A Novel Dibenzoxazepine Attenuates Intracellular Salmonella Typhimurium Oxidative Stress Resistance.

Salmonella enterica serovar Typhimurium is the leading cause of invasive nontyphoidal salmonellosis. Additionally, the emergence of multidrug-resistant S. Typhimurium has further increased the difficulty of controlling its infection. Previously, we showed that an antipsychotic drug, loxapine, suppressed intracellular Salmonella in macrophages. To exploit loxapine's antibacterial activity, we simultaneously evaluated the anti-intracellular Salmonella activity and cytotoxicity of newly synthesized loxapine derivatives using an image-based high-content assay. We identified that SW14 exhibits potent suppressive effects on intramacrophagic S. Typhimurium with an 50% effective concentration (EC50) of 0.5 µM. SW14 also sensitized intracellular Salmonella to ciprofloxacin and cefixime and effectively controlled intracellular multidrug- and fluoroquinolone-resistant S. Typhimurium strains. However, SW14 did not affect bacterial growth in standard microbiological broth or minimal medium that mimics the phagosomal environment. Cellular autophagy blockade by 3-methyladenine (3-MA) or shATG7 elevated the susceptibility of intracellular Salmonella to SW14. Finally, reactive oxygen species (ROS) scavengers reduced the antibacterial efficacy of SW14, but the ROS levels in SW14-treated macrophages were not elevated. SW14 decreased the resistance of outer membrane-compromised S. Typhimurium to H2O2. Collectively, our data indicated that the structure of loxapine can be further optimized to develop new antibacterial agents by targeting bacterial resistance to host oxidative-stress defense. IMPORTANCE The incidence of diseases caused by pathogenic bacteria with resistance to common antibiotics is consistently increasing. In addition, Gram-negative bacteria are particularly difficult to treat with antibiotics, especially those that can invade and proliferate intracellularly. In order to find a new antibacterial compound against intracellular Salmonella, we established a cell-based high-content assay and identified SW14 from the derivatives of the antipsychotic drug loxapine. Our data indicate that SW14 has no effect on free bacteria in the medium but can suppress the intracellular proliferation of multidrug-resistant (MDR) S. Typhimurium in macrophages. We also found that SW14 can suppress the resistance of outer membrane compromised Salmonella to H2O2, and its anti-intracellular Salmonella activity can be reversed by reactive oxygen species (ROS) scavengers. Together, the findings suggest that SW14 might act via a virulence-targeted mechanism and that its structure has the potential to be further developed as a new therapeutic against MDR Salmonella.

Effect of Novel Antipsychotics on Energy Metabolism - In Vitro Study in Pig Brain Mitochondria.

The identification and quantification of mitochondrial effects of novel antipsychotics (brexpiprazole, cariprazine, loxapine, and lurasidone) were studied in vitro in pig brain mitochondria. Selected parameters of mitochondrial metabolism, electron transport chain (ETC) complexes, citrate synthase (CS), malate dehydrogenase (MDH), monoamine oxidase (MAO), mitochondrial respiration, and total ATP and reactive oxygen species (ROS) production were evaluated and associated with possible adverse effects of drugs. All tested antipsychotics decreased the ETC activities (except for complex IV, which increased in activity after brexpiprazole and loxapine addition). Both complex I- and complex II-linked respiration were dose-dependently inhibited, and significant correlations were found between complex I-linked respiration and both complex I activity (positive correlation) and complex IV activity (negative correlation). All drugs significantly decreased mitochondrial ATP production at higher concentrations. Hydrogen peroxide production was significantly increased at 10 µM brexpiprazole and lurasidone and at 100 µM cariprazine and loxapine. All antipsychotics acted as partial inhibitors of MAO-A, brexpiprazole and loxapine partially inhibited MAO-B. Based on our results, novel antipsychotics probably lacked oxygen uncoupling properties. The mitochondrial effects of novel antipsychotics might contribute on their adverse effects, which are mostly related to decreased ATP production and increased ROS production, while MAO-A inhibition might contribute to their antidepressant effect, and brexpiprazole- and loxapine-induced MAO-B inhibition might likely promote neuroplasticity and neuroprotection. The assessment of drug-induced mitochondrial dysfunctions is important in development of new drugs as well as in the understanding of molecular mechanism of adverse or side drug effects.

Loxapine, an antipsychotic drug, suppresses intracellular multiple-antibiotic-resistant Salmonella enterica serovar Typhimurium in macrophages.

BACKGROUND: The emergence of multiple-antibiotic-resistant (MAR) Salmonella has been a serious threat worldwide. Salmonella can invade into host cells and evade the attacks of host humoral defenses and antibiotics. Thus, a new antibacterial agent capable of inhibiting intracellular Salmonella is highly needed. METHODS: The anti-intracellular activity and cytotoxicity of drugs on intracellular bacteria and macrophages were assayed using intracellular CFU assay and MTT cell viability assay, respectively. The uptake of gentamicin into macrophage and the effect of autophagy inhibitor on loxapine's anti-intracellular Salmonella activity were assessed by using image-based high-content system. The expression of bacterial genes was measured by real-time PCR. The efflux pump activity of bacteria was measured by Hoechst accumulation assays. RESULTS: With our efforts, an antipsychotic drug, loxapine, was identified to exhibit high potency in suppressing intracellular MAR S. Typhimurium, Staphylococcus aureus, Shigella flexneri or Yersinia enterocolitica. Subsequent investigations indicated that loxapine's anti-intracellular bacteria activity was not associated with increased penetration of gentamicin into bacteria and macrophages. Loxapine didn't inhibit bacterial growth in broth at concentration up to 500 µM and has no effect on Salmonella's type III secretion system genes' expression. Blockage of autophagy also didn't reverse loxapine's anti-intracellular activity. Lastly, loxapine suppressed bacterial efflux pump activity in all bacteria tested. CONCLUSION: Altogether, our data suggested that loxapine might suppress intracellular bacteria through inhibiting of bacterial efflux pumps. In light of its unique activity, loxapine represents a promising lead compound with translational potential for the development of a new antibacterial agent against intracellular bacteria.

Contrasting loxapine to its isomer isoloxapine--the critical role of in vivo D2 blockade in determining atypicality.

BACKGROUND: Loxapine is a typical antipsychotic while isoloxapine, its 8Cl-isomer, shows atypicality in some animal models. The basis for this difference is not well understood. The purpose of this study was to systematically compare the two drugs in in vitro and in vivo animal models, and to understand mechanisms underlying their differential typical/atypical profiles. METHODS: The in vitro and in vivo receptor profiles as well as the action of loxapine and isoloxapine on rat conditioned avoidance response (CAR), catalepsy (CAT), striatal FOS expression and prolactin levels were determined. To understand loxapine's typical profile, we added MDL100,907, to provide loxapine+MDL the same in vivo 5-HT2/D2 ratio as isoloxapine, while holding its D2 component constant. RESULTS: Isoloxapine behaved as an "atypical" antipsychotic demonstrating CAR inhibition, low CAT, no significant prolactin elevation, and minimal FOS expression in the dorsolateral striatum. Loxapine behaved like a typical antipsychotic, showing unexpectedly high in vivo D2 occupancy. Addition of MDL100,907, which resulted in a very high 5-HT2/D2 in vivo ratio, did not alter loxapine + MDL's typical profile. CONCLUSIONS: Loxapine's behaviour as a typical antipsychotic is most likely due to its disproportionately high D2 occupancy. Appropriate action at D2 receptors in vivo, rather than the high 5-HT2/D2 ratio, seems to be critical in determining why isoloxapine behaves like an atypical antipsychotic.

Differential inverse agonist efficacies of SB-258719, SB-258741 and SB-269970 at human recombinant serotonin 5-HT7 receptors.

Recombinant 5-hydroxytryptamine 5-HT7 receptors are known to express constitutive, i.e., agonist-independent activity. Nonselective ligands, like methiothepin, ritanserin or clozapine behave as full inverse agonists at 5-HT7 receptors. The aim of the present study was to evaluate the degree of inverse agonist activity of three selective 5-HT7 receptor antagonists ((R)-3,N-dimethyl-N-[1-methyl-3-(4-methyl-piperidin-1-yl)propyl]benzene sulfonamide or SB-258719, R-(+)-1-(toluene-3-sulfonyl)-2-[2-(4-methylpiperidin-1-yl)ethyl]-pyrrolidine or SB-258741 and (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolidine-1-sulfonyl)-phenol or SB-269970) in the same model. cAMP accumulation was measured in intact Chinese hamster ovary (CHO) cells expressing human recombinant 5-HT7a receptors. In these cells, 5-HT stimulated cAMP levels and a series of ligands antagonized the effect of 5-HT with a 5-HT7 receptor-like profile. SB-258719 had no inverse agonist activity, SB-258741 behaved as a partial inverse agonist and SB-269970 was a quasi-full inverse agonist (as compared to methiothepin). The inverse agonist effect of SB-269970 was antagonized in a concentration-dependent manner by SB-258719. The widespread spectrum of inverse agonist activities shown by these compounds should help assessing the physiological relevance of constitutive 5-HT7 receptor activity in native tissues.

Pharmacologic and pharmacokinetic considerations in choosing an antipsychotic.

Recent advances in our understanding of schizophrenia along with neuroscience insights into antipsychotic medication mechanisms of action have led to a renaissance in new drug development, including an expanded therapeutic spectrum encompassing more of the symptoms encountered in schizophrenia. Atypical antipsychotics, or new generation therapies, also demonstrate greater selectivity for therapeutic actions than for extrapyramidal symptoms (EPS). Our modern armamentarium of drugs spans a wide range of pharmacologies, and it is more accurate to envision shades of gray rather than a black-and-white description for typical versus atypical properties of medications. As our paradigms for antipsychotic efficacy have shifted, a reexploration of the "older" neuroleptics is warranted to determine if they possess pharmacologic attributes that might have been overlooked during the era of high-dose neuroleptic therapy. Loxapine appears to be in the center of this spectrum, somewhere between haloperidol and risperidone. Dosing implications for drugs with a more even serotonin-2A (5-HT2A) receptor and dopamine-2 (D2) receptor blocking effect are discussed. Loxapine might have a window of partial atypicality at doses < or = 50 mg/day. These lower doses might have potential as both monotherapy in responsive patients with persistent psychotic disorders and as an adjunctive treatment in partially responding patients on concurrent atypical antipsychotic treatments. The pharmacologic properties of loxapine within its usable dosage range are quite complex and are the net sum of the parent's plus metabolites' contributions (demethylation and hydroxylation by cytochrome P450 enzymes). These pharmacologic effects include alpha-adrenergic blockade, inhibition of the noradrenergic transporter protein (reuptake inhibition), and antimuscarinic effects. Drug interactions and cigarette smoking might alter the parent-to-metabolite concentration ratios, affecting the relative atypicality of this antipsychotic therapy. Moreover, with the intramuscular formulation, which does not undergo first-pass metabolism, the parent compound of loxapine, i.e., not its metabolites, is predominantly detected in the plasma of patients, reducing the likelihood for EPS during emergency interventions in patients with positive symptoms. Further study is warranted to determine loxapine's place in our treatment of schizophrenia.

Comparative oxidation of loxapine and clozapine by human neutrophils.

The clozapine-induced agranulocytosis could be due to the formation of a reactive intermediate formed in polymorphonuclear neutrophils and granulocyte precursors with the myeloperoxidase-hydrogen peroxide system. On the contrary, no case of agranulocytosis has been described for loxapine, an other neuroleptic drug with a very close structural analogy. We have compared the clozapine and loxapine interaction with the oxidative burst and particularly with this enzymatic complex. On the one hand, the assay of the oxidative species demonstrated a different impact for the two neuroleptics. The 50% inhibitory concentration was 92 microM for hydrogen peroxide and 40 microM for hypochlorous acid for loxapine. The loxapine target is located before the myeloperoxidase-hydrogen peroxide system in the oxidative stream, whereas clozapine diverts the chlorination pathway of the enzyme. On the other hand, the in vitro metabolism of drugs by the myeloperoxidase-hydrogen peroxide system has been investigated by mass spectrometry. Loxapine remains inert but clozapine undergoes the oxidation. The glutathione or ascorbate addition in the medium leads to a removal of the oxidation. Glutathione is able to trap the toxic intermediate and could avoid its formation.

Effect of cloxazepine on mitochondria.

Cloxazepine is a neuroleptic drug synthesized at the Research Chemical Pharmaceutical Institute, Sofia. Chemically it represents 2-chloro-11 (4-methyl-1-piperazinyl)-dibenz (1,4)-oxazepine succinate. The effect of cloxazepine on respiration and phosphorylation of isolated brain mitochondria as well as on the swelling of rat liver mitochondria was examined. It has been demonstrated that the drug is a very poor inhibitor of the respiratory chain, acting mainly on its substrate end. At concentration of 0.5 mM it causes a 50 per cent inhibition of the soluble mitochondrial ATPase. It has a marked membrane action: it uncouples oxidative phosphorylation and causes swelling of the mitochondria. The results show that pharmacological action of cloxazepine is not connected with influence on the energy metabolism but with membrane action of the drug.