Prochlorperazine interaction with melanin and melanocytes.

Prochlorperazine is a phenothiazine-class antipsychotic drug usually used to treat nausea, vomiting and schizophrenia. Phenothiazine derivatives have been known to cause serious side effects, like extrapyramidal symptoms, but also skin disorders which mechanism has not been fully established. The aim of this study was to examine the interaction between prochlorperazine and melanin as well as to estimate the effect of prochlorperazine on cell viability, melanogenesis and antioxidant defense system in normal human melanocytes. We have demonstrated that prochlorperazine forms stable complexes with melanin, characterized by two classes of independent binding sites with the association constants K1∼106 M-1 and K2∼102 M-1. It has been shown that prochlorperazine induces concentration-dependent loss in cell viability. The value of EC50 was calculated to be 18.49 µM. Prochlorperazine in a concentration of 0.001 µM stimulated melanogenesis, while in concentrations 1.0 and 10.0 µM melanization process was inhibited. Furthermore, the drug in concentrations of 0.1, 1.0 and 10.0 µM caused changes in cellular antioxidant defense system, what indicated the induction of oxidative stress. The observed changes in cell viability, melanization and antioxidant defense system in normal human melanocytes after prochlorperazine treatment may explain a potential role of melanin, oxidative stress and melanocytes in mechanisms of undesirable side effects after accumulation of this drug in pigmented tissues.

Viability of Human Melanocytes HEMa-LP Exposed to Amikacin and Kanamycin.

Aminoglycosides, such as amikacin and kanamycin, are powerful broad-spectrum antibiotics used for the treatment of many bacterial infections. The widely used aminoglycosides have the unfortunate side effects of targeting sensory hair cells of the inner ear, so that treatment often results in permanent hair cell loss. The aim of the study was to evaluate the influence of incubation time and drug concentration on viability of melanocytes cultured in the presence of amikacin or kanamycin. The normal human melanocytes HEMa-LP and the different concentrations of amikacin (0.075, 0.75 and 7.5 mmol/l) and kanamycin (0.06, 0.6 and 6.0 mmol/l), were used. The estimations were performed after 24, 48 and 72 h. The observed decrease in melanocytes viability may be an explanation for the mechanisms involved in aminoglycosides toxicity on pigmented tissues during high-dose and/or long-term therapy.

Interaction of neomycin, tobramycin and amikacin with melanin in vitro in relation to aminoglycosides-induced ototoxicity.

The aim of this study was to examine in vitro the interaction between aminoglycoside antibiotics displaying 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 demonstrated 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 the association constant K1 approximately 0.2-2.0 x 10(5) M(-1) and weak binding sites (n2) with K2 approximately 1.0-4.9 x 10(3) M(-1). 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 aminoglycoside antibiotics to form complexes with melanin in vitro may be one of the reasons for their ototoxicity in vivo, as a result of their accumulation in melanin in the inner ear.

Melanin potentiates kanamycin-induced inhibition of collagen biosynthesis in human skin fibroblasts.

Ototoxicity is one of the well known side effects of kanamycin. The mechanism underlying the organ specificity of the side effect is not understood. Since many pharmacologic agents are known to form complexes with melanin and melanin is an abundant constituent of the inner ear, we investigated whether kanamycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. We found that kanamycin forms complexes with melanin. The amount of kanamycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the kanamycin-melanin complex formation: strong binding sites with the association constant K1 - 3 x 10(5) M(-1), and the weak binding sites with K2 - 4 x 10(3) M(-1). The number of total binding sites (n1 + n2) was calculated as about 0.64 micromol kanamycin per 1 mg melanin. We found that kanamycin induced inhibition of collagen and DNA biosynthesis (IC50 - 5 microM). Melanin at 100 microg/ml produced about 25% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to kanamycin-treated cells (5 microM) augmented the inhibitory action of kanamycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in cell growth and collagen metabolism, may be one of the targets for kanamycin-induced inhibition of these processes. As shown by Western immunoblot analysis melanin augmented kanamycin-induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2. The obtained results demonstrate that melanin potentiates the inhibitory effect of kanamycin on IGF-I receptor-dependent signaling pathway in cultured fibroblasts. The data suggest a potential mechanism for the organ specificity of kanamycin-induced hearing loss in patients which may result from melanin-induced augmentation of the inhibitory effects of kanamycin on collagen and DNA biosynthesis.

Melanin potentiates daunorubicin-induced inhibition of collagen biosynthesis in human skin fibroblasts.

One of the recognized side effects of antineoplastic anthracyclines is poor wound healing, resulting from an impairment of collagen biosynthesis. The most affected tissue is skin. The mechanism underlying the tissue specificity of the side effects of anthracyclines has not been established. In view of the fact that a number of pharmacologic agents are known to form complexes with melanin and melanins are abundant constituents of the skin, we determined whether daunorubicin interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. Results indicated that daunorubicin forms complexes with melanin. Scatchard analysis showed that the binding of daunorubicin to melanin was heterogeneous, suggesting the presence of two classes of independent binding sites with K1 = 1.83 x 10(5) M(-1) and K2 = 5.52 x 10(3) M(-1). The number of strong binding sites was calculated as n1 = 0.158 micromol/mg of melanin and the number of weak binding sites as n2 = 0.255 micromol/mg of melanin. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracycline-induced inhibition of collagen synthesis. We found that daunorubicin induced inhibition of prolidase activity (IC50 = 10 microM), collagen biosynthesis (IC50 = 70 microM) and DNA biosynthesis (IC50= 10 microM) in human skin fibroblasts. Melanin (100 microg/ml) by itself produced about 25% inhibition of DNA synthesis and prolidase activity but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to daunorubicin-treated cells (at IC50 concentration) augmented the inhibitory action of daunorubicin on collagen and DNA biosynthesis without having any effect on prolidase activity. The same effect was achieved when the cells were treated with daunorubicin at one-fourth of the IC50 given at 0, 6, 12 and 18 h during a 24-h incubation. The data suggest that the melanin-induced augmentation of the inhibitory effects of daunorubicin on collagen and DNA biosynthesis may result from: (i) accumulation of the drug in the extracellular matrix, (ii) gradual dissociation of the complex, and (iii) constant action of the released drug on cell metabolism. The phenomenon may explain the potential mechanism for the organ specificity of daunorubicin-induced poor wound healing in patients administered this drug.