ORIGINAL ARTICLES Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Sosnowiec, Poland Interaction of neomycin, tobramycin and amikacin with melanin in vitro in relation to aminoglycosides-induced ototoxicity E. Buszman, D. Wrzes´niok, J. Trzcionka Received July 13, 2006, accepted August 2, 2006 Prof. Ewa Buszman, Ph.D, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Jagiellon´ska 4, PL-41-200 Sosnowiec, Poland [email protected] Pharmazie 62: 210–215 (2007) doi: 10.1691/ph.2007.3.6651 The aim of this study was to examine in vitro the interaction between aminoglycoside antibiotics dis- playing adverse ototoxic effects and melanin which is a constituent of the inner ear. The binding of neomycin, tobramycin and amikacin to model synthetic melanin was studied. It has been demon- strated that all the investigated aminoglycosides form stable complexes with melanin biopolymer. The obtained results show that the amount of drug bound to melanin increases with the increase of initial drug concentration and the incubation time. An analysis of drugs binding to melanin by the use of Scatchard plots has shown that at least two classes of independent binding sites must be implicated in the studied aminoglycoside antibiotic-melanin complexes formation: strong binding sites (n1) with 5 À1 the association constant K1 0.2–2.0 Á 10 M and weak binding sites (n2) with K2 1.0– 4.9 Á 103 MÀ1. Based on the values of association constants the following order of drugs affinity to DOPA-melanin was found: tobramycin > amikacin neomycin. The ability of the analyzed aminogly- coside antibiotics to form complexes with melanin in vitro may be one of the reasons for their ototoxi- city in vivo, as a result of their accumulation in melanin in the inner ear. 1. Introduction also metal ions (Andrzejczyk and Buszman 1992; Liu et al. 2004; Hong et al. 2004), herbicides, dyes, alkaloids The aminoglycosides are a family of structurally diverse etc. (Larsson 1993). Among the drugs showing the great- antibiotics that are effective against a broad spectrum of est affinity to melanin pigment in vitro are policyclic clinically important pathogenic organisms. This family of amines, including chloroquine and aminoglycoside anti- compounds, which includes the clinically relevant drugs biotics (Wa¨sterstro¨m 1984; Wrzes´niok et al. 2002; Pilawa tobramycin, kanamycin, gentamicin, neomycin and amika- et al. 2002; Wrzes´niok et al. 2005). cin, consists of a central aminocyclitol ring with two or Since melanins are present in external and internal tissues, three substituted aminoglycan rings attached at different their capacity to bind to a wide number of drugs may positions (Forge and Schacht 2000). Aminoglycosides are result in various toxic effects due to lesions of pigmented believed to exert their bactericidal effects by binding to tissues. This factor is of importance in the pathogenesis the 16S rRNA of the 30S ribosomal fragment. The amino- associated with long-term and/or high dose therapy with a glycoside antibiotics are known to affect renal tissues and number of drugs, including aminoglycoside antibiotics sensory cells of the inner ear (O’Grady et al. 1997). The (Larsson 1993). It has been earlier suggested that the affi- precise mechanism underlying the organ specificity of nity of melanin pigment for aminoglycosides may cause aminoglycoside-induced toxicity has not been fully estab- these drugs to bind preferentially to the pigmented inner lished. ear, producing greater ototoxicity than in the amelanotic The melanin pigment is a high molecular mass polymer albino cochlea (Wa¨sterstro¨m 1984). The mechanism be- that occurs widely in living organisms and particularly in hind the development of lesions in the pigmented cells is the skin, hair, eye, ear and brain (Ings 1984; Zucca et al. probably a combination of selective retention, due to mel- 2004; Wielgus and Sarna 2005, Liu et al. 2005). It is anin binding, and toxicity, i.e., substances with low toxi- known that various drugs and other chemicals are bound city may scarcely induce lesions, in spite of high melanin (Ibrahim and Aubry 1995; Lowrey et al. 1997; Ma˚rs and affinity, while those with a more expressed or specific Larsson 1999) and retained for long periods in pigmented toxicity may induce the adverse effects (Larsson 1993). tissues due to melanin affinity. The accumulated com- The evidence of greater ototoxicity in albinos has led to pounds are very heterogeneous in structure: drugs of dif- the hypothesis that melanin inhibits the toxicity of amino- ferent categories –– psychotropics, drugs for rheumatoid glycoside antibiotics in the pigmented inner ear. On the arthritis and malaria, local anaesthetics (Larsson 1993; other hand, ototoxicity in the pigmented animals may sim- Buszman et al. 2003; Buszman and Ro´z˙an´ska 2003a), ply be delayed relative to albinos, only to become equal antiarrhythmic drugs (Buszman and Ro´z˙an´ska 2003b), and or even more severe with time (Conlee et al. 1995). 210 Pharmazie 62 (2007) 3 ORIGINAL ARTICLES Under certain circumstances the possible protection me- chanism may be a threat to the pigmented cells. Chronic exposure to certain toxic substances with melanin affinity 0.6 NEOMYCIN ultimately causes adverse effects in the cells. These effects are mainly related to high dose, long-term exposure, and a prominent feature of the lesions is that the histologic ] 0.4 changes are initially found in the pigmented cells, and -1 successively in the adjacent tissues, such as receptor cells. The onset of the adverse effects may be delayed, and the entire manifestation of the lesions may occur even years mol mg • µ after cessation of the offending substances. It is also possi- r [ 0.2 ble that various toxic substances, which are retained in the melanin-containing tissues, are causing additive effects (Larsson 1993). The physiological meaning and the mechanism of drug- melanin binding are still not fully understood. The aim of the presented studies was to examine in vitro the interac- 0 2 4 6 8 10 -4 -3 tion between aminoglycoside antibiotics displaying ad- c o [10 mol • dm ] verse reactions in the inner ear, that is neomycin, tobramy- cin and amikacin, and melanin. For these studies synthetic DOPA-melanin was used because of its similarity to natur- al eumelanins. TOBRAMYCIN 0.6 2. Investigations and results The binding capacity of three aminoglycoside antibiotics: neomycin, tobramycin and amikacin to synthetic DOPA- ] 0.4 -1 mol • mg 100 NEOMYCIN µ r [ 0.2 80 60 -4 % co= 2.5•10 M -4 40 co= 5.0•10 M -4 co= 7.5•10 M 20 -3 co= 1.0•10 M 0 2 4 6 8 10 0 5 10 15 20 25 30 35 40 45 50 -4 -3 time [h] co [10 mol • dm ] TOBRAMYCIN 100 -4 AMIKACIN co= 2.5•10 M 0.6 80 -4 co= 5.0•10-4 M co= 7.5•10-3 M 60 co= 1.0•10 M % 40 20 ] 0.4 -1 0 5 10 15 20 25 30 35 40 45 50 time [h] mol mg • µ r [ 0.2 AMIKACIN -4 100 co= 2.5•10 M 80 -4 co= 5.0•10 M 60 % -4 co= 7.5•10 M 40 -3 0 2 4 6 8 10 co= 1.0•10 M 20 -4 -3 co [10 mol • dm ] 0 5 10 15 20 25 30 35 40 45 50 time [h] Fig. 2: Binding isotherms for neomycin-melanin, tobramycin-melanin and amikacin-melanin complexes. r –– amount of drug bound to mela- nin; co –– initial drug concentration. Fig. 1: Effect of incubation time and initial drug concentration (c0) on the Mean values Æ SD from three independent experiments are pre- amount of neomycin, tobramycin and amikacin bound to DOPA- sented. Points without error bars indicate that SD was less than the melanin (in %). size of the symbol Mean values Æ SD from three independent experiments are pre- sented. Points without error bars indicate that SD was less than the size of the symbol Pharmazie 62 (2007) 3 211 ORIGINAL ARTICLES melanin was analyzed. The effect of the incubation time and initial drugs concentration on the amount of drugs NEOMYCIN bound to melanin is presented in Fig. 1. The results are given for four different initial aminoglycosides concentra- 2.0 À4 À4 À4 tions (c0): 2.5 Á 10 M, 5 Á 10 M, 7.5 Á 10 M and ] 1 Á 10À3 M and for six different incubation times: 1, 3, 6, -1 12, 24, and 48 h. It can be seen that the amount of drug • mg 1.5 3 bound to melanin increases with the prolongation of incu- dm bation time and after 12–24 h it attains a plateau. It has -3 1.0 been also shown that the amounts of antibiotics bound to [10 A melanin increase with increasing initial drug concentra- r/c tion. Simultaneously, the decrease of complex formation 0.5 efficiency, expressed in % as the ratio of the amount of drug bound to melanin to the initial amount of drug added to melanin, was observed with the increase of the initial antibiotic concentration (Fig. 1.) 0.0 0.5 1.0 1.5 2.0 2.5 Dependence of the amount of aminoglycosides bound to melanin after 24 h of incubation as a function of the initial r [10-7mol • mg-1] drug concentration is presented in Fig. 2 as binding iso- therms. All the examined samples demonstrate an increase of bound antibiotics with the increase of the amount of added drug. It can be seen from binding isotherms that the 20 TOBRAMYCIN amount of drugs bound to a constant amount of DOPA-mel- anin reaches a plateau at about 0.19 mmol neomycin per ] 1 mg melanin, which reflects the initial neomycin concen- -1 À4 15 tration 7 Á 10 M, about 0.47 mmol tobramycin/mg mela- • mg nin for the initial tobramycin concentration 7.5 Á 10À4 M 3 dm 3 and about 0.31 mmol amikacin/mg melanin for the initial - À4 10 amikacin concentration 5 Á 10 M.