TY - JOUR
T1 - In vitro and in vivo evidence for active brain uptake of the GHB analogue HOCPCA by the monocarboxylate transporter subtype 1
AU - Thiesen, Louise
AU - Kehler, Jan
AU - Clausen, Rasmus P
AU - Frølund, Bente
AU - Bundgaard, Christoffer
AU - Wellendorph, Petrine
N1 - The American Society for Pharmacology and Experimental Therapeutics.
PY - 2015
Y1 - 2015
N2 - γ-Hydroxybutyric acid (GHB) is a recreational drug, a clinically prescribed drug in narcolepsy and alcohol dependence, and an endogenous substance which binds to both high and low affinity sites in the brain. For studying the molecular mechanisms and the biological role of the GHB high-affinity binding sites, ligands with high and specific affinity are essential. The conformationally restricted GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) 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 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 co-administration of increasing doses of the MCT inhibitor [(R)-5-(3-hydroxypyrrolidine-1-carbonyl)-1-isobutyl-3-methyl-6-(quinolin-4-ylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione] (AR-C141990) 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.
AB - γ-Hydroxybutyric acid (GHB) is a recreational drug, a clinically prescribed drug in narcolepsy and alcohol dependence, and an endogenous substance which binds to both high and low affinity sites in the brain. For studying the molecular mechanisms and the biological role of the GHB high-affinity binding sites, ligands with high and specific affinity are essential. The conformationally restricted GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) 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 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 co-administration of increasing doses of the MCT inhibitor [(R)-5-(3-hydroxypyrrolidine-1-carbonyl)-1-isobutyl-3-methyl-6-(quinolin-4-ylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione] (AR-C141990) 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.
U2 - 10.1124/jpet.115.224543
DO - 10.1124/jpet.115.224543
M3 - Journal article
C2 - 25986445
VL - 354
SP - 166
EP - 174
JO - Journal of Pharmacology and Experimental Therapeutics
JF - Journal of Pharmacology and Experimental Therapeutics
SN - 0022-3565
IS - 2
ER -