Thiesen Louise, Kehler Jan, Clausen Rasmus P, Frølund Bente, Bundgaard Christoffer, Wellendorph Petrine
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.).
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.)
J Pharmacol Exp Ther. 2015 Aug;354(2):166-74. doi: 10.1124/jpet.115.224543. Epub 2015 May 18.
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.
γ-羟基丁酸（GHB）是一种消遣性药物，是发作性睡病和酒精依赖症的临床处方药，也是一种能与大脑中高亲和力和低亲和力位点结合的内源性物质。为了研究GHB高亲和力结合位点的分子机制和生物学作用，具有高亲和力和特异性的配体至关重要。构象受限的GHB类似物HOCPCA（3-羟基环戊-1-烯羧酸）就是这样一种化合物。本研究的目的是在体外和体内研究HOCPCA穿过血脑屏障的转运情况，并研究HOCPCA与GHB一样是单羧酸转运体（MCTs）底物的假说。对于体外摄取研究，MCT1、-2和-4在非洲爪蟾卵母细胞中重组表达，并使用先前报道的放射性配体[³H]HOCPCA作为底物。HOCPCA抑制内源性MCT底物l-[¹⁴C]乳酸的摄取，并且[³H]HOCPCA被证明是MCT1和2的底物（Km值在低至中毫摩尔范围内）。在对MCT功能至关重要的位置引入单点氨基酸突变支持了HOCPCA与MCTs的内源性底物口袋结合。通过共同给予递增剂量的MCT抑制剂AR-C141990[（R）-5-（3-羟基吡咯烷-1-羰基）-1-异丁基-3-甲基-6-（喹啉-4-基甲基）噻吩并[2,3-d]嘧啶-2,4（1H，3H）-二酮]，在小鼠体内进一步证实了MCT1介导的HOCPCA（10mg/kg皮下注射）进入大脑，该抑制剂以剂量依赖性方式抑制HOCPCA的脑渗透（ID50 = 4.6mg/kg）。总体而言，我们的研究提供了证据表明MCT1是HOCPCA进入大脑的重要位点，并有资格用于未来HOCPCA的体内研究。
