Enhanced delivery of fixed-dose combination of synergistic antichagasic agents posaconazole-benznidazole based on amorphous solid dispersions

Accepted Manuscript Enhanced delivery of fixed-dose combination of synergistic antichagasic agents posaconazole-benznidazole based on amorphous solid dispersions

Camila Bezerra Melo Figueirêdo, Daniela Nadvorny, Amanda Carla Quintas de Medeiros Vieira, Giovanna Christinne Rocha de Medeiros Schver, José Lamartine Soares Sobrinho, Pedro José Rolim Neto, Ping I. Lee, Monica Felts de La Roca Soares PII: DOI: Reference:

S0928-0987(18)30189-1 doi:10.1016/j.ejps.2018.04.024 PHASCI 4487

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

9 January 2018 26 March 2018 17 April 2018

Please cite this article as: Camila Bezerra Melo Figueirêdo, Daniela Nadvorny, Amanda Carla Quintas de Medeiros Vieira, Giovanna Christinne Rocha de Medeiros Schver, José Lamartine Soares Sobrinho, Pedro José Rolim Neto, Ping I. Lee, Monica Felts de La Roca Soares , Enhanced delivery of fixed-dose combination of synergistic antichagasic agents posaconazole-benznidazole based on amorphous solid dispersions. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), doi:10.1016/j.ejps.2018.04.024

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ACCEPTED MANUSCRIPT

Enhanced delivery of fixed-dose combination of synergistic antichagasic agents posaconazole-benznidazole based on amorphous solid dispersions

Camila Bezerra Melo Figueirêdoa; Daniela Nadvornyb; Amanda Carla Quintas de Medeiros Vieiraa; Giovanna Christinne Rocha de Medeiros Schverc; José Lamartine Soares Sobrinhob;

a

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Pedro José Rolim Netob; Ping. I. Leec; Monica Felts de La Roca Soaresb*.

CAPES scholarship holder, PVE Program. CAPES Foundation, Ministry of Education of

Brazil, Brasília - DF 70040-020, Brazil.

Department of Pharmaceutical Sciences, Universidade Federal de Pernambuco, Avenida

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Professor Arthur de Sá, SN – Cidade Universitária. Recife – PE, 50740-52, Brazil.

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*Correspondenting author: [email protected]. Phone: +5581-32721383. Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of

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Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada.

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Abstract

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Posaconazole (PCZ) and benznidazole (BNZ) are known to show synergetic effect in treating the acute and chronic phases of Chagas disease, a neglected parasitic disease. However, as

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both compounds are poorly water soluble, the development of amorphous solid dispersions (ASDs) of a PCZ/BNZ fixed-dose combination in a water-soluble polymer becomes an attractive option to increase their apparent solubility and dissolution rate, potentially improving their oral bioavailability. The initial approach was to explore solvent evaporated solid dispertion (SD) systems for a PCZ/BNZ 50:50 (wt. %) combination at several total drug loading levels (from SD with 10% to 50% drug loading) in water-soluble carriers, including polyvinylpyrrolidone (PVP K-30) and vinylpyrrolidone–vinyl acetate copolymer (PVPVA 64). Based on comparison of non-sink in vitro dissolution performance, ASD systems based

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ACCEPTED MANUSCRIPT on PVPVA was identified as the most effective carrier for a 50:50 (w/w %) fixed-dose combination of PCZ/BNZ to increase their apparent solubility and dissolution rate, mainly at 10 % drug loading, which shows more expressive values of area under the curve (AUC) (7336.04 ± 3.77 min.μL/mL for PCZ and 15795.02 ± 7.29 min.μL/mL for BNZ). Further characterization with polarized microscopy, powder X-ray diffraction, and thermal analysis

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reveals that there exists a threshold drug loading level at about 30% PCZ/BNZ, below which ASDs are obtained and above which a certain degree of crystallinity tends to result. Moreover, infrared spectroscopic analysis reveals the lack of hydrogen bonding interactions between the drugs (PCZ and BNZ) and the polymer (PVPVA) in the ASD, this is also confirmed through molecular dynamics simulations. The molecular modeling results further

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show that even in the absence of meaningful hydrogen bonding interactions, there is a greater tendency for PVPVA to interact preferentially with PCZ and BNZ through electrostatic

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interactions thereby contributing to the stability of the system. Thus, the present SD system has the advantage of presenting a fixed-dese combination of two synergistic antichagasic

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agents PCZ and BNZ together in amorphous form stabilized in the PVPVA matrix with enhanced dissolution, potentially improving their bioavailability and therapeutic activity in

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treating Chagas disease.

Keywords: Trypanosoma cruzi; Chagas disease; amorphous solid dispersion; apparent

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solubility; dissolution rate; fixed-dose combination.

Chemical compounds:

Posaconazole (PubChem CID: 468595); Benznidazole (PubChem CID: 31593); Vinylpyrrolidone – vinyl acetate copolymer – PVPVA (PubChem CID: 270885); Polyvinylpyrrolidone – PVP (PubChem CID: 6917);

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ACCEPTED MANUSCRIPT 1. Introduction

Chagas disease or American trypanosomiasis is a neglected disease, which is caused by the parasite Trypanosoma cruzi (T. cruzi) affecting about 6 to 7 million people worldwide mostly in Latin America. The current therapy for the Chagas disease has been relying on the

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antiprotozoal drug benznidazole (BNZ), the only commercially available medicine supplied by the Pernambuco State Pharmaceutical Laboratory (LAFEPE) of Brazil (DNDi, 2016). This drug is effective only during the acute phase of the disease (De Andrade et al., 1996), but its serious side effects have reduced patient compliance to treatment and caused temporary discontinuation of treatment in many cases (Da Silva et al., 2012). In addition, BNZ is poorly

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water-soluble drug with an aqueous solubility of 0.4 mg/mL (Kasim et al., 2004) and a variable oral bioavailability. Thus, the development of a suitable pharmaceutical system to

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enhance the apparent solubility and dissolution rate of the drug becomes highly desirable in providing more effective therapy. The increased bioavailability should allow this drug to

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achieve more optimal concentrations at therapeutic targets with greater exposure in the affected tissue, thereby decreasing the required dose and the toxicological effects associated

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with this drug (Lamas et al., 2006; Morilla et al., 2005). However, the restrictive biological action of BNZ during the acute phase of the Chagas disease is still a limitation of this drug.

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On the other hand, Posaconazole (PCZ) is a known antifungal agent, which has also

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been investigated against T. cruzi. This drug has demonstrated in vitro and in vivo efficacy during the acute and chronic phase of the disease, acting as an inhibitor to ergosterol biosynthesis, which is necessary for the survival, development and growth of this parasite (Da Silva et al., 2012; Ferraz, 2005). The antitrypanosomal activity of PCZ is also reported in studies conducted in patients in the chronic phase of Chagas disease with a dose of up to 400 mg (Molina et al., 2014). Furthermore, a combined therapy of PCZ with BNZ on T. cruzi infection has been envisioned as an ideal approach since it may improve treatment efficacy whilst decreasing toxicity and the likelihood of resistance development. This therapeutical

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ACCEPTED MANUSCRIPT combination was more efficacious in reducing parasitemia levels than the individual drugs given alone, suggesting a synergistic activity of these drugs (Diniz et al., 2013). PCZ can be partially dissolved in a strongly acidic aqueous solution at pH 1 or lower, where it has a solubility of about 790 μg/mL. In contrast, at pH > 4, PCZ has a low aqueous solubility of less than 1 μg/mL (Fang et al., 2011). Due to this compound’s extremely low

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aqueous solubility and the promising activity against T. cruzi, mainly at the chronic phase of the Chagas disease for which there is no satisfactory treatment available, it would be highly desirable to provide this compound together with BNZ in a suitable pharmaceutical formulation capable of increasing the solubility and bioavailability of both drugs, thus providing a combination therapy capable of promoting greater antichagasic action in acute

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and chronic phases.

Although pharmaceutical dosage forms containing either PCZ or BNZ individually

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are known, a suitable pharmaceutical composition capable of delivering these two drugs together with enhanced dissolution is still lacking. Recently, the enhancement of dissolution

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rate through forming eutectic mixture and solid solution of PCZ and BNZ has been proposed (Figueirêdo et al., 2017), however the development of suitable formulations for these drug

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combinations is still required. The use of an amorphous solid dispersion (ASD) system of these two poorly water-soluble drugs in an appropriate polymer matrix provides an efficient

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alternative to enhance the apparent solubility and dissolution rate of both drugs together.

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Thus, the objective of this study is to investigate the impact of widely used watersoluble polymeric excipient polyvinylpyrrolidone (PVP K-30) or vinylpyrrolidone – vinyl acetate copolymer (PVPVA 64) as solid dispersion (SD) carriers on the apparent solubility and dissolution rate of solid dispersions of a fixed-dose combination of PCZ and BNZ. Although ternary solid dispersion systems involving one drug and two different polymers have been investigated in the literature (e.g. Prasad et al., 2014; 2016), to the best of our knowledge a solid dispersion system containing a fixed-dose combination of two drugs with a single polymer has not been reported. The SD samples were prepared by the conventional solvent evaporation method. To correlate in vitro dissolution results with the corresponding

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ACCEPTED MANUSCRIPT solid-state properties, a comprehensive characterization of solid dispersion samples using powder X-ray diffraction, thermal analysis, polarized microscopy, spectroscopic and molecular modeling methods was carried out with a goal to identify an ASD having the greatest improvement in apparent solubility and dissolution rate. The present study has the potential advantage of being able to provide a PCZ/BNZ fixed-dose combination dosage form

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with enhanced dissolution potentially leading to improved absorption of both poorly soluble drugs thus providing a more effective therapeutic arsenal for treating Chagas disease.

2. Material and methods

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2.1 Materials

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Posaconazole (PCZ, batch No. PO-20150201-01) was purchased from ScinoPharm Shangai Biochemical Technoloy, Ltd. (China). Benznidazole (BNZ, batch No. 301045) was

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kindly provided by Nortec Química (Brazil). Copovidone, vinylpyrrolidone-vinylacetate copolymer (PVPVA 64; Kollidon® VA 64 Fine), and polyvinylpyrrolidone (Kollidon® PVP

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K-30) were kindly provided by BASF. All other chemicals and solvents were reagent-grade

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2.2 Methods

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obtained commercially and used as received.

2.2.1 Preparation of SD systems and physical mixtures

Appropriate amounts of the polymer and the antichagasic drugs were first dissolved in methanol to prepare SDs of PCZ/BNZ at a combination ratio of 50:50 (w/w %) in the polymeric carriers (PVP K-30 and PVPVA 64) with different drug loading levels (in weight percentages of drug in SD). The fixed-dose combination of 50:50 (w/w %) PCZ/BNZ was selected as a reference drug composition for this study. It is understood that other ratios of

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ACCEPTED MANUSCRIPT drug combination can be similarly examined. The obtained drug/polymer solution was then spread in a Teflon dish and the solvent removed in a drying oven under elevated temperature (≤70 ºC) for 6 h. The resulting SD in thin cast film was first analyzed by polarized microscopy, and then ground with mortar and pestle in the presence of a small amount of liquid nitrogen to avoid heat-induced crystallization. The resulting powder with particle size

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in 75-150 μm was collected by sieving and further analyzed by X-ray diffraction (XRD), in vitro dissolution, differential scanning calorimetry (DSC), and Infrared (IR) spectroscopy. A molecular dynamics simulation was also used to analyze the potential chemical interactions between the SD components (drugs and polymer).

As reference materials, physical mixtures (PMs) were prepared by mixing the drug

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PCZ/BNZ and the selected polymer in appropriate amounts corresponding to the respective

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SDs.

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2.2.2 Polarized Microscopy

The morphology and microstructure of the SD and the PM samples were analyzed at

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10x magnification using a polarizing microscope (Motic BA400®) equipped with a Dino-eye eyepiece camera controlled by the software DinoCapture® 2.0. This technique allows the

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visualization of crystallite formation thus identifying the maximum loading level of

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PCZ/BNZ that can remain dissolved in each carrier.

2.2.3 X-ray diffraction (XRD)

The physical states of prepared SD and PM powder samples were analyzed on a Rigaku Miniflex II XRD system (Rigaku Co., Tokyo, Japan). The experimental parameters included a scan rate of 2 ° per min over a 2θ range of 2.1–50 °. This XRD analysis provides a quantitative measure of the resulting crystallinity and a confirmation of the maximum concentration of PCZ/BNZ that can remain amorphous in each polymeric carrier.

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2.2.4 Dissolution testing

Dissolutions profiles of SDs of PCZ/BNZ (50:50) in polymeric carriers were compared with that of the crystalline drugs and their respective PMs, all at 75-150 μm (as

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mentioned in Sec. 2.2.1). In order to evaluate the true performance of each SD system, a nonsink condition was maintained during dissolution to build up the supersaturation as commonly encountered under finite volume conditions in the gastrointestinal tract, and to allow for nucleation and crystallization events to proceed (Sun et al., 2012).

The in vitro dissolution was conducted in a small vessel with 10 mL of dissolution

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medium (Hydrochloric Acid Buffer pH 1.6; USP 39), stirred with a magnetic stirring bar at 60 rpm, and maintained at 37 ºC ± 0.5 °C in an incubator. The pH 1.6 dissolution medium was

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selected as it corresponds to a biorelevant condition at the stomach pH (Jantratid et al., 2009) and PCZ has a higher solubility in this medium favoring its quantification, since at pH> 4 this

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drug has a solubility less than 1 μg / mL (Fang et al., 2011), below the quantification limit by UV spectrophotometric analysis. A first-derivative UV spectroscopic method was employed

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to resolve overlapping bands between PCZ and BNZ (Owen, 1996; Saakov et al., 2013). In this case, a wavelength of 345 nm was used for quantifying BNZ and 265 nm for PCZ. At

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each time interval, an aliquot of 100 μL from the dissolution medium was removed and then

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centrifuged at 14000 rpm for 5 min. The supernatant was removed and diluted with dissolution medium before scanning on a Cary 50 UV-VIS spectrophotometer from 200-600 nm at a scan rate of 60 nm/min with an average data acquisition time of 0.1 s. Since samples removed were diluted, the removed aliquots were not returned to the dissolution medium. Triplicate measurements were run and average values with standard deviations are presented for all dissolution results. Standard solutions of 2.5, 5.0, 7.5, 10.0 and 15.0 μg/mL PCZ/BNZ (50:50) were prepared by dissolving the drugs in a small volume of acetonitrile followed by diluting it with the dissolution medium.

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ACCEPTED MANUSCRIPT A dimensionless Sink Index (SI) defined by the equation SI = CSV/(dose) was utilized here to quantify the extent of deviation from the sink condition in all dissolution experiments, where ‘CS’ is the equilibrium solubility of crystalline drug in the dissolution medium, ‘V’ the volume of dissolution medium and ‘dose’ the total amount of drug in the test sample (Sun et al., 2012). In this case, an SI of 10 or greater represents a “perfect sink” condition, whereas

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lower SI values indicate “less sink” or “non-sink” conditions. For all ASDs containing PCZ/BNZ (50:50), the SI was maintained at a constant value of 0.03 for PCZ and 0.07 for BNZ in all dissolution experiments by adjusting the total dose size while recognizing differences in the measured equilibrium solubility of these two drugs in the dissolution medium (see Section 2.2.5). Thus, depending on the drug loading level, the typical sample

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size of a SD prepared at 10% drug loading is 320.0 mg (i.e., 288 mg of polymer + 32 mg of drugs, being 16 mg of PCZ + 16 mg of BNZ), at 20% is 160.0 mg (i.e., 128 mg of polymer +

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32 mg of drugs), at 30% is 106.6 mg (i.e., 74.62 mg of polymer + 32 mg of drugs), at 40% is 80.0 mg (i.e., 48 mg of polymer + 32 mg of drugs) and at 50% is 64.0 mg (i.e., 32 mg of

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polymer + 32 mg of drugs), all having the same dose of 16 mg of PCZ + 16 mg of BNZ, in a

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constant dissolution volume of 10 mL.

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2.2.5 Determination of equilibrium solubility of PCZ and BNZ

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The measurements of equilibrium solubility of PCZ and BNZ were conducted in the dissolution medium (Hydrochloric Acid Buffer pH 1.6; USP 39) with or without polymers at 37 ºC ± 0.5 °C. An excess of a selected drug was added to a glass vial containing 1 mL of dissolution medium either with added polymer (at the maximum concentration of the polymer used in a SD) or without polymer. The vials were hermetically sealed and shaken in an incubator at 37 ºC ± 0.5 °C for 5 days. The experiments were carried out in triplicate. Afterward, the resulting suspension was centrifuged at 14000 rpm for 5 min and the supernatant removed and diluted with the dissolution medium before being quantified by the UV spectrophotometric analysis as described in the previous section.

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2.2.6 Differential Scanning Calorimetry (DSC)

A TA Instrument Q200 differential scanning calorimeter (DSC, TA Instruments, Delaware, USA) was used to analyze the thermal properties of the most promising SDs

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selected based on the dissolution experiment results. Approximately 5 mg of SD or PM prepared as described above was weighed into an aluminum pan on a Mettler Toledo Precision Scale and cold-sealed. The reference pan was left empty and sealed the same way. The sample and reference were heated from ambient temperature to 225 °C at a heating rate of 10 °C/min. A nitrogen blanket at a flow rate of 50 mL/min was used for each run. The

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ºC) and Zinc (419.6 ± 0.3 ºC) standards.

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melting measurement of the equipment was previously calibrated using Indium (156.6 ± 0.3

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2.2.7 Fourier-Transform Infrared Spectroscopy (FTIR)

The most promising SDs identified based on the dissolution results were also

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investigated for the intermolecular interactions between the drugs (PCZ and BNZ) and the

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polymer, by comparing the FTIR spectra of the parent components with that of the SD and its corresponding PM. The analysis was carried out using an FTIR spectrophotometer (Spectrum

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400®, Perkin Elmer) with Attenuated Total Reflectance (ATR) attachment. Each result was obtained from an average of ten scans, from 600 to 4000 cm-1 at a resolution of 4 cm-1.

2.2.8 Molecular modeling

The interactions of PCZ and BNZ with the selected polymer (based on the most promising SD from the dissolution test) were also evaluated theoretically by molecular dynamics (MD). To perform the MD simulations, the chemical structures of the molecules involved were generated from the HyperChem (TM) Professional 7.51 program. The topology

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ACCEPTED MANUSCRIPT files required for the molecular dynamics calculations were obtained through the PRODRG online server (Schüttelkopf and Van Aalten, 2004). In order to perform the calculations, a simulation box containing 100 PVPVA dimers, 4 PCZ molecules and 9 BNZ molecules was generated, thus reflecting the same molecular composition of the most promising ASD identified in Sec. 3 of this study [i.e. SD in PVPVA containing 10% PCZ/BNZ (50:50

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w/w%)]. Initially the system was subjected to a process of energy minimization followed by the production stage (i.e. molecular dynamics run). The simulation was performed for 200 ns, at constant temperature and pressure of 300 K and 1 bar, respectively. In order to carry out the calculations, the program GROMACS 4.6.5 parameterized with force field GROMOS 53A6 (Berendsen et al., 1995; Oostenbrink et al., 2005; Oostenbrink et al., 2004) was used. The

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results were analyzed for Root Mean Square Deviation (RMSD), time and number of hydrogen bonds and interaction energy (between drugs and with the polymer). The VMD

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Program (Visual Molecular Dynamics, Theoretical and Computational Biophysics Group, University of Illinois and Beckman Institute, Urbana, IL) and Xmgrace (Humphrey et al.,

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1996) were used to analyze the results. Density functional theory (DFT) calculations were also performed to find the lower energy geometry of BNZ, as well as in the evaluation of its

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Van der Waals ray.

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3. Results and Discussion

3.1 Characterization of solid dispersions by polarized microscopy and XRD

For the present study, widely used water-soluble polymers PVP K-30 and PVPVA 64 were initially selected as SD carriers for amorphous drugs since an enhanced release of both PCZ and BNZ was desired. These polymers also exhibit high glass transition temperatures (Tg) (e.g. PVP K-30: Tg=164 ºC; PVPVA: Tg=101 ºC) (BASF, 2008) thus reducing the mobility of entrapped drugs. Their high solublility in water also improves the drug’s wettability when the SDs are dispersed in water (Vo et al., 2013) and the high Tg reduces the

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ACCEPTED MANUSCRIPT molecular mobility thereby enhancing the physical stability of SDs (Van Duong and Van den Mooter, 2016). Similar molecular weights (MW) of these carrier polymers were selected since polymer MW plays an important role in affecting the molecular mobility and the recrystallization tendency of the drug in a SD: PVP K-30 – 54.000 g/mol, and PVPVA –

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45.000 g/mol (BASF, 2008; Meng et al., 2015). Typically, in any given SD system there exists a threshold drug loading level (or the miscibility limit) in the carrier polymer above which amorphous to crystalline transition tends to occur thus affecting the physical stability of the SD (Sun et al., 2012). In this study, SD samples were prepared containing PCZ/BCZ (50:50 w/w%) at a total drug loading level of

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10-50 % to facilitate the determination of such threshold drug loading levels. The SDs so obtained were analyzed by polarized microscopy and XRD to determine the limiting or

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threshold drug concentration below which the drugs can be maintained in a dissolved or amorphous state. Here, the formation of a transparent and homogeneous film in the resulting

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SD sample prepared by the solvent evaporation method indicates miscibility between the drug and the polymer carrier forming an amorphous solid solution, as confirmed by the lack of any

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crystalline birefringence under polarized microscopy. Micrograph images under polarized microscopy comparing the SD with the

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corresponding PM samples at different total drug loading levels are represented in Fig. 1. It

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can be seen clearly that crystalline PCZ and BNZ, either individually or in combination, show the typical crystalline birefringence whereas the amorphous polymer background lacks such birefringence. Thus, the presence of crystalline drugs can be easily differentiated from the background polymer in the SD and PM samples by this technique.

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Fig. 1. Micrograph images obtained using polarized microscopy on SD of PVP K-30 and PVPVA, and their corresponding PM samples containing PCZ/BNZ (50:50) at various total

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drug loading levels. Magnification:10 X.

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ACCEPTED MANUSCRIPT The crystalline drugs can be clearly visualized in the PM samples at all drug concentrations (only the lowest and highest concentrations are shown) and in the corresponding SD samples at the highest level of PCZ/BNZ loading (Fig. 1). The presence of the crystalline drugs in the PM samples was also confirmed by XRD analysis showing characteristic peaks over the 2-theta range with varying intensities (see bottom two XRD

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spectra in Fig. 2), where an overlapping of the X-ray diffraction patterns of the three components (PCZ, BNZ and polymer) can be seen with the intensity of crystalline drug peaks

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increasing with the loading level of PCZ/BNZ.

Fig. 2. Comparison of XRD spectra of PCZ, BNZ, and SDs of PCZ/BNZ (50:50) combination

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in PVP K-30 and PVPVA and their corresponding PM samples at various PCZ/BNZ total drug loading levels.

As shown in the polarized micrographs of Fig. 1, SDs with intermediate drug loading levels of 10% to 30% in PVP K-30 and PVPVA, do not show any crystalline birefringence indicating the existence of amorphous PCZ and BNZ dispersed in these polymer carriers. On the other hand, SDs with high drug loading of 50% in PVP K-30 and 40% in PVPVA, all show the appearance of some crystalline drug under polarized microscopy (Fig. 1), and such

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ACCEPTED MANUSCRIPT crystalline characteristics is further revealed in the corresponding PCZ and BNZ peaks in their respective XRD spectra (Fig. 2). It is observed that at an intermediate 40% drug loading level in PVP K-30, very limited birefringence is observed suggesting the existence of only a few crystals (Fig. 1) while the majority area of the sample appears to be free of any crystallized drug as supported by the lack of crystalline peaks in the corresponding XRD

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spectra in Fig. 2. The XRD results also reveal a characteristic amorphous halo in SD samples with 10% to 40% drug loadings in PVP K-30, and 10% to 30% drug loadings in PVPVA (Fig. 2). This suggests the formation of amorphous solid dispersion (ASD), i.e. the antichagasic compounds PCZ and BNZ in the SD samples are mostly present in the amorphous state. From the present

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analysis, as is also visualized under polarized microscopy, the threshold loading level of the PCZ/BNZ (50:50) fixed-dose combination in SDs is about 40% in PVP K-30 and about 30%

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in PVPVA.

It is generally known that an increase in polymer concentration in a SD causes a delay

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in recrystallization of the dissolved drug due to an increase in the kinetic barrier, thereby preventing phase separation between the drug and polymer (Janssens and Van den Mooter,

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2009). Thus, both the polarized microscopy and XRD techniques employed here give similar threshold limits of PCZ/BCZ loading for maintaining SDs in the amorphous state in selected

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polymers, however the polarized microscopy is more sensitive in detecting the first

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appearance of drugs crystals in a SD system.

3.2 In vitro Dissolution

In order to compare dissolution profiles of obtained SD samples, in vitro dissolution experiments were conducted under non-sink conditions, since it takes into consideration the supersaturation capacity of a SD sample in a limited volume of dissolution medium, allowing one to track the supersaturation, nucleation and crystallization events during drug dissolution. Such dissolution conditions commonly occur under finite volume dissolution in the

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ACCEPTED MANUSCRIPT gastrointestinal track (Sun et al., 2012). Under non-sink dissolution conditions, both the dissolution rates and the supersaturation levels obtained from ASDs have been reported to be typically higher with water-soluble (hydrophilic) carriers compared to systems with waterinsoluble (hydrophobic) carriers (Sun and Lee, 2015a). However, the fastest dissolving system does not necessarily produce the best in vivo performance as rapid generation of a

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highly supersaturated solution could lead to rapid crystallization, therefore non-sink dissolution conditions are essential to provide a rational comparison of different SD polymer carriers (Augustijns and Brewster, 2012; Newman et al., 2012; Sun and Lee, 2015b). The dose of the SD and PM used in this analysis was selected based on considerations of equilibrium drug solubility determined in the pH 1.6 buffer, which is 54.52 μg/mL for PCZ

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and 115.89 μg/mL for BNZ. Thus, the SI of the dissolution experiments in all systems (SD and PM) was maintained at predetermined levels for PCZ and BNZ by adjusting the dose as

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described in Section 2.2.4.

The resulting non-sink dissolution profiles of SD samples at different drug loadings

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and their corresponding PM samples at 10% PCZ/BNZ loading versus that of the reference

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crystalline PCZ and BNZ are compared in Fig. 3.

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Fig. 3. Comparison of non-sink dissolution profiles of SD samples of PCZ/BNZ (50:50) in

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PVP K-30 and PVPVA (from top to bottom) at various total drug loading levels, with their corresponding PM samples at 10% total drug loading level. The Cmax (maximum

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concentration) in each graph is highlighted with a red arrow.

In order to establish a ranking order as to the overall in vitro performance of different SD formulations, area under the curve (AUC0–24h) values of the kinetic supersaturation concentration-time profiles of Fig. 3 have been calculated and compared in Fig. 4. This AUC is indicative of the supersaturation maintenance (Dinunzio et al., 2008; Miller et al., 2008), and has been used as a surrogate for the in vivo bioavailability (Brouwers et al., 2009; Lee et al., 2011; Sun and Lee, 2013; Han and Lee, 2017). The higher the AUC value here, the better is its performance in the in vitro dissolution test.

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Fig. 4. Comparison of AUC0–24h values and their respective standard deviation from the non-

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sink in vitro dissolution of SDs having different PCZ/BNZ (50:50) loading levels and their

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correspondent PMs at 10% PCZ/BNZ loading.

Considering the AUC0–24h values of the crystalline PCZ (91390.35 ± 4.99

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min.μL/mL) and BNZ (2811.65 ± 5.12 min.μL/mL) as references, it is evident from Fig. 3 and 4 that all tested SD samples exhibit an increase in dissolution rate and a higher AUC compared to that of the crystalline PCZ and BNZ, and to their corresponding PMs at 10% drug loading irrespective of the SD polymer carrier used. Additional drug loading levels in the PM were not fully evaluated since their dissolution profiles were similarly slow as that of the 10% samples because the same amount of crystalline drugs were present in each of these PM samples thereby exhibiting a similarly slow dissolution as compared to the corresponding SD samples.

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ACCEPTED MANUSCRIPT As expected, these amorphous SD systems based on different water-soluble polymers (PVP and PVPVA) releases the drugs quickly during dissolution and achieved a maximum supersaturation (Cmax) for both PCZ and PBZ in less than 1 h, as indicated by the red narrows in Fig. 3. This initial surge of supersaturation is followed by a decline in drug supersaturation due to supersaturation-induced drug precipitation before approaching a quasi-

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steady state drug concentration, often above saturation. Depending on the polymer used, such precipitation may be retarded to different degrees. It is also seen that, in general, the higher the drug loading (or the lower the polymer content) in the SD system, the poorer its dissolution performance (i.e. lower initial rate of drug release and lower AUC). In other words, the SD with the lowest drug load (i.e. 10%) exhibits the fastest supersaturation buildup

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(Fig. 3) with the highest AUC (Fig. 4). However, SD samples with 20%, 30%, 40% and 50% drug loading can be seen to exhibit a progressive decline in dissolution performance (lower

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AUC) comparing with the SD of 10% drug loading.

This observed reversal of drug loading effect on drug release (i.e., slower release and

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lower AUC at higher drug loadings) is similar to that reported for SD systems based on other hydrophilic polymer carriers (Law et al., 2004; Sun et al., 2012). From a mechanistic point of

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view, when the drug is present as a minor component in the SD, the release of the drug would be dominated by the dissolution behavior of the water-soluble polymer carrier (Craig, 2002)

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leading to a carrier-controlled dissolution with fast initial drug release at a lower drug loading

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of 10%. However, at higher loading levels of the poorly soluble PCZ/BNZ (50:50), the hydrophobicity of the resulting SD sample increases and potential phase separation may be induced by the penetrating aqueous medium, thereby leading to a drug-controlled dissolution process showing an overall reduced initial dissolution rate and AUC at higher drug loadings (Craig, 2002; Law et al., 2004; Higashi et al., 2015). This trend with respect to increasing drug loading is clear for all SDs studied in Fig. 3 and 4, except for the PCZ SDs in PVP K-30 where such trend is less obvious (top figure in Fig. 4). This could be attributed to the unusual closeness between the plateaued PCZ SD dissolution profiles in the later precipitation phase

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ACCEPTED MANUSCRIPT (top left figure in Fig. 3), where a very small variation in the PCZ plateau concentration (~200 μg/mL) could give rise to these observed fluctuations in AUC (top figure in Fig. 4). Although the dissolution profiles show poorer performance for PMs than SDs, most of their AUC values in Fig. 4 are significantly better than that with individual drugs alone. This observation suggests that the presence of the polymer may have modified the drug

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solubility and this has been verified by measuring the solubility of PCZ and BNZ in the dissolution medium containing a test polymer. To illustrate this, in the case of PCZ, its solubility in pH 1.6 buffer without polymer is 54.52 μg/mL and it is enhanced to 113.62 μg/mL in PVP K-30 and to 124.53 μg/mL in PVPVA. For BNZ, its solubility is increased from 115.89 μg/mL in pH 1.6 buffer without polymer to 149.67 μg/mL in PVP K-30 and to

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165.91 μg/mL in PVPVA. In addition, the interfacial tension and the wettability of PCZ and BNZ are probably also modified by the polymer solution, promoting the drug dissolution.

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From the analysis of the non-sink dissolution results of Fig. 3 and 4, it is evident that the interplay between the supersaturation generation and precipitation processes during the

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dissolution of the SD system is responsible for the observed kinetic supersaturation profiles. Depending on the nature of the polymer carrier, supersaturation can be temporarily

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maintained in solution until decreasing to a quasi-steady state drug concentration in the desupersaturation phase, often elevated above saturation. Although such elevated kinetic

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solubility could be the result of formation of a metastable polymorph with higher aqueous

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solubility during precipitation such as in the case of indomethacin (Sun and Lee, 2013), it is less likely to be the reason in the present PCZ and BNZ system for the following reasons: (1) Fig. 3 shows that in almost all SD samples the drug concentration in the de-supersaturation phase eventually drops to the same level of that of the PM sample which is higher than the saturation concentration of the crystalline drug because of the solubility enhancement effect due to dissolved polymer as discussed above; and (2) even if metastable polymorphs were produced, there might not be significant differences in solubility between different polymorphs as reported for BNZ (Honorato et al., 2014).

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ACCEPTED MANUSCRIPT 3.3 Differential Scanning Calorimetry (DSC)

Since the most promising SDs selected from the dissolution experiment were those based on PVPVA, DSC was used in this study to verify the physical state of the drugs (amorphous or crystalline) in these SDs, and also to verify the miscibility between these

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components. The DSC thermograms of PCZ/BNZ (50:50) SD and PM samples based on

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PVPVA at different total drug loading levels are shown in Fig. 5.

Fig. 5. DSC thermograms of solid dispersion (SD) and physical mixture (PM) samples of PCZ/BNZ (50:50) in PVPVA as a function of total drug loading. Arrows indicate the melting events of drugs in PM samples.

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In Fig. 5, the crystalline melting behavior of the drugs can be verified by the characteristic melting endotherms at around 169.81 ºC for PCZ and around 192.2 ºC for BNZ. The DSC thermogram of PCZ also shows a minor melting peak at 135 ºC attributable to an impurity in the bulk PCZ drug substance, which corresponds to about 1.9 % of the raw

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material (measured according to the method described by Garcia et al., 2012). The crystalline behavior of the drugs is also seen in the DSC thermogram of the physical mixture (PM) of 40% PCZ/BNZ (50:50) with PVPVA, revealing the minor melting peak of the PCZ impurity (left arrow in its DSC scan near the bottom of Fig. 5) and a single broad melting peak around 154 °C (second arrow in its DSC scan of Fig. 5) attributable to the melting endotherm of the

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PCZ/BNZ eutectic mixture (Figueirêdo et al., 2017). These two melting events are also seen discretely but with less intensity in the DSC scan of the PM sample of 10% PCZ/BNZ (50:50)

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with PVPVA.

The DSC scan of PVPVA exhibits a broad endothermic event between 37 °C and 96

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°C (Fig. 5) relating to the residual water evaporation. This is commonly detected in the DSC analyzes of PVPVA as it is a hygroscopic polymer (Van Duong, Van den Mooter, 2016). This

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same endothermic event is also seen in SD samples, suggesting it is mostly due to moisture absorption from the environment and not from the organic solvent drying process during SD

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preparation. The glass transition temperature (Tg) of PVPVA, which is commonly shown at

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101 ºC (BASF, 2008), appears to be overwhelmed by the water evaporation peak in DSC scans of PVPVA and all SD samples. Similar effect due to moisture absorption also exists in PVP K-30. This makes it difficult to ascertain whether a single Tg corresponding to a single amorphous phase in the SD sample exists so as to infer miscibility. In this case, the evaluation of miscibility from Tg determination will have to be combined with other methods to obtain the true miscibility (Meng et al., 2015). Based on the present observation, it is not possible to confirm directly if these SDs exist as solid solution systems with one single phase. Nevertheless, when combined with earlier results of polarized light microscopy (Fig. 1) and XRD (Fig. 2), these results suggest

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ACCEPTED MANUSCRIPT the formation of one phase amorphous solid dispersion of PCZ and BNZ in PVPVA, and confirm the threshold loading level of the PCZ/BNZ (50:50) drug combination to be up to 30 %, below which PCZ and BNZ are amorphous in PVPVA. Thus, it can be inferred that PVPVA formulations with PCZ/BNZ (50:50) between 10% and 30% are amorphous; thereby giving rise to the highest increase of apparent solubility and dissolution rate for this fixed-

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dose drug combination with this PVPVA polymer in the SD samples, as evidenced in the in vitro dissolution results (Fig. 3).

3.4 Fourier-Transform Infrared (FTIR) Spectroscopy

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FTIR absorption spectrophotometry was used to investigate the physicochemical interactions between the drugs and the polymer carrier in SD and PM samples of PCZ/BNZ in

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PVPVA. This intermolecular interaction is known to improve the compatibility between the drug and the polymer, which may affect the resulting dissolution, physical and stability

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properties of binary solid dispersions (Sun et al., 2012; Kestur and Taylor, 2010; Karavas et al., 2007; Konno and Taylor, 2006) as well as ternary solid dispersions involving one drug

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and two polymers (Prasad et al., 2014; 2016). As indicated earlier, a solid dispersion system containing a fixed-dose combination of two drugs with a single polymer as described here has

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not been studied previously.

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The FTIR spectra of SD and PM systems of PCZ/BNZ in PVPVA are shown in Fig. 6. The PVPVA molecule has only hydrogen bond acceptor groups, including its cyclic amide C=O stretching band (lactam) (at 1668 cm-1) and its ester C=O stretching band (at 1731 cm-1). The BNZ molecule has one hydrogen bond acceptor group with C=O stretching at 1658 cm-1 (amide band I), and one hydrogen bond donor group corresponding to the N-H stretching at 3270 cm-1 (amide band II). The PCZ molecule also has one hydrogen bond acceptor group with C=O stretching at 1685 cm-1, and one hydrogen bond donor group, with the O-H stretching at 3281 cm-1.

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Fig. 6. FTIR spectra of SD and PM samples of PCZ/BNZ (50:50) in PVPVA as a function of total drug loading.

It is clear from Fig. 6 that the above identified vibrational modes relating to possible hydrogen bonding interactions between the drugs and the polymer show no appreciable band shift in all SD samples, suggesting a lack of significant hydrogen bonding interactions between the acceptor and the donor groups of the drugs (PCZ and BNZ) and the polymer (PVPVA) in the ASDs. The molecular dynamic simulation to be discussed in Sec. 3.5 support this result. In this case, other types of interactions between the component molecules may be

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ACCEPTED MANUSCRIPT at play. Non-hydrogen bonding polymers have been used to obtain stable solid dispersions in which dispersive interactions (Van der Waals forces) and electrostatic (polar or induced dipole) interactions may play a bigger role in determining the stability properties of the amorphous solid dispersions (Page et al., 2016). This aspect will be examined with molecular

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modeling in the next section.

3.5 Molecular modeling

In order to elucidate the chemical interactions in the most promising SDs systems prepared in PVPVA, the theoretical analysis based on molecular dynamics (MD) simulation

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was performed for the ASD containing 10% of drug loading in the same ratio of PCZ/BNZ 50:50 w/w % as used in the experiments. The RMSD was calculated to gauge the stability of

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the system throughout the simulation. The resulting RMSD graph (Fig. 7) illustrates that the simulation reaches a stable state around 50,000 ps, remaining steady at around 4.8 nm RMSD

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afterwards.

Fig. 7. RMSD graph during 200 ns (200,000 ps) of MD computer simulation of the system SD PVPVA- 10% PCZ/BNZ 50:50 (w/w %).

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ACCEPTED MANUSCRIPT As shown in Sec. 3.4, the experimental analysis by FTIR absorption spectrophotometry did not reveal any significant hydrogen bonding between the polymer and the drugs (Fig. 6). In fact, the absence of any meaningful hydrogen bonding here can also be

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verified by the theoretical analysis based on molecular dynamics simulation (Fig. 8).

Fig. 8. Number and average half-life of hydrogen bond from over 200 ns of MD simulation of

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the system SD PVPVA-10% PCZ/BNZ 50:50 (w/w %).

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The results of the MD calculation as shown in Fig. 8 suggest an insignificant contribution from hydrogen bonding, as demonstrated by the low values in both the number

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and average half-life of hydrogen bond between the polymer and the drugs (values around 1.0). Considering the example of BNZ molecule, a steric shielding effect exists with the BNZ

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rings which are positioned in the opposite direction of the NH group, preventing it from forming any meaningful hydrogen bonding with another species (polymer PVPVA, PCZ and even BNZ). This shielding effect in the BNZ molecular structure is better visualized through the optimization of its geometry by the DFT calculation and Van der Waals ray analysis (see Fig. 9). Potential hydrogen bonding interactions involving other components of this SD appear to be even less significant (see Fig. 8).

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Fig. 9. Lower energy geometry of BNZ calculated by DFT (left) and representation of the Van der Waals ray for BNZ structure (right).

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Examining visually the structures of the simulated system in Fig.10, it is noted that at the end of the 200 ns simulation the drugs become fully associated with the polymer mesh,

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which works as a substrate for the drug molecules to be dispersed in.

Fig. 10. Structures of the initial (A) and final (B) stage of the simulated system of SD PVPVA-10% PCZ/BNZ 50:50 (w/w %).

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ACCEPTED MANUSCRIPT These finding led to the investigation of possible electrostatic (polar or induced dipole) interactions. The results computed through theoretical calculations are shown in Table 1. Electrostatic energy (Kcal.mol-1)

Groups

-3359.6

PCZ-BNZ

-136.7

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PCZ/BNZ-Polymer (PCZ-PVPVA + BNZ-PVPVA)

PCZ-PCZ

-545.6

BNZ-BNZ

-5.5

Table 1. Electrostatic energy values between components in the system SD PVPVA-10%

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with PCZ/BNZ 50:50 (w/w%).

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Based on Table 1, the large electrostatic interaction of the two drugs with the polymer is the one that promotes the lowering of the total energy of the system (more

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negative values), contributing more to the stability of the system. Thus, the main element responsible for the lowering of the electrostatic interaction energy of the system is the

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electrostatic interaction of PCZ/BNZ with the polymer, which is 25 times more negative compared to that of the two drugs with each other (PCZ-BNZ), 610 times more negative than

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the BNZ-BNZ interaction, and 6 times more negative than the PCZ-PCZ interaction. The

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interpretation of these data therefore suggests that even in the absence of significant hydrogen bonds in the system, there is a greater tendency for molecularly dispersed PCZ and BNZ to be accommodated and stabilized electrostatically in the polymer mesh of PVPVA. The occurrence of the electrostatic polymer-drug interactions realized from this theoretical analysis, as well as the final state of drug association with the polymer mesh for 10% drug loading is also expected to apply similarly to SDs of PVPVA with PCZ/BNZ 50:50 (w/w %) at other drug loading levels (e.g. 20%, 30%, 40%).

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ACCEPTED MANUSCRIPT 4. Conclusions

From the physicochemical characterization results as well as the comparison of in vitro dissolution performance of different amorphous solid dispersion formulations (via the area under the curve - AUC - values of the kinetic supersaturation concentration-time

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profiles), amorphous solid dispersion (ASD) system based on PVPVA was identified as the most effective carrier for ASDs of a 50:50 (w/w %) fixed-dose combination of PCZ/BNZ, the two synergetic drugs for treating acute and chronic phases of Chagas disease, to increase their apparent solubility and dissolution rate. Based on further characterization with polarized microscopy and XRD, it was identified that there exists a drug loading threshold at about

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30%, below which amorphous solid solution is obtained and above which the loaded PCZ/BNZ tends to result in a certain degree of crystallinity. Also, FTIR analysis reveals the

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lack of significant hydrogen bonding interactions between the drugs (PCZ and BNZ) and the polymer (PVPVA) in the ASD. It is worth noting that molecular dynamics (MD) simulation

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confirms the absence of meaningful hydrogen bonding interactions in this system and the theoretical analysis further shows that the electrostatic interaction of PCZ/BNZ with PVPVA

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is the main element responsible for lowering of the total energy, thus contributing more to the stability of the system. In other words, even in the absence of meaningful hydrogen bonding

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in the system, there is a greater tendency for PVPVA to interact preferentially with PCZ and

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BNZ through electrostatic interactions. Therefore, the present SD system has the advantage of providing two synergistic antichagasic agents together in the amorphous form stabilized in a polymer matrix with enhanced dissolution, potentially improving their bioavailability and therapeutic activity in treating Chagas disease.

Acknowledgements

This work was supported in part by funding from PVE Program of CAPES Foundation, Ministry of Education of Brazil.

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Graphical abstract

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