Life Sciences,Vol. 59, No. 14, pp.

1141-1147,1996
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THE ANTIOXIDATIVE ACTIVITY OF THE MUCOREGULATORY

AGENTS: AMBROXOL, BROMHEXINE AND N-ACETYL-L-
CYSTEINE.
A PULSE RADIOLYSIS STUDY

Klaus Felixl*, Michel Pairetz and Rainer Zimmerman+

1 Institut fiir Klinische Molekularbiologie und Tumorgenetik, GSF-Forschungszentrum,

Hgmatologikum, D-81377 Miinchen, Germany,
2 Biologische Forschung, Dr. K. Thomae GmbH, D-88397 Biberach (Riss), Germany.

*Present address: Laboratory of Genetics, National Cancer Institute, NIH, Bethesda, MD 20892

(Received in final form July 29, 1996)

Summarv

Ambroxol and bromhexine are shown to be scavengers of both superoxide and hydroxyl
radicals as determined by pulse radiolysis experiments. The dismutation of superoxide
was accelerated 3-fold by bromhexine and 2.5fold by ambroxol over the rate of
spontaneous dismutation. The reaction constants of hydroxyl radicals with bromhexine
and ambroxol were determined by competition kinetics to be 1.58 * 0.15 x 1010 M-ls-1
and 1.04 + 0.1 x 1010 M-Is-*, respectively. N-acetyl-L-cysteine also reacted with
hydroxyl radicals (1.28 f 0.14 x 1010 M-1.~1) but not with superoxide radical. These
effects may be clinically relevant in the treatment of oxidant-associated lung damage
induced by inflammatory agents and/or environmental pollutants.

Key Words: reactive oxygen species, pulse radiolysis, ambroxol, bromhexine, N-acetyl-L-cysteine

Cells are continually exposed to reactive oxygen intermediates, and a specific system that maintains
the balance between the generation of and the detoxification of oxygen radicals is required. An
imbalance that favors the accumulation of reactive oxygen intermediates leads to a state known as
oxidative stress (1). Presumably this is the result of an overproduction of reactive substances or a
deficiency in anti-oxidative defenses. Oxidative stress is detrimental to the cell because it can result
in mutations, chromosomal alterations, cytotoxicity, carcinogenesis and/or cellular degeneration.
Knowledge of the source(s) of the damaging species, as well as a thorough understanding of the
processes that lead to radical-induced pathology, is crucial in designing effective therapies. Some
strategies that have been developed to enhance anti-oxidant capacity include superoxide
scavenging, increase of plasma and intracellular antioxidative defense and incorporation of
lipophilic antioxidants into membranes and lipids [for review see (2)].

Oxidant-associated damage plays a critical role in the pathogenesis of respiratory diseases, since
the lungs are continually exposed to environmental pollutants (e.g., tobacco smoke, diesel exhaust
gases, ozone and nitrous oxides).
1142 Antioxidative Activity of Ambroxol & Bromhexine Vol. 59, No. 14, 1996

Mucoregulators such as ambroxol (N-A 872). bromhexine (N-A 274) and N-acetyl-L-cysteine
(NAC), are used in the treatment of respiratory diseases characterized by impaired mucus
clearance. In addition to their participation in mucus production and clearance (3-5). these
compounds have been shown to possess antioxidative activity in vitro (5,6) as well as in vi~to (7).
However, only NAC has been shown to have a direct scavenging effect by pulse radiolysis
studies (5). The pulse radiolysis technique (8) satisfies the basic requiremenLs for demonstrating a
direct action on radicals: (a) the generation of radicals in a short period of time; (h) efficient radical
production to allow accurate monitoring; and (c) the specific production of individual radicals,
which is necessary to avoid interference between multple reactive species.

In this study, we examined the interactions of the mucoregulatory agents ambroxol and
bromhexine with pulse radiolytically generated superoxide (Oz.-) and hydroxyl (.OH) radicals and
compared these results with those of the mucoactive NAC (9). We present evidence that ambroxol
and bromhexine can improve antioxidative defenses, especially in pulmonary tissues exposed to
elevated reactive oxygen intermediates, such as those generated during ischemia. reperfusion or
inflammation.

Methods

Drum: Bromhexine (N-A 274 = 2-Amino-3,5-dibromo-N-cyclohexyl-N-methylben~cnc-

methanamine, BisolvonR), ambroxol (N-A 872 = (4-[[(2-Amino-3,5-dibromo-phenyl)-methyl]
amino] cyclohexanol , MucosolvanR) were from Dr. Karl Thomae GmbH, Biberach/Riss,
Germany. N-acetyl-L-cysteine (NAC) and other chemicals were purchased in analytical grade from
Merck (Darmstadt, Germany) and Sigma (Deisenhofen, Germany). All experiments were carried
out in aqueous solutions, which were prepared with quartz-distilled and pyrolysed water.

Pulse adiolvsis: Pulse radiolysis experiments were performed on a Fehetron 705 electron
accelerat&. The optical detection system was composed of an Osram xenon lamp XB0450 W4, a
Schoeffel monochromator and an EM1 9659 photomultiplier unit. The signals were recorded on a
Hewlett-Packard HP 5 182 Waveform Recorder/Generator and subsequently stored and analysed
on a computer. Air saturated aqueous solution of KSCN (10 mM) was used for monitoring the
dose delivered per pulse, assuming G~=21522 dm-jmol-lcm-1 per 100 eV at 500 nm for the
transient (SCN)2.- species (8).

02.- radicals were produced by a 100 ns radiation pulse of 1.8 MeV electrons in oxygen-saturated
solutions containing 0.1 M formate. Yield of 02.. molecules per 100 eV of absorbed energy under
these conditions rose to G = 6.1. Oz.- radical concentrations were determined from the dose of
radiation pulse and the UV absorption of the radical at h = 250 nm (E~~ = 2250 M-‘cm-l) (12).

The .OH radical reactions were studied in nitrous oxide-saturated aqueous solutions which
converts e-aq into .OH [N20 + e-aq ---> N2 + .OH + OH-] with G = 5.6. The rates of reaction
of the test compounds with .OH were determined by competition kinetic studies using KSCN as
the standard solute and by monitoring the absorbance of (SCN)z.- species at 500 nm for various
concentrations of test-compound/SCN- (10,ll). The rate constants were calculated using the
equation:

A0 ktest-comp./.OH x [test-comp.1
Eqn. 1 ---- _ l= _____________________-_______----------
A kscN/.oH X [SCN-1

Values for ktest_comp,.O~~were obtained from linear regression analysis of (&/A) - 1 vs. [test-
comp.]/[SCN-] where the slope is the ktest_comp,.OH/ksCN_,.~~~. A,, and A represent the initial
absorbances of (SCN)2.-, generated by an electron pulse of 40 Gy absorbed energy in an aqueous
KSCN and N20-saturated solution in the absence and presence of test compound.
Vol. 59, No. 14, 1996 Antioxidative Activity of Ambroxol & Bromhexine 1143

Results
Interaction with superoxide radicals The analysis of the decay kinetics revealed second
order reactions for the compounds tested. There was no measurable reaction between NAC and
Oz.-. The electronic absorption-time curves at 250 nm of Oz.- in the presence of NAC were
congruent with those of spontaneous Oz.- dismutation.

The rate constants for the reaction of 02. with bromhexine and for ambroxol were 3-fold and 2.5
higher, respectively, than that of the uncatalysed reaction (table I).

Table I: Calculated second order rate constants for the reaction of Oz.- with N-acetylcysteine,
bromhexine and ambroxol.
___________________________________________________________________________________________________
Compound Averaged Rate Constant
k2 [M-ts-t]

Oz.- + 02: (spontaneous) (8.10 z!z0.21) x 105

NAC (N-acetyl-L-cysteine) (8.10 zb 0.21) x 105
N-A 274 (bromhexine) (2.41 f 0.34) x 106
N-A 872 (ambroxol) (1.97 f 0.23) x 106

The measurements were carried out in oxygen-saturated solutions containing 0.1 M sodium
formate, pH 7.15, T = 22 oC, pulse length: 100 ns, dose 40 Gy (25 PM Oz.=). Each compound
was tested at 6 different concentrations over the range 5-300 PM. Data represent mean f SD
values of four independent measurements.

To distinguish between catalytic Oz.- turnover and stoichiometric Oz.- scavenging, both
compounds were pulsed repetitively. No change in the decay kinetics was seen after multiple
pulses that resulted in a high excess of Oz.-. indicating that they are genuine catalysts. Transient
electronic absorption spectra obtained on pulse radiolysis of aqueous solutions of bromhexine and
ambroxol showed that with the decrease of absorption at 250 nm, a time shifted slight transient
decrease with a minimum at 305-310 nm was measurable when the concentration of 02.. was 3 or
more times higher than that of the compound tested (e.g., 20 PM 02= and 5 pM ambroxol). These
transient electronic absorption-time curves at 305-310 nm probably result from the first step in the
dismutation reaction during which electrons are transferred from 02.. to the aromatic moiety of the
molecules. To test this hypothesis, the compounds were pulsed in nitrogen-saturated formatc
solutions, a converting system for .OH elimination in which electrons are the main product of the
water radiolysis. Again the same species at 305-310 nm were observed, but in contrast to the
results in oxygen-saturated formate solutions, these species were stable during the maximum
recording time of two minutes. A reoxygenation showed a decline with time in the amount of
transient species until it reached the level corresponding to the initial absorption (before pulse).

Table II: Rate constants for the reaction of .OH radicals with N-acetylcystcinc, hromhexine and
ambroxol.

Compound k2 compound/.OH
(M-Is-‘)

NAC (N-acetyl-L-cysteine) (1.28 f 0.14) x 1010

N-A 274 (bromhexine) (1.58 f 0.15) x 1010
N-A 872 (ambroxol) (1.04 f 0.09) x 1010

Data represent mean f SD values of three measurements for seven different concentrations of
each compound between 10 and 100 l.tM. Measurements were performed in a N20-saturated 100
l.tM KSCN aqueous solution at pH 7.0 and 20 Oc.
1144 Antioxidative Activity of Ambroxol & Bromhexine Vol. 59, No. 14, 1996

0.14
1.4-
O.lZ’! . a d
1.2-
a

I
0.8-

4 0.08 - a
a
E 0 0
5 0.06 -
l

0.04 ., ., , ., 1
0 20 40 60 80 100 120 0.0 0.2 0.4 0.6 0.8 1.0 1.2
N-acetyl-L-cysteine [ @U ] [N-ace@+L-cysteine]/[ SCN-]

l b
0.12
I I

.
0.06 I. , , , r

0 20 40 60 80 100 120 0.0 0.2 0.4 0.6 0.8 1.0 1.2

ambroxol [ @I] [ambroxol]/[SCN-]

0 20 40 60 80 100 120 0.0 0.2 0.4 0.6 0.8 1.0 1.2

bromhexine [ t&I ] [ bromhexine]/[SCN-]

Fig. 1
Competition kinetics of N-acetyl-L-cysteine/.OH - SCN-/.OH; ambroxol/.OH - SCN-
/.OH; bromhexine/.OH - SCN-/.OH. Fig.1 a-c: initial absorbance of (SCN)z.- measured
at 500 nm in the absence (Aa) and presence (A) of different concentrations of compounds
tested. The measurements were performed in 0.1 mM KSCN, pH 7.0 and NzO-
saturated solutions exposed to 40 Gy/pulse. Each point is an average of four
measurements. F&l: d-f linear regression plots. Competition kinetics values for the
reaction of the three compounds and thiocyanate with .OH radicals were calculated
according to Eqn. 1 . The slope represents k testcornp/o~kc~/~~. The rate constant
kzsC~-/.on is 1.1 x 10tuM-t~~l.
Vol. 59, No. 14, 1996 AntioxidativeActivityof Ambroxol & Bromhexine 1145

Interaction with hvd oxvl a&c& The rates of the reactions of .OH radicals with NAC,
bromhexine and ambrox; were Established by competition studies using 100 FM KSCN as the
standard solute. Assuming the value k.OH/sCN- = 1.1 x 1010 M-Is-1 (13), apparent bimolecular rate
constants (k.OH/t&cOrr,Pund) of the order of lOteM-Is-1 at 2OoC and pH 7.0 were obtained (Fig. 1
and Table II). The initial absorbance of (SCN)z- radical, measured at 500 nm in the absence and
presence of different concentrations of each of the three compounds tested, is given in Fig. la-c.

in
vivo (7).

Confirmation that a direct scavenger effect is involved in the therapeutic activities of ambroxol,
bromhexine and NAC requires further investigations. In the case of NAC, a direct effect is unlikely
after oral administration, since the compound has a bioavailability of only 9% (23) and does not
penetrate unchanged into the bronchoalveolar fluid (24). A direct effect, however, is possible after
topical (inhalative) administration, since the compound is delivered directly to the endangered
tissue.

Another possible explanation for the efficient antioxidant protection afforded by ambroxol and
bromhexine is that even when parenteralIy administered these drugs accumulate in the lungs,
leading to a high bioavailability in lung tissue. Additional protective effects can he expected from
the fact that both compounds react with 02.. in the extracellular compartment where there is
normally a low amount of SOD-activity. Ambroxol and bromhexine distribute selectively to the
lung in animals and humans (25, 26). Although the mechanism of this pulmonary tropism is
unknown, the drugs may preferentially distribute to lung tissue because of their chemical
characteristics (amphiphilic structure) or because they specifically bind to a pulmonary constituent.
The antioxidative properties of ambroxol and bromhexine in vitro, associated with the pulmonary
tropism, may explain their protective effects in vivo in various animal models of free-radical-
induced lung damage. e.g. bleomycin-induced pulmonary fibrosis in the rat (27), ozone-induced
airway hyperresponsiveness in the dog (28), paraquat toxicity in the rat (29) and endotoxin-
induced lipid peroxidation in mice (7). In humans, ambroxol has been shown to concentrate in
pulmonary tissue at levels up to 20 times that in blood (26). After the administration of 1 g of
ambroxol as a slow intravenous infusion over 3 hours, the area under the concentration curve was
374.3 l.tg g-*h-t for lung tissue and 18.5 l.tg ml-lh-1 for blood. Mean concentrations of amhroxol
1146 Antioxidative Activity of Ambroxol & Bromhexine Vol. 59, No. 14, 1996

in lung tissue were 36.6, 36.3 and 34.1 pglg one, two and four hours post-administration,
respectively. It is difficult to compare such tissue concentrations with the concentrations used in
vitro, since the impact of extraction procedures and analytical methods on the results cannot be
evaluated with precision. Furthermore, potentially important differences in the intracellular
distribution of the compound cannot be determined. Nevertheless, ambroxol concentrations in the
lungs of approximately 30 l.tg/g are in the range of the concentrations tested in rjitro in the present
study. Lower concentrations of ambroxol in bronchial secretion (368 rig/g)) and bronchoalveolar
lavage fluid (135 t&ml) were obtained when ambroxol was administered orally at a dose of 60 mg
twice daily for 2 weeks (30). However, a dilution factor of approximately 50 in bronchoalvcolar
lavage accounting for the epithelial lining fluid should be taken into account to accurately evaluate
the tissue distribution of the compound.

In conclusion, in addition to their effects on mucus production and clearance, the mucoregulators
ambroxol and bromhexine also exhibit anti-oxidative activities in vitro which may be clinically
relevant in the treatment of oxidant-associated lung damage induced by inflammatory states and/or
environmental pollutants.

Acknowledements

We greatfully acknowledge Prof. E. Lengfelder of the Radiation Biology Institute, University of

Munich for permitting the pulse radiolysis measurements to be completed in his laboratory.

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