Main Research Findings

1) When administered within 24 hours of onset of septic shock, methylene blue resulted in a reduction in time to vasopressor discontinuation and a reduced length of hospitalization.

2) Administration of methylene blue has the potential to treat age-related conditions such as neurodegeneration, premature age-related diseases, memory loss, and skin aging.

 

Selected Data

1) The research team of Ibarra-Estrada et al conducted a parallel, double-blinded, randomized controlled trial conducted in the medical-surgical intensive care unit (ICU) of an academic reference center. Patients eligible for the study were those aged 18 years or older, diagnosed with septic shock as defined by Sepsis-3 criteria. These criteria included a highly suspected or confirmed infection, the requirement of norepinephrine to maintain a mean arterial pressure of 65 mmHg or higher, and a serum lactate level greater than 2 mmol/L despite adequate fluid resuscitation [1].

Patients were excluded if they had been receiving norepinephrine for more than 24 hours, were pregnant, had a high probability of death within 48 hours, or had a history of glucose-6-phosphate dehydrogenase deficiency. Additionally, patients with concurrent hemorrhagic, obstructive, or hypovolemic shock, those requiring damage control surgery, or those with allergies to methylene blue or related substances were excluded. The trial also excluded patients with COVID-19 during its early stages due to uncertainties regarding the pathophysiology of the disease and its potential interaction with methylene blue.

The randomization process involved assigning patients to either the methylene blue group or the control group in a 1:1 ratio using a computer-generated randomization sequence. The randomization was performed by critical care physicians, and both patients and outcome assessors were blinded to the treatment received. Patients in the MB group received an intravenous infusion of 100 mg of methylene blue diluted in 500 ml of 0.9% sodium chloride, administered over six hours once daily for three doses. The control group received the same infusion, but without the inclusion of methylene blue. To ensure blinding, the infusion bags and lines were prepared by the central pharmacy and kept opaque [1].

The study site employed dynamic methods for fluid resuscitation in patients with septic shock, using techniques such as aortic velocity-time integral change after passive leg raising, arterial pulse pressure variation, tidal volume challenge, and respiratory variation of carotid peak flow velocity to predict volume responsiveness. Adequate fluid resuscitation was defined as the administration of at least 500 ml of balanced crystalloid followed by negative volume responsiveness as confirmed by at least two methods. Adjunctive vasopressin was administered at a dose of 0.03 IU/min if the norepinephrine dose reached 0.25 mcg/kg/min. Hydrocortisone, at 200 mg/day, was continuously infused as a standard protocol until 6 hours after all vasopressors were discontinued [1].

Demographic, ventilatory, and laboratory data were collected at the time of randomization, including the diagnosis of acute respiratory distress syndrome. The norepinephrine dose was recorded at multiple intervals: randomization, immediately after each intervention dose, and at 24 and 48 hours post-treatment. A comprehensive multiorgan point-of-care ultrasound was performed to assess organ function, including the calculation of ejection fraction. Additionally, methemoglobin saturation was continuously monitored using pulse co-oximetry throughout the intervention.

The primary outcome of the study was time to vasopressor discontinuation, defined as the cessation of all vasopressors for at least 48 consecutive hours. Secondary outcomes included vasopressor-free days at 28 days, all-cause mortality at 28 days, changes in serum lactate levels, days on mechanical ventilation, length of ICU and hospital stay, and changes in serum creatinine, bilirubin, aspartate/alanine aminotransferase levels, PaO2/FIO2 ratio, and ejection fraction after the intervention [1].

In summary, this trial is designed to evaluate the potential benefits of methylene blue in patients with septic shock, specifically its effects on vasopressor requirements, organ function, and overall recovery. The randomized controlled design, rigorous inclusion and exclusion criteria, and comprehensive outcome measures indicate that the results will contribute valuable insights into the use of methylene blue as a treatment option in critically ill patients [1].

2) The research team of Xue et examined the potential usage of methylene blue as an anti-aging compound. Methylene blue is a well-known chemical compound that was first synthesized as a textile dye in 1876. Due to its small molecular weight, methylene blue can quickly penetrate tissues, making it effective for various medical applications. Methylene blue can be chemically reduced into leucomethylene blue, a form that possesses strong antioxidant properties. Over the years, methylene blue has gained approval from the U.S. Food and Drug Administration and has been widely utilized in clinical settings, including surgical staining, treatment of malaria, and therapy for methemoglobinemia [2].

The free radicals theory of aging proposes that aging results from accumulated damage caused by free radicals. In human cells, reactive oxygen species, a type of free radical, are predominantly produced in the mitochondria. Aging is associated with a decline in mitochondrial mass, respiration capacity, and efficiency across different tissues. Dysfunctional mitochondria produce less ATP while generating more reactive oxygen species, leading to further mitochondrial damage. This results in significant cellular injury and accelerates the aging process. The antioxidative properties of methylene blue allow it to enhance mitochondrial function and disrupt this damaging cycle. As a result, methylene blue is increasingly recognized as a promising candidate for anti-aging therapies.

The review focuses on the structure, biological functions, and diverse applications of methylene blue. Mitochondria are the primary source of cellular energy in eukaryotic organisms, and the mitochondrial electron transport chain is essential for ATP production. The electron transport chain is located in the inner mitochondrial membrane and involves a series of electron transporters that move electrons from NADH and FADH₂ to molecular oxygen. It is organized into four complexes: the first two complexes NADH-ubiquinone oxidoreductase and succinate dehydrogenase serve as entry points for electrons. Complexes three and four, CoQ-cytochrome c reductase and cytochrome c oxidase further transport the electrons, with the assistance of coenzyme Q10 and cytochrome c. Finally, Complex IV transfers the electrons to molecular oxygen, resulting in the formation of water. During this process, Complex V, ATP synthase, uses the energy generated to phosphorylate ADP into ATP [2].

Under normal physiological conditions, approximately 0.4% to 4% of oxygen is only partially reduced, leading to the generation of reactive oxygen species as a byproduct. Although reactive oxygen species can be produced at multiple sites along the electron transport chain, Complex I is the main source. Mitochondrial dysfunction often impairs Complex I and Complex IV, leading to excessive reactive oxygen species production, oxidative stress, and further cellular damage. In this context, methylene blue plays a critical role in protecting mitochondrial function [2].

Structurally, methylene blue is a phenothiazine derivative that can exist in both oxidized and reduced forms. The amphipathic nature of methylene blue enables it to easily cross biological membranes. Furthermore, methylene blue is positively charged, which helps it accumulate in mitochondria. Its redox potential is relatively low at 11 millivolts, facilitating its ability to cycle between oxidized and reduced states within mitochondria. This redox cycling ability allows MB to act as a catalytic redox cycler, enhancing the activity of cytochrome oxidase and promoting ATP production.

In addition to boosting ATP synthesis, methylene blue can reduce the production of reactive oxygen species by bypassing the dysfunctional activities of Complex I and III. Methylene blue accepts electrons from NADH at Complex I and is reduced to leucomethylene blue, which can then directly donate electrons to cytochrome c, thus being oxidized back to methylene blue. This bypass mechanism helps maintain efficient electron flow even when parts of the electron transport chain are impaired, decreasing reactive oxygen species generation and protecting cells from oxidative stress under pathological conditions [2].

Beyond its mitochondrial effects, methylene blue has been used for various medical purposes. It has long been employed in surgical procedures for staining tissues and in treating conditions like malaria and methemoglobinemia. Early studies from as far back as 1928 demonstrated the ability of methylene blue to photo-inactivate bacteria, and subsequent research revealed potent antiviral properties. Methylene blue has also been found to be effective against fungal and parasitic infections, commonly being used as an aquarium disinfectant even at high concentrations [2].

Additionally, methylene blue has been explored in photodynamic therapy for several types of cancer, including lung, breast, and prostate cancers. In photodynamic therapy, methylene blue acts as a photosensitizer that, when activated by light, produces cytotoxic species that can kill cancer cells. It is important to note, patients who received methylene blue alongside standard chemotherapy reported no cases of COVID-19 infection among the treated individuals, suggesting a possible protective effect against viral infections.

Importantly, studies have shown that low doses of methylene blue, ranging from 0.5 to 4 mg/kg, are effective in stimulating mitochondrial respiration and are safe in both animal models and human subjects. These findings reinforce methylene blue as a safe and versatile drug with broad applications, including potential use as an anti-aging therapy due to its capacity to enhance mitochondrial function, reduce oxidative stress, and interrupt cellular damage pathways associated with aging [2].

 

Discussion

1) Between March 16, 2017, and July 30, 2022, a total of 308 patients were screened for eligibility in a randomized controlled trial evaluating the effects of methylene blue in septic shock. Out of these, 92 patients met inclusion criteria and were randomized into two groups: 45 patients were assigned to the MB group, and 46 to the control group. One patient in the methylene blue group withdrew consent after receiving the first dose and was not included in the final analysis. As a result, 91 patients were included in the analysis, with all but one receiving the complete three-dose regimen of the assigned treatment. Every participant in the trial also received hydrocortisone as part of the standard care protocol [1].

The median age of the participants was 46 years, with an interquartile range of 35 to 55 years. Sixty percent of the study population were men. Acute kidney injury was present in 46% of the patients. The most frequently identified sources of sepsis were pulmonary infections, accounting for 49.5% of the cases, followed by intra-abdominal infections, which made up 38.5%. All patients received appropriate antimicrobial therapy within three hours of septic shock diagnosis. Most participants required mechanical ventilation and began their assigned intervention after the initial six hours following shock identification.

Baseline characteristics, including demographics, clinical status, and interventions received, were comparable between the two groups, ensuring an even distribution of confounding variables. The median daily dose of methylene blue in the intervention group was 1.2 mg/kg. Notably, no additional vasopressors such as phenylephrine, angiotensin II, epinephrine, or midodrine or additional inotropes like milrinone or dobutamine were administered during the study [1].

The primary outcome measured was the time to vasopressor discontinuation, which significantly favored the methylene blue group. The median time to discontinue all vasopressors in the MB group was 69 hours, compared to 94 hours in the control group. This represented a statistically significant median difference of 29.4 hours. Furthermore, norepinephrine requirements decreased more markedly in the methylene blue group over the initial four-day treatment window. Although not statistically significant, 11% fewer patients in the methylene blue group required re-initiation of norepinephrine within 48 hours of discontinuation compared to 28% of those in the control group, suggesting a trend toward better sustained shock reversal [1].

Several secondary outcomes also showed favorable results for the methylene blue group. Patients who received methylene blue experienced one additional vasopressor-free day at day 28. The cumulative fluid balance in the methylene blue group was lower by an average of 741 ml compared to the control group. Moreover, patients in the methylene blue group had a shorter length of stay in both the ICU and the hospital overall. The ICU stay was shorter by 1.5 days, and hospital stay was reduced by 2.7 days. A proportional hazards analysis demonstrated that patients in the methylene blue group had a 2.7 times higher hazard ratio for shock reversal within 28 days compared to controls, emphasizing the efficacy of methylene blue in promoting recovery from septic shock.

It is important to note that certain outcomes did not differ significantly between the two groups. Lactate levels over the first three days, the number of days on mechanical ventilation, and 28-day all-cause mortality rates were similar in both the methylene blue and control groups. These findings suggest that while methylene blue may expedite hemodynamic stabilization and hospital discharge, it may not significantly impact respiratory support duration or short-term survival [1].

Regarding safety and tolerability, the most commonly observed side effect in the methylene blue group was a characteristic green-blue discoloration of the urine, which occurred in 93% of the patients. Though harmless and expected, this effect is a known consequence of methylene blue administration. Additionally, methemoglobin saturation was significantly higher in the methylene blue group, with a median value of 2.9% compared to 0.5% in the control group. However, this elevation did not result in clinically significant methemoglobinemia in any patient. Importantly, no differences were found between the two groups regarding changes in cardiac ejection fraction, oxygenation as measured by the PaO2/FiO2 ratio, serum creatinine levels, bilirubin levels, or liver enzyme concentrations following the intervention. This suggests that methylene blue did not adversely affect renal, hepatic, or pulmonary function [1].

In conclusion, this randomized controlled trial demonstrated that methylene blue, when administered as an adjunctive therapy in septic shock, significantly shortened the duration of vasopressor use, improved fluid balance, and reduced ICU and hospital lengths of stay without causing serious adverse effects. While it did not significantly affect ventilator dependency or 28-day mortality, its overall benefit in promoting earlier hemodynamic stability and hospital discharge positions methylene blue as a potentially valuable therapeutic option in the management of septic shock [1].

2) Oxidative metabolism is crucial for brain activity, and mitochondrial dysfunction has been linked to neuronal loss during brain aging, as well as to brain diseases such as Alzheimer’s disease, Parkinson’s disease , and brain injuries. The review study conducted by Xue et al reported that methylene blue, due to its lipophilic nature, effectively crosses the blood-brain barrier and accumulates in the brain at higher concentrations than in plasma after administration. Methylene blue has a strong affinity for mitochondria and reduces free radical production by bypassing Complex I/III activity rather than scavenging free radicals directly. Methylene blue can partially restore membrane potential in Complex III-inhibited mitochondria and acts as an electron donor, increasing cytochrome oxidase expression and oxygen consumption. Methylene blue also inhibits nitric oxide, which otherwise suppresses cytochrome c oxidase activity. These properties make methylene blue a promising therapeutic agent for brain diseases [2].

Alzheimer’s disease is a neurodegenerative disease characterized by amyloid-β aggregation and neurofibrillary tangles, and mitochondrial dysfunction may serve as a critical link between aging and Alzheimer’s disease. Elevated oxidative stress from mitochondria is reported early in the progression of Alzheimer’s disease, along with mitochondrial size reduction and impaired movement. Mitochondrial dysfunction in Alzheimer’s disease also leads to impaired energy metabolism, disruptions in oxidative phosphorylation, calcium imbalance, and increased mitochondrial DNA mutations. Low-dose methylene blue treatment can reduce reactive oxygen species production, benefiting Alzheimer’s patients by potentially mitigating mitochondrial damage.

Studies have shown that mitochondrial dysfunction is associated with abnormal amyloid-β and tau processing. Amyloid precursor protein can become trapped in mitochondrial membranes, impairing function, while overexpression of tau also exacerbates mitochondrial damage by reducing ATP and increasing oxidative stress. Conversely, mitochondrial damage can increase abnormal amyloid-β production and tau phosphorylation. Methylene blue has been reported to prevent or dissolve amyloid-β and tau aggregates through autophagic clearance, potentially improving mitochondrial function in neurons related to Alzheimer’s disease. Additionally, methylene blue may directly or indirectly influence β-secretase activity to regulate amyloid-β production. Given that cytochrome oxidase activity declines in Alzheimer’s disease and methylene blue enhances this enzyme’s activity, the nootropic could further support neuronal oxidative metabolism [2].

Clinical studies investigating the efficacy of methylene blue for Alzheimer’s treatment are ongoing. In transgenic mouse models, methylene blue inhibited amyloid-β production and improved cognitive deficits. Low-doses of methylene blue was found to result in an 81% reduction in the rate of cognitive decline over 50 weeks in patients with mild to moderate Alzheimer’s disease, as well as improved cognition and cerebral blood flow. More recent cohort studies suggested a decrease in brain atrophy rates in patients treated with methylene blue, although further research should be conducted.

In addition to its effects on the brain, treatment with methylene blue is beneficial for skin aging. The skin, composed of the epidermis, dermis, and subcutis, serves as the body’s primary defense. Skin aging involves elasticity loss, thinning, extracellular matrix degradation, and oxidative stress. Intrinsic aging results from natural processes, while extrinsic aging stems from environmental factors like UV radiation. Oxidative stress plays a role in both types. Methylene blue, as an antioxidant, protects skin by reducing oxidative damage, stimulating cell proliferation, and decreasing aging markers. Studies have shown that treatment with methylene blue increased lifespan and oxygen consumption in fibroblasts, reversed premature senescence, and outperformed traditional antioxidants like vitamin C and retinol. Methylene blue also upregulated collagen and elastin expression, improved skin thickness and hydration, and protected against UV-induced DNA damage [2].

Administration of methylene blue also facilitates wound healing. Aging reduces fibroblast proliferation and collagen production, impairing skin repair. Methylene blue promotes fibroblast migration and proliferation during wound healing and has been shown to reduce tissue necrosis in rat burn models by decreasing oxidative stress. Additionally, methylene blue has been shown to decrease microbial burden and hyper-granulation while promoting tissue viability with minimal irritation [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Ibarra-Estrada, M., Kattan, E., Aguilera-González, P., Sandoval-Plascencia, L., Rico-Jauregui, U., Gómez-Partida, C. A., Ortiz-Macías, I. X., López-Pulgarín, J. A., Chávez-Peña, Q., Mijangos-Méndez, J. C., Aguirre-Avalos, G., & Hernández, G. (2023). Early adjunctive methylene blue in patients with septic shock: a randomized controlled trial. Critical care (London, England), 27(1), 110. https://doi.org/10.1186/s13054-023-04397-7

[2] Xue H, Thaivalappil A, Cao K. The Potentials of Methylene Blue as an Anti-Aging Drug. Cells. 2021 Dec 1;10(12):3379. doi: 10.3390/cells10123379. PMID: 34943887; PMCID: PMC8699482.

 

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