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  • Based on the role of NMDA receptors in

    2021-09-06

    Based on the role of NMDA receptors in initiation and propagation of epileptic seizures and LY3009120 on potentiation of NMDA function by glycine, it has been hypothesized that glycine will have proconvulsant activity (Foster and Kemp, 1989). Indeed, there is some evidence that human epileptogenic foci, removed surgically in an attempt to stabilise intractable seizures, contain elevated concentrations of glycine (Perry and Hansen, 1981), while reduction in glycine concentration is associated with rigidity (Homma et al., 1979). Furthermore, a selective antagonist of the strychnine-insensitive glycine site, 7-chlorokynorenic LY3009120 (7-CLKYNA), has anticonvulsant efficacy in several models of epilepsy, such as NMDA-induced convulsions (Chiamulera et al., 1990, Koek and Colpaert, 1990, Singh et al., 1991), amygdala seizures (Croucher and Bradford, 1990, Croucher and Bradford, 1991) and audiogenic seizures in DBA/2 mice (Singh et al., 1990). Since this effect is reversed by a selective agonist at the receptor, d-serine (Singh et al., 1990), it has been suggested that endogenous glycine, acting on strychnine-insensitive glycine binding sites, is proconvulsant (Chiamulera et al., 1990, Koek and Colpaert, 1990, Singh et al., 1990). On the other hand, there is large body of evidence indicating that systemically administered glycine can be anticonvulsant in variety of seizures, such as those induced by kynurenine (Lapin, 1981), strychnine (Seiler and Sarhan, 1984, Halsey et al., 1989), 3-mercaptopropionic acid (3-MPA; Seiler and Sarhan, 1984, Toth et al., 1983) or by auditory stimuli in seizure-prone DBA/2 mice (Toth et al., 1983). Furthermore, glycine potentiates the anticonvulsant activity of clinically active anticonvulsants, such as phenobarbital, carbamazepine and phenytoin (Peterson, 1986, Peterson, 1991, Peterson et al., 1990) and drugs acting via GABA receptors (Seiler and Sarhan, 1984, Seiler et al., 1985). For example, glycine potentiates anticonvulsant effects of diazepam in seizures induced by amygdala kindling (Peterson, 1986) or by electroshock (Peterson and Frye, 1987). The affinity of glycine for the strychnine-insensitive binding site varies between 0.1 and 3.0µM, depending on the NMDA receptor NR2 subunit composition (Danysz and Parsons, 1998). Since glycine levels in cerebrospinal fluid are between 7 and 10µM, it was initially thought that under physiological conditions these glycine binding sites are saturated. However, later it was found that in the vicinity of its binding sites, glycine exists at non-saturating concentrations. Reuptake of glycine by high affinity glycine transporter (GlyT) proteins into nerve terminals or glia is the mechanism that reduces the extracellular levels of glycine and terminates the actions of glycine. There are two types of GlyT proteins, GlyT1 and GlyT2. GlyT1 is associated closely with the NMDA receptor and has been shown to play an important role in controlling glycine modulation of NMDA receptor function (Eulenburg et al., 2005, Cubelos et al., 2005). There is a large body of evidence that selective inhibitors of GlyT1 increase glycine concentration in the vicinity of the NMDA receptor and potentiate its function (Sur and Kinney, 2007). For example, a potent and selective inhibitor of GlyT1, N-[3-(4′-Fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine (NFPS) potentiated NMDA receptor-mediated current in vivo shown by patch-clamp recordings from hippocampal pyramidal neurons (Atkinson et al., 2001, Aubrey and Vandenberg, 2001). Also, using in vivo microdialysis, it was shown that NFPS causes significant increases in extracellular glycine levels in prefrontal cortex (Atkinson et al., 2001). Our initial hypothesis, based on their potentiation of NMDA receptor-mediated activity, was that GlyT1 inhibitors would produce proconvulsant effects. However, our preliminary experiments showed a clear lack of proconvulsant properties and the presence of anticonvulsant properties. Hence, a study involving a comprehensive evaluation of anticonvulsant properties of several, chemically distinct GlyT1 inhibitors was undertaken. Here we report the effects of the GlyT1 inhibitors NFPS (Atkinson et al., 2001), SSR 504734 (Depoortere et al., 2005), Lu AA21279 (Smith et al., 2004), Org 25935 (Walker et al., 2001), SB-710622 (Rahman et al., 2007) and GSK931145 (Passchier et al., 2010) (see Fig. 1 for structures), as well as those of the glycine agonist d-serine, in the maximum electroshock threshold (MEST) test in the rat. After initial characterisation of anticonvulsant efficacies of GlyT1 inhibitors, the effects of Org 25935, SB-710622 and GSK931145 were investigated further, including narrower dose ranges of these compounds and a time-course of the effect of GSK931145. Furthermore, we used a separate cohort of satellite animals to investigate concentrations of Org 25935 in blood and in the brain associated with anticonvulsant activity. In each experiment we used the clinically active anticonvulsant drug lamotrigine as a positive control.