NMDA glutamate receptor antagonist MK-801 induces proteome changes in adult human brain slices which are partially counteracted by haloperidol and clozapine.

Deciphering the molecular pathways associated with N-methyl-D-aspartate receptor (NMDAr) hypofunction and its interaction with antipsychotics is necessary to advance our understanding of the basis of schizophrenia, as well as our capacity to treat this disease. In this regard, the development of human brain-derived models that are amenable to studying the neurobiology of schizophrenia may contribute to filling the gaps left by the widely employed animal models. Here, we assessed the proteomic changes induced by the NMDA glutamate receptor antagonist MK-801 on human brain slice cultures obtained from adult donors submitted to respective neurosurgery. Initially, we demonstrated that MK-801 diminishes NMDA glutamate receptor signaling in human brain slices in culture. Next, using mass-spectrometry-based proteomics and systems biology in silico analyses, we found that MK-801 led to alterations in proteins related to several pathways previously associated with schizophrenia pathophysiology, including ephrin, opioid, melatonin, sirtuin signaling, interleukin 8, endocannabinoid, and synaptic vesicle cycle. We also evaluated the impact of both typical and atypical antipsychotics on MK-801-induced proteome changes. Interestingly, the atypical antipsychotic clozapine showed a more significant capacity to counteract the protein alterations induced by NMDAr hypofunction than haloperidol. Finally, using our dataset, we identified potential modulators of the MK-801-induced proteome changes, which may be considered promising targets to treat NMDAr hypofunction in schizophrenia. This dataset is publicly available and may be helpful in further studies aimed at evaluating the effects of MK-801 and antipsychotics in the human brain.

Body mass index, waist circumference, insulin, and leptin plasma levels differentiate between clozapine-responsive and clozapine-resistant schizophrenia.

BACKGROUND: Between 25% and 50% of patients suffering from treatment-resistant schizophrenia fail to achieve a clinical response with clozapine. The rapid identification and treatment of this subgroup of patients represents a challenge for healthcare practice. AIMS: To evaluate the relationship between metabolic alterations and the clinical response to clozapine. METHODS: A multicenter, observational, case-control study was performed. Patients diagnosed with schizophrenia treated with clozapine were eligible (minimum dose 400 mg/d for at least 8 weeks and/or clozapine plasma levels ⩾ 350 µg/mL). According to the Positive and Negative Syndrome Scale (PANSS) total score, patients were classified as clozapine-responsive (CR) (<80 points) or clozapine non-responsive (CNR) (⩾80 points). Groups were compared based on demographic and treatment-related characteristics, together with body mass index (BMI), waist circumference, insulin, leptin, and C-reactive protein plasma levels. Plasma levels of clozapine and its main metabolite, nor-clozapine, were measured in all the participants. In addition, the potential relationship between PANSS scores and leptin or insulin plasma levels was assessed. RESULTS: A total of 46 patients were included: 25 CR and 21 CNR. BMI and waist circumference, fasting insulin and leptin plasma levels were lower in the CNR group, while C-reactive protein was not different. Moreover, significant negative correlations were observed between PANSS positive and general psychopathology subscores, on one hand, and insulin and leptin plasma levels, on the other hand, as well as between PANSS negative subscores and leptin plasma levels. CONCLUSIONS: Our results suggest that the lack of metabolic effect induced by clozapine is associated with the lack of clinical response.

Clozapine induces astrocyte-dependent FDG-PET hypometabolism.

PURPOSE: Advances in functional imaging allowed us to visualize brain glucose metabolism in vivo and non-invasively with [18F]fluoro-2-deoxyglucose (FDG) positron emission tomography (PET) imaging. In the past decades, FDG-PET has been instrumental in the understanding of brain function in health and disease. The source of the FDG-PET signal has been attributed to neuronal uptake, with hypometabolism being considered as a direct index of neuronal dysfunction or death. However, other brain cells are also metabolically active, including astrocytes. Based on the astrocyte-neuron lactate shuttle hypothesis, the activation of the glutamate transporter 1 (GLT-1) acts as a trigger for glucose uptake by astrocytes. With this in mind, we investigated glucose utilization changes after pharmacologically downregulating GLT-1 with clozapine (CLO), an anti-psychotic drug. METHODS: Adult male Wistar rats (control, n = 14; CLO, n = 12) received CLO (25/35 mg kg-1) for 6 weeks. CLO effects were evaluated in vivo with FDG-PET and cortical tissue was used to evaluate glutamate uptake and GLT-1 and GLAST levels. CLO treatment effects were also assessed in cortical astrocyte cultures (glucose and glutamate uptake, GLT-1 and GLAST levels) and in cortical neuronal cultures (glucose uptake). RESULTS: CLO markedly reduced in vivo brain glucose metabolism in several brain areas, especially in the cortex. Ex vivo analyses demonstrated decreased cortical glutamate transport along with GLT-1 mRNA and protein downregulation. In astrocyte cultures, CLO decreased GLT-1 density as well as glutamate and glucose uptake. By contrast, in cortical neuronal cultures, CLO did not affect glucose uptake. CONCLUSION: This work provides in vivo demonstration that GLT-1 downregulation induces astrocyte-dependent cortical FDG-PET hypometabolism-mimicking the hypometabolic signature seen in people developing dementia-and adds further evidence that astrocytes are key contributors of the FDG-PET signal.

Cannabidiol prevents disruptions in sensorimotor gating induced by psychotomimetic drugs that last for 24-h with probable involvement of epigenetic changes in the ventral striatum.

Cannabidiol (CBD), a major non-psychotomimetic component of the Cannabis sativa plant, shows therapeutic potential in several psychiatric disorders, including schizophrenia. The molecular mechanisms underlying the antipsychotic-like effects of CBD are not fully understood. Schizophrenia and antipsychotic treatment can modulate DNA methylation in the blood and brain, resulting in altered expression of diverse genes associated with this complex disorder. However, to date, the possible involvement of DNA methylation in the antipsychotic-like effects of CBD has not been investigated. Therefore, this study aimed at evaluating in mice submitted to the prepulse inhibition (PPI) model: i) the effects of a single injection of CBD or clozapine followed by AMPH or MK-801 on PPI and global DNA methylation changes in the ventral striatum and prefrontal cortex (PFC); and ii). if the acute antipsychotic-like effects of CBD would last for 24-h. AMPH (5 mg/kg) and MK-801 (0.5 mg/kg) impaired PPI. CBD (30 and 60 mg/kg), similar to clozapine (5 mg/kg), attenuated AMPH- and MK801-induced PPI disruption. AMPH, but not MK-801, increased global DNA methylation in the ventral striatum, an effect prevented by CBD. CBD and clozapine increased, by themselves, DNA methylation in the prefrontal cortex. The acute effects of CBD (30 or 60 mg/kg) on the PPI impairment induced by AMPH or MK-801 was also detectable 24 h later. Altogether, the results show that CBD induces acute antipsychotic-like effects that last for 24-h. It also modulates DNA methylation in the ventral striatum, suggesting a new potential mechanism for its antipsychotic-like effects.

Evaluation of NDEL1 oligopeptidase activity in blood and brain in an animal model of schizophrenia: effects of psychostimulants and antipsychotics.

Nuclear distribution element-like 1 (NDEL1) enzyme activity is important for neuritogenesis, neuronal migration, and neurodevelopment. We reported previously lower NDEL1 enzyme activity in blood of treated first episode psychosis and chronic schizophrenia (SCZ) compared to healthy control subjects, with even lower activity in treatment resistant chronic SCZ patients, implicating NDEL1 activity in SCZ. Herein, higher NDEL1 activity was observed in the blood and several brain regions of a validated animal model for SCZ at baseline. In addition, long-term treatment with typical or atypical antipsychotics, under conditions in which SCZ-like phenotypes were reported to be reversed in this animal model for SCZ, showed a significant NDEL1 activity reduction in blood and brain regions which is in line with clinical data. Importantly, these results support measuring NDEL1 enzyme activity in the peripheral blood to predict changes in NDEL1 activity in the CNS. Also, acute administration of psychostimulants, at levels reported to induce SCZ-like phenotype in normal rat strains, increased NDEL1 enzyme activity in blood. Therefore, alterations in NDEL1 activity after treatment with antipsychotics or psychostimulants may suggest a possible modulation of NDEL1 activity secondary to neurotransmission homeostasis and provide new insights into the role of NDEL1 in SCZ pathophysiology.

Ubiquitin-proteasome system, lipid metabolism and DNA damage repair are triggered by antipsychotic medication in human oligodendrocytes: implications in schizophrenia.

Schizophrenia is a chronic, severe and disabling psychiatric disorder, whose treatment is based on psychosocial interventions and the use of antipsychotic drugs. While the effects of these drugs are well elucidated in neuronal cells, they are still not so clear in oligodendrocytes, which play a vital role in schizophrenia. Thus, we aimed to characterize biochemical profiles by proteomic analyses of human oligodendrocytes (MO3.13) which were matured using a protocol we developed and treated with either haloperidol (a typical antipsychotic), clozapine (an atypical antipsychotic) or a clozapine + D-serine co-treatment, which has emerged lately as an alternative type of treatment. This was accomplished by employing shotgun proteomics, using nanoESI-LC-MS/MS label-free quantitation. Proteomic analysis revealed biochemical pathways commonly affected by all tested antipsychotics were mainly associated to ubiquitination, proteasome degradation, lipid metabolism and DNA damage repair. Clozapine and haloperidol treatments also affected proteins involved with the actin cytoskeleton and with EIF2 signaling. In turn, metabolic processes, especially the metabolism of nitrogenous compounds, were a predominant target of modulation of clozapine + D-serine treatment. In this context, we seek to contribute to the understanding of the biochemical and molecular mechanisms involved in the action of antipsychotics on oligodendrocytes, along with their possible implications in schizophrenia.

Clozapine and the Course of Bipolar Disorder in the Systematic Treatment Enhancement Program for Bipolar Disorder (STEP-BD).

OBJECTIVE: The potential of clozapine in severe bipolar disorder is suggested by its efficacy in refractory schizophrenia, but the evidence is limited thus far. This report utilizes data from the standard care pathway of the Systematic Treatment Enhancement Program to examine the clinical impact of clozapine in bipolar disorder, comparing it to two groups, one that received olanzapine and an additional group that received neither drug. METHOD: A total of 4,032 outpatients were available for this analysis. Groups for longitudinal analyses are based on the medication used at each visit. Outcomes assessed were clinical status, symptoms subscales, hospitalizations, and death. We utilized mixed models and generalized estimating equations to adjust for baseline differences and investigate longitudinal differences in symptoms, clinical status, and hospitalization rates between groups. RESULTS: During the study, 1.1% (n = 43) of the patients used clozapine at any time. Those on clozapine had significantly fewer manic and depressive symptoms during follow-up as compared with those on neither clozapine nor olanzapine, while those on olanzapine had more symptoms. The use of clozapine was not associated with an increased risk of hospitalization. No deaths were recorded for clozapine group during the trial. CONCLUSIONS: Although prescribed to very few patients, the impact of clozapine was notable, with fewer symptoms in patients who had more severe illnesses at baseline. Clozapine could prove to be as successful an intervention for late-stage bipolar disorder as it has been in schizophrenia.

In vitro evidence that endothelium-dependent vasodilatation induced by clozapine is mediated by an ATP-sensitive potassium channel.

BACKGROUND: There is a definite association between antipsychotic drugs and arterial hypertension. However, endothelium functions are scarcely considered. This investigation was carried out to study the mechanisms involved in clozapine endothelium-dependent vascular reactivity. METHODS: The experimental animals were male Wistar rats with a mean age of 70-90 days (250-300 g). The endothelium-dependent vascular reactivity was studied by measuring the isometric force and then constructing clozapine concentration-response curves. The force registrations were obtained in the aorta rings with and without the endothelium precontracted with phenylephrine (PE10-6M) treatment; this followed incubation for 30 min in "organ chambers" with different inhibitors: l- NAME (nitric oxide/cGMP); indomethacin (PGI2/cAMP); tetraethylammonium (TEA), and specific hyperpolarization blockers (paxillin, apamin, glibenclamide). The data were presented as the mean ± standard error of the mean (SEM) and were compared by one-way ANOVA or two-way ANOVA followed by the Bonferroni post-test. RESULTS: The primary outcomes were: 1) Clozapine-induced endothelium-dependent relaxation was not inhibited by indomethacin, l-NAME, ODQ, and methylene blue (MB); 2) The combination of l-NAME + indomethacin partially prevented the relaxation; 3) Clozapine did not induce relaxation in vessels contracted with KCl; 4) TEA did not block the clozapine-induced relaxation in vessels precontracted with PE (10-6 M); 5) The potassium channel blockers paxillin and apamin did not prevent relaxation but glibenclamide did. CONCLUSION: Concerning the mechanisms involved in clozapine endothelium-dependent vascular reactivity, the present study suggests that there is synergistic participation that probably occurs through a crosstalk mechanism of the cAMP, cGMPpathways and hyperpolarization.

Impact of nuclear distribution element genes in the typical and atypical antipsychotics effects on nematode Caenorhabditis elegans: Putative animal model for studying the pathways correlated to schizophrenia.

The nuclear distribution element genes are conserved from fungus to humans. The nematode Caenorhabditis elegans expresses two isoforms of nuclear distribution element genes, namely nud-1 and nud-2. While nud-1 was functionally demonstrated to be the worm nudC ortholog, bioinformatic analysis revealed that the nud-2 gene encodes the worm ortholog of the mammalian NDE1 (Nuclear Distribution Element 1 or NudE) and NDEL1 (NDE-Like 1 or NudEL) genes, which share overlapping roles in brain development in mammals and also mediate the axon guidance in mammalian and C. elegans neurons. A significantly higher NDEL1 enzyme activity was shown in treatment non-resistant compared to treatment resistant SCZ patients, who essentially present response to the therapy with atypical clozapine but not with typical antipsychotics. Using C. elegans as a model, we tested the consequence of nud genes suppression in the effects of typical and atypical antipsychotics. To assess the role of nud genes and antipsychotic drugs over C. elegans behavior, we measured body bend frequency, egg laying and pharyngeal pumping, which traits are controlled by specific neurons and neurotransmitters known to be involved in SCZ, as dopamine and serotonin. Evaluation of metabolic and behavioral response to the pharmacotherapy with these antipsychotics demonstrates an important unbalance in serotonin pathway in both nud-1 and nud-2 knockout worms, with more significant effects for nud-2 knockout. The present data also show an interesting trend of mutant knockout worm strains to present a metabolic profile closer to that observed for the wild-type animals after the treatment with the typical antipsychotic haloperidol, but which was not observed for the treatment with the atypical antipsychotic clozapine. Paradoxically, behavioral assays showed more evident effects for clozapine than for haloperidol, which is in line with previous studies with rodent animal models and clinical evaluations with SCZ patients. In addition, the validity and reliability of using this experimental animal model to further explore the convergence between the dopamine/serotonin pathways and neurodevelopmental processes was demonstrated here, and the potential usefulness of this model for evaluating the metabolic consequences of treatments with antipsychotics is also suggested.

N-3 polyunsaturated fatty acids and clozapine abrogates poly I: C-induced immune alterations in primary hippocampal neurons.

The viral mimetic polyinosinic:polycytidylic acid (poly I:C) is an important tool to study the consequences of viral infection to the development of neuropsychiatric disorders. Here, based on the premise of omega-3 polyunsaturated fatty acids (n3 PUFAs) as supplemental treatment to antipsychotics in schizophrenia, we investigated the involvement of NFkB pathway in the effects of n3 PUFAs or of the atypical antipsychotic clozapine in hippocampal poly I:C-challenged neurons. Primary hippocampal neuronal cultures were exposed to n3 PUFAs (DHA4.35 µM/EPA7.10 µM, DHA 8.7 µM/EPA14.21 µM or DHA17.4 µM/EPA28.42 µM) or clozapine (1.5 or 3 µM) in the presence or absence of poly I:C. MTT assay revealed that poly I:C-induced reduction in cell viability was prevented by n3 PUFAs or clozapine. N3 PUFAs (DHA 8.7 µM/EPA14.21 µM) or clozapine (3 µM) significantly reduced poly I:C-induced increase in iNOS, NFkB (p50/p65), IL-6 and nitrite when compared to non-treated cells. Only n3 PUFAs prevented poly I:C-induced deficits in BDNF. On the other hand, poly I:C caused a marked reduction in DCX immunoexpression, which was prevented only by clozapine. Thus, n3 PUFAs and clozapine exert in vitro neuroprotective effects against poly I:C immune challenge in hippocampal neurons, by mechanisms possibly involving the inhibition of canonical NFkB pathway. The present study adds further evidences to the mechanisms underlying n3 PUFAs and clozapine neuroprotective effects against viral immune challenges. Since n3 PUFAs is a safe strategy for use during pregnancy, our results also add further evidence for the use of this supplement in order to prevent alterations induced by viral hits during this developmental period.

Prelimbic NMDA receptors stimulation mimics the attenuating effects of clozapine on the auditory electrophysiological rebound induced by ketamine withdrawal.

Ketamine (KET) is a non-competitive N-Methyl-d-aspartate (NMDA) receptors antagonist that intensifies sensory experiences, prompts hallucinations and delusions, exacerbates previously installed psychosis and disrupts physiological evoked potentials (AEPs). Pharmacologically, KET stimulates glutamate efflux in the medial prefrontal cortex, mainly in the prelimbic (PrL) sub-region. Efferences from this region exert a top-down regulatory control of bottom-up sensory processes either directly or indirectly. In the midbrain, the central nucleus of the inferior colliculus (CIC) plays a fundamental role in the processing of auditory ascending information related to sound localization, sensorimotor gating, and preattentive event-related potentials. Auditory hallucinations elicited during a psychotic outbreak are accompanied by CIC neural activation. Thus, it is possible that NMDA-mediated glutamate neurotransmission in the PrL indirectly modulates CIC neuronal firing. The aim of the present study was to assess the effects of KET on the latency and amplitude of AEPs elicited in the CIC of rats tested during KET effects and following withdrawal from the chronic administration. Changes on emotionally induced by KET treatment were evaluated with the use of the elevated zero maze (EZM). Unlike typical neuroleptics, the atypical antipsychotic clozapine (CLZ) potently blocks the disruption of the sensorimotor gating induced by NMDA antagonists. Therefore, the effects of KET withdrawal on AEPs were challenged with a systemic injection of CLZ. In addition, we further investigated the role of NMDA receptors of the PrL on the AEPs expression recorded in the CIC through intra-PrL infusions of NMDA itself. Our results showed that the processing of sensory information in the CIC is under indirect control of PrL. These data suggest that the long-term KET treatment disrupts the collicular auditory field potentials, possibly through influencing PrL glutamate activity on intrinsic 5-HT mechanisms in the dorsal raphe and CIC.

Clozapine blockade of MK-801-induced learning/memory impairment in the mEPM: Role of 5-HT1A receptors and hippocampal BDNF levels.

Cognitive impairment associated with schizophrenia (CIAS) is highly prevalent and affects the overall functioning of patients. Clozapine (Clz), an atypical antipsychotic drug, significantly improves CIAS although the underlying mechanisms remain under study. The role of the 5-HT1A receptor (5-HT1A-R) in the ability of Clz to prevent the learning/memory impairment induced by MK-801 was investigated using the modified elevated plus-maze (mEPM) considering the Transfer latency (TL) as an index of spatial memory. We also investigated if changes in hippocampal brain-derived neurotrophic factor (BDNF) levels underlie the behavioral prevention induced by Clz. Clz (0.5 and 1mg/kg)- or vehicle-pretreated Wistar rats were injected with MK-801 (0.05mg/kg) or saline. TL was evaluated 35min later (TL1, acquisition session) while learning/memory performance was measured 24h (TL2, retention session) and 48h later (TL3, long-lasting effect). WAY-100635, a 5-HT1A-R antagonist, was pre-injected (0.3mg/kg) to examine the presumed 5-HT1A-R involvement in Clz action. At TL2, another experimental group treated with Clz and MK-801 and its respective control groups were added to measure BDNF protein levels by ELISA. TL1 and TL3 were not significantly modified by the different treatments. MK-801 increased TL2 compared to control group leading a disruption of spatial memory processing which was markedly attenuated by Clz. WAY-100635 suppressed this action supporting a relevant role of 5-HT1A-R in the Clz mechanism of action to improve spatial memory dysfunction. Although a significant decrease of hippocampal BDNF levels underlies the learning/memory impairment induced by MK-801, this effect was not significantly prevented by Clz.

Clozapine and olanzapine are better antioxidants than haloperidol, quetiapine, risperidone and ziprasidone in in vitro models.

Although the etiopathogenic mechanisms of schizophrenia (SCZ) are unknown, evidences suggest that excessive free radical production or oxidative stress may be involved in the pathophysiology of SCZ. Antipsychotics are the drugs used in the treatment of SCZ but it remains controversial the impact that typical vs. atypical antipsychotics has on the oxidative stress status in SCZ patients. In vitro, the antioxidant capacity of six antipsychotics was assessed by their ability to: decrease or scavenge reactive oxygen species in the neutrophil respiratory burst; donate hydrogen and stabilize the free radical 2,2-diphenyl-1-picryl-hydrazyl (DPPH); and scavenge 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS(+)). This study demonstrated that both clozapine and olanzapine have antioxidant effects, in vitro, by scavenging superoxide anion on the respiratory burst, donating electron in the ABTS(+) assay and stabilizing the radical DPPH. Ziprasidone significantly scavenged ABTS(+) and stabilized the radical DPPH whereas risperidone significantly reduced the respiratory burst. Haloperidol and quetiapine lacked antioxidant effects. The chemical structure-related antioxidant capacity suggests a possible neuroprotective mechanism of these drugs on the top of their antipsychotic mechanism of action.

LASSBio-579, a prototype antipsychotic drug, and clozapine are effective in novel object recognition task, a recognition memory model.

Previous studies on the N-phenylpiperazine derivative LASSBio-579 have suggested that LASSBio-579 has an atypical antipsychotic profile. It binds to D2, D4 and 5-HT1A receptors and is effective in animal models of schizophrenia symptoms (prepulse inhibition disruption, apomorphine-induced climbing and amphetamine-induced stereotypy). In the current study, we evaluated the effect of LASSBio-579, clozapine (atypical antipsychotic) and haloperidol (typical antipsychotic) in the novel object recognition task, a recognition memory model with translational value. Haloperidol (0.01 mg/kg, orally) impaired the ability of the animals (CF1 mice) to recognize the novel object on short-term and long-term memory tasks, whereas LASSBio-579 (5 mg/kg, orally) and clozapine (1 mg/kg, orally) did not. In another set of experiments, animals previously treated with ketamine (10 mg/kg, intraperitoneally) or vehicle (saline 1 ml/100 g, intraperitoneally) received LASSBio-579, clozapine or haloperidol at different time-points: 1 h before training (encoding/consolidation); immediately after training (consolidation); or 1 h before long-term memory testing (retrieval). LASSBio-579 and clozapine protected against the long-term memory impairment induced by ketamine when administered at the stages of encoding, consolidation and retrieval of memory. These findings point to the potential of LASSBio-579 for treating cognitive symptoms of schizophrenia and other disorders.

Low expression of Gria1 and Grin1 glutamate receptors in the nucleus accumbens of Spontaneously Hypertensive Rats (SHR).

The Spontaneously Hypertensive Rat (SHR) strain is a classical animal model for the study of essential hypertension. Recently, our group suggested that this strain could be a useful animal model for schizophrenia, which is a severe mental illness with involvement of glutamatergic system. The aim of this study is to investigate glutamatergic receptors (Gria1 and Grin1) and glycine transporter (Glyt1) gene expression in the prefrontal cortex (PFC) and nucleus accumbens (NAcc) of SHR animals. The effects in gene expression of a chronic treatment with antipsychotic drugs (risperidone, haloperidol and clozapine) were also analyzed. Animals were treated daily for 30 days, and euthanized for brain tissue collection. The expression pattern was evaluated by Real Time Reverse-Transcriptase (RT) PCR technique. In comparison to control rats, SHR animals present a lower expression of both NMDA (Grin1) and AMPA (Gria1) gene receptors in the NAcc. Antipsychotic treatments were not able to change gene expressions in any of the regions evaluated. These findings provide evidence for the role of glutamatergic changes in schizophrenia-like phenotype of the SHR strain.

Antitumor activity of histamine and clozapine in a mouse experimental model of human melanoma.

BACKGROUND: Functional presence of histamine H4 receptor (H4R) was demonstrated in human melanoma cell lines and biopsies. OBJECTIVE: The purposes of this work were to investigate signal transduction pathways and biological responses triggered by the activation of H4R in human primary (WM35) and metastatic (M1/15) melanoma cell lines and to evaluate the in vivo antitumor activity of histamine (HA) and clozapine (CLZ) on human M1/15 melanoma xenografts. METHODS: Clonogenic assay, incorporation of BrdU, cell cycle distribution, phosphorylation levels of ERK1/2 and cAMP production were evaluated in vitro. An experimental human melanoma model was developed into athymic nude mice. Tumor growth, survival and histochemical studies were performed in order to investigate the expression levels of H4R, HA, PCNA, mitotic index (MI), and angiogenesis. RESULTS: The results indicate that H4R agonists inhibited forskolin-induced cAMP levels only in M1/15 cells while increased phosphorylation levels of ERK1/2 and decreased proliferation in both cell types. In vivo studies show that HA and CLZ (1mgkg(-1), sc) significantly increased median survival and decreased tumor volume. These effects were associated to a reduction in MI, in the expression of proliferation marker and in intratumoral neovascularization. CONCLUSIONS: We conclude that HA and CLZ exhibit an antitumoral effect in vitro and in vivo on human melanoma, suggesting the therapeutic potential of these compounds for the treatment of malignant melanoma.

Therapeutic potential of histamine H4 receptor agonists in triple-negative human breast cancer experimental model.

BACKGROUND AND PURPOSE: The presence of the histamine H4 receptor (H4R) was previously reported in benign and malignant lesions and cell lines derived from the human mammary gland. The aim of this work was to evaluate the effects of H4R ligands on the survival, tumour growth rate and metastatic capacity of breast cancer in an experimental model. EXPERIMENTAL APPROACH: Xenograft tumours of the highly invasive human breast cancer cell line MDA-MB-231 were established in immune deficient nude mice. The following H4R agonists were employed: histamine (5 mg kg⁻¹), clozapine (1 mg kg⁻¹) and the experimental compound JNJ28610244 (10 mg kg⁻¹). RESULTS: Data indicate that developed tumours were highly undifferentiated, expressed H4R and exhibited high levels of histamine content and proliferation marker (PCNA) while displaying low apoptosis. Mice of the untreated group displayed a median survival of 60 days and a tumour doubling time of 7.4 ± 0.6 days. A significant decrease in tumour growth evidenced by an augment of the tumour doubling time was observed in the H4R agonist groups (13.1 ± 1.2, P < 0.01 in histamine group; 15.1 ± 1.1, P < 0.001 in clozapine group; 10.8 ± 0.7, P < 0.01 in JNJ28610244 group). This effect was associated with a decrease in the PCNA expression levels, and also reduced intratumoural vessels in histamine and clozapine treated mice. Histamine significantly increased median survival (78 days; Log rank Mantel-Cox Test, P = 0.0025; Gehan-Breslow-Wilcoxon Test, P = 0.0158) and tumoural apoptosis. CONCLUSIONS AND IMPLICATIONS: Histamine through the H4R exhibits a crucial role in tumour progression. Therefore, H4R ligands offer a novel therapeutic potential as adjuvants for breast cancer treatment.

Clozapine administration reverses behavioral, neuronal, and nitric oxide disturbances in the neonatal ventral hippocampus rat.

Clozapine is widely used in the treatment of schizophrenia; however its complete mechanism of action is not fully established. The neonatal ventral hippocampal lesion (nVHL) has emerged as a model of schizophrenia-related behavior. Our group has previously shown hyperresponsiveness to novel environment, neuronal atrophy in prefrontal cortex (PFC) and nucleus accumbens (NAcc) neurons as well as abnormal levels of nitric oxide (NO) in the PFC of the nVHL rat. In the present study, we aimed to investigate the role of repeated clozapine administration (2 mg/kg/day for 21 days) in a novel environment, neuronal rearrangement in PFC, NAcc and basolateral amygdala (BLA) as well as NO levels in this model. Clozapine administration reversed the hyperlocomotion observed in a novel environment in the nVHL rat with no effect on locomotion in sham animals. Quantitative morphological analysis demonstrated a retracted neuronal arborization and decreased spinogenesis in the NAcc, PFC and BLA in nVHL rat. Interestingly, clozapine administration also rescued neuronal atrophy in these brain regions. The nVHL also displayed increased NO levels in PFC, striatum and occipital cortex. Clozapine administration selectively reversed these abnormal levels of NO in striatum in nVHL rat while NO levels were increased in the PFC of sham animals. Our results further extend the usefulness of the nVHL as a model of schizophrenia-related behavior and suggest that clozapine reverses behavioral deficits in these animals by modulating neuronal reorganization and NO levels in the brain.

Rescue of social behavior impairment by clozapine and alterations in the expression of neuronal receptors in a rat model of neurodevelopmental impairment induced by GRPR blockade.

We have previously shown that pharmacological blockade of the gastrin-releasing peptide receptor (GRPR) during the neonatal period in rats produces behavioral features of developmental neuropsychiatric disorders. Here, we show that social interaction deficits in this model are reversed by the atypical antipsychotic clozapine given in the adulthood. In addition, we analyzed the mRNA expression of three neuronal receptors potentially involved in the etiology of disorders of the autism spectrum. Rats were injected with the GRPR antagonist RC-3095 or saline (SAL) from postnatal days 1-10, and tested for social behavior and recognition memory in the adulthood. One hour prior to the behavioral testing, rats were given a systemic injection of clozapine or saline. The mRNA expression of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor, the epidermal growth factor receptor (EGFR), and GRPR was measured in the hippocampus and cortex of a separate set of rats given RC-3095 or SAL neonatally. Rats given neonatal RC-3095 showed decreased social interaction and impaired object recognition memory. Clozapine rescued the social interaction impairment. Neonatal treatment with RC-3095 also resulted in dose-dependent decreases in the expression of GRPR, NR1, and EGFR in the cortex, whereas all three receptor mRNAs were increased in the hippocampus in rats treated with the lower dose of RC-3095. The results contribute to further validate the novel rat model of neurodevelopmental disorders induced by GRPR blockade, and shows alterations in the expression of neuronal receptors in this model.

Effects of antipsychotics with different weight gain liabilities on human in vitro models of adipose tissue differentiation and metabolism.

Weight gain and metabolic abnormalities are serious side effects associated with the use of several second generation antipsychotics (SGA). The adipose tissue has been considered a direct SGA target involved in the development of these adverse effects. Recent studies, mainly using murine cells, have suggested that SGA increase both adipogenesis of preadipocytes and lipid accumulation in mature adipocytes. However, to date there has been little research comparing the effects of antipsychotics with different propensities to induce weight gain on human in vitro models of white adipose tissue neoformation and metabolism. The present study aimed to investigate the effects of antipsychotics either strongly associated with weight gain, such as the SGA clozapine and olanzapine, or not, such as the SGA ziprasidone and the classical antipsychotic haloperidol, on proliferation and adipocyte differentiation of human adipose-derived stem cells (ADSCs) and lipogenesis in human mature adipocytes. Whereas ziprasidone induced elevated levels of cell death during adipogenesis and could not be investigated further, we observed that clozapine, olanzapine and haloperidol had slight stimulatory effects on the transcriptional program of ADSCs adipogenesis. However, the observed changes in adipocyte-specific genes were not accompanied by a significant increase in triglyceride accumulation within differentiated adipocytes. Our data also showed that these three antipsychotics displayed inhibitory effects on the proliferation rates of undifferentiated ADSCs. Regarding mature adipocyte metabolism, we observed that olanzapine slightly inhibited insulin-stimulated lipogenesis at the highest concentration used, and haloperidol exerted the strongest inhibitory effects on both basal and insulin-stimulated lipogenesis. Taken together, our results suggest that a direct and potent effect of clozapine and olanzapine on adipose tissue biology is not an important mechanism by which these SGA induce metabolic disturbances in humans. On the other hand, the haloperidol-mediated downregulation of the lipogenic capacity of human adipose tissue may be a possible mechanism contributing to its lower propensity to induce serious metabolic side effects.