INTRODUCTION
Methylene blue (MB) or methylthionine chloride is a cationic heterocyclic compound with a photosensitizing nature and is widely used in clinical medicine, including surgery [1-3]. MB has a maximum absorption wavelength of 668 nm and exists as a redox couple in equilibrium between oxidized blue MB and reduced colorless leucomethylene blue (LMB) [4]. MB is a promising lead compound for developing therapeutics for various diseases, including viral infections, cancer and dementia [5-7]. The long history of the applications of MB can be traced back to the early studies of Paul Ehrlich on its use for tissue staining and the development of chemotherapy basics [8] and includes research on the use of MB as an anti-malarial [9] and as an antidote for the toxic side effects of methemoglobin, cyclophosphamide, and cyanide poisoning, mostly owing to the physiochemical potential of MB in redox chemistry [10-12]. Additionally, MB can reverse hypotension during septic shock and has been successfully applied in patients [13]. The light spectrum properties of MB allow its application in photodynamic therapy (PDT) for excisional wounds and psoriasis and as a diagnostic tool for oral and breast cancer [14-17]. The low cost and versatility of MB allow further repurposing for different clinical therapies.
MB can be administered as an oral formulation or intravenously and subcutaneously in a 1% solution form (26.74 mM) [18]. The safe dosage range for MB is <2 mg/kg [19]. For aqueous oral administration of 100 mg MB, a maximum plasma concentration of 8 µM (25 ng/mL) was reached after 2 hours, with a plasma half-life of approximately 20 hours [20]. Upon intravenous administration, MB can reach the maximum concentration 1 to 2 hours post-injection with a half-life of approximately 5 hours and is reported to exhibit almost double the concentration in whole blood compared to that shown in oral administration [21]. It is recommended that intravenous MB dosages be limited to 1 to 2 mg/kg since a higher dosage of MB (>5 mg/kg) can cause serotonin toxicity and, rarely, anaphylactic reactions. Intravenous administration should be slow, usually over 5 to 10 minutes [22,23]. Interestingly, an elevated concentration of MB in the brain was observed upon intravenous administration, indicating that MB can penetrate the blood-brain barrier (BBB) [21]. This could be explained by the equal charge distribution on the surface of the MB molecules [24]. Following administration, MB is cleared through the kidneys and reduced to LMB and demethylated metabolites [25]. Currently, MB is approved by the U.S. Food and Drug Administration (FDA) for intravenous and oral administration in methemoglobinemia therapy and as a surgical tracing dye. As of 2022, 225 interventional studies have been registered worldwide (clinicaltrials.gov) to investigate the clinical utility of MB in areas ranging from oncology to depression, and only 102 clinical trials have been completed.
Our review explores the pharmacological aspects of MB and its applicability to various diseases. The clinical trials registry clinicaltrials.gov was queried for all results published or registered before May 2022 with the search terms ‘methylene blue’ and ‘methylthionine chloride.’ Of 171 study records identified, 81 were complete. An analysis of the clinical studies (listed in Table 1) was conducted to explain the therapeutic potential of MB.
CLINICAL STUDIES AND PERSPECTIVES OF METHYLENE BLUE-BASED THERAPY
Methylene blue as a neuroprotective agent
Some of the most prevalent, devastating, and poorly treated diseases are brain diseases such as central nervous system disorders. Because of the complex nature of the brain in contrast to other anatomical areas, the development of drugs for the treatment of brain disease has poor success rates. Researchers have found that MB has antidepressant, anxiolytic, and neuroprotective properties in animal studies as well as in human studies [26,27]. It has a stabilizing effect on mitochondrial function and a therapeutic effect on the production of reactive oxygen species (ROS). Because of these features, MB holds promise as a neuroprotective agent and treatment for neurodegenerative disorders.
The antidepressant-like activity of MB has been effective in preventing and treating cognitive disorders caused by psychosis, both in preclinical and clinical studies [28]. The nitric oxide-cyclic guanosine monophosphate (NO-cGMP) cascade modulates the extracellular levels of serotonin, dopamine, glutamate, and acetylcholine, which may contribute to the pathophysiology of depression. NO production is elevated in depression, emphasizing the importance of the NO-cGMP cascade as a biomarker for depression. MB can induce an antidepressant-like response by directly inhibiting NO synthase and guanylate cyclase [29]. However, only a few clinical trials have reported these results. In one study, a moderate cognitive-enhancing effect was seen after three months in 26 patients with posttraumatic stress disorder treated daily with 260 mg of MB compared with 16 participants awaiting treatment [27]. In another study, MB was found to have significant effects on the symptoms of depression and anxiety in patients with bipolar disorder treated with a dosage of 195 mg, with no signs of serotonergic toxicity in these patients [30].
Mitochondrial dysfunction and oxidative stress are key to the progressive nature of neurodegenerative disorders, including traumatic brain injury, Alzheimer’s disease (AD), and Parkinson’s disease (PD). Downstream of mitochondrial dysfunction, electron transfer impairment occurs, resulting in energy deficits and the release of ROS [31]. Therefore, improving mitochondrial respiration is important for the development of new therapies. The redox chemistry of MB, in addition to its safety, may have therapeutic benefits. MB demonstrated an alternative donor/acceptor supporting role by reducing electron leakage in mitochondria, inhibiting ROS, and improving neuronal energy production [32,33]. These results suggest that MB is a promising candidate for the treatment of neurodegenerative diseases.
The pathology of tau protein aggregation correlates with clinical dementia in patients with AD. Therefore, inhibitors of tau aggregation may have therapeutic potential. The phenothiazine ring in MB was found to be essential for the inhibition of heparin-induced tau filament formation. MB’s inhibitory activity on tau filament formation was found to be dependent on the first and fourth repeat domains of tau protein [34]. Monotherapy with MB, which inhibits tau protein aggregation, has been tested in clinical trials [7]. Wischik et al. [35] reported that 321 patients with mild or moderate disease were treated with MB in clinical trials. Based on the cognitive subscale of the Alzheimer’s Disease Scale, 138 mg daily had a moderate effect after 6 months [35]. The reduced form of MB, LMB, which retains tau-aggregation inhibitor properties, is more soluble and has an improved pKa compared to MB. However, clinical studies involving LMB as a supplemental treatment for patients with mild to moderate AD have shown negative results [36].
As a brain-active drug, MB has many desirable properties. Several factors make it worth considering as a potential therapeutic agent, such as its high solubility in aqueous media, low toxicity, ability to cross the BBB and cellular membranes, and its approval for human use. The impact of MB on mitochondrial function and neurofibrillary tangles suggests that it may be a promising neuroprotective candidate. The development of MB as a multi-tasking therapy for depression in neurodegenerative diseases, such as AD and PD, is essential. A novel therapeutic strategy can be applied when multitargeting bullet drugs are used for the treatment of depression by using currently available analogs of MB because of their physiochemical and pharmacokinetic properties. Furthermore, analogs may provide better response and reduce the risk of side effects. Despite a relatively small number of patients, recent clinical trials have shown promising results. Nevertheless, more extensive studies are required to confirm these findings.
Methylene blue in clinical practice of cancer identification
Currently, ultrasound, frozen section analysis, and visual palpation are methods used to detect tumors during surgery. All three methods are time-consuming and prone to errors [37,38]. Therefore, employing an intraoperative imaging method during surgery can be useful for identifying breast tumors and locate suspicious lesions in the resected tissues. The FDA has approved near-infrared (NIR) fluorescence imaging by MB for intraoperative applications because it allows the accurate identification of tumor margins and sentinel lymph nodes without ionizing radiation [39]. Several studies have demonstrated the feasibility of utilizing MB-based imaging during surgery to identify tumor lesions, such as diagnosing solitary fibrous tumors in the pancreas and identifying parathyroid adenomas and paragangliomas [40]. According to a study by Tummers et al. [6], van der Vorst et al. [41], Tummers et al. [42], and an MB-based imaging approach was used to identify breast cancer. An intravenous injection of MB at a dose of 1.0 mg/kg either immediately before surgery or 3 hours before surgery produced fluorescent signals in 20 out of 24 patients with breast cancer. Even though MB does not cause serious allergic reactions, it is not without risks. Adverse reactions to MB were reported by Stradling et al. [43] in patients with breast cancer. There also have been reports of skin, fat, and parenchymal necrosis [44,45]. Due to its toxic effects on local tissues, MB should not be injected intradermally. Instead, it should be injected deep into the parenchyma.
Anti-infectious and antiviral therapy in clinical studies using methylene blue
Since the first reports of MB being used against malaria nearly a century ago, it has been replaced by chloroquine and other FDA-approved drugs owing to toxicity issues. However, MB remains an affordable and effective anti-malarial agent, particularly in patients resistant to chloroquine. MB showed the potential to inhibit glutathione reductase and reverse chloroquine resistance (CQ-sensitizing action), including the prevention of heme polymerization [46]. The daily dose of 36 to 72 mg/kg was the most effective. Apart from its well-known anti-malarial activity, it also has the potential to prevent methemoglobinemia as a complication of malarial anemia [9].
Recently, MB has been reported as a treatment option for coronavirus disease 2019 (COVID-19). COVID-19 is a natural viral lung infection that leads to increased fatality in the older generation. Sharing a common feature of the immune response to sepsis, COVID-19 activates a systemic immune response by inducing a pro-thrombotic environment enriched with proinflammatory cytokines and free radicals [47]. The application of anti-cytokine or antiviral drugs alone did not show high efficiency in COVID-19 therapy, thus leaving an unmet need for a therapeutic agent that could inhibit both cytokines and free radicals. The application of MB in COVID-19 treatment was launched in April 2020 as a parallel, randomized, interventional clinical trial [48]. Investigations into the mechanism of action of MB focused mainly on the inhibition of toxic NO generation by blocking NO synthase as well as free radical production [5]. Several ongoing clinical studies on this topic are presented in Table 1 [27,30,35,36,48-68].
Methylene blue in the therapy of cardiovascular diseases
MB has the potential to inhibit guanylate cyclase and improve mean arterial pressure and cardiac function in septic shock, resulting in decreased cGMP and smooth vascular muscle relaxation. Additionally, MB demonstrates an improvement in arterial pressure and systemic vascular resistance [13,69,70]. A dosage of 2 mg/kg/hr of MB added to the cardiopulmonary bypass prime composition was successfully applied to prevent refractory hypotension in septic endocarditis [71]. Mortality rates in post-cardiac surgery patients with vasoplegia have been decreased by applying MB [72-74]. A single dose of MB 1.5 to 2 mg/kg was intravenously administered to patients at high risk of vasoplegic syndrome during cardiac surgery [72,75-77]. According to another case report, patients with hepatopulmonary syndrome (a pulmonary condition caused by increased endogenous NO production followed by elevated cGMP levels in the liver of patients with cirrhosis [78]) experienced improvements in alveolar-arterial oxygen pressure after MB injection over 15 minutes at a dosage of 3 mg/kg body weight [79].
METHYLENE BLUE-BASED PHOTODYNAMIC THERAPY
MB is a dye commonly used in PDT that triggers apoptosis via ROS production under NIR light [80]. Owing to its rapid absorption and clearance properties, MB is suitable for topical PDT because it reduces the risk of skin photosensitivity, a common problem after PDT. As the FDA has approved MB for clinical use, it is considered a potential photosensitizer candidate for PDT applications in cancerous and non-cancerous diseases [81,82]. PDT of tumors and nonmalignant diseased tissues is a promising alternative to chemotherapy because diseased cell death is caused by selectively locating photosensitive compounds in the target and generating cytotoxic ROS under light. An activated form of oxygen, 1O2, called singlet oxygen, is produced by a photosensitizer by directly absorbing energy from a light source. Known as the main cytotoxic agent associated with PDT, 1O2 is a highly electrophilic compound capable of oxidizing the electron-rich double bonds in biological molecules and macromolecules [83,84]. While PDT works well within a spectral window of 600 to 950 nm, it shows poor penetration into deep tissue, which may adversely affect its efficiency. In terms of the application of MB for PDT, instability and bioaccumulation in endothelial cells are major impediments. It may be possible to alleviate these issues by fabricating nanoparticles that can protect and transport MB for accumulation in the target tissue, for example, by enhancing PDT at tumor sites. In addition, nanoparticles have been found to be effective in modifying pharmacokinetics and reducing adverse side effects [85].
Several studies have evaluated the PDT effects of MB in fungal diseases. In 2012, Scwingel et al. [86] assessed the effectiveness of PDT for oral candidiasis in 21 patients. A single radiation dose was applied with 450 µg/mL MB at 660 nm, 30 mW, and 7.5 J/cm2 for 10 seconds. While conventional treatment failed to prevent the occurrence of candidiasis in the short term, all patients in the PDT group were free from candida colonies with no recurrence of candidiasis for up to 30 days after radiation [86]. Another study demonstrated that patients with onychomycosis undergoing MB PDT showed greater improvements in the short and long-term post-PDT compared to underwent conventional therapy without PDT [87]. The optimal parameters for MB PDT in human infectious diseases need to be determined, and there is no consensus on the standardization of MB PDT protocols. To date, only a few studies have been conducted on the adjunctive use of MB PDT to treat conditions including onychomycosis, oral candidiasis, and diabetic foot infections.
MB PDT was applied to treat truncal and facial acne vulgaris, a chronic inflammatory disease of the pilosebaceous unit that has multiple pathophysiological factors [88-90]. The topical and oral treatments currently available for acne vulgaris have a limited effect, particularly in mild to moderate cases [91]. Antibiotic therapy has led to the implementation of PDT for the treatment of acne vulgaris. By targeting some of the main pathophysiological factors, topical PDT application may reduce inflammation associated with acne lesions in areas of the body which are difficult to reach with conventional therapy [92]. To overcome the problem of inadequate penetration of photosensitizers into the skin during topical treatment with PDT, nanocarriers containing MB have been developed [88-90]. MB has a high affinity for melanin, making this particularly relevant to pigmented cancer tissues in melanoma lesions [93,94]. Malignant melanomas occur when melanocytes, the pigment-producing cells found primarily in the skin, undergo malignant transformation [95]. A study reported that mitochondrial dysfunction causes apoptotic cell death when PDT with MB derivatives is specifically directed at mitochondrial membranes. It is plausible that mitochondria are involved in MB PDT-induced tumor regression because MB is likely to bind to the mitochondrial matrix in a negative electrochemical environment [96]. Researchers have shown that MB PDT is effective against melanoma in both human and animal studies where large melanoma lesions that could not be surgically removed were effectively treated [81,97,98]. The clinical applications of MB PDT may still be limited, particularly for deeply seated hypoxic tumors. Clinical MB PDT can be more effectively utilized when targeted approaches are used, particularly in precision medicine. In recent years, many studies have focused on improving the specificity and effectiveness of MB PDT in specific cancer cells [99]. Researchers have demonstrated that using nanoparticles can increase the specificity of treatment and decrease adverse systemic effects [100-104]. MB PDT is also favorable when combined with other therapies, including immunotherapy. Of note, there are no clinical trials registered on the clinicaltrials.gov database for cancer MB PDT. However, the current preclinical interest in developing nanoparticles for MB PDT is likely to lead to the development of more effective and advanced therapies. Therefore, it can be concluded that MB PDT has tremendous potential for use in both cancerous and non-cancerous diseases.
CONCLUSION
MB is known for its accessibility, extensive safety profile, and proven versatility and has been used in medicine for decades. The fluorescence properties of this compound give it tremendous potential as a NIR fluorophore. MB imaging with NIR radiation is a promising surgical technique that requires further research. The studies included in the present review did not identify any potentially lethal side effects of PDT, suggesting that it is a safe procedure for treating mild infections if performed under supervision.
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