Intermittent repeated stress but not ketamine changes mice response to antidepressants.

Discovery of the rapid antidepressant effect of ketamine has been considered one of the most important advances in major depressive disorder treatment. Several studies report a significant benefit to patients that lasts up to 19 days after treatment. However, concerns arise from the long-term use of ketamine, thus a safe and effective strategy for maintaining its antidepressant effect is still necessary. To this end, our work assessed the effects of imipramine and fluoxetine after repeated ketamine treatment in male mice. Ketamine (30 mg/kg/day for 14 days) induced an anti-immobility effect in the forced swimming (FS) paradigm, detected 1 and 3 days after treatment. Seven days after the last ketamine injection, mice received imipramine (20 mg/kg) or fluoxetine (30 mg/kg). Imipramine and fluoxetine did not change mice's immobility time, regardless of the pre-treatment (saline or ketamine). Since both drugs' anti-immobility effect was demonstrated in the classical FS test, we can assume that repeated exposure to intermittent stress inhibited the antidepressant drugs' anti-immobility effects. Moreover, pre-exposure to ketamine did not counteract stress-induced changes in mice response to antidepressants. Since exposure to forced swim and i.p. injections are stressful to rodents, each stressor's contribution to the blunted response to antidepressants was investigated. Our data demonstrated that both stressors (FS and i.p. injections) influenced the reported effect. In summary, our results showed that exposure to intermittent repeated stress inhibited the anti-immobility effect of imipramine and fluoxetine in mice and corroborated findings demonstrating that exposure to stress can blunt patients' response to antidepressants.

Is Forced Swimming Immobility a Good Endpoint for Modeling Negative Symptoms of Schizophrenia? - Study of Sub-Anesthetic Ketamine Repeated Administration Effects

ABSTRACT Immobility time in the forced swimming has been described as analogous to emotional blunting or apathy and has been used for characterizing schizophrenia animal models. Several clinical studies support the use of NMDA receptor antagonists to model schizophrenia in rodents. Some works describe the effects of ketamine on immobility behavior but there is variability in the experimental design used leading to controversial results. In this study, we evaluated the effects of repeated administration of ketamine sub-anesthetic doses in forced swimming, locomotion in response to novelty and novel object recognition, aiming a broader evaluation of the usefulness of this experimental approach for modeling schizophrenia in mice. Ketamine (30 mg/kg/day i.p. for 14 days) induced a not persistent decrease in immobility time, detected 24h but not 72h after treatment. This same administration protocol induced a deficit in novel object recognition. No change was observed in mice locomotion. Our results confirm that repeated administration of sub-anesthetic doses of ketamine is useful in modeling schizophrenia-related behavioral changes in mice. However, the immobility time during forced swimming does not seem to be a good endpoint to evaluate the modeling of negative symptoms in NMDAR antagonist animal models of schizophrenia.

Is Forced Swimming Immobility a Good Endpoint for Modeling Negative Symptoms of Schizophrenia? - Study of Sub-Anesthetic Ketamine Repeated Administration Effects.

Immobility time in the forced swimming has been described as analogous to emotional blunting or apathy and has been used for characterizing schizophrenia animal models. Several clinical studies support the use of NMDA receptor antagonists to model schizophrenia in rodents. Some works describe the effects of ketamine on immobility behavior but there is variability in the experimental design used leading to controversial results. In this study, we evaluated the effects of repeated administration of ketamine sub-anesthetic doses in forced swimming, locomotion in response to novelty and novel object recognition, aiming a broader evaluation of the usefulness of this experimental approach for modeling schizophrenia in mice. Ketamine (30 mg/kg/day i.p. for 14 days) induced a not persistent decrease in immobility time, detected 24h but not 72h after treatment. This same administration protocol induced a deficit in novel object recognition. No change was observed in mice locomotion. Our results confirm that repeated administration of sub-anesthetic doses of ketamine is useful in modeling schizophrenia-related behavioral changes in mice. However, the immobility time during forced swimming does not seem to be a good endpoint to evaluate the modeling of negative symptoms in NMDAR antagonist animal models of schizophrenia.

LASSBio-1422: a new molecular scaffold with efficacy in animal models of schizophrenia and disorders of attention and cognition.

Aiming to identify new antipsychotic lead-compounds, our group has been working on the design and synthesis of new N-phenylpiperazine derivatives. Here, we characterized LASSBio-1422 as a pharmacological prototype of this chemical series. Adult male Wistar rats and CF1 mice were used for in-vitro and in-vivo assays, respectively. LASSBio-1422 [1 and 5 mg/kg, postoperatively (p.o.)] inhibited apomorphine-induced climbing as well as ketamine-induced hyperlocomotion (1 and 5 mg/kg, p.o.), animal models predictive of efficacy on positive symptoms. Furthermore, LASSBio-1422 (5 mg/kg, p.o.) prevented the prepulse impairment induced by apomorphine, (±)-2,5-dimethoxy-4-iodoamphetamine, and ketamine, as well as the memory impairment induced by ketamine in the novel object-recognition task at the acquisition, consolidation, and retrieval phases of memory formation. Potential extrapyramidal side-effects and sedation were assessed by catatonia, rota-rod, locomotion, and barbiturate sleeping time, and LASSBio-1422 (15 mg/kg, p.o.) did not affect any of the parameters observed. Binding assays showed that LASSBio-1422 has a binding profile different from the known atypical antipsychotic drugs: it does not bind to AMPA, kainate, N-methyl-D-aspartate, glicine, and mGluR2 receptors and has low or negligible affinity for D1, D2, and 5-HT2A/C receptors, but high affinity for D4 receptors (Ki=0.076 µmol/l) and, to a lesser extent, for 5-HT1A receptors (Ki=0.493 µmol/l). The antagonist action of LASSBio-1422 at D4 receptors was assessed through the classical GTP-shift assay. In conclusion, LASSBio-1422 is effective in rodent models of positive and cognitive symptoms of schizophrenia and its ability to bind to D4 and 5-HT1A receptors may at least in part explain its effects in these animal models.

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.

Determination of pharmacological interactions of uliginosin B, a natural phloroglucinol derivative, with amitriptyline, clonidine and morphine by isobolographic analysis.

Uliginosin B is a natural phloroglucinol derivative, obtained from Hypericum species native to South America. Previous studies have shown that uliginosin B presents antidepressant-like and antinociceptive effects. Although its mechanism of action is still not completely elucidated, it is known that it involves the activation of monoaminergic neurotransmission. The aim of the current study was to further investigate the antinociceptive mechanism of action of uliginosin B by combining it with different drugs used for treating pain in clinical practice. The intraperitoneal administration of uliginosin B, morphine, amitriptyline and clonidine, alone or in mixture, produced a dose-dependent antinociceptive effect in the hot-plate assay in mice. The effect of the mixtures of drugs was studied using an adapted isobologram analysis at the effect level of 50% of the maximal effect observed. The analysis showed that the interactions between uliginosin B and morphine was synergistic, while the interactions between uliginosin B and amitriptyline or clonidine were additive. These findings point to uliginosin B as a potential adjuvant for pain pharmacotherapy, especially for opioid analgesia.

Synthesis and pharmacological evaluation of new N-phenylpiperazine derivatives designed as homologues of the antipsychotic lead compound LASSBio-579.

In an attempt to increase the affinity of our antipsychotic lead compound LASSBio-579 (1-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)-4-phenylpiperazine; (2)) for the 5-HT(2A) receptor, we synthesized five new N-phenylpiperazine derivatives using a linear synthetic route and the homologation strategy. The binding profile of these compounds was evaluated for a series of dopaminergic, serotonergic and alpha-adrenergic receptors relevant for schizophrenia, using classical competition assays. Increasing the length of the spacer between the functional groups of (2) proved to be appropriated since the affinity of these compounds increased 3-10-fold for the 5-HT(2A) receptor, with no relevant change in the affinity for the D2-like and 5-HT(1A) receptors. A GTP-shift assay also indicated that the most promising derivative (1-(4-(1-(4-chlorophenyl)-1H-pyrazol-4-yl) butyl)-4-phenylpiperazine) (LASSBio-1635) (6) has the expected efficacy at the 5-HT(2A) receptors, acting as an antagonist. Intraperitoneal administration of (6) prevented apomorphine-induced climbing behavior and ketamine-induced hyperlocomotion in mice, in a dose dependent manner. Together, these results show that (6) could be considered as a new antipsychotic lead compound.

Biotransformation of LASSBio-579 and pharmacological evaluation of p-hydroxylated metabolite a N-phenylpiperazine antipsychotic lead compound.

Using a combination of docking and molecular dynamics simulations, we predicted that p-hydroxylation by CYP1A2 would be the main metabolic pathway for the 1-[1-(4-chlorophenyl)-1H-4pyrazolylmethyl] phenylhexahydropiperazine, LASSBio-579 (3). As the result of a screening process with strains of filamentous fungi, Cunninghamella echinulata ATCC 9244 was chosen to scale up the preparation of the p-hydroxylated metabolite (4). About 30 min after i.p. administration of (3) to rats was identified as the p-hydroxylated metabolite, confirming our in silico previsions. Chemical synthesis of the metabolite was performed and allowed its pharmacological evaluation in binding assays revealing its high affinity for D2 and D4 receptors, indicating that this metabolite should participate to the antipsychotic effect of (3) in vivo. Furthermore, we report here that both (3) and its p-hydroxylated metabolite (4) have a much lower affinity than clozapine for two receptors involved in adverse reactions. Voltammetric assays were useful to understand the redox profile of (3).

New insights into pharmacological profile of LASSBio-579, a multi-target N-phenylpiperazine derivative active on animal models of schizophrenia.

Previous behavioral and receptor binding studies on N-phenylpiperazine derivatives by our group indicated that LASSBio-579, LASSBio-580 and LASSBio-581 could be potential antipsychotic lead compounds. The present study identified LASSBio-579 as the most promising among the three compounds, since it was the only one that inhibited apomorphine-induced climbing (5 mg/kg p.o.) and apomorphine-induced hypothermia (15 mg/kg p.o.). Furthermore, LASSBio-579 (0.5 mg/kg p.o.) was effective in the ketamine-induced hyperlocomotion test and prevented the prepulse inhibition deficits induced by apomorphine, DOI and ketamine with different potencies (1 mg/kg, 0.5 mg/kg and 5 mg/kg p.o., respectively). LASSBio-579 also induced a motor impairment, catalepsy and a mild sedative effect but only at doses 3-120 times higher than those with antipsychotic-like effects. In addition, LASSBio-579 (0.5 and 1 mg/kg p.o.) reversed the catalepsy induced by WAY 100,635, corroborating its action on both dopaminergic and serotonergic neurotransmission and pointing to the contribution of 5-HT(1A) receptor activation to its pharmacological profile. Moreover, co-administration of sub-effective doses of LASSBio-579 with sub-effective doses of clozapine or haloperidol prevented the apomorphine-induced climbing without induction of catalepsy. In summary, our results characterize LASSBio-579 as a multi-target ligand active in pharmacological animal models of schizophrenia, confirming that this compound could be included in development programs aiming at a new drug for treating schizophrenia.

Searching for multi-target antipsychotics: Discovery of orally active heterocyclic N-phenylpiperazine ligands of D2-like and 5-HT1A receptors.

We described herein the design, synthesis, and pharmacological evaluation of N-phenylpiperazine heterocyclic derivatives as multi-target compounds potentially useful for the treatment of schizophrenia. The isosteric replacement of the heterocyclic ring at the biaryl motif generating pyrazole, 1,2,3-triazole, and 2-methylimidazole[1,2-a]pyridine derivatives resulted in 21 analogues with different substitutions at the para-biaryl and para-phenylpiperazine positions. Among the compounds prepared, 4 (LASSBio-579) and 10 (LASSBio-664) exhibited an adequate binding profile and a potential for schizophrenia positive symptoms treatment without cataleptogenic effects. Structural features of this molecular scaffold are discussed regarding binding affinity and selectivity for D(2)-like, 5-HT(1A), and 5-HT(2A) receptors.