Comparative evaluation of two glycine transporter 1 radiotracers [11C]GSK931145 and [18F]MK-6577 in baboons.

Glycine transporter type-1 (GlyT1) has been proposed as a target for drug development for schizophrenia. PET imaging with a GlyT1 specific radiotracer will allow for the measurement of target occupancy of GlyT1 inhibitors, and for in vivo investigation of GlyT1 alterations in schizophrenia. We conducted a comparative evaluation of two GlyT1 radiotracers, [(11) C]GSK931145, and [(18) F]MK-6577, in baboons. Two baboons were imaged with [(11) C]GSK931145 and [(18) F]MK-6577. Blocking studies with GSK931145 (0.3 or 0.2 mg/kg) were conducted to determine the level of tracer specific binding. [(11) C]GSK931145 and [(18) F]MK-6577 were synthesized in good yield and high specific activity. Moderately fast metabolism was observed for both tracers, with ∼ 30% of parent at 30 min post-injection. In the brain, both radiotracers showed good uptake and distribution profiles consistent with regional GlyT1 densities. [(18) F]MK-6577 displayed higher uptake and faster kinetics than [(11) C]GSK931145. Time activity curves were well described by the two-tissue compartment model. Regional volume of distribution (VT ) values were higher for [(18) F]MK-6577 than [(11) C]GSK931145. Pretreatment with GSK931145 reduced tracer uptake to a homogeneous level throughout the brain, indicating in vivo binding specificity and lack of a reference region for both radiotracers. Linear regression analysis of VT estimates between tracers indicated higher specific binding for [(18) F]MK-6577 than [(11) C]GSK931145, consistent with higher regional binding potential (BPND ) values of [(18) F]MK-6577 calculated using VT from the baseline scans and non-displaceable distribution volume (VND ) derived from blocking studies. [(18) F]MK-6577 appears to be a superior radiotracer with higher brain uptake, faster kinetics, and higher specific binding signals than [(11) C]GSK931145.

Regulación de la neurotransmisión glicinérgica en procesos de dolor inflamatorio: una nueva vía de acción de la prostaglandina E2 en médula espinal / Regulation of the glycinergic neurotransmission during inflammatory pain: A new pathway in the action of Prostaglandin E2 in the spinal cord

La acción de la glicina como neurotransmisor inhibidor es finalizada por su recaptación del espacio sináptico a través de dos transportadores específicos, GlyT1 (isoforma glial) y GlyT2 (isoforma neuronal). En este trabajo describimos un mecanismo mediante el cual la unión de la prostaglandina E2 (un importante mediador del dolor inflamatorio) a sus receptores EP3 activa la recaptación de glicina llevada a cabo por GlyT2. Esta activación coincide con una disminución de la ubiquitinación del transportador, modificación post-traduccional necesaria para su correcto tráfico intracelular. Una menor ubiquitinación de GlyT2 produce una acumulación del transportador en la superficie neuronal, lo que explica la activación observada. Por tanto, los resultados de este trabajo sugieren que GlyT2 es una interesante diana terapéutica cuya inhibición podría contribuir a la reducción del dolor inflamatorio (AU)

Glycinergic inhibitory neurotransmission is terminated by reuptake through specific transporters, GlyT1 (glial isoform) and GlyT2 (neuronal isoform). In this work we describe that Prostaglandin E2 (PGE2, an important mediator of inflammatory pain) activates GlyT2-mediated recapture of glycine via interaction with the EP3 receptor. Moreover, in these conditions a diminished ubiquitination of GlyT2 is observed. Ubiquitination is an important modification for the correct trafficking of this transporter. We propose that the reduction of ubiquitination leads to accumulate GlyT2 in the neuronal surface, which could explain the PGE2-mediated activation of GlyT2. Therefore, our results suggest that GlyT2 is an interesting therapeutic target and its inhibition could contribute to reduce inflammatory pain (AU)

El receptor postsináptico de glicina media de forma transitoria y sostenida la inhibición glicinérgica en la retina de los vertebrados / The post-synaptic glycine receptor mediates in a transitory and sustained way the glycinergic inhibition in the vertebrate retina

Introducción. La glicina y el ácido g-aminobutírico son los principales neurotransmisores inhibidores en la retina de los vertebrados. La acción inhibidora de la glicina es mediada por el receptor postsináptico de glicina, que es un canal selectivo al cloruro, constituido por tres subunidades β y dos α (α1 - α4 ) que se antagoniza por el alcaloide estricnina. En la retina se conoce que las cuatro isoformas de la subunidad α se expresan en la capa sináptica interna y que en muy raras ocasiones se localizan en la misma terminal sináptica. Los receptores de glicina formados por las isoformas α1 o α3 poseen cinéticas rápidas, mientras que los receptores α2 o α4 responden tónicamente. El empleo de ratones transgénicos que tienen eliminada (knock-out) o disminuida (knock-down) la expresión de alguno de los genes que codifican para las diferentes isoformas de la subunidad α del receptor de glicina ha permitido estudiar la participación de estas subunidades en la transmisión glicinérgica de la retina de los mamíferos. Objetivo. Describir la participación del receptor de glicina en la neurotransmisión glicinérgica, particularmente en la retina. Desarrollo. En esta revisión se describen los experimentos que han permitido localizar e identificar la participación de los diferentes subtipos del receptor de glicina en circuitos de neurotransmisión específicos en la retina de los vertebrados. Conclusiones. La localización de receptores de glicina constituidos por diferentes isoformas de la subunidad α, en tipos neuronales específicos, indica la presencia de circuitos glicinérgicos que codifican de manera distinta el paso de información en la retina (AU)

Introduction. Glycine and the g-aminobutyric acid are the principal inhibitory neurotransmitters in the vertebrate retina. The inhibitory action of glycine is mediated by the post-synaptic glycine receptor, a chloride-selective channel, constituted by three β and two α subunits (α1 - α4 ), which is antagonized by the alkaloid strychnine. In the retina, it is known that all α isoforms are expressed at the level of the inner synaptic layer with a very low colocalization. The glycine receptor formed by either α1 or α3 shows rapid kinetics, whereas α2 or α4 receptors respond tonically. The use of transgenic mice has allowed the study of the different glycine receptor α subunits in the glycinegic neurotransmission of the mammalian retina. Aim. To describe the participation of the glycine receptor in the inhibitory neurotransmission particularly in the retina. Development. In this review we describe the experiments that have allowed the localization and the involvement of the α subunit isoforms in specific transmission circuits of the vertebrate retina. Conclusions. The localization of the glycine receptor conformed by different isoforms of the α subunit in specific neuronal types, indicate the presence of glycinergic circuits that encode information differently in the retina (AU)

Pharmacokinetics, metabolism and excretion of the glycine antagonist GV150526A in rat and dog.

1. The pharmacokinetics, metabolic fate and excretion of 3-[-2(phenylcarbamoyl) ethenyl-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, in rat and dog following intravenous administration of [C14]-GV150526A were investigated. 2. Studies were also performed in bile duct-cannulated animals to confirm the route of elimination and to obtain more information on metabolite identity. 3. Metabolites in plasma, urine and bile were identified by HPLC-MS/MS and NMR spectroscopy. 4. GV150526A was predominantly excreted in the faeces via the bile, with only trace metabolites of radioactivity in urine (< 5%). Radioactivity in rat bile was predominantly due to metabolites, whereas approximately 50% of the radioactivity in dog bile was due to parent GV150526. 5. The principal metabolites in bile were identified as glucuronide conjugates of the carboxylic acid, whereas in rat urine the main metabolite was a sulphate conjugate of an aromatic oxidation metabolite. Multiple glucuronide peaks were observed and identified as isomeric glucuronides and their anomers arising from acyl migration and muta-rotation.

Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection.

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine.

Neither GABA(A) nor strychnine-sensitive glycine receptors are the sole mediators of MAC for isoflurane.

UNLABELLED: Inhaled anesthetics produce immobility (a cardinal aspect of general anesthesia) by an action on the spinal cord, possibly by potentiating the responses of gamma-amino-n-butyric acid (GABA(A)) and glycine receptors to GABA and glycine. In this study, we antagonized GABA(A) and glycine responses by intrathecal administration of picrotoxin (a noncompetitive GABA(A) antagonist), strychnine (a competitive glycine antagonist), or combinations of these drugs. We measured the capacity of antagonist infusion to increase isoflurane MAC (the minimum alveolar concentration of anesthetic that prevents movement in response to noxious stimuli in 50% of subjects). We found that these potent GABA(A) and glycine receptor antagonists had a ceiling effect, either alone or in combination increasing the MAC of isoflurane by at most 47%. IMPLICATIONS: gamma-amino-n-butyric acid and glycine receptors may in part be responsible for the immobilizing action of isoflurane. They are not, however, the only receptors that contribute to isoflurane-induced immobility (i.e., that determine the MAC of isoflurane).

Pharmacokinetics of a glycine site antagonist (gavestinel) following multiple dosing in patients with acute stroke.

OBJECTIVE: The objective of this study was to characterize the pharmacokinetics of gavestinel in patients with acute stroke. METHODS: Gavestinel was administered as an 800-mg loading dose and followed by either 100-, 200-, or 400-mg maintenance doses given every 12 h for five doses. Blood and urine samples were collected for pharmacokinetic evaluation. The pharmacokinetics of gavestinel were determined using compartmental analysis. RESULTS: The mean clearance (CL) and central (Vc) and steady-state (Vss) volumes of distribution across the dose groups were 0.31-0.40 l x h(-1), 3.3-3.9 l, and 9.8-17 l, respectively. The mean terminal half-life ranged from 29 h to 56 h. Gavestinel was extensively bound to plasma protein (median percentage free <0.01). During gavestinel administration, some patients exhibited elevated levels of bilirubin, which may be the result of shared mechanisms of elimination (glucuronide conjugation and excretion in bile). CONCLUSIONS: This study characterized the pharmacokinetics of gavestinel following multiple doses in acute stroke patients and showed that the pharmacokinetics are similar for increasing maintenance doses. The high protein binding of gavestinel was confirmed in acute stroke patients. A pharmacokinetic interaction between gavestinel and bilirubin may contribute to the increase in bilirubin.

First time in human for GV196771: interspecies scaling applied on dose selection.

The utility of interspecies scaling in early drug development has been extensively debated. The authors discuss the dose selection strategy for a first time into man (FTIM) study for GV196771, a new glycine antagonist, using techniques of interspecies scaling. The FTIM dose selection strategy was based on predicted plasma profiles of GV196771 in humans using allometric scaling and considerations of safety and pharmacological activity in animals. Allometric techniques were first retrospectively applied to data obtained in humans and animals for GV150526, a glycine antagonist with similar pharmacokinetic characteristics to GV196771. GV196771 and GV150526 are extensively protein bound; thus, protein binding differences among species were considered in the scaling. Using the scaled pharmacokinetic parameters, compartmental modeling was performed to prospectively simulate concentration profiles for the oral administration of GV196771. This article will discuss the outcome of the prospective dose selection strategy for GV196771 compared to the actual concentration profiles observed in the FTIM study.

Safety and tolerability of GV150526 (a glycine site antagonist at the N-methyl-D-aspartate receptor) in patients with acute stroke.

BACKGROUND AND PURPOSE: GV150526 is a novel glycine site antagonist at the N-methyl-D-aspartate receptor complex. It is a potent neuroprotective agent in animal models of stroke, including permanent middle cerebral artery occlusion in the rat. Unlike antagonists at the glutamate ligand binding site, GV150526 appears to be free of hemodynamic and central nervous system adverse effects. The purpose of this study was to assess the safety, tolerability, and pharmacokinetics of loading and maintenance infusions of GV150526 in patients with acute stroke. METHODS: This was a randomized, placebo-controlled, parallel-group, ascending-dose study conducted in 2 phases. In part A of the study, loading doses of 50, 100, 200, 400, or 800 mg were administered. In part B, the maximum loading dose from part A was followed by maintenance infusions (5 infusions at 12-hour intervals), aiming to maintain neuroprotective levels. Safety data were collected throughout. The study was not designed to test efficacy, but outcome data (Barthel Index and National Institutes of Health Stroke Scale) were collected. RESULTS: Sixty-six patients were recruited to the study over 11 months; 18 patients received placebo. GV150526 was well tolerated by the 48 patients who received it. There was no excess of central nervous system or hemodynamic adverse events compared with placebo. Minor abnormalities in liver function tests were observed in association with the higher maintenance doses tested. Four of 7 patients receiving the 800-mg loading dose followed by 400 mg BID and 1 of 6 patients who received the 200-mg BID maintenance dose showed a small rise in bilirubin, and 3 patients had increases in transaminases; the mean values at 72 hours remained under twice the upper limit of normal. These changes were asymptomatic and resolved within 10 days. CONCLUSIONS: GV150526 is an emerging neuroprotective agent, with no apparent significant central nervous system or hemodynamic effects. Dose-limiting effects appear to be restricted to mild transient and asymptomatic rises in bilirubin and/or transaminases, primarily observed at high maintenance doses, and there were no findings that should preclude further clinical development.

A.E. Bennett Research Award. Reversal of phencyclidine-induced effects by glycine and glycine transport inhibitors.

BACKGROUND: Phencycline (PCP, "angel dust") and other noncompetitive antagonists of N-methyl-D-aspartate (NMDA)-type glutamatergic neurotransmission induce psychotic effects in humans that closely resemble positive, negative, and cognitive symptoms of schizophrenia. Behavioral effects of PCP in rodents are reversed by glycine (GLY) and other NMDA augmenting agents. In rodents, behavioral effects of PCP are mediated, in part, by secondary dysregulation of subcortical dopaminergic neurotransmission. This study evaluates effects of GLY and GLY transport antagonists on behavioral and neurochemical consequences of PCP administration in rodents. METHODS: Two separate experiments were performed. In the first, effects of GLY on PCP-induced stimulation of dopaminergic neurotransmission in nucleus accumbens were evaluated using in vivo microdialysis in awake animals. In the second, effects of a series of GLY transport antagonists were evaluated for potency in inhibiting PCP-induced hyperactivity. RESULTS: In microdialysis studies, GLY significantly inhibited PCP-induced stimulation of subcortical DA release in a dose-dependent fashion. In behavioral studies, the potency of a series of GLY transport antagonists for inhibiting PCP-induced hyperactivity in vivo correlated significantly with their potency in antagonizing GLY transport in vitro. CONCLUSIONS: These findings suggest, first, that GLY reverses not only the behavioral, but also the neurochemical, effects of PCP in rodents. Second, the findings suggest that GLY transport antagonists may induce similar effects to GLY, and may therefore represent an appropriate site for targeted drug development.

Toxicokinetics of acute strychnine poisoning.

BACKGROUND: Strychnine competes with the inhibitory neurotransmitter glycine producing an excitatory state characterized clinically by hyperreflexia, severe muscle spasms, and convulsions. However, the kinetics after overdose have not been well described. CASE REPORT: A 34 year-old male presented to the emergency department 20 minus after ingesting half of a 250-mL container of 2% strychnine sulfate (2.25 g). The reported lethal dose is 100-120 mg. He was alert and oriented and experiencing muscle spasms. His condition deteriorated prompting sedation, muscle paralysis, and tracheal intubation. He was given activated charcoal 100 g per nasogastric tube. He was admitted to intensive care where he was managed with diazepam, pentobarbital, and pancuronium. Despite mild rhabdomyolysis, he recovered and was extubated on day three. Although receiving prophylactic heparin therapy, a massive fatal pulmonary embolus ensued. Eighteen blood specimens for strychnine analysis were obtained from 20 minutes to 51 hours after ingestion. Serum concentrations were determined with gas chromatography-mass spectroscopy. Disappearance followed a first-order process with a t 1/2 of 16 hours (r, = 0.97). DISCUSSION: Our results confirm the findings of an earlier case report of 19 strychnine levels obtained between 4 and 19 hours which described first-order kinetics with a similar t 1/2 of 10 hours. CONCLUSION: Strychnine disappearance in this overdose was well described by a first-order process with a t 1/2 of 10-16 hours.

Ketamine and strychnine treatment of an infant with nonketotic hyperglycinaemia.

UNLABELLED: Non-ketotic hyperglycinaemia (NKH) is a severe seizure disorder associated with high glycine levels. Glycine is a major inhibitory neurotransmitter in the CNS, but has also modulating effects at one of the glutamate receptors, the N-methyl-D-aspartate-(NMDA) receptor. Based on this knowledge we treated a female newborn suffering from severe NKH with the NMDA receptor blocker ketamine in association with strychnine and magnesium supplementation. This treatment led to cessation of seizures, reappearance of swallowing and sucking and improved the neurological status. Some pharmacokinetic data of strychnine and ketamine in the infant are given. CONCLUSION: Ketamine in combination with strychnine may be beneficial in non-ketotic hyperglycinaemia.