Reflections

Science in the cultural context

n 1906, Alois Alzheimer, together

with Emil Kraepelin and two Italian
colleagues, studied the brain of a
deceased patient suffering from profound short-term memory loss and
dementia. They observed the cerebral
cortex was smaller than normal and
identified, in stained sections, fibrous
aggregates of protein, more commonly called amyloid plaques. Later
Alois Alzheimer
that year, Alzheimer presented the first
evidence of observable brain pathology associated with malfunctioning cognition and dementia, yet it was Kraepelin who
first referred to the pathophysiology as Alzheimers disease
(AD) (1).

268

An estimated 26.6million people worldwide have AD;

this number may quadruple by 2050 (1); in the US alone,
there are thought to be 4.5 million afflicted patients (2).
Establishing a therapy that would delay the onset of AD by
only one year would save $9 billion in the US alone (3).
Recently, results from a clinical trial, presented at the 2008
International Conference on Alzheimers Disease (ICAD),
showed that an investigational drug was capable of arresting
progression in mild and moderate AD (4, 5). Enthusiasm must
be tempered, as the study involved relatively few patients
and the results must be confirmed by more extensive trials.
Nevertheless, this was the first intervention ever to arrest the
inexorable progression of AD.

Something Old... Something Blue

On reading this news, the first question to come to mind was:

what is the identity of the investigational drug? The answer
came as a stunning surprise. Not a bioengineered protein,
not a monoclonal antibody, not a new synthetic compound
specially designed to fit
N
snugly into an enzymes
CH3
H3C
pocket, the drug canN
S+
N
didate is a dye known
Cl
CH3
CH3
since 1876: methylMethylene blue
thioninium chloride, or
methylene blue (MB).
Despite a long and varied history, MB has fallen into obscurity. Its rebirth might come soon: if further clinical research
yields favorable results, MB may become a compound of surpassing interest to scientists and lay persons alike. It therefore
seems appropriate to review its 130-year medical history and
explore the rationale for its possible effectiveness against AD.

Early History of Methylene Blue

Methylene blue is made by reacting N,N-Dimethyl-pphenylenediamine in acidic medium with hydrogen sulfide
and ferric chloride. The sulfur atom bridges two molecules
of the phenylenediamine, forming a phenothiazine, and is
oxidized by the ferric ion to a positively charged thionium
ion. For industrial use as a dye, MB is supplied as a double
chloride with zinc; for bacteriologic and medicinal uses the
zinc-free form is employed.

Credit: Smithsonian Institution

First synthesized in 1876, concomitant with the beginnings

of bacteriology, MB found immediate use in the staining of
microorganisms, and its effect on bacterial metabolic behaviors was also studied. MB became an early research interest
of Paul Ehrlich, who would later participate in developing
diphtheria antitoxin, concocting the first chemotherapy for
syphilis (salvarsan), and enunciating the receptor theory of
antibody action. Ehrlich made the important observation that
in some tissues, MB was reversibly
reduced to a colorless (leuco-) form.
He also found that it possessed
a selective affinity for nerve cells
and nerve fiber endings, a finding which may have significance
in todays context. Reasoning that
it might therefore interfere with
conduction of pain impulses by the
nerves, Ehrlich obtained a supply
Paul Ehrlich
of purified MB and, together with
a physician named Lippmann, conducted a clinical trial in
patients with neuralgia and arthritis (6). MB produced an
analgesic effect but it tended to damage the kidneys if used
continuously (7).

MB as an Antimalarial
Noting that malaria-causing Plasmodia were stained by MB
and that the dye seemed safe for short-term administration,
Ehrlich and Guttmann, in 1891, administered MB as a treatment for malaria, on two patients in a Berlin hospital. The
results were positive, though not equal to those with quinine
(6). Still, the desirability of another antimalarial, lacking quinines side effects and usable by patients unable to tolerate
quinine, led to further trials by other physicians. In 1904,
Horatio C. Wood, Jr. performed what today would be called
a meta-analysis of
the clinical results
published as of that
date. Reviewing
425 cases (including
Ehrlichs two) gleaned
from eleven published
reports, he concluded
that 362 (85%) were
final cures, whereas
thirty-one suffered
Anopheles gambiae, bearer of malaria
relapses (8).
Although the search for antimalarials focused on synthetic
substitutes for quinine, physicians still turned to MB under certain conditions. Thus, a report from World War IIera Soviet
Union described the treatment of forty patients with a type
of malaria that had proved refractory to repeated courses
of quinine. Treatment consisted of five or six intravenous (iv)
infusions of 15-20 mL of 1% MB in 25% glucose. In most of
these patients, fever was terminated after the first or second
infusion and attacks did not recur. In twenty of the thirty-five
patients in whom plasmodia had been seen, the organisms disappeared from the peripheral blood. The author
concluded that MB has a definite role when the patient is
quinine-intolerant or does not respond to quinacrine and plasmoquine (9).
More recently, the antiplasmodial activity of MB was confirmed in vitro by Vennerstrom et al. whereby several dyes
were tested against three different strains of P. falciparum,
each having a different drug
susceptibility/resistance profile. Dyes of the xanthine,
azine, oxazine, and thiazine
categories were tested. The
thiazine (phenothiazine) dyes
were found to be the most
active antimalarials while
showing low cytotoxicityMB
was among the most potent
of this group (10).
Plasmodium falciparum

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Reflections

MB and Antimalarial Synthesis

MBs main contribution to the cure of malaria lies not in its
direct use in therapy, but in serving as a template for the
synthesis of substitutes for quinine. Sneader (11) relates that
Ehrlich had suggested making derivatives of MB as possibly
improved antimalarials. He was prevented from pursuing his
idea, first, because no animal model for malaria existed at
the time on which compounds could be tested, and second,
because he became involved in research on diphtheria antitoxin [see (12) for historical details of mushing the antitoxin
to Nome].
Only in 1924 did a screening method, using canaries,
become available. Werner Schuleman and colleagues at I. G.
Farben then took up Ehrlichs suggestion. By substituting a
diethylaminoethyl (DEAE) side chain for one of MBs methyl
groups, they obtained a promising compound, but as it was
deeply colored, they felt it might be unacceptable to patients.
Schulemans group substituted quinoline for the phenothiazine
nucleus, retained the DEAE side chain, and added some features of the quinine structure. Of the hundreds of compounds
prepared, the first clinically tested and marketed compound
was Plasmoquine (pamaquine). Thus, the first synthetic drug
designed as an antimalarial derives partly from MB (11).

A Urinary Antiseptic Prescription

Because it possesses antibacterial activity and is secreted into
the urine, MB is a key ingredient in a combination product
for treating infections and painful disorders of the urinary
tract. This product is remarkable for its longevity: it predates
the New Drug Application requirement of the 1938 Food,
Drug, and Cosmetic Act, and is still on the market today.
Originating in an era when physicians were accustomed to
writing multi-ingredient prescriptions (termed polypharmacy),
the product resembles such a prescription. In addition to 5.4
mg of MB, each tablet contains atropine sulfate 0.03 mg,
hyoscyamine 0.03 mg, methenamine 40.8 mg, benzoic acid
5.4 mg, and phenyl salicylate 18.1 mg (13). The atropine
and hyoscyamine, both parasympatholytic alkaloids, were
intended to relieve the pain of smooth muscle spasms in the
urinary tract. Methenamine, a complex compound made by
condensing formaldehyde with ammonia, was at one time
highly regarded. In acid medium methenamine breaks down
into its components; thus, if the urine is acidified (e.g., by
benzoic acid) the antibacterial action of free formaldehyde
would be released. In the 1930s, methenamine was recommended for many conditions, including pyelitis, cystitis,
prostatitis, urolithiasis, gonorrhea, meningitis, and polio
(14)truly a wonder drug, had it worked! The rationale
for adding phenyl salicylate (salol) resided in its antiseptic
action, as it contains insufficient salicylate for any analgesic

270

effect. This formula is on the market as Urised and Uriseptic,

and in generic form as Urinary Antiseptic #2 (13).

Treatment of Methemoglobinemia
MB is used therapeutically for a relatively uncommon condition wherein high concentrations of methemoglobin are present in the blood. Methemoglobin, an oxidation product of
hemoglobin in which the iron atom is in the ferric (trivalent)
state, gives the blood a brown color but does not combine
with oxygen, thus reducing the oxygen-carrying capacity
of the blood. Methemoglobinemia may be congenital, or
may be acquired, for example, from well-water containing
nitrites or from drugs such as benzocaine and sulfonamides.
MB acts through the MBleuco-MB redox system. Upon iv
injection MB reacts
N
N
H
H
with reductases in
N+
Cl
erythrocytes, forming its
H3C
CH3
dihydro derivative, colS
N
N
orless leuco-MB, which
CH3
CH3
can reduce methemoLeucomethylene blue
globin back to normal
(colorless, protonated form)
hemoglobin (15).
This use of MB was discovered long ago, and was well
established by the 1940s. In 1947, Bodansky and Gutmann
induced varying degrees of methemoglobinemia severity in
dogs. By correlating their animal data with results published
in the clinical literature they confirmed that an iv dose of
1-2 mg/kg of MB is rapidly effective against the symptoms
of severe methemoglobinemia (16). In 1993, Eldadah and
Fitzgerald recommended that methemoglobinemia be treated
by iv administration of 1-2 mg/kg of MB as a 1% solution,
infused over fifteen minutes. A second dose may be given,
up to a maximum of 7 mg/kg/24 hrs (17). Methylene Blue
Injection, a 1% sterile solution of MB, is officially recognized
in the United States Pharmacopeia (USP).

Drug Treatment of AD:

Present and Future
Currently Approved Drugs
The drugs currently used to treat AD patients act on brain
neurotransmitters, although it is recognized that they do not
address the cause. Some of the signs and symptoms of AD
can be attributed to a deficiency of cholinergic neurotransmissionpart of the cholinergic system theory of AD. Thus,
enhancing cholinergic function by inhibition of cholinesterase
could be helpful. Four AD drugs based on this mode of
action have been developed. Cholinesterase inhibitors do
not alter the course of the dementia but act to slow cognitive
demise. As AD progresses and fewer cholinergic neurons
remain functional, the effect of these drugs diminishes (18).

Something Old... Something Blue

NH2

Credit: Jerry Mason/

Photo Researchers, Inc.

The first centrally acting cholinesterase inhibitor to become

available was tacrine (Cognex). Its clinical effect was small,
and was further limited by its large intra-individual variation
in oral bioavailability and its potential liver toxicity, as evidenced by an increase in blood liver enzymes in about 50%
of patients (19).
Three additional cholinesterase inhibitors are approved in the
United States. The oldest, donezepil (Aricept), has been in
use since 1997. Rivastigmine (Exelon) also acts by the same
mechanism. It is structurally related to physostigmine, an alkaloid first isolated in 1864 from Calabar beans. Physostigmine
is a potent cholinesterase inhibitor, administered by injection or
by mouth, but it is too
short-acting to be very
useful (20). Exelon,
in capsules and oral
solution, must be taken
twice daily. Recently,
an Exelon transderCalabar Beans
mal patch has become
available, requiring
application only once
per day. The final
approved cholinesterase inhibitor of plant
origin is galantamine
(Razadyne). Isolated
in the 1950s from
Caucasian snowdrops
and from species of
Caucasian Snowdrops
Narcissus, its structure
and methods of synthesis were quickly worked out. In immediate release form it must be taken twice a day. A once-daily
capsule (Razadyne ER) is also available (21, 22).
Memantine (Namenda), a non-cholinesterase inhibitor, is
approved for AD and antagonizes N-Methyl-d-aspartate
receptors (NMDARs). Persistent activation of NMDARs in
the central nervous system (CNS) by glutamate contributes,
according to one hypothesis, to symptoms of AD. Memantine
acts by binding to NMDAR-operated cation channels (23).

Methylene Blue to the Rescue?

As noted, none of the above drugs attacks the cause of AD,
which is believed to involve brain proteins and peptides
rather than small-molecule neurotransmitters. Autopsy studies
have revealed that the brains of patients sufering from the
dementia of AD exhibit an accumulation of plaques, which
are deposits of the peptide beta-amyloid, and neurofibrillary

Tacrine

O
MeO

MeO
CH3

H3C
N

Donepezil

H3C
O

H 3C
O

Rivastigmine

CH3

CH3

N
H

H3C

N
H3C

CH3

Physostigmine

NH2

MeO

HCI

Galantamine

OH
H3C

CH3

Memantine

tangles (NFTs), composed of abnormal microtubules (24, 25).

The number of tangles correlates more closely with neuron
loss and dementia than do the numbers of plaques (26). It
is not known whether the plaques and NFTs cause AD, are
inactive by-products of the disease process, or are actually protective. There has been considerable research effort
aimed at dissolving beta-amyloid plaques or preventing their
accumulation, but thus far, no potential therapeutic agent has
emerged.
Another school of thought posits that the NFTs are a more
appropriate target. The microtubules which constitute the
tangles are composed of a polypeptide, tubulin, and a heatstable tubule assembly protein, named tau, which catalyzes
the polymerization of tubulin into microtubules (27). Tau
protein has been suggested as a therapeutic target for drugs
intended to combat AD. A significant study was recently
carried out in a mouse model of AD. Mice genetically engineered to develop beta-amyloid plaques and age-related
AD-like symptoms were crossed with mice in which the tau
gene had been inactivated, resulting in offspring with lower
levels of tau. In these mice a reduction of endogenous tau by

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Reflections

as little as 50% prevented memory loss and other AD symptoms (28). An earlier in vitro study showed MB blocks the
tau-tau interaction that would lead to the formation of NFTs,
whereas other phenothiazines (i.e., neuroleptic drugs such as
chlorpromazine or fluphenazine) were inactive in this respect
(29). The principal investigator of this study is also a principal in the start-up firm that conducted the clinical trial on MB
cited at the beginning of this article (1).
Will MB prove to be the first medicine to make a real difference in AD? There are formidable obstacles. The failure
rate is high in clinical Phase III among compounds which
pass Phase II successfully. The extent to which MB crosses
the blood-brain barrier is unknown. Further, its rapid excretion via the urine and the absorption problem hinted at in
the ICAD report point to additional potential difficulties.
Thus, there are good reasons for expectations to be muted.
Nevertheless, if its early promise in AD should be fulfilled,
MB would not be the first medicinal to come back from
obscurity. Arsenic trioxide, in use for centuries but deemed
worthless in the second half of the twentieth century, is now
approved for a rare form of leukemia. Thalidomide, the poster-child of disastrously dangerous drugs, has been rehabilitated as an orphan drug for multiple myeloma and erythema
nodosum leprosum, a painful inflammatory complication of
leprosy. A more benign example is provided by the vitamin
folic acid, which can mask pernicious anemia and hamper its
timely diagnosis. This finding, in 1947, prompted folic acids
exclusion from multivitamin products. For about thirty years,
the vitamin was relegated to insignificance, until research
in the 1980s and 1990s established that it prevented birth
defects such as spina bifida. As a result, fortification of all
enriched grain products with folate has been a governmentmandated requirement since 1998.
MB has been in use as a medicinal, to a greater or lesser
extent, continuously throughout the past century. It may now
return to play an important therapeutic role, either per se or
as a structural model for the synthesis of more effective molecules to combat AD.
doi:10.1124/mi.8.6.1

272

References
1.

en.wikipedia.org/wiki/Alzheimers_disease (Accessed Dec 3 2008)

2.

www.nia.nih.gov/Alzheimers/Publications/adfact.htm (Accessed Dec 3

2008)

3.

www.healthline.com/galecontent/alzheimers-disease-3 (Accessed Dec 3

2008)

4.

Wischik, C.M., Bentham, P., Wischik, D.J., and Seng, K.M. Tau aggregation inhibitor (TAI) with Lember arrests disease progression in
mild and moderate Alzheimers disease over 50 weeks. Presented at
International Conference on Alzheimers disease 2008 (ICAD), Chicago,
July 29, 2008.

5.

Marchione, M. Test drug shows promise in halting Alzheimers.

Philadelphia Inquirer, July 30, 2008, p. A6.

6.

Dale, H. Paul Ehrlich. Brit. Med. J. 1, 659665 (1954).

7.

Sneader, W. Drug Discovery: The Evolution of Modern Medicines.

Chichester, UK, John Wiley & Sons, p. 250 (1985).

8.

Wood, H.C., Jr. The use of methylene blue in malarial fevers. Proc. Phila.
Co. Med. Soc. 25, 281286 (1904).

9.

Grabenko, J.K. Combined methylene blue and glucose intravenous

therapy of malaria. Vrachevna Delo., Kharkov, p. 214. Abstract in JAMA
132, 481 (1946).

10. Vennerstrom, J.L., Mackler, M.T., Angerhoffer, C.K., and Williams, J.A.
Antimalarial dyes revisited: Xanthines, azines, oxazines and thiazines.
Antimic. Agents Chemother. 39, 26712677 (1995).
11. Sneader, W. Drug Discovery: A History. Chichester, UK, John Wiley &
Sons, p. 379 (2005).
12. Scheindlin S. The drug that launched a thousand sleds. Mol. Interv. 8,
152158 (2008).
13. American Drug Index, 51st Ed. St. Louis, MO, Walters Kluwers Health, p.
861 (2007).
14. The Merck Index, 5th Ed. Rahway, NJ, Merck & Co. Inc., p. 353 (1940).
15. Osol, A. and Pratt, R., Editors. United States Dispensatory 27th Ed.
Philadelphia, J.B. Lippincott Company, p. 745 (1973).
16. Bodansky, O. and Gutmann, H. Treatment of methemoglobinemia. J.
Pharmacol. Exp. Ther. 90, 4656 (1947).
17. Eldadah, M. and Fitzgerald, M. Methemoglobinemia due to skin application of benzocaine. Clin. Pediatr. (Phila.) 32, 687688 (1993).
18. Physicians Desk Reference Edition 60. Montvale, NJ, Thomson PDR, p.
1086 (2006).
19. Sweetman, S.C., Editor. Martindale: The Complete Drug Reference 35th
Ed. London, Pharmaceutical Press, pp. 331332 (2007).
20. Memory enhancement with oral physostigmine in Alzheimers disease.
New Eng. J. Med. 308, 720721 (1983).
21. The Merck Index 13th Edition. Whitehouse Station, NJ, Merck & Co. Inc.,
p. 771 (2001).
22. Physicians Desk Reference Edition 60. Montvale, NJ, Thomson, p. 2434
(2006).
23. Physicians Desk Reference Edition 60. Montvale, NJ, Thomson, p. 1199
(2006).

Something Old... Something Blue

24. Brunton, L.L., Lazo, J.S., Parker, K.L., Editors. Goodman & Gilmans The
Pharmacological Basis of Therapeutics 11th Edition. New York, McGraw
Hill, pp. 538540 (2006).
25. Tiraboschi, P., Hansen, L.A., Thal, L.J., and Corey-Bloom, J. The importance of neuritic plaques and tangles to the development and evolution
of AD. Neurology 62, 19841989 (2004).
26. Tanzi, R.E. Tangles and neurodegenerative diseasea surprising twist.
New Eng. J. Med. 353, 18531855 (2005).
27. Weingarten, M.D., Lockwood, A.H., Hwo, S.-Y., and Kirschner, M.W. A
protein factor essential for microtubule asssembly. Proc. Nat. Acad. Sci.
U.S.A. 72, 18581862 (1975).
28. Ashe, K.H. A tale about tau. New Eng. J. Med. 357, 933935 (2007).
29. Wischik, C.M., Edwards, P.C., Lai, R.Y. et al. Selective inhibition of
Alzheimers disease-like tau aggregation by phenothiazines. Proc. Nat.
Acad. Sci. U.S.A. 93, 1121311218 (1996).

)MPROVINGRESEARCH
PROTECTIONSMEANS
IMPROVINGRESEARCH
QUALITY

!!(200&OUNDING-EMBER

Stanley Scheindlin, DSc,

holds a BS in pharmacy from
Temple University and graduate
degrees in pharmaceutical
chemistry from Philadelphia
College of Pharmacy and
Science (now University of
Sciences in Philadelphia). His
academic research dealt with
plant constituents and chemical
interactions of vitamins. In his pharmaceutical industry career,
he handled new drug formulation developments, and later
regulatory affairs, presiding over the filing of about 100
generic new drug applications and two innovative drug
applications. Now retired, his activities include volunteer
work, consulting, and writing Reflections pieces for this
journal. E-mail stansch@verizon.net

!!(200ACCREDITATIONx
s IMPROVESYOURHUMAN
RESEARCHPROTECTION
PROGRAM
s ASSISTSYOURORGANIZATION
INACHIEVINGREGULATORY
COMPLIANCE
s HELPSYOURORGANIZATION
COMPETEFORSPONSORED
RESEARCH
s hRAISESTHEBARvINOVERALL
RESEARCHQUALITY
s BUILDSANDENHANCES
PUBLICTRUST
&ORINFORMATIONCALL
  WWWAAHRPPORG

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