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Physico-chemical and biological properties of ambroxol under irradiation
Radiation Physics and Chemistry 60 (2001) 43–52
Physico-chemical and biological properties of ambroxol under irradiation Maurizio Tambaa,*, Armida Torreggianib b
a Istituto F.R.A.E. (C.N.R.), via P. Gobetti, 101, 40129-Bologna, Italy Centro di Spettroscopia Raman, Dipartimento di Biochimica, Universita` di Bologna, via Belmeloro 8/2, 40126 Bologna, Italy
Received 3 May 2000; accepted 29 July 2000
Abstract Physico-chemical properties of Ambroxol (AM), a potential antioxidant drug from the expectorant class, were investigated by radiation chemical and spectroscopic studies. The pulse radiolysis experiments showed that AM is a good scavenger of the primary water radical species, particularly eÿ aq and OH radicals. The OH attack, preferentially addressed to the ring positions activated by the –NH2 group and occupied by bromine atoms, leads to hydroxycyclohexadienyl radicals. The molecule stabilisation may be achieved by a dehalogenation reaction to give phenoxyl radicals. The OH attack to AM is not affected by Cu(II) ions, which interact only weakly with the drug as evidenced by IR and Raman spectroscopy. Survival experiments on E. coli B/r cells irradiated in the presence of AM showed a radiosensitising effect of AM in anoxia. Some possible mechanisms of radiosensitisation are outlined. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Pulse radiolysis; Ambroxol; Antioxidant
1. Introduction Cells are continually exposed to reactive oxygen intermediates which, if accumulated, lead to a state known as oxidative stress. Presumably, this is the result of an overproduction of reactive substances or a deficiency in antioxidative defences (Sies, 1985). Oxidant-associated damage plays a critical role in the pathogenesis of respiratory diseases. In fact, pulmonary emphysema, adult respiratory distress syndrome, and lung cancer moistly involve free-radicalmediated reactions (Pryor, 1986). Thus, it seems that pharmacological enhancement of pulmonary antioxidative defence may be one way of preventing these diseases. Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxy-cyclohexyl]benzylamine) (AM) (Fig. 1) is a drug from the expectorant class widely used to increase surfactant secretion in lung and to decrease mucus *Corresponding author. Tel.: +39-051-6399787; fax: +39051-6399844. E-mail address: [email protected] (M. Tamba).
viscosity (Disse, 1987). AM has been reported to protect a-l-proteinase inhibitor from oxidative inactivation and to inhibit the generation of reactive oxygen species from activates phagocytes in vitro (Rozniecki and Nowak, 1987; Winsel and Becher, 1992). Recently, it has been suggested that AM may scavenge some reactive oxygen species (ROS) and protect lipids from peroxidative attack. In fact, intraperitoneal administration of AM seems to protect heart and lung lipids from oxidative stress provoked by intravenous injection of lipopolysaccharide in mice (Nowak et al., 1994, 1993). In addition, AM seems to exert some anti-inflammatory effects that may be due to a concentration-related antagonism towards prooxidants generated by white blood cells, such as hypochlorous acid (HOCl) and monochloramine (NH2Cl). In this regard, AM has been proved to be effective in the respiratory distress syndrome where neuthrophil-derived oxidants play a crucial pathophysiological role (Lapenna, 1994). Moreover, it is to be considered that transition metals, such as copper and iron, may catalyse ROS formation (i.e.
0969-806X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 6 X ( 0 0 ) 0 0 3 3 0 - 3
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M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
Fig. 1. Chemical structure of Ambroxol (AM).
OH), so contributing to the radical-induced oxidative damage (Halliwell and Gutteridge, 1990). Thus, it may be of interest to investigate the potential ability of AM to bind copper(II) ions so preventing the coppercatalyzed generation of ROS and, of consequence, protecting the biological targets from oxidative stress. The aim of the present work is to obtain a wider insight into the general physico-chemical properties of this drug and its antioxidant role as radical scavenger and metal ion binder, mainly by radiation chemical and spectroscopic studies. It also appeared appropriate to assess, at biological level, the role of the radiationinduced transients of AM on the survival curves of bacterial cells.
the total number of scans for each spectrum was 3000. On the sample, the laser power was 100 mW. All the radiation biological experiments were performed on E. coli B /r cells in stationary phase obtained by overnight growth with aeration in Difco nutrient broth. Cells were then collected on a Millipore filter, washed on the filter with the appropriate M/15 phosphate buffer at pH 7.0 and suspended in the same buffer. The bacterial concentration in samples to be irradiated was adjusted to about 107 cells mlÿ1. Irradiation were performed with a 60Co-Gamma Cell (Nordion Corp., Ltd, Canada) at a dose rate of about 9 Gy minÿ1, as measured by Fricke dosimetry. Bacterial suspensions were saturated with the appropriate gas (N2 or O2) before and during irradiations. AM was added to cell suspensions just before irradiation. At the concentration used (1 10ÿ3 dm3 molÿ1), AM has been tested to be moderately toxic to E. coli B/r up to 3 h contact time. Cell survival was determined by spreading appropriate dilutions of bacterial suspensions on the surface of Difco nutrient agar in plastic dishes and counting the visible colonies after overnight incubation at 378C.
2. Experimental 3. Results and discussion Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxy-cyclohexyl]benzylamine; Sigma) (AM), CuCl22H2O (Fluka) and all other chemicals of Analar grade were used as supplied. All solutions were made up with triple distilled water. The Cu(II)/AM systems were obtained by mixing freshly prepared solutions of CuC12 and AM in different ratios just before experiment. Solutions were saturated, immediately prior to irradiation, with N2O or varying percentages in N2O/O2 by flushing gas mixtures. The pH was adjusted by means of HClO4 or NaOH. Pulse radiolysis experiments were performed using a 12 MeV electron linear accelerator (LINAC). Spectral cells of 0.2 and 0.5 dm optical pathlength were used throughout. Optical filters were employed to minimise photochemical effects. Radiation doses were determined with the use of KCNS dosimetry at 480 nm assuming G=0.3 mmol Jÿ1 and e ¼ 710 m2 molÿ1. The signals from the photomultiplier were digitised by a Tektronix AD7912 and analysed by software running on a PC. The infrared spectra were recorded with a JASCO FT/ IR-300E using the KBr-pellet technique. In order to obtain good quality spectra, 1000 scans for each spectrum were accumulated and computer averaged. The spectral resolution was 4 cmÿ1 and the error in wavenumber was about 0.4 cmÿ1. The Raman spectra were obtained by a Bruker IFS 66 spectrometer equipped with a FRA-106 Raman module and a cooled Ge-diode detector. The excitation source was a Nd3+-YAG laser (1064 nm) in the backscattering (1808) configuration. The spectral resolution 4 cmÿ1 and
3.1. Pulse radiolysis The reaction of AM with eÿ aq was studied in N2saturated aqueous solutions containing a large excess of t-BuOH as a scavenger of .OH radicals which are produced simultaneously, with the hydrated electrons (Jonah, 1995). The small contribution by H atoms can be usually neglected. The rate constant for the reaction of AM with hydrated electrons, measured from the effect of the solute concentration on the lifetime of eÿ aq at 720 nm, resulted to be 2.0 1010 dm3 molÿ1 sÿ1 at pH 6.0 (Fig. 2). On the basis of the behaviour observed for other halogenated aromatic compounds (Koster and Asmus, 1973; Mohan et al., 1992), the reaction of eÿ aq with AM may involve interaction with the p orbitals (1a) of the aromatic ring or with the substituent bromine atoms (lb). When the eÿ aq attack occurs at the level of the aromatic ring, a molecular anion (I) is formed. Conversely, if the sites of the attack are the halogen substituents, one bromide ion can be released via a dissociation electron capture process and an aryl radical (II) is formed. The molecular anion (I) is a bound state and, depending upon the relative magnitude of the bond energy and electron affinity, may cross over to the repulsive state leading to dissociation similar to the process lb. In fact, most of chloro- , bromo- , and iodoaromatic compounds result in the scission of C–X bond, meanwhile fluoro compounds mainly form molecular
M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
45
anions (Lian and Mittal, 1984; Koster and Asmus, 1973).
(1a)
(1b)
where
A dehalogenation reaction has also been found to take place for radical anions derived from nitroaromatic and benzonitrile compounds containing an halogen atom at various positions on the ring (Behar and Neta, 1981; Geppert and Getoff, 1998). The transient formed in the reaction of the eÿ aq with AM shows no characteristic absorption bands in the UV /visible region (Fig. 3). The absence of characteristic absorption band is in agreement with the results of pulse radiolysis studies on bromo- and chloro-aromatic compounds which react quantitatively with eÿ aq, leading
Fig. 3. Absorption spectrum (taken 20 ms after the pulse) of the product of the reaction of AM (1 10ÿ4 mol dmÿ3) with eÿ aq at pH 8.0; N2-saturated, containing t-BuOH (1 10ÿ1 mol dmÿ3). Path length=0.2 dm; dose 34 Gy.
to the splitting of the C–X bond (Lichtsheidl and Getoff, 1976; Mohan et al., 1992). The resulting aryl radical does not probably exhibit any absorption in the above spectral region, since the unpaired electron is orthogonal to p orbital system of the ring. Therefore, the lack of transient absorption bands in our system suggest that the dehalogenation process is probably the predominant pathway for the reaction of eÿ aq with AM. Using COÿ (E8=ÿ1.9 V) (Wardman, 1989), a 2 reducing agent weaker than eÿ aq, no electron transfer reaction (2) takes place; in fact, the decay of COÿ 2, monitored at 250 nm, remained unaffected in the presence of AM: COÿ 2 þAM
CO2 þ AM ÿ
ð2Þ
Fig. 4A shows the time-resolved optical absorption spectrum obtained by pulse irradiation of aqueous N2Osaturated solutions containing 1 10ÿ4 mol dmÿ3 AM at pH 7.5. Under these experimental conditions, essentially all the eÿ aq (598%) are converted to OH radicals according to the reaction: H2 O
ÿ eÿ aq þ N2 O ÿÿÿ! N2 þ OH þ OH:
ð3Þ
The transient spectrum exhibits a maximum at about 330 nm and a less intensive absorption in the 390– 450 nm region. On the basis of the literature, the spectrum can be attributed to the OH-adducts (III) which are formed by addition to the aromatic ring (taking the example of the 3-hydroxyl adduct):
ð4Þ
Fig. 2. The plot of the observed rate constants for the decay of eÿ aq with AM at pH 6.0.
In fact, the spectrum is quite similar to that of the hydroxylcyclohexadienyl radical derived from the
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M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
reaction of OH with benzene and its derivatives (Mohan and Mittal, 1995; Ko¨ster and Asmus, 1973). It is most likely a composite spectrum of several isomeric hydroxylcyclohexadienyl species corresponding to the addition of OH to different ring positions. The OH attack is preferentially addressed to those positions (ortho and para) which are activated by the electrodonating –NH2, group. The presence of the two bromine atoms in the above positions makes the corresponding bromohydroxylcyclohexadienyl radicals (i.e. III) quite unstable, as observed for other halogen substituted OHadducts (Mohan and Mittal, 1995). A molecule stabilisation may be achieved by a hydrogen bromide elimination which leads to a corresponding resonance stabilised phenoxyl radical (IV) (Neta and Fessenden, 1974).
ð5Þ
With respect to the addition at the NH2 position, it can be followed by loss of NH3 leading to a phenoxyl radical (reaction (6)) or by water elimination to produce an anilino radicals (reaction (7)).
(6) Fig. 4. (A) Time-resolved absorption spectrum in the pulse radiolysis of aqueous N2O-saturated solution of AM (1 10ÿ4 mol dmÿ3) at pH 7.5 (B) Time-resolved absorption spectrum of N2O/air (4 : 1) saturated AM solution (1 10ÿ4 mol dmÿ3) at pH 7.5 Path length=0.5 dm; dose 15 Gy.
(7) Unfortunately, both the phenoxyl- and anilinoradicals are difficult to identify directly by pulse radiolysis since a lot of them absorb in a region ( 330 and 400 nm) where there is a considerable contribution from OH-adduct (O’Neill et al., 1978; Christensen, 1972; Land and Ebert, 1967). The absorption bands at 330 and 400 nm decayed at the same rates, indicating that both absorptions probably belong to the same species. Two different kinetic components are however present: one faster and mainly of first-order, followed by a slower bimolecular process. The observed first-order component (k 1.3 103 sÿ1 at pH 7.5), probably due to the unimolecular bromide elimination process to form the phenoxyl radical (reaction (5)), becomes faster and mixed-order increasing dose/pulse. This dose-rate effect may be explained by the occurrence of competitive
bimolecular radical–radical reaction involving the primarily formed OH-adducts. 2 AM ÿ OH ! P:
ð8Þ
A further increase in rate is observed at basic pH (k 2.3 103 sÿ1 at pH 9.5) and is probably related to an OHÿ catalysis of both reactions (5) and (7). In fact, an OHÿ catalysed water elimination, leading to an anilino radical (reaction (7)) has also been reported in the literature (Neta and Fessenden, 1974; Steenken, 1987). With respect to the bimolecular decay component which follows the first-order radical disappearance, it is ascribed to disproportionation and dimerization reactions of the phenoxyl and anilino type radicals. Similar reactions have been suggested for aniline derivatives and fluorinated benzenes (Ko¨ster and Asmus, 1973; Neta and Fessenden, 1974). The rate constants of the transient absorption formation at pH 7.5 are similar both at 330 and
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400 nm (8.5 109 and 7.5 109 dm3 molÿ1 sÿ1, respectively). These data, as well as those obtained from the decay kinetics at the above wavelengths, further support the formation of only one type of radical (i.e. III) although the OH attack can occur at different positions on the aromatic ring. The OH reactivity was evaluated also by a competition method using KCNS as a standard. N2O-saturated solutions of KCNS (1 10ÿ3 mol dmÿ3) were pulse irradiated in the presence of different concentrations of AM (from 2.5 10ÿ4 to 1 10ÿ3 molÿ1 dmÿ3) at pH 7.0. In the presence of CNSÿ ions, the following reactions take place: OH þ CNSÿ ! CNS þOHÿ ; ÿ
ð9Þ
ÿ
CNS þCNS Ð ðCNSÞ2 ðl ¼ 500 nmÞ;
ð10Þ ÿ
where the rate constant of the reaction of CNS with OH (k9 ) has been reported to be 1.1 1010 dm3 molÿ1 sÿ1 (Willson et al., 1971). The rate constants of the reaction between AM and OH radicals (k11 ) were determined following the decrease of the absorption at 500 nm due to (CNS)2ÿ at different [AM]/[CNSÿ] ratios: OH þ AM ! AM ÿ OH:
ð11Þ
Under these conditions, the following competition equation was applied: OD0 =OD ¼ 1 þ
k11 ½AM : k9 ½CNSÿ
ðIÞ
From the slope of the linear regression plot (Fig. 5) a rate constant of 1.8 1010 dm3 molÿ1 sÿ1 (k11 ) was
Fig. 5. Indirect method for the determination of k(AM+OH). Plot pf OD0/OD (from (CNS)ÿ 2 measured at 500 nm) against [AM] at pH 7.0. Dose 9 Gy.
47
calculated, a value similar to that reported in the literature (Felix et al., 1996). The value obtained by the competition method is higher than that obtained by the direct one. Besides to a possible experimental error, the discrepancy may be explained considering the possibility that uncomplexed thiocyanate radical (CNS.) could react with AM in competition with reaction (10) causing an overtime of the OD0/OD ratio with a consequent increase of the k11 value. To test the possible interaction of the primary formed OH-radical adducts with naturally occurring antioxidants, N2O-saturated solutions of AM (5 10ÿ4 mol dmÿ3) were irradiated in the presence of ascorbate (1 10ÿ4 mol dmÿ3) at pH 7.0. Ascorbic acid is known to have reducing properties (E8=+0.30 V) (Wardman, 1989) and it has been chosen for its important antioxidant role played in biological systems (Halliwell, 1996). The occurrence of an interaction between the OH-AM adducts and ascorbate was evidenced by the faster absorption decay of the above adducts at 400 nm, as well as by the slow formation of an additional absorption at 360 nm due to the ascorbyl radical Aÿ. Unfortunately, the overlap of the absorption of both radicals in the 360–400 nm region allows only an estimate of the magnitude order for such interaction (107 dm3 molÿ1 sÿ1). This result suggests a relatively low oxidising capability of the OH-induced AM radicals, a property which may be useful in supporting the potential antioxidant role of the drug itself. In accordance with the aim of this study to explore the scavenging ability of AM towards ROS, the possible reaction with superoxide anions, O2ÿ, was investigated. The measurements were carried out in oxygen-saturated solutions containing 0.1 mol dmÿ3 sodium formate and AM at different concentrations (10ÿ4–10ÿ5 mol dmÿ3). Under these experimental conditions, all the primary water radicals are finally converted into superoxide. The kinetic analysis was performed at 250 nm where the O2ÿ radicals are reported to absorb (Bielski et al., 1985). No significant changes in the decay kinetics of superoxide were evidenced in the presence of variable AM concentrations, suggesting that, if a reaction occurs, the relative rate constant will be 4106 dm3 molÿ1 sÿ1. This is in agreement with the low reactivity of AM toward O2ÿ reported in the literature (Felix et al., 1996; Nowak et al., 1994). The reaction of hydroxyl radicals with AM in the presence of oxygen was investigated in solutions containing N2O/air gas mixtures. Under these experimental conditions, essentially all eÿ aq are converted to OH (reaction (3)). By a comparison of the spectra in the presence and the absence of oxygen (Fig. 4A and B), it is evident that the transient spectrum due to the initially formed OH-adducts (i.e. III) is scarcely affected by O2,
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M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
although the formation of the corresponding peroxyl radicals (V) is expected (Bekbo¨let and Getoff, 1999).
ð12Þ It is worth considering that often the parent radical (III) has a well characterised absorption in the UV/VIS region while the corresponding peroxyl radical (V) absorbs more weakly in the UV and has usually very low absorption also in the visible part of the spectrum (Neta et al., 1990). In fact, hydrocyclohexadienyl radicals absorb strongly near 310 nm, a region where the corresponding peroxyl radicals are reported to absorb only weakly (von Sonntag and Schuchmann, 1997). In agreement, the reaction of OH-adducts of AM with O2 results in a transient with an absorption spectrum almost coincident with that of the parent hydrocyclohexadienyl radical. The rate constant for the oxygen addition to substituted hydrocyclohexadienyl radicals is reported to be significantly below the typical value of the other carbon-centred radicals (k 2 109 dm3 molÿ1 sÿ1) and furthermore, it is decreased by the presence of halogen substituents on the ring (von Sonntag and Schuchmann, 1997). A low reactivity towards O2 has also been observed for phenoxyl radicals which, in our system, can be formed by reaction (5) (von Sonntag and Schuchmann, 1997). After 1.6 ms (Fig. 4B) a small increase in residual absorption can be observed in the UV region. Since this increase does not occur in the absence of oxygen (Fig. 4A), it is reasonable to ascribe it to transient products (P) derived from the corresponding peroxyl radicals (V). In order to investigate how the interactions of AM with transition metal ions may affect the behaviour of the molecule in the scavenging of OH radicals, the Cu(II)/AM systems in N2O saturated aqueous solutions were pulsed irradiated. Transient spectra were obtained at different metal/ligand ratios (from 1 : 1 to 1 : 4) and pHs (5.0 and 7.5). Fig. 6 shows, as an example, the transient spectrum at pH 7.5 and the Cu(II)/AM ratio of 1 : 4. The profile of the spectra resulted to be similar to that observed in the absence of Cu(II) in all cases, thus suggesting that, even if an interaction takes place, this is not able to modify the main sites of the OH attack to the molecule. Therefore, from the above radiation chemical data, the biological activity of AM does not appear to involve copper chelation mechanism.
Fig. 6. Time-resolved absorption spectrum in the pulse radiolysis of N2O-saturated solutions of Cu(II)/AM (1 : 4) at pH 7.5 [Cu(II)]=0.25 10ÿ4 mol dm3, [CpSH]=1 10ÿ4 mol dm3. Path length=0.5 dm; dose 15 Gy.
3.2. Vibrational spectroscopy To verify the eventual coordination of Cu(II) ions, both AM and the Cu(II)-AM system were investigated at different metal/ligand ratios (from 1 : 1 to 1 : 4) by IR and Raman spectroscopy. The IR spectra of AM at pH 5.0 and 7.5 are very similar and only slight differences can be evidenced (Fig. 7). In particular, at neutral pH the secondary aliphatic amine group is present both in the protonated form (i.e. 2789, 1584 and 873 cmÿ1) and in the neutral one (i.e. 3284, 3197 and 1547 cmÿ1) (Colthup et al., 1975). The presence of the >NH group can be evidenced also in the Raman spectrum where the NH vibrations are responsible of the shoulders at 1608 and 3300 cmÿ1 (Fig. 8 and Table 1). In the 3500–2500 cmÿ1 IR spectral region there is a good deal of overlapping between the OH, –NH2 and >NH vibrations making difficult the assignments of the bands. However, it is possible to ascribe the bands at 3406, 3330 and 3243 cmÿ1 in the spectrum at pH 5.0 to the stretching modes of the OH and –NH2 groups and the weaker submaxima, which appear by increasing pH, to the >NH group. In the presence of Cu(II) ions (metal/ligand ratios 1 : 1–1 : 2), the IR and Raman spectra at both pH values are very similar to those of AM alone, suggesting that, if an interaction between AM and Cu(II) occurs, it is very weak.
M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
49
Fig. 7. IR spectra of AM at pH 7.5 (a) and pH 5.0 (b), and of the Cu(II)/AM (1 : 4) system at pH 5.0 (c).
Fig. 8. Raman spectra of AM pH 7.5 (a) and pH 5.0 (b), and of the Cu(II)/AM (1 : 4) system at pH 5.0 (c).
By adding an excess of AM (metal/ligand 1 : 4), some sensitive changes take place, particularly in the high wavenumber IR region (Fig. 7c) whose overall appearance suggests that the coexistence of a variety of hydrogen bonds, with different strength, causes the complex broadening. As a consequence of the interaction with the Cu(II) ions, the bands due to the OH vibrations change in wavenumbers (i.e. from 3406 to 3396 cmÿ1), indicating the involvement of this functional group in metal chelation at pH 5.0 (Table 1). Also the amine groups take part to the copper-drug interaction, as shown both by the perturbation of the NH2 and >NH stretching region ( 3300 cmÿ1), as well as by the modifications of the bands due to dNH2 (i.e. 1631, 982 cmÿ1, etc.) and to na C–N (i.e. 1379 and 1284 cmÿ1) (Fig. 7c and Table 1). Analogously to the IR spectrum, the presence of the metal slightly alters the Raman spectral features (intensity, shape and wavenumber) of the bands due to the N–H and C–N stretching, and the NH deformation modes (Fig. 7 and Table 1).
Under neutral experimental conditions (pH 7.5), both the IR and Raman spectra show modifications similar to those observed at pH 5.0, supporting the involvement of the same functional groups in metal chelation. From the spectroscopic data, it can be concluded that a complex between AM and Cu(II) ions is formed in the presence of an AM excess, and the hydroxyl and amino groups act as coordination sites. However, the slight spectral changes indicate that the interaction between AM and Cu(II) is quite weak as reported for amine complexes which are, generally, stable in air but can be easily hydrolysed by water (Biswas et al., 1983). 3.3. Radiation biological studies The pulse radiolysis experiments have shown as AM is very active in the scavenging of primary water radicals species, particularly eÿ aq and OH radicals. Several AM intermediates are hypothised to be formed through the above reactions and these species could significantly affect the response of biological systems to radiations. In
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M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
Table 1 Wavenumbers and assignments of the main IR and Raman bands of AM and Cu (II)/AM (1 : 4) system at different pHa AM pH 7.5
AM
Cu(II)-AM (1 : 4)
pH 5.0
Assignments
pH 5.0
IR
Raman
IR
Raman
IR
Raman
3403 s 3330 s 3284 sh 3237 sh 3197 sh 2789 m 1635 s } 1584 sh 1547 w 1457 s } 1363 m 1280 w 1205 m 1115 m 1069 m } } 964 w 873 m 729 vw 682 w
3406 vw 3331 w 3301 sh } } 2792 sh 1634 sh 1608 sh 1589 m 1550 sh 1446 m } 1362 w 1282 w 1201 vw 1120 m 1065 sh 1039 s 999 w } 857 vw } 686 w
3406 s 3330 s } 3243 sh } 2788 s 1635 s } 1587 w } 1458 s 1377 w 1365 w 1279 w 1211 m } 1070 s } 998 w 962 w 873 m 730 w 691 w
3404 vw 3332 w } 3245 vw } 2791 sh 1637 w } 1588 m } 1446 m 1377 w 1362 sh 1280 w 1208 vw 1121 m 1062 w 1037 s 999 w } 858 w } 697 w
3396 s 3329 s 3285 s 3233 s 3195 s 2792 s 1631 s } 1585 m } 1457 s 1379 vw 1364 w 1284 w 1205 w } 1066 s } 982 w 964 w 866 m 731 vw 690 vw
} 3329 w 3286 w 3238 w 3191 w 2795 vw 1634 w 1607 sh 1588 m 1549 sh 1447 m 1372 m 1359 sh 1287 w 1200 vw 1129 m 1060 w 1038 s 995 w 964 w 857 sh } 698 w
nOH na NH2 nNH ns NH2 nNH nNH+ 2 dNH2 dNH dNH+ 2 dNH dCH2 na C–N na C–N na C–N gOH na C–N dOH dOH rNH rNH rNH+ 2 gNH gNH
a
Assignments: n=stretching (na and ns =asymmetric and symmetric stretching, respectively), d=in plane deformation, g=out of plane deformation, and r=rocking. Intensities: w=weak, m=medium, s=strong, v=very, and sh=shoulder.
Fig. 9. Survival curves of E. coli B/r irradiated with g-rays in the absence and in the presence of AM (1 10ÿ3 mol dmÿ3) under oxygen and nitrogen saturation.
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fact, the phenomena of modification of cellular or tissue radiosensitivity by endogenous or exogenous chemicals are well documented (Breccia et al., 1979). In order to gain such information for AM, survival experiments on E. coli B/r cells irradiated with g-rays in the presence of AM 10ÿ3 mol dmÿ3 were performed. Fig. 9 shows the survival curves of E. coli B/r irradiated in different gas phases (N2 or O2) in the presence or the absence of AM. These curves show that AM decreases the sensitising effect of oxygen, meanwhile increases considerably the radiation lethality of anoxically irradiated E. coli cells. These preliminary data, showing a large radiosensitising effect in the absence of oxygen, are promising for a more focused screening of this compound as a radiosensitizer. With respect to the possible molecular mechanisms involved in the radiosensitising ability of AM, it is worth pointing out some aspects reported in the above pulse radiolysis section. The majority of eÿ aq attacks the drug preferentially on the activated positions occupied by bromine atoms, leading to dealogenation (reaction (lb)). Conversely, the OH radicals react with AM by attachment to ring positions occupied by bromine atoms (reaction (5)) in addition to the attachment to other positions (reactions (6) and (7)). Therefore, the yield of the bromide elimination caused by OH should be less than that of eÿ aq. The release of bromide ions could be responsible for the radiation chemical mechanism of sensitisation. In fact, Brÿ may be converted into oxidising Brÿ 2 radical species (reactions (11) and (12)) which are known to be more selective than OH in inactivating enzymes, damaging DNA constituents and, probably, more active in killing cells (Saha et al., 1995; von Sonntag, 1987). Similar radiosensitisation mechanism has been reported for iodine-containing compounds for which the release of iodine, as iodine radicals such as I and I2ÿ or as non radical species such as Iÿ have been proved in irradiated solutions of iodine-containing reagents (Quintiliani, 1987). Brÿ þ OH ! Br þOHÿ ; ÿ
Br þBr !
Brÿ 2 ;
ÿ ÿ Oÿ 2 þBr2 ! O2 þ 2 Br :
51
ð15Þ
4. Conclusions The pulse radiolysis investigations have evidenced the high reactivity of AM towards both eÿ aq and OH radicals. The transient absorption spectrum due to the eÿ aq attack suggests as the dehalogenation reaction is the main process leading to an aryl-type radical. With respect to the OH radical, its reaction with AM gives rise to a primarily formed OH-adducts which are probably converted into a phenoxyl- or anilino- radicals. The above suggested reactions for both both eÿ aq and OH are supported by the literature data regarding similar halogen-substituted aromatic compounds. The OH-adducts have shown to posses a relatively low oxidising property and to be quite stable in the presence of oxygen. As far as the interaction with Cu(II) is concerned, both IR and Raman spectra have indicated the establishment of only a weak interaction with AM which, however, does not affect the main sites of the OH attack on the drug. In addition, the low chelating ability of AM indicates that the complexation reaction is not relevant to the antioxidant activity of the drug. Finally, radiation biological data have shown an interesting radiosensitising effect of AM in anoxia, which stimulates to carry out deeper studies on this behaviour of the drug.
Acknowledgements We thank Dr. Paola Taddei for the helpful suggestions and Ing. A. Martelli and Mr. A. Monti for their assistance to Linac accelerator.
ð11Þ ð12Þ
In the presence of oxygen, the radiosensitising effect of AM disappears and a small radioprotection is evidenced. Under these conditions, only a small fraction of eÿ aq ( 10%) escapes from reaction with O2 and react with AM, meanwhile the majority of OH radicals will be scavenged by AM instead of bacterial cells. The bromine species produced by the reactions of eÿ aq and OH with AM can interfere with superoxide radicals, O2ÿ, so limiting the sensitisation caused by bromine radicals: ÿ eÿ aq þ O2 ! O2 ;
ð13Þ
ÿ Oÿ 2 þBr ! O2 þ Br ;
ð14Þ
References Behar, D., Neta, P., 1981. Intramolecular electron transfer and dehalogenation of anion radicals. 2. Halonitroaromatic compounds. J. Phys. Chem. 85, 690–693. Bekbo¨let, M., Getoff, N., 1999. Radiation decomposition of chlorinated benzaldehydes in aqueous solution. Radiat. Phys. Chem. 56, 333–339. Bielski, B.H.J., Cabelli, D.E., Arudi, R.L., Ross, A.B., 1985. Reactivity of HO2/Oÿ 2 radicals in aqeuous solution. J. Phys. Chem. Ref. Data 14, 1041–1100. Biswas, A.K., Hall, J.R., Schweinsberg, D.P., 1983. The synthesis and characterization by infrared and Raman spectroscopy of some ammine, methylammine, di- and trimethylamine and aniline complexes of group VB (As, Sb, and Bi) trihalides. Inorg. Chim. Acta 75, 57–64.
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Breccia, A., Rimondi, C., Adams, G.E., 1979. Radiosensitizers of hypoxic cells. Elsevier/North-Holland Biomedical Press, Amsterdam. Christensen, H., 1972. Pulse radiolysis of aqueous solutions of aniline and substituted anilines. Int. J. Radiat. Phys. Chem. 4, 311–333. Colthup, N.B., Daly, L.H., Wiberly, S.E., 1975. Introduction to Infrared and Raman Spectroscopy, 2nd Edition. Academic Press, New York. Disse, B.G., 1987. The pharmacology of ambroxol: review and new results. Eur. J. Respir. Dis. 153, 255–262. Felix, K., Pairet, M., Zimmermann, R., 1996. The antioxidative activity of the mucoregolatory agents: ambroxol, bromhexine, and N-acetyl-L-cysteine. A pulse radiolysis study. Life Sci. 59, 1141–1147. Geppert, W.D., Getoff, N., 1998. Radiolysis of 2-, 3- and 4chlorobenzonitrile in aqueous solution and product analysis. Radiat. Phys. Chem. 51, 281–292. Halliwell, B., 1996. Vitamin C: antioxidant or pro-oxidant in vivo? Free. Radic. Res. 25, 439–454. Halliwell, B., Gutteridge, J.M.C., 1990. Role of free-radicals and catalytic metal ions in human disease: an overview. Meth. Enzymol. 186, 1–85. Jonah, C.D., 1995. A short history on the radiation chemistry of water. Radiat. Res. 144, 141–147. Ko¨ster, R., Asmus, K.D., 1973. Reactions of fluorinated benzenes with hydrated electrons and hydroxyl radicals in aqueous solutions. J. Phys. Chem. 77, 749–755. Land, E.J., Ebert, M., 1967. Pulse radiolysis studies of aqueous phenol. Trans. Farad. Soc. 63, 1181–1190. Lapenna, D., De Gioia, S., Ciofani, G., Cuccurullo, F., 1994. Ambroxol is a scavenger of hypochlorous acid and monochloramine. Pharmacology 49, 132–135. Lian, C.T.S., Mittal, J.P., 1984. Matrix isolation studies of transients produced in the gamma radiolysis of decafluoro benzophenone. Radiat. Phys. Chem. 24, 209–212. Lichtsheidl, J., Getoff, N., 1976. Radiolysis of halogenated aromatic compounds in aqeuorus solutions. I. Conductometric pulse radiolysis and steady-state studies of the reaction of eÿ aq. Int. J. Radiat. Phys. Chem. 8, 661–665. Mohan, H., Mittal, J.P., 1995. Formation and redox properties of radical ions of iodopentafluorobenzene in aqueous solution: a pulse radiolysis study. J. Phys. Chem. 99, 12559–12564. Mohan, H., Mudaliar, M., Rao, B.S.M., Mittal, J.P., 1992. Reactions of hydrated electron and alcohol radicals with haolgenated aromatic compounds: a pulse radiolysis study. Radiat. Phys. Chem. 40, 513–517.
Neta, P., Fessenden, W., 1974. Hydroxyl radical reactions with phenols and anilines as studied by Electron Spin Resonance. J. Phys. Chem. 78, 523–529. Neta, P., Huie, R.E., Ross, A., 1990. Rate constants for reactions of peroxyl radicals in fluid solutions. J. Chem. Ref. Data 19, 413–513. Nowak, D., Antczak, A., Kro`l, M., Bialasiewicz, P., Pietras, T., 1994. Antioxidant properties of ambroxol. Free Rad. Biol. Med. 16, 517–522. Nowak, D., Pietras, T., Antczak, A., Kro`l, M., Piasecka, G., 1993. Ambroxol inhibits endotoxin-induced peroxidation in mice. Pot. J. Pharmacol. 45, 317–322. O’Neill, P., Schulte-Frohlinde, D., Steenken, S., 1978. Formation of radical cations and zwitterions versus demethoxylation in the reaction of OH with a series of methoxylated benzenes and benzioc acids. Faraday Discuss. Chem. Soc. 63, 141–148. Pryor, M.A., 1986. Oxy-radicals and related species: their formation, lifetimes, and reactions. Ann. Rev. Physiol. 48, 657–667. Quintiliani, M., 1987. Physico-chemical basis of radiosensitization by iodine compounds. Radiat. Phys. Chem. 30, 409–422. Rozniecki, J., Nowak, D., 1987. Effect of ambroxol on the chloramine T-induced decreased of serum elastase inhibitor capacity in vitro. Lug Resp. 4, 14–15. Saha, A., Mandal, P.C., Bhattacharyya, S.N., 1995. Radiationinduced inactivation of enzymes } a review. Radiat. Phys. Chem. 46, 123–145. Sies, H., 1985. Oxidative Stress. Academic Press, London. Steenken, S., 1987. Addition-elimination paths in electrontransfer reactions between radicals and molecules. J. Chem. Soc. Faraday Trans. 1, 83, 113–124. von Sonntag, C., 1987. Pulse radiolysis of nucleic acids and their base constituents: an updating review. Radiat. Phys. Chem. 30, 313–330. von Sonntag, C., Schuchmann, H-P., 1997. Peroxyl radicals in aqueous solutions. In: Alfassi, Z.B. (Ed.), Peroxyl Radicals. Wiley, Chichester, England, pp. 173–234. Wardman, P., 1989. Reduction potential of one electron couple involving free-radicals in aqueous solution. J. Phys. Chem. Ref. Data 18, 1637. Willson, R.L., Greenstock, C.L., Adams, G.E., Wageman, R., Dorfman, M.L., 1971. The standardization of hydroxyl radical rate data from radiation chemistry. Int. J. Rad. Phys. Chem. 3, 211–220. Winsel, K., Becher, G., 1992. Effect of ambroxol on chemiluminescence of phagocytic cells and Na-arachidonate induced broncho-constriction in guinea pigs. Eur. Resp. J. 5, 289.
Physico-chemical and biological properties of ambroxol under irradiation Maurizio Tambaa,*, Armida Torreggianib b
a Istituto F.R.A.E. (C.N.R.), via P. Gobetti, 101, 40129-Bologna, Italy Centro di Spettroscopia Raman, Dipartimento di Biochimica, Universita` di Bologna, via Belmeloro 8/2, 40126 Bologna, Italy
Received 3 May 2000; accepted 29 July 2000
Abstract Physico-chemical properties of Ambroxol (AM), a potential antioxidant drug from the expectorant class, were investigated by radiation chemical and spectroscopic studies. The pulse radiolysis experiments showed that AM is a good scavenger of the primary water radical species, particularly eÿ aq and OH radicals. The OH attack, preferentially addressed to the ring positions activated by the –NH2 group and occupied by bromine atoms, leads to hydroxycyclohexadienyl radicals. The molecule stabilisation may be achieved by a dehalogenation reaction to give phenoxyl radicals. The OH attack to AM is not affected by Cu(II) ions, which interact only weakly with the drug as evidenced by IR and Raman spectroscopy. Survival experiments on E. coli B/r cells irradiated in the presence of AM showed a radiosensitising effect of AM in anoxia. Some possible mechanisms of radiosensitisation are outlined. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Pulse radiolysis; Ambroxol; Antioxidant
1. Introduction Cells are continually exposed to reactive oxygen intermediates which, if accumulated, lead to a state known as oxidative stress. Presumably, this is the result of an overproduction of reactive substances or a deficiency in antioxidative defences (Sies, 1985). Oxidant-associated damage plays a critical role in the pathogenesis of respiratory diseases. In fact, pulmonary emphysema, adult respiratory distress syndrome, and lung cancer moistly involve free-radicalmediated reactions (Pryor, 1986). Thus, it seems that pharmacological enhancement of pulmonary antioxidative defence may be one way of preventing these diseases. Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxy-cyclohexyl]benzylamine) (AM) (Fig. 1) is a drug from the expectorant class widely used to increase surfactant secretion in lung and to decrease mucus *Corresponding author. Tel.: +39-051-6399787; fax: +39051-6399844. E-mail address: [email protected] (M. Tamba).
viscosity (Disse, 1987). AM has been reported to protect a-l-proteinase inhibitor from oxidative inactivation and to inhibit the generation of reactive oxygen species from activates phagocytes in vitro (Rozniecki and Nowak, 1987; Winsel and Becher, 1992). Recently, it has been suggested that AM may scavenge some reactive oxygen species (ROS) and protect lipids from peroxidative attack. In fact, intraperitoneal administration of AM seems to protect heart and lung lipids from oxidative stress provoked by intravenous injection of lipopolysaccharide in mice (Nowak et al., 1994, 1993). In addition, AM seems to exert some anti-inflammatory effects that may be due to a concentration-related antagonism towards prooxidants generated by white blood cells, such as hypochlorous acid (HOCl) and monochloramine (NH2Cl). In this regard, AM has been proved to be effective in the respiratory distress syndrome where neuthrophil-derived oxidants play a crucial pathophysiological role (Lapenna, 1994). Moreover, it is to be considered that transition metals, such as copper and iron, may catalyse ROS formation (i.e.
0969-806X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 6 X ( 0 0 ) 0 0 3 3 0 - 3
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M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
Fig. 1. Chemical structure of Ambroxol (AM).
OH), so contributing to the radical-induced oxidative damage (Halliwell and Gutteridge, 1990). Thus, it may be of interest to investigate the potential ability of AM to bind copper(II) ions so preventing the coppercatalyzed generation of ROS and, of consequence, protecting the biological targets from oxidative stress. The aim of the present work is to obtain a wider insight into the general physico-chemical properties of this drug and its antioxidant role as radical scavenger and metal ion binder, mainly by radiation chemical and spectroscopic studies. It also appeared appropriate to assess, at biological level, the role of the radiationinduced transients of AM on the survival curves of bacterial cells.
the total number of scans for each spectrum was 3000. On the sample, the laser power was 100 mW. All the radiation biological experiments were performed on E. coli B /r cells in stationary phase obtained by overnight growth with aeration in Difco nutrient broth. Cells were then collected on a Millipore filter, washed on the filter with the appropriate M/15 phosphate buffer at pH 7.0 and suspended in the same buffer. The bacterial concentration in samples to be irradiated was adjusted to about 107 cells mlÿ1. Irradiation were performed with a 60Co-Gamma Cell (Nordion Corp., Ltd, Canada) at a dose rate of about 9 Gy minÿ1, as measured by Fricke dosimetry. Bacterial suspensions were saturated with the appropriate gas (N2 or O2) before and during irradiations. AM was added to cell suspensions just before irradiation. At the concentration used (1 10ÿ3 dm3 molÿ1), AM has been tested to be moderately toxic to E. coli B/r up to 3 h contact time. Cell survival was determined by spreading appropriate dilutions of bacterial suspensions on the surface of Difco nutrient agar in plastic dishes and counting the visible colonies after overnight incubation at 378C.
2. Experimental 3. Results and discussion Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxy-cyclohexyl]benzylamine; Sigma) (AM), CuCl22H2O (Fluka) and all other chemicals of Analar grade were used as supplied. All solutions were made up with triple distilled water. The Cu(II)/AM systems were obtained by mixing freshly prepared solutions of CuC12 and AM in different ratios just before experiment. Solutions were saturated, immediately prior to irradiation, with N2O or varying percentages in N2O/O2 by flushing gas mixtures. The pH was adjusted by means of HClO4 or NaOH. Pulse radiolysis experiments were performed using a 12 MeV electron linear accelerator (LINAC). Spectral cells of 0.2 and 0.5 dm optical pathlength were used throughout. Optical filters were employed to minimise photochemical effects. Radiation doses were determined with the use of KCNS dosimetry at 480 nm assuming G=0.3 mmol Jÿ1 and e ¼ 710 m2 molÿ1. The signals from the photomultiplier were digitised by a Tektronix AD7912 and analysed by software running on a PC. The infrared spectra were recorded with a JASCO FT/ IR-300E using the KBr-pellet technique. In order to obtain good quality spectra, 1000 scans for each spectrum were accumulated and computer averaged. The spectral resolution was 4 cmÿ1 and the error in wavenumber was about 0.4 cmÿ1. The Raman spectra were obtained by a Bruker IFS 66 spectrometer equipped with a FRA-106 Raman module and a cooled Ge-diode detector. The excitation source was a Nd3+-YAG laser (1064 nm) in the backscattering (1808) configuration. The spectral resolution 4 cmÿ1 and
3.1. Pulse radiolysis The reaction of AM with eÿ aq was studied in N2saturated aqueous solutions containing a large excess of t-BuOH as a scavenger of .OH radicals which are produced simultaneously, with the hydrated electrons (Jonah, 1995). The small contribution by H atoms can be usually neglected. The rate constant for the reaction of AM with hydrated electrons, measured from the effect of the solute concentration on the lifetime of eÿ aq at 720 nm, resulted to be 2.0 1010 dm3 molÿ1 sÿ1 at pH 6.0 (Fig. 2). On the basis of the behaviour observed for other halogenated aromatic compounds (Koster and Asmus, 1973; Mohan et al., 1992), the reaction of eÿ aq with AM may involve interaction with the p orbitals (1a) of the aromatic ring or with the substituent bromine atoms (lb). When the eÿ aq attack occurs at the level of the aromatic ring, a molecular anion (I) is formed. Conversely, if the sites of the attack are the halogen substituents, one bromide ion can be released via a dissociation electron capture process and an aryl radical (II) is formed. The molecular anion (I) is a bound state and, depending upon the relative magnitude of the bond energy and electron affinity, may cross over to the repulsive state leading to dissociation similar to the process lb. In fact, most of chloro- , bromo- , and iodoaromatic compounds result in the scission of C–X bond, meanwhile fluoro compounds mainly form molecular
M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
45
anions (Lian and Mittal, 1984; Koster and Asmus, 1973).
(1a)
(1b)
where
A dehalogenation reaction has also been found to take place for radical anions derived from nitroaromatic and benzonitrile compounds containing an halogen atom at various positions on the ring (Behar and Neta, 1981; Geppert and Getoff, 1998). The transient formed in the reaction of the eÿ aq with AM shows no characteristic absorption bands in the UV /visible region (Fig. 3). The absence of characteristic absorption band is in agreement with the results of pulse radiolysis studies on bromo- and chloro-aromatic compounds which react quantitatively with eÿ aq, leading
Fig. 3. Absorption spectrum (taken 20 ms after the pulse) of the product of the reaction of AM (1 10ÿ4 mol dmÿ3) with eÿ aq at pH 8.0; N2-saturated, containing t-BuOH (1 10ÿ1 mol dmÿ3). Path length=0.2 dm; dose 34 Gy.
to the splitting of the C–X bond (Lichtsheidl and Getoff, 1976; Mohan et al., 1992). The resulting aryl radical does not probably exhibit any absorption in the above spectral region, since the unpaired electron is orthogonal to p orbital system of the ring. Therefore, the lack of transient absorption bands in our system suggest that the dehalogenation process is probably the predominant pathway for the reaction of eÿ aq with AM. Using COÿ (E8=ÿ1.9 V) (Wardman, 1989), a 2 reducing agent weaker than eÿ aq, no electron transfer reaction (2) takes place; in fact, the decay of COÿ 2, monitored at 250 nm, remained unaffected in the presence of AM: COÿ 2 þAM
CO2 þ AM ÿ
ð2Þ
Fig. 4A shows the time-resolved optical absorption spectrum obtained by pulse irradiation of aqueous N2Osaturated solutions containing 1 10ÿ4 mol dmÿ3 AM at pH 7.5. Under these experimental conditions, essentially all the eÿ aq (598%) are converted to OH radicals according to the reaction: H2 O
ÿ eÿ aq þ N2 O ÿÿÿ! N2 þ OH þ OH:
ð3Þ
The transient spectrum exhibits a maximum at about 330 nm and a less intensive absorption in the 390– 450 nm region. On the basis of the literature, the spectrum can be attributed to the OH-adducts (III) which are formed by addition to the aromatic ring (taking the example of the 3-hydroxyl adduct):
ð4Þ
Fig. 2. The plot of the observed rate constants for the decay of eÿ aq with AM at pH 6.0.
In fact, the spectrum is quite similar to that of the hydroxylcyclohexadienyl radical derived from the
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reaction of OH with benzene and its derivatives (Mohan and Mittal, 1995; Ko¨ster and Asmus, 1973). It is most likely a composite spectrum of several isomeric hydroxylcyclohexadienyl species corresponding to the addition of OH to different ring positions. The OH attack is preferentially addressed to those positions (ortho and para) which are activated by the electrodonating –NH2, group. The presence of the two bromine atoms in the above positions makes the corresponding bromohydroxylcyclohexadienyl radicals (i.e. III) quite unstable, as observed for other halogen substituted OHadducts (Mohan and Mittal, 1995). A molecule stabilisation may be achieved by a hydrogen bromide elimination which leads to a corresponding resonance stabilised phenoxyl radical (IV) (Neta and Fessenden, 1974).
ð5Þ
With respect to the addition at the NH2 position, it can be followed by loss of NH3 leading to a phenoxyl radical (reaction (6)) or by water elimination to produce an anilino radicals (reaction (7)).
(6) Fig. 4. (A) Time-resolved absorption spectrum in the pulse radiolysis of aqueous N2O-saturated solution of AM (1 10ÿ4 mol dmÿ3) at pH 7.5 (B) Time-resolved absorption spectrum of N2O/air (4 : 1) saturated AM solution (1 10ÿ4 mol dmÿ3) at pH 7.5 Path length=0.5 dm; dose 15 Gy.
(7) Unfortunately, both the phenoxyl- and anilinoradicals are difficult to identify directly by pulse radiolysis since a lot of them absorb in a region ( 330 and 400 nm) where there is a considerable contribution from OH-adduct (O’Neill et al., 1978; Christensen, 1972; Land and Ebert, 1967). The absorption bands at 330 and 400 nm decayed at the same rates, indicating that both absorptions probably belong to the same species. Two different kinetic components are however present: one faster and mainly of first-order, followed by a slower bimolecular process. The observed first-order component (k 1.3 103 sÿ1 at pH 7.5), probably due to the unimolecular bromide elimination process to form the phenoxyl radical (reaction (5)), becomes faster and mixed-order increasing dose/pulse. This dose-rate effect may be explained by the occurrence of competitive
bimolecular radical–radical reaction involving the primarily formed OH-adducts. 2 AM ÿ OH ! P:
ð8Þ
A further increase in rate is observed at basic pH (k 2.3 103 sÿ1 at pH 9.5) and is probably related to an OHÿ catalysis of both reactions (5) and (7). In fact, an OHÿ catalysed water elimination, leading to an anilino radical (reaction (7)) has also been reported in the literature (Neta and Fessenden, 1974; Steenken, 1987). With respect to the bimolecular decay component which follows the first-order radical disappearance, it is ascribed to disproportionation and dimerization reactions of the phenoxyl and anilino type radicals. Similar reactions have been suggested for aniline derivatives and fluorinated benzenes (Ko¨ster and Asmus, 1973; Neta and Fessenden, 1974). The rate constants of the transient absorption formation at pH 7.5 are similar both at 330 and
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400 nm (8.5 109 and 7.5 109 dm3 molÿ1 sÿ1, respectively). These data, as well as those obtained from the decay kinetics at the above wavelengths, further support the formation of only one type of radical (i.e. III) although the OH attack can occur at different positions on the aromatic ring. The OH reactivity was evaluated also by a competition method using KCNS as a standard. N2O-saturated solutions of KCNS (1 10ÿ3 mol dmÿ3) were pulse irradiated in the presence of different concentrations of AM (from 2.5 10ÿ4 to 1 10ÿ3 molÿ1 dmÿ3) at pH 7.0. In the presence of CNSÿ ions, the following reactions take place: OH þ CNSÿ ! CNS þOHÿ ; ÿ
ð9Þ
ÿ
CNS þCNS Ð ðCNSÞ2 ðl ¼ 500 nmÞ;
ð10Þ ÿ
where the rate constant of the reaction of CNS with OH (k9 ) has been reported to be 1.1 1010 dm3 molÿ1 sÿ1 (Willson et al., 1971). The rate constants of the reaction between AM and OH radicals (k11 ) were determined following the decrease of the absorption at 500 nm due to (CNS)2ÿ at different [AM]/[CNSÿ] ratios: OH þ AM ! AM ÿ OH:
ð11Þ
Under these conditions, the following competition equation was applied: OD0 =OD ¼ 1 þ
k11 ½AM : k9 ½CNSÿ
ðIÞ
From the slope of the linear regression plot (Fig. 5) a rate constant of 1.8 1010 dm3 molÿ1 sÿ1 (k11 ) was
Fig. 5. Indirect method for the determination of k(AM+OH). Plot pf OD0/OD (from (CNS)ÿ 2 measured at 500 nm) against [AM] at pH 7.0. Dose 9 Gy.
47
calculated, a value similar to that reported in the literature (Felix et al., 1996). The value obtained by the competition method is higher than that obtained by the direct one. Besides to a possible experimental error, the discrepancy may be explained considering the possibility that uncomplexed thiocyanate radical (CNS.) could react with AM in competition with reaction (10) causing an overtime of the OD0/OD ratio with a consequent increase of the k11 value. To test the possible interaction of the primary formed OH-radical adducts with naturally occurring antioxidants, N2O-saturated solutions of AM (5 10ÿ4 mol dmÿ3) were irradiated in the presence of ascorbate (1 10ÿ4 mol dmÿ3) at pH 7.0. Ascorbic acid is known to have reducing properties (E8=+0.30 V) (Wardman, 1989) and it has been chosen for its important antioxidant role played in biological systems (Halliwell, 1996). The occurrence of an interaction between the OH-AM adducts and ascorbate was evidenced by the faster absorption decay of the above adducts at 400 nm, as well as by the slow formation of an additional absorption at 360 nm due to the ascorbyl radical Aÿ. Unfortunately, the overlap of the absorption of both radicals in the 360–400 nm region allows only an estimate of the magnitude order for such interaction (107 dm3 molÿ1 sÿ1). This result suggests a relatively low oxidising capability of the OH-induced AM radicals, a property which may be useful in supporting the potential antioxidant role of the drug itself. In accordance with the aim of this study to explore the scavenging ability of AM towards ROS, the possible reaction with superoxide anions, O2ÿ, was investigated. The measurements were carried out in oxygen-saturated solutions containing 0.1 mol dmÿ3 sodium formate and AM at different concentrations (10ÿ4–10ÿ5 mol dmÿ3). Under these experimental conditions, all the primary water radicals are finally converted into superoxide. The kinetic analysis was performed at 250 nm where the O2ÿ radicals are reported to absorb (Bielski et al., 1985). No significant changes in the decay kinetics of superoxide were evidenced in the presence of variable AM concentrations, suggesting that, if a reaction occurs, the relative rate constant will be 4106 dm3 molÿ1 sÿ1. This is in agreement with the low reactivity of AM toward O2ÿ reported in the literature (Felix et al., 1996; Nowak et al., 1994). The reaction of hydroxyl radicals with AM in the presence of oxygen was investigated in solutions containing N2O/air gas mixtures. Under these experimental conditions, essentially all eÿ aq are converted to OH (reaction (3)). By a comparison of the spectra in the presence and the absence of oxygen (Fig. 4A and B), it is evident that the transient spectrum due to the initially formed OH-adducts (i.e. III) is scarcely affected by O2,
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M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
although the formation of the corresponding peroxyl radicals (V) is expected (Bekbo¨let and Getoff, 1999).
ð12Þ It is worth considering that often the parent radical (III) has a well characterised absorption in the UV/VIS region while the corresponding peroxyl radical (V) absorbs more weakly in the UV and has usually very low absorption also in the visible part of the spectrum (Neta et al., 1990). In fact, hydrocyclohexadienyl radicals absorb strongly near 310 nm, a region where the corresponding peroxyl radicals are reported to absorb only weakly (von Sonntag and Schuchmann, 1997). In agreement, the reaction of OH-adducts of AM with O2 results in a transient with an absorption spectrum almost coincident with that of the parent hydrocyclohexadienyl radical. The rate constant for the oxygen addition to substituted hydrocyclohexadienyl radicals is reported to be significantly below the typical value of the other carbon-centred radicals (k 2 109 dm3 molÿ1 sÿ1) and furthermore, it is decreased by the presence of halogen substituents on the ring (von Sonntag and Schuchmann, 1997). A low reactivity towards O2 has also been observed for phenoxyl radicals which, in our system, can be formed by reaction (5) (von Sonntag and Schuchmann, 1997). After 1.6 ms (Fig. 4B) a small increase in residual absorption can be observed in the UV region. Since this increase does not occur in the absence of oxygen (Fig. 4A), it is reasonable to ascribe it to transient products (P) derived from the corresponding peroxyl radicals (V). In order to investigate how the interactions of AM with transition metal ions may affect the behaviour of the molecule in the scavenging of OH radicals, the Cu(II)/AM systems in N2O saturated aqueous solutions were pulsed irradiated. Transient spectra were obtained at different metal/ligand ratios (from 1 : 1 to 1 : 4) and pHs (5.0 and 7.5). Fig. 6 shows, as an example, the transient spectrum at pH 7.5 and the Cu(II)/AM ratio of 1 : 4. The profile of the spectra resulted to be similar to that observed in the absence of Cu(II) in all cases, thus suggesting that, even if an interaction takes place, this is not able to modify the main sites of the OH attack to the molecule. Therefore, from the above radiation chemical data, the biological activity of AM does not appear to involve copper chelation mechanism.
Fig. 6. Time-resolved absorption spectrum in the pulse radiolysis of N2O-saturated solutions of Cu(II)/AM (1 : 4) at pH 7.5 [Cu(II)]=0.25 10ÿ4 mol dm3, [CpSH]=1 10ÿ4 mol dm3. Path length=0.5 dm; dose 15 Gy.
3.2. Vibrational spectroscopy To verify the eventual coordination of Cu(II) ions, both AM and the Cu(II)-AM system were investigated at different metal/ligand ratios (from 1 : 1 to 1 : 4) by IR and Raman spectroscopy. The IR spectra of AM at pH 5.0 and 7.5 are very similar and only slight differences can be evidenced (Fig. 7). In particular, at neutral pH the secondary aliphatic amine group is present both in the protonated form (i.e. 2789, 1584 and 873 cmÿ1) and in the neutral one (i.e. 3284, 3197 and 1547 cmÿ1) (Colthup et al., 1975). The presence of the >NH group can be evidenced also in the Raman spectrum where the NH vibrations are responsible of the shoulders at 1608 and 3300 cmÿ1 (Fig. 8 and Table 1). In the 3500–2500 cmÿ1 IR spectral region there is a good deal of overlapping between the OH, –NH2 and >NH vibrations making difficult the assignments of the bands. However, it is possible to ascribe the bands at 3406, 3330 and 3243 cmÿ1 in the spectrum at pH 5.0 to the stretching modes of the OH and –NH2 groups and the weaker submaxima, which appear by increasing pH, to the >NH group. In the presence of Cu(II) ions (metal/ligand ratios 1 : 1–1 : 2), the IR and Raman spectra at both pH values are very similar to those of AM alone, suggesting that, if an interaction between AM and Cu(II) occurs, it is very weak.
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49
Fig. 7. IR spectra of AM at pH 7.5 (a) and pH 5.0 (b), and of the Cu(II)/AM (1 : 4) system at pH 5.0 (c).
Fig. 8. Raman spectra of AM pH 7.5 (a) and pH 5.0 (b), and of the Cu(II)/AM (1 : 4) system at pH 5.0 (c).
By adding an excess of AM (metal/ligand 1 : 4), some sensitive changes take place, particularly in the high wavenumber IR region (Fig. 7c) whose overall appearance suggests that the coexistence of a variety of hydrogen bonds, with different strength, causes the complex broadening. As a consequence of the interaction with the Cu(II) ions, the bands due to the OH vibrations change in wavenumbers (i.e. from 3406 to 3396 cmÿ1), indicating the involvement of this functional group in metal chelation at pH 5.0 (Table 1). Also the amine groups take part to the copper-drug interaction, as shown both by the perturbation of the NH2 and >NH stretching region ( 3300 cmÿ1), as well as by the modifications of the bands due to dNH2 (i.e. 1631, 982 cmÿ1, etc.) and to na C–N (i.e. 1379 and 1284 cmÿ1) (Fig. 7c and Table 1). Analogously to the IR spectrum, the presence of the metal slightly alters the Raman spectral features (intensity, shape and wavenumber) of the bands due to the N–H and C–N stretching, and the NH deformation modes (Fig. 7 and Table 1).
Under neutral experimental conditions (pH 7.5), both the IR and Raman spectra show modifications similar to those observed at pH 5.0, supporting the involvement of the same functional groups in metal chelation. From the spectroscopic data, it can be concluded that a complex between AM and Cu(II) ions is formed in the presence of an AM excess, and the hydroxyl and amino groups act as coordination sites. However, the slight spectral changes indicate that the interaction between AM and Cu(II) is quite weak as reported for amine complexes which are, generally, stable in air but can be easily hydrolysed by water (Biswas et al., 1983). 3.3. Radiation biological studies The pulse radiolysis experiments have shown as AM is very active in the scavenging of primary water radicals species, particularly eÿ aq and OH radicals. Several AM intermediates are hypothised to be formed through the above reactions and these species could significantly affect the response of biological systems to radiations. In
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Table 1 Wavenumbers and assignments of the main IR and Raman bands of AM and Cu (II)/AM (1 : 4) system at different pHa AM pH 7.5
AM
Cu(II)-AM (1 : 4)
pH 5.0
Assignments
pH 5.0
IR
Raman
IR
Raman
IR
Raman
3403 s 3330 s 3284 sh 3237 sh 3197 sh 2789 m 1635 s } 1584 sh 1547 w 1457 s } 1363 m 1280 w 1205 m 1115 m 1069 m } } 964 w 873 m 729 vw 682 w
3406 vw 3331 w 3301 sh } } 2792 sh 1634 sh 1608 sh 1589 m 1550 sh 1446 m } 1362 w 1282 w 1201 vw 1120 m 1065 sh 1039 s 999 w } 857 vw } 686 w
3406 s 3330 s } 3243 sh } 2788 s 1635 s } 1587 w } 1458 s 1377 w 1365 w 1279 w 1211 m } 1070 s } 998 w 962 w 873 m 730 w 691 w
3404 vw 3332 w } 3245 vw } 2791 sh 1637 w } 1588 m } 1446 m 1377 w 1362 sh 1280 w 1208 vw 1121 m 1062 w 1037 s 999 w } 858 w } 697 w
3396 s 3329 s 3285 s 3233 s 3195 s 2792 s 1631 s } 1585 m } 1457 s 1379 vw 1364 w 1284 w 1205 w } 1066 s } 982 w 964 w 866 m 731 vw 690 vw
} 3329 w 3286 w 3238 w 3191 w 2795 vw 1634 w 1607 sh 1588 m 1549 sh 1447 m 1372 m 1359 sh 1287 w 1200 vw 1129 m 1060 w 1038 s 995 w 964 w 857 sh } 698 w
nOH na NH2 nNH ns NH2 nNH nNH+ 2 dNH2 dNH dNH+ 2 dNH dCH2 na C–N na C–N na C–N gOH na C–N dOH dOH rNH rNH rNH+ 2 gNH gNH
a
Assignments: n=stretching (na and ns =asymmetric and symmetric stretching, respectively), d=in plane deformation, g=out of plane deformation, and r=rocking. Intensities: w=weak, m=medium, s=strong, v=very, and sh=shoulder.
Fig. 9. Survival curves of E. coli B/r irradiated with g-rays in the absence and in the presence of AM (1 10ÿ3 mol dmÿ3) under oxygen and nitrogen saturation.
M. Tamba, A. Torreggiani / Radiation Physics and Chemistry 60 (2001) 43–52
fact, the phenomena of modification of cellular or tissue radiosensitivity by endogenous or exogenous chemicals are well documented (Breccia et al., 1979). In order to gain such information for AM, survival experiments on E. coli B/r cells irradiated with g-rays in the presence of AM 10ÿ3 mol dmÿ3 were performed. Fig. 9 shows the survival curves of E. coli B/r irradiated in different gas phases (N2 or O2) in the presence or the absence of AM. These curves show that AM decreases the sensitising effect of oxygen, meanwhile increases considerably the radiation lethality of anoxically irradiated E. coli cells. These preliminary data, showing a large radiosensitising effect in the absence of oxygen, are promising for a more focused screening of this compound as a radiosensitizer. With respect to the possible molecular mechanisms involved in the radiosensitising ability of AM, it is worth pointing out some aspects reported in the above pulse radiolysis section. The majority of eÿ aq attacks the drug preferentially on the activated positions occupied by bromine atoms, leading to dealogenation (reaction (lb)). Conversely, the OH radicals react with AM by attachment to ring positions occupied by bromine atoms (reaction (5)) in addition to the attachment to other positions (reactions (6) and (7)). Therefore, the yield of the bromide elimination caused by OH should be less than that of eÿ aq. The release of bromide ions could be responsible for the radiation chemical mechanism of sensitisation. In fact, Brÿ may be converted into oxidising Brÿ 2 radical species (reactions (11) and (12)) which are known to be more selective than OH in inactivating enzymes, damaging DNA constituents and, probably, more active in killing cells (Saha et al., 1995; von Sonntag, 1987). Similar radiosensitisation mechanism has been reported for iodine-containing compounds for which the release of iodine, as iodine radicals such as I and I2ÿ or as non radical species such as Iÿ have been proved in irradiated solutions of iodine-containing reagents (Quintiliani, 1987). Brÿ þ OH ! Br þOHÿ ; ÿ
Br þBr !
Brÿ 2 ;
ÿ ÿ Oÿ 2 þBr2 ! O2 þ 2 Br :
51
ð15Þ
4. Conclusions The pulse radiolysis investigations have evidenced the high reactivity of AM towards both eÿ aq and OH radicals. The transient absorption spectrum due to the eÿ aq attack suggests as the dehalogenation reaction is the main process leading to an aryl-type radical. With respect to the OH radical, its reaction with AM gives rise to a primarily formed OH-adducts which are probably converted into a phenoxyl- or anilino- radicals. The above suggested reactions for both both eÿ aq and OH are supported by the literature data regarding similar halogen-substituted aromatic compounds. The OH-adducts have shown to posses a relatively low oxidising property and to be quite stable in the presence of oxygen. As far as the interaction with Cu(II) is concerned, both IR and Raman spectra have indicated the establishment of only a weak interaction with AM which, however, does not affect the main sites of the OH attack on the drug. In addition, the low chelating ability of AM indicates that the complexation reaction is not relevant to the antioxidant activity of the drug. Finally, radiation biological data have shown an interesting radiosensitising effect of AM in anoxia, which stimulates to carry out deeper studies on this behaviour of the drug.
Acknowledgements We thank Dr. Paola Taddei for the helpful suggestions and Ing. A. Martelli and Mr. A. Monti for their assistance to Linac accelerator.
ð11Þ ð12Þ
In the presence of oxygen, the radiosensitising effect of AM disappears and a small radioprotection is evidenced. Under these conditions, only a small fraction of eÿ aq ( 10%) escapes from reaction with O2 and react with AM, meanwhile the majority of OH radicals will be scavenged by AM instead of bacterial cells. The bromine species produced by the reactions of eÿ aq and OH with AM can interfere with superoxide radicals, O2ÿ, so limiting the sensitisation caused by bromine radicals: ÿ eÿ aq þ O2 ! O2 ;
ð13Þ
ÿ Oÿ 2 þBr ! O2 þ Br ;
ð14Þ
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