title: In vitro bidirectional permeability studies identify pharmacokinetic limitations of NKCC1 inhibitor bumetanide.
abstract: Recently, it has been suggested that bumetanide, an inhibitor of the Na-K-2Cl co-transporter (NKCC1), may be useful in the treatment of central nervous system (CNS) disorders. However, from a physicochemical perspective, bumetanide may not cross the blood-brain barrier to the extent that is necessary for it to be an effective brain NKCC1 inhibitor in vivo. High plasma-protein binding, potentially high brain-tissue binding and putative efflux transporters including organic anion transporter 3 (OAT3) contribute to the poor pharmacokinetic profile of bumetanide. Bidirectional permeability assays are an in vitro method to determine the impact of plasma-protein/brain tissue binding, as well as efflux transport, on the permeability of a compound. We established and validated a cell line stably overexpressing human OAT3 using lentiviral cloning techniques for use in in vitro bidirectional permeability assays. Using efflux transport studies, we show that bumetanide is a transported substrate of human OAT3, exhibiting a transport ratio of ≥1.5, which is attenuated by OAT3 inhibitors. Bidirectional permeability assays were carried out in the presence and absence of either albumin or brain homogenate to elucidate the effect of plasma-protein/brain tissue binding. These tests confirmed the pharmacokinetic limitations for brain delivery of bumetanide. In this experiment, bumetanide is 53% bound to albumin, 77% bound to brain tissue and accumulates in brain cells. Moreover, we conclusively established that bumetanide is a transported substrate of OAT3. Taken together, these bidirectional permeability studies highlight the potential of efflux transporter inhibition as an augmentation strategy for enhanced delivery of bumetanide to the CNS.
authors.affiliation: Pharmacodelivery group, School of Pharmacy, University College Cork, Cork, Ireland; Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland.
authors.forename: Maria D
authors.initials: MD
authors.lastname: Donovan
authors.affiliation: Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland; Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland.
authors.forename: Harriët
authors.initials: H
authors.lastname: Schellekens
authors.affiliation: Department of Paediatrics and Child Health, University College Cork, Cork, Ireland; Irish Centre for Fetal and Neonatal Translational Research, University College Cork and Cork University Maternity Hospital, Cork, Ireland.
authors.forename: Geraldine B
authors.initials: GB
authors.lastname: Boylan
authors.affiliation: Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland; Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland.
authors.forename: John F
authors.initials: JF
authors.lastname: Cryan
authors.affiliation: Pharmacodelivery group, School of Pharmacy, University College Cork, Cork, Ireland. Electronic address: brendan.griffin@ucc.ie.
authors.forename: Brendan T
authors.initials: BT
authors.lastname: Griffin
mesh_terms: D001812:Blood-Brain Barrier / Q000378:metabolism; D002034:Bumetanide / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D029362:Organic Anion Transporters, Sodium-Independent / Q000235:genetics / Q000378:metabolism; D010539:Permeability; D064506:Solute Carrier Family 12, Member 2 / Q000378:metabolism*
chemical_list: D029362:Organic Anion Transporters, Sodium-Independent; D064506:Solute Carrier Family 12, Member 2; C114338:organic anion transport protein 3; D002034:Bumetanide
keywords: Bidirectional permeability assay; Blood–brain barrier; Bumetanide; Bumetanide (PubChem CID: 2471); Efflux transport; Neonatal seizures; Penicillin G Sodium (PubChem CID: 23668834); Pharmacokinetics; Probenecid (PubChem CID: 4911)