysis of irinotecan and p-nitrophenyl acetate by mouse plasma carboxylesterase and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a(-/-), but not Abcb1a/1b(-/-), Abcg2(-/-), or Abcb1a/1b;Abcg2(-/-)mice, displayed highly (>5-fold) increased oral availability of everolimus.
  
  
  CONCLUSIONS
  Brain accumulation of everolimus was restricted by Abcb1, but not Abcg2, suggesting the use of coadministered ABCB1 inhibitors to improve brain tumor treatment. Cyp3a, but not Abcb1a/1b, restricted everolimus oral availability, underscoring drug-drug interaction risks via CYP3A. Upregulated Ces1c likely mediated the tight binding and stabilization of everolimus, causing higher plasma retention in knockout strains. This Ces upregulation might confound other pharmacologic studies.
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Seng Chuan
authors.initials: SC
authors.lastname: Tang
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Rolf W
authors.initials: RW
authors.lastname: Sparidans
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Ka Lei
authors.initials: KL
authors.lastname: Cheung
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Fukami
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Selvi
authors.initials: S
authors.lastname: Durmus
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Els
authors.initials: E
authors.lastname: Wagenaar
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yokoi
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Bart J M
authors.initials: BJ
authors.lastname: van Vlijmen
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, JapanAuthors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Jos H
authors.initials: JH
authors.lastname: Beijnen
authors.affiliation: Authors' Affiliations: Division of Molecular Oncology, the Netherlands Cancer Institute; Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam; Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht; Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands; and Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan a.schinkel@nki.nl.
authors.forename: Alfred H
authors.initials: AH
authors.lastname: Schinkel
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000502:physiology*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D000818:Animals; D001682:Biological Availability; D001692:Biological Transport; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002265:Carboxylic Ester Hydrolases / Q000097:blood*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D000068338:Everolimus; D006801:Humans; D007166:Immunosuppressive Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D051379:Mice; D018345:Mice, Knockout; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D014018:Tissue Distribution; D008565:Membrane Proteins
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D002265:Carboxylic Ester Hydrolases; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System; D000068338:Everolimus; D007166:Immunosuppressive Agents; D020123:Sirolimus; C501412:Abcg2 protein, mouse; C545375:carboxylesterase 1, mouse; C507084:Abcb1b protein, mouse; C496508:Cyp3a11 protein, mouse; D008565:Membrane Proteins