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J Antimicrob Chemother 2010; 65: 399 – 409
doi:10.1093/jac/dkp466 Advance publication 6 January 2010
N-chlorotaurine, a natural antiseptic with outstanding tolerability
Waldemar Gottardi and Markus Nagl*
Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology,
Innsbruck Medical University, A-6020 Innsbruck, Austria
*Corresponding author. Tel: þ43-512-9003-70708; Fax: þ43-512-9003-73700; E-mail: [email protected]
N-chlorotaurine, the N-chloro derivative of the amino acid taurine, is a long-lived oxidant produced by activated
human granulocytes and monocytes. Supported by a high number of in vitro studies, it has mainly antiinflammatory properties and seems to be involved in the termination of inflammation. The successful synthesis
of the crystalline sodium salt (Cl-HN-CH2-CH2-SO3Na, NCT) facilitated its development as an endogenous antiseptic. NCT can be stored long-term at low temperatures, and it has killing activity against bacteria, fungi,
viruses and parasites. Transfer of the active chlorine to amino groups of molecules of both the pathogens
and the human body (transhalogenation) enhances rather than decreases its activity, mainly because of the
formation of monochloramine. Furthermore, surface chlorination after sublethal incubation times in NCT
leads to a post-antibiotic effect and loss of virulence of pathogens, as demonstrated for bacteria and
yeasts. Being a mild oxidant, NCT proved to be very well tolerated by human tissue in Phase I and II clinical
studies. A 1% aqueous solution can be applied to the eye, skin ulcerations, outer ear canal, nasal and paranasal
sinuses, oral cavity and urinary bladder, and can probably be used for inhalation. Therapeutic efficacy in Phase
II studies has been shown in external otitis, purulently coated crural ulcerations and keratoconjunctivitis, so far.
Based upon all presently available data, NCT seems to be an antiseptic with a very good relation between
tolerability and activity. Recently, C-methylated derivatives of NCT have been invented, which are of interest
because of improved stability at room temperature.
Keywords: chloramines, active chlorine compounds, monochloramine, biocide, disinfection
Introduction
Historical survey and functions of NCT in vivo
It has been known for .40 years that oxidants are involved in
the action of the human defence system.1 – 3 Among them,
so-called long-lived oxidants (N-chloro derivatives of amino
acids and peptides) were identified to contribute not only to
the killing of pathogens invading the body, but also to controlling
the inflammatory response.4 – 7
An important representative of this class of compounds is
N-chlorotaurine (NCT), which plays an essential role in vivo
because of both its relatively high concentration and its superior
stability.4,8,9 The compound is also cited as ‘taurine chloramine’,
a term that, however, does not conform to the International
Union of Pure and Applied Chemistry (IUPAC) nomenclature recommendation. It was a logical challenge to try to make NCT available for practical use, because its well-documented occurrence in
the human body and proved germicidal activity suggest it as an
antiseptic, which implies a minimal potential of intolerance.
Subsequent to the successful synthesis of NCT in form of the
sodium salt,10 an endogenous compound was available in ample
quantities, which, indeed, revealed an optimal compromise
between sufficient disinfecting power and good tissue compatibility. Both are basic requirements for an antiseptic in human
medicine.
The first extended studies on active chlorine compounds
(bearing O-Cl or N-Cl functions) date from the First World
War, and refer to the activity of chloramine T (CAT) and hypochlorous acid in vitro and in vivo.11 – 13 In 1930, the killing of
spores of Bacillus anthracis by N-chloroglycine in its dichlorinated form was described.14 The first explicit quotation of
NCT (denoted as ‘taurine chloramine’) was in 1971 by researchers from Poland, who found the chlorination of amino acids by
the myeloperoxidase system.15,16
In the following years, the properties and possible functions of
NCT as the main long-lived oxidant produced by leucocytes were
investigated, which comprise the killing of pathogenic organisms
and immune-modulatory reactions.
The first systematic quotation of NCT’s killing activity was in
1987, against larvae of the helminth Schistosoma mansoni.17
Some preliminary data indicating antibacterial and antifungal
activity were published by Thomas and Wagner et al. 18 – 20 Comprehensive studies followed, facilitated by the availability of NCT
as a pure sodium salt since 1989 and by the assessment of
its intrinsic chemical properties.10 In vitro, NCT proved to kill
bacteria,21 – 24 fungi (yeasts and moulds),23,25,26 viruses27 – 29
and protozoa.30
# The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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399
Review
In the 1990s, it was found that concentrations of NCT of up to
0.5 mM were not cytotoxic but caused a down-regulation of
mainly proinflammatory chemokines, cytokines and enzymes,
i.e. prostaglandin E2, tumour necrosis factor a, nitric oxide, interleukins 1b, 2, 6, 8 and 12, collagenase, and neopterin.31 – 44 As
one basic mechanism, Met45 oxidation of ikB-a followed by inhibition of the activation of NF-kB have been described.45 – 47
Another mechanism is the induction of heme oxygenase-1, a
stress-inducible enzyme with anti-inflammatory properties, at
mRNA and protein levels.48,49 Additionally, NCT induces a proliferative arrest independent from the cell cycle, followed by apoptotic cell death.50 – 52 All these reports support the concept that
NCT is involved in the termination of inflammation,5,6 although
activation of the complement system has also been
reported.53,54 Moreover, an anticoagulant activity of NCT was
found due to oxidation of fibrinogen, factor V, factor VIII and
factor X.55
Chemical issues
Formation in vivo
NCT is the N-chloro derivative of the b-aminosulphonic acid
taurine, a conditionally essential amino acid not involved in the
set-up of peptides. The substitution of the amine function of
taurine by a chlorine atom, e.g. by reaction with HOCl (Equation
3), results in the abolition of its basic properties, and is why it
loses the betaine structure. The emerging acid NCT-H disproportionates very fast, forming taurine and the strong acid
N,N-dichlorotaurine (NDCT-H) (Equation 4):
H3 Nþ CH2 CH2 SO
3 þ HOCl ! ClHNCH2 CH2 SO3 H þ H2 O
By a cascade of enzyme-catalysed reactions
!HOCl) known as the respiratory (or oxidative) burst, special leucocytes (neutrophilic and eosinophilic granulocytes, monocytes
and, in small amounts, probably macrophages) are able to synthesize the very strong oxidant and chlorinating agent hypochlorous acid (HOCl) from oxygen, hydrogen ions and chloride
ions.56
In turn, HOCl reacts with N-H compounds occurring in the
intra- and extracellular environment of leucocytes:
R-NH2 þ HOCl ! R-NHCl þ H2 O
Completely different is the sodium salt of NCT, whose anion
undergoes hydrolysis (Equation 5), forming initially a weak alkaline solution (pH 9) that soon, partly by the action of airborne
CO2, is neutralized, equilibrating between pH 8.2 (1% NCT) and
pH 7.1 (0.1% NCT):10
ð5Þ
Since disproportionation is largely suppressed in neutral and, particularly, in weak alkaline solution, NCT self-actingly produces the
right milieu (pH 7 –8) for an optimal stability.
NCT sodium salt forms colourless crystals that decompose at
135– 1408C. Already, at the temperature of a normal refrigerator
(2 –48C) both the salt and aqueous solution demonstrate sufficient stability for practical use. The decrease of oxidation
capacity [c(Ox)] was only 10% within 12 months.10
ð1Þ
Because taurine amounts to 50% of the amino acid pool of
neutrophilic granulocytes,57 NCT is the main reaction product,
which, moreover, exhibits surprising stability.10,16 The above reaction (Equation 1) is also understood as a detoxification of the
very strong oxidant HOCl, which would destroy living cells.3
Oxidizing properties
The oxidizing properties of NCT can be attributed to the N-Cl
function according to the general equation:
. N-Cl þ Hþ þ 2e !.N-H þ Cl
A typical oxidizing reaction of NCT, e.g. with a thiol (Equation 6),
reads as:
Synthetic approach
Although aqueous solutions of NCT can be obtained easily by
reaction of taurine with chlorinating agents such as hypochlorite,58 the isolation of the pure substance did not succeed in
the aqueous system. Unexpectedly, this was possible by a heterogeneous one-step reaction of taurine with an ethanolic solution of CAT.10 It yielded the only slightly soluble sodium salt
of NCT (NCT-Na, mol. wt 181.57), which could easily be separated from the well-soluble by-product toluenesulphonamide:
H3 Nþ CH2 CH2 SO
3 þ CH3 -C6 H5 -SO2 NClNa !
ClHNCH2 CH2 SO3 Na þ CH3 -C6 H5 -SO2 NH2
ð3Þ
2ClHNCH2 CH2 SO3 H $ Cl2 NCH2 CH2 SO3 H þ H3 Nþ CH2 CH2 SO
3 ð4Þ
þ
ClHNCH2 CH2 SO
3 þ H2 O $ ClH2 N CH2 CH2 SO3 þ OH
(O2!O2
2 !H2O2
2R-SH þ NCT ! R-SS-R þ taurine þ Cl
ð2Þ
ð6Þ
and shows that the final products of its reduction are taurine and
chloride.59,60
Against activated aromatic (Ar) C-H functions (phenols, histidine), NCT acts as a chlorinating agent according to Equation (7):
Ar-H þ NCT þ Hþ ! Ar-Cl þ taurine
The reaction implies the simultaneous transfer of sodium and
chlorine from CAT to taurine, and represents a special feature,
since it failed with connatural compounds such as
2,2-dimethyltaurine, b-alanine and g-aminopropanesulphonic
acid.10
400
Chemical characteristics
ð7Þ
NCT is a weak oxidant and lies on the lower end of the
following sequence, which is ranked according to decreasing
oxidizing potency: HOCl.chloroisocyanuric acids.chloramides.
chloramines.
Accordingly, unlike HOCl or chloroisocyanuric acids, NCT does
not react with amides and alcohols. Of particular importance
are reactions with amines, which are discussed below.
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Review
Transhalogenation equilibria
An essential property of N-chloro compounds, which was named
‘transhalogenation’ and ‘transchlorination’,60,61 designates the
transfer of the halogen of an N-chloro compound to another
N-H compound:
R-NHCl þ R0 -NH2 $ R-NH2 þ R0 -NHCl
ð8Þ
In general, it deals with an equilibrium that is reached at room
temperature within minutes and that needs no catalyst. It
suggests that in the presence of N-H compounds (amino acids,
peptides) not only one but diverse chlorinating agents can be
responsible for a defined reaction. The following three transhalogenation reactions of NCT are important for understanding its
activity.
Disproportionation
The above-mentioned Equation (5) discloses that a hydroxyl ion
is produced with the effect that the pH of the solution increases
and disproportionation is additionally slowed down. This reaction
brings about a dynamic pH regulation important for the longterm stability of NCT solutions, e.g. by neutralizing the hydrochloric acid that emerges at the intramolecular redox decay
reaction shown in Equation (10).
Equilibration with amino acids
An important feature of NCT concerns the increase of microbicidal activity in the presence of N-H compounds such as glycine
and a- and b-alanine, which can override consumption and
degradation effects.18,23,25,62 – 64 The origin of this effect, which
increases in the order glycine a-alanine ,b-alanine, is the formation of the corresponding N-chloro derivatives, whose bactericidal activity is higher than that of NCT alone. One can derive that
at pH 7 the portion of not dissociated, i.e. not charged, free
amino acid is 400 times higher for glycine (pKa¼ 2.36) and
a-alanine (pKa ¼ 2.35) and 7000 times higher for b-alanine
(pKa ¼ 3.55) compared with NCT (pKa¼ 20.3). This could
explain a fostered penetration of the uncharged molecules,
resulting in an improved bactericidal activity.
Monochloramine formation
A similar reaction, however, with a really dramatic increase
in bactericidal and fungicidal activity takes place in the
presence of ammonium, whereby monochloramine is produced
(Equation 9):
þ
þ
ClHNCH2 CH2 SO
3 þ NH4 $ H3 N CH2 CH2 SO3 þ NH2 Cl
ð9Þ
The mass-law constant of this reaction (5.8+1.21023) shows
that the equilibrium of Equation (9) lies on the left side.62
The specificity of this reaction is that at room temperature
and without any buffer it yields only monochloramine. If
strongly chlorinating agents such as CAT are used, also diand trichloramine, NHCl2 and NCl3, very unpleasantly smelling
by-products, are formed, which precludes application in
practice.62
The combination of NCT and ammonium chloride offers for
the first time the chance to use NH2Cl as an antiseptic agent
in human and veterinary medicine. Its enhanced bactericidal
activity originates from its small bulk and the absence of
charge (different to NCT), which both facilitate the penetration
of cell walls.4,18 Principally, every non-toxic ammonium salt
could be used for NH2Cl production. Because of the ubiquitous
presence of chloride in the human body, ammonium chloride
was applied.
Stability
The N-chloro derivatives of a-aminocarbonic acids (e.g. glycine
and alanine) disintegrate very fast at room temperature, i.e.
within hours to some days.16 The reasons for this are the
reactions presented in the following paragraph, which finally
lead to the complete loss of c(Ox). In the case of the baminosulphonic acid NCT the same reactions take place, but
extremely slowly. Both the pure compound (sodium salt of
NCT) and its aqueous solution lose only 10% activity per year if
stored in a refrigerator (2 –48C).10 This is an acceptable value in
view of application in medical practice.
Degradation and transformation products
All N-chloro compounds containing the structure element
R-CH2NHCl spontaneously undergo a degradation by first splitting off HCl followed by hydrolysis of the intermediary imine,
which yields an aldehyde and ammonia:
ClHN-CH2 -R ! HNvC-R þ Hþ þ Cl
ð10Þ
HNvCH-R þ H2 O ! OvCH-R þ NH3
ð11Þ
R ¼ COO ; CH2 -CO
2 ; CH2 -SO3
The loss of c(Ox) is very fast with N-chloro-a-aminocarbonic
acids,16,58 decreases with N-chloro-b-alanine and is slowest with
65
NCT (final product: sulphoacetaldehyde, O¼CH-CH2-SO2
The
3 ).
observed order can be explained by the dissimilarity of the
carboxylate and sulphonate of the isosteric molecules N-chlorob-alanine and NCT, while the interjacent CH2 group improves the
stability of N-chloro-b-alanine compared with N-chloro-a-alanine.
Another reaction affecting stability concerns transformation
to NDCT by disproportionation (see above). Since NDCT exhibits
antiseptic properties, too,66 this reaction is not connected with
a remarkable decrease of microbicidal activity.
General formulation of NCT use-solutions
Differing from common pharmaceutical practice, use-solutions
containing NCT are made without a buffer. One reason is that
NCT spontaneously brings forth a weak alkaline pH where disproportionation is widely repelled (see the ‘Chemical characteristics’
section above); a second reason is that the commonly employed
phosphate buffer increases disproportionation. The same applies
also to mixtures of NCT and NH4Cl. A buffer would thwart the
benefits of the dynamic pH regulation illustrated in the ‘Chemical
characteristics’ section and is therefore not used. Since NCT has
401
Review
no buffering capacity, it exerts only a marginal change of the
prevailing pH upon addition to a buffered system.
Microbicidal properties
Reactions with biological material
From the foregoing it can be derived that there are three
pathways for NCT to react with biological material, which differ
concerning reaction rates and the fate of c(Ox).10,58 – 60
NCT reacts
† with thiols very fast—c(Ox) is completely lost;
† with activated aromatic compounds (phenol, histidine) moderately fast—c(Ox) is completely lost;
† with amines (amino acids, ammonium)—an equilibrium is
established within minutes, c(Ox) remains intact; but
† not with amides and alcohols (as opposed to HOCl and
chloroisocyanuric acids).
All these reactions contribute not only to the microbicidal
activity, but they are also the reason for unwanted reactions
such as tissue irritation and consumption effects. The latter
effects are only weakly pronounced, based on NCT’s moderate
and restricted oxidizing power.
Antimicrobial activity
NCT without additives
A summary of the log10 reduction of viable counts by 1% NCT
(55 mM) in phosphate buffer solution is provided in Tables 1
and 2. Staphylococcus aureus, including methicillin-resistant
strains, Escherichia coli, Proteus mirabilis, Pseudomonas
aeruginosa, Streptococcus pyogenes, Diplococcus pneumoniae,
Klebsiella spp. and Helicobacter pylori are killed within a
few minutes to 20 min at 378C and within 20 –60 min at
208C21 – 23,67 (also M. Nagl, unpublished results). Incubation
times of 7 days are needed for a 3 log10 reduction of mycobacteria at 208C.63
Candida albicans, Candida krusei, Candida glabrata, Candida
tropicalis and Candida dubliniensis are killed within 2 –5 h at
378C,22,23,26 and Aspergillus spp., Penicillium spp., Fusarium spp.
and Alternaria spp. after 4 –8 h at 378C.25 Herpes simplex virus
types 1 and 2, all relevant types of adenovirus, and HIV-1 are
inactivated within 5– 60 min at 208C,27 – 29 and influenza viruses
within a few minutes according to presently performed investigations (M. Nagl, unpublished results). Acanthamoebae, leishmaniae and trichomonads are killed within 1 h at 378C, while cysts
of acanthamoebae are more resistant.30,68
In the study of 1987, S. mansoni larvae were killed after
18 h by physiological concentrations of 50 mM NCT.17
Bactericidal activity against staphylococci, E. coli, P. mirabilis
and P. aeruginosa could be found even by 12.5 mM.24
Table 1. Bactericidal and fungicidal activity of 1% NCT in phosphate buffer at pH 7.1 and 378C
Log10 reduction in cfu
Incubation time
Staphylococcus aureus ATCC 25923
Staphylococcus aureus ATCC 6538
Staphylococcus epidermidis ATCC 12228
Staphylococcus aureus Smith diffuse
MRSAa clinical isolate
Streptococcus pyogenes d68
Escherichia coli ATCC 11229
Proteus mirabilis ATCC 14153
Pseudomonas aeruginosa ATCC 27853
Candida albicans
Candida dubliniensis
Candida krusei
Candida glabrata
Candida tropicalis
Candida parapsilosis
Aspergillus flavus
Aspergillus fumigatus
Alternaria alternata
Fusarium moniliforme
Penicillium commune
a
b
402
Methicillin-resistant Staphylococcus aureus.
Not significant (P.0.05).
10 min
2.5–5.0
5.0
3.6–3.8
.6.7
2.0–2.3
.5.0
3.8 to .5.0
.5.0
3.8–5.0
0
0
0
0
0
0
0
0
0
0
0
1h
2h
4h
1.3–4.1
4.5
4.3–4.4
0 –0.2b
0.6–1.1
3.8– 4.7
.5.0
.5.0
1.6– 1.9
.4.0
.3.8
1.4– 2.6
1.4– 2.6
.5.0
.5.0
.5.0
.6.7
.5.0
.5.0
.5.0
.5.0
.5.0
0.2–2.3
2.0–3.5
1.0–2.5
0.2b
0.6–1.0
1.0–1.8
0.0–0.2b
0.0–0.2b
.1.1
0.2–2.0
0.0–0.9b
3.0– 3.4
2.1– 3.1
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Review
Table 2. Virucidal and protozoocidal activity of 1% NCT in phosphate buffer at pH 7.1 and 208C
Log10 reduction in pfu and viable cells, respectively
Incubation time
15 min
Herpes simplex virus type 1
Herpes simplex virus type 2
Adenovirus 5
Influenza virus A (H1N1, Hong Kong, 2000)
HIV-1
3.0–3.6
2.9–4.1
0.3–0.9
.5.0
.5.0
Acanthamoeba hatchetti trophozoites
Acanthamoeba polyphaga trophozoites
Leishmania donovani and infantum promastigotesa
Trichomonas vaginalis ATCC 30001 and ATCC 50138
1h
3h
3.1– 5.3
3.1– 5.1
3.6– 4.4
.5.0
.5.0
.5.0
.5.0b
a
Amastigotes were largely inactivated by 0.036% NCT at 378C.
378C.
b
NCT and monochloramine
Tests with bacteria and fungi performed with NCT in the presence
of ammonium chloride with both compounds in the range of
0.1% –1.0% disclosed a drastic reduction of killing times.62 The
most impressive results were obtained with both mycobacteria
and moulds. Mycobacterium terrae was reduced below the
detection limit within 20 min,63 while hyphae and spores of
moulds were killed within surprisingly short incubation times of
a few minutes.25,69 Moreover, cysts of acanthamoebae could
be inactivated,30 and there is a significant reduction even of bacterial spores (M. Nagl, unpublished results). The reason for this
increased activity can clearly be attributed to the formation of
monochloramine, which demonstrated a high activity as a pure
compound without NCT.69 – 71
pH effects
The microbicidal activity against bacteria and yeasts shows a significant pH effect, with shorter killing times at pH 4– 6 compared
with the normal environment at pH 7,22,24 which can be
explained as follows. Increasing acidity favours the formation
of the protonated form NCTHþ where the chlorine atom bears
a positive partial charge. This species is more likely to come
into reaction with nucleophilic agents such as R-NH2 than it is
the case with non-protonated NCT. The entity NCTHþ, therefore,
can be regarded as the active form in chlorination reactions.10
The increase of its portion at pH 5, at any rate, makes plausible
the higher chlorinating and bactericidal activity in the more
acidic milieu.
Influence of reducing substrates
Body fluids
The interaction with body fluids has an ambivalent impact, as
they can both decrease and increase the antiseptic activity of
NCT.23,25 On the one hand, complete consumption of c(Ox), up
to a concentration of 1% NCT, is possible, e.g. in human whole
blood (C. Martini and M. Nagl, unpublished results). Mainly
responsible for that are the very rapidly reacting S-H and S-CH3
functions belonging to free or protein-bound cysteine and
methionine.58,59
On the other hand, with less-reducing fluids such as blood
plasma and diverse inflammation samples, an increase of microbicidal activity was found against bacteria and, particularly,
fungi.21,23,25 This can be explained by the formation of fasteracting chloramines by transhalogenation (see above), whereby
the effect of reduction is counteracted. In large part, monochloramine is responsible for the enhancement of activity, formed from
ammonium ubiquitously present in these substrates.
Culture media
Nutrient solutions contain considerable amounts of reducing
substrates, which should not be used in killing tests with active
chlorine compounds. Therefore, MIC and MBC testing according
to protocols standardized for antibiotics reveals too high values
and hardly reasonable results.
Formation of bacterial chlorine covers
Chlorine cover designates a superficial c(Ox) attached on microorganisms, which is not an adsorption phenomenon but the
result of covalent N-Cl bonds fixed to the proteinaceous matrix
of the surface.72,73 This phenomenon, to date proven for bacteria
and fungi, was also discovered on skin surfaces after disinfection
with active halogen compounds.74,75 Surprisingly, bacteria and
fungi furnished with a chlorine cover are viable and can be
recultivated after the oxidizing coating is reduced again, e.g. by
thiosulphate, which exerts an immediate and non-destructive
elimination. Upon ice cooling, the chlorine cover persisted for
8 h after a 30% reduction during the first 2 h.72 The formation
of a chlorine cover without loss of viability, which typically develops upon incubation in 1% NCT for 1 min, can be considered the
initial step in the antimicrobial action of active chlorine compounds. Though not lethal, it exerts a lag of regrowth76 and a
loss of virulence (see the next section).
A striking effect was observed when bacteria (S. aureus) with
a chlorine cover were incubated in saline or in 5% NH4Cl. While in
saline no decrease in cfu was observed, a decrease of .5 log10
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Review
was observed within 120 min in NH4Cl.72 This can be explained
by a transhalogenation from the bacterial surface to ammonium
ions, forming the highly bactericidal monochloramine. In the
presence of reducing organic material (serum, nutrient solution),
the superficial c(Ox) vanishes very quickly.
Interestingly, the discovery of the viability of bacteria bearing
a chlorine cover was not possible until the mild oxidant NCT was
available. With other, stronger oxidants such as CAT or hypochlorite a high-level chlorine cover is formed, which obviously
exerts an immediate destructive impact on the bacterial
surface, whereby a rapid penetration of chlorinating molecules
leads to instantaneous killing.
Post-antibiotic effect of NCT
Interactions with sublethal times or concentrations (e.g. 1% NCT
for 1 min), which lead to measurable chlorine covers, cause a
post-antibiotic effect (delay of regrowth) connected with a loss
of virulence.72,76 As a consequence, eradication of bacteria by
the body’s own immune system can be fostered. This was
impressively demonstrated by survival in mice challenged
intraperitoneally with S. aureus strain Smith diffuse and
S. pyogenes.24,67 In line with that, a decrease of virulence
factors, i.e. secretory aspartyl proteinases of C. albicans and
C. dubliniensis as well as gliotoxin of Aspergillus fumigatus, was
found before viability of these fungi was impaired.26,69 As a consequence from these data and from earlier considerations
concerning the chemistry of chloramines,77 there is strong evidence that penetration of oxidative activity into the pathogens
is necessary for a microbicidal effect. Indeed, after incubation
times in NCT leading to a significant reduction in cfu, changes
within the cytosolic compartment were seen in electron
microscopy of S. aureus and changes of multiple intracellular
proteins of E. coli were found with proteomics.24,78
inflamed human eye.81 Patients suffering from bacterial conjunctivitis were cured within 3 –5 days in a Phase IIa open pilot
study.81
Viral conjunctivitis
Adenoviral conjunctivitis (epidemic keratoconjunctivitis), which,
to date, lacks a well-tolerated therapy, was investigated in a
Phase IIb clinical study.82 The symptoms could be attenuated
in severe infections, while the rate of subepithelial infiltrates
was not influenced. This could be due to a late onset of
therapy in some cases, but also to the low penetration of NCT
into the cornea. The latter could be enhanced by the addition
of ammonium chloride, with which NCT equilibrates to the
more lipophilic monochloramine.29 Indeed, in a rabbit epidemic
keratoconjunctivitis model the combination demonstrated
superior activity.29 Fewer positive viral cultures and shorter duration of viral shedding were found. Recently, 0.1% NCT plus
0.1% NH4Cl was well tolerated in a Phase I study,83 and presently
this formulation is investigated in viral conjunctivitis in humans.
Otorhinolaryngology
Two animal studies dealt with the safety of NCT in the ear.84,85
When injected to the middle ear of mice via the transtympanal
route, 1% and, to a higher extent, 10% NCT caused a transient
increase of the auditory brainstem response threshold due to
local irritation around the artificial perforation of the tympanic
membrane, but no damage of the inner ear.84 For testing
repeated dosing to the middle ear, a novel guinea pig model
with an implanted catheter system was used. Injection of low
volumes of 0.1% and 1% NCT, which did not lead to elevated
middle ear pressure and damage of the membranes, did not
cause toxic inner ear effects.85 The only side effect in some
animals was moderate thickening of the middle ear mucosa.
Microbicidal activity at physiological concentrations
In view of the estimated in vivo NCT concentrations of 10–
50 mM,3,4,9 which are 2750- to 550-fold lower than the generally
clinically used concentration of 55 mM (1% NCT), the question
arises how the killing of invaded pathogens proceeds in vivo.
In vitro experiments with these concentrations disclosed that
incubation times of many hours are necessary.24 Given the
reduction of NCT by the body’s own sulphur compounds and
transhalogenation to less stable N-chloramines, a sufficient
level of c(Ox) for an extended period can only be maintained if
continuous production by arriving granulocytes and monocytes
leads to a dynamic equilibrium between formation and loss.
Clinical studies and case reports
The antimicrobial and immune-modulatory features encouraged
investigations of the usability of 1% (55 mM) aqueous NCT solution as a topical antiseptic for the treatment of infections in
medicine.
Ophthalmology
The good tolerability of NCT up to 1% –2% was confirmed by
tests in the healthy rabbit and human eye,79,80 and also in the
404
External otitis
These results enabled the investigation of 1% NCT ear strips for
the treatment of external otitis in a Phase IIb study.86 It was significantly more effective than a combination of neomycin, polymyxin B and hydrocortisone. The time needed for disappearance
of the symptoms was 5.6+1.6 days in the NCT group, compared
with 7.4+1.6 days in the control group (P,0.01). According to
these studies, NCT seems to be a very well-tolerated and highly
effective medication in external otitis.
Rhinitis and sinusitis
Regarding these diseases, application of NCT as a nasal spray and
irrigation solution, respectively, is near at hand. In vitro tests with
human nasal tissue demonstrated that 1% NCT had hardly an
influence on the ciliary beat function of the epithelial cells in contrast to a deswelling solution used daily in practice.87 In a Phase
IIa study, a novel catheter (YAMIK) was used to irrigate the nasal
and paranasal sinuses three times a week for 1 month in
patients suffering from chronic rhinosinusitis. There were no
toxic side effects but some positive effects, i.e. mucosal deswelling, improved nasal breathing and olfaction, of which it is
unclear up to now if they were related to the irrigations per se
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or to NCT.88 An immunosuppressed patient who experienced
repeated maxillary bacterial sinusitis caused by P. aeruginosa
despite antibiotic treatment could be cured by irrigations with
1% NCT.89
purification steps of the viruses were necessary, which may be
a decisive advantage regarding preparation and efficiency.27
Arthritis
Dermatology
Crural ulcers
Particularly, patients with leg perfusion problems develop crural
ulcerations, which frequently become infected and inflamed. A
Phase IIb clinical study has been performed comparing the
effect of 1% NCT and 1% CAT in patients suffering from crural
ulcers.90 CAT was chosen as it is a well-known antiseptic,
which had already been used to treat infected wounds during
the First World War.11,13 Twice daily irrigations and bandages
soaked with these antiseptics for 5 days were sufficient to
remove the clinical signs of infection in both groups. However,
pain was significantly lower, and granulation and re-epithelialization
occurred significantly earlier in the NCT group.90
Due to the down-regulation of proinflammatory cytokines in
synoviocytes in vitro, NCT has been considered an interesting
medication in arthritis.38 In a collagen-induced arthritis mouse
model, administration of NCT (2 mM, 0.5 mL) for 21 days after
the first immunization resulted in a delay of the onset of symptoms, while similar application after the booster immunization
diminished the incidence of the disease.94 Since NCT was
applied subcutaneously in this study and did not achieve a sufficient concentration in the joints, the mechanism for the seen
effects remains unclear. In a septic arthritis mouse model,
locally (1 – 100 mM, 20 mL) but not intraperitoneally applied
NCT diminished the number of arthritic lesions.95
Pharmacokinetics
Other fields
Urinary tract infections
In these infections, antiseptics, in general, have been used
topically with only partial success, depending on the localization
of the infection. NCT (55 mM) proved to be well tolerated upon
repeated irrigations via a urinary catheter. Also, in one of three
cases the attempt to eradicate an omni resistant P. aeruginosa
from the urinary bladder was sufficient, although NCT was bactericidal in vivo.64
Oral cavity
In 2004, NCT was mentioned to inhibit the production of interleukin 6 and other proinflammatory substances, which may
play a role in periodontitis.91 Recently, the influence of 2% and
3% NCT mouth rinse on dental plaque was assessed. Rinsing
with 10 mL of the test solution two times daily for 4 days did
not inhibit plaque growth, but reduced its vitality.92
Bovine mammary gland
Naturally, not only human but also veterinary medicine may be a
field of interest. Irrigation of the bovine mammary gland for
5 days with 0.1% –2.0% NCT caused a transient increase of leucocytes in the milk, similar to 0.9% saline, but no severe side
effects.93 The absence of both systemic resorption and c(Ox) in
the milk for .5 h after lavage can be conceived as an advantage
of NCT compared with other antimicrobial agents.
HIV
A further interesting approach has arisen recently. According
to its properties as an antiseptic, NCT kills also HIV27 (also
H. Stoiber and M. Nagl, unpublished results). The viruses
became inactivated but remained immunogenic in a recent
study.27 In a mouse model, HIV treated with 2 –5 mM NCT
acted as a whole-killed highly active vaccine against murine
AIDS. The authors stressed that no harsh inactivation or
Activity in clinical studies is in good correlation with the pharmacokinetics of NCT. In human inflammation samples, c(Ox)
could be detected for a few hours in vitro, if started with
1% (55 mM).23 In vivo, c(Ox) was present for 15 min after
the application of one drop of a 1% solution into the eye.80
After irrigation of the maxillary sinus with the 1% solution,
c(Ox) could be detected for 3 –4 h.88 In the urinary bladder
it has been found for .1 h (90% decomposition within 1 h)
after the instillation of a 1% solution, if the catheter was
clamped.64
General reflections on active halogen
compounds and the position of NCT
as an antiseptic
The rate of microbial killing by an active halogen compound
(-O-Cl, .N-Cl) correlates with its oxidative power. Accordingly,
hypochlorous acid, the strongest active chlorine compound
that is stable in an aqueous system, will surpass all weaker oxidants (CAT, NCT and others) in this view, provided that tests are
done with washed bacteria. However, this approach does not
consider the conditions prevailing in practice, most notably the
presence of proteinaceous material (body fluids, corpuscular
material such as living and dead bacteria) and the contact
with body surfaces (skin, mucous membranes). The result of
these interactions is unwanted side reactions that lead to: (i)
consumption of c(Ox); and (ii) irritation of tissue. Since the
(wanted) microbicidal activity and the quoted (unwanted) side
reactions are based on the same fundamental reactions (oxidation and transhalogenation), a compromise has to be made
between sufficient microbicidal activity and a minimum of side
reactions. Depending on the sensitivity of the treated body site,
the adequate agent has to be chosen. A rather strong oxidant
such as CAT might be qualified for the disinfection of intact
skin, while a mild agent like NCT should be preferred for
mucous membranes and other delicate sites (for instance
eyes, ears, vagina, wounds). NCT can be regarded as an agent
of choice for irritable body sites because of the following reasons.
405
Review
NCT represents a native endogenous agent, which is
produced in vivo by leucocytes upon human host defence
The production of N-chloramines by the immune system is not
restricted to NCT, but encompasses the N-chloro derivatives of
all amine compounds (amino acids, peptides, ammonia)
present in the intra- and extracellular environment of white
blood cells capable of the respiratory burst.3,4,9,15,16,96 The
relevance of NCT originates both from its high in vivo
concentration—nearly 50% of these amines is made up of
taurine4—and the superior stability of NCT.4,15,16,58 Its creation
by the immune system suggests that the body has developed
tolerance not only towards NCT, but also to its possible
conversion products (e.g. NDCT) or metabolites (e.g.
sulphoacetaldehyde).
NCT fulfils the requirement of sufficient microbicidal
activity combined with good tissue tolerability
Microbicidal activity was assessed in vitro for a broad spectrum of
pathogens, and in vivo for bacteria and viruses (Tables 1 and 2).
Clinical studies revealed the applicability of NCT for the therapy of
several diseases and confirmed its tissue tolerability.
The final products of NCT after reaction with reducing
substrates are the non-toxic compounds taurine
and chloride
Taurine and chloride are present in the human body at relatively
high concentrations (the human body is made up of 0.1%
taurine;97 human serum contains 96 –110 mM chloride), so the
therapeutic application of NCT does not lead to measurable
changes. This is a unique feature in disinfection and antiseptic
practice. For instance, for iodine-containing preparations, which
are known for their excellent antiseptic properties, this cannot
be asserted in view of a potential thyroidal disorder induced by
iodide.98
The innate status of monochloramine, the efficacy
booster of NCT
As a consequence of the ubiquitous presence of ammonium in
the human body, monochloramine is formed whenever NCT
emerges.3,4,99,100 For the antiseptic combination of NCT with
ammonium chloride, therefore, the same assumption concerning the tolerance developed by the body can be stated as for
NCT alone (see above). The omnipresent appearance of
ammonium explains also the surprisingly high microbicidal
activity of NCT when it comes in contact with body fluids, in
spite of their proven reducing properties.23,25
NCT produces neither resistance nor allergic reactions
Due to the unspecific oxidative mechanism of action, in contrast
to antibiotics, development of secondary resistance can be
excluded, which was proven with four strains of bacteria and
C. albicans.23 Treatment with 1% NCT at a non-lethal exposure
time did not change the susceptibility of the tested strains.
Being an uncomplicated and, in the biological system, almost
permanently occurring ‘long-lived’ molecule of low molecular
406
weight also suggests the absence of allergic reactions during
therapeutic use. They were never observed during the hitherto
clinical studies.
In vitro anti-inflammatory properties of NCT might play
a role in clinical application
As mentioned in the ‘Historical survey’ section, there exists a
substantial number of studies in which a down-regulation of
proinflammatory cytokines produced mainly by stimulated
leucocytes was found in the presence of NCT (for review see
Koprowski and Marcinkiewicz,5 and Marcinkiewicz6). It may be
that such properties are responsible for the therapeutic
success, besides the antimicrobial activity in treatment of infections. This is very difficult to differentiate clinically and has never
been investigated in detail. In a small pilot study, NCT was
applied to the outer ear canal for post-operative care after tympanoplasty. The operation wound and the canal dried earlier in
the NCT group than in the control group, although there were
no signs of infection.101 Recently, the in situ expression of
some cytokines was measured in healthy pigs that inhaled
NCT, NCT plus ammonium, or saline as placebo. No differences
in the cytokine levels were found between the groups, but standard deviations were high.102 Therefore, the question of whether
anti-inflammatory effects of NCT play a role remains open to
date, although the signs from previous in vitro and in vivo
studies are rather supportive.
N-chloro derivatives of 2,2-dimethyltaurine
Recently, the synthesis of C-methylated derivatives of NCT,
which were promoted as possible substitutes for NCT, has
been reported.103 – 105 Mainly, the monochloro-dimethyltaurine
(MCDMT) and dichloro-dimethyltaurine (DCDMT) were suggested
as suitable candidates, because they show microbicidal properties and, at the same time, higher stability than NCT. The latter
assertion is quite plausible because no hydrogen atom exists
adjacent to the N-Cl function, which is necessary for splitting
off HCl (see the ‘Stability’ section above).
This advantage, however, is counteracted by the fact that
MCDMT and DCDMT cannot be designated as the body’s own
compounds. As a consequence, the premise about the developed
tolerance of the body against NCT and its transformation products cannot be similarly assigned to the new C-methylated
derivatives of NCT. This drawback has to be considered, since in
delicate antiseptic applications (inhalation,102 application in
mammary glands, disinfection of mucosa and large-area
wounds), a resorption of the compound or its reaction products
is conceivable by all means.
The main difference between NCT and its methylated derivatives is that the developed tolerance of the body against NCT is a
matter of fact, which allows no toxic side effects to be expected,
at physiological concentrations. This important feature could
also apply for MCDMT and DCDMT, but it remains to be proven.
Conclusions
Hitherto published literature reveals the man-made sodium salt
of the endogenous compound NCT as a very valuable antiseptic
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Review
in human and veterinary medicine. It offers a well-balanced
compromise between sufficient microbicidal activity and excellent tissue tolerability. In combination with ammonium chloride,
very potent preparations containing monochloramine, as well an
endogenous compound, are available, which kill even mycobacteria and moulds after very short incubation times. Not only NCT
but also its reaction products taurine and chloride are the body’s
own compounds, a fact that is unique in disinfection practice
since it points to an unrivalled tolerability.
Funding
W. G. and M. N. have received grant support from the Austrian Science
Fund (FWF), the Austrian National Bank and the Innsbruck Medical
University. Furthermore, they received support from the companies
Pathogenics, Acuity, Metasys and Agepha.
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Transparency declarations
None to declare.
22 Nagl M, Gottardi W. In vitro experiments on the bactericidal action of
N-chlorotaurine. Hyg Med 1992; 17: 431–9.
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