Synthesis and evaluation in vitro and in vivo of a 11C-labeled cyclooxygenase-2 (COX-2) inhibitor

The radiosynthesis and radiopharmacological evaluation of 1-[(11)C]methoxy-4-(2-(4-(methanesulfonyl)phenyl)cyclopent-1-enyl)-benzene [(11)C]5 as novel PET radiotracer for imaging of COX-2 expression is described. The radiotracer was prepared via O-methylation reaction with [(11)C]methyl iodide in 19% decay-corrected radiochemical yield at a specific activity of 20-25GBq/mumol at the end-of-synthesis within 35 min. The radiotracer [(11)C]5 was evaluated in vitro using various pro-inflammatory and tumor cell lines showing high functional expression of COX-2 at baseline or after induction. In vivo biodistribution of compound [(11)C]5 was characterized in male Wistar rats. Compound [(11)C]5 was rapidly metabolized in rat plasma, and more pronounced, in mouse plasma. In vivo kinetics and tumor uptake were demonstrated by dynamic small animal PET studies in a mouse tumor xenograft model. Tumor uptake of radioactivity was clearly visible overtime. However, radioactivity uptake in the tumor could not be blocked by the pre-injection of nonradioactive compound 5. Therefore, it can be concluded that radioactivity uptake in the tumor was not COX-2 mediated.     

Recovery of functional peptide transporter PepT1 in budded baculovirus fraction

Transporters play a critical role in many physiological and pathological states and expression of the functional transporter protein is essential in exploring its kinetics and developing effective drugs. We describe here the recovery of functional transporter protein in the baculovirus fraction. We introduced a gene encoding human peptide transporter PepT1, important for the absorption of protein hydrolytic products or peptide-mimetic drugs, into a baculovirus vector. After infection, a large amount of PepT1 appeared in the budded virus fraction compared with Sf9 cells. Uptake of [14C]glycylsarcosine was markedly increased in an acidic condition and showed a clear overshoot in PepT1-expressing virus fraction. The apparent Michaelis constant for [14C]glycylsarcosine was 0.55 +/- 0.06 mM. [14C]Glycylsarcosine uptake was inhibited by di- and tripeptides and orally active beta-lactam antibiotics. These results suggest that functional PepT1 recovers efficiently in a budded virus fraction, and, thus, this expression system will be a useful tool for characterization and screening of peptide-mimetic drugs in drug discovery.     

Transport of the phosphonodipeptide alafosfalin by the H+/peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells.

The interaction of the antibacterial phosphonodipeptide alafosfalin with mammalian H(+)/peptide cotransporters was studied in Caco-2 cells, expressing the low-affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high-affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [(14)C]glycylsarcosine with K(i) values of 0.19 +/- 0.01 mm and 0.07 +/- 0.01 mm for PEPT1 and PEPT2, respectively. Saturation kinetic studies revealed that in both cell types alafosfalin affected only the affinity constant (K(t)) but not the maximal velocity (V(max)) of glycylsarcosine (Gly-Sar) uptake. The inhibition constants and the competitive nature of inhibition were confirmed in Dixon-type experiments. Caco-2 cells and SKPT cells were also cultured on permeable filters: apical uptake and transepithelial apical to basolateral flux of [(14)C]Gly-Sar across Caco-2 cell monolayers were reduced by alafosfalin (3 mm) by 73%. In SKPT cells, uptake of [(14)C]Gly-Sar but not flux was inhibited by 61%. We found no evidence for an inhibition of the basolateral to apical uptake or flux of [(14)C]Gly-Sar by alafosfalin. Alafosfalin (3 mm) did not affect the apical to basolateral [(14)C]mannitol flux. Determined in an Ussing-type experiment with Caco-2 cells cultured in Snapwells trade mark, alafosfalin increased the short-circuit current through Caco-2 cell monolayers. We conclude that alafosfalin interacts with both H(+)/peptide symporters and that alafosfalin is actively transported across the intestinal epithelium in a H(+)-symport, explaining its oral availability. The results also demonstrate that dipeptides where the C-terminal carboxyl group is substituted by a phosphonic function represent high-affinity substrates for mammalian H(+)/peptide cotransporters.     

Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1.

The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N-acetyl-Phe (Ac-Phe), phe-amide (Phe-NH2), N-acetyl-Phe-amide (Ac-Phe-NH2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [3H]-D-Phe-L-Gln. In an equivalent set of experiments, the blocked peptides Ac-Phe-Tyr, Phe-Tyr-NH2 and Ac-Phe-Tyr-NH2 were compared with the parent compound Phe-Tyr. Comparing amino acids and derivatives, only Ac-Phe was an effective inhibitor of peptide uptake (Ki = 1.81+/- 0.37 mM). Ac-Phe-NH2 had a very weak interaction with PepT1 (Ki = 16.8+/-5.64 mM); neither Phe nor Phe-NH2 interacted with PepT1 with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (Ki = 0.10+/-0.04 mM). The blocked C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relatively high affinity (Ki = 0.94+/-0.38 mM). Both Ac-Phe-Tyr and Ac-Phe-Tyr-NH2 interacted weakly with PepT1 (Ki = 8.41+/-0.11 and 9.97+/-4.01 mM, respectively). The results suggest that the N-terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C-terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.     

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT_So, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT_So represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.     

H+-peptide cotransport in the human bile duct epithelium cell line SK-ChA-1

This study describes for the first time the presence of H+-peptide cotransport in cells of the bile duct. Uptake of [glycine-1-14C]glycylsarcosine ([14C]Gly-Sar) in human extrahepatic cholangiocarcinoma SK-ChA-1 cells was stimulated sevenfold by an inwardly directed H+ gradient. Transport was mediated by a low-affinity system with a transport constant (Kt) value of 1.1 mM. Several dipeptides, cefadroxil, and delta-aminolevulinic acid, but not glycine and glutathione, were strong inhibitors of Gly-Sar uptake. SK-ChA-1 cells formed tight, polarized monolayers on permeable membranes. The transepithelial electrical resistance was 856 +/- 29 omega x cm(2). The transepithelial flux of [14C]Gly-Sar in apical-to-basolateral direction exceeded the basolateral-to-apical flux 11-fold. Uptake was 20-fold higher from the apical side. RT-PCR analysis using primer pairs specific for the intestinal-type peptide transporter (PEPT1) or kidney-type (PEPT2) revealed that the transport system expressed in SK-ChA-1 and also in cells of the native rabbit bile duct is PEPT1. Immunohistochemistry localized PEPT1 to the apical membrane of cholangiocytes of mouse extrahepatic biliary duct. We conclude that the cells of the mammalian extrahepatic biliary tract epithelium express the intestinal-type H+-peptide cotransporter in their apical membrane. SK-ChA-1 cells represent a convenient model to study the physiological and clinical aspects of peptide transport in cholangiocytes.     

Targeted disruption of the peptide transporter Pept2 gene in mice defines its physiological role in the kidney

The peptide transporter PEPT2 mediates the cellular uptake of di- and tripeptides and selected drugs by proton-substrate cotransport across the plasma membrane. PEPT2 was functionally identified initially in the apical membrane of renal tubular cells but was later shown to be expressed in other tissues also. To investigate the physiological importance of PEPT2 and for a detailed analysis of the protein expression sites, we generated a Pept2 knockout mouse line in which the Pept2 gene was disrupted by insertion of a beta-galactosidase gene under the control of the PEPT2 promoter. The Pept2(-/-) mice showed no obvious phenotypic abnormalities but also no adaptive upregulation in the expression level of related genes in the kidney. The importance of PEPT2 in the reabsorption of filtered dipeptides was demonstrated in knockout animals by significantly reduced renal accumulation of a fluorophore-labeled and a radiolabeled dipeptide after in vivo administration of the tracers. This indicates that PEPT2 is the main system responsible for tubular reabsorption of peptide-bound amino acids, although this does not lead to major changes in renal excretion of protein or free amino acids.     

Renal catabolism of human glucagon-like peptides 1 and 2

The renal catabolism of [125I]glucagon-like peptide 1 (GLP-1) and [125I]glucagon-like peptide 2 (GLP-2) has been studied both in vivo, by the disappearance of these peptides from the plasma of bilaterally nephrectomized (BNX), ureteral-ligated (BUL) or normal rats, and in vitro, analyzing their catabolism by the isolated, perfused rat kidney. Results from in vivo studies demonstrated that half-disappearance time for both peptides was lower in controls than in BUL rats, and this value in BUL rats was not significantly different from that in BNX rats. In addition, metabolic clearance rate of GLP-1 was higher in control rats than in the other two groups of animals. Urinary clearance rate of both peptides was negligible. In isolated kidney experiments, values for organ clearance of both [125I]GLP-1 and [125I]GLP-2 were similar to those of inulin clearance, which represents the glomerular filtration rate. Urinary clearance of trichloroacetic acid precipitable radioactivity represented less than 1% of total clearance. In conclusion, these results demonstrate a significant role for the kidney in the plasma removal of [125I]GLP-1 and [125I]GLP-2 by a mechanism that involves glomerular filtration and tubular catabolism.     

Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells

Aims

The purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells.

Main methods

Real-time PCR analysis, uptake study of [³H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells.

Key findings

The expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [³H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [³H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [³H]Gly-Sar uptake were observed in type I-like cells. The maximal [³H]Gly-Sar uptake was observed at pH 6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [³H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC₅₀ value being 4.3 μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [³H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0 μM.

Significance

PEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.     

Relation of Na-K-ATPase to acute changes in renal tubular sodium and potassium transport

Renal Na-K-ATPase activity changes adaptively in response to chronic alterations in sodium reabsorption or potassium secretion, but the role of this enzyme in rapid adjustments of renal tubular Na and K transport is not known. To evaluate this question, microsomal Na-K-ATPase specific activity and kinetics were determined in the rat and guinea pig kidney after massive but short-term (3 h) sodium or potassium loading. In other experiments renal sodium handling was evaluated in hydropenic and saline-loaded rats in which enzyme synthesis was prevented by the concurrent administration of actinomycin D or cycloheximide. Saline loading increased net sodium reabsorption in both rats and guinea pigs, but microsomal Na-K-ATPase from the outer medulla (where the reabsorptive increment is greatest) did not change significantly in either species. In vitro [3H]ouabain bidint to guinea pig microsomes and apparent Km for sodium of rat microsomal Na-K-ATPase, both from outer medulla, were also unaltered. Actinomycin D and cycloheximide failed to increase sodium excretion and microsomal Na-K-ATPase remained unchanged. KCL loading resulted in a 10-fold increase in K excretion but again Na-K-ATPase specific activity (in cortex, outer medulla, and papilla), and its apparent Km for potassium were not affected. Taken together these results suggest that rapid adjustments in remal tubular Na or K transport are mediated by mechanisms that do not involve the Na-K-ATPase enzyme system.     

寡肽转运蛋白PepT2及其药物转运

PepT2（peptide transporter 2）是一种高亲和力、低容量的转运载体.它在人体中分布广泛,不仅能转运二、三肽,也可以识别和转运许多仿肽类药物,如β-内酰胺抗生素、血管紧张素转换酶抑制剂、贝他定等.研究表明,PepT2的转运机制不同于氨基酸的吸收机制,其底物的仿肽结构特征影响其转运速率. 本文综述了PepT2与药物相互作用的研究以及PepT2转运药物底物结构特征的研究进展.     

PepT1-mediated fMLP transport induces intestinal inflammation in vivo

In the presentstudy, the effect of H⁺/peptide transporter(PepT1)-mediatedN-formylmethionyl-leucyl-phenylalanine (fMLP)transport on inflammation in vivo in the rat small intestine, whichexpresses high PepT1 levels, and in the rat colon, which does notexpress PepT1, were investigated using myeloperoxidase (MPO) activity and histological analysis. We found that 10 µM fMLP perfusion in thejejunum for 4 h significantly increased MPO activity and alteredthe architecture of jejunal villi. In contrast, 10 µM fMLP perfusionin the colon for 4 h did not induce any inflammation. In addition,we have shown that 50 mM Gly-Gly alone did not affect basal MPOactivity but completely inhibited the MPO activity induced by 10 µMfMLP in the jejunum. Together, these experiments demonstrate that1) the differential expression of PepT1 between the small intestine and the colon plays an important role inepithelial-neutrophil interactions and 2) the inhibition offMLP uptake by jejunal epithelial cells (expressing PepT1) reduces theneutrophil ability to move across the epithelium, in agreement with ourpreviously published in vitro study. This report constitutes the firstin vivo study showing the implication of a membrane transporter (PepT1)in intestinal inflammation.

     

Increased renal tubular sodium reabsorption during exercise-induced hypervolemia in humans.

We tested the hypothesis that renal tubular Na(+) reabsorption increased during the first 24 h of exercise-induced plasma volume expansion. Renal function was assessed 1 day after no-exercise control (C) or intermittent cycle ergometer exercise (Ex, 85% of peak O(2) uptake) for 2 h before and 3 h after saline loading (12.5 ml/kg over 30 min) in seven subjects. Ex reduced renal blood flow (p-aminohippurate clearance) compared with C (0.83 +/- 0.12 vs. 1.49 +/- 0.24 l/min, P < 0.05) but did not influence glomerular filtration rates (97 +/- 10 ml/min, inulin clearance). Fractional tubular reabsorption of Na(+) in the proximal tubules was higher in Ex than in C (P < 0.05). Saline loading decreased fractional tubular reabsorption of Na(+) from 99.1 +/- 0.1 to 98.7 +/- 0.1% (P < 0.05) in C but not in Ex (99.3 +/- 0.1 to 99.4 +/- 0.1%). Saline loading reduced plasma renin activity and plasma arginine vasopressin levels in C and Ex, although the magnitude of decrease was greater in C (P < 0.05). These results indicate that, during the acute phase of exercise-induced plasma volume expansion, increased tubular Na(+) reabsorption is directed primarily to the proximal tubules and is associated with a decrease in renal blood flow. In addition, saline infusion caused a smaller reduction in fluid-regulating hormones in Ex. The attenuated volume-regulatory response acts to preserve distal tubular Na(+) reabsorption during saline infusion 24 h after exercise.     

Synthesis and in vivo evaluation of a new PET radioligand for studying sigma-2 receptors

The cyclohexyl piperazine 1 (1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-1-yl)-propyl]-piperazine) has been shown to be a potent and selective sigma-2 receptor ligand. In the present study, we prepared [(11)C]1 by O-alkylation of the phenolic precursor 2 with [(11)C]CH(3)I. [(11)C]1 was obtained in a 29% non-decay corrected yield and specific activity of 9299 mCi/micromol calculated at end-of-synthesis. The biodistribution of [(11)C]1 in mouse brain demonstrated rapid and homogenous concentration in all brain structures, which included the cortex, thalamus, cerebellum and striatum. Co-administration of unlabelled 1 (1 mg/kg) or the sigma-2 selective ligand SM-21 (1 mg/kg) failed to show any significant inhibition of [(11)C]1 uptake in the mouse brain. The evaluation of this radioligand in vivo in the mouse clearly indicates that it does not possess the required properties for studying sigma-2 receptors in the brain using PET.     

Renal Response to L-Arginine in Diabetic Rats. A Possible Link between Nitric Oxide System and Aquaporin-2

The aim of this study was to evaluate whether L-Arginine (L-Arg) supplementation modifies nitric oxide (NO) system and consequently aquaporin-2 (AQP2) expression in the renal outer medulla of streptozotocin-diabetic rats at an early time point after induction of diabetes. Male Wistar rats were divided in four groups: Control, Diabetic, Diabetic treated with L-Arginine and Control treated with L-Arginine. Nitric oxide synthase (NOS) activity was estimated by [¹⁴C] L-citrulline production in homogenates of the renal outer medulla and by NADPH-diaphorase staining in renal outer medullary tubules. Western blot was used to detect the expression of AQP2 and NOS types I and III; real time PCR was used to quantify AQP2 mRNA. The expression of both NOS isoforms, NOS I and NOS III, was decreased in the renal outer medulla of diabetic rats and L-Arg failed to prevent these decreases. However, L-Arg improved NO production, NADPH-diaphorase activity in collecting ducts and other tubular structures, and NOS activity in renal homogenates from diabetic rats. AQP2 protein and mRNA were decreased in the renal outer medulla of diabetic rats and L-Arg administration prevented these decreases. These results suggest that the decreased NOS activity in collecting ducts of the renal outer medulla may cause, at least in part, the decreased expression of AQP2 in this model of diabetes and constitute additional evidence supporting a role for NO in contributing to renal water reabsorption through the modulation of AQP2 expression in this pathological condition. However, we cannot discard that another pathway different from NOS also exists that links L-Arg to AQP2 expression.     

Atrial natriuretic factor stimulates renal dopamine uptake mediated by natriuretic peptide-type A receptor

To determine the effects of atrial natriuretic factor (ANF) on renal dopamine (DA) metabolism, 3H-DA and 3H-L-DOPA uptake by renal tubular cells was measured in experiments carried out in vitro in Sprague-Dawley rats. The receptor type involved was also analyzed. The results indicate that ANF increased at 30 min, DA uptake in a concentration-response fashion having 10 pM ANF as the threshold concentration. Conversely, the uptake of the precursor L-DOPA was not modified by the peptide. ANF effects were observed in tissues from external and juxtamedullar cortex and inner medulla. On this basis, 100 nM ANF was used to continue the studies in external cortex tissues. DA uptake was characterized as extraneuronal uptake, since 100 microM hydrocortisone blocked ANF-induced increase of DA uptake. Renal DA uptake was decreased at 0 degrees C and in sodium-free medium. The effects of ANF in these conditions were not present, confirming that renal DA uptake is mediated by temperature- and sodium-dependent transporters and that the peptide requires the presence of the ion to exhibit its actions on DA uptake. The biological natriuretic peptide type A receptor (NPR-A) mediates ANF effects, since 100 nM anantin, a specific blocker, reversed ANF-dependent increase of DA uptake. The natriuretic peptide type C receptor (NPR-C) is not involved, since the specific analogous 100 nM 4-23 ANF amide has no effect on renal DA uptake and does not alter the effects of 100 nM ANF. In conclusion, ANF stimulates DA uptake by kidney tubular cells. ANF effects are mediated by NPR-A receptors coupled to guanylate cyclase and cGMP as second messenger. The process involved was characterized as a typical extraneuronal uptake, and characterized as temperature- and sodium-dependent. This mechanism could be related to DA effects on sodium reabsorption and linked to ANF enhanced natriuresis in the kidney. The increment of endogenous DA into tubular cells, as a consequence of increased DA uptake, would permit D1 receptor recruitment and Na+,K+-ATPase activity inhibition, which results in decreased sodium reabsorption and increased natriuresis.     

The Peptide Transporter PepT2 Mediates the Uptake of the Glutathione Precursor CysGly in Astroglia-Rich Primary Cultures

Abstract: The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cultures to utilize cysteinylglycine (CysGly) for glutathione synthesis. After a 24-h starvation period in the absence of glucose and amino acids, CysGly was able to substitute for cysteine plus glycine in the restoration of glutathione. Glutathione restoration from CysGly plus glutamate was only slightly affected by the dipeptides carnosine or serylglycine in a 200-fold excess. Captopril, a substrate of the peptide transporter PepT1, had almost no effect on glutathione restoration. In contrast, with increasing concentrations of alanylalanine or cefadroxil, known substrates of the peptide transporter PepT2, the amount of glutathione restored in the presence of CysGly and glutamate was strongly reduced. Cefadroxil in a 200-fold excess totally prevented the utilization of CysGly for glutathione restoration. The presence of mRNA for PepT2 in astroglia-rich primary cultures was demonstrated by application of RT-PCR. These results demonstrate that PepT2 is expressed in astroglia-rich primary cultures and that this transporter is highly likely to be responsible for the uptake of CysGly in these cultures.     

Evaluation of 18F-labeled acetylcholinesterase substrates as PET radiotracers

Four 18F-labeled acetylcholinesterase (AChE) substrates, (S)-N-[18F]fluoroethyl-2-piperidinemethyl acetate (1), (R)-N-[18F]fluoroethyl-3-pyrrolidinyl acetate (2), N-[18F]fluoroethyl-4-piperidinyl acetate (3), and (R)-N-[18F]fluoroethyl-3-piperidinyl acetate (4), were evaluated for in vivo blood and brain metabolism in mice, brain pharmacokinetics in rats monkeys (M. nemistrina) using PET imaging. All 18F-labeled compounds were compared to N-[11C]methyl-4-piperidinyl propionate (PMP). Compound 1 was completely metabolized within 1 min in mouse blood and brain. This compound had relatively fast regional brain pharmacokinetics and poor discrimination between brain regions with different AChE concentration. Compound 4 showed relatively slower blood metabolism and slower pharmacokinetics than compound 1 but again poor discrimination between brain regions. Both compounds 1 and 4 showed different kinetic profiles than PMP in PET studies. Compound 3 had the slowest blood metabolism and slower pharmacokinetics than PMP. Compound 2 showed highly encouraging characteristics with an in vivo metabolism rate, primate brain uptake, and regional brain pharmacokinetics similar to [11C]PMP. The apparent hydrolysis rate constant k3 in primate cortex was very close to that of [11C]PMP. This compound has potential to be a good PET radiotracer for measuring brain AChE activity. The longer lifetime of 18F would permit longer imaging times and allows preparation of radiotracer batches for multiple patients and delivery of the tracer to other facilities, making the technique more widely available to clinical investigators.     

Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[¹¹C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([¹¹C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC₅₀: 26?nM) for mGluR2 overexpressed in human cells. [¹¹C]1 was synthesized by O-[¹¹C]methylation of the phenol precursor 2 with [¹¹C]methyl iodide. After the reaction, HPLC purification and formulation, [¹¹C]1 of 7.4?±?2.8?GBq (n?=?8) was obtained from [¹¹C]carbon dioxide of 22.5?±?4.8?GBq (n?=?8) with >99% radiochemical purity and 70?±?32?GBq/μmol (n?=?8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [¹¹C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [¹¹C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [¹¹C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.