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Tolerability of N-chlorotaurine plus ammonium chloride in the rabbit and human eye - A phase 1 clinical study

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N-chlorotaurine (NCT), an endogenous mild antiseptic, is well-tolerated by application to the human conjunctiva and has been shown to offer beneficial effects in infectious conjunctivitis. Animal tests revealed improved efficacy of a combination of NCT with ammonium chloride in adenoviral conjunctivitis. The aim of this study was to evaluate the tolerability of NCT plus ammonium chloride in the healthy rabbit and human eye. First, a tolerability study was performed in rabbits. In a blinded and randomized fashion, one eye was treated with the test medication, the other one with 0.9% saline. Twenty-one animals (three per concentration) were treated with one drop every 2 hours for 6 days. Second, in two volunteers one drop of a defined concentration was applied to one eye every 15 min for 1 hour, saline to the control eye. Four different concentrations were tested on different days. Third, a double-blind, randomized phase 1 study in 13 healthy volunteers was performed. One drop of 0.1% NCT plus 0.1% NH(4)Cl versus saline was applied every 15 min within the first hour, followed by four drops every 2 hours. This regimen was done daily for 5 days. In rabbits, no side effects were seen with 0.1% NCT plus 0.1% NH(4)Cl, while higher concentrations sometimes caused short-time and minimal conjunctival injection and secretion after dosing. By 1% NCT plus 1% NH(4)Cl, these effects were moderate, but disappeared again without any detectable residues. In the pilot study with two volunteers, treatment with 0.5% NCT plus 0.1% NH(4)Cl caused medium-scale eye burning for 30 seconds, while 0.1% NCT plus 0.1% NH(4)Cl was very well-tolerated, with no or minimal burning for a few seconds. In the subsequent phase 1 study, 0.1% NCT plus 0.1% NH(4)Cl was well-tolerated by all subjects except for minimal eye burning for a few seconds after dropping. No objective signs of eye changes could be detected in the human beings. The results of this study clearly demonstrate the good tolerability of a promising NCT formulation with improved activity.
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MEDICAL OPHTHALMOLOGY
Tolerability of N-chlorotaurine plus ammonium chloride
in the rabbit and human eye - a phase 1 clinical study
Barbara Teuchner &Eduard Schmid &Hanno Ulmer &
Waldemar Gottardi &Markus Nagl
Received: 12 April 2008 / Revised: 24 June 2008 /Accepted: 28 June 2008
#Springer-Verlag 2008
Abstract
Background N-chlorotaurine (NCT), an endogenous mild
antiseptic, is well-tolerated by application to the human
conjunctiva and has been shown to offer beneficial effects
in infectious conjunctivitis. Animal tests revealed improved
efficacy of a combination of NCT with ammonium chloride
in adenoviral conjunctivitis. The aim of this study was to
evaluate the tolerability of NCT plus ammonium chloride in
the healthy rabbit and human eye.
Methods First, a tolerability study was performed in
rabbits. In a blinded and randomized fashion, one eye was
treated with the test medication, the other one with 0.9%
saline. Twenty-one animals (three per concentration) were
treated with one drop every 2 hours for 6 days. Second, in
two volunteers one drop of a defined concentration was
applied to one eye every 15 min for 1 hour, saline to the
control eye. Four different concentrations were tested on
different days. Third, a double-blind, randomized phase 1
study in 13 healthy volunteers was performed. One drop of
0.1% NCT plus 0.1% NH
4
Cl versus saline was applied
every 15 min within the first hour, followed by four drops
every 2 hours. This regimen was done daily for 5 days.
Results In rabbits, no side effects were seen with 0.1%
NCT plus 0.1% NH
4
Cl, while higher concentrations
sometimes caused short-time and minimal conjunctival
injection and secretion after dosing. By 1% NCT plus 1%
NH
4
Cl, these effects were moderate, but disappeared again
without any detectable residues. In the pilot study with two
volunteers, treatment with 0.5% NCT plus 0.1% NH
4
Cl
caused medium-scale eye burning for 30 seconds, while
0.1% NCT plus 0.1% NH
4
Cl was very well-tolerated, with
no or minimal burning for a few seconds. In the subsequent
phase 1 study, 0.1% NCT plus 0.1% NH
4
Cl was well-
tolerated by all subjects except for minimal eye burning for
a few seconds after dropping. No objective signs of eye
changes could be detected in the human beings.
Conclusion The results of this study clearly demonstrate
the good tolerability of a promising NCT formulation with
improved activity.
Keywords N-chlorotaurine .Monochloramine .
Ammonium chloride .Active chlorine compounds .
Conjunctivitis .Oxidants .Tolerability .Rabbit model .
Clinical trial
Introduction
N-chlorotaurine (Cl-HN-CH
2
-CH
2
-SO
3
, NCT) is a mild
long-lived oxidant produced by human granulocytes during
the oxidative burst [3,24]. The chemical synthesis of the
crystalline sodium salt (Cl-HN-CH
2
-CH
2
-SO
3
Na) was
successful a few years ago [2]. It acts like an antiseptic
and is bactericidal against Gram-positive and Gram-
negative bacteria [6,7,10,11], virucidal [12]and
Graefes Arch Clin Exp Ophthalmol
DOI 10.1007/s00417-008-0900-x
B. Teuchner :E. Schmid
Department of Ophthalmology, Innsbruck Medical University,
Innsbruck, Austria
H. Ulmer
Department of Medical Statistics, Informatics, and Health
Economics, Innsbruck Medical University,
Innsbruck, Austria
W. Gottardi :M. Nagl (*)
Department of Hygiene, Microbiology and Social Medicine,
Division of Hygiene and Medical Microbiology,
Innsbruck Medical University,
Fritz-Pregl-Str. 3,
A-6020 Innsbruck, Austria
e-mail: m.nagl@i-med.ac.at
fungicidal [7,9,13]. Activity against a broad spectrum of
adenoviruses causing epidemic keratoconjunctivitis has
been proved [22,23].
Tolerability of 1% NCT (55 mM) by human tissue, for
instance in the healthy human eye [14], is very good, so the
substance seems to provide an optimal compromise
between antimicrobial activity and tolerability. In a phase
2a study on bacterial conjunctivitis, the substance was also
very well tolerated, and the inflammation could be healed
rapidly [17]. Recent phase 1 and 2 studies revealed very
good tolerability and good efficacy in local therapy of
crural ulcers [15] and external otitis [19]. Moreover, NCT
was very well tolerated upon irrigations of recurrent
sinusitis (Phase 2a) [18]. In one of three patients suffering
from cystitis caused by omniresistant Pseudomonas aerugi-
nosa, it was possible to eradicate the pathogen [16,26].
Viral conjunctivitis, particularly epidemic keratoconjunc-
tivitis (EKC) which is caused by adenoviruses, is a highly
infectious, in part severe eye disease, for which no well-
tolerated causative therapy exists to date [5]. Even in viral
conjunctivitis, efficacy of 1% NCT is very probable in the
sense of attenuation of severe courses of inflammation,
according to the results of our recent double-blind,
randomized phase 2 study [25]. This was confirmed in an
animal model using rabbits whose eyes were infected with
adenoviruses [23]. However, the development of subepi-
thelial infiltrates in man can probably not be inhibited by
NCT. These infiltrates are frequent (ca. 40% in the test and
control group in our study [25]), and may lead to vision
disorders for several months. Because of this fact, there is
the need to further improve the efficacy of NCT in viral
conjunctivitis.
It is known that addition of ammonium chloride (NH
4
Cl)
markedly enhances the activity of NCT against bacteria and
fungi [7,8,13,16]. This can be explained by transfer of the
oxidative chlorine atom to ammonium, at which mono-
chloramine is formed in equilibration, which is more
lipophilic than NCT and penetrates pathogens better [2,3].
Cl HN CH2CH2SO
3þNHþ
4
$H3NþCH2SO
3þNH2Cl ð1Þ
This is a natural process, as well, which occurs during
every inflammation upon which a mixture of chloramines is
formed after the oxidative burst. The chlorine transfer leads
to an improved microbicidal activity of NCT in body fluids
and inflammatory exudates compared to buffer solution [7,
13,16]. This can be regarded as an advantage, in the sense
that a mild substance is enhanced exactly at the place of
action. Our clinical studies prove clearly that these
processes do not cause toxic effects.
Although the direct virucidal effect of NCT is enhanced by
ammonium chloride only to a small extent [Nagl M,
unpublished], the combination may be of advantage in viral
conjunctivitis because of the following facts. Plain NCT hardly
permeates the cornea [23], and attack of subepithelially placed
viruses might be difficult. In vitro, significantly more
oxidative activity permeates the cornea if ammonium chloride
is added [23]. Actually, in the EKC rabbit model the efficacy
of NCT plus ammonium chloride proved to be significantly
higher than that of NCT alone, demonstrated by higher in
vivo antiviral activity and shorter periods of viral shedding.
Concentrations of 0.1% NCT +0.1% NH
4
Cl were equally
effective to 1% NCT +0.1% NH
4
Cl or 0.1% NCT +1%
NH
4
Cl [23]. This seems surprising, but may be explained by
the fact that the concentrations of the formed monochloramine
(NH
2
Cl) differ, at maximum, by about a factor of 3, with
31.5, 91.2, and 75.0 ppm respectively [1]. It was the aim of
this study to evaluate the tolerability of NCT plus ammonium
chloride in the healthy eye in the rabbit and in man.
Materials and methods
Reagents
Pure NCT as a crystalline sodium salt (molecular weight
181,57) was prepared according to [2]. Purity was proved
by spectrophotometry and standardized quality controls.
Ammonium chloride at the highest available purity was
purchased from Merck (Darmstadt, Germany). Both
reagents were dissolved in sterile and pyrogen-free distilled
water to a final concentration of 0.1% NCT (5.5 mM) and
0.1% NH
4
Cl (18.7 mM). In a pilot study, higher concen-
trations were applied also (see below). Solutions were
prepared before the beginning of the study and stored at
24°C. They were contained in flasks of similar appearance
to ensure double-blinding, and all eye drops were numbered
consecutively in accordance with the randomization code.
Animal study
The animal experiments were approved by the Austrian
Federal Government Department for Science and Research
and followed the Principles of laboratory animal care.
Twenty-one rabbits (from SAVO, Kissleg/Germany) were
randomized and divided into groups of three. One eye was
treated with the test medication, the control eye with 0.9%
NaCl. The schedule of application was one single dose of
one drop five times per day (every 2 hours) for 6
consecutive days in total.
To find out well-tolerated and irritating concentrations, the
following test medications were applied to three animals each:
1% NCT +1% NH
4
Cl, 1% NCT +0.1% NH
4
Cl, 0.1% NCT
+1% NH
4
Cl, 0.1% NCT +0.1% NH
4
Cl, and 1% NH
4
Cl
(control without NCT). One week later, 0.2% NCT +0.2%
NH
4
Cl as well as 0.3% NCT +0.3% NH
4
Cl were tested.
Graefes Arch Clin Exp Ophthalmol
Applications and examinations were performed in a
blind fashion. The external parts of the eye (conjunctiva,
cornea, sclera, iris) were examined daily by usage of a slit
lamp and magnifying glasses. The grading of adverse
effects was similar to that used in human subjects described
below. Examinations were performed daily during the
treatment period, 1 and 2 weeks later and 4 months later
to exclude long-time adverse effects.
Phase 1 - study design
Both a previous pilot study in two healthy volunteers and
the main study in 13 healthy volunteers were designed as a
double-blind, randomized, placebo-controlled phase 1
study. This university trial was in accordance with the
Declaration of Helsinki, and it was approved by the Ethics
Committee of the University of Innsbruck. All subjects
gave written informed consent.
Pilot study
A pilot study with two male subjects (39 and 71 years old)
was done to identify the tolerated dose and to get
pharmacodynamic data. Because it had been tolerated
without any side effects in the rabbit study, we started with
a test concentration of 0.1% NCT +0.1% NH
4
Cl. Subse-
quently, 0.5% NCT +0.1% NH
4
Cl, then 0.3% NCT +0.1%
NH
4
Cl, and finally 0.1% NCT +0.2% NH
4
Cl were applied
to one eye, isotonic saline solution (0.9%, placebo) to the
other one. It was the intention to perform frequent dosing
connected with practicable and close monitoring. There-
fore, each concentration was dripped on one different day,
and five single doses of one drop were administered within
1 hour, i.e. one drop every 15 min. Both subjects were
examined as described below 3 to 4 hours after the last
single dose on each day of dosing.
For qualitative examination of pharmacodynamics, lac-
rimal fluid was collected 1, 3, 5, 10, and 15 min subsequent
to application of 0.1% NCT +0.1% NH
4
Cl using a strip of
filter paper (Schleicher & Schuell, width of 5 mm) placed
onto the conjunctiva bulbi and palpebrae inferioris for
5 seconds. One drop each of potassium iodide and starch
solution was applied onto the wet strip, which caused the
formation of blue color [21] in the presence of NCT (NCT
+2I
+H
+
taurine +I
2
+Cl
;I
2
+starch blue complex).
Phase 1 study
Subjects
Thirteen volunteers (six female, seven male, ranging in age
from 22 to 71 years, mean 33.3 years, standard deviation
15.1) participated. Medical status was determined by
evaluation of the medical history and medication and by
detailed ophthalmologic examination by usage of a slit
lamp and an indirect ophthalmoscope. Included were
healthy volunteers who particularly did not suffer from a
disease of the eye. Exclusion criteria were nonage (<19a),
eye disease, any acute disease, chronic disease with
involvement of the eye, medication with side effects on
the eye, other treatment of the eye at the same time, contact
lenses during the study, participation in another clinical
study at the same time, and pregnancy. Subjects were
numbered consecutively, and test and control eyes were
assigned in accordance with the randomization code. No
subjects had to be withdrawn after randomisation.
Treatment
The test eye of each subject was treated with 0.1% NCT
plus 0.1% NH
4
Cl, the concentration which was best
tolerated in the rabbit study and human pilot study and
which had shown efficacy in the EKC rabbit model. The
control eye was dripped with 0.9% saline in a randomized
and double-blind manner. The dosing schedule was planned
to simulate treatment for viral conjunctivitis, to warrant a
high frequency and to be practicable for the volunteers.
Therefore, the period of dosing was 5 days. On each day,
treatment was started with an intensive phase of five single
doses at an interval of 15 minutes to warrant oxidative
action for 1 hour, followed by four single doses at an
interval of 2 hours starting 1 hour later.
Clinical evaluation
All subjects were examined daily subsequent to the last
single dose during the treatment cycle, as well as 14 days
later. The baseline investigation was performed on day 1
before the beginning of treatment. The following parts of
the eye were evaluated: conjunctiva, caruncula, cornea,
sclera, glandula lacrimalis, iris, lens, choroidea, and retina.
Any changes were rated as objective signs of adverse
effects (palpebral edema, conjunctival hyperemia, petechial
hemorrhages, exudation, chemosis, pseudomembranes, cor-
neal stippling, iritis, cataract, choroiditis, and changes of the
ocular fundus). Subjective symptoms evaluated were pain
(eye burning, headache, pain of the eyelid), tearing, itching,
foreign-body sensation, andimpairedvision(unsharp
vision, photophobia, colored vision, impaired visual field)
and other impairments, as well as their intensity, duration
and time. Objective signs and subjective symptoms were
scaled absent, mild, moderate, and severeand rated 0, 1,
2, and 3 points respectively, similar to what has been
reported previously [4,20]. Mild were hardly noticeable
subjective effects like short-time minimal eye-burning, and
objective ones like discrete corneal stippling or discrete
Graefes Arch Clin Exp Ophthalmol
vascular conjunctival injection. Moderate were subjective
effects with impairment of condition such as more
pronounced eye burning, and more pronounced objective
effects, but self-limited and not threatening health. Severe
were intense subjective effects such as hardly tolerable eye-
burning, and objective effects threatening health or requir-
ing therapeutic intervention. Objective and subjective
scores were calculated by addition of the mentioned single
points of signs and symptoms on all days as the primary
criteria of evaluation.
To exclude long-term adverse effects, an additional
clinical examination was performed 4 months after the
end of the study in 12 of the 13 subjects, since one was no
more available because of emigration. Because the corneal
endotheliuma monolayer of hexagonally shaped cells
without regenerative propertiesis very sensitive to toxic
effects, the corneal endothelium was photographed with an
endothelial specular microscope (ESM 2001, Rhine-Tec, D-
41179 Mönchengladbach, Germany) before starting treat-
ment and 4 months later. Endothelial cell density was
determined with the SeaEagle® software (Rhine-Tec,
Mönchengladbach, Germany) and the values of the test
and control eye as well as the values before and after the
study were compared.
Statistical analyses
Adverse effects were evaluated by descriptive statistical
analysis. Frequency of each side effect was calculated in
relation to the number of subjects as well as to the
number of days of treatment. McNemars test was used
to test for statistical significance. The number of corneal
endothelial cells was calculated as the mean value from
three different photographs from each eye in each
subject. The mean values of all subjects were compared
by Studentspairedt-test. Pvalues of less than 0.05 were
considered significant.
Results
Animal study
During the treatment with five daily doses for 6 days, 0.1%
NCT + 0.1% NH
4
Cl caused no detectable adverse effects
(Table 1). Conjunctival injection, edema, and secretion
occurred in higher concentrations (Table 1). These side
effects were minimal and temporary, and in no case did
discontinuation of the application become a consideration.
Only the highest concentration used, i.e. 1% NCT + 1%
NH
4
Cl, caused moderate effects (Table 1). Therefore,
dosing was stopped after 1 day in these three animals.
After 2 days, effects had decreased to minimal and
disappeared within 7 days without treatment.
The threshold concentration tolerated in the rabbit was
0.3% NCT + 0.3% NH
4
Cl, at which temporary minimal to
moderate injection, particularly of the nictitating mem-
brane, became apparent.
The inner parts of the eye of all animals remained
normal, and Tyndall tests were negative on all days. No
adverse effects occurred in the control eyes treated with
saline or 1% NH
4
Cl. No long-term adverse effects were
found, including in those rabbits challenged with 1% NCT
+1%NH
4
Cl.
Table 1 Tolerability of NCT + NH
4
Cl in the rabbit eyes
NCT NH
4
Cl Animal Side effects observed
1% 1% 1* injection, edema, secretion (moderate each)
2* injection (moderate), edema and secretion (minimal)
3* injection and edema (moderate), secretion (minimal)
0.3% 0.3% 1 edema (minimal)
2 injection (minimal - moderate), edema (minimal)
3 injection (minimal - moderate), edema (minimal)
0.2% 0.2% 1 secretion (minimal)
2 none
3 injection (minimal)
1% 0.1% 1 none
2 injection, edema, secretion (minimal each)
3 injection, edema, secretion (minimal each)
0.1% 1% 1 none
2 none
3 injection (minimal)
0.1% 0.1% 1,2,3 none
0% 1% 1,2,3 none
*application terminated after the first day
Graefes Arch Clin Exp Ophthalmol
In an extra experiment, the three rabbits who had
received 0.1% NCT + 0.1% NH
4
Cl were treated with one
drop of this concentration every 5 min for half an hour, i.e.
seven applications. This intensive treatment was tolerated
virtually without side effects.
Pilot study
In the pilot test with two volunteers, different concen-
trations were administered every 15 min within 1 hour on
1 day each. 0.1% NCT plus 0.1% NH
4
Cl was very well-
tolerated, with mild eye burning for 5 seconds in the first
subject and with mild foreign-body sensation for 5 to
10 seconds in the other one (subjective score 1).
0.3% NCT plus 0.1% NH
4
Cl caused moderate burning
for about 10 seconds. This was followed by mild foreign-
body sensation for 5 min in the second subject (score 2).
No objective changes were observed at the ophthalmologic
examination.
0.5% NCT plus 0.1% NH
4
Cl caused moderate to strong
burning after the second to fifth dose for about 20 seconds
immediately after application. The burning sensation dis-
appeared after 5 min, and was rated a score of 2. A mild
foreign-body sensation was present for 15 min subsequent
to the last dosing. Both subjects had a minimal conjunctival
injection on the nasal side (objective score 1), but in the
first subject this was detected in the control eye treated with
saline. No disorders came up during the following time.
0.1% NCT plus 0.2% NH
4
Cl led to moderate burning for
approximately 10 seconds in the second subject, while this
sensation was only mild in the first subject (score 2 and 1
respectively). Minimal temporary corneal stippling could be
seen in the eye treated with the test substances in the second
subject (score 1).
Corneal endothelial cell counts measured 6 months after
the beginning of the study did not change. Mean values ±
SD of two measurements of the first volunteer were 2788 ±
74 before and 2718±83 after the study in the test eye (P=
0.41), and 2723±16 before and 2739±173 afterwards in the
control eye (P=0.91). Values of the second volunteer were
2449±64 before and 2382±133 after the study in the test
eye (P=0.57), and 2387±42 before and 2266±45 after-
wards in the control eye (P= 0.058).
Summing up, no serious adverse effects occurred. Eye
burning would have become a limiting factor at concen-
trations exceeding 0.5% NCT + 0.1% NH
4
Cl. Since 0.1%
NCT + 0.1% NH
4
Cl was tolerated with no or minimal
burning, and because this dose had shown good antiviral
efficacy in the rabbit model [23], it was chosen for the main
study.
Pharmacodynamics Oxidative activity was detectable for 4
to 5 min after application of one drop of 0.1% NCT + 0.1%
NH
4
Cl in both subjects.
Phase 1 study
All 13 subjects completed the study according to the 5-day
dosing protocol. No severe adverse effects occurred.
Chronological appearance of adverse effects in each subject
is noted in Tables 2and 3. The only subjective side effect
(Table 2) significantly related to 0.1% NCT plus 0.1%
NH
4
Cl was mild to moderate eye-burning for a few seconds
Table 2 Chronological appearance and overlaps of subjective adverse effects during the five-day treatment with NCT + NH
4
Cl
Subject Test-eye Placebo-eye
Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 Day 2 Day 3 Day 4 Day 5
1 bb,fbbb_f ___
2 bbbbb_____
3 bbbb,f____f _
4 bbb_______
5 bbbbb_____
6 bbbbb_____
7 bbb_b_____
8 bbbbb_____
9 bb,fbbb_____
10_b,fbbb,f_f ___
11bbbbb_____
12bbbbb_____
13bbbbbf f f f f
b burning (P<0.01)
f foreign-body sensation (P>0.1)
Graefes Arch Clin Exp Ophthalmol
immediately after dosing (P<0.01 compared to the control
eye). Minimal foreign-body sensation occurred also in the
control group at a similar frequency (P> 0.1). Most of the
time the burning was mild, and it was rated less intensive
than the anesthetic eye drops used before applanation
tonometry by most of the subjects, including those who
rated the burning as moderate. In 12 participants, it
disappeared within 5 to 10 seconds, in one (no. 13), who
was known to be particularly sensitive to all kinds of eye
drops, within 30 seconds. In six persons, burning was not
present after the first daily dose, but appeared during the
intensive phase of applications within the first hour. Besides
that, it did not increase with duration of the study but
sometimes disappeared. Subjective sensations were approx-
imately similar to the entry of sweat to the conjunctiva, and
of such small magnitude and short duration that no subject
asked for premature treatment discontinuation.
Except for sporadic foreign-body sensation, no adverse
effects occurred in the eyes treated with placebo. Impaired
vision and other impairments were not noted.
Objective side effects were mild corneal stippling and
mild conjunctival injection. Stippling was detected on 15 of
65 days (13 subjects x 5 days) of treatment in total (23.1%)
in the test, and on four of 65 days (6.2%) in the control
group (P=0.0026). Conjunctival injection occurred in
15.4% in the test and in 1.5% in the control group (P=
0.0077). All these effects were minimal, self-limited and in
part disappeared again even during the treatment period
(Table 3). When their appearance was calculated in relation
to the number of subjects, there was no statistical difference
to the controls (P=0.125 for stippling and P= 0.5 for
injection). The control examinations 14 days after the end
of dosing revealed no disorders, except for bilateral eye
itching and mild conjunctival injection in one subject which
was obviously related to solar irradiation and dry eyes. No
further adverse effects occurred, and no alterations of the
middle and internal parts of the bulbus oculi were noted.
No long-term adverse effects appeared during follow-up, as
confirmed at the examination 4 months after the end of
treatment.
There was no difference in the ocular pressure between
test and control eyes measured on day 1 (baseline), day 5,
day 19, and 4 months later (P>0.2 in all cases: data not
shown). The baseline values were minimally higher
because they were measured in the morning, the other ones
in the late afternoon. Visual acuity, which was determined
on the same days, did not change in any subject.
The number of endothelial cells per mm
2
did not change
above the natural rate. Mean values calculated from single
mean values derived from three measurements each of 13
test persons decreased from 2558.5±151.5 at the baseline to
2537.2±163.8 after 4 months in the test group (P= 0.188)
and from 2539.6±177.7 to 2498.0±169.1 in the control
group (P=0.078). Values of the test group were not
different from those of the control group at the baseline
(P=0.445) and 4 months later (P=0.169).
Discussion
As an endogenous mild antiseptic, NCT has proved to be
very well-tolerated and effective in clinical studies. Patients
suffering from bacterial conjunctivitis were cured rapidly in
a phase 2a trial [17], and also signs and symptoms of severe
adenoviral conjunctivitis were attenuated by 1% NCT eye
drops in a phase 2b trial [25]. Improved penetration of
Table 3 Chronological appearance and overlaps of objective adverse effects during the five-day treatment with NCT + NH
4
Cl
Subject Test-eye Placebo-eye
Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 Day 2 Day 3 Day 4 Day 5
1 _s__s ____s
2 __________
3 _ss _______
4 __________
5 __________
6 c____c____
7 __________
8 ___ss__sss
9 _ssss_____
10_s s s ______
11_c,sc,scc _____
12__________
13ccccc_____
c conjunctival injection (P<0.01)
s corneal stippling (P<0.01)
Graefes Arch Clin Exp Ophthalmol
activity into the cornea is desirable to avoid or successfully
treat subepithelial viral infiltrates. Penetration can be
improved by addition of ammonium chloride, since the
formed monochloramine is more lipophilic than NCT [2,
3]. Basically it deals with a natural process, since
ammonium chloride is present ubiquitously in the human
body and body fluids. Artificial addition of ammonium
chloride makes it possible to steer the activity of NCT in a
controlled way. This was confirmed in adenoviral conjunc-
tivitis using the Ad5/NZW rabbit ocular model, where NCT
+NH
4
Cl demonstrated superior reduction of viral load
compared to 1% NCT [23].
In our tolerability tests in healthy rabbits, after repeated
application, 0.1% NCT + 0.1% NH
4
Cl was excellently
tolerated, while 0.3% + 0.3% was the threshold concentra-
tion leading to mild conjunctival injection and swelling for
a few hours. Since all signs (even after 1% + 1%)
disappeared without long-term adverse effects, safety
proved to be high enough to test the combination in man.
The results of the pilot study revealed similar tolerability
of NCT + NH
4
Cl in man and in the rabbit. The combination
of 0.1% + 0.1% seems to be optimal because of the
following reasons. First, it is tolerated without considerable
adverse subjective or objective side effects. Concentrations
of about 0.5% NCT + 0.1% NH
4
Cl may lead to marked
burning and irritation, but not to serious events, so that the
safety-distance to toxic effects is high. Second, it has
oxidative activity in the eye for 4 to 5 min, so that long-
term adverse effects are very improbable and have actually
not been observed. Third, it has excellent activity in vitro
against pathogens [1], and in vivo it was as effective as are
higher concentrations in the viral conjunctivitis rabbit
model [23]. Fourth, it is as well-tolerated in the healthy
eye as is 1% NCT alone [14]. Since the latter was tolerated
very well in the inflamed human eye [17,25], this has to be
expected for 0.1% NCT + 0.1% NH
4
Cl as well.
The short period of oxidative activity (a few minutes)
after dosing on the one hand probably makes repeated
dosing necessary to achieve sufficient efficacy; on the other
hand, penetration to the inner parts of the eye and
considerable side effects are avoided. Adaptations of
dosing, for instance frequent application at the beginning
of treatment followed by longer intervals, can be done
easily and without risks of side effects.
A regimen such as this has been simulated in this phase
1 study. The subjective effects (eye burning, foreign-body
sensation) are of such short duration and low extent such
that they absolutely do not counteract application. By
contrast, anesthetic eye drops used in daily practice seem to
cause more intensive burning, as noticed by most of the
subjects. The detected objective effects do not differ from
everyday changes of the eye, and sometimes they even
disappeared during the treatment cycle. No long-term
adverse effects were found even with the highly sensitive
method of in vivo microscopy of corneal endothelial cells.
Therefore, the good tolerability of NCT + NH
4
Cl in this
study clearly confirms the expectations derived from
considerations mentioned above.
Conclusion
The combination of NCT and NH
4
Cl at a respective
concentration of 0.1% + 0.1% proved to be very well-
tolerated and safe during intensive treatment of the healthy
eye for 5 days. Subjective side effects are mild and limited
to seconds; objective ones are within a range of everyday
minimal irritations. Further development of this medication
is justified.
Acknowledgements Patient insurance was provided by the Tiroler
Landeskrankenanstalten GmbH (TILAK, Innsbruck, Austria). We
thank Jeffrey Nau, MMS, Denis OShaughnessy, PhD, and Nathan
R.F. Beeley, PhD (Opko Ophthalmics, Miami, USA) for their support.
Clinical trial registration EudraCT number 2006-002701-32
Animal tests approval no. GZ 66.011/79-BrGT/2003 (Austrian Federal
Government of Science and Research).
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Graefes Arch Clin Exp Ophthalmol
... Since the reaction with thio groups leads to immediate inactivation of NCT, the oxidative activity decreased quickly and could not be measured in situ approximately 30 min after the last dosing. The short decomposition time in vivo is in accordance with previous studies in the eye (5–15 min, [36,43]). Longer times of a few hours are only possible if a fluid level containing NCT is kept in body cavities [37]. ...
... This leads to formation of about 0.025% (4.9 mM) monochloramine (NH 2 Cl) which is only little more reactive but significantly more lipophilic than the hydrophilic NCT [46] . The consequence is increased penetration of pathogens but also body cells and tissue, which may be wanted in some indications , e.g. in the eye for treatment of viral conjunctivitis [11,43]. The concentration of 1% of both constituents is very high and cannot be applied to the eye, but a tenfold dilution is very well tolerated [43]. ...
... The consequence is increased penetration of pathogens but also body cells and tissue, which may be wanted in some indications , e.g. in the eye for treatment of viral conjunctivitis [11,43]. The concentration of 1% of both constituents is very high and cannot be applied to the eye, but a tenfold dilution is very well tolerated [43]. As expected, in the pig model oxygenation and perfusion parameters changed after inhalation with 1% NCT + 1% NH 4 Cl compared to saline and NCT. ...
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N-chlorotaurine, a long-lived oxidant produced by human leukocytes, can be applied in human medicine as an endogenous antiseptic. Its antimicrobial activity can be enhanced by ammonium chloride. This study was designed to evaluate the tolerability of inhaled N-chlorotaurine (NCT) in the pig model. Anesthetized pigs inhaled test solutions of 1% (55 mM) NCT (n = 7), 5% NCT (n = 6), or 1% NCT plus 1% ammonium chloride (NH4Cl) (n = 6), and 0.9% saline solution as a control (n = 7), respectively. Applications with 5 ml each were performed hourly within four hours. Lung function, haemodynamics, and pharmacokinetics were monitored. Bronchial lavage samples for captive bubble surfactometry and lung samples for histology and electron microscopy were removed. Arterial pressure of oxygen (PaO2) decreased significantly over the observation period of 4 hours in all animals. Compared to saline, 1% NCT + 1% NH4Cl led to significantly lower PaO2 values at the endpoint after 4 hours (62 +/- 9.6 mmHg vs. 76 +/- 9.2 mmHg, p = 0.014) with a corresponding increase in alveolo-arterial difference of oxygen partial pressure (AaDO2) (p = 0.004). Interestingly, AaDO2 was lowest with 1% NCT, even lower than with saline (p = 0.016). The increase of pulmonary artery pressure (PAP) over the observation period was smallest with 1% NCT without difference to controls (p = 0.91), and higher with 5% NCT (p = 0.02), and NCT + NH4Cl (p = 0.05).Histological and ultrastructural investigations revealed no differences between the test and control groups. The surfactant function remained intact. There was no systemic resorption of NCT detectable, and its local inactivation took place within 30 min. The concentration of NCT tolerated by A549 lung epithelial cells in vitro was similar to that known from other body cells (0.25-0.5 mM). The endogenous antiseptic NCT was well tolerated at a concentration of 1% upon inhalation in the pig model. Addition of ammonium chloride in high concentration provokes a statistically significant impact on blood oxygenation.
... Tolerability was very good in the eye [50,51], on skin [52], and mucous membranes [53,54]. NCT was effective in external otitis [55], purulent coated crural ulcerations [52], and bacterial and viral conjunctivitis [51,56,57]. Tolerability in the paranasal sinuses was good, too, with some hints for improvement of symptoms in a phase IIa study [58]. ...
... Pickl, M. Nagl). To avoid eye irritation, the concentration of NCT plus ammonium chloride has to be reduced to 0.1% [57], while 1% plain NCT is tolerated in the eye [56]. ...
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N-Chlorotaurine (NCT) is a mild long-lived oxidant that can be applied to sensitive body regions as an endogenous antiseptic. Enhancement of its microbicidal activity in the presence of proteinaceous material because of transchlorination, a postantibiotic/postantifungal effect and antitoxic activity renders it interesting for treatment of fungal infections, too. This is confirmed by first case applications in skin and mucous membranes of different body sites. Recent findings of good tolerability of inhaled NCT suggest further investigations of this substance for treatment of bronchopulmonary diseases, where microorganisms play a role, particularly multi-resistant ones. The availability of a well-tolerated and effective inhaled antiseptic with anti-inflammatory properties could be a significant progress, in particular for chronic pulmonary diseases, such as chronic obstructive pulmonary disease or cystic fibrosis.
... Monochloramine (NH 2 Cl), the smallest N-chloro compound, has been in use for water disinfection since decades (Brodtmann and Russo 1979). Recently, it aroused interest in human medicine as it is formed from a combination of N-chlorotaurine (NCT) and ammonium chloride (eqn 1) (Gottardi et al. 2007), which seems to be useful for topical treatment of infections, for instance of the skin and the eye (Teuchner et al. 2008;Gottardi and Nagl 2010). ...
... On the other hand, usability of NH 2 Cl as antiseptic only became feasible by a very convenient in situ procedure, the reaction of NCT with ammonium chloride (eqn 1; Gottardi et al. 2007). The available sodium salt of NCT (Gottardi and Nagl 2002) combined with NH 4 Cl offers a very effective antiseptic formulation, which excels NCT alone and even CAT, however, without the irritating potential of the latter one (Romanowski et al. 2006;Teuchner et al. 2008). An important feature concerns the innate incidence of both NCT and NH 4 + , which suggests that NH 2 Cl plays also an essential role in the human immune system. ...
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Unlabelled: Recently, we showed that monochloramine (NH2 Cl) has a significantly stronger bactericidal and fungicidal activity than chloramine T despite its lower oxidizing power. This phenomenon was explained by increased penetration because of the higher lipophilicity and smaller bulk of NH2 Cl. As iodine (I2 ) has an even fivefold higher bulk than NH2 Cl, a comparison of both compounds regarding their microbicidal activity became the aim of this study. Aqueous solutions of I2 at a concentration of 10·7 μmol l(-1) killed 10(6) colony forming units per millilitre (CFU ml(-1) ) of Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa to the detection limit of 10(2) CFU ml(-1) within 1 min at 20°C and pH 7·1, while a concentration of 36-355 μmol l(-1) of NH2 Cl was needed to achieve the same effect. Aspergillus fumigatus was inactivated within 5 min by 36 μmol l(-1) I2 and by 355 μmol l(-1) NH2 Cl, Candida albicans within 1 min by 10·7 μmol l(-1) I2 and by 355 μmol l(-1) NH2 Cl. The lipophilicity of I2 , determined with the octanol/water method, was three powers of 10 higher than that of NH2 Cl. The at least 10-fold stronger microbicidal activity of iodine suggests that the hindrance of penetration of the bulky molecule is outweighed by enhanced lipophilicity. Significance and impact of the study: The microbicidal activity of active halogen compounds increases not only with their reactivity, but also with higher lipophilicity and lower bulk, as shown recently. In this study, iodine showed a higher microbicidal activity than monochloramine and a 1000-fold higher lipophilicity. Therefore, the lipophilicity of a disinfectant may be more important than the bulk for bactericidal activity. These facts should be considered upon the design of new antiseptics and their clinical application.
... Despite the great importance of this research stage, since it links preclinical studies and the first human exposure to a certain formulation, the helpful information provided by phase 1 studies is not usually published or made available in any way to both the rest of the scientific community and general public. Even when recently there has been a slight increase in the number of published phase 1 trials, [5][6][7][8] these are mostly cancer trials, leaving a significant gap of available data regarding the development of other specialty's drugs during this essential stage of initial exposure of the target species of such medications. 9 In order to present a wider view of NHV who have undergone ophthalmology trials, our study describes relevant elements of phase 1 clinical trials, including drugs used for common eye disorders: dry eye therapy, glaucoma, and antibiotic agents. ...
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Purpose: The purpose of this study was to evaluate the safety and tolerability profile of drugs used for treating common eye disorders when applied to normal healthy volunteers (NHVs) as explored in phase 1 trials. Subjects and methods: A total of 166 NHVs were identified in six phase 1 trials, examined in a retrospective analysis. The primary endpoints were visual comfort (by ocular comfort index, OCI) and safety (laboratory evaluations, vital signs (VS), visual acuity (VA), intraocular pressure (IOP), lissamine green and fluorescein staining, conjunctival hyperemia, chemosis, and adverse events' incidence (AE)). Results: Compared to baseline, 75.9%, 40.4% and 73.7% of NHV (for lubricant, hypotensive and antibiotic treatments, respectively) improved their OCI score by their final visit. Laboratory evaluations and VS were within normal ranges in 88% of NHV. Similar results were found for VA, corneal and conjunctival staining, and chemosis. IOP decreased significantly in the hypotensive agents' group, trace to mild hyperemia was reported in 32.1%, 27.1%, and 6.8%, respectively. Additionally, lubricant and hypotensive investigational drugs (ID) had a lower risk of incidence of AE than approved drugs (OR 0.856, 95% CI [0.365, 1.999] and 0.636, 95% CI [0.096, 4.197], respectively). Meanwhile, on antibiotic drugs, the risk for ID-related AE was higher (OR 1.313, 95% CI [0.309, 5.583]). Conclusion: Phase 1 trials are important in order to ensure the safety and tolerability of ophthalmic medications. This study demonstrates that NHVs do not face a significant risk of harm in these studies, since 98% of the reported AE were mild, and all AE were resolved by the end of the study in which they appeared. Trial registration: This is a retrospective study of six previously conducted clinical trials, registered on clinicaltrials.gov with the following registration IDs: NCT04081610, NCT03524157, NCT03520348, NCT03966365, NCT03965052 and, NCT03519516.
... 6 This could be enhanced by the addition of ammonium chloride to NCT, 6 whereby the more lipophilic monochloramine (NH 2 Cl) is formed in equilibrium, 20,21 which also inactivates bacteria, fungi, and amoebae more rapidly than NCT. 13,20,22,23 Accordingly, we tested the combination of NCT and ammonium chloride in the corneal model against amoebae at a concentration of 0.1% each, which is well tolerated in vivo in the rabbit and human eye 24 and has an even higher microbicidal activity than 1% NCT against bacteria and fungi in buffer solution. 20,25 Interestingly, in the cornea model with amoebae, a higher volume (4 mL instead of 1 mL) of 0.1% NCT plus 0.1% NH 4 Cl was needed to achieve a similar antimicrobial effect as that of 1 mL of 1% NCT. ...
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Purpose: N-chlorotaurine (NCT) is an anti-infective belonging to the class of chloramines and an investigative drug for the topical treatment of keratoconjunctivitis. The aim of the present study was to demonstrate its efficacy against Acanthamoeba and Candida in corneas infected ex vivo. Methods: Corneal buttons from porcine eyes were contaminated with Acanthamoeba castellanii trophozoites or Candida albicans Centraalbureau voor Schimmelcultures 5982 and incubated for 7 and 3 days, respectively. Subsequently, they were treated with 1% NCT for 5 to 120 minutes. After further incubation for 2 days in the absence of NCT in tests with A. castellanii, the buttons were homogenized, and the amoebae grown for a further 5 days before they were counted in a light microscope. For C. albicans, quantitative cultures were performed from corneal homogenates. Results: Incubation of 120 minutes in NCT completely inhibited the regrowth of A. castellanii and reduced the number of C. albicans colony-forming unit counts by 4 log10. In addition, at 60 minutes, significant reductions of both pathogens could be observed. Histology showed penetration of pathogens into the stroma of the corneal buttons. Conclusions: NCT inactivates A. castellanii and C. albicans in corneal tissue.
... The latter is more lipophilic and has a higher bactericidal and fungicidal activity than NCT because of increased penetration into tissue and bacteria and fungi (Gottardi et al. 2007;Gottardi & Nagl 2010). Recently, a combination of 0.1% NCT and 0.1% NH 4 Cl demonstrated activity superior to that of 1% NCT in the rabbit model of epidemic keratoconjunctivitis (Romanowski et al. 2006), and the tolerability of this combination has been proven in humans (Teuchner et al. 2008). A specific advantage of NCT plus NH 4 Cl is the non-formation of irritating trichlor-amines, which is not the case with other active chlorine compounds in the presence of NH 4 Cl (Gottardi et al. 2007). ...
Article
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... 29 Fewer positive viral cultures and shorter duration of viral shedding were found. Recently, 0.1% NCT plus 0.1% NH 4 Cl was well tolerated in a Phase I study, 83 and presently this formulation is investigated in viral conjunctivitis in humans. ...
Article
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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 anti-inflammatory properties and seems to be involved in the termination of inflammation. The successful synthesis of the crystalline sodium salt (Cl-HN-CH(2)-CH(2)-SO(3)Na, 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.
... One explanation is that those concentrations are substantially reduced within few minutes in vivo by the tear film. Indeed, the ocular tolerability of the topical application of NCT has been demonstrated in clinical trials [23,32] . Our finding that much lower NCT concentrations also significantly reduced spread of the more endurable poxviruses in vitro suggests that weak chlorinating agents might be promising as a possible local treatment option for a broader spectrum of viruses. ...
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Weak oxidants produced by activated human leukocytes are proven antimicrobial substances. We tested whether N-chlorotaurine (NCT, taurine chloramine), the chlorinated metabolite of the amino acid taurine, in addition to direct virucidal effects on viral suspensions, has the capability to prevent cell-to-cell spread of viruses in human corneal epithelium. Human corneal grafts were infected in vitro with poxvirus (vaccinia virus, VV) and herpesvirus. Different NCT dilutions were added to prevent viral spread within the corneal epithelium as detected by immune-staining and microscopy of cytopathic effects. Additionally, virus release was measured by cell culture. Addition of NCT significantly reduced the number of VV-infected epithelial cells at concentrations as low as 0.01% in culture medium, which was far beyond cytotoxic concentrations in long-term cultures. The release of virus by the infected corneal grafts was reduced by 2-3 log(10 )as well. As expected, herpesvirus infection was also positively affected. Smallpox has been known as a major cause of blindness in historical outbreaks. NCT could therefore provide an additional supportive means for treating orthopoxvirus-associated keratitis. Additionally, biocompatible local antiseptics like NCT could also serve as an experimental treatment in other keratitis of suspected viral origin.
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N-chlorotaurine (NCT), a well-tolerated endogenous long-lived oxidant that can be applied topically as antiseptic, was tested on its fungicidal activity against Scedosporium and Lomentospora, opportunistic fungi that cause severe infections with limited treatment options mainly in immunocompromised patients.In quantitative killing assays, both hyphae and conidia of Scedosporium apiospermum, Scedosporium boydii, and Lomentospora prolificans (former Scedosporium prolificans) were killed by 55 mM (1.0%) NCT at pH 7.1 and 37 °C with a log10 reduction in CFU of 1 - 4 after 4 h and of 4 to > 6 after 24 h. Addition of ammonium chloride to NCT markedly increased this activity. LIVE/DEAD staining of conidia treated with 1.0% NCT for 0.5 to 3 h disclosed increased permeability of the cell wall and membrane. Pre-incubation of the test fungi in 1.0% NCT for 10 - 60 min delayed the time to germination of conidia by 2 h - >12 h and reduced their germination rate by 10.0 - 100.0%. Larvae of Galleria mellonella infected with 1.0 × 10E7 conidia of S. apiospermum and S. boydii died at a rate of 90.0 - 100% after 8-12 days. The mortality rate was reduced to 20 - 50.0% if conidia were pre-incubated in 1.0% NCT for 0.5 h or if heat-inactivated conidia were used.Our study demonstrates fungicidal activity of NCT against different Scedosporium and Lomentospora species. A postantifungal effect connected with loss of virulence occurs after sublethal incubation times. The augmenting effect of ammonium chloride can be explained by formation of monochloramine. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Article
Human adenoviral conjunctivitis is a highly contagious eye infection affecting millions of people world-wide. If untreated, it can further develop into keratitis, corneal ulceration, scarring and possible blindness. Despite the significant patient morbidity and socio-economic costs, it is an unmet medical need with no FDA approved treatment. Here, we demonstrate the virucidal activity of NVC-422 (N,N-dichloro-2,2-dimethyltaurine) against adenovirus type 5 (Ad5) and investigated its mechanism of action of Ad5 inactivation. NVC-422 inhibits Ad5-induced loss of cell viability in vitro with 50% inhibitory concentration (IC(50)) ranging from 9 to 23 μM. NVC-422 does not cause any cytotoxicity at concentrations as high as 250 μM. Invitro, NVC-422 inactivates Ad5 but does not interfere with viral replication, indicating that NVC-422 acts on the extracellular adenovirus as a virucidal agent. NVC-422 inactivates Ad5 by oxidative inactivation of key viral proteins such as fiber and hexon as evidenced by SDS-PAGE, Western blotting and reversed-phase HPLC. These data, combined with measurements of the kinetics of the NVC-422 reactivity with selected amino acids, indicate that the changes in the viral proteins are caused by the selective oxidation of sulfur-containing amino acids. The conformational changes of the viral proteins result in the destruction of the viral morphology as shown by transmission electron microscopy. In summary, NVC-422 exhibits virucidal activity against Ad5 by the oxidative inactivation of key viral proteins, leading to the loss of viral integrity and infectivity.
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The influence of organic matter on the antibacterial activity of the endogenous substance N-chlorotaurine was examined. In contrast to other active N-chlorine compounds (e.g. hypochlorite, chloramine T) the efficacy of N-chlorotaurine was enhanced in the presence of the amine compounds α- and β-alanine, glycine and especially ammonium chloride. This remarkable effect was found to result from an equilibration between N-chlorotaurine and the amino acids, resp. ammonium, and formation of the corresponding N-chlorine derivatives by transhalogenation. In human exudates, too, the efficacy of N-chlorotaurine increased, which can be explained by an over-compensation of consumption effects by generation of these highly bactericidal N-chlorine derivatives. Moreover, repeated treatment at sublethal doses did not cause a decrease in efficacy of N-chlorotaurine. This suggests that application of N-chlorotaurine for local treatment of topical infections will be successful.
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