title: In Vitro and In Vivo Evidence for Active Brain Uptake of the GHB Analog HOCPCA by the Monocarboxylate Transporter Subtype 1.
abstract: γ-Hydroxybutyric acid (GHB) is a recreational drug, a clinically prescribed drug in narcolepsy and alcohol dependence, and an endogenous substance that binds to both high- and low-affinity sites in the brain. For studying the molecular mechanisms and the biologic role of the GHB high-affinity binding sites, ligands with high and specific affinity are essential. The conformationally restricted GHB analog HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid) is one such compound. The objective of this study was to investigate the transport of HOCPCA across the blood-brain barrier in vitro and in vivo and to investigate the hypothesis that HOCPCA, like GHB, is a substrate for the monocarboxylate transporters (MCTs). For in vitro uptake studies, MCT1, -2, and -4 were recombinantly expressed in Xenopus laevis oocytes, and the previously reported radioligand [(3)H]HOCPCA was used as substrate. HOCPCA inhibited the uptake of the endogenous MCT substrate l-[(14)C]lactate, and [(3)H]HOCPCA was shown to act as substrate for MCT1 and 2 (Km values in the low- to mid-millimolar range). Introducing single-point amino acid mutations into positions essential for MCT function supported that HOCPCA binds to the endogenous substrate pocket of MCTs. MCT1-mediated brain entry of HOCPCA (10 mg/kg s.c.) was further confirmed in vivo in mice by coadministration of increasing doses of the MCT inhibitor AR-C141990 [(R)-5-(3-hydroxypyrrolidine-1-carbonyl)-1-isobutyl-3-methyl-6-(quinolin-4-ylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione], which inhibited brain penetration of HOCPCA in a dose-dependent manner (ID50 = 4.6 mg/kg). Overall, our study provides evidence that MCT1 is an important brain entry site for HOCPCA and qualifies for future in vivo studies with HOCPCA.
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (L.T., R.P.C., B.F., P.W.); and Discovery Chemistry and DMPK, H. Lundbeck A/S, Ottiliavej, Valby, Denmark (J.K., C.B.).
authors.forename: Louise
authors.initials: L
authors.lastname: Thiesen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (L.T., R.P.C., B.F., P.W.); and Discovery Chemistry and DMPK, H. Lundbeck A/S, Ottiliavej, Valby, Denmark (J.K., C.B.).
authors.forename: Jan
authors.initials: J
authors.lastname: Kehler
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (L.T., R.P.C., B.F., P.W.); and Discovery Chemistry and DMPK, H. Lundbeck A/S, Ottiliavej, Valby, Denmark (J.K., C.B.).
authors.forename: Rasmus P
authors.initials: RP
authors.lastname: Clausen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (L.T., R.P.C., B.F., P.W.); and Discovery Chemistry and DMPK, H. Lundbeck A/S, Ottiliavej, Valby, Denmark (J.K., C.B.).
authors.forename: Bente
authors.initials: B
authors.lastname: Frølund
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (L.T., R.P.C., B.F., P.W.); and Discovery Chemistry and DMPK, H. Lundbeck A/S, Ottiliavej, Valby, Denmark (J.K., C.B.).
authors.forename: Christoffer
authors.initials: C
authors.lastname: Bundgaard
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (L.T., R.P.C., B.F., P.W.); and Discovery Chemistry and DMPK, H. Lundbeck A/S, Ottiliavej, Valby, Denmark (J.K., C.B.) pw@sund.ku.dk.
authors.forename: Petrine
authors.initials: P
authors.lastname: Wellendorph
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D003517:Cyclopentanes / Q000737:chemistry / Q000378:metabolism*; D004285:Dogs; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D051379:Mice; D027501:Monocarboxylic Acid Transporters / Q000378:metabolism*; D051381:Rats; D012978:Sodium Oxybate / Q000031:analogs & derivatives* / Q000378:metabolism*; D027981:Symporters / Q000378:metabolism*; D014982:Xenopus laevis; D000099233:Monocarboxylate Transport Protein 1
chemical_list: D002264:Carboxylic Acids; D003517:Cyclopentanes; D027501:Monocarboxylic Acid Transporters; D012978:Sodium Oxybate; D027981:Symporters; D000099233:Monocarboxylate Transport Protein 1; C586846:3-hydroxycyclopent-1-enecarboxylic acid