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.

Investigation of the photolysis and TiO2, SrTiO3, H2O2-mediated photocatalysis of an antipsychotic drug loxapine - Evaluation of kinetics, identification of photoproducts, and in silico estimation of properties.

The photolytic and photocatalytic transformation of loxapine with the use of H2O2, TiO2 and SrTiO3 under the simulated solar radiation was studied. A micro-scale method for simultaneous irradiation of multiple samples in photostability chamber was applied. RP-UHPLC-DAD coupled with ESI-Q-TOF mass spectrometer was used for the quantitative and qualitative analysis of the processes. Influence of catalysts concentration on kinetic parameters of loxapine photodecomposition was evaluated, and TiO2 at medium concentration (100 mg L-1) turned out to be the most effective. Sixteen transformation products were detected and their structures were elucidated. On the basis of the elucidated structures, computational evaluation of toxicity, bioconcentration and bioaccumulation factors as well as biodegradability of transformation products were conducted. The multivariate chemometric method (principal component analysis) was used to compare the calculated properties as well as the applied methods. Most of the transformation products were generally less toxic and more biodegradable than the parent compound.

A complementary experimental and computational study of loxapine succinate and its monohydrate.

The crystal structures of loxapine succinate [systematic name: 4-(2-chlorodibenzo[b,f][1,4]oxazepin-11-yl)-1-methylpiperazin-1-ium 3-carboxypropanoate], C18H19ClN3O(+)·C4H5O4(-), and loxapine succinate monohydrate {systematic name: bis[4-(2-chlorodibenzo[b,f][1,4]oxazepin-11-yl)-1-methylpiperazin-1-ium] succinate succinic acid dihydrate}, 2C18H19ClN3O(+)·C4H4O4(2-)·C4H6O4·2H2O, have been determined using X-ray powder diffraction and single-crystal X-ray diffraction, respectively. Fixed cell geometry optimization calculations using density functional theory confirmed that the global optimum powder diffraction derived structure also matches an energy minimum structure. The energy calculations proved to be an effective tool in locating the positions of the H atoms reliably and verifying the salt configuration of the structure determined from powder data. Crystal packing analysis of these structures revealed that the loxapine succinate structure is based on chains of protonated loxapine molecules while the monohydrate contains dispersion stabilized centrosymmetric dimers. Incorporation of water molecules within the crystal lattice significantly alters the molecular packing and protonation state of the succinic acid.

Loxapine inhalation powder: a review of its use in the acute treatment of agitation in patients with bipolar disorder or schizophrenia.

Loxapine is a well-established, first-generation antipsychotic agent. Loxapine inhalation powder (Adasuve(®)) was recently approved in the USA and the EU for use in the acute treatment of agitation in patients with bipolar disorder or schizophrenia. Inhaled loxapine is delivered by a hand-held, single-dose, single-use device that uses the Staccato(®) drug delivery system. With Adasuve(®), maximum plasma loxapine concentrations are reached in a median of 2 min. In two randomized, double-blind, placebo-controlled, multicentre trials, inhaled loxapine 5 or 10 mg significantly reduced agitation (assessed using Positive and Negative Syndrome Scale-Excited Component scores) in patients with bipolar I disorder or schizophrenia, with the onset of effect seen within 10 min of administration. Inhaled loxapine was generally well tolerated in phase III trials (which excluded patients with clinically significant acute or chronic pulmonary disease), with the most commonly occurring adverse events including dysgeusia and sedation. Inhaled loxapine is contraindicated in patients with airways disease associated with bronchospasm or acute respiratory signs or symptoms. In conclusion, inhaled loxapine provides a novel new option for use in the acute treatment of agitation in patients with bipolar disorder or schizophrenia, combining a rapid onset of effect with a noninvasive route of administration.

An investigation into the morphology of loxapine in a thermal aerosolization process from crystalline to amorphous.

A highly pure aerosol of the antipsychotic drug, loxapine, can be thermally generated through vaporization from a thin coating of loxapine on a stainless steel substrate with the formation of a condensation aerosol. Because loxapine can exist in two polymorphic forms, the morphological time course from loxapine drug substance to coating on the substrate (intermediate product) and ultimately to the aerosol was investigated using differential scanning calorimetery, X-ray diffraction (XRD), Fourier transform infrared, and Raman spectroscopy. Monoclinic and orthorhombic crystalline forms of loxapine were confirmed by single crystal and powder XRD. A mixture of both loxapine crystalline polymorphs was formed on the substrate, independent of the initial loxapine crystalline morphology, and demonstrated to be stable. The loxapine aerosols generated from the thermal aerosolization process were demonstrated to be amorphous, regardless of the initial polymorph of loxapine active pharmaceutical ingredient used. In humans, the amorphous aerosol was reported to be rapidly absorbed and the particle size resulted in rapid delivery to the deep lung.

In vitro aerosol characterization of Staccato(®) Loxapine.

Medicinal aerosol products (metered dose and dry powder inhalers) require characterization testing over a wide range of use and pre-operating stress scenarios in order to ensure robust product performance and support submissions for regulatory approval. Aerosol characterization experiments on Staccato(®) Loxapine for inhalation (Staccato Loxapine) product (emitted dose, particle size, and purity) were assessed at different operating settings (flow rates, ambient temperature and humidity, altitude, and orientation) and at nominal test conditions following exposure to various stresses on the device (mechanical shock, vibration, drop, thermal cycling, and light exposure). Emitted dose values were approximately 90% of the coated dose at every condition, meeting target specifications in each case. Aerosol purity was consistently >99.5% for every test setting, with no reportable impurities according to ICH standards (>0.1%). Particle size averaged 2µm (MMAD) and was independent of the different test conditions with the exception of different airflow rates. Particle size decreased slightly with airflow, which may assist in maintaining constant deep lung deposition. The combination of high emitted dose efficiency and a particle size range ideally suited for lung deposition, along with the consistency of these key aerosol attributes, suggests that the Staccato system has distinct advantages over more traditional aerosol systems.

Interaction of clozapine and its nitrenium ion with rat D2 dopamine receptors: in vitro binding and computational study.

The interaction of diazepine analogues like clozapine or olanzapine with D2 receptor was greatly affected by a mixture of HRP/H(2)O(2) known to induce the formation of nitrenium ion. Unlike diazepine derivatives, the oxidative mixture had low impact on the affinity of oxa- and thiazepine derivatives such as loxapine, clothiapine or JL13 for the D2 receptor. Molecular docking simulations revealed a huge difference between the mode of interaction of clozapine nitrenium ion and the parent drug. Electronic and geometric changes of the tricyclic ring system caused by the oxidation appeared to prevent the compound finding the correct binding mode and could therefore explain the difference observed in binding affinities.

A novel prodrug approach for tertiary amines. 2. Physicochemical and in vitro enzymatic evaluation of selected N-phosphonooxymethyl prodrugs.

Quaternary amine prodrugs resulting from N-phosphonooxymethyl derivatization of the tertiary amine functionality of drugs represents a novel approach for improving their water solubility. Separate reports have demonstrated the synthetic feasibility and rapid and quantitative prodrug to parent drug conversion in rats and dogs. This work is a preliminary evaluation of the physicochemical and in vitro enzymatic reversion properties of selected prodrugs. The loxapine prodrug had over a 15 000-fold increase in aqueous solubility relative to loxapine free base at pH 7.4. The loxapine prodrug was also shown to be quite stable at neutral pH values. The time for degradation product (parent drug) precipitation from an aqueous prodrug formulation would be expected to dictate the shelf life. Using this assumption, together with solubility and elevated temperature chemical stability studies, the shelf life of a parenteral formulation of the loxapine prodrug was projected to be close to 2 years at pH 7.4 and 25 degrees C. In addition, the prodrugs of cinnarizine and loxapine have been shown to be substrates for alkaline phosphatase, an enzyme found throughout the human body, and revert to the parent compound in its presence. The results from these evaluations demonstrate that the derivatives examined have many of the ideal properties required for potential clinical application.

A novel prodrug approach for tertiary amines. 3. In vivo evaluation of two N-phosphonooxymethyl prodrugs in rats and dogs.

N-phosphonooxymethyl derivatives of tertiary amine containing drugs have been identified as a novel prodrug approach for improving aqueous solubility. The in vivo reversion of two prodrugs to the corresponding parent compounds following iv and im administration to rats and dogs was investigated. Equimolar doses of parent drugs (loxapine or cinnarizine) and the corresponding prodrugs were each administered via a rapid iv infusion to rats and dogs. Equimolar doses of loxapine and its prodrug were each administered im to rats only. Blood samples were collected over 12 h, and plasma was assayed for both parent drug and intact prodrug by HPLC. Comparison of the plasma AUC for the parent drugs following administration of the parent drugs and prodrugs allowed estimation of the apparent bioavailability of parent drug from prodrug dosing. Plasma levels of the prodrugs fell below the limit of detection 5 min after iv infusion with an approximate half-life of 1 min. The mean AUCs following iv and im dosing of parent drugs were not statistically different from the parent drug AUCs obtained after prodrug dosing. The results are consistent with rapid and quantitative prodrug to parent drug reversion following administration of the phosphonooxymethyl prodrugs to the rats and dogs. This information, together with previous studies on the synthesis and physicochemical evaluation of the prodrugs, suggests that this novel prodrug strategy is a very promising approach for overcoming solubility limitations seen with many tertiary amine containing drugs at physiological pH values.

Novel prodrug approach for tertiary amines: synthesis and preliminary evaluation of N-phosphonooxymethyl prodrugs.

The synthesis and preliminary evaluation of a novel prodrug approach for improving the water solubility of drugs containing a tertiary amine group are reported. The prodrug synthesis involves a nucleophilic substitution reaction between the parent tertiary amine and a novel derivatizing reagent, di-tert-butyl chloromethyl phosphate, resulting in formation of the quaternary salt. The tertiary butyl groups are easily removed under acidic conditions with trifluoroacetic acid giving the N-phosphonooxymethyl prodrug in the free phosphoric acid form, which can subsequently be converted to the desired salt form. The synthesis was successfully applied to a model compound (quinuclidine) and to three tertiary amine-containing drugs (cinnarizine, loxapine, and amiodarone). The prodrugs were designed to undergo a two-step bioreversion process. The first step was an enzyme-catalyzed rate-determining dephosphorylation followed by spontaneous chemical breakdown of the N-hydroxymethyl intermediate to give the parent drug. Selected prodrugs were shown to be substrates for alkaline phosphatase in vitro. A preliminary in vivo study confirmed the ability of the cinnarizine prodrug to be rapidly and completely converted to cinnarizine in a beagle dog following iv administration.

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.