Joep Titulaer1, Monica Marcus1, Alessia Perrone2, Giesbert Alken2, Torgny H

P.625 Sodium nitroprusside but not molsidomine may be used for antipsychotic augmentation in treatment resistant schizophrenia Joep Titulaer1, Monica Marcus1, Alessia Perrone2, Giesbert Alken2, Torgny H. Svensson1 1: Department of Physiology and Pharmacology, Section of Neuropsychopharmacology, Karolinska Institutet, Stockholm, Sweden. 2: BiRDS Pharma GmbH Neuenhagen Germany [email protected] Background Conclusion Recently, a single injection of the nitric oxide donor sodium nitroprusside Our behavioral results clearly indicate that SNP has a more favorable antipsychotic profile than molsidomine, which (SNP) was found to induce a rapid (within 4 hours) and sustained (several showed no antipsychotic-like effect in the CAR test either given alone or in combination with risperidone. In contrast, weeks) antipsychotic effect in treatment-resistant schizophrenic patients on SNP significantly enhanced the antipsychotic-like effect of a sub-effective dose of risperidone in analogy with Hallak et al a stable antipsychotic medication [1]. Previous preclinical studies have also [1]. The CAR test is an extensively used preclinical model with excellent predictive validity in determining clinical antipsy- shown that a single injection of SNP in rats can produce a prolonged block chotic potential of drugs or drug combinations [6]. Hence, SNP but not molsidomine may be considered as a highly pro- of the psychotomimetic effects of phencyclidine and ketamine that lasted at mising add-on treatment for TRS. least a week [2]). SNP also induced rapid and persisting changes in brain Moreover, molsidomine increased risperidone-induced dopamine release in the NAc, but not the mPFC, whereas SNP, in synaptic function and morphology, including enhancements in excitatory stark contrast, generated increased risperidone-induced dopamine release in the mPFC, but not in the NAc. Importantly, postsynaptic current (EPSC) responses and spine morphology in layer V schizophrenic patients show increased dopamine release in the NAc but a reduced dopamine release in the PFC, which pyramidal cells in medial prefrontal cortex (mPFC)[3]. in turn relates to impaired working memory and negative symptoms [7]. Thus, antipsychotic drugs should preferentially enhance dopamine release in the PFC, but not in the NAc, as produced by the combination of SNP and risperidone but Furthermore, like a low dose of risperidone, the NO donor molsidomine re- not by add-on molsidomine, which showed the opposite effect profile. The increased prefrontal dopamine release versed cognitive impairment induced by MK-801 in rats [4,5]. Molsidomine induced by the combination of SNP and risperidone may improve cognition as indicated by previous preclinical and is a prodrug that is metabolized in the liver to the active substance SIN-1, clinical studies and, furthermore, via enhanced synaptic spine function and morphology in the PFC generate a both rapid which subsequently releases NO. and prolonged antipsychotic and pro cognitive effect. Here we have compared the antipsychotic-like effects of SNP and molsido- The differential effects of SNP vs. molsidomine may be related to the fact that molsidomine is a prodrug that needs to be mine using behavioral techniques, both when given alone and in combina- metabolized in the liver and, therefore, may not generate a rapid increase in brain NO levels, which has been considered tion with a sub-effective dose of risperidone. Correlative biochemical stud- essential for the SNP induced antipsychotic augmentation in TRS (c.f. Introduction). In conclusion, our results delineate ies of regional dopamine release in brain were also performed. SNP but not molsidomine as a highly promising new treatment option for schizophrenia when added to a stable antipsychotic medication to improve efficacy at maintained or even reduced antipsychotic dosage with less side effects. Conditioned avoidance response test results - SNP had no antipyschotic-like effect on its own, but significantly enhances the antipsychotic-like effect of a sub-effective dose of risperidone to a clinically relevant level, which is 70-80% avoidance reduction. - Molsidomine does not enhance the antipsychotic-like effect of risperidone.

The effects on CAR behavior in rats 20 min after i.p. administration of risperidone/vehicle and SNP/saline. The The effects on CAR behavior in rats 40 min after i.p. administration of risperidone/vehicle and molsidomine/ saline. The results results are presented as median (avoidance %) ± semi-interquartile range. Statistics were performed by Friedman two- are presented as median (avoidance %) ± semi-interquartile range. Statistics were performed by Friedman two-way ANOVA way ANOVA followed by Wilcoxon matched-pairs signed-ranks test. n=10, * compared to vehicle + saline, # compared followed by Wilcoxon matched-pairs signed-ranks test. n=11, * compared to vehicle + saline to risperidone + saline. Microdialysis results - Addition of 1 and 1.5 mg/kg SNP significantly enhanced the risperidone-induced dopamine release in the mPFC, but not in the NAc. - Addition of 2 mg/kg molsdiomine significantly enhanced the risperidone-induced dopamine release in the NAc, but not in the mPFC. Dopamine release Dopamine release

The effects of risperidone (0.25 mg/kg i.p.) and SNP (1.0 and 1.5 mg/kg i.p.) alone and incombination on dopamine release The effects of risperidone (0.25 mg/kg i.p.) and molsidomine (2.0 mg/kg i.p.) alone and the combination on dopamine re- in the mPFC and NAc over time. Arrows indicate injections of SNP/saline and risperidone/vehicle, respectively. The dotted lease in the mPFC and NAc over time. Arrows indicate injections of molsidomine/saline and risperidone/vehicle, respec- line represents baseline (100%). Inserted figs show the effects calculated as mean output during 60-150 min (mPFC) and tively. The dotted line represents baseline (100%). Inserted figs show the effects calculated as mean output during 60-150 45-150 min (NAC), i.e. after the second injection. The results are presented as mean ± SEM. *p<0.05, **p<0.01, min (mPFC) and 45-150 min (NAc), i.e. after the second injection. The results are presented as mean ± SEM. *p<0.05, ***p<0.001 vs. control, i.e. vehicle + saline. #p<0.05, &&p<0.01 as indicated in the figures. For mPFC and NAc respectively **p<0.01, ***p<0.001 vs. control, i.e. vehicle + saline. #p<0.05 as indicated in the figure. For mPFC and NAc respectively n=7 and 5 for vehicle + saline, 9 and 8 for risperidone + saline, 6 and 4 for 1 mg/kg SNP + vehicle, 8 and 5 for 1 mg/kg SNP n=7 and 5 for vehicle + saline, 9 and 8 for risperidone + saline, 3 and 4 for 2 mg/kg molsidomine + vehicle, 6 and 5 for 2 + risperidone, 5 and 6 for 1.5 mg/kg SNP + vehicle and 8 and 6 for 1.5 mg/kg SNP + risperidone. mg/kg molsidomine+ risperidone. Materials and Methods Microdialysis Animals Anesthetized rats were implanted with dialysis probes into the mPFC (with an angle of 12 degrees) or the NAc; AP +2.5, +1.6; ML -1.4, -1.4; DV -6.0, Adult male Wistar rats (~300g) were used. They were kept under standard laboratory conditions with food and water available ad libitum. For the -8.2, respectively, relative to bregma and dural surface (in mm) in accordance with the atlas of Paxinos and Watson 1998. Dialysis occurred through a CAR test, the animals were kept on a reversed 12/12h light/dark cycle (lights of at 6:00 AM). Experiments were approved by, and conducted in accor- semipermeable membrane (AN69 Hospal) with an active surface length of 5.5 mm (mPFC) or 2 mm (NAc). Dialysis dance with the local Animal Ethics Committee, Stockholm North and the Karolinska Institutet, Sweden. experiments were conducted approximately 48 h after surgery in freely moving rats. The dialysis probe was perfused with a physiological perfusion solution at a rate of 2.5 μl/min set by a microinfusion pump. Online quantification of monoamines in the dialysate was accomplished by high perfor- Conditioned avoidance response (CAR) mance liquid chromatography (HPLC) coupled to electrochemical detection, with a detection limit of ~0.2 fmol/min. The location of the probe was Rats were trained in a conventional two-way active avoidance (shuttlebox) apparatus. The boxes were divided into two compartments of equal size later verified in slices stained with neutral red. To analyze difference between different treatments, area under curve during 60-150 min (mPFC) and by a partition with one opening. Upon presentation of a 80 dB white noise conditioned stimulus, rats had10 s to move from one compartment of 45-150 min (NAc) was calculated and statistical evaluation was performed by one-way ANOVA followed by planned comparisons of Least Squares the shuttlebox into the other. If the rat remained in the same compartment for more than 10 s, the unconditioned stimulus was presented as an in- means. termittent electric shock (~0.5 mA) from the grid floor until an escape was performed. If it did not respond within 60 s, including the first 10 s, the trial was terminated (escape failure). CAR experiments were analyzed with Friedman’s ANOVA, followed up by Wilcoxon signed ranks test.

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