Evaluation of 18F-flutemetamol amyloid PET image analysis parameters on the effect of verubecestat on brain amlyoid load in Alzheimer's disease.

PURPOSE: The BACE inhibitor verubecestat was previously found to reduce amyloid load as assessed by 18F-flutemetamol positron emission tomography (PET) composite cortical standard uptake value ratio (SUVr) in patients with mild-to-moderate Alzheimer's disease (AD) in a substudy of the EPOCH trial. Here, we report on additional analyses relevant to the EPOCH PET data, to help inform on the use of PET for assessing amlyloid load in AD clinical trials. PROCEDURES: The analyses addressed (1) identification of an optimal 18F-flutemetamol reference region, (2) determination of the threshold to characterize the magnitude of the longitudinal change, and (3) the impact of partial volume correction (PVC). Pons and subcortical white matter were evaluated as reference regions. The SUVr cutoffs and final reference region choice were determined using 162 18F-flutemetamol PET scans from the AIBL dataset. 18F-flutemetamol SUVrs were computed at baseline and at Week 78 in EPOCH participants who received verubecestat 12 mg (n = 14), 40 mg (n = 20), or placebo (n = 20). Drug effects on amyloid load were computed using either Meltzer (MZ), or symmetric geometric transfer matrix (SGTM) PVC and compared to uncorrected data. RESULTS: The optimal subcortical white matter and pons SUVr cutoffs were determined to be 0.69 and 0.62, respectively. The effect size to detect longitudinal change was higher for subcortical white matter (1.20) than pons (0.45). Hence, subcortical white matter was used as the reference region for the EPOCH PET substudy. In EPOCH, uncorrected baseline SUVr values correlated strongly with MZ PVC (r2 = 0.94) and SGTM PVC (r2 = 0.92) baseline SUVr values, and PVC did not provide improvement for evaluating treatment effects on amyloid load at Week 78. No change from baseline was observed in the placebo group at Week 78, whereas a 0.02 and a 0.04 decrease in SUVr were observed in the 12 mg and 40 mg arms, with the latter representing a 22% reduction in the amyloid load above the detection threshold. CONCLUSIONS: Treatment-related 18F-flutemetamol longitudinal changes in AD clinical trials can be quantified using a subcortical white matter reference region without PVC. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov NCT01739348.

MK-8719, a Novel and Selective O-GlcNAcase Inhibitor That Reduces the Formation of Pathological Tau and Ameliorates Neurodegeneration in a Mouse Model of Tauopathy.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT: MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

A multinational study distinguishing Alzheimer's and healthy patients using cerebrospinal fluid tau/Aß42 cutoff with concordance to amyloid positron emission tomography imaging.

INTRODUCTION: Changes in cerebrospinal fluid (CSF) tau and amyloid ß (Aß)42 accompany development of Alzheimer's brain pathology. Robust tau and Aß42 immunoassays were developed to establish a tau/Aß42 cutoff distinguishing mild-to-moderate Alzheimer's disease (AD) subjects from healthy elderly control (HC) subjects. METHODS: A CSF tau/Aß42 cutoff criteria was chosen, which distinguished the groups and maximized concordance with amyloid PET. Performance was assessed using an independent validation cohort. RESULTS: A tau/Aß42 = 0.215 cutoff provided 94.8% sensitivity and 77.7% specificity. Concordance with PET visual reads was estimated at 86.9% in a ∼50% PET positive population. In the validation cohort, the cutoff demonstrated 78.4% sensitivity and 84.9% specificity to distinguish the AD and HC populations. DISCUSSION: A tau/Aß42 cutoff with acceptable sensitivity and specificity distinguished HC from mild-to-moderate AD subjects and maximized concordance to brain amyloidosis. The defined cutoff demonstrated that CSF analysis may be useful as a surrogate to imaging assessment of AD pathology.

Visualization of mitotic arrest of cell cycle with bioluminescence imaging in living animals.

PURPOSE: Visualization of the cell cycle in living subjects has long been a big challenge. The present study aimed to noninvasively visualize mitotic arrest of the cell cycle with an optical reporter in living subjects. PROCEDURES: An N-terminal cyclin B1-luciferase fusion construct (cyclin B-Luc) controlled by the cyclin B promoter, as a mitosis reporter, was generated. HeLa or HCT116 cells stably expressing cyclin B-Luc reporter were used to evaluate its cell cycle-dependent regulation and ubiquitination-mediated degradation. We also evaluated its feasibility to monitor the mitotic arrest caused by Taxotere both in vitro and in vivo. RESULTS: We showed that the cyclin B-Luc fusion protein was regulated in a cell cycle-dependent manner and accumulated in the mitotic phase (M phase) in cellular assays. The regulation of cyclin B-Luc reporter was mediated by proteasome ubiquitination. In the present study, in vitro imaging showed that antimitotic reagents like Taxotere upregulated the reporter through cell cycle arrest in the M phase. Noninvasive longitudinal bioluminescence imaging further demonstrated an upregulation of the reporter consistent with mitotic arrest induced in tumor xenograft models. Induction of this reporter was also observed with a kinesin spindle protein inhibitor, which causes cell cycle blockage in the M phase. CONCLUSIONS: Our results demonstrate that the cyclin B-Luc reporter can be used to image whether compounds are capable, in vivo, of causing an M phase arrest and/or altering cyclin B turnover. This reporter can also be potentially used in high-throughput screening efforts aimed at discovering novel molecules that will cause cell cycle arrest at the M phase in cultivated cell lines and animal models.

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo.

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.

Ex vivo imaging of pancreatic beta cells using a radiolabeled GLP-1 receptor agonist.

PURPOSE: The purpose of this study was to evaluate the binding specificity of the radiolabeled glucagon-like peptide 1 receptor (GLP-1R) agonist (Lys4°(DOTA)NH2)Exendin-4 in the pancreas using a combination of ex vivo autoradiography and immunohistochemistry. PROCEDURES: Sprague-Dawley rats were administered [64Cu](Lys4°(DOTA)NH2)Exendin-4 i.v. with or without unlabeled Exendin (9-39) to determine binding specificity. Similar experiments were performed using Zucker diabetic fatty (ZDF) and Zucker lean (ZLC) rats. Animals were euthanized and the pancreas was extracted, immediately frozen, and sectioned. The sections were apposed to phosphor imaging plates, scanned, and immunostained for insulin. RESULTS: Co-registration of the autoradiographic and immunohistochemical images revealed that [64Cu] (Lys4°(DOTA)NH2)Exendin-4 specific binding was restricted to islet cells. This binding was blocked by the co-administration of Exendin(9-39) indicating that the radiotracer uptake is mediated by GLP-1R. Uptake of [64Cu](Lys4°(DOTA)NH2)Exendin-4 was greatly decreased in the pancreas of ZDF rats. CONCLUSIONS: Ex vivo autoradiography results using [64Cu](Lys4°(DOTA)NH2)Exendin-4 suggest that GLP-1R agonists based on Exendin-4 are attractive PET ligands for the in vivo determination of ß-cell mass.

Pharmacological properties of MK-3207, a potent and orally active calcitonin gene-related peptide receptor antagonist.

Calcitonin gene-related peptide (CGRP) has long been hypothesized to play a key role in migraine pathophysiology, and the advent of small-molecule antagonists has clearly demonstrated a clinical link between blocking the CGRP receptor and migraine efficacy. 2-[(8R)-8-(3,5-Difluorophenyl)-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(2R)-2'-oxo-1,1',2',3-tetrahydrospiro[indene-2,3'-pyrrolo[2,3-b]pyridin]-5-yl]acetamide (MK-3207) represents the third CGRP receptor antagonist to display clinical efficacy in migraine trials. Here, we report the pharmacological characterization of MK-3207, a potent and orally bioavailable CGRP receptor antagonist. In vitro, MK-3207 is a potent antagonist of the human and rhesus monkey CGRP receptors (K(i) = 0.024 nM). In common with other CGRP receptor antagonists, MK-3207 displays lower affinity for CGRP receptors from other species, including canine and rodent. As a consequence of species selectivity, the in vivo potency was assessed in a rhesus monkey pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging. MK-3207 produced a concentration-dependent inhibition of dermal vasodilation, with plasma concentrations of 0.8 and 7 nM required to block 50 and 90% of the blood flow increase, respectively. The tritiated analog [3H]MK-3207 was used to study the binding characteristics on the human CGRP receptor. [3H]MK-3207 displayed reversible and saturable binding (K(D) = 0.06 nM), and the off-rate was determined to be 0.012 min(-1), with a t(1/2) value of 59 min. In vitro autoradiography studies on rhesus monkey brain slices identified the highest level of binding in the cerebellum, brainstem, and meninges. Finally, as an index of central nervous system penetrability, the in vivo cerebrospinal fluid/plasma ratio was determined to be 2 to 3% in cisterna magna-ported rhesus monkeys.

Dual in vivo quantification of integrin-targeted and protease-activated agents in cancer using fluorescence molecular tomography (FMT).

PURPOSE: Integrins, especially α(v)ß(3) and α(v)ß(5), are upregulated in tumor cells and activated endothelial cells and as such, serve as cancer biomarkers. We developed a novel near-infrared-labeled optical agent for the in vivo detection and quantification of α(v)ß(3)/α(v)ß(5). PROCEDURES: A small peptidomimetic α(v)ß(3) antagonist was synthesized, coupled to a near-infrared fluorescent (NIRF) dye, and tested for binding specificity using integrin-overexpressing cells, inhibition of vitronectin-mediated cell attachment, binding to tumor and endothelial cells in vitro, and competition studies. Pharmacokinetics, biodistribution, specificity of tumor targeting, and the effect of an antiangiogenic treatment were assessed in vivo. RESULTS: The integrin NIRF agent showed strong selectivity towards α(v)ß(3/)α(v)ß(5) in vitro and predominant tumor distribution in vivo, allowing noninvasive and real-time quantification of integrin signal in tumors. Antiangiogenic treatment significantly inhibited integrin signal in vivo but had no effect on a cathepsin-cleavable NIR agent. Simultaneous imaging revealed different patterns of distribution reflecting the underlying differences in integrin and cathepsin biology during tumor progression. CONCLUSIONS: NIRF-labeled integrin antagonists allow noninvasive molecular fluorescent imaging and quantification of tumors in vivo, improving and providing more refined approaches for cancer detection and treatment monitoring.

Inhibition of glycine transporter 1 attenuates nicotine- but not food-induced cue-potentiated reinstatement for a response previously paired with sucrose.

RATIONALE: Compounds which decrease NMDA receptor functioning, such as PCP and ketamine have abuse liability, whereas co-agonists of the NMDA receptor attenuate some of the behavioral and neurochemical effects of stimulant drugs. Here we examined the effects of a glycine transporter (GlyT1) inhibitor, which elevates glycine and hence NMDA signaling, on the behavioral effects of nicotine. OBJECTIVES: To examine the influence of a novel potent, selective, and brain penetrant GlyT1 inhibitor, compound 5 {(2-chloro-N-[1-(ethylsulfonyl)-4-isobutylpiperidin-4-yl]methyl)}-4-(trifluoromethyl)benzamide; human IC(50)=22 nM; rat=30 nM), on nicotine-induced potentiation of progressive ratio responding for a food reward and nicotine- and food-induced cue-potentiated reinstatement for a response previously paired with sucrose. RESULTS: Compound 5 (33 mg/kg; p.o.; achieving approximately 62% GlyT1 blockade) significantly attenuated nicotine-, but not food-induced cue-potentiated reinstatement for a response previously paired with sucrose whereas a lower dose (11 mg/kg, which achieved approximately 34% GlyT1 blockade) did not. The effect of the higher dose was similar to that observed for mecamylamine (1mg/kg i.p.), a non-selective nicotinic receptor antagonist. CONCLUSIONS: These results suggest that compound 5 influences the ability of nicotine to promote reinstatement in the presence of a cue embedded with incentive motivation. Given the hypothesized contribution of reinstatement and conditioned stimuli to drug abuse and relapse, these findings suggest that GlyT1 inhibitors could have utility for treating nicotine addiction.

Inhibition of tumor growth progression by antiandrogens and mTOR inhibitor in a Pten-deficient mouse model of prostate cancer.

Androgen receptors have been shown to play a critical role in prostate cancer. We used ultrasound imaging techniques to track tumor response to antiandrogen and rapamycin treatment in a prostate-specific Pten-deleted mouse model of cancer. Depletion of androgens by either surgical or chemical castration significantly inhibited tumor growth progression without altering the activation of Akt and mammalian target of rapamycin (mTOR). We also showed for the first time that targeting mTOR along with antiandrogen treatment exhibited additive antitumor effects in vivo when compared with single agents. Our preclinical data suggest that combination of antiandrogens with mTOR inhibitors might be more effective in treating androgen-dependent prostate cancer patients.

A quantitative volumetric micro-computed tomography method to analyze lung tumors in genetically engineered mouse models.

Two genetically engineered, conditional mouse models of lung tumor formation, K-ras(LSL-G12D) and K-ras(LSL-G12D)/p53(LSL-R270H), are commonly used to model human lung cancer. Developed by Tyler Jacks and colleagues, these models have been invaluable to study in vivo lung cancer initiation and progression in a genetically and physiologically relevant context. However, heterogeneity, multiplicity and complexity of tumor formation in these models make it challenging to monitor tumor growth in vivo and have limited the application of these models in oncology drug discovery. Here, we describe a novel analytical method to quantitatively measure total lung tumor burden in live animals using micro-computed tomography imaging. Applying this methodology, we studied the kinetics of tumor development and response to targeted therapy in vivo in K-ras and K-ras/p53 mice. Consistent with previous reports, lung tumors in both models developed in a time- and dose (Cre recombinase)-dependent manner. Furthermore, the compound K-ras(LSL-G12D)/p53(LSL-R270H) mice developed tumors faster and more robustly than mice harboring a single K-ras(LSL-G12D) oncogene, as expected. Erlotinib, a small molecule inhibitor of the epidermal growth factor receptor, significantly inhibited tumor growth in K-ras(LSL-G12D)/p53(LSL-R270H) mice. These results demonstrate that this novel imaging technique can be used to monitor both tumor progression and response to treatment and therefore supports a broader application of these genetically engineered mouse models in oncology drug discovery and development.

Bioluminescence imaging of hollow fibers in living animals: its application in monitoring molecular pathways.

We have applied noninvasive optical imaging technology to the in vivo hollow fiber assay, using tumor cell lines in which optical reporters are expressed in response to activation/inhibition of a specific molecular pathway. In vivo noninvasive imaging of molecular pathways in cells within hollow fibers enables a rapid and accurate evaluation of drug targets and provides useful insights to guide novel drug discovery. In this protocol we show, as an example, that a luciferase reporter, driven by the responsive element of nuclear factor NF-kappaB, was induced in cells within hollow fibers implanted in living mice, and a detailed procedure for in vivo bioluminescence imaging of hollow fibers is described. This approach can, in principle, be applied to image any molecular pathways of interest when appropriate reporter cells are generated. Hollow fiber encapsulation and implantation takes 2 d, and in vivo validation of reporter takes 1-2 weeks.

A novel radioligand for glycine transporter 1: characterization and use in autoradiographic and in vivo brain occupancy studies.

INTRODUCTION: 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.

Optical imaging of tumor cells in hollow fibers: evaluation of the antitumor activities of anticancer drugs and target validation.

The in vivo hollow fiber assay, in which semipermeable hollow fibers filled with tumor cells, are implanted into animals, was originally developed to screen for anticancer compounds before assessment in more complex tumor models. To enhance screening and evaluation of anticancer drugs, we have applied optical imaging technology to this assay. To demonstrate that tumor cells inside hollow fibers can communicate with the host mice, we have used fluorescence imaging in vivo and CD31 immunostaining ex vivo to show that angiogenesis occurs around cell-filled hollow fibers by 2 weeks after subcutaneous implantation. Bioluminescence imaging has been used to follow the number of luciferase-expressing tumor cells within implanted hollow fibers; proliferation of those cells was found to be significantly inhibited by docetaxel or irinotecan. We also used bioluminescence imaging of hollow fibers to monitor the nuclear factor kappaB (NFkappaB) pathway in vivo; NFkappaB activation by lipopolysaccharide and tumor necrosis factor-alpha was evaluated in tumor cell lines genetically engineered to express luciferase controlled by an NFkappaB-responsive element. These results demonstrate that optical imaging of hollow fibers containing reporter tumor cells can be used for the rapid and accurate evaluation of antitumor activities of anticancer drugs and for measurement of molecular pathways.

Effects of typical and atypical antipsychotics on human glycine transporters.

Augmentation strategy in the treatment of schizophrenia with the NMDA receptor co-agonist glycine has demonstrated significant improvement in patient symptoms. Interestingly, the therapeutic efficacy of glycine was more consistent among patients that were not co-administered clozapine suggesting that clozapine modulates glycine levels in brain. Since cerebral glycine concentration in the vicinity of NMDA receptors is thought to be controlled by the glia expressed glycine transporter type 1 (GlyT1), the effects of several typical and atypical antipsychotics on glycine uptake were examined in human placenta choriocarcinoma (JAR) cells expressing human GlyT1a. The selectivity of these compounds was investigated by measuring their inhibitory potency at the closely related glycine transporter type 2 (GlyT2). Typical antipsychotics haloperidol, thioridazine and chlorpromazine non-selectively inhibited [(14)C]glycine uptake mediated by GlyT1a and GlyT2 with potency of 9-21 microM. The atypical antipsychotic, clozapine antagonized glycine transport by human GlyT1a with an IC(50) of 100 microM and was weaker at recombinant GlyT2. Its main metabolites, N-desmethylclozapine and clozapine N-oxide were very weak inhibitors at all glycine transporters. Similarly, olanzapine did not potently block GlyT1a- and GlyT2-mediated uptake. Detailed kinetic analysis of hGlyT1a in the presence and absence of haloperidol and clozapine revealed that both drugs were not competitive inhibitors of glycine uptake. Data also indicated that these compounds did not interact with the Na(+) and Cl(-) sites of hGlyT1a. Our results have revealed the existence of an inhibitory interaction between some antipsychotics and hGlyT1a and raise the possibility that these drugs could interact with GlyT1 function at therapeutic doses.

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity.

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.

The glycine transporter type 1 inhibitor N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine potentiates NMDA receptor-mediated responses in vivo and produces an antipsychotic profile in rodent behavior.

Glycine acts as a necessary coagonist for glutamate at the NMDA receptor (NMDAR) complex by binding to the strychnine-insensitive glycine-B binding site on the NR1 subunit. The fact that glycine is normally found in the brain and spinal cord at concentrations that exceed those required to saturate this site has led to the speculation that glycine normally saturates NMDAR-containing synapses in vivo. However, additional lines of evidence suggest that synaptic glycine may be efficiently regulated in synaptic areas by the glycine transporter type 1 (GlyT1). The recent description of a potent and selective GlyT1 inhibitor (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine [NFPS]) provides a tool for evaluation of the hypothesis that inhibition of GlyT1 may increase synaptic glycine and thereby potentiate NMDAR function in vivo. In the present study, we found that (+)-NFPS demonstrated >10-fold greater activity in an in vitro functional glycine reuptake assay relative to the racemic compound. In vivo, (+/-)-NFPS significantly enhanced long-term potentiation in the hippocampal dentate gyrus induced by high-frequency electrical stimulation of the afferent perforant pathway. Furthermore, (+)-NFPS induced a pattern of c-Fos immunoreactivity comparable with the atypical antipsychotic clozapine and enhanced prepulse inhibition of the acoustic startle response in DBA/2J mice, a strain with low basal levels of prepulse inhibition. Collectively, these data suggest that selective inhibition of GlyT1 can enhance NMDAR-sensitive activity in vivo and also support the idea that GlyT1 may represent a novel target for developing therapeutics to treat disorders associated with NMDAR hypofunction.