To develop an LC-MS/MS method for simultaneous quantification of thioridazine and its metabolites in rat plasma, samples were prepared by protein precipitation and filtration using thioridazine-d3 as an internal standard. Chromatographic separation was achieved on a reversed-phase C18 UPLC column with 0.1% formic acid in acetonitrile and 10-mM ammonium acetate in water at 0.7 mL/min. Quantification was performed using multiple reaction monitoring in positive ion mode with the following precursor-product ion transitions: m/z 371.1 → 126.1 (thioridazine), m/z 387.1 → 126.1 (mesoridazine), m/z 403.1 → 126.1 (thioridazine-2-sulfone), m/z 387.2 → 126.1 (thioridazine-5-sulfoxide), and 374.1 → 129.1 (thioridazine-d3). This method showed good linearity: 0.1-1000 ng/mL for thioridazine (r2 = 0.9980) and 0.5-1000 ng/mL for mesoridazine (r2 = 0.9981), thioridazine-2-sulfone (r2 = 0.9976), and thioridazine-5-sulfoxide (r2 = 0.9967). The accuracy of the LLOQ and QC samples for thioridazine and its metabolites was within 85%-115%, with intrabatch and interbatch precisions (CV%) below 15%. The average recovery ranged from 98% to 109%, and no significant matrix effect was observed, with values ranging from 93% to 110%. All analytes were stable under the conditions used in this study. This method was applied in a pharmacokinetic study following the intravenous administration of thioridazine to rats.

Because there is no published experience with mesoridazine during breastfeeding, other antipsychotic agents are preferred.

Mesoridazine and metoclopramide are cationic drugs that are distributed in the human brain despite being substrates of multidrug resistance protein 1 (MDR1), an efflux transporter expressed at the blood-brain barrier (BBB). We investigated their transport mechanisms at the BBB using hCMEC/D3, a human cerebral microvascular endothelial cell line often used as an in vitro BBB model. The cells exhibited time- and concentration-dependent uptake of mesoridazine and metoclopramide, with Km values of 34 and 277 µM, respectively. The uptake of both drugs significantly decreased in the presence of typical inhibitors and/or substrates of the H[+]-coupled organic cation (H[+]/OC) antiporter but not in the presence of inhibitors or substrates of organic cation transporters (OCTs), OCTN2, OATPs, SLC35F2, or the plasma membrane monoamine transporter (PMAT). Furthermore, metoclopramide uptake by hCMEC/D3 cells was pH- and energy-dependent, whereas mesoridazine uptake was unaffected by intracellular acidification and treatment with metabolic inhibitors. These results suggest that the H[+]/OC antiporter is involved in the influx of mesoridazine and metoclopramide into the brain across the BBB.

Tartary buckwheat (TB) sprout is a kind of novel nutritional vegetable, but its consumption was limited by low biomass and thin hypocotyl. The tetraploid TB sprouts was considered to be able to solve this issue. However, the nutritional quality of tetraploid TB sprouts and differences between conventional (diploid) and tetraploid TB sprouts remain unclear. In this study, the morphological traits, nutrient compositions and metabolome changes of diploid and tetraploid TB sprouts were analyzed. The water, pigments and minerals contents of TB sprouts increased during sprouting, while the contents of total soluble protein, reducing sugar, cellulose, and total phenol decreased. Compared with diploid sprouts, tetraploid sprouts had higher biomass and thicker hypocotyl. Tetraploid sprouts had higher ash and carotenoid contents, but had lower phenol and flavonoid accumulation. 677 metabolites were identified in TB sprouts by UPLC-MS analysis, including 62 diseases-resistance metabolites and 43 key active ingredients. Some key bioactive metabolites, such as rimonabant, quinapril, 1-deoxynojirimycin and miglitol, were identified. 562 differential expressed metabolites (DEMs) were identified during sprouting with seven accumulation patterns, and five hormones were found to be involved in sprout development. Additionally, 209 DEMs between diploid and tetraploid sprouts were found, and some key bioactive metabolites were induced by chromosome doubling such as mesoridazine, amaralin, atractyloside A, rhamnetin and Qing Hau Sau. This work lays a basis for the development and utilization of TB sprouts and provides evidence for the selection of tetraploid varieties to produce sprouts with high biomass and quality.

The worldwide infection with the new Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) demands urgently new potent treatment(s). In this study we predict, using molecular docking, the binding affinity of 15 phenothiazines (antihistaminic and antipsychotic drugs) when interacting with the main protease (Mpro) of SARS-CoV-2. Additionally, we tested the binding affinity of photoproducts identified after irradiation of phenothiazines with Nd:YAG laser beam at 266 nm respectively 355 nm. Our results reveal that thioridazine and its identified photoproducts (mesoridazine and sulforidazine) have high biological activity on the virus Mpro. This shows that thioridazine and its two photoproducts might represent new potent medicines to be used for treatment in this outbreak. Such results recommend these medicines for further tests on cell cultures infected with SARS-CoV-2 or animal model. The transition to human subjects of the suggested treatment will be smooth due to the fact that the drugs are already available on the market.

The lack of studies on the fate and effects of drug metabolites in the environment is of concern. As their parent compounds, metabolites enter the aquatic environment and are subject to biotic and abiotic process. In this regard, photolysis plays an important role. This study combined experimental and in silico quantitative structure-activity relationship (QSAR) methods to assess the fate and effects of Mesoridazine (MESO), a pharmacologically active human drug and metabolite of the antipsychotic agent Thioridazine, and its transformation products (TPs) formed through a Xenon lamp irradiation. After 256min, the photodegradation of MESO⋅besylate (50mgL-1) achieved 90.4% and 6.9% of primary elimination and mineralization, respectively. The photon flux emitted by the lamp (200-600nm) was 169.55Jcm-2. Sixteen TPs were detected by means of liquid chromatography-high resolution mass spectrometry (LC-HRMS), and the structures were proposed based on MSn fragmentation patterns. The main transformation reactions were sulfoxidation, hydroxylation, dehydrogenation, and sulfoxide elimination. A back-transformation of MESO to Thioridazine was evidenced. Aerobic biodegradation tests (OECD 301 D and 301F) were applied to MESO and the mixture of TPs present after 256min of photolysis. Most of TPs were not biodegraded, demonstrating their tendency to persist in aquatic environments. The ecotoxicity towards Vibrio fischeri showed a decrease in toxicity during the photolysis process. The in silico QSAR tools QSARINS and US-EPA PBT profiler were applied for the screening of TPs with character of persistence, bioaccumulation, and toxicity (PBT). They have revealed the carbazole derivatives TP 355 and TP 337 as PBT/vPvB (very persistent and very bioaccumulative) compounds. In silico QSAR predictions for mutagenicity and genotoxicity provided by CASE Ultra and Leadscope® indicated positive alerts for mutagenicity on TP 355 and TP 337. Further studies regarding the carbazole derivative TPs should be considered to confirm their hazardous character.

Tricyclic antidepressants (TCAs) are found to have an analgesic action for relieving cutaneous pain associated with neuropathies. The aim of this study was to assess cutaneous absorption and analgesia of topically applied TCAs. Percutaneous delivery was investigated using nude mouse and pig skin models at both infinite and saturated doses. We evaluated the cutaneous analgesia in nude mice using the pinprick scores. Among five antidepressants tested in the in vitro experiment, mesoridazine, promazine and doxepin showed a superior total absorption percentage. The drug with the lowest total absorption percentage was found to be fluphenazine (<7%) either at an infinite dose or at saturated solubility. The follicular pathway was important for mesoridazine and promazine delivery. Mesoridazine showed stronger skin analgesia than the other TCAs although the in vivo skin absorption of mesoridazine (0.34nmol/mg) was less than that of promazine (0.80nmol/mg) and doxepin (0.74nmol/mg). Mesoridazine had a prolonged duration of pain relief (165min) compared to promazine (83min) and doxepin (17min). The skin irritation test demonstrated an evident barrier function deterioration and cutaneous erythema by promazine and doxepin treatment, whereas mesoridazine caused no obvious adverse effect by topical application for up to 7days.

CYP2D6, a cytochrome P450 isoform, significantly contributes to the metabolism of many clinically important drugs. Thioridazine (THD) is one of the phenothiazine-type antipsychotics, which exhibit dopamine D2 antagonistic activity. THD shows characteristic metabolic profiles compared to other phenothiazine-type antipsychotics such as chlorpromazine. The sulfur atom attached to the phenothiazine ring is preferentially oxidized mainly by CYP2D6, that is, the 2-sulfoxide is a major metabolite, and interestingly this metabolite shows more potent activity against dopamine D2 receptors than THD. On the other hand, the formation of this metabolite causes many serious problems for its clinical use. Wójcikowski et al. (Drug Metab. Dispos. 2006, 34, 471) reported a kinetic study of THD formed by CYP2D6. Recently, Wang et al. (J. Biol. Chem. 2012, 287, 10834 and J. Biol. Chem. 2015, 290, 5092) revealed the crystallographic structure of THD with CYP2D6. In the current study, the binding and reaction mechanisms at the atomic and electronic levels were computationally examined based on the assumption as to whether or not the different crystallographic binding poses correspond to the different metabolites. The binding and oxidative reaction steps in the whole metabolic process were investigated using molecular dynamics and density functional theory calculations, respectively. The current study demonstrated the essential importance of the orientation of the substrate in the reaction center of CYP2D6 for the metabolic reaction.

NADPH oxidases (NOXs) constitute a family of enzymes generating reactive oxygen species (ROS) and are increasingly recognized as interesting drug targets. Here we investigated the effects of 10 phenothiazine compounds on NOX activity using an extensive panel of assays to measure production of ROS (Amplex red, WST-1, MCLA) and oxygen consumption. Striking differences between highly similar phenothiazines were observed. Two phenothiazines without N-substitution, including ML171, did not inhibit NOX enzymes, but showed assay interference. Introduction of an aliphatic amine chain on the N atom of the phenothiazine B ring (promazine) conferred inhibitory activity toward NOX2, NOX4, and NOX5 but not NOX1 and NOX3. Addition of an electron-attracting substituent in position 2 of the C ring extended the inhibitory activity to NOX1 and NOX3, with thioridazine being the most potent inhibitor. In contrast, the presence of a methylsulfoxide group at the same position (mesoridazine) entirely abolished NOX-inhibitory activity. A cell-free NOX2 assay suggested that inhibition by N-substituted phenothiazines was not due to competition with NADPH. A functional implication of NOX-inhibitory activity of thioridazine was demonstrated by its ability to block redox-dependent myofibroblast differentiation. Our results demonstrate that NOX-inhibitory activity is not a common feature of all antipsychotic phenothiazines and that substitution on the B-ring nitrogen is crucial for the activity, whereas that on the second position of the C ring modulates it. Our findings contribute to a better understanding of NOX pharmacology and might pave the path to discovery of more potent and selective NOX inhibitors.

The object of the present study was to develop and validate an assay method of mesoridazine in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples from rats were prepared by simple protein precipitation and injected onto the LC-MS/MS system for quantification. Mesoridazine and chlorpromazine as an internal standard (IS) were separated by a reversed phase C18 column. A mobile phase was composed of 10mM ammonium formate in water and acetonitrile (ACN) (v/v) by a linear gradient system, increasing the percentage of ACN from 2% at 0.4min to 98% at 2.5min with 4min total run time. The ion transitions monitored in positive-ion mode [M+H](+) of multiple-reaction monitoring (MRM) were m/z 387>126 for mesoridazine and m/z 319>86 for IS. The detector response was specific and linear for mesoridazine at concentrations within the range 0.001-4μg/ml and the correlation coefficient (R(2)) was greater than 0.999 and the signal-to-noise ratios for the samples were ≥10. The intra- and inter-day precision and accuracy of the method were determined to be within the acceptance criteria for assay validation guidelines. The matrix effects were approximately 101 and 99.5% from rat plasma for mesoridazine and chlorpromazine, respectively. Mesoridazine was stable under various processing and/or handling conditions. Mesoridazine concentrations were readily measured in rat plasma samples after intravenous and oral administration. This assay method can be practically useful to the pharmacokinetic and/or toxicokinetic studies of mesoridazine.