Perphenazine and prochlorperazine induce concentration-dependent loss in human glioblastoma cells viability.

Phenothiazine derivatives possess biological properties very useful for cancer therapy, such as antiemetic and sedative activity as well as good blood-brain barrier permeability. Our goal was to determine if perphenazine and prochlorperazine are possessing cytotoxic activity towards U87-MG cells. It has been shown that the analyzed drugs induce concentration-dependent loss in cell viability, what correlates with their chemical structure. The calculated EC50 values for perphenazine (0.98 µM) and prochlorperazine (0.97 µM) are related to their toxic concentrations in human plasma. The obtained results suggest that perphenazine and prochlorperazine may have a potential for the development of new and effective anticancer therapies.

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 chlorpromazine, fluphenazine and trifluoperazine with ocular and synthetic melanin in vitro.

The aim of this study was to examine in vitro the binding capacity of three phenothiazine derivatives--chlorpromazine, fluphenazine and trifluoperazine--causing adverse effects in the eye structures, to natural melanin isolated from pig eyes as well as to synthetic DOPA-melanin used as a model polymer. The amount of drug bound to melanin was determined by UV spectrophotometry. The analysis of results for the kinetics of drug-melanin complex formation showed that the amount of drug bound to melanin increases with increasing initial drug concentration and longer incubation time, attaining an equilibrium state after about 24 h. Binding parameters, i.e. the number of binding sites (n) and association constants (K), were determined on the basis of Scatchard plots. For neuroleptic-ocular melanin and neuroleptic-DOPA-melanin complexes two classes of independent binding sites were found, with association constants K1 approximately 10(4) and K2 approximately 10(2) M (-1) for chlorpromazine and fluphenazine complexes, and K1 approximately 10(5) and K2 approximately 10(3) M(-1) for trifluoperazine complexes. The numbers of strong (n1) and weak (n2) binding sites indicate lower affinity of the drugs examined to ocular melanin compared with DOPA-melanin. The ability of chlorpromazine, fluphenazine and trifluoperazine to interact with melanin, especially the ocular melanin, in vitro is discussed in relation to the ocular toxicity of these drugs in vivo.

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.

Pharmacokinetics of fluconazole in the cerebrospinal fluid of children with hydrocephalus.

BACKGROUND: The aim of this study was to examine the pharmacokinetics of fluconazole in the CSF of children with hydrocephalus during CNS infection treatment after intravenous and/or intraventricular drug administration. Direct fluconazole administration into the ventricular CSF of patients to treat serious CNS infections is an aggressive therapy, and data on the pharmacokinetics of fluconazole in CSF are limited. METHODS: A method of fluconazole quantification in CSF by solid-phase extraction (SPE)-high-performance liquid chromatography (HPLC) was developed to conduct pharmacokinetic studies. The population of patients included 2 children with hydrocephalus. Fluconazole was administered intravenously at average multiple doses of 12.5 mg/kg/24 h and intraventricularly at doses of 4, 5 and 7.5 mg/24 h, and 7.5 and 10 mg/12 h. The CSF samples were taken 2-24 h after administration of fluconazole. The concentrations of fluconazole in CSF specimens were assessed, and after pharmacokinetic studies the fluconazole dosage was modified. RESULTS: The method of fluconazole determination in CSF using the SPE-HPLC method is specific, precise and accurate. After intravenous fluconazole administration, the concentration of this antifungal drug was not detected in the ventricular CSF. The pharmacokinetic parameters determined after intraventricular fluconazole administration were: steady-state peak CSF fluconazole concentration (19.54 +/- 5.63 mg/l); trough CSF fluconazole concentration (0.0-0.3 mg/l); elimination rate constant (0.4654 +/- 0.2097 h(-1)), and half-life (1.84 +/- 0.93 h). CONCLUSIONS: The authors developed a method to determine fluconazole in CSF by SPE-HPLC. After intravenous fluconazole administration, the drug was not detected in the examined CSF samples. The intraventricular multidose pharmacokinetic data suggest the necessity of fluconazole monitoring in children with hydrocephalus during the treatment of shunt infection.

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.

Interaction of quinidine, disopyramide and metoprolol with melanin in vitro in relation to drug-induced ocular toxicity.

The aim of this work was to evaluate binding capacity of quinidine, disopyramide and metoprolol to melanin in vitro. The antiarrhythmics studied cause adverse reactions to the eye. Synthetic DOPA-melanin was used in the studies and a UV spectrophotometric method was employed to determine the drugs. The studies of the kinetics of the formation of quinidine-melanin, disopyramide-melanin and metoprolol-melanin complexes indicate that for all the complexes investigated the maximum time to reach reaction equilibrium is 24 h. Binding parameters, i.e., the numbers of independent binding sites and the association constants were determined on the basis of the Scatchard plots. An analysis of the binding curves obtained supports our conclusion that both strong (n1) and weak (n2) binding sites are involved in the formation of the complexes investigated. The total numbers of binding sites in synthetic DOPA-melanin complexes with quinidine, disopyramide and metoprolol were 0.525, 0.493 and 0.387 micromol/mg, respectively. The quinidine-melanin complex is characterized by greater stability (K1 = 3.00 x 10(5) M(-1), K2 = 1.75 x 10(3) M(-1)) in comparison with biopolymer complexes with disopyramide (K1 = 1.12 x 10(4) M(-1), K2 = 6.04 x 10(2) M(-1)) and metoprolol (K1 = 1.42 x 10(4) M(-1), K2 = 7.89 x 10(2) M(-1)). The ability of these drugs to form complexes with melanin in vitro may be one of the reasons for their ocular toxicity in vivo, as a result of their accumulation in melanin in the eye.

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.

The effect of endo- and exogenous melanin on Zn2+ and Co2+ elimination and distribution in mice.

The content of endogenous melanin in white Balb c and black C-57 Bl. mice affected Zn2+ and Co2+ accumulation, elimination and distribution following intraperitoneal administration of these ions. Elimination of Co2+ ions was significantly greater in white mice than in black mice. Accumulation of Zn2+ was higher than that of Co2+ and did not depend on melanin content in mice. The same response was observed upon administration of exogenous melanin. Both ions accumulated mainly in spleen and heart irrespective of melanin content.

Interaction of methotrexate with melanins and melanosomes from B16 melanoma.

It has been demonstrated that methotrexate forms stable complexes with melanin and melanosomes isolated from B16 melanoma. The number of binding sites and binding constants for methotrexate binding by intact melanosomes and melanin were n = 0.046 mumol/mg, K = 0.32 x 10(4) M-1 and n = 0.063 mumol/mg, K = 1.08 x 10(4) M-1, respectively. Binding of methotrexate to synthetic DOPA-melanin used for comparison also shows a single class of binding sites, n = 0.060 mumol/mg with binding constant K = 2.34 x 10(4) M-1. The possibility of side effects caused by methotrexate-melanin interactions after treatment of neoplasms is discussed.

Spectroscopic studies of chemically modified synthetic melanins.

The infrared and electron spin resonance spectra of synthetic 3,4-dihydroxyphenylalanine (DOPA) and tyrosine melanins and chemically modified melanin samples were determined, and it was shown that unmodified and reduced DOPA melanins exhibited similar ir spectra. Oxidized DOPA melanins showed a higher number of carboxy groups in the sample. A significant increase of free radical content in reduced DOPA melanin and a decrease of free radical content in oxidized DOPA melanin in comparison to unmodified samples were demonstrated by the use of ESR methodology. Methylation of tyrosine melanin with an excess of diazomethane gave very rich ir spectra as compared to melanins methylated with methanol saturated by gaseous HCl. In tyrosine melanin samples the esterification of carboxy groups with methanol caused a decrease in the free radical content. When diazomethane was used, the methylated melanin samples had free radical levels reduced to only about 4% of the total observed for unmodified tyrosine melanin.

Electron spin resonance studies of chloroquine-melanin complexes.

Electron spin resonance (ESR) studies of chloroquine complexes with synthetic DOPA-melanin, synthetic catechol-melanin, melanin isolated from bananas and humic acid were performed. Two chloroquine concentrations (5 X 10(-5) M and 1 X 10(-3) M) were used for chloroquine-melanin complex formation. ESR studies showed differences in free radicals concentration depending on melanin origin. It was demonstrated that addition of chloroquine to melanin results in broadening of the melanin ESR signal and is accompanied by changes in the integrated intensity of the analyzed melanin samples.

The effect of pH on colchicine conformation and structure.

The effect of various pH values between 0 and 14 on the structure and conformation of colchicine was examined using UV-vis spectrophotometry at a concentration of 1.7 x 10(-5) M and NMR techniques at a colchicine concentration of 0.1M. The complete interpretation of the colchicine NMR spectrum in D2O is given. A stable structure of the colchicine molecule in aqueous solutions at pH from 2 to 12 was demonstrated. However, during incubation at 40 degrees C colchicine was found to be stable only at pH values between 2 and 10. The significance of these data for reactions of cholchicine in regard to metabolism and interaction with macromolecules is discussed.

Interaction of colchicine with DNA molecules.

Colchicine (7.5 X 10-6M or 3.3 X 10-5M) was incubated at pH 7.0, 10.0, or 12.0 with high-molecular DNA from salmon sperm (7 X 10-5M or 3.5 X 10-5M DNA-P) at 40 degrees C. Interaction was monitored by UV spectrophotometry; Amax/Amin ratios, difference and additive spectra, and the quantity of colchicine bound to DNA were presented as functions of time, ionic strength and DNA concentration. Using NMR techniques, delta deltav 1/2/deltac values, chemical shifts and half-line widths of colchicine protons were analyzed as a function of the DNA/colchicine ratio, thus proving the interaction between alkaloid and DNA. An intercalation of the tropolone moiety of colchicine between the nitrogenous bases of DNA is suggested.