Plasmodium

Methylene blue functions as a substitute electron acceptor/donor, which helps to repair mitochondria, enhances neural energy generation, and prevents superoxide from forming[1]. Cytochrome P450 (CYP) isozymes are inhibited by methylene blue. When combined with water, methylene blue, an odorless, water-soluble, dark blue-green crystalline powder, turns blue. A vasopressor called methylene blue inhibits inducible NOS, which in turn inhibits the activation of sGC, which is how it affects the NO synthesis pathway. Methylene blue also inhibits the buildup of cyclic GMP (cGMP) by attaching to the iron heme moiety of sGC and directly competing with NO for the ability to activate soluble guanylyl cyclase[3].

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LPS-activated BV2 microglia can alter their immunological features and have their levels of CD14, IL-1β, TNF-α, and CCL2 mRNA decreased by methylene blue (Basic Blue 9) (4.5 μM; BV2 microglia) [3].

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Methylene blue (MB), the first lead chemical structure of phenothiazine and other derivatives, is commonly used in diagnostic procedures and as a treatment for methemoglobinemia. We have previously demonstrated that MB could function as an alternative mitochondrial electron transfer carrier, enhance cellular oxygen consumption, and provide protection in vitro and in rodent models of Parkinson's disease and stroke. In the present study, we investigated the structure-activity relationships of MB in vitro using MB and six structurally related compounds. MB reduces mitochondrial superoxide production via alternative electron transfer that bypasses mitochondrial complexes I-III. MB mitigates reactive free radical production and provides neuroprotection in HT-22 cells against glutamate, IAA and rotenone toxicity. Distinctly, MB provides no protection against direct oxidative stress induced by glucose oxidase. Substitution of a side chain at MB's 10-nitrogen rendered a 1000-fold reduction of the protective potency against glutamate neurototoxicity. Compounds without side chains at positions 3 and 7, chlorophenothiazine and phenothiazine, have distinct redox potentials compared to MB and are incapable of enhancing mitochondrial electron transfer, while obtaining direct antioxidant actions against glutamate, IAA, and rotenone insults. Chlorophenothiazine exhibited direct antioxidant actions in mitochondria lysate assay compared to MB, which required reduction by NADH and mitochondria. MB increased complex IV expression and activity, while 2-chlorphenothiazine had no effect. Our study indicated that MB could attenuate superoxide production by functioning as an alternative mitochondrial electron transfer carrier and as a regenerable anti-oxidant in mitochondria[4].

In male Sprague-Dawley rats (7-week-old, 200-250 g), methylene blue (1, 5, and 25 μg/rat) dramatically reduces brain cyclic guanosine monophosphate (cGMP) content and sevoflurane minimum alveolar anesthetic concentration (MAC) in a dose-dependent manner[2]. Methylene blue is sprayed into the gastrointestinal tract mucosa during chromoendoscopy as a dye to detect dysplasia, or precancerous lesions[2]. Mean arterial pressures (MAP) can be normalized, vascular tone can be restored, and the need for vasopressors can be decreased with methylene blue[3].

Mitochondria Membrane Potential Analysis[4]
Mitochondrial membrane potential was analyzed by FRET using TMRE/NAO as described previously. TMRE quenches the NAO fluorescence under normal mitochondria membrane potential. As the membrane potential collapses, the TMRE fluorescence decreases, which results in an increase in NAO fluorescence. The increased NAO fluorescence is interpreted as a decrease in the mitochondria membrane potential. Cells were incubated with glutamate and Methylene blue (MB) or related compounds for 12 hours. The media was then removed and the cells were washed once with PBS, then incubated in PBS containing 1 µM NAO and 1 µM TMRE for 30 minutes at 37°C. The NAO/TMRE was removed and cells were incubated for an additional 15 minutes at 37°C in KRH. Cells were washed twice in PBS and NAO fluorescence was measured using a Tecan Infinite F200 plate reader (excitation 485, emission 530). Raw data are represented as RFU. The NAO fluorescence was then standardized based on control and Calcein AM cell viability.

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Reactive Oxygen Species Analysis[4]
Changes in cellular ROS were measured by the ROS reactive fluorescent indicator H2DCFDA (Anaspec) using a fluorescent microplate reader, flow cytometry, and fluorescent microscopy. For the microplate experiment, HT-22 cells were plated overnight at a density of 3,000 cells/well in a 96-well plate. Cells were incubated with drug and 20 mM glutamate for 12 hours at 37°C and 5% CO2. The media was then removed and the cells were washed once with PBS then incubated in PBS containing 10 µM H2DCFDA for 30 minutes at 37°C. The PBS was removed and cells were incubated for an additional 15 minutes at 37°C in KRH. Cells were washed twice in PBS and DCF fluorescence was measured using a Tecan Infinite F200 plate reader (excitation 485, emission 530). Raw data are represented as RFU. The DCF fluorescence was then standardized based on control and Calcein AM cell viability. For fluorescent microscopy, HT-22 cells were plated at a density of 10,000 cells/well in a 6-well plate. Cells were incubated for 8 hours in glutamate and indicated drug. After 8 hours, media was replaced with KRH media containing 10 µM H2DCFDA. Cells were incubated at 37°C for 15 minutes, washed once with KRH and incubated an additional 10 minutes in fresh KRH at 37°C. The media was replaced with fresh KRH buffer and the cells imaged. For flow cytometry, HT-22 cells were seeded at a density of 50,000 cells/well in 6-well dishes and attached overnight. Media was removed and replaced with fresh DMEM (high glucose, 1 mM pyruvate, 10% FBS) containing vehicle, 10 µM Methylene blue (MB), 20 mM glutamate, or 10 µM Methylene blue (MB) and20 mM glutamate. Cells were incubated for 8 hours at 37°C and 5% CO2. Following the incubation, the media was removed, the cells were washed once with PBS, and incubated in PBS containing 10 µM H2DCFDA for 15 minutes at 37°C. The PBS was removed and cells were incubated for an additional 10 minutes at 37°C in PBS. The PBS was replaced with fresh PBS and the DCF fluorescence was determined with a Beckman Coulter FC-500.

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Mitochondria Lysate Oxidation Assay[4]
Four compounds (Methylene blue (MB), NR, 2-chlorophenothiazine, and chlorpromazine) were assayed in 10 mM phosphate buffer (pH = 7.4) with 500 µM H2O2, 10 µM DCF and in the presence or absence of 165 µM NADH and mitochondria lysate (19.4 µg/ml). Assay took place in Greiner 96-well black plates for 30 minutes at 37°C, at which time the DCF fluorescence was measured with a Tecan Infinite F200 plate reader (excitation 485, emission 530).

Cell Viability Assay[4]
Cell viability was determined by Calcein AM and MTT assays. For the Calcein AM assay, HT-22 cells were seeded at a density of 3,000 cells/well and were incubated overnight in 96-well plates in 100 µl of DMEM (high glucose with 1 mM pyruvate and 10% FBS). Varying concentrations of Methylene blue (MB) or its derivatives and 20 mM glutamate were added to each well and incubated for 12 hours at 37°C with 5% CO2. After 12 hours, media was removed and replaced with a 1 µM solution of Calcein AM in PBS. Cells were incubated for 5 minutes at 37°C and fluorescence was measured using a Tecan Infinite F200 plate reader (excitation 485 emission 530). For the MTT assay, HT-22 cells were seeded into 96-well, flat-bottomed plates at a density of 3000 cells/well in 100 µl DMEM (high glucose, 1 mM pyruvate, 10% FBS) and allowed to attach overnight. Varying concentrations of drug and 20 mM glutamate (or media for control wells) was then added to each well. Plates were incubated for 12 hours at 37°C with 5% CO2. Plates were removed from the incubator and 20 µl MTT (5 mg/ml in PBS) was added per well. The plates were agitated gently to mix the MTT into the media and then returned to the incubator for 2 hours. After 2 hours the media was removed and 100 µl of DMSO was added to each well. The plate was mixed by gentle agitation and the absorbance was measured (560 nm with a reference of 670 nm) with a Tecan Infinite F200 plate reader.

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Rotenone Neurotoxicity Assay[4]
HT-22 cells were seeded into 96-well flat-bottomed plates at a density of 3000 cells/well in 100 µl DMEM (high glucose, 1 mM pyruvate, 10% FBS) and allowed to attach overnight. Varying concentrations of Methylene blue (MB) or its derivatives and 5 µM rotenone (or media for control wells) was then added to each well. Plates were incubated for 24 hours at 37°C with 5% CO2. Viability was determined by Calcein AM assay.

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Glucose Oxidase Neurotoxicity Assays[4]
HT-22 cells were seeded into 96-well flat-bottomed plates at a density of 3000 cells/well in 100 µl DMEM (high glucose, 1 mM pyruvate, 10% FBS) and allowed to attach overnight. Varying concentrations of Methylene blue (MB) or its derivatives and 2 U glucose oxidase (or media for control wells) was then added to each well. Plates were incubated for 3 hours at 37°C with 5% CO2. Viability was determined by Calcein AM assay.

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Iodoacetic Acid (IAA) Neurotoxicity Assays[4]
HT-22 cells were seeded into 96-well flat-bottomed plates at a density of 3000 cells/well in 100 µl DMEM (high glucose, 1 mM pyruvate, 10% FBS) and allowed to attach overnight. Varying concentrations of Methylene blue (MB) or its derivatives and 20 µM IAA (or media for control wells) was then added to each well. Plates were incubated for 2 hours at 37°C with 5% CO2. After 2 hours, all media was removed and replaced with fresh media containing drugs, but not IAA. The plates were incubated an additional 22 hours at 37°C with 5% CO2. Viability was determined by Calcein AM assay.

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Western Blot[4]
HT-22 cells were plated at a density of 150000/well in a 6-well plate. Cells attached overnight and either Methylene blue (MB) or 2-chlorophenothiazine was added to the cells the following day at the indicated concentrations. Cells were grown for 3 days and lysed in radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitors. Cell lysate was loaded onto a 10% polyacrylamide gel and transferred onto nitrocellulose. Nitrocellulose was incubated with primary antibody overnight at 4°C at the indicated concentrations (Cox1, 1∶500; Actin, 1∶3000). Secondary antibody linked to horseradish peroxidase was incubated for 2 hours at room temperature (1∶2000 dilution). Chemiluminescence was detected with a UVP Biospectrum 500.

The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signal pathway plays an important role in anesthetic and analgesic effects. We sought to determine the involvement of inhibition of soluble guanylyl cyclase (sGC) in the anesthetic mechanism and site of action of volatile anesthetics. We examined the effect of intracerebroventricular (ICV) administration of methylene blue (MB), a sGC inhibitor, on the minimum alveolar anesthetic concentration (MAC) of sevoflurane and the brain cGMP content in rats in vivo. We also investigated the effect of sevoflurane on NO-stimulated sGC activity in vitro. The rats were divided into three groups. After the ICV administration of MB, sevoflurane MAC and brain cGMP contents were measured in the first group and the second group, respectively. In the third group, brain cGMP contents were determined after sevoflurane anesthesia without the ICV administration of MB to examine the direct effect of sevoflurane on brain cGMP contents. MB significantly decreased sevoflurane MAC and brain cGMP content in a dose-dependent manner. Sevoflurane itself also dose-dependently decreased cGMP contents in brain in vivo and inhibited the NO-stimulated sGC activity in vitro. These results suggest that the inhibition of the NO-cGMP signal pathway at the sGC level could be involved in anesthetic or analgesic effects, and the inhibitory effect of sevoflurane on sGC would be one of the sites of action of this anesthetic. Implications: Because the nitric oxide-cyclic guanosine monophosphate signal pathway mediates nociception and the site of action of halogenated volatile anesthetics in uncertain, we examined the possible involvement of inhibition of soluble guanylyl cyclase in the anesthetic mechanism. The inhibitory effect of sevoflurane on soluble guanylyl cyclase could be one of sites of this anesthetic.[3]

Absorption, Distribution and Excretion
Excreted primarily in urine and bile. Approximately 75% of the oral dose is excreted in the urine, mainly as a stable, colorless form of methylene blue. 10 mg/kg (rat). 3.0 ± 0.7 L/min. …The concentrations of methylene blue in whole blood were determined by high-performance liquid chromatography (HPLC) in seven volunteers after intravenous and oral administration of 100 mg methylene blue (with or without mesna). Distribution of methylene blue in different tissues was determined after intraduodenal and intravenous administration in rats. Following intravenous administration, the concentration-time curve of methylene blue in whole blood showed a multiphasic change, with an estimated terminal half-life of 5.25 hours. After oral administration, the area under the concentration-time curve was significantly reduced (9 nmol/min/mL vs 137 nmol/min/mL). Co-administration with mesna (a drug that may affect drug distribution through ion-pair interactions) did not alter the pharmacokinetics of methylene blue. Following intravenous administration, the urinary excretion of methylene blue and its leukotriene forms was only slightly higher than that of intravenous administration (18% vs 28% dose). In rats, duodenal administration resulted in higher drug concentrations in the intestinal wall and liver than intravenous administration, but lower drug concentrations in whole blood and brain tissue. The difference in organ distribution of methylene blue is the primary reason for the different pharmacokinetic profiles after oral and intravenous administration. …Methylene blue is well absorbed in the gastrointestinal tract, reaching peak plasma concentrations approximately 1–2 hours after oral administration. …After distribution to tissues, methylene blue is rapidly reduced to leukotriene methylene blue (leukotriene methyl sulfonium chloride). Neonates may metabolize leukotriene methylene blue less efficiently than adults. Methylene blue is excreted in urine and bile. Approximately 75% of orally administered methylene blue is excreted in urine, primarily as the stable, colorless form of methylene blue (white methylene blue). Urine exposed to air turns green or blue due to the presence of the oxidation product methylene blue sulfone (methylene blue sulfone). Some unmetabolized drug is also excreted in urine. Background: Although methylene blue dye is routinely used for lymphatic tracing, it is not used for lymphatic tracing of pregnancy-associated breast cancer due to concerns about fetal risks. Methods: To investigate the safety of methylene blue dye for lymphatic tracing of breast cancer during pregnancy, we conducted a pharmacokinetic study of methylene blue dye in 10 non-pregnant women and extrapolated the results to estimate the maximum fetal exposure to the dye. Results: Plasma and urine tests showed that the dye rapidly distributed from the breast injection site into the bloodstream, with 32% of the total dose excreted in the urine within 48 hours. Based on existing data on organ distribution of methylene blue, the maximum fetal dose was estimated to be 0.25 mg (5% of the administered dose), and other pregnancy-related physiological factors may further reduce this dose. Conclusion: The analysis indicates that methylene blue dye can be used for lymphatic tracing of pregnancy-associated breast cancer with extremely low fetal risk. Pharmacokinetics of methylene blue distribution in vivo and urinary excretion were determined after intravenous injection of 15 mg/kg methylene blue in adult female sheep. The effects of methylene blue alone versus injection of 50 mg/kg sodium nitrite followed by methylene blue were compared. The overall elimination rate constant (β) of methylene blue was 0.0076 ± 0.0016 min⁻¹, unaffected by prior sodium nitrite injection. However, sodium nitrite significantly altered the distribution rate of methylene blue. Despite its relatively short half-life, very little methylene blue is excreted in urine as unchanged or colorless form. ...

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Metabolism/Metabolites
After distribution to tissues, methylene blue is rapidly reduced to methylene blue (methylene blue chloride). Neonates may metabolize methylene blue less efficiently than older adults.
Methylene blue can be reduced to colorless methylene blue; these compounds together constitute a reversible redox system. Low concentrations of methylene blue can accelerate the conversion of methemoglobin to hemoglobin. In patients with methemoglobinemia, methylene blue is reduced to methylene blue by methemoglobin reductase in erythrocytes; methylene blue then reduces methemoglobin to hemoglobin. High concentrations of methylene blue can oxidize the ferrous iron in reduced hemoglobin to ferric iron, thereby converting hemoglobin into methemoglobin. After distribution to tissues, methylene blue is rapidly reduced to leucomycete methylene blue (leucomycete methylene blue chloride). Neonates may metabolize leucomycete methylene blue less efficiently than adults. Biological half-life: 5-6.5 hours (after intravenous injection). The estimated half-life of methylene blue after intravenous injection is 5-6.5 hours. …The concentration of methylene blue in whole blood was determined in 7 volunteers after intravenous and oral administration of 100 mg methylene blue (with or without mesna) using high-performance liquid chromatography. The distribution of methylene blue in different tissues in rats was determined after intraduodenal and intravenous injection. The concentration change of methylene blue in whole blood after intravenous injection showed a multiphasic process, with an estimated terminal half-life of 5.25 hours.

Protein Binding
Methylene blue has been reported to bind strongly to rabbit plasma (71-77% of the drug is bound). Identification and Uses: Methylene blue is a solid. Its aqueous solution is deep blue. It is used as a bacteriological stain, mixing indicator, dye, redox colorimetric agent, and melanoma targeting agent. It is also used to treat drug-induced methemoglobinemia. Human Studies: Methemoglobinemia is commonly treated with methylene blue. However, in patients with glucose-6-phosphate dehydrogenase deficiency, methylene blue can induce methemoglobinemia. Preclinical studies have shown that low-dose methylene blue can increase the activity of mitochondrial cytochrome oxidase in the brain and improve memory retention after learning tasks, including fear extinction. Intraocular injection of 1% methylene blue has cytotoxic effects on corneal endothelium and iris epithelium. Several suspected cases of serotonin syndrome have been reported among patients receiving methylene blue in combination with serotonin-active drugs. Other reports indicate allergic hypersensitivity reactions following transfusion of methylene blue-treated plasma. One premature infant developed methemoglobinemia and hemolytic anemia after enteral administration of methylene blue. Epidemiological evidence suggests that methylene blue is teratogenic, and its use during pregnancy may harm the fetus. The use of methylene blue during amniocentesis has been associated with neonatal ileal and jejunal atresia, ileal obstruction, and other adverse reactions. Use of methylene blue during pregnancy can lead to hemolytic anemia, hyperbilirubinemia, methemoglobinemia, respiratory distress, skin staining, and phototoxicity in newborns. Animal studies: Methylene blue treatment leads to methemoglobin formation and oxidative damage to erythrocytes, resulting in regenerative anemia and a series of tissue and biochemical changes secondary to erythrocyte damage. One early change is a dose-dependent increase in methemoglobin levels, with similar responses in rats and mice. Mice appear to be more prone to Heinz body formation and anemia than rats, characterized by decreased hemoglobin, hematocrit, and erythrocyte count. Splenomegaly was observed at necropsy in all tested mice and rats in the 100 mg/kg (male only) and 200 mg/kg dose groups. Methylene blue is embryotoxic in rats. Methylene blue caused mice to give birth before day 18 of gestation (full term). This response was observed in 45%, 50%, and 83% of animals treated with 50, 60, and 85 mg/kg methylene blue, respectively. Under cell-free conditions, methylene blue induces DNA damage. This is characterized by the production of large amounts of base modifications sensitive to the repair endonuclease FPG protein (formamide pyrimidine-DNA glycosidase). Methylene blue is mutagenic in cultured mammalian cells. In contrast, the results of the mouse micronucleus assay showed that its genotoxicity was not significant in vivo. The greatest concern regarding the use of methylene blue in veterinary medicine is the potential for Heinz body anemia or other red blood cell morphological changes, methemoglobinemia, and shortened red blood cell lifespan. Cats are often very sensitive to these effects, and some believe that methylene blue is contraindicated in felines. However, dogs and horses may also experience adverse hematologic reactions at relatively low doses. Ecotoxicity studies: Methylene blue has teratogenic effects on angelfish. Non-human toxicity values: Mouse intravenous LD50: 77 mg/kg; Mouse intraperitoneal LD50: 150 mg/kg; Mouse oral LD50: 3500 mg/kg; Rat intravenous LD50: 1250 mg/kg

[1]. Methylene blue and its analogues as antidepressant compounds. Metab Brain Dis. 2017 Oct;32(5):1357-1382.

[2]. Methylene blue, a soluble guanylyl cyclase inhibitor, reduces the sevoflurane minimum alveolar anesthetic concentration and decreases the brain cyclic guanosine monophosphate content in rats. Anesth Analg. 1999 Aug;89(2):484-9.

[3]. Intraoperative vasoplegia: methylene blue to the rescue! Curr Opin Anaesthesiol. 2018 Feb;31(1):43-49.

[4]. Neuroprotective actions of methylene blue and its derivatives. PLoS One. 2012;7(10):e48279.

Methylene blue trihydrate is a tasteless or nearly tasteless dark green crystal with a bronze luster, or a dark green crystalline powder. The pH (1% aqueous solution) is 3 to 4.5. (NTP, 1992)
Methylene blue is a synthetic basic dye. Methylene blue can stain negatively charged cellular components (such as nucleic acids); in tumor surgery, when injected into the tumor lymph bed, methylene blue can stain the lymph nodes draining the tumor, thus aiding in the imaging localization of the tumor sentinel lymph nodes. At low doses intravenously, the drug can convert methemoglobin to hemoglobin.
The compound consists of dark green crystals or crystalline powder with a bronze luster. Its aqueous or alcoholic solution is deep blue. Methylene blue is used as a bacterial stain and indicator. It inhibits guanylate cyclase and has been used to treat cyanide poisoning and reduce methemoglobin levels. Methylene blue is an organochloride salt with its counterion 3,7-bis(dimethylamino)phenothiazine-5-onium. It is a commonly used dye with antioxidant, antimalarial, antidepressant, and cardioprotective effects. It is used as an EC 1.4.3.4 (monoamine oxidase) inhibitor, acid-base indicator, fluorescent dye, antidepressant, cardioprotective agent, EC 3.1.1.8 (cholinesterase) inhibitor, histological dye, EC 4.6.1.2 (guanylate cyclase) inhibitor, antioxidant, antibacterial agent, neuroprotective agent, physical tracer, and antimalarial drug. It contains 3,7-bis(dimethylamino)phenothiazine-5-onium. Methylene blue is a redox agent. Intravenous methylene blue is FDA approved for the treatment of acquired methemoglobinemia in children and adults. Historically, it was widely used in Africa to treat malaria, but it has been phased out with the availability of chloroquine (CQ) and other drugs. Its use as a urinary tract disinfectant has also been studied. Methylene blue chloride (INN, or methylene blue, with the proposed trade name Rember) is an investigational drug being developed by the University of Aberdeen and TauRx Therapeutics. Early clinical trials have shown that it can inhibit the aggregation of Tau protein. This drug has potential value in treating Alzheimer's disease patients. Methylene blue is a synthetic basic dye. Methylene blue can stain negatively charged cellular components (such as nucleic acids); in tumor surgery, injecting methylene blue into the tumor lymph bed can stain the lymph nodes draining the tumor, thus aiding in the imaging and localization of the tumor sentinel lymph nodes. At low doses intravenously, the drug can convert methemoglobin to hemoglobin. Methylene blue is a compound consisting of dark green crystals or crystalline powder with a bronze luster. Its aqueous or alcoholic solution is deep blue. Methylene blue can be used as a bacterial stain and indicator. It inhibits guanylate cyclase and has been used to treat cyanide poisoning and lower methemoglobin levels. Drug Indications For the treatment of acquired methemoglobinemia in children and adults. Other clinical applications of methylene blue include improving hypotension associated with various clinical conditions, as a disinfectant for urinary tract infections, treating hypoxia and hyperdynamic circulation caused by cirrhosis and severe hepatopulmonary syndrome, and treating neurotoxicity caused by ifosfamide. Lumeblue is indicated as a diagnostic agent to enhance the imaging of colorectal lesions in adult patients undergoing screening or monitoring colonoscopy. For the acute symptomatic treatment of acute methemoglobinemia caused by drugs and chemicals. Methylthiocyanate Chloride Proveblue is indicated for adults, children, and adolescents (0 to 17 years of age). Mechanism of Action The primary mechanism of action involves the inhibition of nitric oxide synthase and guanylate cyclase. In Alzheimer's disease: A mechanistic study found that methylene blue oxidizes cysteine sulfhydryl groups on tau protein to maintain its monomeric state. A preclinical treatment study in mice with tau protein disease reported that methylene blue can mediate anti-inflammatory or neuroprotective effects through Nrf2/antioxidant response elements (AREs); another study reported that early administration reduced insoluble tau protein and improved learning and memory. In methemoglobinemia: Methylene blue exerts its effect by reacting with erythrocytes to generate leukocyte methylene blue, a reducing agent that reduces ferric ions (Fe+++) in oxidized hemoglobin to oxygen-carrying ferrous ions (Fe++). As an antimalarial drug: Methylene blue is a specific inhibitor of glutathione reductase in Plasmodium falciparum, with the potential to reverse chloroquine resistance. Its mechanism of action is similar to that of 4-aminoquinoline antimalarial drugs, preventing heme from polymerizing into heme crystals. Regarding ifosfamide-induced neurotoxicity: Methylene blue acts as an alternative electron acceptor. It reverses NADH inhibition induced by hepatic gluconeogenesis and blocks the conversion of chloroethylamine to chloroacetaldehyde. Furthermore, it inhibits the activity of various amine oxidases, thereby preventing the formation of chloroacetaldehyde.
This study investigated the mechanism by which methylene blue (a potential inhibitor of soluble guanylate cyclase) regulates cyclic guanosine monophosphate (cGMP) accumulation in cultured rabbit pulmonary artery smooth muscle cells. In short-term co-culture, control or methylene blue-pretreated rabbit pulmonary artery smooth muscle cells were stimulated with sodium nitroprusside, nitrosothiols, or endothelial relaxing factor basally released from bovine pulmonary artery endothelial cells. The hypothesized endothelial relaxants S-nitroso-L-cysteine, stable deamination analogs of S-nitroso-L-cysteine, S-nitroso-3-mercaptopropionic acid, and sodium nitroprusside all increased the level of cyclic guanosine monophosphate (cGMP) in rabbit pulmonary artery smooth muscle cells in a concentration-dependent manner (1–100 μM) (by 1.5–12-fold). Methylene blue pretreatment inhibited S-nitroso-L-cysteine and sodium nitroprusside-induced cGMP accumulation by 51%–100%, but had no effect on the S-nitroso-3-mercaptopropionic acid-mediated response. The inhibitory effect of methylene blue on nitrovasodilator-induced cGMP accumulation could be quantitatively reproduced by the extracellular superoxide anion generation from xanthine (100 μM) and xanthine oxidase (5 mU). Similar to methylene blue pretreatment, superoxide anion generation had no effect on basal cGMP levels or S-nitroso-3-mercaptopropionic acid-induced cGMP responses. Furthermore, methylene blue dose- and time-dependently induced superoxide anion generation in rabbit pulmonary artery smooth muscle cells, as confirmed by spectrophotometric determination of cytochrome c reduction. The inhibitory effect of methylene blue on S-nitroso-L-cysteine and sodium nitroprusside-induced cGMP accumulation was completely blocked by superoxide dismutase but not by catalase. Selective pretreatment of endothelial cells with methylene blue before co-culturing with untreated rabbit pulmonary artery smooth muscle reduced cyclic guanosine monophosphate (cGMP) levels in rabbit pulmonary artery smooth muscle to a degree comparable to that observed when methylene blue-pretreated rabbit pulmonary artery smooth muscle was co-cultured with untreated endothelial cells, and this reduction was partially inhibited by superoxide dismutase. Schizophrenia is a major public health problem affecting approximately 1% of the global population. Phenylopridine (PCP) is a drug with significant psychogenic properties that can induce schizophrenia-like symptoms in humans. PCP disrupts pre-pulse inhibition of the vocal startle reflex in rodents, an inhibition also confirmed in patients with schizophrenia. Nitric oxide synthase (NOS) inhibitors block this effect, suggesting that nitric oxide plays a crucial role in this effect of PCP. Methylene blue, an inhibitor of guanylate cyclase and nitric oxide synthase, has shown efficacy as adjunctive therapy to conventional antipsychotics in the treatment of schizophrenia. This study aimed to investigate whether the disruption of pre-pulse inhibition induced by PCP (4 mg/kg) in mice was affected by methylene blue (50 or 100 mg/kg). Furthermore, this study investigated the effect of methylene blue (50 mg/kg) on the kinetic hyperactivity induced by PCP (4 mg/kg). The results showed that PCP readily disrupted pre-pulse inhibition in mice without affecting the impulse-only stimulation test. The study also found that methylene blue inhibited the reduction of pre-pulse inhibition induced by PCP in a dose-dependent manner. In addition, methylene blue pretreatment can reduce the hyperkinetic effects induced by phencyclidine. The results of this study further support the view that the pharmacological and behavioral effects of phencyclidine are related to the nitric oxide synthase/guanylate cyclase pathway. Since phencyclidine also has psychotic-like features, drugs that interfere with the nitric oxide synthase/guanylate cyclase pathway may also have therapeutic value for the treatment of schizophrenia. [1]

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Introduction: Neurofibrillary tangles (NFTs) composed of Tau protein are a hallmark of neurodegenerative changes in Alzheimer's disease. Transgenic mice expressing full-length pro-aggregating human Tau protein (2N4R Tau-ΔK280, or Tau(ΔK)) or its repeating domain (TauRD-ΔK280, or TauRD(ΔK)) gradually develop Tau protein pathological changes, including missorting, phosphorylation, aggregation, synaptic loss and functional defects of Tau protein. TauRD(ΔK) assembles into neurofibrillary tangles (NFTs) and is associated with neuronal death, while Tau(ΔK) aggregates to form Tau proteotangles without causing significant neuronal loss. Both forms of Tau protein lead to similar cognitive decline (onset at 10 and 12 months, respectively), and cognitive decline is reversible upon shutting down transgenic expression in both cases. Since methylene blue (MB) can inhibit Tau protein aggregation in vitro, we investigated whether MB could prevent or improve Tau protein-induced cognitive impairment in our mouse model. We administered MB orally to two types of mice using different prophylactic and therapeutic regimens, starting before or after disease onset. Cognitive status in mice was assessed using behavioral tests (open field test, Morris water maze test) to determine the optimal therapeutic intervention. Results: Neither prophylactic nor therapeutic MB application prevented or improved learning and memory deficits in TauRD(ΔK) mice. Similarly, therapeutic MB treatment initiated after the onset of cognitive impairment was ineffective in Tau(ΔK) mice. Conversely, prophylactic use of methylene blue (MB) before the onset of functional impairment protected cognitive function in Tau(ΔK) mice. In addition to improvements in learning and memory, MB-treated Tau(ΔK) mice also showed significant reductions in insoluble Tau protein, conformationally altered Tau protein (MC1) and phosphorylated Tau protein (AT180, PHF1), and upregulation of protein degradation systems (autophagy and proteasome). This suggests that MB has other pleiotropic effects in addition to its properties as a Tau protein aggregation inhibitor. Conclusion: Our data support the use of Tau protein aggregation inhibitors as potential drugs for the treatment of Alzheimer's disease and other tau protein diseases and highlight the need for prophylactic treatment before the onset of cognitive impairment. [2]

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Traumatic brain injury (TBI) is associated with cerebral edema, blood-brain barrier disruption and neuroinflammation, all of which can affect the severity of the injury and functional recovery. Unfortunately, there are currently no effective prophylactic treatments to limit the short-term or long-term consequences of TBI. Therefore, this study aimed to determine the efficacy of the antioxidant methylene blue (MB) in reducing inflammation and behavioral complications associated with diffuse brain injury. We found that intravenous injection of MB immediately after midline fluid impact injury in mice (15–30 minutes after traumatic brain injury) reduced cerebral edema, inhibited microglia activation, reduced neuroinflammation, and improved behavioral recovery. Specifically, MB significantly reduced the expression of traumatic brain injury-related cerebral edema and inflammatory genes in the hippocampus one day after injury. Furthermore, MB intervention attenuated the expression of traumatic brain injury-induced inflammatory genes (interleukin [IL]-1β, tumor necrosis factor α) in enriched microglia/macrophages one day after injury. Lipopolysaccharide-activated BV2 microglia culture experiments confirmed that MB treatment directly reduced IL-1β levels and increased IL-10 levels in microglia. Finally, researchers assessed functional recovery and depressive-like behaviors within one week after brain injury. MB intervention did not prevent the decline in body weight or motor coordination within 1–7 days after brain injury. However, MB reduced the development of acute depressive-like behavior 7 days after traumatic brain injury. In conclusion, immediate intervention with MB can effectively reduce neuroinflammation and improve behavioral recovery after diffuse brain injury. Therefore, MB intervention may reduce life-threatening complications of traumatic brain injury, including cerebral edema and neuroinflammation, and prevent the occurrence of neuropsychiatric complications. [3]

C16H24CLN3O3S

373.90

373.122

C, 51.40; H, 6.47; Cl, 9.48; N, 11.24; O, 12.84; S, 8.57

7220-79-3

Methylene blue trihydrate;7220-79-3;Methylene blue hydrate;122965-43-9; 61-73-4 (Cl)

104827

Light brown to black solid powder

0.98 g/mL at 25 °C

190 °C (dec.)(lit.)

14 °C

3

7

1

24

483

0

XQAXGZLFSSPBMK-UHFFFAOYSA-M

InChI=1S/C16H18N3S.ClH.3H2O/c1-18(2)11-5-7-13-15(9-11)20-16-10-12(19(3)4)6-8-14(16)17-13;;;;/h5-10H,1-4H3;1H;3*1H2/q+1;;;;/p-1

[7-(dimethylamino)phenothiazin-3-ylidene]-dimethylazanium;chloride;trihydrate

Methylene Blue trihydrate; 7220-79-3; Phenothiazin-5-ium, 3,7-bis(dimethylamino)-, chloride, trihydrate; C.I. Basic Blue 9 trihydrate; Methylthionine chloride; Basic Blue 9 trihydrate; 3,7-Bis(dimethylamino)phenothiazin-5-ium chloride trihydrate; C.I. Basic Blue 9, trihydrate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100 mg/mL (267.45 mM)

10% DMSO+ 40% PEG300+ 5% Tween-80+ 45% saline : ≥ 2.5 mg/mL (6.69 mM) (Please use freshly prepared in vivo formulations for optimal results.)