A novel radioligand for glycine transporter 1: characterization and use in autoradiographic and in vivo brain occupancy studies.
BACKGROUND
In an effort to develop agents to test the NMDA hypofunction hypothesis of schizophrenia, benchmark compounds from a program to discover potent, selective, competitive glycine transporter 1 (GlyT1) inhibitors were radiolabeled in order to further study the detailed pharmacology of these inhibitors and the distribution of GlyT1 in brain. We here report the in vitro characterization of [35S](S)-2-amino-4-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl)benzamide ([35S]ACPPB), a radiotracer developed from a potent and selective non-sarcosine-derived GlyT1 inhibitor, its use in autoradiographic studies to localize (S)-2-amino-6-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl)benzamide (ACPPB) binding sites in rat and rhesus brain and for in vivo occupancy assays of competitive GlyT1 inhibitors.


METHODS
Functional potencies of unlabeled compounds were characterized by [14C]glycine uptake into JAR (human placental choriocarcinoma) cells and synaptosomes. Radioligand binding studies were performed with tissue homogenates. Autoradiographic studies were performed on tissue slices.


RESULTS
ACPPB is a potent (Kd=1.9 nM), selective, GlyT1 inhibitor that, when radiolabeled with [35S], is a well-behaved radioligand with low nondisplaceable binding. Autoradiographic studies of rat and rhesus brain slices with this ligand showed that specific binding sites were plentiful and nonhomogeneously distributed, with high levels of binding in the brainstem, cerebellar white matter, thalamus, cortical white matter and spinal cord gray matter. In vivo studies demonstrate displaceable binding of [35S]ACPPB in rat brain tissues following iv administration of this radioligand.


CONCLUSIONS
This is the first report of detailed anatomical localization of GlyT1 using direct radioligand binding, and the first demonstration that an in vivo occupancy assay is feasible, suggesting that it may also be feasible to develop positron emission tomography tracers for GlyT1.
Zeng
Zhizhen
Z
Imaging, Merck Research Laboratories, West Point, PA 19486, USA. zhizhen_zeng@merck.com
O'Brien
Julie A
JA
Lemaire
Wei
W
O'Malley
Stacey S
SS
Miller
Patricia J
PJ
Zhao
Zhijian
Z
Wallace
Michael A
MA
Raab
Conrad
C
Lindsley
Craig W
CW
Sur
Cyrille
C
Williams
David L
DL
D000818:Animals; D001345:Autoradiography; D001549:Benzamides / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics*; D001667:Binding, Competitive; D001921:Brain / Q000000981:diagnostic imaging*; D002250:Carbon Radioisotopes / Q000493:pharmacokinetics; D045744:Cell Line, Tumor; D002822:Choriocarcinoma / Q000000981:diagnostic imaging; D005260:Female; D005998:Glycine / Q000493:pharmacokinetics; D050487:Glycine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006801:Humans; D008253:Macaca mulatta; D011247:Pregnancy; D011869:Radioligand Assay; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013449:Sulfonamides / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics*; D013462:Sulfur Radioisotopes / Q000493:pharmacokinetics; D013574:Synaptosomes / Q000000981:diagnostic imaging; D014018:Tissue Distribution
C530439:2-amino-4-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl)benzamide; D001549:Benzamides; D002250:Carbon Radioisotopes; D050487:Glycine Plasma Membrane Transport Proteins; D019275:Radiopharmaceuticals; D013449:Sulfonamides; D013462:Sulfur Radioisotopes; C037689:benzamide; D005998:Glycine