Functional profile of a novel modulator of serotonin, dopamine, and glutamate neurotransmission

Abstract

Rationale: Schizophrenia remains among the most prevalent neuropsychiatric disorders, and current treatment options are accompanied by unwanted side effects. New treatments that better address core features of the disease with minimal side effects are needed.

Objectives: As a new therapeutic approach, 1-(4-fluoro-phenyl)-4-((6bR, 10aS)-3-methyl-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3',4':4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-butan-1-one (ITI-007) is currently in human clinical trials for the treatment of schizophrenia. Here, we characterize the preclinical functional activity of ITI-007.

Results: ITI-007 is a potent 5-HT2A receptor ligand (K i = 0.5 nM) with strong affinity for dopamine (DA) D2 receptors (K i = 32 nM) and the serotonin transporter (SERT) (K i = 62 nM) but negligible binding to receptors (e.g., H1 histaminergic, 5-HT2C, and muscarinic) associated with cognitive and metabolic side effects of antipsychotic drugs. In vivo it is a 5-HT2A antagonist, blocking (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI)-induced headtwitch in mice with an inhibitory dose 50 (ID50) = 0.09 mg/kg, per oral (p.o.), and has dual properties at D2 receptors, acting as a postsynaptic D2 receptor antagonist to block D-amphetamine hydrochloride (D-AMPH) hyperlocomotion (ID50 = 0.95 mg/kg, p.o.), yet acting as a partial agonist at presynaptic striatal D2 receptors in assays measuring striatal DA neurotransmission. Further, in microdialysis studies, this compound significantly and preferentially enhances mesocortical DA release. At doses relevant for antipsychotic activity in rodents, ITI-007 has no demonstrable cataleptogenic activity. ITI-007 indirectly modulates glutamatergic neurotransmission by increasing phosphorylation of GluN2B-type N-methyl-D-aspartate (NMDA) receptors and preferentially increases phosphorylation of glycogen synthase kinase 3β (GSK-3β) in mesolimbic/mesocortical dopamine systems.

Conclusion: The combination of in vitro and in vivo activities of this compound support its development for the treatment of schizophrenia and other psychiatric and neurologic disorders.

Fig. 1

The structure of 1-(4-fluoro-phenyl)-4-((6bR,10aS)-3-methyl-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-butan-1-one (ITI-007), the tosylate salt of IC200056

Fig. 2

Dose–response curve for inhibition of DOI-induced headtwitch behavior by ITI-007 in mice. The 5-HT_2A agonist, DOI, was used to elicit stereotyped headtwitch behavior in mice. Mice (N = 4/group) were given a specified oral dose of ITI-007 (0.001–1 mg/kg in 0.5 % methylcellulose in water) or vehicle (0.5 % methylcellulose). Thirty minutes later, the mice were injected with vehicle (saline) or with the 5-HT_2A agonist, DOI (2.5 mg/kg, i.p., in saline). Headtwitches were then counted for 5 min, starting 10 min after DOI injection. The mean (±SEM) number of headtwitches recorded in vehicle-treated mice was 13.7 ± 0.67. An ID₅₀ for inhibition of DOI-induced headtwitch was calculated using a four-parameter logistical fit (Excel Fit software, IDBS)

Fig. 3

Dose–response curve for inhibition of AMPH-induced hyperlocomotion by ITI-007 in rats. The psychostimulant drug D-amphetamine was used to elicit hyperlocomotion in rats. Sprague–Dawley rats (N = 4/group) were habituated to locomotor activity chambers (AccuScan, Columbus, OH) for 60 min then given a specified oral dose of ITI-007 (0.3–10 mg/kg, in 0.5 % methylcellulose in water, p.o.) or vehicle (0.5 % methylcellulose in water). Thirty minutes later, the rats were injected with vehicle (saline, i.p.) or with D-amphetamine (D-AMPH) (1 mg/kg, in saline, i.p.) and locomotor activity monitored for an additional 2 h. Total distance traveled was quantitated and averaged for each treatment group. The mean (+SEM) total activity (centimeters traveled) recorded for vehicle-treated rats given D-AMPH was 21,583 ± 4,153. Percent inhibition of each ITI-007 treatment group compared with D-AMPH group was calculated. The activity level in the D-AMPH + vehicle group was used to determine 0 % inhibition. Data were analyzed to determine an ID₅₀ using a four-parameter logistical fit (Excel Fit software, IDBS)

Fig. 4

Comparison of the effect of antipsychotic medications with ITI-007 on the phosphorylation state of striatal TH in vivo. Mice (N = 6/treatment group) were treated acutely with behaviorally efficacious doses of ITI-007 (3 mg/kg, p.o.), clozapine (5 mg/kg, i.p.), aripiprazole (10 mg/kg, p.o.), quetiapine (10 mg/kg, i.p.), olanzapine (1 mg/kg, i.p.), risperidone (3 mg/kg, p.o.), or haloperidol (1 mg/kg, i.p.) then killed 15, 30, or 60 min later. The change in phosphorylation state at serine (S) 40 of tyrosine hydroxylase (TH) was determined in striatal samples by Western blotting using a phosphorylation-state specific S40 antibody. Phosphoprotein levels were normalized for the total level of phosphoprotein in the sample as detected by a pan-TH antibody. Integrated changes in phosphorylation state were calculated, relative to control samples, over the 60-min period after drug treatment for each compound. *p < 0.01; ***p < 0.001 compared with control, ^‡ p < 0.001 compared with ITI-007, clozapine, and aripiprazole; ^† p < 0.05 compared with aripiprazole, ANOVA with Newman–Keuls post hoc test

Fig. 5

Effect of chronic (21 day) daily administration of haloperidol, risperidone, aripiprazole, or ITI-007 on striatal dopamine metabolism in vivo. Mice (N = 6/dosing group) received an oral dose of vehicle (5 % gum arabic in water, 6.7 ml/kg volume, p.o.) or vehicle solution containing either haloperidol (1 or 3 mg/kg), risperidone (1 or 10 mg/kg), aripiprazole (3 or 30 mg/kg), or ITI-007 (1, 3, or 10 mg/kg) once daily for 21 days. Animals were killed by focused cranial microwave irradiation 2 h after the last drug dose. Striatum was collected for analysis of levels of dopamine and dopamine metabolites, DOPAC and HVA, using HPLC-EC. DOPAC/DA ratio, used as an index of dopamine synthetic rate, is shown. *p < 0.05 compared with vehicle alone; ^# p < 0.05 compared with ITI-007 (3); ^{^} p < 0.05 compared with ITI-007 (10)

Fig. 6

Effect of haloperidol and ITI-007 on motor performance as measured by forelimb catalepsy. Forelimb catalepsy was measured in mice using the bar grip test. Animals received a single oral dose of vehicle (Veh) (0.5 % methylcellulose in water, 6.7 ml/kg volume, p.o.) or haloperidol (3 mg/kg) or ITI-007 (1–30 mg/kg) in vehicle solution. Catalepsy was then measured in mice (N = 4/dose/drug) by recording the latency (in seconds) to step both front paws down to the floor of the cage up to a maximum time of 120 s. Catalepsy scores were recorded for each mouse at 120, 180, 240, and 360 min after drug administration. Mean forelimb catalepsy time (in seconds) was calculated across each group and time point. Data were analyzed using ANOVA with Newman–Keuls post hoc test. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01 compared with vehicle treatment. ^‡ p < 0.01, statistically significant difference between haloperidol and ITI-007 treatments

Fig. 7

Effect of acute administration of haloperidol, aripiprazole, or ITI-007 on extracellular dopamine and DOPAC levels in rat striatum and medial prefrontal cortex, as measured by in vivo microdialysis. Adult, male Wistar rats were surgically prepared with microdialysis probes for collection of dialysate from both medial prefrontal cortex (mPFC) and striatum. Following establishment of baseline DA and DOPAC levels, the rats received (at t = 0 min, designated by arrow) an acute dose of vehicle solution (0.5 % methylcellulose in water, 1 ml/kg volume, p.o.; N = 8–9 rats; filled box), haloperidol (0.3 mg/kg in acidified water, 1 ml/kg, s.c.; N = 6–10 rats; filled triangle), aripiprazole (30 mg/kg, p.o.; N = 5–6 rats; open red triangle), or ITI-007 (3 or 10 mg/kg, p.o.; N = 6–10 rats each; open green box and cross, respectively). Striatal and mPFC dialysates were collected every 20 min for 3 h for measurement of dopamine (top panels) and DOPAC (bottom panels). Analysis of variance with Newman–Keuls post hoc tests revealed significant effects, compared with vehicle control, of haloperidol on DA efflux in mPFC (p < 0.01) and striatum (p < 0.001) and DOPAC efflux in mPFC and striatum (p < 0.001). ITI-007 (3 mg/kg) induced a significant increase in DA efflux, compared to vehicle control, in mPFC (p < 0.05). The increase in DA efflux in mPFC induced by ITI-007 (3 mg/kg) was significantly larger than that induced by aripiprazole (30 mg/kg) (p < 0.05)

Fig. 8

Effect of chronic administration of ITI-007 on social behavior following repeated social defeat. Mice (N = 8–12/treatment group) were subjected to exposure to an aggressive resident mouse in the social defeat/resident intruder paradigm. They were then dosed once daily for 28 days, with either vehicle (5 % DMSO/5 % Tween 20/15 % PEG400/75 % water, 6.7 ml/kg volume) or ITI-007 (1 mg/kg, ip.) in vehicle solution. On the day after the last drug or vehicle treatment, mice were placed in the open field in the presence of a resident mouse (enclosed in a smaller cage) and the animal’s behavior recorded by videotape for 150 s. Videotracking software was employed to calculate the time spent by each mouse in specified open-field quadrants, defined schematically in a. The total time (s) spent by each drug treatment group in the interaction zone (b) in proximity to the resident mouse or in the corner zones, at a distance from the resident mouse (c) was expressed as a mean (±SEM). *p < 0.05; **p < 0.01 compared with control vehicle; NS not significantly different from drug-treated control