Benznidazole self-emulsifying delivery system: A novel alternative dosage form for Chagas disease treatment

Journal Pre-proof

Benznidazole Self-Emulsifying Delivery System: A Novel Alternative Dosage Form For Chagas Disease Treatment Ana Lia MAZZETI , Liliam Teixeira OLIVEIRA , ´ Karolina R. GONC ¸ ALVES , Gessica C. SCHAUN , Vanessa Carla Furtado MOSQUEIRA , Maria Terezinha BAHIA PII: DOI: Reference:

S0928-0987(20)30023-3 https://doi.org/10.1016/j.ejps.2020.105234 PHASCI 105234

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

24 July 2019 9 January 2020 21 January 2020

Please cite this article as: Ana Lia MAZZETI , Liliam Teixeira OLIVEIRA , Karolina R. GONC ¸ ALVES , ´ Gessica C. SCHAUN , Vanessa Carla Furtado MOSQUEIRA , Maria Terezinha BAHIA , Benznidazole Self-Emulsifying Delivery System: A Novel Alternative Dosage Form For Chagas Disease Treatment, European Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.ejps.2020.105234

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

BENZNIDAZOLE SELF-EMULSIFYING DELIVERY SYSTEM: A NOVEL ALTERNATIVE DOSAGE FORM FOR CHAGAS DISEASE TREATMENT

Ana Lia MAZZETI1,2, Liliam Teixeira OLIVEIRA 1, Karolina R. GONÇALVES1; Géssica C. SCHAUN2; Vanessa Carla Furtado MOSQUEIRA2*, Maria Terezinha BAHIA1

1

Laboratório de Doenças Parasitárias, Escola de Medicina & Núcleo de Pesquisas em

Ciências Biológicas, Universidade Federal de Ouro Preto, Campus Universitário Morro do Cruzeiro, Ouro Preto, MG, 35400-000, Brazil 2

Laboratório de Desenvolvimento Galênico e Nanotecnologia, Escola de Farmácia,

Universidade Federal de Ouro Preto, Campus Universitário Morro do Cruzeiro, Ouro Preto, MG, 35400-000, Brazil

*Corresponding author: Professor Vanessa Carla Furtado Mosqueira Laboratório de Desenvolvimento Galênico e Nanotecnologia CiPharma, Escola de Farmácia, Universidade Federal de Ouro Preto, Morro do Cruzeiro Ouro Preto, MG, 35400-000, Brazil Phone: +55 31 3559-1032 E-mail: [email protected], [email protected]

1

Abstract Benznidazole (BZ) tablets are a unique form of treatment available for treating Chagas disease. Development of a liquid formulation containing BZ easy to administer orally for the treatment of paediatric patients, particularly for newborns is urgently required, with the same efficacy, safety and suitable biopharmaceutical properties as BZ tablets. Self-emulsifying drug delivery systems (SEDDS) may improve bioavailability of drugs such as BZ, which have poor water solubility and low permeability. In this context, the aim of this work was to develop a liquid BZ-SEDDS formulation as an alternative to tablets and to evaluate its cytotoxicity in different host cell lines and its efficacy in experimental Trypanosoma cruzi infection in mice. The optimized SEDDS formulation (25 mg/ml of BZ) induced no cytotoxicity in H9c2, HepG2 and Caco2 cells in vitro at 25 μM level. BZ-SEDDS and free-BZ showed similar in vitro trypanocidal activity in H9c2 cells infected by T. cruzi Y strain, with IC50 values of 2.10 ± 0.41 μM and 1.29 ± 0.01 μM for BZ and BZ-SEDDS, respectively. A follow up of efficacy in an acute model of infected mice resulted in the same percentage of cure (57%) for both free-BZ and BZ-SEDDS- groups according to established parameters. Furthermore, no additional in vivo toxicity was observed in animals treated with BZ-SEDDS. Taken together, in vitro and in vivo data of BZ-SEDDS showed that the incorporation of BZ into SEDDS does not alter its potency, efficacy and safety. Thus, BZ-SEDDS can be a more practical and personalized orally administered liquid dosage form compared to suspension of crushed BZ-tablets to treat newborn and young children by emulsifying SEDDS in different aqueous liquids with advantage of dosing flexibility. Keywords: Trypanosoma cruzi; benznidazole; self-emulsifying drug delivery system; Chagas disease; cytotoxicity; efficacy; release kinetics

2

1. Introduction Chagas disease affects 6 to 7 million people worldwide and is an important public health problem, especially in Latin America, causing approximately 7.000 deaths annually (WHO, 2019, Cucunubá et al., 2016). The disease contributes significantly to the global burden of cardiovascular disease, as the main cause of infectious cardiomyopathy in the world (Martins-Melo et al., 2012). BZ and nifurtimox (NFX) are the first line drugs for treating Chagas disease, although they present several toxic effects, variable and limited efficacy, mainly in the chronic phase of the disease. Currently the treatment is recommended for children, acute cases (congenital transmission included), laboratory accidents, and reactivation of the disease due to immunosuppression (Andrade et al., 2011). During the chronic phase the treatment should be offered to adults aged 19–50 years without symptomatic cardiomyopathy (Dias et al, 2016). BZ is the most used drug and is given orally twice a day for 60 days following standard clinical protocol, as 100 mg tablets for adults (Dias et al., 2016). However, for treatment of paediatric patients, there is no liquid formulation and the tablets have to be divided or crushed, with toxicity risk, compromising the optimal treatment dose resulting in non-suitable drug release in the gastrointestinal tract (GIT) after administration. In 2011, after Drugs for Neglected Diseases Initiative (DNDi) partnership with the public manufacturer, Pharmaceutical Laboratory of Pernambuco, LAFEPE®(Recife, Brazil), a paediatric BZ tablet was registered as affordable for paediatric patients (DNDi, 2011). BZ nano- and microparticles in powder dosage form were also developed and suggested as an alternative for paediatric treatment of Chagas disease (Seremeta et al., 2019). However, solid powder formulations or dispersible tablets are undesirable for very young children, and newborns. As such, the

3

development of a new alternative paediatric form of drug administration where BZ is in a pre-dissolved state that could be dose-modulated following child’s bodyweight or surface area, facilitating dose adjustment and administration could be very interesting and useful. Extemporaneous suspensions prepared from dispersible tablets proved that dosage measurement by paediatric intensive care nurses led to significant deviations from the intended dose (Lee et al., 2004 and 2005). These inaccurate dosages are less likely to occur in the case of a liquid oral formulation. Plasma BZ concentrations in healthy human peaked at 3.5 h, with maximal concentrations of 2.2 µg/mL and with a terminal half-life of 12.1 h after 100 mg tablet administration (Molina et al., 2017). Human trypanocidal concentration in blood is classically 3-6 mg/L and equivalent to 23 µM in plasma, concentrations reached at steady-state with 150 mg/12-h or 100 mg/8h regimen. However, BZ was theoretically classified as class III based on its cLogP (Kassim et al., 2004) according to the Biopharmaceutical Classification System (BCS) (Amidon et al.,1995). Maximiano et al. (2010) experimentally classified it as BCS class IV, presenting low water solubility and low permeability. Thus, literature reports many attempts to enhance BZ dissolution rate, particularly employing solid dispersions, co-solvents and cyclodextrin inclusion complexes in order to improve biopharmaceutical profile (Simonazzi et al., 2018; Melo et al., 2018). As BZ toxicity is dose-dependent, recently nano-based formulations showed the ability to reduce doses of BZ maintaining the efficacy in experimental mice models, which is an important approach to improve BZ safety (Rial et al, 2017). Lipid-based drug delivery systems promote enhancement of bioavailability of drugs with poor aqueous solubility and low permeability, which may present incomplete and variable absorption. In this line, self-emulsifying drug delivery systems (SEDDS) are homogeneous anhydrous mixtures containing oil, surfactant and drug, which forms a

4

milky emulsion with submicrometric droplet size upon mild agitation in water or in gastrointestinal fluids (Singh et al., 2011). The small globules formed increase the interfacial area promoting faster drug release, which may increase intestinal permeability of a number of drugs by activation of lymphatic transport and bypass hepatic first pass metabolism, consequently improving drug bioavailability (Porter et al., 2008; Singh et al., 2011, O'Driscoll, 2002). Furthermore, the surfactants used in these formulations can inhibit the PgP mediated drug efflux (Li et al., 2017). These versatile oily liquid preparations can be administered orally via filled in gelatin hard or soft capsules to adults or dispersed in water, milk or juice at different doses, which facilitates dosage adjustment and drug administration to newborn and paediatric patients. In this context the aim of this work was to prepare and characterize BZ-SEDDS liquid formulation as an alternative to tablets in an attempt to have a bioequivalent dosage form of treatment for Chagas disease targeted particularly at infected young children. Furthermore, this study was focused on the evaluation of the activity and toxicity of BZ-SEDDS formulation in vitro, toward different host cells and against intracellular amastigotes of the parasite, in vivo toxicity and the efficacy in experimental murine model of infection by T. cruzi, Y strain.

2. Materials and methods 2.1. Drugs and Materials Benznidazole, (N-benzyl-2-nitro-1-imidazolacetamide) was purchased from LAFEPE

(Recife,

Brazil).

Cyclophosphamide

(N,N-bis(2-chloroethyl)-1,3,2-

oxazaphosphinan-2-amine 2-oxide (Genuxal) was purchased from Asta Medica Oncologica (Germany). HPLC-grade acetonitrile, acetone, ethanol and dimethyl

5

sulfoxide (DMSO) were provided by Tedia® (Rio de Janeiro, Brazil). Miglyol®810N (medium chain triglycerides) was provided by Sasol GmbH (Germany). Food-grade soy and sunflower oils were purchased from Liza® (Brazil). Lipoid S75® containing approximately 75% phosphatidylcholine was purchased from ipoid Gm H Germany . ween 80 sodium phosphate mono asi

sodium hydroxide hydro hlori a id N,N-

dimethylacetamide (DMA) and N-methyl pyrrolidone (NMP) were purchased from Vetec® (Brazil). Labrasol® and Capryol 90® were purchased from Gattefossé (France) and Cremophor® EL was provided by BASF (Germany). Ultra-purified water was obtained from Symplicity 185® system (Millipore, Bedford, USA). Trypsin, fetal bovine serum (FBS), l-glutamine, penicillin/streptomycin and cell culture medium Dul e o’s Modified Eagle’s Medium DMEM were pur hased from Sigma-Aldrich Co (St Louis, MO, USA).

2.2. HPLC-UV analytical method for benznidazole quantification BZ quantification was performed according to Moreira da Silva et al. (2012) using high performance liquid chromatography (HPLC) system Waters Alliance e2695 (Waters, Manchester, UK) and it was validated for quantification of BZ in SEDDS formulation as shown in Table I of Supplementary Materials. A Phenomenex® C18 olumn Gemini NX 150 mm x 4.6 mm 5 μm with a pre-column C18 Phenomenex AJO-7597 2 mm x 4.6 mm 3 μm at 40°C was used. he mo ile phase onsisted of acetonitrile:water (30:70, v/v), which was pumped with isocratic flow (1.0 ml/min). BZ was detected by monitoring the absorbance of the column eluent at 324 nm in Ultraviolet-Visible detector (Waters 2489).

2.3. Selection of excipients, preparation and characterization of BZ-SEDDS

6

SEDDS development was performed based on BZ solubility in different oils, cosolvents and surfactants. For this, an excess of BZ was added to test tubes containing 1 mL of different types of oil (Miglyol®, soy oil and sunflower oil), surfactants (Capryol 90®, Cremophor®EL, Labrasol®, Tween 80®) and co-solvents (ethanol, DMA and NMP). The mixtures were vortexed (IKA® Vortex Genius 3) for 1 min, followed by stirring at 37°C for 48 h on a magnetic plate (SP-10209/A – SPLABOR). All samples were centrifuged at 5000 rpm for 20 min to separate the non-dissolved drug; the supernatant was withdrawn and filtered through a 0.45 µm syringe filter (Millipore®). Aliquots of supernatant were diluted in acetonitrile and quantified by HPLC system, as described in the session 2.2. All studies were conducted in triplicate. After screening of various SEDDS excipients in preliminary experiments, optimal SEDDS formulation containing Miglyol®810N: Capryol® 90: Lipoid® S75: Labrasol®: NMP (30: 15: 20: 15: 20) (% v/v) was selected. Blank SEDDS (no drug) preparation was performed in three steps. First, a mixing of Lipoid®S75, Miglyol®810N and Capryol®90 under agitation and mild heating (40°C) was performed until complete dissolution of Lipoid®S75. In parallel, NMP and Labrasol® were mixed under agitation. Finally, the solution prepared in step 2 was added to the solution prepared in step 1 and was kept under magnetic stirring at 40°C for 20 min to ensure the incorporation of all excipients. BZ-SEDDS was prepared via the addition of 25 mg of BZ into the mixture from step 2 until complete drug dissolution. The drug content was confirmed after dilution of BZ-SEDDS in acetonitrile and analyzed by HPLC-UV method. Blank-formulation was analyzed in the same conditions to verify the occurrence of interferences. Values reported are the mean ± standard deviation of three samples.

7

2.4. SEDDS physicochemical characterization, stability and robustness upon dilution Hydrodynamic diameter (globule size), polydispersity index (PdI), and zeta potential were determined using PN3702 Zetasizer Nano ZS (Malvern Instruments, UK). For this characterization, all formulations were previously emulsified in ultrapurified water at 37°C at 1:1000 dilution and vortexed for 20s. Values reported are the mean ± standard deviation of three readings of at least three formulations batches just after preparation and after 60 days. The stability of the emulsions formed was evaluated by the centrifugation test (Gupta, 2011). Blank-SEDDS and BZ-SEDDS were diluted 10-fold in ultra-purified water, centrifuged at 3500 rpm (5415D Eppendorf®, USA) for 30 min at room temperature and then visually examined for evidence of creaming, coalescence, phase separation or BZ precipitation. All studies were conducted in triplicate. The robustness towards dilution of the formulations was also evaluated similarly as described (Gupta, 2011). Blank-SEDDS and BZ-SEDDS were diluted 50, 100, 250 and 1000 times with ultra-purified water and evaluated for changes in globule size, PdI and BZ precipitation or crystallization during 48 h after dilution under light microscopy (Olympus microscope CX40).

2.5. In vitro benznidazole release studies Drug release kinetics in vitro were determined in simulated gastric (pH 1.2 SGF) and intestinal (pH 7.5 SIF) fluids without enzymes, prepared according to United States Pharmacopeia (United States Pharmacopeia Convention, 2006). Initially, the BZ equilibrium solubility was determined in both fluids as described in the solubility studies for selection of excipients above. Release kinetic studies were conducted by

8

direct dialysis method (Zhang et al., 2008) using cellulose dialysis membrane (6.4 mm, SpectraPor®, Spectrum Labs MWCO 14,000) that was previously hydrated in ultrapurified water for 24 h. BZ-SEDDS (25 mg/mL) was previously emulsified in water (1:4) and then 200µL of this dispersion added to the dialysis bag, with a BZ equivalent mass of 1.25 mg. The amount of drug inserted was in accordance with the sink condition, 0.03125 mg/ml (approximately 10% of saturation solubility). The dialysis bags were inserted in 40 mL of different external media at 37°C under constant stirring (Zhang et al., 2008). The same procedure was performed with the suspension of crushed BZ-tablets (with the same BZ concentration) and free-BZ (powder of raw material). As crushed tablets suspension in water sediments quickly, we added methylcellulose 0.5% (w/v) to this coarse suspension of crushed tablets in order to have dose homogeneity for further gavages of mice. External medium samples (500 µl) were collected at 0, 5, 15, 30, 60, 120, 180, 240, 300, 360, 420, and 480 minutes, diluted in acetonitrile (1:1), mixed, centrifuged, the supernatant filtered (0.45 µm) and quantified by HPLC-UV. The volume withdrawn was replaced with fresh media (Zhang et al., 2008). The experiments were performed in triplicate.

2.6. SEDDS cytotoxicity in vitro The cytotoxicity of free-BZ (powder of raw material), Blank-SEDDS and BZSEDDS was determined in H9c2 cells (American Type Culture Collection, ATCC: CRL 1446), a cardiomyoblast lineage from rat, in HepG2 cells (ATCC: HB 8065), a lineage derived from human hepatocarcinoma cells and in Caco-2 cells (ATCC: HTB-37), a lineage derived from human colon adenocarcinoma. All cell cultures were maintained in DMEM medium supplemented with 10% fetal bovine serum, 1 mM l-glutamine, and 100 μg/m

peni illin/streptomy in and stored in stoves with 5% CO2 atmosphere at

9

37°C. For in vitro cytotoxicity and activity studies, stock solutions of free-BZ were prepared in DMSO and stored at -20 ºC. The stock solutions were further diluted to appropriate working concentrations using culture medium (DMEM). To avoid toxicity to host cells, the final DMSO concentration never exceeded 0.1% (v/v). The cells were seed at 1×103 cells/well for H9c2 and Caco-2, and 5×103 cells/well for HepG2 cells in 96-well plates with 100 μ of media and in u ated for 24h. Afterwards the ells were treated with free BZ, BZ-SEDDS or the equivalent amount of Blank-SEDDS diluted in the culture media. Eight successive serial dilutions (1:2) were made starting from 100 μM of drug. The cells were kept at 37ºC, 5% CO2 with final volume of 100 μl. Cell viability was measured after 72 hours, by the Resazurin colorimetric assay (Riss et al, 2004). The absorbance was determined using plate reader-spectrophotometer (Biochrom Anthos 2010) at wavelengths of 570 nm (oxidized state) and 600 nm (reduced state). All tests were performed in triplicate at least in two independent experiments. The reduction of more than 30% of cell viability was considered cytotoxic, as recommended by the International Organization for Standardization (ISO10993-5, 2009). The cytotoxic concentration for 50% (CC50) of cells values were calculated using Calcusyn software (Biosoft, United Kingdom).

2.7. BZ-SEDDS anti-T. cruzi activity in vitro The in vitro anti-T. cruzi activity assays were performed using the H9c2 infected by trypomastigotes of T. cruzi Y strain, which were obtained from the supernatant of infected cells (Diniz et al. 2018). In 24-well culture plates covered with 13 mm cover slips, 1×104 cells/well were seeded and incubated at 37 ºC and 5 % CO2. After 24 hours, the cells were infected with T. cruzi trypomastigotes at a 20:1 ratio of parasites to host cells. After 24 h incubation, non-adherent parasites were removed by washing with

10

DMEM and the cultures were exposed to different concentrations of free-BZ (powder of raw material), Blank-SEDDS and BZ-SEDDS. he highest on entration was 20 μM of BZ for all groups, and subsequently seven serial dilutions (1:2) were performed. After 72 h of incubation at 37°C and 5% CO2, the cells were washed and fixed with methanol and stained with Giemsa. Anti-T. cruzi activity was calculated based on inhibition of infection compared to the untreated control by microscopic analysis, counting at least 200 cells per sample. All experiments were performed in duplicate, with at least two replicates. IC50 values were calculated using Calcusyn software (Biosoft, United Kingdom).

2.8. BZ-SEDDS anti-T. cruzi efficacy in vivo Female Swiss mice (18–24 g) from the animal facility of the Federal University of Ouro Preto, Minas Gerais, Brazil, were maintained at 22 ± 2 °C in a controlled temperature room with access to water and food ad libitum under 12 h day/night cycles. The Ethics Committee in Animal Research at UFOP approved all procedures and experimental conditions (number 2014/56). Swiss mice were inoculated by intraperitoneal route with 5.000 trypomastigotes of the T. cruzi Y strain. The different treatments started on the 4 th day of infection. The infected mice were divided into 4 groups: infected control, treated with BZ at 100 mg/kg/day of suspension of crushed BZ-tablets, treated with 100 mg/kg/day of BZSEDDS and treated with Blank-SEDDS at equivalent excipient doses for 20 days. Two groups of not infected mice were used for toxicity assay, one group was treated with Blank-SEDDS and the other one received no treatment (non-infected control). The animals were examined at least once daily to observe the occurrence of mortality and changes in clinical signs: appearance (twisting, piloerection, dirty eyes), pain (torsion,

11

spasm) and behavior change (withdrawal, vocalization, scratching, reluctance to move, irritability, anorexia, abnormal posture, ataxia). In addition, the toxicity of the new formulation and the free drug was evaluated by hepatic enzyme dosages in mouse serum collected in the last day of treatment. Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) were determined by colorimetric assay using an autoanalyzer (Wiener Lab model CM200 - kinetic analysis) and commercial Bioclin® kit according to the manufacturer's instructions. Efficacy of the treatment was determined following the protocol described by Caldas et al. (2008). Briefly, mortality was checked daily until 30 days after treatment. The parasitaemia was estimated daily by fresh blood examination according to Brener (1962) during and up to 30 days after the end of treatment. Animals with negative results in the fresh blood examination were intraperitoneally immunosuppressed with cyclophosphamide (Baxter Oncology, Germany) at the dose of 50 mg/kg/day in 3 cycles of 4 consecutive doses with an interval of 3 days between each cycle. The parasitaemia was checked daily during and up to 10 days after the end of the cycles. Blood qPCR analyses were performed 30 and 180 days after end of the treatment in samples from mice with negative fresh blood examination results. For the isolation and purifi ation of genomi DNA of 200 μ of lood samples Wizard genomi DNA purification kit (Promega Corp., Madison, WI) was used according to the manufacturer's instructions. PCR analyses were performed as described by Caldas et al. (2012), using the primers: TCZ-F (5=-GCTCTTGCCCACAMGGGTGC-3=, where M indicates A or C) and TCZ-R (5=-CCAAGCAGCGGATAGTTCAGG-3=), as described by Cummings and Tarleton (2003). The murine TNF-α gene sequen e was amplified separately using the primers TNF-5241(5=-TCCCTCTCATCAGTTCTATGGCCCA3=)

and

TNF-5411

(5=-CAGCAAGCATCTATGCACTTAGACCCC-3=).

The

12

presence of T. cruzi in blood samples was evaluated by amplifying a 195 bp tandem repeat in genomic DNA (Cummings and Tarleton, 2003). For reactions, 2 μ template DNA, specific primers at concentration of 10 μM and Sy r-Green PCR Master Mix in a total volume of 10 μ were used. DNA amplifications were carried out in an ABI 7300 real-time PCR system (Applied Biosystems, Life Technologies). After the initial denaturation step of 10 min at 95°C, amplifications were carried out for 40 cycles (94°C for 15 s). Fluorescence data collection was performed at 64.3°C for 1 min at the end of each cycle. Amplification was immediately followed by a melting program with initial denaturation for 15 s at 95°C, cooling to 60°C for 1 min, and then a stepwise temperature increase from 60 to 95°C at 0.3°C/s. All samples were analyzed in duplicate and negative samples and reagent controls were processed in parallel in each assay. Animals showing negative results in all tests were considered cured.

2.9. Statistical analysis Statistical data analyses were performed using Graph Pad Prisma 5.01 statistical software (GraphPad Software Inc., San Diego, CA, USA). The results were expressed as mean ± standard deviation. Parametri data were analyzed with Student’s t test and nonparametric data with the Mann–Whitney test. Statistical significance was established with 95% confiden e intervals and p < 0.05.

3. Results and Discussion There is no commercial suspension or liquid formulation of BZ available for use in paediatrics. There are always many inconvenient in use tablets to administer to children, related to absence of uniformity of dose, high risk of toxicity and the impossibility of administration to newborns that are infected via the congenital route. In

13

these cases, a more individualized dose is difficult to obtain with dispersible tablets. SEDDS are liquid formulations and can be dosed in syringe and dispersed in water, milk or juice and administered to small children or in soft gelatin capsules to adults. So we propose a versatile formulation, anhydrous, with BZ in pre-dissolved form. Reformulation of already used drugs in order to improve their biopharmaceutical profile and to design paediatric oral dosage forms is a useful approach. In this work we develop a safe and stable new liquid formulation for oral administration of BZ, as SEDDS, particularly adapted to be diluted in aqueous vehicles before administration or filled in gelatin capsules. The optimized BZ-SEDDS formulation was designed from a previous work where in vivo low toxicity of similar SEDDS formulation was shown (Spósito, et al 2017), which demonstrated that the SEDDS with Labrasol 15% (w/w) was safe for mice upon 20-day oral treatment given once a day. The Table II of Supplementary Information shows the complete data about the SEDDS formulation development based on the variation of percentage of oil phase, type and percentage of surfactant, type of co-solvent and drug concentration (mg/ml). This data showed that medium chain triglycerides were essential to inhibit BZ crystallization and Labrasol, co-solvents (NMP) and co-surfactant (Capryol) were essential to prevent phase separation of anhydrous SEDDS under storage. Additionally, to design SEDDS incorporating BZ, solubility tests were performed in several excipients generally used in lipid-based oral formulations in order to select the most suitable constituents to optimize drug loading without the instability of BZ crystal growth. To quantify BZ a method previously published was used (Moreira da Silva et al, 2012) and it was also validated to determine BZ in SEDDS as shown in Table I of supplementary material, which presents the validation parameters. The

14

method was selective, precise and linear in the BZ concentration range of 0.5-100 µg/ml at 324 nm, a wavelength where no absorption was detected for the SEDDS excipients (Figure 1 in Supplementary information). Miglyol®810N and soybean oil showed similar ability to solubilize BZ: 0.37 and 0.42 mg/mL, respectively (Table 1). Medium-chain triglycerides, such as Migliol®810N are generally regarded as safe category by oral, parenteral and topical administration (Moss, 2009). Miglyol-type triglycerides present good fluidity, suitable self-emulsification properties and it is fully digestible. (Porter & Charman, 2001; Cornaire et al. 2004;). To improve BZ dissolution in lipid phase Capryol 90® was used, due to its co-surfactant properties. Capryol 90® is frequently employed in the development and optimization of lipid-based formulations loading various poorly watersoluble drugs (Shakeel et al. 2013). A strategy to load more BZ was utilized via the inclusion of a co-solvent in the SEDDS. Among all co-solvents tested, NMP exhibited highest capacity to solubilize BZ, to improve incorporation in the SEDDS and to inhibit BZ crystallization. Furthermore, NMP is a water miscible, aprotic solvent and it is accepted as solubilizing excipient in parenteral and oral medications (Chang et al., 2009). Among the surfactants tested, Cremophor® EL, Labrasol® and Tween 80®, no significant difference in solubility was observed (Table 1). Therefore, their cytotoxic properties were decisive in an appropriate surfactant selection. Although Cremophortype surfactants are widely used in pharmaceutical preparations, their use in high concentrations has been avoided because of adverse effects, mainly due to its low metabolism by the organism (Gelderblom et al., 2001). Ujhelyi et al. (2011) showed that Tween® 20, 60, 80 present high cytotoxicity. Differently, the degree of esterification and lack of sorbitan component of Labrasol® decreased its toxicity in Caco-2 cells

15

(Ujhelyi et al., 2011). Thus, Labrasol® was then selected as surfactant. Furthermore, the phosphatidylcholine (Lipoid®S75) included herein in BZ-SEDDS preparation was shown to dramatically reduce the toxicity of lipid-based formulations, increasing biocompatibility, as already observed and discussed (Jumaa & Müller, 2000; Spósito et al., 2017). From preliminary experiments to develop a BZ-SEDDS, drug loading and selfemulsification properties were considered as critical parameters to select the best composition (Table II in SI). Thus, the optimized SEDDS formulation composition was Miglyol®810N, Capryol 90®, Lipoid S75, Labrasol®, NMP at following proportions (30:15:20:15:20) loaded at 25 mg/ml of BZ. The actual drug concentration was confirmed by HPLC-UV to be 26.5 mg/ml (Table 2). The total incorporated mass of the BZ added remained dissolved in the SEDDS and no macro or microscopic drug crystallization or precipitation was observed over 6 months at room temperature. The physicochemical aspects and stability (60 days) of emulsions formed after self-emulsification of SEDDS in water are shown in Table 2. The emulsions are formed spontaneously and were polydisperse (PdI > 0.3), indicating that several droplet size populations were formed during self-emulsification. The size of emulsion droplets formed were around 300 nm for blank-SEDDS and around 500 nm for BZ loaded SEDDS, indicating a strong effect of BZ on droplet surface tension that induces an increase in size. After storage during 60 days no significant alterations were observed in droplet sizes after emulsification, PdI or BZ content (Table 2). The size and charge of the droplets formed may affect the stability of the colloidal dispersion and the kinetics of drug release (Cherniakov et al., 2014). The droplet surface available to interact with bile salts to form mixed micelles and with enterocytes are major factors affecting lipids and lipophilic drug transport in the

16

membrane of the GIT. The zeta potential of Blank-SEDDS and BZ-SEDDS nanodroplets are approximately -45 mV (Table 2), indicating that the incorporation of BZ did not alter the droplet surface charge. This result indicates that BZ could be associated with the oily core of the emulsion droplet. High zeta potential values induce electrostatic repulsion between the droplets, preventing coalescence and improving emulsion stability (Müller et al., 2001). However, since SEDDS form emulsions only upon exposure to gastrointestinal fluids, these characteristics should be further evaluated in the extremely complex physiological dispersion media. Although, the stability data after 60 days showed no alterations in droplet sizes formed, zeta potential was reduced in BZ-SEDDS indicating a reorganization of the drug at the droplet surface (Table 2) or an increase in negative phospholipids or free acid chains at the surface upon storage. Robustness towards dilution was monitored by diluting the formulations 50, 100, 250 and 1000 times in ultra-pure water (Table 3). Upon dilution the droplets formed from blank-SEDDS maintain their mean size with no significant alterations, which indicates that emulsion droplets are kinetic or thermodynamic stable, in accordance with the formation of type IIIA nanoemulsions (Pouton, 2006 and Porter et al., 2008). However, when the BZ-SEDDS were diluted the droplet sizes vary largely, indicating an effect of BZ on droplet interfacial tension and on droplet polydispersion. These larger sizes of BZ-SEDDS in the submicrometric range are, however, suitable to maintain the BZ pre-solubilized state in the GIT. The stability of emulsions formed at different dilutions of SEDDS is important in order to ensure gradual dilution in contact with GI fluids (without precipitation of drug) which could ensure uniform release (Bakhle & Avari, 2015). Even after 24 h, neither precipitation of the drug nor any phase separation was observed when the SEDDS was diluted up to 1000 times, showing the

17

stability of the emulsion droplets formed (Table 2 and 3). Additionally, in stress conditions the SEDDS formulation was stable after centrifugation of the formed emulsion. No sign of creaming, separation or drug precipitation was observed, indicating stability of the formed emulsion (data not shown). In order to estimate and compare dissolution performance between BZ-SEDDS and suspension of BZ crushed tablets release studies were conducted (Figure 1). BZ equilibrium solubility in simulated gastric fluid (SGF) was 0.292 ± 0.003 mg/ml and 0.301 ± 0.030 mg/ml in simulated intestinal fluid (SIF). The in vitro release profile of BZ from the SEDDS, suspension of BZ crushed tablets and free-BZ (raw material) were evaluated in both fluids in sink conditions, i.e. amount of 0.03 mg/ml of BZ in each type of fluid (Figure 1). BZ-SEDDS formulation released about 100% of BZ within 6 hours in SGF medium. At the same condition, approximately 70% of BZ was released from suspension of crushed tablets (Figure 1), with significant difference on drug release profiles (p < 0.05); mainly in the release kinetics between 2 and 6 h was observed. FreeBZ dissolution is not significantly different from suspension of crushed tablets in both pH and media. BZ release from SEDDS is faster and higher compared to the suspension of crushed tablets in gastric fluid. This may be related to size of the particulate dispersed system and the pre-solubilized state of BZ in the oily phase. In the SIF medium, a slower release profile was found for all formulations and no significant difference in the BZ release kinetics was observed in this medium (Figure 1). It may be useful to predict release properties related to BZ ionization with a variation in pH. From our data no dramatic difference in release profile exists in intestinal pH. In this case, possibly the lower ionization of drug in alkaline SIF medium would lead to a slower drug release and retention of the drug within the oily phase of the emulsion. However, in gastric fluid BZ-SEDDS showed improvement of BZ dissolution rate as shown in the figure 1.

18

The study of release is longer (8 h) compared to gastric emptying time. However, it was done to monitor not only release but also BZ stability in the release medium. This may indicate that BZ-SEDDS is probably bioequivalent compared to suspension of crushed tablets and may have even a better profile of absorption. In vitro release tests are important in order to predict the effects of pharmaceutical formulations on drug released in contact with different physiological fluids and to compare them. In this regard, BZ-SEDDS showed a faster BZ release profile than suspension of crushed tablets in gastric media. However, for the SEDDS, not only the dispersion properties in the colloidal state, but also the digestion of lipids by lipases (lipolysis) play an important role maintaining the drug in its solubilized form (Kollipa & Ganhi, 2014). Despite the wide use of these tests, the correlation between in vitro and in vivo is generally controversial, as there are many limitations involved in the in vitro experiments (Bernkop-Schnürch & Jalil, 2018), especially with regard to simulation of the complexity of gastrointestinal fluids and the effects of bile salts on drug absorption. In murine model, after an oral dose of 100 mg/kg, the time to reach plasma maximal concentration (Tmax) reported is 50 min (Périn et al., 2017) for a maximal plasma concentration of 41.6 µg/ml or more than 130 min according to Moreira da Silva and coworkers (2012) for a Cmax of approximately 1.7 µg/ml in Swiss mice. Thus, pharmacokinetic parameters vary widely between studies, even using the same species. From this data, it can be observed that BZ is poorly absorbed (low extent) and even in mice, slow absorption is observed (low rate). These pharmacokinetic data are in accordance the slow BZ dissolution presented in this work in in vitro release studies. Free BZ (powder of raw material), Blank-SEDDS and BZ-SEDDS effects were evaluated on viability of H9c2, HepG2 and Caco-2 cells (Figure 2). The three cell lineages, H9c2, HepG2 and Caco-2 represents the main tissue targets involved in the

19

parasite-drug interaction; the main host cell of T. cruzi in chronic phase of infection, the BZ main site of metabolism and the intestinal cell in contact with SEDDS upon oral administration, all subjected to drug and formulation toxicity, respectively. It can be clearly observed that below 25 µM no cytotoxicity was observed in any type of cell for the free-BZ or BZ-SEDDS formulation, under the same experimental conditions (Figure 2B). This concentration is almost three times higher than the IC90 of BZ in vitro (8.69 ± 1.46 µM), revealing the safety of the SEEDS formulation toward this selected cells. Free-BZ, particularly, showed no cytotoxic effects against host cells at the doses up to 100 µM (Figure 2A). Caco-2 cells were more sensitive to SEDDS formulations at higher concentrations. SEDDS surfactants became more toxic to cells at higher concentrations as previously discussed, particularly for the Caco-2 cell line (Sha et al., 2005). HepG2, that represent liver cells with high metabolic activity, were the less sensitive cell line to BZ and formulations. The cytotoxic concentration for 50% (CC50) of cells calculated in Calcusyn software were showed in Figure 2C. Thus, only non-toxic concentrations were used to assess in vitro anti-T. cruzi activity of the developed-SEDDS. Dose-response curves showed that the Blank-SEDDS formulation did not inhibit infection of H9c2 cells by the T. cruzi Y strain (Figure 3). In addition, incorporation of BZ into SEDDS did not alter the trypanocidal activity of the drug (Figure 3), showing similar response to control group treated with free-BZ (powder of raw material). The IC50 and IC90 values of BZ were 2.10 ± 0.41 μM and 8.69 ± 1.46 μM and slightly lower for BZ-SEDDS 1.29 ± 0.01 μM and 7.24 ± 1.99 μM respectively. Considering the CC50 of BZ and BZ-SEDDS determined in the host cell (H9c2), the selectivity indexes (CC50 divided by IC50) were higher than 50 and 40 for BZ and BZ-SEDDS treatments in vitro, respectively. These values were all lower than

20

plasma concentrations of 23 μM rea hed in human pharma okineti s previously determined (Molina et al, 2017). BZ-SEDDS formulation efficacy was assessed in vivo in experimental mouse model of infection by T. cruzi Y strain. Animals treated with free drug or SEDDS formulations showed no changes in observed clinical signs, appearance, behavior and pain. Infected mice treated with Blank-SEDDS showed similar results to not treated infected group, with no parasitaemia suppression or death prevention (Table 3). Suspension of crushed BZ-tablets (BZ) and BZ-SEDDS had similar efficacy suppressing the parasitaemia of the infected mice, with negative results in fresh blood after administration of 1.0 ± 0.00 and 1.14 ± 0.38 doses, respectively (Table 4). According to cure parameters, 57% (4 of 7) of cure was observed in the suspension of tablets and BZ-SEDDS. Both treatments were able to prevent animal mortality completely (Table 4) observed in 100% of non-treated mice infected with T. cruzi Y strain. The in vivo toxicity of the suspension of crushed BZ-tablets (BZ), Blank SEDDS and BZ-SEDDS was also evaluated in infected mice by dosing liver enzymes (AST and ALT) in serum samples collected on the last day of treatment (Figure 4). Uninfected animals were treated with Blank-SEDDS for 20 days, and this formulation did not induce changes in AST and ALT enzyme levels in relation to untreated uninfected animals (non-infected control). Infected control shows high levels of AST and ALT enzymes at 15 days post infection (Figure 4). Subsequent measurements could not be performed because the infection by T. cruzi Y strain induces 100% of mortality until the 18th day of infection (data not shown). Serum AST levels of infected and untreated animals (infected control) were significantly higher (p <0.0001) than that observed for all other groups, indicating that the parasite has an important role in liver damage.

21

Decreasing and/or elimination of the parasite induced by the treatment reduces AST serum levels. However, AST levels for groups treated with BZ were significantly higher than the untreated uninfected group (non-infected control). In treated groups, ALT levels were similar to those observed in serum samples from non-infected untreated animals except in those treated with BZ (Figure 4). In murine model, liver damage may be the result of both BZ metabolism and specific mechanisms activated by T. cruzi infection (Novaes et al., 2015). In summary, in vitro and in vivo efficacy and toxicity studies of BZ-SEDDS showed that the incorporation of BZ in SEDDS does not alter its potency and safety. The commercial success of products such as Fortovase ® (saquinavir), Norvir® (ritonavir), and Sandimmun Neoral® (cyclosporine) aroused commercial interest in using SEDDS as a drug delivery system (Agrawal et al. 2015). For example, Sandimmun Neoral® is available as capsules and as oral solution, which must be diluted, preferably in orange or apple juice, but other beverages such as these may be used as soft drinks to suit individual taste, according to the manufacturer (Delpharm Huningue SAS, Huningue, France). Additionally, Spósito et al. (2017) developed and characterized SEDDS containing ravuconazole showing better drug potency in vitro against T. cruzi Y strain and no toxic effects in healthy mice. The developed BZ-SEDDS is a stable formulation with a high drug payload and it can be prepared by an affordable simple method of drug dissolution intended for Chagas disease treatment, which presents the same efficacy in murine experimental model and safety compared to a suspension of crushed commercial BZ-tablets. Liquid BZ-SEDDS is a promising delivery dosage form and deserves further studies, especially for paediatric use, allowing proper treatment dose flexibility.

Acknowledgments

22

This work was also supported by NANOBIOMG-Network (# 7–14), FAPEMIG, UFOP, CNPq-research fellowships to MTB and VCFM, BRICS-STI/CNPQ #442351/2017-8 and CAPES (research fellowships to ALM . We also thank the “ a oratório Piloto de Análises Clíni as”

APAC/UFOP for the help with hepati enzymes analyses. We are

grateful to Lauren Ball for English language revising.

Author contributions All authors contributed toward data analyses, interpretation of data, drafting and revising manuscript critically and approved the final version to be published and agree to be accountable for all aspects of this work.

Disclosure The authors report no conflicts of interest in this work.

CRediT author statement Ana Lia Mazzeti, MT Bahia, Vanessa CF Mosqueira: Conceptualization, Methodology,Writing- Original draft preparation, Formal analysis, Visualization, Review & Editing. Ana Lia Mazzeti, Líliam T. Oliveira: Methodology, Method Validation, Investigation, Visualization. Maria T Bahia, Vanessa CF Mosqueira: Supervision, Resources, Project administration, Funding acquisition. Karolina R. Gonçalves, Géssica C. Schaun: Investigation.

23

References Agrawal, A.G., Kumar, A., Gide, P.S., 2015. Formulation of solid self-nanoemulsifying drug delivery systems using N -methyl pyrrolidone as cosolvent. Drug Development and Industrial Pharmacy 41, 594–604. https://doi.org/10.3109/03639045.2014.886695 Amidon, G.L., Lennernäs, H., Shah, V.P., Crison, J.R., 1995. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 12, 413–420. https://doi.org/10.1023/a:1016212804288 Andrade, J.P. de, Marin Neto, J.A., Paola, A.A.V. de, Vilas-Boas, F., Oliveira, G.M.M., Bacal, F., Bocchi, E.A., Almeida, D.R., Fragata Filho, A.A., Moreira, M. da C.V., Xavier, S.S., Oliveira Junior, W.A. de, Dias, J.C.P., 2011. I Diretriz Latino-Americana para o diagnóstico e tratamento da cardiopatia chagásica: resumo executivo. Arq. Bras. Cardiol. 96, 434–442. https://doi.org/10.1590/S0066-782X2011000600002. Bernkop-Schnürch, A., Jalil, A., 2018. Do drug release studies from SEDDS make any sense? J. Control. Release. 271, 55-59. https://doi.org/10.1016/j.jconrel.2017. Brener, Z., 1962. Therapeutic activity and criterion of cure on mice experimentally infected with Trypanosoma cruzi. Rev. Inst. Med. Trop. Sao Paulo 4, 389–396. Caldas, S., Caldas, I.S., Diniz, L. de F., Lima, W.G. de, Oliveira, R. de P., Cecílio, A.B., Ribeiro, I., Talvani, A., Bahia, M.T., 2012. Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection. Acta Tropica 123, 170–177. https://doi.org/10.1016/j.actatropica.2012.05.002 Caldas, S., Santos, F.M., Lana, M. de, Diniz, L.F., Machado-Coelho, G.L.L., Veloso, V.M., Bahia, M.T., 2008. Trypanosoma cruzi: Acute and long-term infection in the vertebrate host can modify the response to benznidazole. Experimental Parasitology 118, 315–323. https://doi.org/10.1016/j.exppara.2007.08.016 Chang, R.K., Qu, W., Shukla, A.J., Trivedi, N., 2009. Pyrrolidone, in: Rowe, R.C., Sheskey, P.J., Owen, S.C., American Pharmacists Association (Eds.), 2009. Handbook of pharmaceutical excipients: edited by Raymond C. Rowe, Paul J. Sheskey, Marian E. Quinn, 6th ed. ed. APhA/Pharma euti al Press ondon ; Chi ago pp. 600-602. Cherniakov, I., Domb, A.J., Hoffman, A., 2015. Self-nano-emulsifying drug delivery systems: an update of the biopharmaceutical aspects. Expert Opinion on Drug Delivery 12, 1121–1133. https://doi.org/10.1517/17425247.2015.999038 Cornaire, G., Woodley, J., Hermann, P., Cloarec, A., Arellano, C., Houin, G., 2004. Impact of excipients on the absorption of P-glycoprotein substrates in vitro and in vivo. International Journal of Pharmaceutics 278, 119–131. https://doi.org/10.1016/j.ijpharm.2004.03.001 Cucunubá, Z.M., Okuwoga, O., Basáñez, M.-G., Nouvellet, P., 2016. Increased mortality attributed to Chagas disease: a systematic review and meta-analysis. Parasites Vectors 9, 42. https://doi.org/10.1186/s13071-016-1315-x

24

Cummings, K.L., Tarleton, R.L., 2003. Rapid quantitation of Trypanosoma cruzi in host tissue by real-time PCR. Mol. Biochem. Parasitol. 129, 53–59. Dias, J.C.P., Ramos, A.N. Jr., Gontijo, E.D., et al.,2016.Second Brazilian consensus on Chagas disease, 2015. Rev Soc Bras Med Trop. 49, 3–60. Diniz, L. de F., Mazzeti, A.L., Caldas, I.S., Ribeiro, I., Bahia, M.T., 2018. Outcome of E1224-Benznidazole Combination Treatment for Infection with a Multidrug-Resistant Trypanosoma cruzi Strain in Mice. Antimicrob Agents Chemother 62, e00401-18. https://doi.org/10.1128/AAC.00401-18 DNDi, 2011. Paediatric Benznidazole | DNDi [WWW Document]. Drugs for Neglected Diseases initiative (DNDi). URL https://www.dndi.org/achievements/paediatricbenznidazole/ (accessed 7.10.19). Gelderblom, H., Verweij, J., Nooter, K., Sparreboom, A., 2001. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation. Eur. J. Cancer 37, 1590–1598. Gupta, S., Chavhan, S., Sawant, K.K., 2011. Self-nanoemulsifying drug delivery system for adefovir dipivoxil: Design, characterization, in vitro and ex vivo evaluation. Colloids and Surfaces A: Physicochemical and Engineering Aspects 392, 145–155. https://doi.org/10.1016/j.colsurfa.2011.09.048 Jumaa, M., Müller, B.W., 2000. Lipid emulsions as a novel system to reduce the hemolytic activity of lytic agents: mechanism of the protective effect. Eur J Pharm Sci 9, 285–290. ISO10993-5. 2009. International standard: Biological Evaluation of Medical Devices Part 5: Tests for Cytotoxicity: in vitro methods. International Organization for Standardization, Geneva. Lee, W.M., Lugo, R.A., Rusho, W.J., Mackay, M., Sweeley, J., 2004. Chemical stability of extemporaneously prepared Lorazepam suspension at two temperatures. J. Pediatr. Pharmacol. Ther. 9 (4), 254–258. https://doi.org/10.5863/1551-6776-9.4.254. Lee,W.M., Lugo, R.A., Cash, J., Rusho,W.J., Mackay, M., Welkie, K., 2005. The accuracy and precision of measuring Lorazepam from three liquid preparations. J. Pediatr. Pharmacol. Ther. 10 (1), 36–42. https://doi.org/10.5863/1551-6776-10.1.36. Li, F., Hu, R., Wang, B., Gui, Y., Cheng, G., Gao, S., Ye, L., Tang, J., 2017. Selfmicroemulsifying drug delivery system for improving the bioavailability of huperzine A by lymphatic uptake. Acta Pharmaceutica Sinica B 7, 353–360. https://doi.org/10.1016/j.apsb.2017.02.002 Martins-Melo, F.R., Ramos Jr, A.N., Alencar, C.H., Heukelbach, J., 2012. Mortality due to Chagas disease in Brazil from 1979 to 2009: trends and regional differences. J Infect Dev Ctries 6, 817–824. https://doi.org/10.3855/jidc.2459 Maximiano, F.P., Costa, G.H.Y., Souza, J. de, Cunha-Filho, M.S.S. da, 2010. Caracterização físico-química do fármaco antichagásico benznidazol. Quím. Nova 33, 1714–1719. https://doi.org/10.1590/S0100-40422010000800018. 25

Melo, P.N., Caland, L.B., Fernandes-Pedrosa, M.F.1, Silva-Junior, A.A., 2018. Designing and monitoring microstructural properties of oligosaccharide/co-solvent ternary complex particles to improve benznidazole dissolution. J Mater Sci. 53, 2472– 2483. https://doi.org/10.1007/s10853-017-1720-3 Molina, I., Salvador F., Sánchez-Montalvá, A., Artaza, M.A., Moreno, R., Perin, L., Esquisabel, A., Pinto, L., Pedraz, J.L., 2017. Pharmacokinetics of benznidazole in healthy volunteers and implications in future clinical trials. Antimicrob. Agents Chemother. 61:e01912-16. https://doi.org/10.1128/AAC.01912-16 Moreira da Silva, R., Oliveira, L.T., Silva Barcellos, N.M., de Souza, J., de Lana, M., 2012. Preclinical Monitoring of Drug Association in Experimental Chemotherapy of Chagas’ Disease y a New HP C-UV Method. Antimicrob. Agents Chemother. 56, 3344–3348. https://doi.org/10.1128/AAC.05785-11 Moss, G., 2009. Medium-chain triglycerides, in: Rowe, R.C., Sheskey, P.J., Owen, S.C., American Pharmacists Association (Eds.), 2009. Handbook of pharmaceutical excipients: edited by Raymond C. Rowe, Paul J. Sheskey, Marian E. Quinn, 6th ed. ed. APhA/Pharma euti al Press ondon ; Chi ago pp. 429–431. Müller, R.H., Jacobs, C., Kayser, O., 2001. Nanosuspensions as particulate drug formulations in therapy. Advanced Drug Delivery Reviews 47, 3–19. https://doi.org/10.1016/S0169-409X(00)00118-6 Novaes, R.D., Santos, E.C., Cupertino, M.C., Bastos, D.S.S., Oliveira, J.M., Carvalho, T.V., Neves, M.M., Oliveira, L.L., Talvani, A., 2015. Trypanosoma cruzi infection and benznidazole therapy independently stimulate oxidative status and structural pathological remodeling of the liver tissue in mice. Parasitol Res 114, 2873–2881. https://doi.org/10.1007/s00436-015-4488-x O’Dris oll C.M. 2002. Lipid-based formulations for intestinal lymphatic delivery. Eur J Pharm Sci 15, 405–415. Perin, L., Moreira da Silva, R., Fonseca, K.D., Cardoso, J,M., Mathias, F.A., Reis, L.E., Molina, I., Correa-Oliveira, R., Vieira, P.M., Carneiro, C.M., 2017. Pharmacokinetics and Tissue Distribution of Benznidazole after Oral Administration in Mice. Antimicrob. Agents Chemother. 61(4) e02410-16. https://doi.org/10.1128/AAC.02410-16. Pouton CW., 2006. Formulation of poorly water-soluble drugs for oral administration: physicochemical and physiological issues and the lipid formulation classification system. Eur J Pharm Sci. 29(3–4):278–287. Porter, C.J.H., Charman, W.N., 2001. In vitro assessment of oral lipid based formulations. Advanced Drug Delivery Reviews 50, S127–S147. https://doi.org/10.1016/S0169-409X(01)00182-X Porter, C.J.H., Pouton, C.W., Cuine, J.F., Charman, W.N., 2008. Enhancing intestinal drug solubilisation using lipid-based delivery systems. Advanced Drug Delivery Reviews 60, 673–691. https://doi.org/10.1016/j.addr.2007.10.014 Rial, M.S., Scalise, M.L., ArruÂa, E.C., Esteva, M.I., Salomon, C.J., Fichera, L.E., 2017 Elucidating the impact of low doses of nano-formulated benznidazole in acute 26

experimental Chagas disease. PLoS Negl https://doi.org/10.1371/journal.pntd.0006119

Trop

Dis

11(12),

e0006119.

Riss, T.L., Moravec, R.A., Niles, A.L., Duellman, S., Benink, H.A., Worzella, T.J., Minor, L., 2004. Cell Viability Assays, in: Sittampalam, G.S., Coussens, N.P., Brimacombe, K., Grossman, A., Arkin, M., Auld, D., Austin, C., Baell, J., Bejcek, B., Caaveiro, J.M.M., Chung, T.D.Y., Dahlin, J.L., Devanaryan, V., Foley, T.L., Glicksman, M., Hall, M.D., Haas, J.V., Inglese, J., Iversen, P.W., Kahl, S.D., Kales, S.C., Lal-Nag, M., Li, Z., McGee, J., McManus, O., Riss, T., Trask, O.J., Weidner, J.R., Wildey, M.J., Xia, M., Xu, X. (Eds.), Assay Guidance Manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda (MD). Seremeta, K.P., Arrúa, E.C., Okulik, N.B., Salomon, C.J., 2019. Development and characterization of benznidazole nano- and microparticles: A new tool for pediatric treatment of Chagas disease? Colloids and Surfaces B: Biointerfaces 177, 169–177. https://doi.org/10.1016/j.colsurfb.2019.01.039 Sha, X., Yan, G., Wu, Y., Li, J., Fang, X., 2005. Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells. European J. Pharm. Sci. 24, 477–486. https://doi.org/10.1016/j.ejps.2005.01.001 Shakeel, F., Haq, N., Alanazi, F.K., Alsarra, I.A., 2013. Impact of various nonionic surfactants on self-nanoemulsification efficiency of two grades of Capryol (Capryol-90 and Capryol-PGMC). Journal of Molecular Liquids 182, 57–63. https://doi.org/10.1016/j.molliq.2013.03.011 Simonazzi, A., Davies, C., Cid, A.G., Gonzo, E., Parada, L., Bermúdez, J.M., 2018 Preparation and Characterization of Poloxamer 407 Solid Dispersions as an Alternative Strategy to Improve Benznidazole Bioperformance. J Pharm Sci. 107(11), 2829-2836. https://doi.org/10.1016/j.xphs.2018.06.027. Singh, A., Worku, Z.A., Van den Mooter, G., 2011. Oral formulation strategies to improve solubility of poorly water-soluble drugs. Expert Opinion on Drug Delivery 8, 1361–1378. https://doi.org/10.1517/17425247.2011.606808 Spósito, P.Á.F., Mazzeti Silva, A.L., de Oliveira Faria, C., Urbina, J.A., Pound-Lana, G., Bahia, M.T., Mosqueira, V.C.F., 2017. Ravuconazole self-emulsifying delivery system: in vitro activity against Trypanosoma cruzi amastigotes and in vivo toxicity. IJN Volume 12, 3785–3799. https://doi.org/10.2147/IJN.S133708 Ujhelyi, Z., Fenyvesi, F., Váradi, J., Fehér, P., Kiss, T., Veszelka, S., Deli, M., Vecsernyés, M., Bácskay, I., 2012. Evaluation of cytotoxicity of surfactants used in self-micro emulsifying drug delivery systems and their effects on paracellular transport in Caco-2 cell monolayer. European Journal of Pharmaceutical Sciences 47, 564–573. https://doi.org/10.1016/j.ejps.2012.07.005 United States Pharmacopeial Convention (Ed.), 2006. The United States Pharmacopeia: the National Formulary. United States Pharmacopeial Convention, Rockville, Md. WHO, 2019. Chagas disease (American trypanosomiasis) [WWW Document]. URL https://www.who.int/news-room/fact-sheets/detail/chagas-disease-(americantrypanosomiasis) (accessed 7.10.19). 27

Zhang, P., Liu, Y., Feng, N., Xu, J., 2008. Preparation and evaluation of selfmicroemulsifying drug delivery system of oridonin. International Journal of Pharmaceutics 355, 269–276. https://doi.org/10.1016/j.ijpharm.2007.12.026

28

Figure Legends

Figure 1 - Benznidazole release profile from BZ-SEDDS, BZ-free and BZ-crushed tablet. In simulated gastric fluid (SGF) (A) and simulated intestinal fluid (B).* p < 0.05, 29

difference in the release kinetics of benznidazole from SEDDS compared to the suspension of crushed commercial benznidazole tablets.

Figure 2 - Cytotoxicity of benznidazole and BZ-SEDDS. Effects of benznidazole, BZ-SEDDS and Blank-SEDDS on viability of Caco-2, H9c2, and HepG2 cells upon 96 h incubation (A). Bar graphic comparing cell viability at 25µM concentration of BZ (or corresponding volume of Blank-SEDDS) in the three cell lines (B). The cytotoxic concentration for 50% (CC50) of cells (Calcusyn software) (C). The results are the average of two independent experiments, performed in triplicate.

30

Figure 3 – Anti-T. cruzi activity. In vitro dose-response curves of H9c2 cells infected by Trypanosoma cruzi Y strain incubated for 72 h with free-benznidazole, BZ-SEDDS and Blank-SEDDS at initial concentration of 20 μM followed y seven sets of dilution (1:2). Each point of the dose-response curves corresponds to the mean of two independent experiments.

31

Figure 4 - Liver enzymes levels of treated mice. (A) Aspartate aminotransferase (AST) and (B) alanine aminotransferase (ALT) serum levels of non-infected and infected mice by Trypanosoma cruzi Y strain treated for 20 days with benznidazole, BZ-SEDDS or Blank-SEDDS. Samples were collected on the last day of treatment. The levels of AST and ALT in infected and non-treated control group were measured on the 15th day of infection. *Different compared to non-infected control, with p < 0.01.

32

Table 1 - Solubility of benznidazole in different SEDDS excipients

Oil

Surfactant

Co-solvents 1

Excipient

Benznidazole solubility (mg/ml)

HLB1

Miglyol 810®

0.37 ± 0.02

-

Soy oil

0.42 ± 0.03

-

Sunflower oil

0.01 ± 0.00

-

Capryol-90®

2.43 ± 0.04

5

Cremophor® EL

11.58 ± 0.11

12 -14

Labrasol®

11.61 ± 2.86

14

Tween 80®

11.76 ± 0.43

15

Lipoid®S75

ND

-

Ethanol

10.04 ± 3.74

-

N,N dimethylacetamide

105.88 ± 15.86

-

N-methyl-pyrrolidone

287.50± 3.17

-

HLB= Hydrophilic Lipophilic Balance, ND= Not determined

Table 2 – Physicochemical characterization and stability of SEDDS droplets after selfemulsification just after preparation and after 60 days Blank-SEDDS

BZ-SEDDS

Initial

After 60 days

Initial

After 60 days

BZ visual precipitation

NA

NA

no ppt

no ppt

Droplet size (nm)

282.7 ± 13.0

299.9 ± 11.93

496.5 ± 25.5*

485.8 ± 51.44

Polydispersity index (PdI)

0.498

0.480

0.548

0.524

Zeta potential (mV)

-46.6 ± 1.81

-48.1 ± 3.78

-45.4 ± 2.75

-60.9 ± 1.01**

Benznidazole (mg/ml)

NA

NA

26.50 ± 1.56

26.10 ± 1.23

Anhydrous SEDDS formulations were previously emulsified in ultra-pure water at 37°C at 1:1000 dilution and stirred for 20 seconds. NA= Not applicable, ppt: BZ precipitates.* (p<0.05) compared with Blank-SEDDS; **(p<0.05) compared with initial time.

33

Table 3: Robustness towards dilution of self-emulsifying drug delivery system in water Dilution

Blank-SEDDS

BZ-SEDDS Mean PdI

Labrasol final conc. (% v/v)

BZ precipitation

319.7 ± 5.1

0.641

0.3000

No

0.498

427.8 ± 11.5

0.647

0.1500

No

252.7 ± 0.6

0.374

283.3 ± 2.9

0.322

0.0600

No

282.7 ± 13.0

0.498

496.5 ± 25.5

0.548

0.0150

No

v/v *

Size (nm)

1:50

298.4 ± 24.9

0.521

1:100

286.4 ± 7.4

1:250 1:1000

Mean PdI

Size (nm)

*SEDDS formulations were previously emulsified in ultra-purified water at 37°C at different dilution and mixed for 20 seconds. NA= Not applicable. Sign of BZ precipitation or crystallization on diluted dispersion was verified up to 48 h in light microscope.

Table 4 - Effect of benznidazole and BZ-SEDDS on the course of mice infection with Y strain of Trypanosoma cruzi 1 Group (Infection status/Treatment)

Parasitaemia Negative results – clearance/ (days of FBE and treatment) PCR/animals 2

Number of surviving/ animals3

Non-infected control

-

6/6 (100%)

6/6 (100%)

Non-infected – Blank-SEDDS

-

6/6 (100%)

6/6 (100%)

Infected control

0/7

0/7 (0%)

0/7 (0%)

Infected treated BZ

7/7 (1.00 ± 0.00)

4/7 (57.1%)

7/7 (100%)

Infected treated – Blank SEDDS

0/7 ND

0/7 (0%)

0/7 (0%)

Infected treated SEDDS-BZ

7/7 (1.14 ± 0.38)

4/7 (57.1%)

7/7 (100%)

1

Swiss mice (18 to 22 g) were inoculated with 5×103 trypomastigotes of Trypanosoma cruzi Y strain. Treatment stared 4 days after infection and continued for 20 days. 2 FBE (Fresh Blood Examination) before and after immunosuppression with cyclophosphamide. PCR (Polymerase Chain Reaction) assays were performed 30 and 180 days after treatment. BZ: benznidazole.

34

Graphical Abstract

35