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Pharmacology and therapeutics

Phenytoin induces in vitro melanocyte proliferation
Daniela Porojan1, MD, Ioana Ba ˆ ldea2, MD, PhD, Marcela Achim3, PhD and Rodica Cosgarea1, MD, PhD

Departments of 1Dermatology, 2 Physiology, and 3Pharmaceutical Technology and Biopharmaceutics, ‘‘Iuliu Hatieganu’’ University of Medicine and Pharmacy, Cluj-Napoca, Romania Correspondence Dr Rodica Cosgarea, MD, PhD Department of Dermatology, ‘‘Iuliu Hatieganu’’ University of Medicine and Pharmacy 3–5, Clinicilor Street Cluj-Napoca 400006 Romania E-mail: cosgarear@yahoo.com

Abstract
Background Phenytoin (5,5 diphenylhydantoin), a widely-used anticonvulsant, has been employed both topically and systemically in the treatment of some dermatological disorders. Immunological and non-immunological defects suggests that phenytoin could be therapeutically effective for vitiligo repigmentation. Aim of study The first objective was to evaluate the effect of phenytoin on cultures of normal human melanocytes and to assess cytotoxicity of the substance using colorimetric methods. The second objective was to select the topical product that releases phenytoin best and fastest, for further clinical studies in vitiligo patients. Materials and methods In the first part of the study, we exposed normal human epidermal melanocyte cultures at 5,5 diphenylhydantoin at different concentrations. Melanocyte proliferation was evaluated using colorimetric assays (MTS assay), by measuring the absorbance at 490 nm with an ELISA plate reader. In the second part of the study, we formulated and obtained three types of phenytoin topical preparations: gel, hydrophilic cream, and ointment, for evaluation of the in vitro phenytoin release. Results The statistical analysis revealed that at low concentrations (0.1, 0.5 lg/ml), phenytoin stimulates growth and cellular proliferation in melanocyte cultures. The in vitro study of drug release indicated that gel formulation gives the highest phenytoin release. Conclusions Our result indicates that topical applications of low concentrations of phenytoin may have beneficial effects in vitiligo, which justifies its further testing on clinical trials. Our gel formulation occurred to be the most suitable for the clinical studies.

Introduction Phenytoin (5,5-diphenylhydantoin) is a highly effective and widely prescribed anticonvulsant, which has been employed both topically and systemically in the treatment of some dermatological disorders, like epidermolysis bullosa, ulcers, inflammatory conditions;1 it has been used to treat linear scleroderma in children; it increases insulin sensitivity and might ameliorate acanthosis nigricans; orally and topically, it has been used to treat lichen planus.2 Vitiligo is an idiopathic, acquired, depigmenting disorder that affects approximately 0.1–2% of the general population worldwide3 and is characterized by circumscribed, depigmented macules that enlarge centrifugally as time goes on. The etiopathogenesis of vitiligo is still unclear, and the most accepted hypothesis is that the autoimmune destruction of melanocytes is triggered by genetic and non-genetic factors.4 Although it is asymptomatic, vitiligo can determine lesions with major consequences on the mental health of the patient. Several therapeutic alternatives were tried in vitiligo, with more
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or less beneficial effects.5 A meta-analysis of published studies reveals that the most efficient non-surgical therapeutic options are represented by phototherapy and topical corticotherapy.6 Because none of the vitiligo therapies has been followed by complete cure, finding new compounds with stimulatory effects on repigmentation and/or the suppressive effect of the local immune reaction represents an important requirement at present. Previous reports7 suggest that phenytoin may be an option in the treatment of vitiligo, considering its immunomodulatory activity and its secondary effect of facial pigmentation observed during the therapy of neurological conditions. In vitro experiments testing the effect of phenytoin on melanocytes have not been carried out so far. In addition, a recent study performed on a small vitiligo group from Iran tested only the efficiency of a high concentration of topical phenytoin (20%) in combination with topical-PUVA,8 and experiments using phenytoin monotherapy have not been performed so far. In the present study, the authors aim to show that low concentrations of phenytoin induce in vitro melanocyte proliferation as
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Japan) and documented photographically. 18. in a humidified environment.5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay.10. the cells were washed and exposed to 5.2 g of product was applied on the membrane surface. Ludwigshafen. Carbopol 940 polymer (B.1. and water. Romania). IL. Results were considered significant for P £ 0. After incubation. 12. WI. Untreated controls were exposed to Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum. cetyl alcohol (BASF. lanolin (BASF). All the experiments were conducted in subdued light. further clinical studies are intended to be carried out with this formulation. At specified time intervals.9 Morphology The morphological aspect of melanocytes was observed by microscopic examination (Nikon Eclipse T 100. using a 3-(4. humidified environment. and an electron acceptor agent. triethanolamine. 20. 5. Results The results of the in vitro study conducted in triplicate. For every specimen. and water. USA).0-Statistical Software Package (SPSS. either in proliferation or in chemosensitivity tests. compared with the control group. Austria). phenazine methosulfate (PMS. were recorded in ª 2012 The International Society of Dermatology International Journal of Dermatology 2012. 8. the absorbance at 490 nm was recorded using an ELISA plate ¨ dig.1380 Pharmacology and therapeutics Assessment of the effect of phenytoin on human melanocytes Porojan et al. and 1000 lg/ml). growth. We used progressively increasing Formulation of topical preparations of phenytoin (a) and in vitro evaluation of phenytoin release (b) We carried out a study of in vitro evaluation of phenytoin release. respectively. Heidelberg. phenytoin powder in constant concentrations was added. 1379–1384 .1–1000 lg/ml). Materials and methods Study of phenytoin effects on normal human melanocyte cultures The aim of the study was the assessment of cell survival. USA). The hydrophilic cream (oil-in-water emulsion) was prepared from cetyl alcohol. Taufkirchen. using the diffusion through semipermeable membrane method. Chicago. melanocytes were seeded at 104 per well in ELISA 96-well microtitration flat bottom plaques (TPP. 30.5-diphenylhydantoin. propyl p-hydroxybenzoate. however. OH. Bucharest. 10.5-diphenylhydantoin powder (SigmaAldrich. After incubation at 37 °C. sodium lauryl sufhate (Merck). methyl p-hydroxybenzoate. The quantity of the formazan produced is directly proportional to the number of living cells in the culture. The ointment has a white petrolatum-lanolin base. 50. Cellular proliferation We studied melanocyte proliferation with colorimetric methods that assess the number of viable cells. in triplicate. 2.F. Germany). The quantity of diffused phenytoin. was calculated using a calibration curve (5–25 lg/ml). Tokyo. Germany). The bioreduction of MTS generates a formazan product that strongly absorbs light at 490 nm. we used a blank. for different phenytoin concentrations. NJ.11 For phenytoin measurement. Promega. Whitehouse Station. Normal human melanocyte cultures We used a line of normal human epidermal melanocytes obtained from newborn foreskin (PromoCell. compound of a donor and a receptor compartment. 5% CO2. we sampled specimens for phenytoin measurements from the receptor compartment (represented by methanol : water 55 : 45 [v/v]). white petrolatum (BASF). and proliferation when exposed to various concentrations of 5. we used the Franz-type diffusion cells. Germany) of different concentrations (0. Switzerland) and incubated for 24 hours in 200 ll/well MGM (Cell Systems). 1. Cells were exposed to a MTS/PMS mixture (2 ml/100 ll) for 1–4 hours. 40. 5% CO2. The materials used are: phenytoin (Merck. methyl p-hydroxybenzoate. Zurich. is based on a tetrazolium compound. Goodrich. The experiment was fulfilled at room temperature. glycerin. CellSystems. A uniform layer of 0. sodium lauryl sulfate. This signal can be measured directly in a microculture plate with standard microplate readers. To this composition. USA). We studied melanocyte proliferation with the CellTiter 96â AQueous Non-Radioactive Cell Proliferation Assay (Promega). (b) For the in vitro evaluation of phenytoin release. Cells were cultured in melanocyte growth medium (MGM. Cells were suspended in tissue culture test plates with 96 wells and incubated for 24 hours and maintained at 37 °C in a humidified atmosphere containing 5% CO2. and propyl p-hydroxybenzoate (Merck) (a) We obtained a Carbopol 940 polymer (polyacrylic acid) gel compound of Carbopol 940. at 37 °C. 100. with a cellulose acetate membrane mounted between them. Avon Lake. glycerin. The culture medium was changed every three days. 16.05. In the proliferation assays. Hamburg. concentrations of phenytoin (0. Gro We analyzed the data with the help of non-parametric methods and the SPSS package version 13.5. triethanolamine (Merck). propyl p-hydroxybenzoate. MTS. 0. 51. 14. Germany). The method. glycerin (Chimopar. the phenytoin percentage released from the entire amount of phenytoin contained in the specimen. obtained in the same conditions as the sample but without phenytoin. methyl p-hydroxybenzoate. reader (Tecan. USA). we used a spectrophotometric method of determining the absorbance at 220 nm. We found out that the gel formulation was the most suitable vehicle for phenytoin. Fitchburg.

00 1000.00 30.341 0. this is a work hypothesis made plausible and consistent by the relationship between the two variables.00 1. Therefore.00 30.269 0.00 10. The progressive increase of phenytoin release started during the first hour.00 50. P = 0.35 0.1 and 0.00 0.00 50.356 0.282 0.346 0.338 0.00 5. the variable correlation that measures viability.345 0.5 lg/ml).00 1000. we considered that we are in a situation comparable to a test–retest design measurement.289 0.00 12.00 0. however. By increasing phenytoin concentration. The linear regression line determines the trend modulation.294 0. Table 2). cell viability decreases with every unit of concentration increase.00 18.365 0.327 0. has a value diminished with the control cases (Fig.352 0.00 30.10 0. but the trend is maintained. But despite the fact that the relationship between cell viability and the concentration of phenytoin is significantly negative.307 0.00 100.00 40.353 0.283 0. the simultaneous covariation of the two variables (phenytoin concentration and viability.00 8. ª 2012 The International Society of Dermatology International Journal of Dermatology 2012. has an absolute value closer to 1.00 5.296 0. 4). 3).323 0. Statistical analysis of the recorded data of in vitro experimental testing of phenytoin Table 1 Viability of melanocytes recorded at 490 nm by ELISA plate reader.385 0. without considering it is due to errors. at low values (0.00 40.00 12.43 0.00 1000.00 16. every increase of one unit of phenytoin concentration shows a decrease in viability of 0.50 1.00 14.00 14.00 14.285 0.00 50.385 0.292 0.00 40.357 0.32 0. 51. Assessment of the effect of phenytoin on human melanocytes Pharmacology and therapeutics 1381 Table 1 and the viability recorded by the ELISA plate reader was represented in Figure 1 The statistical analysis revealed that at low concentrations (0.00 16. the linear relationship hides the fact that by increasing concentration at low values of phenytoin (0.385 0.00 0.341 0. viability increases too.331 0.00 8.00 8.00 20.329 0. respectively).352 0. Hence.365 0.307 For statistical interpretation.299 0.319 0. we used the linear regression method (SPSS). 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Viability 0.00 16.313 0.281 0.39 0.00 20.382 0.373 0.Porojan et al. phenytoin stimulates growth and cellular proliferation in melanocyte cultures. In vitro release of phenytoin from semisolid formulations The in vitro evaluation of phenytoin release study revealed percentages of released phenytoin as a function of time (see the representation in Fig. Correlation r (x.00 10.00 20.50 0. when reaching higher concentrations.10 0.317 0. 1379–1384 .395 0.10 0.00 10.00 Measurement no.00 18. melanocyte viability increases too.00 1. the cell viability value of all cases corresponding to melanocytes that were not exposed to phenytoin was subtracted.365 0.001. according to phenytoin concentrations Concentration (lg/ml) 0.34 0.50 0.492.00 100.4 0.338 0.0000. The research design also involved the use of melanocytes as control cases.31 0.00 12.323 0. but the sustainability of this increasing viability is consistent and provides new perspectives.29 0. The inverse proportion of the two variables determines the negative sign. which gives statistical significance (Figures 1 and 2).00 5.5 lg/ml).1 and 0.5 lg/ml).311 0.369 0. This relationship cannot be considered statistically significant because there were only three measurements for every concentration. compared with controls (melanocytes in MGM. used in conjunction with phenytoin.1 and 0.346 0. therefore the value can be generalized to the entire population.00 2. The linear relationship is clear.302 0.00 2.00 18. y) = )0.392 0.00 2.362 0.00 100.

it significantly decreases suppressor T-cells and increases the helper/suppressor ratio. Discussion In the present study. this preparation will be selected for further clinical studies. 51. after eight hours.54% for the hydrophilic cream.1 lg/ml 0.368 0.15 Phenytoin inhibits the production of superoxide anion by immune cells16 and. which may also be reached by the topical application. 14.12 This agent possesses some effects that suggest that it may be beneficial in vitiligo. at high concentrations. and 1. so. 1379–1384 .391 0.25% for the gel. Additionally.378 0. and inhibits the activity of monoamine oxidase.25% for the ointment.1382 Pharmacology and therapeutics Assessment of the effect of phenytoin on human melanocytes Porojan et al. We found out that the gel formulation could be a suitable vehicle for phenytoin.0 lg/ml Viability 0. inhibits the release of norepinephrine.14 and cell-mediated immunity. we identified that low concentrations of phenytoin stimulate melanocyte proliferation. and antibacterial activity. including stimulation of fibroblast proliferation. Figure 1 Melanocyte proliferation after phenytoin exposure Table 2 The average melanocyte viability corresponding to phenytoin concentrations with the highest stimulating activity Concentration Control 0. it has also been proposed that the agent may interact with membrane lipids (and with ª 2012 The International Society of Dermatology Figure 2 Average of cell viability measurements after phenyt- oin exposure International Journal of Dermatology 2012. The analysis of this representation reveals that the gel releases the substance faster than the hydrophilic cream and ointment.5 lg/ml 1. Phenytoin is documented to significantly suppress the mitogen-induced activation of lymphocytes. giving a significantly higher drug release than the other vehicles and. glucocorticoid antagonism. facilitation of collagen deposition.13 cytotoxic T-lymphocyte activity.303 the values found being 47. Phenytoin may promote wound healing through multiple mechanisms.

including site of application. a hypothesis sustained by a doubleblind. acne.Porojan et al. because skin thickness and blood flow in the skin vary with age. Transdermal drug absorption can significantly alter drug kinetics and depends on a variety of factors.17 There are a few common cutaneous side effects occurring with the oral administration of phenytoin. In some situations. hirsutism. size of the molecule. 1379–1384 Phenytoin released (%) . 51. the level of copper-zinc superoxide dismutase and malondialdehyde being significantly increased. phenytoin may be effective against vitiligo. which could promote the stabilization of the membranes. At higher concentrations of phenytoin. and its ability to stimulate melanocytes at low concentrations (0. placebocontrolled study within the use of twice-daily topical phenytoin 20% that could not enhance the therapeutic response of PUVA. the most frequent being gingival hyperplasia. this may be an advantage. Phenytoin use can alter vitamin and mineral levels. but the glutathione level is significantly decreased in patients using phenytoin monotherapy compared with those of the controls.2 In addition.18 ª 2012 The International Society of Dermatology Studies of the pharmacokinetics of phenytoin suggest that the immunomodulatory effect of this agent may be employed topically in relatively high concentrations without producing the unwanted systemic effects. bilateral-comparison. International Journal of Dermatology 2012. if phenytoin will show efficiency in vitiligo. viability decreases.1 and 0. Given its inhibitory effect on cell-mediated immunity. especially in children. randomized. toxic epidermal necrolysis.5 lg/ml). norepinephrine release and monoamine oxidase activity.8 The administration of drugs by transdermal or transmucosal routes offers the advantage of being relatively painless. vasculitis. cutaneous hyperpigmentation. its further utilization would also allow the reduction of topical steroids concentration and thus their frequently-occurring dermatological side effects. drug-induced lupus. thickness and integrity of the stratum corneum epidermidis. and dermatomyositis. Assessment of the effect of phenytoin on human melanocytes Pharmacology and therapeutics 1383 Figure 3 Cell viability (control subtracted) according to phenytoin concentrations 50 40 30 20 10 0 0 2 4 6 Gel Ointment Cream 8 10 Time (h) Figure 4 In vitro release of topical preparations melanosome membrane lipids as well). The potential for toxic effects of the drug and difficulty in limiting drug uptake are major considerations for nearly all transdermal delivery systems. while in others systemic toxicity may result.

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