Further characterization of the GlyT-1 inhibitor Org25935: anti-alcohol, neurobehavioral, and gene expression effects

Abstract

The glycine transporter-1 inhibitor Org25935 is a promising candidate in a treatment concept for alcohol use disorder targeting the glycine system. Org25935 inhibits ethanol-induced dopamine elevation in brain reward regions and reduces ethanol intake in Wistar rats. This study aimed to further characterise the compound and used ethanol consumption, behavioral measures, and gene expression as parameters to investigate the effects in Wistar rats and, as pharmacogenetic comparison, Alko-Alcohol (AA) rats. Animals were provided limited access to ethanol in a two-bottle free-choice paradigm with daily drug administration. Acute effects of Org25935 were estimated using locomotor activity and neurobehavioral status. Effects on gene expression in Wistar rats were measured with qPCR. The higher but not the lower dose of Org25935 reduced alcohol intake in Wistar rats. Unexpectedly, Org25935 reduced both ethanol and water intake and induced strong CNS-depressive effects in AA-rats (withdrawn from further studies). Neurobehavioral effects by Org25935 differed between the strains (AA-rats towards sedation). Org25935 did not affect gene expression at the mRNA level in the glycine system of Wistar rats. The data indicate a small therapeutic range for the anti-alcohol properties of Org25935, a finding that may guide further evaluations of the clinical utility of GlyT-1 inhibitors. The results point to the importance of pharmacogenetic considerations when developing drugs for alcohol-related medical concerns. Despite the lack of successful clinical outcomes, to date, the heterogeneity of drug action of Org25935 and similar agents and the unmet medical need justify further studies of glycinergic compounds in alcohol use disorder.

Keywords: AA-rats; Alcohol use disorder; Ethanol intake; Glycine; Glycine transporter-1 inhibitors; Wistar-rats.

Fig. 1

Chemical structure of Org25935. For biochemical properties, see “Introduction” section

Fig. 2

Data from 2.5-h limited access alcohol (6% v/v) drinking in Wistar rats. Displayed are effects on a ethanol intake and b water intake, n = 11–12, after treatment with Org25935 (6 mg/kg i.p.) or vehicle 40 min prior to the drinking session for five consecutive days (d 4–8). The last 3 days of seven baseline days are displayed in the graph (d 1–3) and mean ethanol preference during d 1–3 was 49 and 48% for rats receiving Org25935 and controls, respectively. In the same paradigm, effects of treatment with Org25935 (3 mg/kg i.p.) or vehicle for five consecutive days on c ethanol intake and d water intake, n = 10–11 are displayed. The mean ethanol preference of the last three baseline days (d 1–3) was 47 and 43% for rats receiving Org25935 and controls, respectively. Shown are mean ± SEM. For statistics, see the “Results” section

Fig. 3

Data from 2.5-h limited access alcohol (10% v/v) drinking in Alko-Alcohol (AA) rats after vehicle or 2 days of treatment with Org25935 6 mg/kg i.p. (d 4–5) and 6 days (d 6–11) with 3 mg/kg i.p. The drug was administered 40 min prior to the drinking session. The last 3 days of the seven baseline days are displayed in the graph (d 1–3) and mean ethanol preference of d 1–3 was 62 and 61% in AA rats receiving Org25935 and controls, respectively. Shown are the mean ± SEM of effects on a ethanol intake and b water intake in 17–20 animals. For statistics, see the “Results” section

Fig. 4

Effect of Org25935 (6 mg/kg i.p.) or vehicle on locomotor activity. Results are displayed as accumulated counts during the entire 30-min period (a) and as effect over time, where counts every 5 min were recorded for 30 min (b). Shown are the mean ± SEM, n = 10. For statistics, see the “Result”. Effect of Org25935 (6 mg/kg i.p.) or vehicle on rearing activity. Results are displayed as accumulated counts during the entire 30-min period (c) and as effect over time where counts every 5 min were recorded for 30 min (d). Shown are the mean ± SEM, n = 10. For statistics, see the “Results”

Fig. 5

Gene expression in Amygdala of genes involved in glycinergic transmission. Fig a displays Glra1 (GlyR subunit α1), Glra2 (GlyR subunit α2), Glra3 (GlyR subunit α3), Glrb (GlyR subunit β), Slc6a9 (Glycine transporter 1), and Slc6a6 (Taurine transporter). Fig b displays Slc6a1 (GABA transporter 1), Slc6a11 (GABA transporter 3), and Gphn (gephyrin). Treatment with Org25935 for 19 consecutive days did generally not affect expression of glycinergic genes. Exceptions were an up-regulation of Slc6a6 (TauT) (a) and a down-regulation in Slc6a1 (GAT-1) gene expression (b) in Org25935-treated rats. Shown are the mean ± SEM, n = 6. **p < 0.01. Statistics, see “Results”. Negative results from the other brain regions are not presented