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 in urine and bile. About 75% of an oral dose excreted in urine, primarily as stabilized colorless leukomethylene blue.
10 mg/kg (in rats).
3.0 ± 0.7 L/min.
... The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in seven volunteers after IV and oral administration of 100 mg methylene blue with and without mesna. The distribution of methylene blue in different tissues was measured in rats after intraduodenal and IV application. The time course of methylene blue in whole blood after IV administration showed a multiphasic time course with an estimated terminal half-life of 5.25 hr. Following oral administration, the area under the concentration-time curve was much lower (9 nmol/min/mL vs 137 nmol/min/mL). Co-administration of mesna, which could influence distribution by ion-pairing, did not alter the pharmacokinetics. The urinary excretion of methylene blue and its leukoform was only moderately higher after IV administration (18% vs 28% dose). Intraduodenal administration to rats resulted in higher concentrations in intestinal wall and liver but lower concentrations in whole blood and brain than IV methylene blue. Differences in organ distribution of methylene blue are mainly responsible for the different pharmacokinetics after oral and IV administration. ...
Methylene blue is well absorbed from the GI tract, and peak plasma concentrations occur approximately 1-2 hours after an oral dose. ... Following distribution into tissues, methylene blue is rapidly reduced to leukomethylene blue (leucomethylthioninium chloride). Metabolism to leucomethylene blue may be less efficient in neonates than in older individuals. Methylene blue is excreted in urine and bile. About 75% of an oral dose of methylene blue is excreted in urine, mostly as stabilized colorless leukomethylene blue. On exposure to air, the urine turns green or blue, due to the presence of the oxidation product methylene azure (methylene blue sulfone). Some unchanged drug is also excreted in urine.
BACKGROUND: Although blue dye is routinely used for lymphatic mapping, it is not used for lymphatic mapping in pregnancy-associated breast cancer, because of concern of fetal risk. METHODS: To investigate the safety of blue dye for lymphatic mapping in pregnant women, the pharmacokinetics of methylene blue dye were examined in 10 nonpregnant women, and the results were extrapolated to estimate maximal fetal exposure to the dye. RESULTS: Plasma and urine measurements indicated that the dye quickly distributed from the breast injection site to the circulation, with 32% of the total dose excreted in urine within 48 hours. Combined with existing data on organ distribution of methylene blue, the estimated maximal dose to the fetus is 0.25 mg (5% of the administered dose), likely further reduced by other physiologic factors related to pregnancy. CONCLUSIONS: The analysis suggests that methylene blue dye can be used for lymphatic mapping in pregnancy-associated breast cancer with minimal fetal risk.
The disposition and urinary excretion pharmacokinetics of methylene blue were determined after its intravenous administration at 15 mg/kg to mature female sheep. Comparisons were made between methylene blue administered alone or subsequent to 50 mg/kg sodium nitrite. The overall elimination rate constant (beta) of methylene blue, 0.0076 +/- 0.0016 min-1, was not influenced by prior administration of sodium nitrite. However, the distribution rate was significantly altered by sodium nitrite. Very little of the methylene blue was eliminated in the urine either intact or as leukomethylene blue in spite of its relatively short half life. ...

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Metabolism / Metabolites
Following distribution into tissues, rapidly reduced to leukomethylene blue (leucomethylthioninium chloride). Metabolism to leucomethylene blue may be less efficient in neonates than in older individuals.
Methylene blue can be reduced to a colorless form, leukomethylene blue; together, these compounds form a reversible oxidation-reduction system. In low concentrations, methylene blue accelerates conversion of methemoglobin to hemoglobin. In patients with methemoglobinemia, methylene blue is reduced to leukomethylene blue by methemoglobin reductases in erythrocytes; leukomethylene blue then reduces methemoglobin to hemoglobin. In high concentrations, methylene blue oxidizes the ferrous iron of reduced hemoglobin to the ferric state, thereby changing hemoglobin to methemoglobin.
Following distribution into tissues, methylene blue is rapidly reduced to leukomethylene blue (leucomethylthioninium chloride). Metabolism to leucomethylene blue may be less efficient in neonates than in older individuals.

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Biological Half-Life
5–6.5 hours (after IV dose).
Following IV administration, the estimated half-life of methylene blue is 5-6.5 hours.
... The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in seven volunteers after IV and oral administration of 100 mg methylene blue with and without mesna. The distribution of methylene blue in different tissues was measured in rats after intraduodenal and IV application. The time course of methylene blue in whole blood after IV administration showed a multiphasic time course with an estimated terminal half-life of 5.25 hr. ...

Protein Binding
Methylene blue was reported to bind strongly to rabbit plasma (71–77% of bound drug).

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IDENTIFICATION AND USE: Methylene blue is a solid. Water solutions are deep blue. It is used as a stain in bacteriology, as mixed indicator, a dye, a redox colorimetric agent, and a targeting agent for melanoma. It is also used as a medication to treat drug-induced methemoglobinemia. HUMAN STUDIES: Methemoglobinemia is usually 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 increases mitochondrial cytochrome oxidase activity in the brain and improves memory retention after learning tasks, including fear extinction. The intracameral use of 1% methylene blue has a cytotoxic effect on the corneal endothelium and iris epithelium. Several cases of suspected serotonin syndrome have been reported in patients who received methylene blue in combination with serotonin active agents. Allergic hypersensitivity reaction to methylene blue-treated plasma transfusion has also been reported. A preterm infant had methemoglobulinemia and hemolytic anemia after enteral administration of methylene blue. There is epidemiologic evidence that methylene blue is a teratogen, and the drug can cause fetal harm if administered during pregnancy. Use of methylene blue in amniocentesis has been associated with atresia of the ileum and jejunum, ileal occlusion, and other adverse effects in neonates. Use of methylene blue during pregnancy has resulted in hemolytic anemia, hyperbilirubinemia, methemoglobinemia, respiratory distress, skin staining, and phototoxicity in neonates. ANIMAL STUDIES: Methylene blue treatment resulted in methemoglobin formation and oxidative damage to red blood cells, leading to a regenerative anemia and a variety of tissue and biochemical changes secondary to erythrocyte injury. An early change was a dose-related increase in methemoglobin, where the response of rats and mice was similar in magnitude. Mice appeared to be more sensitive than rats to the formation of Heinz bodies and the development of anemia that was characterized by a decrease in hemoglobin, hematocrit, and erythrocyte count. Splenomegaly was apparent in all treated mice and in the 100 mg/kg (males only) and 200 mg/kg rats at necropsy. Methylene blue was embryotoxic in the rat. Methylene blue caused the mice to deliver before gestation day 18 (term gestation). This response was observed in 45%, 50% and 83% of animals receiving methylene blue at 50, 60 or 85 mg/kg, respectively. Under cell-free conditions methylene blue induced DNA damage. It is characterized by a high number of base modifications sensitive to the repair endonuclease FPG protein (formamidopyrimidine-DNA glycosylase). Methylene blue was mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. The greatest concerns with methylene blue therapy in veterinary use are the development of Heinz body anemia or other red cell morphological changes, methemoglobinemia, and decreased red cell life spans. Cats tent to be very sensitive to these effects and some consider it contraindicated in feline patients, but dogs and horses can also develop hematologic adverse effects at relatively low dosages. ECOTOXICITY STUDIES: Methylene blue had a teratogenic effect in angelfish.

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Non-Human Toxicity Values LD50 Mouse iv 77 mg/kg
LD50 Mouse ip 150 mg/kg
LD50 Mouse oral 3500 mg/kg
LD50 Rat iv 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 appears as odorless or almost odorless dark green crystals with bronze luster or dark green crystalline powder. pH (1% solution in water) 3 to 4.5. (NTP, 1992)
Methylene Blue is a synthetic basic dye. Methylene blue stains to negatively charged cell components like nucleic acids; when administered in the lymphatic bed of a tumor during oncologic surgery, methylene blue may stain lymph nodes draining from the tumor, thereby aiding in the visual localization of tumor sentinel lymph nodes. When administered intravenously in low doses, this agent may convert methemoglobin to hemoglobin.
A compound consisting of dark green crystals or crystalline powder, having a bronze-like luster. Solutions in water or alcohol have a deep blue color. Methylene blue is used as a bacteriologic stain and as an indicator. It inhibits GUANYLATE CYCLASE, and has been used to treat cyanide poisoning and to lower levels of METHEMOGLOBIN.

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Methylene blue is an organic chloride salt having 3,7-bis(dimethylamino)phenothiazin-5-ium as the counterion. A commonly used dye that also exhibits antioxidant, antimalarial, antidepressant and cardioprotective properties. It has a role as an EC 1.4.3.4 (monoamine oxidase) inhibitor, an acid-base indicator, a fluorochrome, an antidepressant, a cardioprotective agent, an EC 3.1.1.8 (cholinesterase) inhibitor, a histological dye, an EC 4.6.1.2 (guanylate cyclase) inhibitor, an antioxidant, an antimicrobial agent, a neuroprotective agent, a physical tracer and an antimalarial. It contains a 3,7-bis(dimethylamino)phenothiazin-5-ium.
Methylene blue is an oxidation-reduction agent. The intravenous form of methylene blue is approved by the FDA for the treatment of pediatric and adult patients with acquired methemoglobinemia. Historically, it has been widely used in Africa to treat malaria, but now it disappeared when chloroquine (CQ) and other drugs entered the market. Its use as an urinary tract antiseptic has also been investigated. Methylthioninium chloride (INN, or methylene blue, proposed trade name Rember) is an investigational drug being developed by the University of Aberdeen and TauRx Therapeutics that has been shown in early clinical trials to be an inhibitor of Tau protein aggregation. The drug is of potential interest for the treatment of patients with Alzheimer's disease.
Methylene Blue is a synthetic basic dye. Methylene blue stains to negatively charged cell components like nucleic acids; when administered in the lymphatic bed of a tumor during oncologic surgery, methylene blue may stain lymph nodes draining from the tumor, thereby aiding in the visual localization of tumor sentinel lymph nodes. When administered intravenously in low doses, this agent may convert methemoglobin to hemoglobin.
A compound consisting of dark green crystals or crystalline powder, having a bronze-like luster. Solutions in water or alcohol have a deep blue color. Methylene blue is used as a bacteriologic stain and as an indicator. It inhibits GUANYLATE CYCLASE, and has been used to treat cyanide poisoning and to lower levels of METHEMOGLOBIN.

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Drug Indication
Indicated for the treatment of pediatric and adult patients with acquired methemoglobinemia. Other clinical applications of methylene blue include improvement of hypotension associated with various clinical states, an antiseptic in urinary tract infections, treatment of hypoxia and hyperdynamic circulation in cirrhosis of liver and severe hepatopulmonary syndrome, and treatment of ifofosamide induced neurotoxicity.
Lumeblue is indicated as a diagnostic agent enhancing visualisation of colorectal lesions in adult patients undergoing screening or surveillance colonoscopy.
Acute symptomatic treatment of medicinal and chemical products- induced methaemoglobinaemia. Methylthioninium chloride Proveblue is indicated in adults, children and adolescents (aged 0 to 17 years old).

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Mechanism of Action
* Main mechanism of action involves inhibition of nitric oxide synthase and guanylate cyclase. * In Alzheimers Disease: a mechanistic study found that methylene blue oxidizes cysteine sulfhydryl groups on tau to keep tau monomeric. One preclinical treatment study in tauopathy mice reported anti-inflammatory or neuroprotective effects mediated by the Nrf2/antioxidant response element (ARE); another reported insoluble tau reduction and a learning and memory benefit when given early. * In Methemoglobinemia: Methylene Blue acts by reacting within RBC to form leukomethylene blue, which is a reducing agent of oxidized hemoglobin converting the ferric ion (fe+++) back to its oxygen-carrying ferrous state(fe++). * As antimalarial agent: Methylene Blue, a specific inhibitor of P.falciparum glutathione reductase has the potential to reverse CQ resistance and it prevents the polymerization of haem into haemozoin similar to 4-amino-quinoline antimalarials. * For ifosfamide induced neurotoxicity: Methylene blue functions as an alternate electron acceptor. It acts to reverse the NADH inhibition caused by gluconeogenesis in the liver while blocking the transformation of chloroethylamine into chloroacetaldehyde. In addition, it inhibits various amine oxidase activities, which also prevents the formation of chloroacetaldehyde.
The mechanism of modulation of cyclic guanosine monophosphate accumulation by methylene blue, a putative inhibitor of soluble guanylate cyclase, was investigated in cultured rabbit pulmonary arterial smooth muscle cells. Control or methylene blue pretreated rabbit pulmonary arterial smooth muscle were stimulated with sodium nitroprusside, nitrosothiols or endothelium derived relaxing factor released basally from bovine pulmonary arterial endothelial cells, in short term cocultures. The putative endothelium-derived relaxing factor, S-nitroso-L-cysteine, a stab1e deaminated analog of S-nitroso-L-cysteine, S-nitroso-3-mercaptoproprionic acid and sodium nitroprusside produced concentration-dependent (1-100 uM) increase (1.5- to 12-fold) in rabbit pulmonary arterial smooth muscle cells cyclic guanosine monophospate levels. Methylene blue pretreatment inhibited S-nitroso-L-cysteine and sodium nitroprusside induced cyclic guanosine monophosphate accumulation by 51% to 100%, but S-nitroso-3-mercaptoproprionic acid mediated responses were not altered by methylene blue. The inhibition profile of methylene blue on nitrovasodilator induced cyclic guanosine monophosphate accumulation was quantitatively reproduced by extracellular generation of superoxide anion with xanthine (100 uM) and xanthine oxidase (5 mU). Similarly to methylene blue pretreatment, superoxide anion generation had no effects on base-line cyclic guanosine phosphate levels or cyclic guanosine phosphate responses elicited by S-nitroso-3-mercaptoproprionic acid. Furthermore, methylene blue induced a dose and time dependent generation of superoxide anion from rabbit pulmonary arterial smooth muscle cells, as evidenced from spectrophotometric determination of cytochrome c reduction. Inhibition of cyclic guanosine monophosphate accumulation in response to S-nitroso-L-cysteine and sodium nitroprusside by methylene blue was completely prevented by superoxide dismutase but not catalase. Selective pretreatment of endothelial cells with methylene blue before co-culture with untreated rabbit pulmonary arterial smooth muscle produced a reduction in rabbit pulmonary arterial smooth muscle cyclic guanosine monophosphate levels of a magnitude comparable with that seen in cocultures of methylene blue pretreated rabbit pulmonary arterial smooth muscle with untreated endothelial cells, and which was partially prevented by superoxide dismutase.

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Schizophrenia is a major public health problem that affects approximately 1% of the population worldwide. Schizophrenia-like symptoms can be induced in humans by phencyclidine (PCP), a drug with marked psychotomimetic properties. Phencyclidine disrupts prepulse inhibition of acoustic startle in rodents, a measure which has also been shown to be disrupted in schizophrenic patients. This effect is blocked by nitric oxide synthase (NOS) inhibitors, suggesting that nitric oxide plays an important role in this effect of phencyclidine. Methylene blue, a guanylate cyclase and nitric oxide syntase inhibitor, has shown therapeutic value as an adjuvant to conventional antipsychotics in the therapy of schizophrenia. The aim of the present study was to investigate if phencyclidine-(4 mg/kg)induced disruption of prepulse inhibition could be affected by methylene blue (50 or 100 mg/kg) in mice. Furthermore, the effect of methylene blue (50 mg/kg) on phencyclidine-(4 mg/kg)induced hyperlocomotion was investigated. The present study shows that phencyclidine readily disrupts prepulse inhibition in mice without affecting pulse-alone trials. It was also found that methylene blue prevents the decrease in prepulse inhibition caused by phencyclidine in a dose-related manner. Furthermore, the increase in locomotor activity caused by phencyclidine was reduced by pretreatment with methylene blue. The results from the present study further support the suggestion that the nitric oxide synthase/guanylate cyclase pathway is involved in pharmacological and behavioural effects of phencyclidine. Since phencyclidine as well exerts psychotomimetic characteristics, agents that interfere with the nitric oxide synthase/guanylate cyclase pathway may be of therapeutic value also in the treatment of schizophrenia.[1]

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Introduction: Neurofibrillary tangles (NFT) composed of Tau are hallmarks of neurodegeneration in Alzheimer disease. Transgenic mice expressing full-length pro-aggregant human Tau (2N4R Tau-ΔK280, termed Tau(ΔK)) or its repeat domain (TauRD-ΔK280, TauRD(ΔK)) develop a progressive Tau pathology with missorting, phosphorylation, aggregation of Tau, loss of synapses and functional deficits. Whereas TauRD(ΔK) assembles into NFT concomitant with neuronal death, Tau(ΔK) accumulates into Tau pretangles without overt neuronal loss. Both forms cause a comparable cognitive decline (with onset at 10mo and 12mo, respectively), which is rescued upon switch-off of transgene expression. Since methylene blue (MB) is able to inhibit Tau aggregation in vitro, we investigated whether MB can prevent or rescue Tau-induced cognitive impairments in our mouse models. Both types of mice received MB orally using different preventive and therapeutic treatment protocols, initiated either before or after disease onset. The cognitive status of the mice was assessed by behavior tasks (open field, Morris water maze) to determine the most successful conditions for therapeutic intervention. Results: Preventive and therapeutic MB application failed to avert or recover learning and memory deficits of TauRD(ΔK) mice. Similarly, therapeutic MB treatment initiated after onset of cognitive impairments was ineffective in Tau(ΔK) mice. In contrast, preventive MB application starting before onset of functional deficits preserved cognition of Tau(ΔK) mice. Beside improved learning and memory, MB-treated Tau(ΔK) mice showed a strong decrease of insoluble Tau, a reduction of conformationally changed (MC1) and phosphorylated Tau species (AT180, PHF1) as well as an upregulation of protein degradation systems (autophagy and proteasome). This argues for additional pleiotropic effects of MB beyond its properties as Tau aggregation inhibitor. Conclusions: Our data support the use of Tau aggregation inhibitors as potential drugs for the treatment of AD and other tauopathies and highlights the need for preventive treatment before onset of cognitive impairments.[2]

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Traumatic brain injury (TBI) is associated with cerebral edema, blood brain barrier breakdown, and neuroinflammation that contribute to the degree of injury severity and functional recovery. Unfortunately, there are no effective proactive treatments for limiting immediate or long-term consequences of TBI. Therefore, the objective of this study was to determine the efficacy of methylene blue (MB), an antioxidant agent, in reducing inflammation and behavioral complications associated with a diffuse brain injury. Here we show that immediate MB infusion (intravenous; 15-30 minutes after TBI) reduced cerebral edema, attenuated microglial activation and reduced neuroinflammation, and improved behavioral recovery after midline fluid percussion injury in mice. Specifically, TBI-associated edema and inflammatory gene expression in the hippocampus were significantly reduced by MB at 1 d post injury. Moreover, MB intervention attenuated TBI-induced inflammatory gene expression (interleukin [IL]-1β, tumor necrosis factor α) in enriched microglia/macrophages 1 d post injury. Cell culture experiments with lipopolysaccharide-activated BV2 microglia confirmed that MB treatment directly reduced IL-1β and increased IL-10 messenger ribonucleic acid in microglia. Last, functional recovery and depressive-like behavior were assessed up to one week after TBI. MB intervention did not prevent TBI-induced reductions in body weight or motor coordination 1-7 d post injury. Nonetheless, MB attenuated the development of acute depressive-like behavior at 7 d post injury. Taken together, immediate intervention with MB was effective in reducing neuroinflammation and improving behavioral recovery after diffuse brain injury. Thus, MB intervention may reduce life-threatening complications of TBI, including edema and neuroinflammation, and protect against the development 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.)