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 - 2015/8/1
Y1 - 2015/8/1
N2 - 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 [3H]HOCPCA was used as substrate. HOCPCA inhibited the uptake of the endogenous MCT substrate L-[14C]lactate, and [3H]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 5 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 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 [3H]HOCPCA was used as substrate. HOCPCA inhibited the uptake of the endogenous MCT substrate L-[14C]lactate, and [3H]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 5 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
SN - 0022-3565
VL - 354
SP - 166
EP - 174
JO - Journal of Pharmacology and Experimental Therapeutics
JF - Journal of Pharmacology and Experimental Therapeutics
IS - 2
ER -