The Prodrug 4-Chlorokynurenine Causes Ketamine-Like Antidepressant Effects, but Not Side Effects, by NMDA/GlycineB-Site Inhibition

Abstract

Currently approved antidepressant drug treatment typically takes several weeks to be effective. The noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist ketamine has shown efficacy as a rapid-acting treatment of depression, but its use is associated with significant side effects. We assessed effects following blockade of the glycineB co-agonist site of the NMDA receptor, located on the GluN1 subunit, by the selective full antagonist 7-chloro-kynurenic acid (7-Cl-KYNA), delivered by systemic administration of its brain-penetrant prodrug 4-chlorokynurenine (4-Cl-KYN) in mice. Following administration of 4-Cl-KYN, 7-Cl-KYNA was promptly recovered extracellularly in hippocampal microdialysate of freely moving animals. The behavioral responses of the animals were assessed using measures of ketamine-sensitive antidepressant efficacy (including the 24-hour forced swim test, learned helplessness test, and novelty-suppressed feeding test). In these tests, distinct from fluoxetine, and similar to ketamine, 4-Cl-KYN administration resulted in rapid, dose-dependent and persistent antidepressant-like effects following a single treatment. The antidepressant effects of 4-Cl-KYN were prevented by pretreatment with glycine or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX). 4-Cl-KYN administration was not associated with the rewarding and psychotomimetic effects of ketamine, and did not induce locomotor sensitization or stereotypic behaviors. Our results provide further support for antagonism of the glycineB site for the rapid treatment of treatment-resistant depression without the negative side effects seen with ketamine or other channel-blocking NMDA receptor antagonists.

Fig. 1.

Extracellular levels of 7-chorokynurenic acid studied by microdialysis. (A) Schematic representation of 7-chlorokynurenic acid (7-Cl-KYNA) production from 4-chlorokynurenine (4-Cl-KYN) in the brain. 4-Cl-KYN readily enters the brain from the circulation and is then converted to 7-Cl-KYNA by kynurenine aminotransferase (KAT) in astrocytes. (B) Hippocampal microdialysis in freely-moving mice. The arrow indicates intraperitoneal injection of 7-Cl-KYNA (25 mg/kg) or 4-Cl-KYN (25 mg/kg). Data are the mean ± S.E.M. (n = 6/group). Inset: area under the curve (AUC); unpaired Student’s t-test. ***p < 0.001.

Fig. 2.

Antidepressant effects in the forced-swim, tail-suspension, and novelty-suppressed feeding tests. Mice received intraperitoneal injections of saline (SAL), fluoxetine (FLX), ketamine (KET), or 4-chlorokynurenine (4-Cl-KYN) and were tested in the FST 1-hour post-treatment. (A) Acute administration of FLX (n = 9/group; unpaired Student’s t-test) as well as KET and 4-Cl-KYN (n = 12–16/group; one-way ANOVA followed by Bonferroni’s multiple comparison) significantly reduced immobility in the FST. (B) Glycine pretreatment prevented the antidepressant-like effects of 4-Cl-KYN in the FST (n = 13–15/group; two-way ANOVA followed by Bonferroni’s multiple comparison). (C) Administration of NBQX prevented the antidepressant effects of both KET and 4-Cl-KYN in the FST (n = 11–14/group, two-way ANOVA followed by Bonferroni’s multiple comparison). (D) In the TST, administration of 4-Cl-KYN resulted in antidepressant-like effects 1 hour postinjection (n = 16/group; one-way ANOVA followed by Bonferroni’s multiple comparison). (E) Administration of KET and 4-Cl-KYN significantly reduced latency to feed in the NSF test, (F) without affecting home-cage food consumption (n = 10/group; one-way ANOVA followed by Bonferroni’s multiple comparison). Data are the mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3.

Sustained antidepressant effects in the 24-hour forced-swim test and learned helplessness paradigms. Mice received intraperitoneal injections of saline (SAL), fluoxetine (FLX), ketamine (KET), 7-chlorokynurenic acid (7-Cl-KYNA), or 4-chlorokynurenine (4-Cl-KYN) and were tested in the FST 1 or 24 hours post-treatment, and in the LH paradigm 24 hours and 7 days following treatment. (A) While FLX administration did not induce antidepressant effects, both KET and 4-Cl-KYN significantly reduced immobility time in the FST and decreased the number of escape failures in the LH paradigm at (B) 24 hours or (C) 7 days (n = 10–11/group; one-way ANOVA followed by Bonferroni’s multiple comparison). Injections of FLX or 7-Cl-KYNA did not replicate the effects of 4-Cl-KYN in the LH paradigm 24 hours (D) or 7 days (E) post-treatment (n = 8–9/group; one-way ANOVA followed by Bonferroni’s multiple comparison). Data are the mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

Fig. 4.

Open-field behaviors. (A) Behavioral sensitization protocol. Acute intraperitoneal administration of 4-chlorokynurenine (4-Cl-KYN) did not have effects similar to intraperitoneal ketamine (KET) on (B) acute hyperlocomotion (n = 7–9/group; two-way ANOVA followed by Bonferroni’s multiple comparison) or (C) locomotor sensitization (n = 7–9/group; two-way ANOVA followed by Bonferroni’s multiple comparison). Unlike KET, 4-Cl-KYN administration was not associated with (D) increased stereotypic circling (n = 7–9/group; one-way ANOVA followed by Bonferroni’s multiple comparison) or (E) rearing behavior (n = 7–9/group; two-way ANOVA followed by Bonferroni’s multiple comparison). Data are the mean ± S.E.M. **p < 0.01, ***p < 0.001.

Fig. 5.

Conditioned-place preference and prepulse inhibition. (A) Time spent in the drug-paired compartment during the postconditioning test revealed place preference conditioning to ketamine (KET) but not to 4-chlorokynurenine (4-Cl-KYN) (n = 7–8/group; two-way ANOVA followed by Bonferroni’s multiple comparison). KET caused a (B) dose-dependent disruption of prepulse inhibition (% PPI; two-way ANOVA followed by Bonferroni’s multiple comparison), (C) but did not alter the startle amplitude (one-way ANOVA followed by Bonferroni’s multiple comparison). 4-Cl-KYN did not inhibit (D) % PPI (two-way ANOVA followed by Bonferroni’s multiple comparison) or (E) startle amplitude (one-way ANOVA followed by Bonferroni’s multiple comparison) (n = 11–12/group). Data are the mean ± S.E.M.; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.