Photochemical and photobiological studies of benzydamine hydrochloride

Abstract

Benzydamine hydrochloride [l-benzyl-3-(3-dimethylaminopropoxy)-1Hindazole hydrochloride] is a unique non-steroidal anti-inflammatory drug with local anaesthetic properties. Its photochemistry, photophysics, and photosensitization characteristics have been studied intensively to determine the basis of the adverse photosensitivity reactions associated with the use of its different formulations.

Benzydamine absorbs strongly in the UVB region at 308 nm. The excited state properties of benzydamine were established by fluorescence and phosphorescence measurement. In water, benzydamine gives a strong fluorescence at 345 nm with a quantum yield of 0.17 at pH 7 and 0.10 at pH 11. It also shows a strong phosphorescence emission at 422, 457, 490 nm implying a relatively high population of the triplet state when irradiated.

The photodegradation of benzydamine in acetonitrile, methanol and water solutions, was examined using reverse phase HPLC. The decomposition occurs readily in acetonitrile solution under a nitrogen atmosphere with the formation of 3-dimethylaminopropoxy-lH-indazole as the main primary product. Under aerobic conditions, benzaldehyde is an additional product presumed to result from oxidation of the benzyl radical dissociated from benzydamine. The photodecomposition of benzydamine in methanol and water under aerobic conditions is slower than that in acetonitrile with the formation of the above products plus 1-benzy1-3-(3—methylaminopropoxy)-lH-indazole from N—demethylation and 3-benzyl-lH-indazole as primary main products. The identification of the products was based on data from UV, Cl and EI Mass spectra, and H and C NMR spectra.

The photostability of benzydamine in three commercially available formulations (Difflam cream, gel and mouthwash) has been tested. Photodegradation occurs upon exposure to direct sunlight or artificial UV radiation. The principal reaction in the formulations is the photodegradation of benzydamine, yielding the same photoproducts as found in benzydamine water solution under the UV arc source.

The ability of benzydamine to act as a photosensitizer was examined in terms of its capacity to initiate free radical reactions and generate singlet oxygen in aqueous solution and micellar solutions by measuring the oxygen uptake rate of benzydamine in the presence of singlet oxygen acceptors. The capacity to act as a generator of singlet oxygen species is dependent on pH and the cation form is more efficient than the neutral form. The ability of benzydamine to act as a Type I (fiee radical) photosensitizer on irradiation was tested by the initiation of photopolymerization of acrylamide. The neutral form of benzydamine is more involved in free radical generation than the cationic form.

The involvement of an electron transfer mechanism in benzydamine photosensitization was tested under aerobic and anaerobic conditions by several methods (i) reduction of nitro blue tetrazolium (ii) reduction of cytochrome c (iii) reduction of Cu (11) and formation of copper (1) complex with BCDS in phosphate buffer solutions (iv) the oxidation of tetramethylphenylenediamine to generate TMPD cation radical, in acetonitrile, methanol and water solutions. Benzydamine was active in all these systems, showing the ability to act as both an electron donor and acceptor when irradiated.

Additionally, benzydamine photosensitized the peroxidation of linoleic acid, as monitored by the spectrophotometric detection of dienic hydroperoxide formation. While this photodynamic action was found to be strictly oxygen dependent, the reaction was suppressed completely when the free radical scavengers, reduced glutathione or butylated hydroxyanisole was added before irradiation, but was only partially inhibited by the singlet oxygen quenchers, sodium azide or 1,4-diazabicyclo[2,2,2]octane, suggesting that the free radical pathway is predominant.

Studies have also been performed to elucidate the phototoxic potential of benzydamine and its photoproducts towards biological membranes using human erythrocytes as a model system both under oxygen and nitrogen atmosphere. At a much higher concentration employed compared to that used in the lipid peroxidation experiment, benzydamine photosensitized hemolysis of human erythrocytes. A free radical mechanism was suggested since GSH inhibited the hemolysis under anaerobic conditions. Under aerobic conditions, the hemolysis was inhibited totally by GSH and partially by NaNs, but not by superoxide dismutase (SOD). The hemolysis was completely suppressed by copper(II) with bathocuproinedisulphonic acid (Cu/BCDS) which is a complex with SOD-like activity. The photohemolysis was enhanced when D20 replaced H20 in the buffer solution suggesting singlet oxygen involvement. Preirradiated benzydamine induced hemolysis in the dark indicating that one or more of the photoproducts is toxic.

In summary, benzydamine has been shown to be relatively active in a photochemical sense with free radical mechanisms playing a major role. The results imply that there is a definite risk of phototoxicity when long time sunlight exposure is combined with topical application of a benzydamine formulation.