Drug accumulation in melanin: an affinity chromatographic study

https://doi.org/10.1016/S0378-4347(98)00242-4Get rights and content

Abstract

The affinity of several drugs to melanin has been indirectly assessed using an affinity chromatographic approach based on immobilized melanin. Plots of the retention of the drugs on the affinity column versus the number of molecules applied were fitted best by nonlinear, exponential curves characteristic for each drug. These curves reflect the complexity of the binding behaviour, consisting of a variety of hydrogen bonding, hydrophobic or ionic interactions as well as cooperative or anti-cooperative interactions between the drug molecules and melanin. The nonlinear fitting procedure was based on a descriptive function and allowed to discriminate the binding behaviour according to parameter estimates which specified the investigated drugs.

Introduction

Melanins are biopolymers mainly composed of indole-5,6-quinone units with several dopachrome and 5,6-dihydroxyindole carboxylic acid moieties present in the molecule. Melanin pigments are commonly classified into sulfur-containing pheomelanins and the non-sulfur-containing eumelanins [1]. One biological precursor of both forms is l-DOPA which yields eumelanin after oxidation, subsequent cyclization and polymerization. In the formation of pheomelanin, the addition of cysteine results in cysteinyldopa which then undergoes polymerization. Synthetic melanins prepared enzymatically or chemically from l-DOPA contain more carboxyl functions than natural melanins.

The binding of substances to melanin is of broad biological and pharmacological interest. Melanins are present in external and internal tissues (skin, hair, ear, eye, and brain). Thus, their capacity to bind and release (exogeneous and endogeneous) substances in a dynamic fashion may result in various, possibly pathogenetic effects onto the organism involved 2, 3, 4. These conditions seem important in the pathogenesis of disease states associated with long-term therapy with a number of drugs. Toxic effects have been best recognized in the case of chlorpromazine and chloroquine, both inducing chorioretinopathy by binding to and subsequent release from retina melanin [5].

The occurrence of tardive dyskinesia, a serious movement disorder associated with the chronic administration of antipsychotic drugs like haloperidol or tricyclic neuroleptics, is closely correlated to the neuromelanin affinity of these drugs [6]. A successful and promising causual therapy of tardive dyskinesia may consist in the displacement of the melanin-bound neuroleptic by other substances with high melanin affinity, but without any affinity to dopamine receptors [7].

More recently, research has focused on the role of melanin binding in some forms of cancer and cancer therapy. Various carcinogenic substances have been shown to accumulate selectively in pigmented cells of laboratory animals [8]. The use of melanin–affine medicines, accumulating in pigmented tissues, might be valuable in the treatment of diseases with hyperpigmentation. For instance, a melanin–affine anticancer drug for the selective treatment of malignant melanoma seems conceivable.

Pathophysiologically, melanin or the binding of substances to melanin is suspected to play an important role in Parkinson's disease (PD). This suggestion results from the observation that heavily pigmented dopaminergic nigrostriatal neurons are preferentially lost in PD. A selective destruction of pigmented neurons in the substantia nigra is observed after the application of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Its active metabolite methylphenylpyridine (MPP+) accumulates intraneuronally by the catecholamine uptake system and binds with high affinity to neuromelanin [9]. In addition to this condition, the binding of Fe(II) ions to melanin might be involved in the pathogenesis of PD [10]. Fe(II) ions bound to the redox polymer melanin might cause cell damage by reactive oxygen species like the hydroxyl radical synthesized in the Fenton reaction.

The molecular nature of drug binding to melanin is rather complex. Several parameters, like ionic and aromatic interactions, van der Waals attraction or the formation of charge-transfer complexes determine the affinity of substances to melanin. Both natural and synthetic melanins have been used in binding studies using radiolabelled ligands, and no significant differences have been observed [11]. The use of native instead of radioactively labelled ligands seems preferential since a metabolization of the drug under investigation can not be excluded by measuring radioactivity only. A spectroscopic investigation of the binding to melanin of non-radioactively labelled ligands is difficult since melanins are completely insoluble in water and organic solvents and their molecular weights are not known. In addition, the strong absorption of melanins in the whole spectral range prohibits a spectroscopic detection of free ligand concentrations.

In this study, we investigated the binding of several drugs to melanin using an affinity chromatography method. We employed a stationary phase of immobilized synthetic melanin. The advantages of this procedure are the use of native, non-isotope labelled drugs and the easy spectroscopic determination of the ligands due to the immobilization of melanin.

Section snippets

Experimental

Aminopropyl silica (APS, Polygosil 60-5-NH2) was obtained from Macherey–Nagel, Düren, Germany. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (EDC methiodide), 5,6-dihydroxy phenylalanine (l-DOPA), sodium maleate, chloroquine, haloperidol, sulpiride, and desipramine were purchased from Sigma, Deisenhofen, Germany. Flunitrazepam was a gift of Merckle, Blaubeuren, Germany; desmethylflunitrazepam was a gift of Hoffmann–La Roche, Basel, Switzerland, clonazepam was a gift of

Binding to APS

In order to characterize the retention of the drugs on the HPLC column as a measure of the true affinity to melanin, the binding behaviour of selected drugs with different affinities to melanin was investigated on non-modified APS. Each drug eluted with the void volume of the column and revealed no affinity to this melanin-free stationary phase (Table 1). Thus, the retention on the affinity column may represent the interactions of the drug with immobilized melanin.

Binding to l-DOPA melanin

Chromatograms of various

Discussion

In this study we investigated the binding of drugs to synthetic l-DOPA melanin. We have developed an affinity chromatographic method based on a stationary phase of melanin covalently coupled to aminopropyl silica via amide bonds between the carboxyl functions of melanin and the amino functions of APS. Drug doses were applied to the column to simulate the actual binding behaviour to melanin stores in vivo after administration of a drug bolus to a patient.

Studies on the melanin affinity of

Acknowledgements

We thank Dr. W. Fischer, Rhone–Poulenc Rorer, Köln, Germany for providing zotepin and trimipramine, Dr. C. Rüger, Arzneimittelwerke Dresden, Dresden, Germany for providing clonazepam, Dr. E. Gutknecht, Hoffmann–La Roche, Basel, Switzerland for providing desmethylflunitrazepam, and Dr. F. Schmid, Merckle, Blaubeuren, Germany for providing flunitrazepam. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 505).

References (25)

  • R.J. d'Amato et al.

    Life Sci.

    (1987)
  • H. Ibrahim et al.

    Anal. Biochem.

    (1995)
  • A. Kozik et al.

    Biophys. Chem.

    (1990)
  • G. Schwarz

    Biophys. Chem.

    (1976)
  • G. Schwarz et al.

    Biophys. Chem.

    (1979)
  • P.K. Gessner et al.

    J. Pharm. Sci.

    (1987)
  • H.J. Broxterman et al.

    Cancer Lett.

    (1985)
  • G. Prota

    Med. Res. Rev.

    (1988)
  • B.S. Larsson

    Pigment Cell Res.

    (1993)
  • L. Lyttkens et al.

    Adv. Otorhinolaryngol.

    (1979)
  • M.M. Salazar-Bookaman et al.

    J. Ocul. Pharmacol.

    (1994)
  • N.G. Lindquist et al.

    Acta Pharmacol. Toxicol. (Copenh.)

    (1972)
  • Cited by (40)

    • Anatomy and physiology of human hair

      2023, Toxicologie Analytique et Clinique
    • Global priority multidrug-resistant pathogens do not resist photodynamic therapy

      2020, Journal of Photochemistry and Photobiology B: Biology
    • Chlortetracycline and melanin biopolymer – The risk of accumulation and implications for phototoxicity: An in vitro study on normal human melanocytes

      2019, Chemico-Biological Interactions
      Citation Excerpt :

      Drug ability to induce phototoxic reactions depends on its physical, chemical, pharmacodynamic and pharmacokinetic properties, which involve low molecular mass (200–500 Da), planar, tri – or policyclic structure, the presence of heteroatoms, halogen substituent, high lipophilicity, and affinity to melanin [35]. The problem of drug-melanin complex formation is connected mainly to the possibility of accumulation of drugs in pigmented tissues, which may result in a higher risk of side effects [36–39]. In the currently available publications there is scarce information on reactions between tetracycline antibiotics and melanin polymers.

    • Implications of melanin binding in ocular drug delivery

      2018, Advanced Drug Delivery Reviews
      Citation Excerpt :

      Chloroquine is a good example of a widely studied drug that binds well to melanin both in vitro and in vivo. Chloroquine bound effectively to melanin in vitro when isolated porcine [133], bovine [144,146], Sepia [146,208] and synthetic [113,209] melanin were used (for more references see Appendix A). Moreover, we recently showed the extensive uptake (99% of 0.29–4.1 μM) of chloroquine to pigmented porcine RPE cells [133].

    View all citing articles on Scopus
    View full text