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A New Thermodynamically Favored Flubendazole/Maleic Acid Binary Crystal Form: Structure, Energetics and In Silico PBPK Model-based Investigation Gabriel L. B. de Araujo, Fabio Furlan Ferreira, Carlos E. S. Bernardes, Juliana A. P. Sato, Otávio M. Gil, Dalva L. A. de Faria, Raimar Löbenberg, Stephen R. Byrn, Daniela D. M. Ghisleni, Nádia A. Bou-Chacra, Terezinha J. A. Pinto, Selma G. Antonio, Humberto G. Ferraz, Dmitry Zemlyanov, Débora S. Gonçalves, and Manuel Eduardo Minas da Piedade Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01807 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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Crystal Growth & Design

A New Thermodynamically Favored Flubendazole/Maleic Acid Binary Crystal Form: Structure, Energetics and In Silico PBPK Model-based Investigation Gabriel L. B. de Araujo,*,a Fabio Furlan Ferreira,*,b Carlos E. S. Bernardes,c Juliana A. P. Sato,b Otávio M. Gil,d Dalva L. A. de Faria,d Raimar Loebenberg,e Stephen R. Byrn,f Daniela D. M. Ghisleni,a Nadia A. Bou-Chacra,a Terezinha J. A. Pinto,a Selma G. Antonio,g Humberto G. Ferraz,a Dmitry Zemlyanov,h Débora S. Gonçalves,a Manuel Eduardo Minas da Piedadec a

Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil b

Centro de Ciencias Naturais e Humanas, Universidade Federal do ABC (UFABC), Santo André, SP, Brazil

c

Centro de Química e Bioquímica e Centro de Química Estrutural, Faculdade de Ciências, Universidade de Lisboa, 1649-016 Lisboa, Portugal d

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil. e

Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 f

Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN, United States g

Departamento de Físico-Química, Instituto de Química, Universidade Estadual Paulista, Araraquara, Brazil h

Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana 47907, United States

*corresponding authors: [email protected]; [email protected]

1 ACS Paragon Plus Environment

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ABSTRACT The use of flubendazole (FBZ) in the treatment of lymphatic filariasis and onchocerciasis (two high incidence neglected tropical diseases) has been hampered by its poor aqueous solubility. A material consisting of binary flubendazole/maleic acid crystals (FBZ/MA), showing considerably improved solubility and dissolution rate relative to flubendazole alone, has been prepared in this work through solvent assisted mechanical grinding. The identification of FBZ/MA as a binary crystalline compound with salt character (proton transfer from MA to FBZ) relied on the combined results of X-ray powder diffraction, Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), thermogravimetry (TG), and differential scanning calorimetry (DSC).

Isothermal

solution microcalorimetry studies further suggested that the direct formation of FBZ/MA from its precursors in the solid state is thermodynamically favored. A comparison of the in silico pharmacokinetic performance of the FBZ/MA with that of pure FBZ based on a rat fasted physiology model, indicated that the absorption rate, mean plasma peak concentration, and absorption extension of FBZ/MA were ∼2.6 times, ∼1.4 times, and 60% larger, respectively, than those of FBZ. The results here obtained therefore suggest that the new FBZ/MA salt has a considerable potential for the development of stable and affordable pharmaceutical formulations with improved dissolution and pharmacokinetic properties. Finally, X-ray powder diffraction studies also led to the first determination of the crystal structure of flubendazole.


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Introduction Flubendazol

(FBZ),

methyl

N-[6-(4-fluorobenzoyl)-1H-benzimidazol-2-

yl]carbamate, is an efficient benzimidazole antihelmentic agent developed by Janssen Pharmaceutica in the mid-1970s. Over the past ten years, this drug has been highlighted by the scientific community as a promising candidate to be used in the global control/elimination programs of human lymphatic filariasis (elephantiasis) and onchocerciasis (river blindness), lead by the World Health Organization (WHO).1–3 Together, lymphatic filariasis and onchocerciasis affect over 150 million people living in tropical areas of the World.2,4,5 The marketed flubendazole formulations available have shown, however, low systemic bioavailability due to the poor aqueous solubility of the compound and, consequently, are currently approved only to treat gastrointestinal nematode infections in humans and animals, which do not require the achievement of systemic drug plasma levels.1,6 The considerable potential of flubendazole as a macrofilaricide and the challenges related to its safety and efficacy have been discussed by Mackenzie and Geary1, who also stressed the importance of expediting FBZ reformulation using modern pharmaceutical platforms.3 Albeit promising results against onchocerciasis were obtained in a first clinical trial of an intramuscular FBZ formulation carried out in Mexico, severe inflammation at the injection site was observed.7 Possible relationships between formulation composition and inflammation were not discussed, but the use of solubilizing vehicles could, perhaps, explain, the observed adverse reaction. In recent years, some advances in pre-clinical studies have indicated that flubendazole has the potential to be reformulated through new approaches. Ceballos and co-workers have, for example, developed aqueous cyclodextrin (CD) based formulations with a 75 fold increase in solubility.6 Despite these superior properties, the administration of FBZ to sheep as a cyclodextrin solution, by intra-ruminal (i.r.) or intra-abomasal (i.a.) routes, did not provide a significant pharmacokinetic improvement when compared to a conventional

carboxymethylcelulose

(CMC)

suspension.

These

authors

also

demonstrated that oral and subcutaneous administration of FBZ-CD to rats and jirds produced significantly higher systemic levels than a FBZ-CMC suspension or a polysorbate 80-based solution,1,3,6 and might, therefore, constitute a promising alternative to reformulate flubendazole. Nevertheless, cyclodextrins are expensive



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excipients and they need to be used in significant amounts to promote the solubilization of FBZ (usually dissolving 0.1% FBZ and 10% CD in deionized water). Vialpando et al.8 showed that amorphization approaches, such as those involving ordered mesoporous silica and drug-polymer spray dried dispersions, led to considerable bioavailability improvements in rats when compared to a micronized FBZ suspension. However, the optimal balance of release performance, drug-loading, residual solvents, and stability of the formulation is still a concern.8 When considering the relatively high FBZ dose (750 mg) administered to human volunteers in the intramuscular study mentioned above,7 the cost of production and the challenges for commercial batches (e.g. scalability) associated with the use of cyclodextrin and amorphization approaches may pose significant economic obstacles for developing countries.9 As for all other neglected diseases, the investment on medicines to treat lymphatic filariasis and onchocerciasis is not commercially attractive. Consequently, there is a strong need for the development of formulations that are affordable for low income countries. In the last decade, the use of multicomponent crystals emerged as a promising strategy to improve and optimize key properties of drug substances, such as solubility, dissolution rate, and bioavailability.10,11 Pharmaceutical multicomponent crystals are usually defined as multicomponent single phase crystals that contain a molecular or ionic drug and a coformer (generally in a stoichiometric ratio) that are a solid under ambient conditions.10–12 When the drug and coformer interact by hydrogen bonding different extents of proton transfer may occur: if the proton remains essentially bonded to the original moiety the obtained compound is dubbed a pharmaceutical cocrystal; upon proton transfer a salt is said to be formed. Carbamazepine, meloxicam, lamotrigine, indomethacin, megestrol, gliclazide, tolbutamide, and glipizide are examples of poorly soluble drugs whose solubility has been increased by cocrystal or salt formation.13–16 The present study describes the synthesis and characterization of a new thermodynamically stable binary crystal form consisting of FBZ and maleic acid. Besides being stable, this new material exhibits enhanced pharmaceutical properties relative to FBZ. It offers, therefore, a very interesting potential alternative for the development of affordable pharmaceutical dosage forms based on flubendazole. Additionally the molecular and crystal structure of flubendazole, obtained from powder X-ray diffraction data, is discussed for the first time.


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Experimental Section Materials. Pharmaceutical grade flubendazole (Changzhou Yabang-QH Pharmachem Co., 99.9%), maleic acid (Sigma-Aldrich, >99%), tetrahydrofuran (THF, Merck, HPLC grade), acetonitrile (Merck, HPLC grade), ammonium acetate (Merck, analytical grade), and sodium hydroxide (Merck, analytical grade) were used as received. The DMSO (Sigma ≥99.5%) employed as solvent in the solution calorimetry experiments was not further purified. Water from a Milli-Q system (Millipore, Bedford, MA, USA) was used throughout the work. Flubendazole has very limited solubility in most of common solvents, being only significantly soluble in dimethyl sulfoxide (340.5 g⋅L-1) and dimethylformamide (5.6 g⋅L-1).17 The use of those high boiling point solvents makes cocrystallization from solution inadequate given the impact in the drying step. Consequently, the solvent-drop grinding approach was preferred, since it has proved to be a highly efficient method for cocrystal formation when compared to other techniques.18 To prepare the FBZ/MA crystals, FBZ and maleic acid were mixed in a 1:1 mole ratio, and subjected to solvent assisted mechanical grinding with pestle and mortar for 5 minutes.18–21 The procedure consisted in the addition of 500 µL of THF to 700 mg of an equimolar mixture of API and coformer. The resulting material was dried in an oven at 333 K, with air circulation, for 1 h. Thermal Analysis. Thermogravimetry/derivative thermogravimetry (TG/DTG) experiments in the temperature range 298-873 K, were carried out at a heating rate of 10 K⋅min-1, under a dynamic nitrogen atmosphere (50 cm3⋅min-1), in an Exstar-7200 thermobalance (Hitachi High-Tech Science Corporation). The sample of ~5 mg mass was contained in an open platinum crucible. The calibration of the instrument mass scale was previously verified with a CaC2O4⋅H2O reference standard (Sigma-Aldrich). Differential scanning calorimetry (DSC) studies were performed in an Exstar7020 apparatus (Hitachi High-Tech Science Corporation), using aluminum crucibles containing ∼2 mg of sample. A dynamic nitrogen atmosphere (100 cm3⋅min-1) and a heating rate of 10 K⋅min-1 were used. The temperature and energy scales of the apparatus were calibrated with indium (Tfus = 429.75 K; ∆fush = 28.62 J⋅g-1) and zinc (Tfus = 692.68 K; ∆fush = 108.09 J⋅g-1).



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X-ray Powder Diffraction (XRD) and structure determination. X-ray powder diffraction patterns were recorded at room temperature (298±2 K) on a STADIP powder diffractometer (Stoe®, Darmstadt, Germany) in transmission geometry by using a Kα1 (λ = 1.54056 Å) wavelength emitted by a Cu anode and selected by a curved Ge(111) crystal, with a tube voltage of 40 kV and a current of 40 mA. The X-ray photons were detected by a Mythen 1K linear detector. The measurements were performed in the range from 2º to 61.835º (2θ) with a step size of 0.015º and integration time of 120 s at each 1.05º. The samples were loaded between two acetate-cellulose foils, which were spun during data collection. A rigid body structure of the FBZ chemical structure was built and the simulated annealing approach as implemented in Topas-Academic v.6 package was used to solve its structure.22 Soft bond distance and angle restraints were included as inferred from average values in Mogul.23 After 10,000 iterations, a reliable solution was found and the Rietveld method24,25 was then used to refine the crystal structure. Vibrational Spectroscopy. A Bruker FT-Raman equipment (RFS 100/S) fitted with a liquid nitrogen cooled Ge detector was used in the Raman measurements with excitation at 1064 nm (Nd3+/YAG laser) with laser power of 70 mW at the sample and 4 cm-1 of spectral resolution. The ATR-FTIR spectra were obtained with a Bruker Alpha (DTGS detector and KBr optics), using a single bounce ATR accessory (diamond crystal); spectral resolution was 4 cm-1 and 512 spectra were co-added. The spectra were analyzed with the GRAMS/AI (v. 9.1) package (Thermo Sci.). X-ray Photoelectron Spectroscopy (XPS). XPS data was collected using a Kratos Axis Ultra DLD spectrometer with monochromic Al Kα radiation (1486.6 eV). Pass energies of 20 eV and 160 eV were selected to obtain high-resolution and survey spectra, respectively. A commercial Kratos charge neutralizer was used to avoid nonhomogeneous electric charge of non-conducting powder and to achieve better resolution. The resolution measured as full width at half-maximum of the curve-fitted C 1s and N 1s peaks was approximately 1 eV. The charge correction was done by setting the main C 1s component at 284.8 eV corresponding to C-C or C-H bonds. The position of the main C 1s component was determined through the curve fitting and the resulting standard deviation of peak positions was typical 0.1 eV for all high-resolution regions. XPS data was analyzed with the CasaXPS software version 2.3.13 Dev64 (www.casaxps.com).


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Isothermal Solution Microcalorimetry. Enthalpy of solution measurements at 298.15 K were carried out on a LKB 2277 Thermal Activity Monitor (TAM), equipped with a 15.0 cm3 stainless steel cell including systems for stirring, electrical calibration, and solid sample dissolution.26 The calorimeter was kept in an air-conditioned room whose temperature was regulated to 295±1 K. Instrument control and data acquisition were performed with the CBCAL 3.0 program.27 Three different types of dissolution experiments were made: (i) FBZ/MA in DMSO, (ii) MA in DMSO, and (iii) FBZ in a MA+DMSO solution. The masses of MA and FBZ used in (ii) and (iii) were adjusted so that their combined final state was as similar as possible to that of the FBZ/MA runs. In a typical experiment 11.2±0.9 mg of FBZ-MA, 3.6±0.4 mg of MA, or 8.9±0.2 mg of FBZ (mass ranges correspond to mean deviations) were placed in a silica crucible closed by a detachable lid, and weighed with a precision of ±0.1 µg on a Mettler XP2U ultra-micro balance. The crucible was adapted to the calorimetric cell containing ∼13 g of the calorimetric solvent (DMSO or MA+DMSO solution), which had been weighted to ±10 µg in a Mettler XS 205 balance. The cell was closed, transferred to the thermostat unit, and left to equilibrate. An initial baseline was recorded and the crucible was dropped into the solution. The lid and base separated upon drop, exposing the sample to the calorimetric solvent, and the signal from the dissolution process was monitored until returning to the baseline. The corresponding enthalpy change was calculated from the area A of the measured curve through the equation

∆ sol H mo =

M ε ( A − Ab ) m

(1)

where m and M represent the mass and molar mass of sample, respectively, Ab is the contribution to the measured area from the drop process alone, and ε is the calibration constant. The value of Ab = 1.98±0.76 mV⋅s was determined in a series of four independent experiments where an empty crucible was dropped in the calorimetric solvent. The constant ε = 6.51±0.22 mW⋅mV−1 was obtained from ten electrical calibrations where a potential difference V was applied to a 22 Ω manganin resistance immersed in the calorimetric liquid, causing a current of intensity I to flow during a time t and leading to the dissipation of an amount of heat Q = VIt. The calculation of ε relied on the equation 7 ACS Paragon Plus Environment


∑ V I ∆t i i

ε=

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i

i

(2)

Ac

where Ac is the area of the measured curve, Vi and Ii are the ith voltage and current readings, respectively, during the overall time t of the calibration and ∆ ti is the time difference between two consecutive data acquisitions. The accuracy of the electrical calibration (better than 0.5%) and the efficacy of the dissolution device were previously assessed by measuring the enthalpy of solution of KCl in water.26 Solubility. Solubility studies on pure FBZ, a FBZ+MA physical mixture, and 1:1 FBZ/MA binary crystals, were performed by the saturation shake-flask method28, in simulated gastric fluid without enzymes (pH 1.2)29 and standard phosphate buffer (pH 6.8).30 Briefly, an excess of sample was added to the solubility medium in a sealed vial. The resulting suspension was shaken at 150 rpm and 310 K for 24 h. After this period of time the sample was immediately filtered and diluted in a volumetric flask with a 50:50 (v/v) acetonitrile/water mixture. The filtrate was analyzed by HPLC in a Thermo Scientific Accela apparatus equipped with a Zorbax Eclipse Plus C18 Rapid Resolution HT (100 × 3.0 mm, 1.8 µm). The mobile phase consisted of a buffer solution containing 0.04 M ammonium acetate and a few microliters of acetic acid to adjust the pH at 5.2 (A), and acetonitrile (B). Gradient elution was applied, and the program consisted of 60A:40B (0 min), 60A:40B to 40A:60B (0-8 min), 60A:40B (8-10 min). Separation was performed at 298±2 K using a 0.5 mL⋅min-1 flow rate and a run time of 10 min. The injection volume was 5 µL and the photodiode array detector was adjusted at 250 nm. The results were analyzed with the statistical software Minitab® version 17.1.0. In Silico Comparison of FBZ and FBZ/MA Release Profiles. Gastroplus™ 9.0 (Simulation Plus, Inc.) was used to predict and compare the plasma concentration profiles of hypothetical pharmaceutical immediate-release FBZ and FBZ/MA oral suspensions (5 mg⋅Kg-1). The experimental solubility data was loaded into the program and the model was fitted using the experimental parameters reported by Ceballos et al.3. A default rat fasted physiology model (Opt–logD model) was used.

Results and Discussion 8 ACS Paragon Plus Environment

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Solid State Characterization. The X-ray powder diffraction patterns of FBZ, MA, FBZ/MA, and 1:1 molar ratio FBZ+MA physical mixture, prepared by the same grinding procedure of FBZ/MA but without solvent addition, are shown in Figure 1; it is

Figure 1. Comparison of the X-ray powder diffraction patterns of flubendazole (FBZ), maleic acid (MA), FBZ+MA physical mixture of 1:1 molar ratio, and FBZ/MA sample obtained by liquid-assisted (THF) grinding. For the sake of clarity the X-ray diffractograms are represented using the normalized intensities as a function of Q-space.

clear that the diffraction pattern of FBZ/MA does not correspond to a simple physical mixture of FBZ and MA, thus indicating that a new crystalline form was obtained. The thermal analysis results (see below) also support this observation. Despite the unequivocal crystallinity of FBZ/MA evidenced by the corresponding diffractogram in Figure 1, some degree of peak broadening and background variation is perceptible



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which can potentially indicate the presence of traces of amorphous material. Although the extent and impact of this contribution on the physical properties and dissolution performance of FBZ/MA could not be discriminated in this study, this should be pondered in further investigations. Prior to this work, the crystal structure of FBZ and FBZ/MA had not been reported and crystallization experiments in our laboratories failed to produce crystals suitable for a single crystal X-ray diffraction determination. Vapor diffusion,31 wire induction,32 and crystallization from gels,31 were unsuccessful tried. We therefore resorted to X- ray powder diffraction data to index the FBZ pattern and solve the crystal structure, based on the first 20 reflections and on the Topas-Academic v.6 software.33 The selected region of the powder pattern was fitted by using the fundamental parameters approach34 and the best solution from the indexing procedure corresponded to the monoclinic crystal system, space group P21, with unit cell parameters a = 13.6994(7) Å, b = 3.9714(3) Å, c = 13.3227(6) Å, β = 100.761(2)º, and V = 712.08(7) Å3. The crystal details as well as the statistical indices describing the final refinement are shown in Table 1. The Rietveld plot is shown in the Supporting Information (Figure S4). Due to the inherent complexity of the FBZ/MA compound, the structure determination from powder data was not achieved.

Table 1. Crystal Data and Details of the FBZ Crystal Structure. Formula

C16H12FN3O3

Molecular weight / g⋅mol-1

313.29

Crystal system

Monoclinic

Space group

P21 (nr. 4)

a, b, c / Å

13.6784(9), 3.9725(2), 13.3304(7)

β/°

100.756(2)

Volume / Å3

711.61(7)

Z, Z’

2, 1

ρcalc / g cm

−3

1.4621(2)

Rexp / %

1.062

Rwp / %

3.805

RBragg / %

0.258


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S

3.585



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As shown in Figure 2, the crystal structure of FBZ consists of two formula units per unit cell (Z = 2) and one molecule in the asymmetric unit (Z’ = 1). The molecules are held together by hydrogen bonds (Figure 3; values indicated in Table 2).

Figure 2. Crystal structure of FBZ displaying two formula units per unit cell.

Figure 3. FBZ crystal packing highlighting the main hydrogen bond patterns (cyan lines).

Table 2. Intramolecular and Intermolecular and Hydrogen Bonds for FBZ. D and A Represent Hydrogen Donors and Acceptors, Respectively. D–H ··A N(14)–H(24)···O(4)a

D–H/Å 0.86(1)

H···A/Å 2.07(1)

D···A/Å 2.63(1)

D–H···A/° 122.4(9)

N(14)–H(24)···O(16)b

0.86(1)

2.22(2)

2.85(2)

130(1)

N(5)–H(32)···N(7)c

0.86(1)

2.46(1)

3.28(1)

157(1)

C(23)–H(29)···O(4)d

0.96(1)

1.6(1)

2.50(1)

153(1)


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a

Intramolecular. Symmetry: b −x, −1/2+y, 2−z; c −x, −1/2+y, 1−z; d −x, 1/2+y, 2−z.

Figure 4 shows the TG patterns obtained for FBZ, MA, FBZ/MA, and FBZ+MA physical mixture under a dynamic nitrogen atmosphere, at a heating rate of 10 K⋅min-1. The curves evidence three decomposition steps for FBZ (473-548 K, ∆m1 = 14.8%; 568-625 K, ∆m2 = 12.5%; 643-873 K, ∆m3 = 12.3%) and two thermal events for MA. The TG curve of the FBZ/MA sample exhibits clear differences relative to those of pure FBZ and MA. Most notably: (i) the onset (Ton) of the first mass loss event in FBZ/MA, assigned to MA decomposition, occurs at a substantially higher temperature (Ton = 449 K) than the corresponding one for pure MA or the FBZ+MA physical mixture (Ton = 414 K); (ii) in contrast, the first decomposition event due to FBZ in FBZ/MA, shifts to a considerably lower temperature, namely from Ton =502 K to 452 K. This suggests the presence of a significant FBZ-MA interaction in the binary crystal, leading to an increased thermal stability of the MA coformer and decreased thermal stability of the FBZ moiety. The formation of a new FBZ/MA crystalline form was further indicated by the results of DSC studies (Figure 5). Indeed, the measured DSC curve for FBZ/MA (Figure 5a) shows a single endothermic event at Ton = 439 K, which does not correspond to the fusion of pure maleic acid (Figure 5b; Ton = 400 K) or flubendazole (Figure 5c; Ton = 497 K). This suggests the presence of a new homogeneous phase. Similarly, Lian et al.35 attributed the observation of a single fusion event with the disappearance of the pure maleic acid fusion peak at 408 K to the formation of a novel azelnidipine/maleic acid crystal form. Analogous behaviors were also noted by other groups

for

a

variety



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Figure 4. TG patterns obtained for FBZ, MA, FBZ/MA, and FBZ+MA physical mixture at a heating rate of 10 K⋅min-1.

Figure 5. DSC patterns obtained for (a) FBZ/MA, (b) MA, (c) FBZ, and (d) FBZ+MA physical mixture, at a heating rate of 10 K⋅min-1.

of systems.36–39 The tendency for spontaneous formation of the new FBZ/MA crystalline form from its precursors is also suggested by the DSC pattern of the physical mixture.37–39 As shown in Figure 5d a second peak closely matching the fusion temperature of FBZ/MA is observed after fusion of MA (first peak). This gives a strong 14 ACS Paragon Plus Environment

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indication that FBZ/MA can spontaneously form once melted MA and solid FBZ are able to interact. Additional insights into the nature of the FBZ/MA crystalline form (cocrystal or salt) were obtained from vibrational spectroscopy results. Both ATR-FTIR (Figure 6) and Raman (Figure 7) spectra are dominated by the FBZ features, with the maleic acid presence evidenced as weak bands. A detailed assignment of the FBZ bands is not straightforward mainly because of the molecular complexity, which includes a large number of tautomeric forms40 and mechanical/electronic coupling. Assignment of the bands corresponding to the main FBZ chemical groups and their behavior upon interaction with maleic acid provides, however, a clear evidence about the nature of the interaction. Such bands are ν(N-H)carbamate = 3303 cm-1, ν(C=O)carbamate = 1736 cm-1, ν(C=O)benzoyl = 1645 cm-1, and ν(C=N) + ν(C=C) = 1594 cm-1 in the ATR-FTIR spectrum and at 3300, 1738, 1634 and 1598 cm-1, respectively, in the FT-Raman spectrum.

Figure 6. ATR-FTIR spectra of FBZ/MA, FBZ+MA physical mixture, MA, and FBZ.



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Figure 7. Raman spectra of FBZ/MA, FBZ+MA physical mixture, MA, and FBZ. The high frequency region of the FBZ ATR-FTIR spectrum is dominated by ν(C-H) vibrations but it also exhibits an intense band at ∼3303 cm-1 assigned to ν(NH)carbamate and a very broad band centered at ∼2700 cm-1 that extends from 2000 to 3600 cm-1 (Figure 6). The latter is assigned to a ν(N-H)benz stretch, which interacts through intramolecular hydrogen bonding with the carbonyl group of the carbamate moiety.41 This broad band is also present in the spectra of the FBZ+MA physical mixture. In this case, however, besides the FBZ and MA bands, weak new features are observed at 2971 cm-1 and 2980 cm-1. These changes indicate that some degree of FBZ-MA interaction is promoted by the grinding process. In the FBZ/MA ATR-FTIR spectrum, the intense 3303 cm-1 band typical of FBZ almost disappears and the two weak features observed in the spectrum of the physical mixture (2971 and 2980 cm-1) are observed as strong bands; a new broad band centered at ∼3100 cm-1 is also noted. This band can be tentatively assigned to the ν(N-H)benz which is weakened by the formation of a hydrogen bond with a maleic acid carbonyl group, as observed for mebendazole hydrochloride.42 The broad ν(N-H)carbamate band at ∼2700 cm-1 apparently does not change in position, but seems to decrease in intensity, thus suggesting a decrease in the strength of the hydrogen bond with the C=Ocarbamate


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group. In fact, the ν(C=O)carbamate band shifts from 1736 cm-1 to 1747 cm-1, thus confirming the conclusion based on the ν(N−H)carbamate. The N-H bending modes (carbamate and benzimidazole) are expected in the 1600 – 1650 cm-1 region, which also contains ν(C=C)arom and ν(C=O)benzoyl; considering that all of them are expected to be affected to some extent by the interaction with maleic acid, band assignments in this region are not straightforward. The features observed by ATR-FTIR are confirmed by the FT-Raman results (Figure 7): the high frequency region of the FBZ spectrum is dominated by ν(C-H) vibrations but two weak bands are observed at 3300 cm-1 and 3184 cm-1. As in the case of ATR-FTIR, the 3300 cm-1 band is not observed in the FBZ/MA spectrum. Raman spectroscopy is much less sensitive to hydrogen bonding than FTIR but it easily allows the access to the lattice frequency region (below 400 cm-1). Figure 8 shows that the spectral profile of the FBZ+MA physical mixture does not correspond to a mere sum of the FBZ and MA individual spectra but contains new features which are the dominant ones in the FBZ/MA spectrum. This clearly indicates that, even without solvent, grinding already induces some degree of FBZ/MA formation. Overall, the ATR-FTIR and Raman spectra here reported indicate that in the FBZ/MA crystal form maleic acid and flubendazole interact through a hydrogen bond involving the carbamate and benzimidazole groups (Figure 8). Note that the difference ∆pKa = 1.08 between the pKa s of maleic acid, pKa (MA) = 2.85 and protonated flubendazole, pK a (FBZH + ) = 3.93, estimated by the protonation module of MarvinSketch,43 falls in

the range −1 to 4, where the prediction of preference for cocrystal or salt formation based on a ∆pKa rule44 is difficult.45,46 Nevertheless, a reported correlation that relies on the analysis of a large number of acid–base pairs in the Cambridge Structural Database47 and on ∆pKa data obtained by MarvinSketch suggests a slight advantage towards cocrystal formation in the case of FBZ/MA (46% probability salt and 54% probability cocrystal). The use of the experimental values pK a (FBZH + ) = 3.32 (determined in this work, see Supporting Information) and pKa1 (MA) = 1.9248 in the correlation would, however, raise the probability of salt formation to 52%. This stresses the fact that in the case of the FBZ/MA system a reliable prediction of preference for cocrystal or salt formation based on common ∆pKa rules is not possible.



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The cocrystal or salt nature of the FBZ/MA form was, therefore, assessed through a XPS investigation. Figure 9 shows a comparison of the N 1s high-resolution spectra of FBZ and FBZ/MA. The overlapping peaks in the FBZ spectra (Figure 9a) could be separated by deconvolution into two components (the atom labeling scheme is given in Figure 3): one at a binding energy (BE) of 398.4 eV, assigned to the imine (N7)

Figure 8. Hydrogen bond interactions between maleic acid and FBZ involving the carbamate and benzimidazole groups. and another at 400.0 eV corresponding to the secondary amines (N5 and N14). In the case of FBZ/MA a single peak is observed at 400.8 eV (Figure 9b) for N5, N7, and N14. This binding energy is close to the secondary amines; it shifted, however, by 0.8 eV towards high binding energy. The ΔBE = 2.4 eV increase of the binding energy of N7 on going from pure FBZ to FBZ/MA indicates a decrease in the electron density of the imine environment, which can be explained by the occurrence of proton transfer from MA to FBZ leading to salt formation. A similar behavior has been reported for salts of ambazone49 and lapatinib.50 Since nitrogen is only present in the FBZ moiety and the protonation happens on the amino group, this effect is readily detectable in the N 1s spectra. The detailed analysis of the C 1s and O 1s spectra is consistent with protonation (the spectra are provided in the Supporting Information). The C 1s and O 1s spectra point to strong mutual interaction between FBZ and maleic acid.


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Figure 9. The N 1s high- resolution XPS spectra of FBZ (a) and FBZ/MA (b)



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Energetics of FBZ/MA formation. The energetics of FBZ/MA salt formation from its precursors, in the solid state, according to:

FBZ(cr) + MA(cr) → FBZ/MA(cr)

(3)

was characterized, based on calorimetric measurements of the enthalpies of the following dissolution processes:

MA(cr) + DMSO(l) → [MA + DMSO](sln)

(4)

FBZ(cr) + [MA + DMSO](sln) → [FBZ + MA + DMSO](sln)

(5)

FBZ/MA(cr) + DMSO(l) → [FBZ + MA + DMSO](sln)

(6)

o The standard molar enthalpy of reaction 3, ∆ r H m (3) , is related to the enthalpies of

processes 4-6 through:

∆ r H mo (3) = ∆ sln H mo (4) + ∆ sln H mo (5) − ∆ sln H mo (6)

(7)

o The calorimetric measurements, carried out at 298.15 K, gave ∆ sln H m (4) = o −6.64±0.28 kJ⋅mol-1, ∆ sln H m (5) = 14.5±4.6 kJ⋅mol-1, and ∆ sln H mo (6) = 15.4±2.6

kJ⋅mol-1, where the assigned uncertainties correspond to twice the standard error of the mean of 5, 5 and 6 independent determinations, respectively (see Supporting o Information for details). Based on these results eq 7 leads to ∆ r H m (3) = −7.5±5.3 o kJ⋅mol-1. The fact that ∆ r H m (3) is exothermic indicates that the direct formation of

FBZ/MA from FBZ and MA in the solid state is enthalpically favored. This conclusion is in line with a very recent and extensive theoretical study indicating that, in terms of lattice energy, 95% of the over 300 cocrystals present in the Cambridge Structural Database47 investigated were, on average, stabilized by 8 kJ⋅mol-1 relative to their o separated precursors.51 Moreover, if the entropic term T ∆ r S m (3) was to overcome

∆ r H mo (3) = −7.5±5.3 kJ⋅mol-1, making ∆ r Gmo (3) > 0 at 298 K, then ∆ r S mo (3) should be lower than −25 J⋅K-1 mol-1. Given that entropy contributions for solid-state reactions such as that in eq 3 are likely to be close to zero (0±3 J⋅K-1 mol-1),52 the results here 20 ACS Paragon Plus Environment

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o obtained suggest that reaction 3 will be thermodynamically favored, i.e. ∆ r Gm (3) < 0 .

Note, finally, that, on average the dissolution processes corresponding to eqs 4-6 were completed in 25 min for MA, 121 min for FBZ, and 56 min for FBZ/MA. This indicates that the dissolution rate of FBZ/MA in DMSO is ∼2 times faster than that of FBZ alone. Such observation is in line with the solubility enhancement of FBZ/MA in aqueous media compared to FBZ discussed in the following section. Experimental Solubility and In Silico Dissolution/Absorption Behavior of FBZ/MA Salt. Figure 10 compares the experimental solubilities obtained in this work at 310 K and pH 1.2 and 6.8, for FBZ, FBZ/MA salt and FBZ+MA physical mixture. The FBZ/MA salt shows a three and six fold increase in solubility at pH 1.2 and 6.8, respectively, when compared to the pure FBZ (neutral FBZ).

Figure 10. Comparison of the equilibrium solubility values (n = 6) obtained at pH 1.2 and 6.8 for pure flubendazole (FBZ), flubendazole-maleic acid equimolar physical mixture (FBZ+MA), obtained by grinding without solvent, and 1:1 FBZ/MA binary crystal. Solubility data was subjected to one-way ANOVA and Tukey´s tests. For the same number of asterisks there were no statistically significant difference among the samples within each group (p = 0.05).



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Based on these solubility results, the GastroPlusTM Advanced Compartmental and Transit Model53,54 was applied to estimate the in vivo performance of the FBZ/MA oral suspension comparatively to pure FBZ, using a rat fasted physiology model. This approach is a valuable tool that can significantly minimize (or at least significantly reduce) the need for in vivo experiments in early formulation development.54 The obtained mean plasma concentration-time profiles are shown in Figure 11. The FBZ/MA suspension was predicted to be more rapidly absorbed than the pure drug formulation, achieving a peak concentration of 0.650 µg⋅mL-1 in less than 30 min, while pure FBZ required ∼1.3 h to reach the considerably smaller peak level of 0.468 µg⋅mL-1. Moreover, an impressive 60% increase in the extension of absorption, represented by the area under the concentration–time curve from time zero extrapolated to infinity (AUCinf), is observed for FBZ/MA (AUCinf = 32.7 µg⋅h⋅mL-1) when compared to pure FBZ (AUCinf = 20.4 µg⋅h⋅mL-1). Despite the low solubility profile in basic media of both FBZ/MA and FBZ, no intestinal precipitation was predicted by the simulation. This can be explained by the high permeability and, consequently, high absorption rates of flubendazole observed during the simulations.

Figure 11. Mean flubendazole plasma concentration–time profiles following a single dose (5 mg⋅Kg-1) of a conventional pure flubendazole suspension(FBZ) and a FBZ/MA suspension under fasting conditions.


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Conclusion The immense social burden caused by parasitic helminth diseases in tropical regions is still an un-served public health emergency. Considerable efforts have to be made globally to sufficiently address this crisis. The current study demonstrated through a combination of advanced orthogonal approaches that the new FBZ/MA crystal is a thermodynamically favorable salt with superior solubility performance relative to FBZ alone. Simulations further suggested improved pharmacokinetic features with a 60% increase in drug exposure. This new salt has a considerable potential to be used in the development of pharmaceutical formulations to treat lymphatic filariasis and onchocerciasis.

Acknowledgments: We thank E. J. Barbosa for help in the experimental determination of the pK a of FBZ. This work was supported by the São Paulo Research Foundation (FAPESP; grants #2015/05685-7 and #2015/26233-7) and the National Council for Scientific and Technological Development (CNPq-Brasil; grants #400455/2014-5, #402289/2013-7 and #307664/2015-5). We also acknowledge financial support from Fundação para a Ciência e a Tecnologia (FCT Portugal), through project PEstOE/QUI/UI0612/2013 and a post-doctoral grant awarded to C. E. S. Bernardes (SFRH/BPD/101505/2014). The authors gratefully acknowledge Simulation Plus for providing the software license.

Supporting Information Available: Table S1 with the detailed results of the solution microcalorimetry experiments. Details of the XPS analysis of FBZ/MA (Figures S1 and S2) and of the experimental determination of FBZ pK a (Figure S3). Rietveld plot of the FBZ compound (Figure S4). CCDC ID 1585995 contains de supplementary crystallographic data for FBZ. This material is available free of charge via the Internet at http://pubs.acs.org.



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For Table of Contents Use Only A New Thermodynamically Favored Flubendazole/Maleic Acid Binary Crystal Form: Structure, Energetics and In Silico PBPK Model-based Investigation Gabriel L. B. de Araujo, Fabio Furlan Ferreira, Carlos E. S. Bernardes, Juliana A. P. Sato, Otávio M. Gil, Dalva L. A. de Faria, Raimar Loebenberg, Stephen R. Byrn, Daniela D. M. Ghisleni, Nadia A. Bou-Chacra, Terezinha J. A. Pinto, Selma G. Antonio, Humberto G. Ferraz, Dmitry Zemlyanov, Débora S. Gonçalves, Manuel Eduardo Minas da Piedade

Synopsis A new flubendazole/maleic acid crystal form showing considerably improved solubility and in silico pharmacokinetic profile compared to flubendazole has been prepared and characterized. The compound has salt character, resulting from maleic acid → flubendazole proton transfer, and is thermodynamically favored relative to the individual precursors. The crystal structure of flubendazole is also reported for the first time.


Maleic Acid

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