ARTIGO 3 – AVALIAÇÃO DO POTENCIAL HEPATOTÓXICO, FOTOTÓXICO E FOTOSSENSIBILIZANTE DO CLORIDRATO DE DRONEDARONA E SEUS

COMPLEXOS DE INCLUSÃO COM CICLODEXTRINAS

Publicação científica: Marcolino, A.I.P; Nogueira-Librelotto, D.R.; Mitjans, M.; Vinardell, M.P.; Rolim; C.M.B.. Evaluation of the hepatotoxic, phototoxic and photosensitizing potential of dronedarone hydrochloride and its inclusion complexes with cyclodextrins. Manuscrito em preparação.

INTRODUÇÃO

Nesse estudo, avaliou-se o potencial hepatotóxico, fototóxico e fotossensibilizante do cloridrato de dronedarona e seus complexos de inclusão com β-ciclodextrina e 2- hidroxipropil-β-ciclodextrina utilizando ensaios de citotoxicidade in vitro. Dentre esse ensaios, foram realizados o teste de fototoxicidade in vitro 3T3 NRU, o fotoensaio utilizando a linhagem celular de leucemia monocítica aguda humana (THP-1) e liberação de interleucina-8 e o ensaio de citotoxicidade em células tumorais de hepatoma humano (HepG2). O estudo foi desenvolvido no Departamento de Fisiologia da Universidade de Barcelona, Barcelona, Espanha durante a realização do Doutorado Sanduich no Exterior (SWE), pelo Programa Ciências sem Fronteiras (CNPq), sob a orientação da Prof. Dra. María Pilar Vinardell Martínez-Hidalgo e da Prof. Dra. Montserrat Mitjans.

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Evaluation of the hepatotoxic, phototoxic and photosensitizing potential of dronedarone hydrochloride and its inclusion complexes with cyclodextrins

Ana Isa Pedroso Marcolinoa, Daniele Rubert Nogueira-Librelottoa,b, Montserrat

Mitjansc, María Pilar Vinardellc

and Clarice Madalena Bueno Rolima,b*

a

Postgraduate Program in Pharmaceutical Sciences, Federal University of Santa Maria, Av. Roraima 1000, 97105-900, Santa Maria – RS, Brazil

b

Department of Industrial Pharmacy, Federal University of Santa Maria, Av. Roraima 1000, 97105-900, Santa Maria – RS, Brazil

c

Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Science, University of Barcelona, Joan XXIII 27-31, 08028, Barcelona – Spain

* Corresponding author. Department of Industrial Pharmacy, Federal University of Santa Maria, Santa Maria – RS 97015-900, Brazil. Tel.: (+55) 55 3220 8645. Fax: (+55) 55 3220 8248.

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Abstract

In this study, the phototoxicity, hepatotoxicity and photosensitizing potential of free dronedarone and its inclusion complexes with β-CD and HP-β-CD were investigated by using in vitro cell-based approaches. The results of the 3T3 NRU phototoxicity assay showed that free dronedarone and the inclusion complexes did not present phototoxic potential. However, an exception was the inclusion complex with HP-β-CD prepared through colyophilization, which presented a minor phototoxic effect. The photosensitization was assessed by using THP-1 cells and IL-8 as a biomarker, and the experimental data evidenced that both the free drug and inclusion complexes showed potential to cause skin sensitization, as they were able to induce IL-8 release after irradiation. Nevertheless, the inclusion complex with β-CD obtained by kneading following spray-drying induced a significant lower release of IL-8 and also presented the lowest stimulation index in comparison with free dronedarone, suggesting a reduction in the photosensitizing potential. The free drug and inclusion complexes were also tested for hepatotoxicity by using HepG2 cells. Even though lower IC50 values were found for the inclusion complexes prepared by kneading following spray-drying, there was no significant difference, indicating that the complexation did not alter the hepatotoxic potential of dronedarone. Overall, the data suggest that dronedarone is not phototoxic, however, it presents photosensitizing potential. The inclusion complex prepared by kneading following spray-dryer is suggested as a formulation which might reduce the photoallergic potential of dronedarone.

Keywords: Dronedarone. Cyclodextrins. Inclusion complex. Cytotoxicity. In vitro 3T3

1. Introduction

Dronedarone (DRO) is a new antiarrhythmic agent indicated to reduce the hospitalization rate in patients with atrial fibrillation. This benzofuran derivative was obtained from modifications of amiodarone molecule with the intention to reduce its adverse effects, by reducing its lipophilicity and then the accumulation in tissues [1,2]. DRO is metabolized by cytochrome P450 3A4, and is also a moderate inhibitor [3]. DRO is a biopharmaceutics classification system II compound with pH-dependent aqueous solubility, practically insoluble at pH 7 [4,5]. Regardless its adverse effects, hepatocellular liver injury, even requiring liver transplantation, has been reported in the postmarket setting of DRO tablets [6]. A case of fatal lung toxicity was also reported after DRO use [7]. Photosensitive reactions occurred in a patient taking DRO for one month, showing the drug potential to cause a photodistributed drug eruption, even though this reaction appeared to be uncommon, affecting 1% of the patients [8].

Cyclodextrins (CDs) are pharmaceutical excipients of the family of cyclic oligosaccharides. The natural α-, β- and γ-CDs are formed by 6, 7 and 8 (α-1,4-)- linked D-glucopyranose units, which have limited aqueous solubility. The CD derivative 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) has been synthesized to present higher water-solubility. The CD structure presents a lipophilic central cavity and a hydrophilic outer surface. CDs form inclusion complexes like guest-host, where the guests are hydrophobic drug moieties that are entrapped into the central cavity. As a result of complexation, changes occur in the physicochemical properties of the guest molecule, such as enhanced solubility and bioavailability of poor-water soluble drugs [9–11]. Hydrophilic CDs like HP-β-CD are capable to enhance permeation of lipophilic drugs or to reduce drug permeation through lipophilic membranes by

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reducing the partition from the exterior to the membrane, and could also increase drug chemical stability at the aqueous membrane exterior [12]. In relation to safety and toxicology of CDs, they are practically non-toxic after oral administration, as only negligible amounts are able to permeate lipophilic membranes such as gastrointestinal mucosa 1 . β-CD cannot be used in parenteral administration as it can result in renal toxicity, in contrast, HP-β-CD are suitable and can be found in market parenteral formulations [13,14]. The formation of inclusion complexes could also be described as a micro-encapsulation process, as the guest molecule is surrounded by the cyclodextrin molecules, altering the chemical, physical and biological properties [15], such as stabilization against effects of light degradation [16]; decreasing the biomass and cellular activity of Staphylococcus and toxicity against leucocytes [17]; and enhancing anti-proliferative activity in cancer cells while reducing cytotoxicity in normal lung fibroblast cells (MTC-5) [18]. Then, the purpose of overcome certain limitations has stimulated the investigations into cyclodextrin applications [19].

Safety is a primary concern when developing new pharmaceutical formulations. Thus, toxicological issues of the drug formulation must be investigated and approved according to available legislation procedures, before the intended use. Phototoxic side effects of pharmaceutical formulations are of increasing concern, urging the need of pre-clinical tests for side effects, particularly to detect phototoxic potential of chemicals [20].

Photoreactions to pharmaceutical products are side effects that can be triggered after exposure to environment light, mainly in response to UVA light (range of 315-400 nm), which penetrates deep into epidermis and dermis, possessing mutagenic and carcinogenic activity mediated by oxidative stress [21]. Drug induced

photoirritancy (phototoxicity) is defined as tissue response following topical or systemic administration of pharmaceutical substances. DRO presents a high absorption in UV range, with maximum absorption peaks at 217 and 289 nm (with a shoulder until 350 nm).

In contrast, photoallergy is an immunologically-mediated reaction to a chemical, initiated by the formation of a photoproduct following a photochemical reaction [20,22,23]. The mechanism of photoallergy is consider to be a form of delayed type of hypersensitivity, being immunologically mediated [20]. The first stage of the photosensitizing process is the absorption of photons of the appropriate wavelength (ultraviolet or visible radiation) by the exogenous agent (photosensitizing drug), that reach an excited state. The excited energy is transferred to oxygen molecules, generating reactive oxygen species (ROS), which can induce local oxidative stress and damages to genomic DNA, lipids and proteins in cells [22]. The next step is the uptake of the photochemically converted exogenous agent (in combination with carrier proteins, forming a complete antigen) by the antigen- presenting cells, such as Langerhans cells present in the skin [20,24]. These cells present the antigen to the lymph node, thereby inducing sensitization, and during this phase, they differentiate and mature immunostimulatory cells by up-regulating the expression of several co-stimulatory molecules and secreting various cytokines, such as IL-8 [25].

In order to assess photosafety in vitro with a correlation with in vivo observations, photosafety assays are conducted and cytotoxicity assays as in vitro endpoints are explored, always taking into account the need of biological markers to discriminate allergy and irritation without animal testing [23,25]. The most used assay to evaluate the phototoxic potential of the drug is the 3T3 Neutral Red Uptake

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phototoxic test, the first alternative method accepted by OECD, replacing animal testing [26,27]. The assay is focused on the effect of UVA light exposure on cell viability, which is measured by the inhibition of the capacity of cell cultures to take up the NR dye after a specific time in comparison to non-treated cells [23]. In this validated study, amiodarone hydrochloride is described as phototoxic. In the case of photosensitization and photoallergic reactions, the use of THP-1 cells and the IL-8 release was proposed as a model to identify the potential of chemicals to induce skin sensitization [24].

Regarding investigations on liver toxicity, the mechanisms underlying DRO hepatotoxicity were studied by using isolated rat liver mitochondria, primary human hepatocytes and a well-characterized human hepatoma cell line HepG2. DRO was described to inhibit transport chain and β-oxidation and uncoupling oxidative phosphorylation of liver mitochondria, and the study associated this mechanism with the liver injury reported in patients [28].

In this study, we focused on the safety status of DRO hydrochloride and its inclusion complexes with β-CD and HP-β-CD and hypothesized that DRO toxicity would be lower because of its complexation with CDs. The phototoxic potential of DRO has not been described in the literature up this moment. In order to investigate the mechanisms underlying the photochemical reactivity of DRO and its inclusion complexes, two photosafety analytical studies were used: the 3T3 Neutral Red Uptake phototoxicity test and a photoassay using a human cell line cultured in vitro (THP-1 monocytes), considering the interleukin 8 (IL-8) expression as endpoint. In addition, we aimed to investigate the hepatotoxic effects associated with DRO using HepG2 cells, following by a comparison with those of the inclusion complexes with CDs.

2. Material and methods

2.1. Chemicals

DRO hydrochloride (purity> 98. %), β-CD and HP-β-CD were obtained from Zibo Qianhui Biotechnology Co., Ltd. (Zibo, Shandong, China). Chlorpromazine hydrochloride (CPZ), dimethyl sulfoxide (DMSO), 2,5-diphenyl-3,-(4,5-dimethyl-2- thiazolyl) tetrazolium bromide (MTT), Neutral Red (NR) dye, 2-mercaptoethanol and , ′, , ′-tetramethylbenzidine liquid substrate, supersensitive, for ELISA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Methanol was purchased from Panreac (Barcelona, Spain). Trypsin-EDTA solution (0.5 g/L trypsin and 0.2 g/L EDTA), phosphate buffered saline (PBS), fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM without phenol red, and RPMI-1640 medium, L-glutamine and antibiotic/ antimicotic (100 μg mL of streptomycin sulfate and 100 U/mL potassium penicillin) were purchased from Lonza (Verviers, Belgium). For all analyses, ultrapure water was purified with Millipore Milli-Q Plus Ultra-Pure Water Purifier (Germany).

2.2. Cell culture

The murine fibroblast cell line NIH-3T3 and the human hepatoma cell line HepG2 were maintained in DMEM (with 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin), supplemented with 10% (v/v) of heat inactivated FBS. The human monocytic leukemia cell line THP-1 were cultured at 37°C and 5% CO2 in

RPMI-1640 medium containing 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol, and supplemented with 10% (v/v) of heat

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inactivated FBS. Cells were kept in a cell incubator with 5% CO2 at 37°C and were

harvested by trypsinization when reached around 80% confluence. The cell number was determined using a Neubauer hemacytometer and the viability using the trypan blue exclusion method.

2.3. Test compounds

2.3.1. Preparation of inclusion complexes

The solid inclusion complexes of DRO (molecular weight 9 .2 g mol) with β- CD (molecular weight 1135.0 g/mol) and HP-β-CD (molecular weight 1540.0 g/mol) were prepared by three different methods with a 1:10 molar ratio (drug: cyclodextrin).

Lyophilization. Stoichiometric amount of DRO and β-CD or HP-β-CD (1:10,

M/M) were mixed in a mortar for 10 min. The mixture was dissolved in water at 50°C. Next, the pH of the suspension was adjusted to 4.5 with acetic acid and it was stirred at room temperature for 24 h. The resulting suspension was frozen at -20°C with lactose (10%, p/v) for 24 h and lyophilized for 48 h.

Colyophilization. Appropriate quantities of DRO and β-CD or HP-β-CD (1:10,

M/M) were dissolved in hydroalcoholic solution (1:1, v/v), kept in agitation for 24h. Ethanol was then removed in a rotary evaporator at 50 ± 5ºC. The pH value was then adjusted to 4.5 and lactose (10%, p/v) was added to the resulting suspension, which was frozen and lyophilized.

Kneading and spray-drying. The powders of DRO and β-CD or HP-β-CD were

mixed in a mortar for 20 min. Then, 0.5 mL of water was added and mixed again for 5 min to form a paste, which was solubilized in 25 mL of water at 50°C for 20 min. The pH of the suspension was adjusted to 4.5, following by stirring for 24 h at room

temperature. The suspension was dried in a spray dryer model LM MSD 1.0 (Labmaq, Ribeirão Preto, SP, Brazil) with the following operation conditions: inlet temperature: 120ºC, air pressure: 3 kgf/cm2, feed flow rate: 0.21L/h.

2.3.2. Preparation of sample solutions

The samples were freshly prepared and, accordingly to their solubility, the stock solution of the free DRO was dissolved in methanol, while the solutions of the inclusion complexes were prepared in ultrapure water. The stock solutions were prepared at the concentration of 1 mg/mL DRO. The stock solution of chlorpromazine was diluted in DMSO at the final concentration of 5 mg/mL.

2.4. Irradiation source

The plates were irradiated by using three UVA lamps Actinic BL TL/TL-D/T5 (Philips®, 43 V, 352 nm, 15 W) placed in a photostability chamber (58 × 34 × 28 cm). Irradiance was checked with a photoradiometer Delta OHM equipped with a UVA probe (HD2302- Italy), placed below the plate lid for accurate measurements. Irradiance was determined to be 1.8 mW/cm².

2.5. In vitro cytotoxicity studies

2.5.1. 3T3 Neutral Red Uptake phototoxicity test

The 3T3 Neutral Red Uptake phototoxicity test was conducted according to the OECD TG 432 guideline, with some modifications [26]. First, the fibroblast sensitivity to radiation was tested in different doses of UVA (1.0, 1.7, 1.9, 2.5 and 5.0

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J/cm²). The test was then performed with the UVA dose that provided > 80% cell viability after irradiation. The NIH 3T3 murine fibroblast cell line was seeded in the central 60 wells of 96-well cell culture plates (cell density of 1×105 cells/mL). After 24 h of incubation (5% CO2, 37°C), the cells were washed with 150 µL PBS and the

medium was replaced by 100 µL fresh DMEM (without phenol red) supplemented with 5% FBS containing the free drug and the inclusion complexes in the concentration range from 0.3 to 15.0 µg/mL. The plates were incubated (5% CO2,

37°C) in the dark for 60 min. Then, the selected plate was irradiated with an irradiation dose of 1.7 J/cm² within 15 min of light exposure. In parallel, another plate was prepared and kept in the dark, as a control (non-irradiated). At the end of the exposure period, cells were washed with 150 µL PBS, the medium was replaced and plates were incubated overnight (5% CO2, 37°C). Chlorpromazine was tested as UVA

positive control in the concentration range from 0.35 to 90 µg/mL.

2.5.2. Determination of the photosensitizing potential using THP-1 cells

The evaluation of the photosensitizing potential of DRO and the inclusion complexes with CDs were performed according to a protocol used to identify photoallergenic chemicals [24]. The THP-1 cells were seeded into 24-well plates at a density of 1×106 cells/mL. Each well was filled with 500 µL of RPMI medium supplemented with 10% FBS (v/v), where 5 µL of increasing drug concentration (0.625, 1.25 and 2.50 µg/mL of free DRO and equivalent of inclusion complexes) or vehicle (methanol, ultrapure water and DMSO) were added. CPZ, a known photoallergen, was tested at 0.1 µg/mL. Immediately after applying the chemical treatment, one plate was irradiated with UVA, in order to provide an irradiation dose of 1.9 J/cm². A non-irradiated control plate was prepared in parallel and kept in the

dark. After 24 h of incubation at 37 °C, plates were centrifuged at 1200 rpm for 5 min in order to assess IL-8 release in the free supernatants, which were stored at -20°C until analysis. Stimulation indexes (SI) were used to detect photoallergens and were calculated as the ratio of IL-8 release for treated cells against untreated cells for irradiated (I-SI) and non-irradiated cells (NI-SI). The ratio between these two indexes (I-SI/NI-SI) was the overall stimulation index.

2.5.3. Cytotoxicity in HepG2 cells

HepG2 cells (1×105 cells/ mL) were grown in the central 60 wells of 96-well cell culture plates in DMEM supplemented with 10 % FBS. After 24 h of incubation (5% CO2, 37°C), the media was removed and the samples of free drug and inclusion

complexes were applied, prepared in DMEM containing 5% FBS and in the concentration range from 0.3 to 15.0 µg/mL. Afterward, plates were incubated overnight at 37°C in 5% CO2.

2.6. Cell viability assays

Cell viability of the in vitro phototoxicity assay were measured by the Neutral Red Uptake test. Following treatment, cells were washed with 150 µL PBS and then 100 µL Neutral Red solution at 50 µg/mL were added at each well. After 3 h of incubation (5% CO2, 37°C), cells were washed with 150 µL PBS and 150 µL of NR

desorb solution (water: ethanol: acetic acid; 49:50:1, v/v/v) was added.

Cytotoxicity in THP-1 and HepG2 cells were performed by the MTT test, according to the method of Mosmann [29]. Cell viability was determined by the percentage of tetrazolium salt reduction by viable cells against untreated cells. In the

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photoassay using THP-1 cells, 500 µL of a MTT solution at 0.75 mg/mL were added to each well. The plate was incubated for 3 h (5% CO2, 37°C), centrifuged and then

500 µL of acidified isopropanol was added to lyse the cells. Next, an aliquot of 100 µL of each well was transferred to a 96-well plate. For each sample, the 75% viability was calculated.

For the assessment of cytotoxicity in HepG2 cells, following overnight incubation, 100 µL of MTT solution at 0.5 mg/mL were added to each well and after 3 h of incubation at 37°C in 5% CO2, the formazan product was dissolved with 100 µL

of DMSO.

In all in vitro cytotoxicity assays, after 10 min on a microtitre-plate shaker, absorbance was read at 550 nm using a Tecan Sunrise microplate reader equipped with Magellan (v. 6.6) software (Männedorf, Switzerland). Results were expressed as the percentage of viability compared with control wells (the mean optical density of untreated cells was set as 100 % viability).

2.7. IL-8 release measurements

Human interleukin-8 (IL-8) release from free supernatants was determined using an enzyme-linked immunosorbent assays (ELISA) kit (BD OptEIA™) from BD Biosciences (San Diego, CA, USA). Results are expressed in pg/mL.

Based on the release of IL-8 from cells treated with the respective concentrations of the products, stimulation indexes (SI) were determined, according to Martínez [24]. The stimulation indexes were calculated by the ratio of the treated cells against untreated cells (control cells), for the non-irradiated (NI-SI) and irradiated (I-SI) conditions, and the ratio of the stimulation indexes as determined as the overall stimulation index (I-SI/NI-SI).

2.8. Statistical analysis

Results were expressed as mean ± standard error of least three independent experiments. Statistical analysis were conducted using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons and by two-sample t-test using the Statistica software (v. 7.0; StatSoft. Inc., Tulsa, OK, USA).

3. Results and discussion

3.1. 3T3 Neutral Red Uptake phototoxicity test

By using the 3T3 NRU phototoxic test, the cytotoxicity of the cells treated with increasing concentrations of the compounds and irradiated with non-toxic dose of UVA light was compared to non-irradiated cells. Fig. 1 illustrated the dose response curves in absence and presence of UV light. The statistical analysis by ANOVA did not show significant difference between the reduction in cell viability of irradiated and non-irradiated cells (p > 0.05) for the free dronedarone, suggesting that it may not be phototoxic. This effect was also observed for the inclusion complexes, indicating that the complexation did not alter the phototoxic potential of dronedarone. An exception was the minor phototoxic effect observed for the inclusion complex with HP-β-CD prepared by colyophilization (Fig. 1c), evidenced by the significant difference (p < 0.05) between the cell viability of irradiated and non-irradiated cells for the lower concentration tested, performed with Dunnett’s multiple comparison test (non- irradiated as control). The colyophilization technique involves the use of ethanol in

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the sample preparation, and even though rotary evaporation removes it, residual solvent would remain in the sample and could contribute to the cytotoxic effect.

Firstly, the radiation sensitivity of the cells to the light source was tested following exposure to increasing doses of irradiation (1.0, 1.7, 1.9, 2.5 and 5.0 J/cm²),