Is anyone thinking of doing studies with this drug on PSP patients who only have tau tangles and do not have amyloid plaques at all?

View all comments by joanna connolly

Methylene blue most likely decreases the hyperphosphorylation of tau proteins by inhibiting the formation of peroxynitrites (peroxynitrites form through the combination of superoxides and inducible nitric oxides). Methylene blue accepts electrons from various oxidases, thus limiting the formation of superoxides (and thus peroxynitrites).

Peroxynitrites play a critical role in the progression of Alzheimer disease. Peroxynitrites result in high GSK3 activity, which in turn causes the hyperphosphorylation of tau proteins. By largely inactivating protein kinase B (AKT) through tyrosine nitration and largely inactivating most forms of protein kinase C through cysteine oxidation of G proteins, peroxynitrites inhibit the two pathways by which GSK3 is inactivated. Peroxynitrites also decrease the protein kinase C mediated uptake of choline through muscarinic receptors and choline acetyltransferase activity. Thus, peroxynitrites cause large deficits in the memory storing compound acetylcholine.

Researchers should study the efficacy of other peroxynitrite inhibitors in combination...  Read more

Methylene blue most likely decreases the hyperphosphorylation of tau proteins by inhibiting the formation of peroxynitrites (peroxynitrites form through the combination of superoxides and inducible nitric oxides). Methylene blue accepts electrons from various oxidases, thus limiting the formation of superoxides (and thus peroxynitrites).

Peroxynitrites play a critical role in the progression of Alzheimer disease. Peroxynitrites result in high GSK3 activity, which in turn causes the hyperphosphorylation of tau proteins. By largely inactivating protein kinase B (AKT) through tyrosine nitration and largely inactivating most forms of protein kinase C through cysteine oxidation of G proteins, peroxynitrites inhibit the two pathways by which GSK3 is inactivated. Peroxynitrites also decrease the protein kinase C mediated uptake of choline through muscarinic receptors and choline acetyltransferase activity. Thus, peroxynitrites cause large deficits in the memory storing compound acetylcholine.

Researchers should study the efficacy of other peroxynitrite inhibitors in combination with or separate from methylene blue. Rosemary holds high promise in this regard. Rosemary can be inhaled directly into the brain, it decreases homocysteine levels (high homocysteine levels contribute to the formation of peroxynitrites), it directly scavenges peroxynitrites, and it limits tyrosine nitration. If you stop the formation of peroxynitrites, you stop the progression of Alzheimer disease.

References:
Alkam T, Nitta A, Mizoguchi H, Itoh A, Nabeshima T. A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Abeta(25-35). Behav Brain Res. 2007 Jun 18;180(2):139-45. Abstract

Guermonprez L, Ducrocq C, Gaudry-Talarmain YM. Inhibition of acetylcholine synthesis and tyrosine nitration induced by peroxynitrite are differentially prevented by antioxidants. Mol Pharmacol. 2001 Oct;60(4):838-46. Abstract

Heydrick SJ, Reed KL, Cohen PA, Aarons CB, Gower AC, Becker JM, Stucchi AF. Intraperitoneal administration of methylene blue attenuates oxidative stress, increases peritoneal fibrinolysis, and inhibits intraabdominal adhesion formation. J Surg Res. 2007 Dec;143(2):311-9. Abstract

Zhang YJ, Xu YF, Liu YH, Yin J, Li HL, Wang Q, Wang JZ. Peroxynitrite induces Alzheimer-like tau modifications and accumulation in rat brain and its underlying mechanisms. FASEB J. 2006 Jul;20(9):1431-42. Abstract

View all comments by Lane Simonian

There is just a small handful of information about methylene blue and Alzheimer's (see Atamna et al., 2008; Necula et al., 2007; Taniguchi et al., 2005; Wischik et al., 1996).

As an interesting and somewhat related concept, the use of phenothiazines for prion diseases has been investigated at UC San Francisco. Apparently phenothiazines were derived from methylene blue—not everyone knew that, perhaps.

A press release from UCSF said:

"In [Korth's] current study, he set out by identifying classes of drugs that were known to cross the blood-brain barrier to the brain, and then tested their ability to inhibit prion formation in the cultured mouse neuroblastoma cells.

"He identified only one class that met both criteria: phenothiazines, a group of tricyclic drugs used to treat psychosis. He then determined that a phenothiazine containing a particular side chain structure was the most effective. This was chlorpromazine.

"When he discovered that phenothiazines were derived from methylene blue, a dye used in England in the 1850s, he examined other derivatives of the...  Read more

There is just a small handful of information about methylene blue and Alzheimer's (see Atamna et al., 2008; Necula et al., 2007; Taniguchi et al., 2005; Wischik et al., 1996).

As an interesting and somewhat related concept, the use of phenothiazines for prion diseases has been investigated at UC San Francisco. Apparently phenothiazines were derived from methylene blue—not everyone knew that, perhaps.

A press release from UCSF said:

"In [Korth's] current study, he set out by identifying classes of drugs that were known to cross the blood-brain barrier to the brain, and then tested their ability to inhibit prion formation in the cultured mouse neuroblastoma cells.

"He identified only one class that met both criteria: phenothiazines, a group of tricyclic drugs used to treat psychosis. He then determined that a phenothiazine containing a particular side chain structure was the most effective. This was chlorpromazine.

"When he discovered that phenothiazines were derived from methylene blue, a dye used in England in the 1850s, he examined other derivatives of the dye and determined that one, quinacrine, had a similar tricyclic scaffold and the same side chain structure as chlorpromazine."

References:
Press Release

Korth C, May BC, Cohen FE, Prusiner SB. Acridine and phenothiazine derivatives as pharmacotherapeutics for prion disease. Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9836-41. Abstract

Atamna H, Nguyen A, Schultz C, Boyle K, Newberry J, Kato H, Ames BN. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J. 2008 Mar;22(3):703-12. Epub 2007 Oct 10. Abstract

Necula M, Breydo L, Milton S, Kayed R, van der Veer WE, Tone P, Glabe CG. Methylene blue inhibits amyloid Abeta oligomerization by promoting fibrillization. Biochemistry. 2007 Jul 31;46(30):8850-60. Epub 2007 Jun 27. Abstract

Taniguchi S, Suzuki N, Masuda M, Hisanaga S, Iwatsubo T, Goedert M, Hasegawa M. Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. J Biol Chem. 2005 Mar 4;280(9):7614-23. Epub 2004 Dec 17. Abstract

Wischik CM, Edwards PC, Lai RY, Roth M, Harrington CR. Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):11213-8. Abstract

View all comments by P.F. Jennings

How Does Rember^TM Work?
How exactly does Rember work? We have been puzzling over this in recent days, and are finding it difficult to believe that a drug so remarkably successful (yes, we know the caveats) could act on only one of the many problems in AD brain.

Rember is methylene blue, we are told. Methylene blue is a redox dye, which means it transports electrons. This is what mitochondria do. Methylene blue has been found to restore cognition to animals with dysfunctional cytochrome oxidase (Callaway et al., 2002), which is of great interest because cytochrome oxidase transports electrons in mitochondria and is low in AD brain (Mutisya et al., 1994).

Haem synthesis is another potential target of methylene blue. Very recently Atamna et al. (2008) found that methylene blue delays cellular senescence and improves haem synthesis. Haem is made in mitochondria and involves reduction of iron (III) to iron (II) by the electron transport chain, and specifically by cytochrome oxidase (Williams et al., 1976). In fact, cytochrome oxidase is itself a haem...  Read more

How Does Rember^TM Work?
How exactly does Rember work? We have been puzzling over this in recent days, and are finding it difficult to believe that a drug so remarkably successful (yes, we know the caveats) could act on only one of the many problems in AD brain.

Rember is methylene blue, we are told. Methylene blue is a redox dye, which means it transports electrons. This is what mitochondria do. Methylene blue has been found to restore cognition to animals with dysfunctional cytochrome oxidase (Callaway et al., 2002), which is of great interest because cytochrome oxidase transports electrons in mitochondria and is low in AD brain (Mutisya et al., 1994).

Haem synthesis is another potential target of methylene blue. Very recently Atamna et al. (2008) found that methylene blue delays cellular senescence and improves haem synthesis. Haem is made in mitochondria and involves reduction of iron (III) to iron (II) by the electron transport chain, and specifically by cytochrome oxidase (Williams et al., 1976). In fact, cytochrome oxidase is itself a haem enzyme, which means a defect in haem synthesis could feed back on itself in a vicious circle. Quite possibly the tangles that Rember is targeting would not develop in the first place if mitochondria were working properly to make resources available for breaking down faulty proteins before they become a problem. Rember dissolves tangles in vitro, like some other redox dyes (Wischik et al., 1996). Tau-tau interaction is thought to be the target, but it might not be the only one. According to Yamamoto et al. (2002), tangles isolated from AD brain can be dissolved by reduction of iron (III) to iron (II), which mirrors what methylene blue might be doing in haem synthesis (see above), and in methaemoglobinaemia, where it does indeed reduce iron (III) to iron (II) (Bradberry, 2003). Iron (III) can aggregate hyperphosphorylated tau via the phosphate groups, say Yamamoto et al., but iron (II) cannot. Iron is a problem in AD brain (Smith et al., 1997), and perhaps its ability to aggregate tau is just as important as its promotion of oxidative stress.

The success of Rember might have even wider significance. Very recently Leslie Klevay published a paper in Medical Hypotheses entitled “Alzheimer's disease as copper deficiency” (Klevay, 2008). Klevay is best known for the copper deficiency theory of heart disease (Klevay, 2000). Heart disease shares important characteristics with AD, not least high serum homocysteine (Whincup et al., 1999) and low cytochrome oxidase activity (Burke and Poyton, 1998).

Cytochrome oxidase is a copper enzyme as well as a haem enzyme. Copper is required for other aspects of iron metabolism besides haem synthesis, including iron efflux from the brain (Xu et al., 2004). Homocysteine metabolism, too, is intimately associated with that of copper (Bethin et al., 1995a and 1995b). Methylation reactions are inhibited by S-adenosylhomocysteine (SAH), which is broken down by SAH hydrolase, a copper protein. Another key enzyme in the pathway, methionine synthase, may require copper in addition to vitamin B12 (Tamura et al., 1999). Most significantly, copper and protein methylation are involved in NGF-dependent neurite outgrowth, and so is SAH hydrolase (Birkaya and Aletta, 2005).

High homocysteine means problems with methylation reactions. And here is the link with tangles in AD brain: methylation is needed for assembly of the phosphatase primarily responsible for dephosphorylating P-tau, PP2A (Vafai and Stock, 2002). Obeid et al. (2007) found correlations in neurological patients between CSF P-tau and homocysteine, SAH and the SAM/SAH ratio, and they suggest the link is through PP2A.

Copper is low in the modern diet, being largely removed during refining of grains, and Table 1 of the 2006 paper by Morris et al. shows an astonishing correlation between copper intake and cognitive function. It was recently found, most intriguingly, that Aβ peptides 1-40 and 1-42 are members of the Ecto-nox family of copper-dependent redox oscillators (Markert et al., 2004), which suggests they are not just toxic cellular junk.

Methylene blue is a kind of redox oscillator, too. All kinds of biological processes involve redox oscillations, almost certainly including neurite extension and axonal transport. Tangles are produced when these processes malfunction. Even if Rember doesn't turn out to work quite as well as it appears, it will still have made a major contribution to AD research.

References:
Atamna H, Nguyen A, Schultz C, Boyle K, Newberry J, Kato H, Ames BN. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J. 2008 Mar;22(3):703-12. Abstract

Bethin KE, Petrovic N, Ettinger MJ. Identification of a major hepatic copper binding protein as S-adenosylhomocysteine hydrolase. J Biol Chem. 1995a Sep 1;270(35):20698-702. Abstract

Bethin KE, Cimato TR, Ettinger MJ. Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levels. J Biol Chem. 1995b Sep 1;270(35):20703-11. Abstract

Birkaya B, Aletta JM. NGF promotes copper accumulation required for optimum neurite outgrowth and protein methylation. J Neurobiol. 2005 Apr;63(1):49-61. Abstract

Bradberry SM. Occupational methaemoglobinaemia. Mechanisms of production, features, diagnosis and management including the use of methylene blue. Toxicol Rev. 2003;22(1):13-27. Abstract

Burke PV, Poyton RO. Structure/function of oxygen-regulated isoforms in cytochrome c oxidase. J Exp Biol. 1998 Apr;201(Pt 8):1163-75. Abstract

Callaway NL, Riha PD, Wrubel KM, McCollum D, Gonzalez-Lima F. Methylene blue restores spatial memory retention impaired by an inhibitor of cytochrome oxidase in rats. Neurosci Lett. 2002 Oct 31;332(2):83-6. Abstract

Klevay LM. Dietary copper and risk of coronary heart disease. Am J Clin Nutr. 2000 May;71(5):1213-4. Abstract

Klevay LM. Alzheimer's disease as copper deficiency. Med Hypotheses. 2008;70(4):802-7. Abstract

Markert C, Morré DM, Morré DJ. Human amyloid peptides Abeta1-40 and Abeta1-42 exhibit NADH oxidase activity with copper-induced oscillations and a period length of 24 min. Biofactors. 2004;20(4):207-21. Abstract

Morris MC, Evans DA, Tangney CC, Bienias JL, Schneider JA, Wilson RS, Scherr PA. Dietary copper and high saturated and trans fat intakes associated with cognitive decline. Arch Neurol. 2006 Aug;63(8):1085-8. (Note that the conclusions of this paper appear to be at odds with the data in its Table 1.) Abstract

Mutisya EM, Bowling AC, Beal MF. Cortical cytochrome oxidase activity is reduced in Alzheimer's disease. J Neurochem. 1994 Dec;63(6):2179-84. Abstract

Obeid R, Kasoha M, Knapp JP, Kostopoulos P, Becker G, Fassbender K, Herrmann W. Folate and methylation status in relation to phosphorylated tau protein(181P) and beta-amyloid(1-42) in cerebrospinal fluid. Clin Chem. 2007 Jun;53(6):1129-36. Abstract

Smith MA, Harris PL, Sayre LM, Perry G. Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci U S A. 1997 Sep 2;94(18):9866-8. Abstract

Tamura T, Hong KH, Mizuno Y, Johnston KE, Keen CL. Folate and homocysteine metabolism in copper-deficient rats. Biochim Biophys Acta. 1999 May 24;1427(3):351-6. Abstract

Vafai SB, Stock JB. Protein phosphatase 2A methylation: a link between elevated plasma homocysteine and Alzheimer's Disease. FEBS Lett. 2002 May 8;518(1-3):1-4. Abstract

Williams DM, Loukopoulos D, Lee GR, Cartwright GE. Role of copper in mitochondrial iron metabolism. Blood. 1976 Jul;48(1):77-85. Abstract

Wischik CM, Edwards PC, Lai RY, Roth M, Harrington CR. Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):11213-8. Abstract

Whincup PH, Refsum H, Perry IJ, Morris R, Walker M, Lennon L, Thomson A, Ueland PM, Ebrahim SB. Serum total homocysteine and coronary heart disease: prospective study in middle aged men. Heart. 1999 Oct;82(4):448-54. Abstract

Xu X, Pin S, Gathinji M, Fuchs R, Harris ZL. Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis. Ann N Y Acad Sci. 2004 Mar;1012:299-305. Abstract

View all comments by Jane Karlsson

PubMed lists six peer-reviewed publications showing preclinical research in which methylene blue facilitates memory and one in which it prevents neurodegeneration by its combined action as a brain metabolic enhancer and antioxidant. Below is a list of these publications:

Wrubel KM, Riha PD, Maldonado MA, McCollum D, Gonzalez-Lima F. The brain metabolic enhancer methylene blue improves discrimination learning in rats. Pharmacol Biochem Behav. 2007 Apr;86(4):712-7. Epub 2007 Mar 6. Abstract

Wrubel KM, Barrett D, Shumake J, Johnson SE, Gonzalez-Lima F. Methylene blue facilitates the extinction of fear in an animal model of susceptibility to learned helplessness. Neurobiol Learn Mem. 2007 Feb;87(2):209-17. Epub 2006 Oct 2. Abstract

Zhang X, Rojas JC, Gonzalez-Lima F. Methylene blue prevents neurodegeneration caused by rotenone in the retina. Neurotox Res. 2006 Jan;9(1):47-57. Abstract

Riha PD, Bruchey AK, Echevarria DJ,...  Read more

PubMed lists six peer-reviewed publications showing preclinical research in which methylene blue facilitates memory and one in which it prevents neurodegeneration by its combined action as a brain metabolic enhancer and antioxidant. Below is a list of these publications:

Wrubel KM, Riha PD, Maldonado MA, McCollum D, Gonzalez-Lima F. The brain metabolic enhancer methylene blue improves discrimination learning in rats. Pharmacol Biochem Behav. 2007 Apr;86(4):712-7. Epub 2007 Mar 6. Abstract

Wrubel KM, Barrett D, Shumake J, Johnson SE, Gonzalez-Lima F. Methylene blue facilitates the extinction of fear in an animal model of susceptibility to learned helplessness. Neurobiol Learn Mem. 2007 Feb;87(2):209-17. Epub 2006 Oct 2. Abstract

Zhang X, Rojas JC, Gonzalez-Lima F. Methylene blue prevents neurodegeneration caused by rotenone in the retina. Neurotox Res. 2006 Jan;9(1):47-57. Abstract

Riha PD, Bruchey AK, Echevarria DJ, Gonzalez-Lima F. Memory facilitation by methylene blue: dose-dependent effect on behavior and brain oxygen consumption. Eur J Pharmacol. 2005 Mar 28;511(2-3):151-8. Abstract

Gonzalez-Lima F, Bruchey AK. Extinction memory improvement by the metabolic enhancer methylene blue. Learn Mem. 2004 Sep-Oct;11(5):633-40. Abstract

Callaway NL, Riha PD, Bruchey AK, Munshi Z, Gonzalez-Lima F. Methylene blue improves brain oxidative metabolism and memory retention in rats. Pharmacol Biochem Behav. 2004 Jan;77(1):175-81. Abstract

Callaway NL, Riha PD, Wrubel KM, McCollum D, Gonzalez-Lima F. Methylene blue restores spatial memory retention impaired by an inhibitor of cytochrome oxidase in rats. Neurosci Lett. 2002 Oct 31;332(2):83-6. Abstract

View all comments by Francisco Gonzalez-Lima

Is methylene blue, a rather old drug, finally on the way to becoming a cure? Speculation and criticism come by the dozen.

The blue urine may enhance placebo effects. Therefore it would be worthwhile to investigate human brain penetration before we start to speculate, and well before we inject or swallow it in larger numbers. Iodine-labeled methylene blue did not reach the brain within 14h, but the additional iodine may have interfered with brain penetration (Link et al.,1996). Therefore an 11C-labeled methylene blue would be far more appropriate. Strange enough: 11C-labeled methylene blue has been available at the University of Aberdeen since 2003 (Schweiger et al, 2003)!

So where are the data? Was the brain penetration of methylene blue disclosed at the ICAD?

References:
Link EM, Costa DC, Lui D, Ell PJ, Blower PJ, Spittle MF. Targeting disseminated melanoma with radiolabelled methylene blue: Comparative bio-distribution studies in man and animals. Acta Oncol. 1996;35(3):331-41. Abstract

Schweiger L, Craib S, Welch A, Sharp P. Radiosynthesis of [N-methyl-11C]methylene blue. Journal of Labelled Compounds and Radiopharmaceuticals, 2003 Nov;46,(13):1221-1228. Abstract

View all comments by Boris Schmidt

This report states that we had pooled randomization arms post-hoc in our efficacy analyses, which was not true. All of our analyses respected the original randomization, and the study remained double blind through to the end, i.e., two years. The primary analysis was conducted as pre-specified, and achieved statistical significance at the 24-week and 50-week time points. The effect was about an 84 percent reduction in the observed rate of progression over one year, regardless of how the analysis was conducted and which of several imputation methods was used in the ITT analysis.

View all comments by Claude Wischik