ORIGINAL ARTICLES Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Silesia, Sosnowiec, Poland Interaction of chlorpromazine, fluphenazine and trifluoperazine with ocular and synthetic melanin in vitro E. Buszman, A. Beberok, R. Ro´z˙an´ska (y), A. Orzechowska Received June 19, 2007, accepted August 8, 2007 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 63: 372–376 (2008) doi: 10.1691/ph.2008.7663 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 poly- mer. 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 associa- tion 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 4 2 À1 association constants K1 10 and K2 10 M for chlorpromazine and fluphenazine complexes, 5 3 À1 and K1 10 and K2 10 M 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 mela- nin, especially the ocular melanin, in vitro is discussed in relation to the ocular toxicity of these drugs in vivo. 1. Introduction tions (Baldessarini 1996; Marques et al. 2004; Parfitt 2002; Delgado and Remers 1998). Aliphatic derivatives Chlorpromazine – an aliphatic phenothiazine derivative – exert stronger drug-induced ocular side effects than piper- and fluphenazine and trifluoperazine – piperazine phe- azine derivatives; however these effects can also appear nothiazine derivatives – are neuropsychiatric agents of the with prolonged use of piperazine derivatives (Buszman neuroleptic group (Baldessarini 1996; Delgado and Re- and Ro´z˙an´ska 2003a; Wolf et al. 1993). Therapy with phe- mers 1998). Chlorpromazine was the earliest phenothia- nothiazine derivatives can lead to corneal endothelial zine derivative introduced into therapy, having significant phototoxicity (Hull et al. 1982, 1983; Panzit et al. 2001; sedative and hypotensive properties. Because fluphenazine Hollander and Aldave 2004), and pigmentary changes of and trifluoperazine have both 2-trifluoromethyl and piper- conjunctiva, sclera and lens (Panzit et al. 2001; Rennie azine groups, they are potent antipsychotic agents with 1993; Walsh 2001; Hollander and Aldave 2004). More- low sedative and hypotensive effects (Delgado and Remers over, chlorpromazine, fluphenazine and trifluoperazine can 1998). Phenothiazine derivatives are widely used in the cause drug-induced degenerative retinopathy with histolo- treatment of psychotic disorders including schizophrenia, gical, electrophysiological and symptomatological features psychosis, maniacal states and behaviour disturbances similar to those of primary retinitis pigmentosa (Buszman (Baldessarini 1996; Marques et al. 2004; Parfitt 2002; and Ro´z˙an´ska 2003a; Fornaro et al. 2002; Parfitt 2002; Walsh 2001). Moreover, trifluoperazine, as one of the Toler 2005). Subjective symptoms of neuroleptic-induced most potent calmodulin inhibitors (intracellular mediator retinopathy are as follows: decrease in vision, disturbances for calcium ions), induces an analgesic effect (Golbidi of dark adaptation, night blindness and central scotoma, et al. 2002) and demonstrates fungicidal (Sharma et al. and they generally precede abnormalities of the retina in- 2001) and antimycobacterial (Gadre et al. 1998) activity. cluding degeneration and pigmentation. In serious cases Numerous side effects are due to phenothiazine adminis- this can even cause complete or very severe loss of vision tration, e.g. extrapyramidal side effects and resultant disor- (Fornaro et al. 2002; Rennie 1993; To 2000). The exact ders including acute dystonia, a parkinsonism-like syn- mechanism of phenothiazine retinal toxicity is still un- drome, akathisia and tardive dyskinesia (fluphenazine and known. It is suggested that blockade of retinal dopamine trifluoperazine exert stronger extrapyramidal effects than receptors (localized mainly in the photoreceptor layer and chlorpromazine), neuroleptic malignant syndrome, ocular retinal pigment epithelium – RPE cells), and drug binding toxicity, and alteration of endocrine and metabolic func- to melanin granules in RPE cells (causing alterations of 372 Pharmazie 63 (2008) 5 ORIGINAL ARTICLES retinal enzyme kinetics, loss of photoreceptors, RPE and 2. Investigations and results choriocapillaris with damage to the rods and cones Kinetics of the formation of chlorpromazine, fluphenazine through inhibition of oxidative phosphorylation), can lead and trifluoperazine-melanin complexes shown as the rela- to drug-induced retinopathy (Fornaro et al. 2002; Toler tionship between the amount of drug bound to the poly- 2004). mer and the incubation time are presented in Fig. 1 for Melanin pigments are multifunctional polymers. The phy- three initial drug concentrations (c ). It is demonstrated siological function of melanin is to buffer against photo- 0 that longer incubation time results in an increased amount chemical stress through absorption and dispersing UV ra- of drug bound to synthetic melanin. On the basis of the diation, sequestering metal ions and trapping free radicals results it may be concluded that the maximum time to and reactive oxygen species (Hu 2005; Nofsinger et al. achieve an equilibrium state is 24 h. Complex formation 2002; Tolleson 2005). Biopolymer granules are able to efficiency (the ratio of the amount of drug bound to bind many heterogeneous chemical compounds such as DOPA-melanin and the amount of drug added to form the metal ions, organic amines and cyclic compounds (i.e. complex, expressed as %) decreased with increased initial drugs). By inhibiting or significantly restricting drug ac- drug concentration. cess to cell receptors, they protect the organism against A relationship between the amount of chlorpromazine, flu- drug side effects. On the other hand, long-term exposure phenazine and trifluoperazine bound to melanin after 24 h of and slow release of drugs or their metabolites from bonds incubation and the initial drug concentration for ocular mela- may build up high and long-lasting levels of noxious che- nin and for synthetic-DOPA melanin, as binding isotherms, micals stored by melanin, which may cause degeneration is presented in Fig. 2A and Fig. 3A, respectively. It may be in the melanin-containing cells (especially in the eye, ear, seen from the binding curves that the amount of drug bound skin and brain) and secondary lesions in surrounding tis- to melanin increased with increased initial drug concentra- sues (Hu et al. 2002; Larsson 1993; Mars and Larsson tions. In the concentration range studied (from 5 Â 10À5 Mto 1999). 3 Â 10À3 M) the increase was 12-fold for chlorpromazine The purpose of the studies was to examine in vitro the complexes with both ocular (0.0338 to 0.4189 mmol/mg) binding capacity of the phenothiazine derivatives chlorpro- and synthetic (0.0478 to 0.5259 mmol/mg) melanin, and mazine, fluphenazine and trifluoperazine to both natural 6-fold for fluphenazine-melanin complexes (0.0274 to and synthetic DOPA-melanin. To achieve this, the kinetics 0.1814 mmol/mg for ocular and 0.0359 to 0.2138 mmol/mg of drug-melanin complex formation were examined, and for synthetic melanin). In the case of trifluoperazine the in- the number of independent binding sites and the associa- crease was 3- and 2-fold respectively for drug-eye melanin tion constants were also determined. Natural ocular mela- nin isolated from pig eyes was used with synthetic DOPA- melanin as a model polymer. A B CHLORP ROMAZINE 0.8 ] 2.0 CHLORPROMAZINE -1 ] mg -1 100 0.6 • 1.5 -4 3 co=1 •10 M mg • 80 dm 0.4 -3 1.0 60 co=5 •10-4 M [10 A % r[mol 0.2 0.5 r/c 40 co=1 •10-3 M 20 0 5 10 15 20 25 30 0 1 2 3 4 5 -4 -3 -7 -1 co[10 mol •dm ] r[10 mol • mg ] 0 10 20 30 40 50 FLUPHENAZINE time [h] ] 0.3 -1 1.0 ] -1 mg FLUPHENAZINE • 0.8 100 3 mg • 0.2 dm 0.6 80 -3 0.4 [10 r[mol 0.1 60 -4 A % co=1 •10 M r/c 0.2 40 co=5 •10-4 M 20 co=1 •10-3 M 0 5 10 15 20 25 30 0.0 0.5 1.0 1.5 2.0 -4 -3 -7 -1 co[10 mol •dm ] r[10 mol • mg ] 0 10 20 30 40 50 TRIFLUOPERAZINE time [h] ] ] 0.3 -1 3 -1 mg • mg • 100 TRIFLUOPERAZINE 3 0.2 2 -4 co=1 •10 M dm 80 -3 r[mol 0.1 [10 1 60 A % 40 r/c co=5 •10-4 M 20 co=1 •10-3 M 0 4 8 12 16 0.0 0.5 1.0 1.5 2.0 -4 -3 -7 -1 co[10 mol •dm ] r[10 mol • mg ] 0 10 20 30 40 50 time [h] Fig. 2: Binding isotherms (A) and Scatchard plots (B) for chlorpromazine- ocular melanin, fluphenazine-ocular melanin and trifluoperazine- Fig. 1: Effect of incubation time and initial drug concentration (c0)on ocular melanin complexes. r – amount of drug bound to melanin; amount of chlorpromazine, fluphenazine and trifluoperazine bound c0 – initial drug concentration; cA – concentration of unbound to DOPA-melanin (%). drug. Mean values Æ SD from three independent experiments are pre- Mean values Æ SD from three independent experiments are pre- sented.