Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice.

BACKGROUND: Forty million adults in the US suffer from anxiety disorders, making these the most common forms of mental illness. Transient receptor potential channel canonical subfamily (TRPC) members 4 and 5 are non-selective cation channels highly expressed in regions of the cortex and amygdala, areas thought to be important in regulating anxiety. Previous work with null mice suggests that inhibition of TRPC4 and TRPC5 may have anxiolytic effects. HC-070 IN VITRO: To assess the potential of TRPC4/5 inhibitors as an avenue for treatment, we invented a highly potent, small molecule antagonist of TRPC4 and TRPC5 which we call HC-070. HC-070 inhibits recombinant TRPC4 and TRPC5 homomultimers in heterologous expression systems with nanomolar potency. It also inhibits TRPC1/5 and TRPC1/4 heteromultimers with similar potency and reduces responses evoked by cholecystokinin tetrapeptide (CCK-4) in the amygdala. The compound is >400-fold selective over a wide range of molecular targets including ion channels, receptors, and kinases. HC-070 IN VIVO: Upon oral dosing in mice, HC-070 achieves exposure levels in the brain and plasma deemed sufficient to test behavioral activity. Treatment with HC-070 attenuates the anxiogenic effect of CCK-4 in the elevated plus maze (EPM). The compound recapitulates the phenotype observed in both null TRPC4 and TRPC5 mice in a standard EPM. Anxiolytic and anti-depressant effects of HC-070 are also observed in pharmacological in vivo tests including marble burying, tail suspension and forced swim. Furthermore, HC-070 ameliorates the increased fear memory induced by chronic social stress. A careful evaluation of the pharmacokinetic-pharmacodynamic relationship reveals that substantial efficacy is observed at unbound brain levels similar to, or even lower than, the 50% inhibitory concentration (IC50) recorded in vitro, increasing confidence that the observed effects are indeed mediated by TRPC4 and/or TRPC5 inhibition. Together, this experimental data set introduces a novel, high quality, small molecule antagonist of TRPC4 and TRPC5 containing channels and supports the targeting of TRPC4 and TRPC5 channels as a new mechanism of action for the treatment of psychiatric symptoms.

LC-MS/MS-based quantification of kynurenine metabolites, tryptophan, monoamines and neopterin in plasma, cerebrospinal fluid and brain.

AIM: The kynurenine (KYN) pathway is implicated in diseases such as cancer, psychiatric, neurodegenerative and autoimmune disorders. Measurement of KYN metabolite levels will help elucidating the involvement of the KYN pathway in the disease pathology and inform drug development. METHODOLOGY: Samples of plasma, cerebrospinal fluid or brain tissue were spiked with deuterated internal standards, processed and analyzed by LC-MS/MS; analytes were chromatographically separated by gradient elution on a C18 reversed phase analytical column without derivatization. CONCLUSION: We established an LC-MS/MS method to measure 11 molecules, namely tryptophan, KYN, 3-OH-KYN, 3-OH-anthranilic acid, quinolinic acid, picolinic acid, kynurenic acid, xanthurenic acid, serotonin, dopamine and neopterin within 5.5 min, with sufficient sensitivity to quantify these molecules in small sample volumes of plasma, cerebrospinal fluid and brain tissue.

Duloxetine and 8-OH-DPAT, but not fluoxetine, reduce depression-like behaviour in an animal model of chronic neuropathic pain.

The current study assessed whether antidepressant and/or antinociceptive drugs, duloxetine, fluoxetine as well as (±)-8-hydroxy-2-[di-n-propylamino] tetralin (8-OH-DPAT), are able to reverse depression-like behaviour in animals with chronic neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve in rats was selected as neuropathic pain model. Mechanical hypersensitivity and depression-like behaviour were evaluated 4 weeks after surgery by "electronic algometer" and forced swimming test (FST), which measured the time of immobility, and active behaviours climbing and swimming. The selective noradrenergic and serotonergic uptake blocker duloxetine (20mg/kg) and the selective 5-HT1A agonist 8-OH-DPAT (0.5mg/kg) significantly reversed both mechanical hypersensitivity and depression-like behaviour in CCI animals. Duloxetine significantly reversed depression-like behaviour in CCI rats by increasing the time of climbing and swimming, while 8-OH-DPAT attenuated depression-like behaviour mainly by increasing the time of swimming. However, the selective serotonergic uptake blocker fluoxetine (20mg/kg) failed to attenuate mechanical hypersensitivity and depression-like behaviour, possibly due to confounding pro-nociceptive actions at 5-HT3 receptors. These data suggest to target noradrenergic and 5-HT1A receptors for treatment of chronic pain and its comorbidity depression.

Mouse chronic social stress increases blood and brain kynurenine pathway activity and fear behaviour: Both effects are reversed by inhibition of indoleamine 2,3-dioxygenase.

Psychosocial stress is a major risk factor for mood and anxiety disorders, in which excessive reactivity to aversive events/stimuli is a major psychopathology. In terms of pathophysiology, immune-inflammation is an important candidate, including high blood and brain levels of metabolites belonging to the kynurenine pathway. Animal models are needed to study causality between psychosocial stress, immune-inflammation and hyper-reactivity to aversive stimuli. The present mouse study investigated effects of psychosocial stress as chronic social defeat (CSD) versus control-handling (CON) on: Pavlovian tone-shock fear conditioning, activation of the kynurenine pathway, and efficacy of a specific inhibitor (IDOInh) of the tryptophan-kynurenine catabolising enzyme indoleamine 2,3-dioxygenase (IDO1), in reversing CSD effects on the kynurenine pathway and fear. CSD led to excessive fear learning and memory, whilst repeated oral escitalopram (antidepressant and anxiolytic) reversed excessive fear memory, indicating predictive validity of the model. CSD led to higher blood levels of TNF-α, IFN-γ, kynurenine (KYN), 3-hydroxykynurenine (3-HK) and kynurenic acid, and higher KYN and 3-HK in amygdala and hippocampus. CSD was without effect on IDO1 gene or protein expression in spleen, ileum and liver, whilst increasing liver TDO2 gene expression. Nonetheless, oral IDOInh reduced blood and brain levels of KYN and 3-HK in CSD mice to CON levels, and we therefore infer that CSD increases IDO1 activity by increasing its post-translational activation. Furthermore, repeated oral IDOInh reversed excessive fear memory in CSD mice to CON levels. IDOInh reversal of CSD-induced hyper-activity in the kynurenine pathway and fear system contributes significantly to the evidence for a causal pathway between psychosocial stress, immune-inflammation and the excessive fearfulness that is a major psychopathology in stress-related neuropsychiatric disorders.

CD40-TNF activation in mice induces extended sickness behavior syndrome co-incident with but not dependent on activation of the kynurenine pathway.

The similarity between sickness behavior syndrome (SBS) in infection and autoimmune disorders and certain symptoms in major depressive disorder (MDD), and the high co-morbidity of autoimmune disorders and MDD, constitutes some of the major evidence for the immune-inflammation hypothesis of MDD. CD40 ligand-CD40 immune-activation is important in host response to infection and in development of autoimmunity. Mice given a single intra-peritoneal injection of CD40 agonist antibody (CD40AB) develop SBS for 2-3days characterized by weight loss and increased sleep, effects that are dependent on the cytokine, tumor necrosis factor (TNF). Here we report that CD40AB also induces behavioral effects that extend beyond acute SBS and co-occur with but are not mediated by kynurenine pathway activation and recovery. CD40AB led to decreased saccharin drinking (days 1-7) and decreased Pavlovian fear conditioning (days 5-6), and was without effect on physical fatigue (day 5). These behavioral effects co-occurred with increased plasma and brain levels of kynurenine and its metabolites (days 1-7/8). Co-injection of TNF blocker etanercept with CD40AB prevented each of SBS, reduced saccharin drinking, and kynurenine pathway activation in plasma and brain. Repeated oral administration of a selective indoleamine 2,3-dioxygenase (IDO) inhibitor blocked activation of the kynurenine pathway but was without effect on SBS and saccharin drinking. This study provides novel evidence that CD40-TNF activation induces deficits in saccharin drinking and Pavlovian fear learning and activates the kynurenine pathway, and that CD40-TNF activation of the kynurenine pathway is not necessary for induction of the acute or extended SBS effects.

Selective CB2 receptor agonists. Part 2: Structure-activity relationship studies and optimization of proline-based compounds.

Through a ligand-based pharmacophore model (S)-proline based compounds were identified as potent cannabinoid receptor 2 (CB2) agonists with high selectivity over the cannabinoid receptor 1 (CB1). Structure-activity relationship investigations for this compound class lead to oxo-proline compounds 21 and 22 which combine an impressive CB1 selectivity profile with good pharmacokinetic properties. In a streptozotocin induced diabetic neuropathy model, 22 demonstrated a dose-dependent reversal of mechanical hyperalgesia.

Selective CB2 receptor agonists. Part 3: the optimization of a piperidine-based series that demonstrated efficacy in an in vivo neuropathic pain model.

A novel class of potent cannabinoid receptor 2 (CB2) agonists based on a (S)-piperidine scaffold was identified using ligand-based pharmacophore models. Optimization of solubility and metabolic stability led to the identification of several potent CB2 agonists (e.g., 30) that displayed selectivity over cannabinoid receptor 1 (CB1) and acceptable drug like properties. In rats, compound 30 demonstrated a favorable pharmacokinetic profile and efficacy in a Streptozotocin-induced diabetic neuropathy model, with full reversal of mechanical hyperalgesia.

Tramadol reduces anxiety-related and depression-associated behaviors presumably induced by pain in the chronic constriction injury model of neuropathic pain in rats.

Depression and anxiety are common comorbidities of neuropathic pain (NP). Pharmacological preclinical studies on NP have given abundant information on the effects of drugs on reflex measures of stimulus-evoked pain. However, few preclinical studies focus on relief of comorbidities evoked by NP. In this study, we investigated the effects of tramadol on nociceptive reflex, depression-associated and anxiety-related behaviors in a NP model in rats. We used chronic constriction injury (CCI) of the sciatic nerve as an animal model of neuropathic pain. We performed electronic von Frey tests (evF) to measure mechanical sensitivity, elevated plus maze tests (EPM) to record anxiety-related behaviors and forced swimming tests (FST) to evaluate depression-associated behaviors. In the evF, CCI rats showed a decrease of 82% of the paw withdrawal threshold (PWT) compared to sham (P<0.001). Tramadol increased the PWT by 336% in CCI rats (P<0.001) and by 16% in sham (P<0.05). On the EPM, CCI rats spent 45% less time than sham on the open arms of the maze (P<0.05). Tramadol increased the time spent on the open arms of CCI rats by 67% (P<0.05) and had no significant effect on sham. During the FST, CCI rats showed 28% longer immobility than sham (P<0.01). Tramadol reduced the immobility time in CCI rats by 22% (P<0.001), while having no effect on sham. Tramadol reversed the changes in mechanical sensitivity as well as anxiety-related and depression-associated behaviors that are caused by injury of the sciatic nerve with only minor effects in the absence of injury. These data suggest that tramadol relieves chronic pain and its indirect consequences and comorbidities, and that this study also is a model for pharmacological studies seeking to investigate the effect of drugs on the major disabling symptoms of NP.

Flibanserin, a drug intended for treatment of hypoactive sexual desire disorder in pre-menopausal women, affects spontaneous motor activity and brain neurochemistry in female rats.

Flibanserin, a 5-HT(1A) receptor agonist and 5-HT(2A) receptor antagonist, is being developed for the treatment of hypoactive sexual desire disorder (HSDD) in pre-menopausal women. Here, we investigated the effects of acute administration of flibanserin (15 and 45 mg/kg, p.o.) and the selective 5-HT(1A) receptor agonist (+)-8-OH-DPAT (1 mg/kg, i.p.) on neurotransmitter levels in brain areas of female rats. Specifically, levels of dopamine (DA) and serotonin (5-HT) and neurotransmitter metabolites were examined in prefrontal cortex (PFC), nucleus accumbens, hypothalamus and brain stem using high performance liquid chromatography coupled to electrochemical detection. In addition, spontaneous motor activity was determined in an automated motor activity system. Flibanserin (45 mg/kg) but not (+)-8-OH-DPAT significantly reduced motor activity, when compared to vehicle controls. Specifically, the DA turnover was significantly increased (279%) in the PFC after flibanserin treatment but less pronounced (159%) after 8-OH-DPAT administration. Serotonin tissue levels were not altered in any of the investigated brain regions upon flibanserin treatment. However, flibanserin produced a significant decrease of the major serotonin metabolite 5-hydroxyindoleacetic acid and 5-HT turnover in the PFC, nucleus accumbens, hypothalamus and brain stem similar to (+)-8-OH-DPAT. In conclusion, the present study indicates that flibanserin is able to modulate dopaminergic and serotonergic activity in distinct brain areas. The observed effects in the PFC on dopaminergic markers are different from those induced by (+)-8-OH-DPAT and may contribute to its therapeutic efficacy in HSDD. The effects of flibanserin on spontaneous motor behaviour are in agreement with its receptor profile and underscore that flibanserin is devoid of any locomotor hyperactivity inducing properties.

Acute and repeated flibanserin administration in female rats modulates monoamines differentially across brain areas: a microdialysis study.

INTRODUCTION: Hypoactive sexual desire disorder (HSDD) is defined as persistent lack of sexual fantasies or desire marked by distress. With a prevalence of 10% it is the most common form of female sexual dysfunction. Recently, the serotonin-1A (5-HT(1A)) receptor agonist and the serotonin-2A (5-HT(2A)) receptor antagonist flibanserin were shown to be safe and efficacious in premenopausal women suffering from HSDD in phase III clinical trials. AIM: The current study aims to assess the effect of flibanserin on neurotransmitters serotonin (5-HT), norepinephrine (NE), dopamine (DA), glutamate, and gamma-aminobutyric acid (GABA) in brain areas associated with sexual behavior. METHODS: Flibanserin was administered to female Wistar rats (280-350 g). Microdialysis probes were stereotactically inserted into the mPFC, NAC, or MPOA, under isoflurane anesthesia. The extracellular levels of neurotransmitters were assessed in freely moving animals, 24 hours after the surgery. MAIN OUTCOME MEASURES: Dialysate levels of DA, NE, and serotonin from medial prefrontal cortex (mPFC), nucleus accumbens (NAC), and hypothalamic medial preoptic area (MPOA) from female rats. RESULTS: Acute flibanserin administration decreased 5-HT and increased NE levels in all tested areas. DA was increased in mPFC and MPOA, but not in the NAC. Basal levels of NE in mPFC and NAC and of DA in mPFC were increased upon repeated flibanserin administration, when compared to vehicle-treated animals. The basal levels of 5-HT were not altered by repeated flibanserin administration, but basal DA and NE levels were increased in the mPFC. Glutamate and GABA levels remained unchanged following either repeated or acute flibanserin treatment. CONCLUSIONS: Systemic administration of flibanserin to female rats differentially affects the monoamine systems of the brain. This may be the mechanistic underpinning of flibanserin's therapeutic efficacy in HSDD, as sexual behavior is controlled by an intricate interplay between stimulatory (catecholaminergic) and inhibitory (serotonergic) systems.

Effect of high trait anxiety on mechanical hypersensitivity in male rats.

Recently, we have shown that neuropathic pain is associated with anxiety-like behaviour in rats with sciatic nerve lesion. An enhanced pain perception has also been described in patients with anxiety disorders. However, there is only limited knowledge about the relationship between anxiety and pain in animals. To investigate whether trait anxiety influences nociception, we measured mechanical hypersensitivity following chronic constriction injury (CCI) in rats selectively bred for high (HAB) or low (LAB) anxiety-like behaviour. Pain sensitivity was assessed before surgery and at day 7, 14, 21, 36 and 57 after CCI by determination of withdrawal thresholds. Additionally, we examined pain-induced anxiety-like behaviour using the elevated plus-maze (EPM). HAB and LAB rats exhibited similar levels of mechanical hypersensitivity 7 days post-injury. However, at day 14 and 21 after surgery, mechanical-induced pain thresholds were significantly decreased in HAB rats (p<0.05) in comparison to LAB rats. From day 21 onward HAB rats displayed a faster return of paw withdrawal threshold to baseline level when compared to LAB rats (p<0.01). In the EPM anxiety-like behaviour was observed following CCI injury in HAB and LAB rats on top of low and high trait anxiety reflected in a reduced number of entries in open arms. These findings indicate that trait anxiety increases mechanical hypersensitivity in CCI rats during the chronic phase of pain, thereby suggesting that affective processes modulate even simple pain-related behaviour. Furthermore, we demonstrated that neuropathic pain in the CCI model increases anxiety-like behaviour even in LAB rats.

Depression-like behaviour in rats with mononeuropathy is reduced by the CB2-selective agonist GW405833.

The current study assessed whether the chronic constriction injury (CCI) model of neuropathic pain causes depression-like behaviour in animals, and if this depression-like behaviour can be reversed by anti-nociceptive and/or antidepressant drugs. CCI of the sciatic nerve in rats was selected as a neuropathic pain model, mechanical hypersensitivity was assessed by punctuate mechanical stimuli, and depression-like behaviour was evaluated in the forced swimming test (FST) measuring the time of immobility, climbing and swimming. The CCI rats displayed a significant mechanical hypersensitivity (sham 27+/-2g, CCI 12+/-2g; P<0.001) and a significant increase in time of immobility (sham 133+/-14s, CCI 201+/-9s; P<0.001). As time of swimming was unchanged, immobility was increased at the expense of climbing behaviour (sham 105+/-17s, CCI 63+/-9s; P<0.05). There was no difference in ambulation between sham and CCI animals. In sham and CCI animals, desipramine (20mg/kg) significantly reduced immobility (sham+vehicle 134+/-19s, sham+desipramine 79+/-13s; P<0.01, CCI+vehicle 195+/-8s, CCI+desipramine 140+/-11s; P<0.05) and increased climbing behaviour (sham+vehicle 118+/-21s, sham+desipramine 182+/-16s; P<0.05, CCI+vehicle 59+/-8s, CCI+desipramine 112+/-14s; P<0.05) with little effect on mechanical hypersensitivity. In contrast in CCI animals the cannabinoid CB2-selective agonist GW405833 (2,3-dichloro-phenyl)-[5-methoxy-2-methyl-3-(2-morpholin-4-yl-ethyl)-indol-1-yl]-methanone) (30 mg/kg) significantly attenuated immobility (CCI+vehicle 191+/-7s, GW405833 145+/-14s; P<0.01) and mechanical hypersensitivity (CCI+vehicle 15+/-1g, CCI+GW405833 24+/-1g; P<0.001). Moreover, differently from desipramine, GW405833 did not change the climbing behaviour. These data suggest that rats subjected to the CCI model of neuropathic pain develop depression-like behaviour, which can be reversed by appropriate anti-nociceptive treatment.

Anxiety-like behaviour in rats with mononeuropathy is reduced by the analgesic drugs morphine and gabapentin.

Anxiety has been described as an important comorbidity in patients suffering from chronic pain. However, in animals the connection between persistent pain and anxiety has hardly been investigated. Therefore, in the current study it was assessed whether chronic pain also causes anxiety-like behaviour in animals and if it can be reversed by analgesic or anxiolytic drugs. Neuropathic pain was induced in rats by partial sciatic nerve ligation (PNL) and chronic constriction injury (CCI). Mechanical hypersensitivity was assessed by the "electronic algometer", while anxiety-like behaviour was measured by using the elevated plus maze. In both neuropathic pain models, rats exhibited mechanical hypersensitivity, whereas a significant increase in anxiety-like behaviour was observed only in CCI rats (time spent in open arms decreased significantly from 99+/-15.8s in sham animals to 33.4+/-7.5s in CCI animals). Furthermore, midazolam (0.5mg/kg; i.p.) significantly reduced anxiety-like behaviour in both sham- and CCI-operated animals without influencing mechanical hypersensitivity. Morphine (3mg/kg; i.p.) and gabapentin (30 mg/kg; i.p.) significantly attenuated anxiety-like behaviour in the CCI lesioned rats: morphine increased entries into open arms from 3.0+/-0.4 to 7.7+/-1.4 (P=0.01), gabapentin elevated this value from 4.7+/-1 to 7.5+/-0.9 (P=0.02). These data suggest that rats subjected to neuropathic pain models develop anxiety-like behaviour which can be reversed by appropriate analgesic treatment. Morphine and gabapentin had no anxiolytic-like effect in sham treated animals, thus their effect on anxiety-like behaviour in the neuropathic pain model is likely indirect via their anti-nociceptive properties.