Posted 30 July 2009

Essay on the Medical History of Methylene Blue
by Heiner Schirmer

Biochemistry of MB
MB is a tricyclic phenothiazine drug (Wainwright and Amaral, 2005). Under physiological conditions it is a blue cation that undergoes a catalytic redox cycle: MB is reduced by NADPH or thioredoxin to give leucoMB, an uncharged colorless compound. LeucoMB is then spontaneously reoxidized by O2 (Figure 1). The typical redox-cycling of MB in vivo can be illustrated in vitro using the famous blue bottle experiment: MB is visibly reduced by glucose to give leucoMB and then, by opening the lid of the bottle, one can reoxidize it by atmospheric O2: the colour comes back. Pathologists observe an analogous phenomenon at autopsies (Tan and Rodriguez, 2008; Warth et al., 2009). After closing the lid, one sees a lag phase and then MB is reduced again.

Figure 1. MB as a Redox-cycling Compound
In vivo MB has prominent partners such as NAD(P)H—which is the most important source of reducing antioxidative equivalents in nature—riboflavin-containing enzymes such as dihydrolipoamide dehydrogenase (LipDH = diaphorase), molecular oxygen (O2) or Fe3+-containing heme proteins like methemoglobin. MB is reduced by the flavoenzyme according to the equation NADPH + MB --> NADP+ + leucoMB, and the resulting neutral leucoMB is spontaneously oxidized by molecular oxygen (O2) to give toxic reactive oxygen species (ROS) like superoxide or hydrogen peroxide while MB is formed again. In this way MB is available for the next cycle; it acts—as a functional unit with flavoenzymes and molecular oxygen—as a recycling catalyst against infectious organisms which wastes the precious electrons of NAD(P)H and molecular triplet O2 while producing toxic H2O2 in or close to the pathogen. As an alternative to O2, the ferric iron of methemoglobin can act as the agent which oxidizes leucoMB. There are also pharmacologic activities of MB which do not depend on its redox properties. Image credit: Heiner Schirmer

MB is metabolized in part to yield demethylated molecules like azure B and azure A. These compounds can have pharmacological effects as well: azure B, for example, was found to inhibit the aggregation of tau protein (Taniguchi et al., 2005) or even more efficiently than MB does (Wischik 1996). The IC50 values for inhibition of tau aggregation by MB and azure B are in the same order (0.1 to 5 μM) as for the inhibition of enzymes. Note that both compounds also inhibit the aggregation of Aβ in the μM range (Taniguchi et al., 2005). For azure B and azure A as the active agents, MB would be a prodrug. MB is excreted in the urine as a mixture of MB and leucoMB and other methylated thionins (Gaudette and Lodge, 2005). MB-containing urine is very clear and has, of course, a green or blue color which disappears a few days after the last administration of MB (Guttmann and Ehrlich, 1891).

History of MB
MB was the very first fully synthetic drug used in medicine. In 1891 Paul Guttmann and Paul Ehrlich applied it for the treatment of malaria, and this application has recently been revived (Vennerstrom et al., 1995; Färber et al., 1998; Coulibaly et al., 2009). The famous Giemsa solution for staining and characterizing malaria parasites and blood cells contains MB, eosin A, and azure B as active principals (Fleischer, 2004; Barcia, 2007). Numerous other microscopic discoveries including the identification of Mycobacterium tuberculosis by Robert Koch and the structural organization of nerve tissues (Ehrlich, 1886; Cajal, 1896; Garcia-Lopez et al., 2007) were based on the biochemical properties of MB. Staining with MB was also the beginning of modern drug research (Kristiansen, 1989): Paul Ehrlich argued that if pathogens like bacteria and parasites are preferentially stained by MB, then this staining might indicate a specific harmful effect on the pathogen which could be exploited for fighting disease. This explains why the terms “drug” and “dye” were used synonymously until World War I.

Malaria and methylene blue played a major role also in World War II. In 1943, General Douglas MacArthur, commander of the Allied Forces in the Southwest Pacific theater, stated: "This will be a long war, if for every division I have facing the enemy, I must count on a second division in the hospital with malaria, and a third division convalescing from this debilitating disease." Because of the blue urine (“Even at the loo we see, we pee, navy blue”), MB was not well liked among the soldiers.

Going back to the beginning of the twentieth century, MB was used for a wide variety of medical and hygienic indications (Clark et al., 1925). Inter alia it was added to the medication of psychiatric patients in order to study their compliance which could be monitored by the observable color of the urine. These studies led to the discovery that MB has antipsychotic and other psychotropic effects (Ehrlich and Leppmann, 1890; Bodoni, 1899) improving the condition of many patients. Thus, MB became the lead compound for other drugs including chlorpromazine and the tricyclic antidepressants. In 1925 W. Mansfield Clark, famous for the introduction of the pH electrode and the oxygen electrode, was coauthor of an impressive 80-page review on the application of MB in engineering, industrial chemistry, biology, and medicine (Clark et al., 1925). A remarkable aspect of this article is the reference list of illustrious scientists including several Nobel Prize winners (Santiago Ramon y Cajal, Robert Koch, Paul Ehrlich, Alphonse Laveran, Otto Meyerhof, and Heinrich Wieland), who contributed major papers on MB. Thus, MB is an example for the value of articles that were published 100 years ago. It is obvious that the ideas, observations, actual investigations, and conclusions were superior to many current publications.

Current Medical Indications
By 2009, there are more than 10,000 entries for “methylene blue” in the biomedical library PubMed, not counting the studies which had been published in the era not covered by PubMed. Current indications for MB that are approved by the U.S. Food and Drug Administration (FDA) are enzymopenic hereditary methemoglobinemia and acute acquired methemoglobinemia, prevention of urinary tract infections in elderly patients (Table 1), and intraoperative visualization of nerves, nerve tissues, and endocrine glands as well as of pathologic fistulae.

Of great practical importance is also the administration of MB for the prevention and treatment of ifosfamid-induced neurotoxicity in cancer patients (Kupfer et al., 1994). Recommended doses are three (to six) times 50 mg/d i.v. or p.o. as a treatment and three (to four) times 50 mg/d p.o. given for prophylaxis, starting one day before ifosfamid-infusion and continuing still after oxazaphosphorin-treatment is done (Pelgrims et al., 2000). Concerning inborn enzymopenic methemoglobinemia (Table 1), the treatment of the Blue People of Troublesome Creek in Kentucky (and other persons worldwide) using the blue drug MB was a visible success of knowledge-based medicine (Cawein et al., 1964). The rationale is that MB can be reduced to colorless leucoMB by erythrocyte enzymes and that leucoMB reduces the inactive methemoglobin to give hemoglobin (Figure 1). This conversion turns the bluish tinge of the skin to a rosy complexion—in the early 1960s the right issue for emerging color TV. Furthermore, topical MB is the treatment of choice for priapism (Van der Horst et al., 2003), and for intractable pruritus ani (Wolloch and Dintsman, 1979; Mentes et al., 2004; Sutherland et al., 2009). Recently, MB was introduced against acute catecholamine-refractory vasoplegia and other forms of shock (Shanmugam, 2005).

There are contraindications for MB. This applies, for instance, to patients taking serotonin reuptake inhibitors (Ramsay et al., 2007, Khavandi et al., 2008) and possibly to persons with certain types of hereditary G6PD deficiency. G6PD is the abbreviation for the enzyme glucose-6-phosphate dehydrogenase, which provides antioxidant-reducing equivalents in the form of NADPH. G6PD deficiency in different forms affects more than 500 million persons in the world and is thus the most common potentially hazardous hereditary condition. On the other hand, positive side effects of MB acting as a tonic have also been observed, possibly due to an enhancement of mitochondrial activity (Cardamatis, 1900, Riha et al., 2005, Atamna et al., 2008)

Pleiotropism of MB
There is an amazing number of different molecular targets which have been identified on a molecular level for MB and its demethylated metabolites such as azure B. The most prominent targets are NO synthases, guanylate cyclase, methemoglobin, monoamino-oxidase A, acetylcholine esterases, and disulfide reductases such as glutathione reductase or dihydrolipoamide dehydrogenases (Wikipedia, 2009). As to the interactions with the flavin-dependent disulfide reductases, MB is not only a (non-competitive or uncompetitive) inhibitor but also a substrate (Figure 1). MB is reduced by the flavoenzyme, and the resulting leucoMB is spontaneously oxidized by molecular oxygen (O2) to give toxic reactive oxygen species (ROS) like superoxide or hydrogen peroxide while MB is formed again. In this way, MB is available for the next cycle; it acts as a functional unit with flavoenzymes and molecular oxygen as a recycling catalyst against infectious organisms.

MB as an Analogue of the Phenazine Compound Pyocyanin
An explanation for the pleiotropism of MB could be that it is a thioanalogue (Schirmer et al., 2008) of the blue secondary metabolite pyocyanin, which acts as a signaling compound and a virulence factor (Dietrich et al., 2008). Secondary metabolites (Kossel, 1908) such as caffeine, acetyl salicylate (ACC, aspirin), and most antibiotics have many effects in different organisms, but evolution has primed them nevertheless for biospecific interactions (Dietrich et al., 2006; Ahuja et al., 2008; Dietrich et al., 2008). Synthetic drugs, in contrast, have not gone through this evolutionary training.

Methylene Blue for Falciparum Malaria in Children
The revitalization of MB as an antimalarial drug candidate began in 1998 when we observed that MB interacts with glutathione reductase of the malaria parasite Plasmodium falciparum (Atamna et al., 1996; Färber et al., 1998). The primary goal of our work is to develop an affordable, available, and accessible therapy of uncomplicated falciparum malaria for children under five years of age in Africa. The results of the clinical studies directed by Olaf Müller and Peter Meissner are promising. Methylene blue-based combination therapy is efficacious and safe even for children with the African form of glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency Aminus); this condition affects approximately 15 percent of the male population in West Africa. As shown in an anthropological study, MB-based therapies are accepted by the communities in spite of the blue discoloration; on the contrary, blue washable spots in clothes or diapers indicate patient compliance to caretakers and health workers (Müller et al., 2008; Coulibaly et al., 2009).

Pharmacokinetics and bioavailability of MB given orally have recently been reinvestigated (Walter-Sack et al., 2009); details are shown in the legend of Table 1. MB is active in vitro and in vivo not only against the malaria-causing blood schizonts (Vennerstrom et al., 1995; Akoachere et al., 2005), but also against the gametocytes of P. falciparum which are responsible for disease transmission from patients to mosquitoes (Buchholz et al., 2008; Coulibaly et al., 2009). As a therapy, two oral doses of 12 mg MB/kg body weight are administered for three days, that is, in total 72 mg/kg (Zoungrana et al., 2008). The follow-up period for appropriate clinical and parasito-logic response is 28 days in these studies. Formulations that are suitable for children, a sweet granulate or syrup of MB, have recently been developed (Gut et al., 2008).

Olaf Müller and his team did not observe different effects of MB when using different commercial sources including the 0.1 g MB capsules prepared for us at the University Pharmacy. Since 2007, however, we have had difficulties in obtaining sufficient MB from pharmaceutical companies for the clinical trials in West Africa. In addition, it was said that the available MB preparations were not pure enough, the major contaminants being heavy metals, demethylated MB derivatives, and water. The pros and cons of impurities, if present, cannot be discussed here in detail. I regard the requirements of USP and EP (listed for instance in www.methylen-blue.com) as appropriate. Metals like copper and chromium ions are essential nutrients, and it is interesting to compare their contents in a daily MB dose (Table 1) with their contents in the ingredients of a standard meal. As a conservative physician I am prejudiced against the overblown safety concerns of modern medicine, which too often prevent health- or even life-saving measures.

The explanations cited for the shortage of MB were contradictory; the most plausible one was that there are ongoing market rearrangements. We were privileged to receive MB as part of the DSM Dream Action Award to Wolfgang Schiek and his colleagues from DSM Fine Chemicals, Austria.

In this context it should also be emphasized that MB is an ethical drug, which implies that the drug must be affordable and available everywhere in sufficient dosages for patients who need it, considering that the incidence of malaria exceeds 250 million cases per year. It is indeed debatable if development, production, and distribution of drugs for malaria and other diseases of the poor can be left to the pharmaceutical industry, as there is an enormous need but no demand for these drugs (Schirmer, 2004).

A typical daily dosage is 200 mg MB given orally. The apparent half-life of MB in the human body is approximately 10 hours; the bioavailability has been measured to be 72.3 percent. After the oral intake of 500 mg MB, the concentration in blood peaks at 19 μM; after i.v. administration of 50 mg MB the corresponding value is approximately 2.2 μM (Walter-Sack et al., 2009); the diverging data of Peter et al., (2000) and of other authors are discussed in the same paper.

The distribution among organs depends on the form of administration. When MB is given to rats intravenously, it will accumulate in the brain. MB can permeate the blood-brain-barrier in rats irrespective of the administration route—i.p. (O'Leary et al., 1968), intraduodenally, or i.v. (Peter et al., 2000; Walter-Sack et al., 2009). With patients it should never be given intrathecally.

Concerning MB toxicology, a dose of 7 mg/kg given i.v. leads to severe gastrointestinal symptoms in patients. For sheep the LD50 was found to be 42 mg/kg body weight.

MB for Slowing Down the Development of Alzheimer Disease?
Following the discussions of Claude Wischik’s report in blogs, there is little doubt that patients and their caretakers who suffer now will not wait until MB in the form of RemberTM might be registered as a drug for use against Alzheimer disease in 2012 or later. In order to get additional insights into the efficacy of MB, it might be interesting to retrospectively study possible neurological effects of MB in patients who have taken MB in the form of Urolene Blue for the prevention of urinary tract infections.

Concerning interactions with other drugs against Alzheimer disease such as acetylcholine esterase inhibitors (AChEI), it should be noted that the targets of MB as an inhibitor also include acetylcholine esterase (AChE) (Pfaffendorf et al., 1997). Thus, MB is expected to interfere with the effects of AChE inhibitors presently used in the therapy of Alzheimer disease.

References:
Ahuja, E.G., Janning, P., Mentel, M., Graebsch, A., Breinbauer, R., Hiller, W., Costisella, B., Thomashow, L.S., Mavrodi, D.V., and Blankenfeldt, W. 2008. PhzA/B catalyzes the formation of the tricycle in phenazine biosynthesis. Journal of the American Chemical Society 130(50): 17053-17061. Abstract

Akoachere, M., Buchholz, K., Fischer, E., Burhenne, J., Haefeli, W.E., Schirmer, R.H., and Becker, K. 2005. In vitro assessment of methylene blue on chloroquine-sensitive and -resistant Plasmodium falciparum strains reveals synergistic action with artemisinins. Antimicrobial agents and chemotherapy 49(11): 4592-4597. Abstract

Atamna, H., Krugliak, M., Shalmiev, G., Deharo, E., Pescarmona, G., and Ginsburg, H. 1996. Mode of antimalarial effect of methylene blue and some of its analogues on Plasmodium falciparum in culture and their inhibition of P. vinckei petteri and P. yoelii nigeriensis in vivo. Biochemical pharmacology 51(5): 693-700. Abstract

Barcia, J.J. 2007. The Giemsa stain: its history and applications. International journal of surgical pathology 15(3): 292-296. Abstract

Bodoni, P. 1899. Dell'azione sedativa del bleu di metilene in varie formi di psicosi. Clin Med Ital 21: 217-222.

Buchholz, K., Schirmer, R.H., Eubel, J.K., Akoachere, M.B., Dandekar, T., Becker, K., and Gromer, S. 2008. Interactions of methylene blue with human disulfide reductases and their orthologues from Plasmodium falciparum. Antimicrobial agents and chemotherapy 52(1): 183-191. Abstract

Cajal, S. 1896. El azul de metileno en los centros nerviosos. Rev Trimest Microgr 1: 151-203.

Cawein, M., Behlen, C.H., 2nd, Lappat, E.J., and Cohn, J.E. 1964. Hereditary Diaphorase Deficiency and Methemoglobinemia. Archives of internal medicine 113: 578-585. Abstract

Clark, W.M., Cohen, B., and Gibbs, H.D. 1925. Studies on oxidation-reduction VIII Methylene blue. US Pub Health Rep 40: 1131-1201.

Coulibaly, B., Zoungrana, A., Mockenhaupt, F.P., Schirmer, R.H., Klose, C., Mansmann, U., Meissner, P.E., and Muller, O. 2009. Strong gametocytocidal effect of methylene blue-based combination therapy against falciparum malaria: a randomised controlled trial. PloS one 4(5): e5318. Abstract

Dietrich, L.E., Price-Whelan, A., Petersen, A., Whiteley, M., and Newman, D.K. 2006. The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Molecular microbiology 61(5): 1308-1321. Abstract

Dietrich, L.E., Teal, T.K., Price-Whelan, A., and Newman, D.K. 2008. Redox-active antibiotics control gene expression and community behavior in divergent bacteria. Science (New York, NY 321(5893): 1203-1206. Abstract

Ehrlich, P. 1886. Über die Methylenblaureaktion der lebenden Nervensubstanz. Dtsch Med Wochenschr 12: 49-52.

Ehrlich, P. and Leppmann, A. 1890. Über schmerzstillende Wirkung des Methylenblau. Pergamon Press, London.

Färber, P.M., Arscott, L.D., Williams, C.H., Jr., Becker, K., and Schirmer, R.H. 1998. Recombinant Plasmodium falciparum glutathione reductase is inhibited by the antimalarial dye methylene blue. FEBS letters 422(3): 311-314. Abstract

Fleischer, B. 2004. Editorial: 100 years ago: Giemsa's solution for staining of plasmodia. Trop Med Int Health 9(7): 755-756. Abstract

Garcia-Lopez, P., Garcia-Marin, V., and Freire, M. 2007. The discovery of dendritic spines by Cajal in 1888 and its relevance in the present neuroscience. Progress in neurobiology 83(2): 110-130. Abstract

Gaudette, N.F. and Lodge, J.W. 2005. Determination of methylene blue and leucomethylene blue in male and female Fischer 344 rat urine and B6C3F1 mouse urine. Journal of analytical toxicology 29(1): 28-33. Abstract

Gut F, Schiek W, Haefeli WE, Walter-Sack I, Burhenne J. Cation exchange resins as pharmaceutical carriers for methylene blue: binding and release. Eur J Pharm Biopharm. 2008 Jun;69(2):582-7. Abstract

Guttmann, P. and Ehrlich, P. 1891. Über die Wirkung des Methylenblau bei Malaria. Berlin Klin Woch 28: 953-956.

Hattori, M., Sugino, E., Minoura, K., In, Y., Sumida, M., Taniguchi, T., Tomoo, K., and Ishida, T. 2008. Different inhibitory response of cyanidin and methylene blue for filament formation of tau microtubule-binding domain. Biochemical and biophysical research communications 374(1): 158-163. Abstract

Khavandi, A., Whitaker, J., and Gonna, H. 2008. Serotonin toxicity precipitated by concomitant use of citalopram and methylene blue. The Medical journal of Australia 189(9): 534-535. Abstract

Kossel, A. 1908. Die Probleme der Biochemie. Universitäts-Buchdruckerei, Heidelberg.

Kristiansen, J.E. 1989. Dyes, antipsychotic drugs, and antimicrobial activity. Fragments of a development, with special reference to the influence of Paul Ehrlich. Danish medical bulletin 36(2): 178-185. Abstract

Kupfer, A., Aeschlimann, C., Wermuth, B., and Cerny, T. 1994. Prophylaxis and reversal of ifosfamide encephalopathy with methylene-blue. Lancet 343(8900): 763-764. Abstract

Mentes, B.B., Akin, M., Leventoglu, S., Gultekin, F.A., and Oguz, M. 2004. Intradermal methylene blue injection for the treatment of intractable idiopathic pruritus ani: results of 30 cases. Techniques in coloproctology 8(1): 11-14. Abstract

Müller, O., Meissner, P., and Schirmer, H. 2008. Methylenblau in der Malariatherapie. Eine Alternative für Afrika? Flugmedizin, Tropenmedizin, Reisemedizin (FTR) 14: 33-35.

Necula M, Kayed R, Milton S, Glabe CG. Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem. 2007 Apr 1;282(14):10311-24. Abstract

O'Leary, J.L., Petty, J., Harris, A.B., and Inukai, J. 1968. Supravital staining of mammalian brain with intra-arterial methylene blue followed by pressurized oxygen. Stain technology 43(4): 197-201. Abstract

Pelgrims, J., De Vos, F., Van den Brande, J., Schrijvers, D., Prove, A., and Vermorken, J.B. 2000. Methylene blue in the treatment and prevention of ifosfamide-induced encephalopathy: report of 12 cases and a review of the literature. British journal of cancer 82(2): 291-294. Abstract

Peter, C., Hongwan, D., Kupfer, A., and Lauterburg, B.H. 2000. Pharmacokinetics and organ distribution of intravenous and oral methylene blue. European journal of clinical pharmacology 56(3): 247-250. Abstract

Pfaffendorf, M., Bruning, T.A., Batnik, H.D., and van Zwieten, P.A. 1997. The interaction between methylene blue and the cholinergic system. British journal of pharmacology 122(1): 95-98. Abstract

Riha, P.D., Bruchey, A.K., Echevarria, D.J., and Gonzalez-Lima, F. 2005. Memory facilitation by methylene blue: dose-dependent effect on behavior and brain oxygen consumption. European journal of pharmacology 511(2-3): 151-158. Abstract

Schirmer, R.H., Adler, H., Zappe, H.A., and Gromer, S. 2008. Disulfide reductases as drug targets: methylene blue combination therapies for falciparum malaria in African children. Prensas Universitarias de Zaragoza.

Shanmugam, G. 2005. Vasoplegic syndrome--the role of methylene blue. Eur J Cardiothorac Surg 28(5): 705-710. Abstract

Sutherland, A.D., Faragher, I.G., and Frizelle, F.A. 2009. Intradermal injection of methylene blue for the treatment of refractory pruritus ani. Colorectal Dis 11(3): 282-287. Abstract

Tan, C.D. and Rodriguez, E.R. 2008. Blue dye, green heart. Cardiovasc Pathol. 2008 Aug 12. Abstract

Taniguchi, S., Suzuki, N., Masuda, M., Hisanaga, S., Iwatsubo, T., Goedert, M., and Hasegawa, M. 2005. Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. The Journal of biological chemistry 280(9): 7614-7623. Abstract

Van der Horst, C., Stuebinger, H., Seif, C., Melchior, D., Martinez-Portillo, F.J., and Juenemann, K.P. 2003. Priapism - etiology, pathophysiology and management. Int Braz J Urol 29(5): 391-400. Abstract

Vennerstrom, J.L., Makler, M.T., Angerhofer, C.K., and Williams, J.A. 1995. Antimalarial dyes revisited: xanthenes, azines, oxazines, and thiazines. Antimicrobial agents and chemotherapy 39(12): 2671-2677. Abstract

Wainwright, M. and Amaral, L. 2005. The phenothiazinium chromophore and the evolution of antimalarial drugs. Trop Med Int Health 10(6): 501-511. Abstract

Walter-Sack, I., Rengelshausen, J., Oberwittler, H., Burhenne, J., Mueller, O., Meissner, P., and Mikus, G. 2009. High absolute bioavailability of methylene blue given as an aqueous oral formulation. European journal of clinical pharmacology 65(2): 179-189. Abstract

Warth A, Goeppert B, Bopp C, Schirmacher P, Flechtenmacher C, Burhenne J. Turquoise to dark green organs at autopsy. Virchows Arch 454(3): 341-344. Abstract

Wischik, C.M., Bentham, P., Wischik, D.J., and Seng, K.M. 2008. Tau aggregation inhibitor (TAI) therapy with RemberTM arrests disease progression in mild and moderate Alzheimer's disease over 50 weeks. Alzheimer's and Dementia 4(4): T167.

Wischik, C.M., Edwards, P.C., Lai, R.Y., Roth, M., and Harrington, C.R. 1996. Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proceedings of the National Academy of Sciences of the United States of America 93(20): 11213-11218. Abstract

Wolloch, Y. and Dintsman, M. 1979. A simple and effective method of treatment for intractable pruritus ani. American journal of proctology, gastroenterology & colon & rectal surgery 30(1): 34-36. Abstract