Preparation and Solid-State Characterization of Three Novel

Article pubs.acs.org/crystal

Preparation and Solid-State Characterization of Three Novel Multicomponent Solid Forms of Oxcarbazepine: Improvement in Solubility through Saccharin Cocrystal Renu Chadha,*,† Anupam Saini,† Dharamvir S. Jain,‡ and P. Venugopalan‡ †

University Institute of Pharmaceutical Sciences and ‡Department of Chemistry, Panjab University, Chandigarh-160014, India S Supporting Information *

ABSTRACT: Oxcarbazepine (OXCBZ) is an antiepileptic drug with low aqueous solubility, and its dissolution is the rate limiting step for absorption. The present work investigates three muticomponent solid forms of OXCBZ with watersoluble coformers with an aim to enhance its solubility and in vivo performance. The experiments based on the solution method yielded two cocrystals, with succinic acid (1) and saccharin (2) and a solvate with acetic acid (3). Compound 1 was identified by single crystal X-ray diffraction (XRD) as a solvated cocrystal involving eight molecules of OXCBZ, four molecules of succinic acid, and four molecules of chloroform in the unit cell. The structural changes upon desolvation of cocrystal 1 have also been examined. Single crystals of compounds 2 and 3 could not be obtained in a size suitable for single crystal X-ray analysis and thus was studied by differential scanning calorimetry, thermogravimetric analysis, hot stage microscopy, powder XRD, Fourier-transform infrared spectroscopy, and solid-state nuclear magnetic resonance spectroscopy. Furthermore, the powder dissolution of compounds 1 (desolvated form), 2, 3, and OXCBZ was performed in an acidic aqueous medium and analyzed by high performance liquid chromatography. Their physical stability was also assessed. The cocrystal with saccharin showed a significant improvement in the solubility of OXCBZ in aqueous conditions and exhibited a lower ED50 value as compared to pure OXCBZ.

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INTRODUCTION Over the past few years, crystallizing the active pharmaceutical ingredient (API) as a cocrystal has been an accepted approach to generate solid forms with diverse physical properties as well as for potential expansion of intellectual property.1 Pharmaceutical cocrystals are solid molecular complexes comprising an API with a neutral guest compound coformer within the same crystal lattice.2 This class of engineered supramolecular materials has emerged as an effective means of tailoring physical properties of APIs via supramolecular modification3,4 alongside polymorphs,5,6 solvate7,8 hydrates,9 salts,10 and amorphous materials.11 Salt formation is the primary method for improving the necessary physicochemical properties of APIs. However, salts can only be formed for acidic or basic APIs. Furthermore, crystalline salts with the appropriate properties might be difficult to find.12,13 Therefore, cocrystallization is a viable means to improve the physicochemical properties such as solubility and dissolution14−16 and to expand the potential of non-salt-forming APIs. Pharmaceutical cocrystals are also capable of modifying pharmacokinetic (PK) properties. A recent review has summarized the case studies where the effect of cocrystals on the PK profile has been evaluated,17 many of which support that pharmaceutical cocrystals are a viable option to enhance the clinical performance of a poorly soluble API.18−21 Hickey et al.22 reported a pharmacokinetic study on cocrystals and a marketed form (Tergretol) of carbamazepine in dogs which revealed that © 2012 American Chemical Society

the cocrystal showed higher plasma levels than Tergretol which contained an anhydrous polymorph of carbamazepine. According to a more recent report by Zaworotko et al.,23 a cocrystal of meloxicam and aspirin was prepared, which enabled an approximately 12-fold decrease in the time required to reach a concentration of 0.51 g/mL in rats compared with pure meloxicam at an equivalent dose. Thus, pharmaceutical cocrystals provide an opportunity to fine-tune the physicochemical and PK properties of APIs without the formation/breakage of covalent bonds. Solution-based cocrystallization experiments in ternary systems of the two cocrystal components and a solvent have become increasingly popular for the preparation of pharmaceutical binary compounds.24−28 However, a major risk in this solution-based approach is the recrystallization of the individual components or the possible competition with solvate formation of one of the components. Recently, the use of solvent mixtures has been suggested to thermodynamically suppress the solvate formation as a competing reaction in solution-based cocrystallization experiments.29 Solvent mixtures provide an additional advantage in that they reduce the solubility differences between different compounds as compared to pure solvents and thus help Received: May 25, 2012 Revised: June 20, 2012 Published: June 20, 2012 4211

dx.doi.org/10.1021/cg3007102 | Cryst. Growth Des. 2012, 12, 4211−4224

Crystal Growth & Design

Article

Sample Preparation. OXCBZ−SA−CHCl3 (1). A 1:1 mixture of OXCBZ (50.4 mg, 0.199 mmol) and succinic acid (SA) (23.6 mg, 0.199 mmol) was dissolved in a 1.5 mL solution of chloroform/methanol (2:0.3) in a 10 mL conical flask. The resulting solution was placed at room temperature for slow evaporation. After two days, faintly yellow, hexagonal-shaped platelike crystals of 1 were recovered, filtered, and dried. For slurry experiments, compound 1 was prepared in bulk by seeding 6 mL of clear solution of chloroform and methanol containing stoichiometric amounts of OXCBZ and SA, with the single crystals of 1. A yield of approximately 90% was obtained, and its PXRD pattern was found to be similar to that of single crystals of 1. OXCBZ−SA (1a). Compound 1a was prepared by desolvation of compound 1 by keeping it at a constant temperature of 150 °C for 3 h and was characterized by differental scanning calorimetry/thermogravimetric analysis (DSC/TGA) and powder X-ray diffraction (PXRD) analysis. OXCBZ−SAC (2). Compound 2 was obtained by dissolving a 1:1 mixture of OXCBZ (100.8 mg, 0.399 mmol) and saccharin (SAC) (73.2 mg, 0.399 mmol) in a 6 mL solution of chloroform/methanol (8:0.26). The resulting clear solution was transferred to a round-bottom flask and was subjected to fast evaporation under reduced pressure of 500 mbar using rotovap (Eyela N-1100) with the water bath temperature set at 60 °C (Eyela OSB-2100) until the whole of the solvent evaporated leaving behind a transparent film concentrate in the round-bottom flask (5 min). The clear film of concentrate so obtained was kept at room temperature. Nucleation of compound 2 started within 1 h and growth continued overnight. Yield obtained was sufficient to carry out the slurry experiment. Efforts to prepare quality single crystals of 2 remained unsuccessful. OXCBZ−AA (3). OXCBZ was dissolved in acetic acid (AA) using heat, and compound 3 was obtained as clusters of very fine needle-shaped crystals upon slow evaporation of acetic acid. The crystal size was not sufficient for single crystal X-ray diffraction analysis. The preliminary characterization was performed using DSC, TGA, PXRD, Fourier-transform infrared spectroscopy (FT-IR) and solid-state nuclear magnetic resonance spectroscopy (ssNMR). Differential Scanning Calorimetry (DSC). DSC of all the samples was conducted using DSC Q20 (TA Instruments, USA). The samples (3−5 mg) were placed in sealed nonhermetic aluminum pans and were scanned at a ramping rate of 5 °C/min under a dry nitrogen atmosphere (flow rate 50 mL/min). The data were managed by TA Q series Advantage software (Universal analysis 2000). Thermogravimetric Analysis (TGA). TGA was performed on a Mettler Toledo TGA/SDTA 851e instrument. Approximately 5 mg sample was heated from 10 to 250 °C in open alumina pan at the rate of 10 °C/min under nitrogen purge at flow rate of 50 mL/min. The data were managed by STAR software (9.00). Optical Microscopy. Optical microscopy was performed using a Leica DM 3000 phase contrast microscope with polarizing attachment. Hot Stage Microscopy (HSM). Melting points and physical changes were visually examined at 50× magnification by HSM. The study was carried out using polarized/optical hot stage microscope (Nikon Eclipse LV100 POL, Japan) equipped with a controlled heating and cooling stage (LTS 420E, Linkam) and an imaging system (VTO 232, JVC − Digital camera and Linksys 32 imaging software, Linkam, England). The powder sample was mounted in air and heated from 25 to 250 °C at a rate of 5 °C/min. Powder X-ray Diffraction (PXRD). PXRD patterns were collected on X’Pert PRO diffractometer system (Panalytical, Netherlands) with a Cu Kα radiation (1.54060 Ǻ ). The tube voltage and current were set at 45 kV and 40 mA respectively. The divergence slit and antiscattering slit settings were set at 0.48° for the illumination on the 10 mm sample size. Each sample was packed in an aluminum sample holder and measured by a continuous scan between 5 and 50° in 2θ with a step size of 0.017°. The experimental PXRD patterns were refined using X’Pert High Score software. Fourier Transform-Infrared Spectroscopy (FT-IR). A Spectrum RX I FT-IR spectrometer (Perkin-Elmer, UK) was employed in the KBr diffuse-reflectance mode (sample concentration 2 mg in 20 mg of KBr) for collecting the IR spectra of samples. The spectra were measured over

the different components to crystallize out as a multicomponent single compound.30 In the present work, we have utilized this approach of solvent mixtures in obtaining novel cocrystals of OXCBZ, a carbonylated derivative of carbamazepine (CBZ), which has been a model compound for the study of polymorphs,31,32 solvates,29 and cocrystals.33−38 Oxcarbazepine (OXCBZ, Trileptal) is a modern antiepileptic drug used as both monotherapy and adjunctive therapy for the treatment of partial seizures with or without secondary generalization in adults and children.39 It is a neutral lipophilic compound (molecular weight 252.3) with a melting point of 215−216 °C.40 However, this API is a Biopharmaceutics Classification System (BCS), Class II drug41 with poor water solubility (8.4 mg/100 mL at 25 °C),42 which is a major hurdle in making it bioavailable in the body. The traditional approach of salt formulation has turned out to be unsuccessful as the molecule is not sufficiently acidic/basic to enable the salt formation (pKa of 10.7).43 Therefore, several other formulation strategies have been tried to enhance its dissolution rate in order to improve its systemic absorption, including complexation with hydroxypropyl β-cyclodextrin,44 fast dissolving tablets containing Ac-Di-sol as a superdisintegrant,45 microcrystals with methylcellulose,46 granulation with solubility and release enhancing agents,47 solid dispersions48 and isolation of new polymorphs,49−51 solvates,49 and amorphous forms.52 However, OXCBZ has very rarely been investigated for its cocrystallization/solvation tendencies. This motivated us to explore the potential of OXCBZ to cocrystallize in order to improve its solubility and dissolution profile. Carboxamide moiety, the principal functional unit in OXCBZ, is expected to form multipoint N−H···O hydrogen bond interactions with the cocrystal formers having complementary functional groups. Thus, various coformers such as saccharin, succinic acid, adipic acid, glutaric acid, tartaric acid, acetic acid, and propionic acid were selected from the GRAS list generated by the U.S. FDA (generally regarded as safe additive chemicals by the Food and Drug Administration)53 and investigated for cocrystallization with OXCBZ utilizing a solution-based approach. The multicomponent compounds were identified with only three coformers and are reported herein: a solvated cocrystal of OXCBZ with succinic acid (1), a cocrystal with saccharin (2), and a solvate with acetic acid (3). Solvates and cocrystals are closely related to one another since components within the crystal typically interact by hydrogen bonding, but, to be more specific, we are using here the term “solvate” for compound 3, as the coformer acetic acid is a liquid at room temperature. Out of the three multicomponent forms, the one with saccharin (2) exhibited a dissolution profile that was ∼3.5 fold superior to that of OXCBZ alone. This enhanced solubility of 2 inspired us to test its in vivo potential. Thus, dose−response studies were carried out in mice.54 This cocrystal outperformed the OXCBZ alone by significantly lowering the ED50 value of the parent drug.

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EXPERIMENTAL SECTION

Chemicals. OXCBZ was obtained as complementary sample from Ami Life Science Pvt. Ltd., Baroda, India. Succinic acid (SA) (99% purity) and saccharin (SAC) (99% purity) were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India, and the crystallization solvents including acetic acid were of AR grade and purchased from various commercial suppliers. All of these were used as received. 4212



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the range of 4000−400 cm−1. Data were analyzed using Spectrum software. Solid State Nuclear Magnetic Resonance spectroscopy (ssNMR). The solid state 13C cross-polarization magic angle spinning (CP-MAS) and 1H MAS spectra were acquired using a Bruker DSX-300 NMR spectrometer using a 5 mm double resonance CP-MAS probe. For 1 H/13C CP-MAS, the 13C and 1H resonance frequencies are 75.46 and 300.13 MHz, respectively. For 13C experiment, data were collected at 273 K using a spectral width of 18.8 kHz, 1130 complex data points, acquisition time 30 ms, contact time 2 ms. A total of 1024 scans were acquired with a relaxation delay 4 s. For 1H experiments, data were collected at 273 K using a spectral width of 29.9 kHz, 2092 complex data points, acquisition time 35 ms, contact time 5 μs. A total of 128 scans were acquired with a relaxation delay 5 s. All the 13C spectra were referenced to tetramethylsilane (TMS) using the carbonyl carbon of glycine (176.03 ppm) as a secondary reference. Single Crystal X-ray Diffraction. Intensity data for compound 1 were collected on a Siemens P4 single-crystal diffractometer equipped with a molybdenum sealed tube (λ = 0.71073 Å) and highly oriented graphite monochromator using crystals of dimensions 0.24 × 0.17 × 0.10 mm3 and mounted in Lindeman glass capillaries at room temperature (293 K). The lattice parameters and standard deviations were obtained by least-squares fit to 39 reflections (11.50° < 2θ < 24.49°). The data were collected by the 2θ−θ scan mode with a variable scan speed ranging from 3.0° min−1 to a maximum of 60.0° min−1. Three reflections were used to monitor the stability and orientation of the crystal and were measured after every 97 reflections. Their intensities showed only statistical fluctuations during 32.79 h of X-ray response time. The data were collected for Lorentz and polarization factors and on empirical absorption correction based on the ψ-scan method applied. The structure was solved by the direct methods using SHELX 9755 suite of programs and also refined on F2 using the same. All the non-hydrogen atoms were refined anisotropically. The hydrogen atoms were included in the ideal positions with fixed isotropic U values and were riding with their respective non-hydrogen atoms. A weighting scheme of the form w = 1/[(σ2F2o) + (ap)2 + bp] with a = 0.1052 and b = 0.67 was used. The refinement converged to a final R value of 0.0647 (wR2 = 0.1653 for 1394 reflections) [I > 2σ(I)]. The final difference map was featureless. Analysis of the H-bonding and other noncovalent interactions was carried out using software XP (Bruker). Packing diagrams were generated using Mercury-2.2. Crystallographic .cif file (CCDC No. 843314) is available free of charge at http://www.ccdc.cam.ac.uk/data_request/cif or as part of the Supporting Information. Powder Dissolution Experiment. Powder dissolution of OXCBZ and compounds 1a, 2, and 3 was determined in 0.1 N HCl. For this study, the starting solids were sieved using Gilson mesh sieve to provide samples with an approximate particle size of 150 μm. In each experiment, a flask containing 50 mL of 0.1 N HCl was equilibrated at 37 °C in a constant temperature bath. Excess of solid phase (ca. 100 mg) was added to the flask and the resulting slurry was shaken at 200 rpm. An aliquot of slurry was withdrawn at multiple time points (5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150, 210, 270, and 360 min) and filtered through 0.22 μm membrane filter, diluted suitably, and analyzed by high performance liquid chromatography. After the last aliquot was collected, pH of the slurries was measured and the remaining solids were filtered, air-dried, and analyzed by PXRD. High Performance Liquid Chromatography (HPLC). The solution concentration of OXCBZ in the dissolution experiments on pure commercial sample of OXCBZ and compounds 1a, 2, and 3 was determined by a Waters Alliance HPLC system which includes a Waters 2695 separation module, a Waters 2996 Photodiode Array Detector, and a 4.6 mm × 150 mm SunFire C18, 5 μm column (Waters Corporation, Milford, MA). Standards of OXCBZ, SA, and SAC were prepared in pure ACN and were diluted appropriately with a 70:30 mixture of 0.1 M acetic acid/ACN to obtain various concentrations of calibration standards (500−16000 ng/mL for OXCBZ and SAC, 41800−418000 ng/mL for SA). The samples of the dissolution experiment were also diluted with the mixture of 0.1 M acetic acid/ACN (70:30) and 10 μL of each sample was injected onto the column in triplicate. Separations were

conducted using the mobile phase of a mixture of 0.1 M acetic acid and ACN (75:25) pumped at a flow rate of 1.0 mL/min through the column at a temperature of 35 °C. OXCBZ and SAC were detected at 305 and 268 nm respectively. Data acquisition and analysis were performed using software Empower 2.0. Solid State Stability Studies. Accurately weighed samples (approximately 200 mg) of compounds 1a, 2, and 3 placed in loosely capped glass vials were kept in humidity controlled photostability chamber (Thermolab, model no. TH0000400G) at 25 °C/60% RH and 40 °C/75% RH as well as under the ambient conditions for 4 weeks and then characterized by PXRD. Also, the physical mixtures of OXCBZ and SAC (unmilled) in a 1:1 molar ratio were stored in desiccators and kept under both the ambient as well as accelerated conditions (40 °C/75% RH) for 3 months and monitored for any cocrystal formation by FT-IR and PXRD. Dose−Response Studies in Mice. Male mice (Balb/C; 20−30 g) were used. Animals were weighed and placed in standard cages with free access to food and tap water. After 7 days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups each comprising 10 mice. OXCBZ and cocrystal 2 were prepared as suspensions in 0.5% carboxymethylcellulose in saline and administered via oral gavage (p.o.) in a final volume of 10 mL/kg. Both the forms were observed insoluble in the vehicle. Fresh drug suspensions were prepared on each day of experimentation and administered 45 min before electroconvulsions that were produced by current (fixed current 30 mA, 0.2 s stimulus duration) delivered to saline-wetted eyes via corneal electrodes from an electroshock apparatus (IMCORP, India). The criterion for the occurrence of seizure activity was the tonic hind limb extension (HLE, i.e., the hind limbs of animals outstretched 180° to the plane of the body axis). The protective activity of OXCBZ and its cocrystal was determined as the median effective dose (ED50 value in mg/kg) against maximal electroshock (MES) induced seizures. Sufficient animals were tested over a range of six different doses of both pure OXCBZ and cocrystal 2 (6.0, 9.0, 12.0, 15.0, 18.0, and 21.0 mg/kg of OXCBZ or its equivalent) in order to obtain a variable percentage of protection against MES. This allows the determination of ED50 value of cocrystal 2 (corresponding to the dose of oxcarbazepine) necessary to protect 50% of mice against tonic hind limb extension in the MES test in comparison with ED50 value for pure OXCBZ. ED50 values were calculated by computer probit analysis according to Litchfield and Wilcoxon method56 and were statistically analyzed to obtain 95% confidence limit with the unpaired Student’s t test using SPSS 16.0 software package. This experiment is approved by institutional animal ethical issue committee and has been conducted according to Indian National Science Academy (INSA) guidelines for use and care of experimental animals.

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RESULTS AND DISCUSSION In the present study, three multicomponent solid forms of OXCBZ have been identified. In cocrystallization of compounds 1 and 2, the solubilities of OXCBZ and coformers (SA and SAC) were investigated in various solvents/solvent mixtures in an attempt to find a solvent system in which both starting components have similar, measurable solubilities. Chloroform/ methanol mixtures were finally selected and tried over varying ratios from 40:1 to 10:1; ratios 20:3 and 40:1.3 were found to be suitable for obtaining compounds 1 and 2 respectively. However, in the case of compound 2, the conventional solution-based approach of slow evaporation at room temperature resulted in crystallization of individual components, but a fast evaporation of solvent at 60 °C under reduced pressure resulted in a new crystalline phase as characterized by DSC, TGA, PXRD, and FTIR analysis. Bag et al.57 have also reported the utility of a fast evaporation method for cocrystal screening utilizing a slightly undersaturated solution of components in a single solvent; however, unlike that, the use of a single solvent did not work in the case of OXCBZ and SAC cocrystal. The intimate physical 4213



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contact that developed between drug and coformer by the use of an appropriate solvent mixture and its fast evaporation resulted in cocrystallization. The molecular structures of OXCBZ and all the coformers are given in Scheme 1 and morphology of all three multicomponent forms is shown in Figure 1. The pKa values and melting points of coformers are given in Table 1. Scheme 1. Molecular Structures of Drug and Coformers

Figure 1. Optical micrographs of OXCBZ (5×), compound 1 (5×), 2 (50×), and 3 (50×).

Figure 2. DSC scan of (a) OXCBZ, (b) SA, (c) OXCBZ−SA (physical mixture), (d) compound 1, (e) compound 1a, (f) SAC, (g) OXCBZ− SAC (physical mixture), (h) compound 2, and (i) compound 3.

Table 1. Cocrystal Formers, Their pKa Values and Melting Points and Melting Point Data for New Cocrystalsa

cocrystal 1 2 3

cocrystal former succinic acid/ CHCl3 saccharin acetic acid

molar ratio (drug/ coformer)

cocrystal former pKab

cocrystal former mp (°C)b

repeated with the binary mixture of OXCBZ and SA that exhibited a comparatively broader melting endotherm at a temperature of 161.1 °C which is lower than the melting temperature of cocrystal (169.9 °C). Thus, DSC and TGA results confirmed that compound 1 is a solvated cocrystal. However, the DSC scan of compound 1a did not show any desolvation peak before melting at 169.9 °C suggesting the transformation of solvated cocrystal 1 into its desolvated form 1a without any further phase change. The DSC of 1a also reveals that the crystal packing is intact even after the removal of CHCl3 molecule as its melting point matches that of 1 and this is further confirmed by the comparison of their PXRD patterns. DSC scan of compound 2 showed a single sharp melting endotherm at a temperature of 180.1 °C (OXCBZ, 230.6 °C; SAC, 230.2 °C), while that of the binary mixture of OXCBZ and SAC exhibited a comparatively broader melting endotherm at a temperature of 173.8 °C. A closer inspection of this binary mixture melting peak showed another very small endothermic peak at 179.9 °C almost merged with the broader melting peak. This broader melting endotherm which is different from that of both the starting components, is attributed to eutectic melting followed by the melting of cocrystal (179.9 °C) which might have formed from the eutectic melt. This is in contrast with the DSC thermogram of compound 2 which showed a single sharp melting endotherm free of any eutectic melting, thus, establishing

cocrystal mp (°C)

2:1:1

4.21, 5.64

185−188

169−171

1:1 1:1

4.80 2.32

228−229 16.64 (mp), 118 (bp)

179−181 90−100

Melting point of OXCBZ is 227−230 °C. bObtained from Merck Index.

a

Thermal Analysis (DSC, TGA, and HSM). The first hand information about the existence of new solid phases was obtained by DSC. The DSC thermograms of pure OXCBZ and SA showed single melting endotherms at 230.6 and 189.3 °C respectively (Figure 2). However, the DSC thermogram of compound 1 showed a broad endotherm at 149.7 °C which is accompanied by a mass loss of 15.68% in TGA (Figure 3) in a temperature range of 146−160 °C, before final melting at 169.9 °C. This mass loss in TGA corresponded to a theoretical mass loss of 16.08% suggesting a 2:1:1 stoichiometry for OXCBZ/SA/CHCl3 in compound 1. Further, to confirm that second endotherm is due to melting of cocrystal and not a eutectic, the DSC scan was also 4214



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Figure 3. TGA curve of (a) compound 1 and (b) compound 3.

Figure 4. HSM photographs of compound 1 exhibiting the various events occurring as the temperature increased from 24 to 240 °C.

the existence of pure cocrystal phase. The DSC results of both 1 and 2 also agree well with the observations made by Suryanarayanan58 on different APIs that the eutectic melt temperature of binary mixtures is lower than the corresponding cocrystal melting temperature. Further, no mass loss was observed in the TGA scan of compound 2 before melting in the temperature range of 165−190 °C, suggesting it to be an anhydrous phase. The compound 3 showed a broad endotherm in DSC at 92.88 °C (peak = 94.59 °C) accompanied with a mass loss of 18.96% prior to 140 °C in TGA, indicating the desolvation of 3 releasing one molecule of acetic acid from the crystal lattice as suggested by theoretical mass loss of 19.22%. This endotherm was followed by a sharp endothermic transition at 229.6 °C corresponding to the melting of pure OXCBZ, thus suggesting the transformation of original crystal lattice of 3 after the evaporation of acetic acid molecule to pure OXCBZ. The desolvation of compound 3 was further supported by the various events observed during hot stage microscopy. Figure 4 shows that the needle-shaped crystals of 3, which were transparent at 25 °C, became quite deformed and opaque in the temperature range of 75−120 °C, suggesting the loss of acetic acid molecules present as solvent in these crystals as indicated by

DSC and TGA. These desolvated crystals showed no further change before final melting at 231 °C. Thus, the HSM images proved the homogeneity of sample 3 and also support the desolvation of crystals of 3 to pure OXCBZ. PXRD Analysis. Powder X-ray diffraction is a fingerprint characterization method for cocrystals. If the resulting PXRD of the solid product obtained after cocrystallization experiment is different from that of the reactants, then it may be inferred that a new solid phase has formed. The PXRD pattern of OXCBZ exhibited characteristic reflections at about 2θ 7.48°, 10.49°, 12.19°, 14.65°, 17.88°, 19.09°, 19.29°, 20.27°, 21.34°, 23.09°, 23.24°, 23.81°, 25.26°, 25.43°, 25.83°, and 26.33°, while compound 1 exhibited characteristic reflections at about 2θ 10.13°, 12.85°, 15.13°, 16.82°, 18.29°, 19.31°, 20.26°, 20.98°, 22.13°, 22.58°, 23.63°, 25.38°, 26.13°, 27.33°, and 28.25° (Figure 5). The purity of bulk material of this compound was assessed by comparing the experimental PXRD pattern with the simulated powder diffractiograms obtained from the X-ray crystal structure of 1 which proved it to be pure and homogeneous cocrystal composition. The PXRD pattern of 1a is very much similar to that of compound 1 except that a major peak of 1 at 15.13° has totally disappeared in the PXRD pattern of 1a. This is probably due to 4215



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that compound 3 transforms to the original form of pure OXCBZ after desolvation at 100 °C. FT-IR Spectroscopy. Vibrational spectroscopy is an excellent technique to characterize and study cocrystallization and solvate formation. The IR spectrum of OXCBZ showed peaks at 3467 and 3340 cm−1 (corresponding to free anti-NH and hydrogen bonded syn-NH respectively), 1685 cm−1 (-C O, ketone group vibration), 1654 cm−1 (-CO, carboxamide group vibration), 1591 and 1562 cm−1 (range of −CCvibration and -NH deformation), and 1407 cm−1 (C−N stretch) (Figure 6). The FT-IR spectra of 1 showed distinct shifts in both

Figure 5. The PXRD pattern of (a) OXCBZ, (b) SA, (c) compound 1, (d) compound 1a, (e) compound 1a post dissolution, (f) SAC, (g) OXCBZ−SAC (physical mixture), (h) compound 2, (i) compound 2 post dissolution (symbol ● shows the appearance of peaks corresponding to pure OXCBZ), (j) compound 3, (k) form of pure OXCBZ obtained after desolvation of 3 at 100 °C, (l) compound 3 post dissolution.

the loss of the CHCl3 molecule from the crystal structure of 1. This shows that although the CHCl3 molecule has left the channels of 1 after heating up to 150 °C, the crystal lattice was not disrupted and the hydrogen bonding between OXCBZ and SA is still maintained. Had the structure been broken, the PXRD pattern of 1a would have shown the original peaks of both OXCBZ and SA at their respective positions. The PXRD pattern of compound 2 exhibited unique peaks with reflections at about 2θ 7.07°, 9.36°, 11.12°, 12.72°, 13.47°, 14.00°, 16.69°, 17.58°, 18.66°, 19.61°, 20.21°, 21.13°, 21.80°, 23.96°, 24.68°, 25.95°, 26.90°, 27.58°, 28.06°, and 29.59°. These peaks were found to be significantly different from that of both the crystalline OXCBZ and SAC, where SAC exhibited peaks at about 2θ 9.51°, 15.39°, 16.27°, 19.25°, 25.08°, and 28.81°. The absence of peaks characteristic of pure OXCBZ and SAC in the PXRD pattern of compound 2 implies it to be a pure and homogeneous cocrystal sample. The PXRD pattern of 3 exhibited characteristic reflections at about 2θ 6.38°, 7.54°, 9.54°, 10.84°, 11.96°, 12.58°, 12.94°, 14.91°, 16.68°, 17.46°, 18.94°, 20.04°, 21.93°, 22.82°, 23.50°, 24.44°, 24.98°, and 26.14° which indicated that compound 3 exists in a crystal unique with respect to pure OXCBZ. The PXRD pattern of compound 3 after heating up to 100 °C for 1 h was found to be similar to the pure form of OXCBZ, suggesting

Figure 6. FT-IR spectra of (a) OXCBZ, (b) SA, (c) compound 1, (d) SAC, (e) OXCBZ−SAC (physical mixture), (f) compound 2, (g) AA, and (h) compound 3.

the carboxamide and carboxyl region of OXCBZ and SA respectively. After cocrystallization, the -N−H vibrations of OXCBZ shifted from their original positions to 3461 and 3302 cm−1 respectively, while −CO stretch of carboxyl group of SA shifted from 1688 cm−1 to 1636 cm−1 in 1. The FT-IR peaks of OXCBZ in the -CC- vibration and −NH deformation region 4216



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has also shifted to 1597 and 1533 cm−1 respectively. In addition, two broad stretches appeared in IR spectrum of 1 corresponding to O−H stretch of carboxylic acid hydrogen bonded to oxygen atom of amide group of OXCBZ and the H···O stretch of COOH···O at 2463 and 1948 cm−1 respectively.59 This also suggests that the COOH group of SA is present in its neutral state in compound 1. However, no change is there in the ketone group vibration of OXCBZ in compound 1 suggesting that this -CO group is not participating in any hydrogen bonded interactions. Thus, all these variations in the spectrum of compound 1, from the IR peaks of starting components, suggest it to be a cocrystalline form of OXCBZ and SA. Vibrational spectroscopy also proved to be an efficient technique to identify intermolecular hydrogen bond interactions in compound 2. The spectrum of pure SAC showed peaks at 3092 and 1721 cm−1 corresponding to NH and CO stretch of its secondary amide group respectively. In addition, peaks corresponding to asymmetric and symmetric stretching of −SO2 group were observed at 1335 and 1178 cm−1 respectively. However, the spectrum of 2 showed shifts in the peaks corresponding to carbonyl, amide, and SO2 regions when compared with the spectra of starting components. The NH and CO stretch of the amide group of OXCBZ shifted from 3467 to 3491 cm−1 and from 1653 to 1649 cm−1 respectively, while the CO stretch of the amide group of SAC shifted from 1720 to 1731 cm−1. The asymmetric stretch of SO2 of SAC also shifted from 1335 to 1325 cm−1 in the spectrum of 2. However, no change was observed in the CO stretch of the ketone group of OXCBZ as in the case of 1, indicating that it is not participating in any interactions. All these spectral shifts are quite similar to those observed in the cocrystal of CBZ and SAC by Jayasankar et al.,35 thus giving a good evidence toward formation of cocrystal of OXCBZ with SAC. IR spectra of compound 3 showed peaks at 3429, 3169, 1750, 1679, 1596, 1399, 1109, and 776 cm−1, which differed significantly from those of pure OXCBZ and AA confirming the formation of a newer phase between the starting components. In addition, two broad stretches at 2562 and 1949 cm−1 also appeared, corresponding to the O−H stretch of AA hydrogen bonded to an oxygen atom of the amide group of OXCBZ and the H···O stretch of COOH···O respectively, suggesting that carboxylic group of AA is hydrogen bonded to amide group of OXCBZ in its neutral state. SS NMR Spectroscopy. The ssNMR spectroscopy has been utilized in this study to further support the hydrogen bonding patterns illustrated in Scheme 2 for compound 2. The 13C ssNMR spectra of OXCBZ, SAC, compounds 2 and 3 are shown in Figure 7. The 13C spectra fulfill the first basic criterion of cocrystal formation in that the spectrum of cocrystal varies from the spectra of known phases of input materials. A closer inspection of the 13C ssNMR spectra of compound 2 shows that main spectral perturbations appear in the amide carbon of both OXCBZ and SAC, and in C22 which is adjacent to the -SO2 group of SAC (Table 2). These shifts indicate that the amide group of OXCBZ is most likely interacting with both the amide carbonyl as well as the -SO2 of SAC in compound 2. No change was observed in the carbonyl carbon C7 of OXCBZ indicating that this keto group is not involved in any hydrogen bonding in the complex. The 1H ssNMR spectra (Figure S1 available in Supporting Information) show poor resolution due to homonuclear dipolar couplings. A shift in the amide proton N2H of OXCBZ is observed in the spectrum of complex but is not clearly evident due to overlapping with the other aromatic

Scheme 2. Synthons Observed in CBZ, OXCBZ, and Their Multicomponent Formsa

a

(a) Amide dimer homosynthon in CBZ crystals, (b) amide chains in OXCBZ crystals, (c) identical amide carboxylic acid heterosynthon observed in both CBZ and OXCBZ multicomponent crystals.

Figure 7. 13C CP/MAS ssNMR spectra of OXCBZ, SAC, compounds 2 and 3.

protons. Similarly, a shift is observed in the signal assigned to aromatic protons of SAC including N3H in the spectrum of compound 2 (Table 2). These shifts in the amide protons of both OXCBZ and SAC points toward the direct involvement of these protons in the association between the starting components. Solid state 13C NMR spectrum for 3 illustrates the differences typically observed for solvates as inferred by different chemical 4217



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Table 2. 13C and 1H ssNMR Chemical Shift Values for Pure OXCBZ, SAC, Compounds 2 and 3 compd

C7 (δ)

C8 (δ)

C15 (δ)

OXCBZ SAC 2 Δδ = δ(OXCBZ/SAC)- δ(2) 3 Δδ = δ(OXCBZ)- δ(3)

190.71

44.63

156.09

190.05 0.66 190.20 0.51

48.52 −3.89 47.65 −3.02

159.93 −3.84 157.88 −1.78

C16(δ)

C22 (δ)

C23 (δ)

C24 (δ)

N2H(δ)

3H(δ)

OH(δ)

7.21 163.84 159.93 3.91

138.91 137.86 1.05 176.96

shifts of equivalent 13C nuclei. It is clear from Figure 7 that resonances of C7, C8, and C15 of OXCBZ has significantly shifted in the spectrum of 3 from their respective values in the pure OXCBZ spectrum indicating that both the amide as well as the keto group of OXCBZ is involved in hydrogen bonding. The 13 C resonance characteristics of the acetic acid molecule (C23 and C24) are also observed in the spectrum of 3. The 1H ssNMR spectrum of 3 exhibits the acetic acid protons resonances shifted from their solution state values as well as the amide protons of OXCBZ which have undergone an upfield shift, thus supporting the results obtained by 13C ssNMR spectroscopy. Single Crystal X-ray Diffraction. Compound 1 was obtained from the solution as crystals of suitable size, whereas compounds 2 and 3 could only be obtained as fine crystalline powders. Thus, the single crystal X-ray crystallography has been performed only on compound 1. The powder X-ray diffraction pattern of compound 1 correlated nicely with the simulated pattern derived from crystal structure of 1, thus confirming it to be a CHCl3 solvated cocrystal of OXCBZ and SA (Figure 8). In

21.82

7.67 −0.46 6.08 1.13

8.11 8.56 −0.45 10.87

Figure 9. ORTEP view of an asymmetric unit of compound 1 with atomic numbering scheme. Thermal ellipsoids are drawn at the 40% probability level. Only the major component of the disordered CHCl3 is shown here.

166.16°) and N−H···O hydrogen bonding (N2−H2A···O3, N···O 2.952 (4) Å, O···H 2.149 Å, N−H···O 155.18°) (Figure 10). Each carboxylic acid−amide heteromer connects to the neighboring heteromer via N−H···O hydrogen bonding (N2− H2B···O3, N···O 2.963 (3) Å, O···H 2.404 Å, N−H···O 123.18°), thereby forming corrugated layers that extend along the c-axis (Figure 11). These form voids to incorporate the disordered CHCl3 molecules that connect the inversion symmetry related OXCBZ molecules via C−H···O (C18A− H18A···O1, C···O 3.341(16) Å, O···H 2.409 Å, C−H···O 158.71°) interactions (Figure 12). These hydrogen bond interactions caused the delayed escape of CHCl3 from the host network that explains the observed thermal behavior of compound 1. Upon heating at 150 °C, 1 is desolvated and converted to 1a. The single crystal XRD analysis of 1 showed that the hydrogen bonding and packing arrangement of OXCBZ with SA is identical to that observed in cocrystal of CBZ with SA which also crystallized in a 1:0.5 stoichiometry in the monoclinic crystal system and illustrated the formation of identical carboxylic acid amide heterosynthon between CBZ and SA molecules33,58,62 instead of amide homosynthon between CBZ molecules (Scheme 2). Thus, it can be interpreted from this result that although the single component crystal structure of OXCBZ differs from CBZ in exhibiting a chain motif in contrast to dimer motifs,63 the multicomponent crystal structures of OXCBZ and CBZ with SA are isomorphic. Powder Dissolution Studies. The powder dissolution profiles for compounds 1a, 2, and 3 were obtained in comparison with pure OXCBZ from the slurry experiments performed in 0.1 N HCl, a medium that resembles the gastric conditions. Figure 13 clearly shows an improved dissolution profile of 1a with a

Figure 8. Experimental PXRD patterns (red) of compound 1 compared with its rietveld refined simulated PXRD pattern (blue).

the single crystal structure of OXCBZ as determined by Hempel A et al.,60 OXCBZ crystallizes in a monoclinic space group P21/c; In the present study, compound 1 crystallizes in a monoclinic space group C2/c with the single unit cell consisting of eight molecules of OXCBZ, four molecules of SA, and four molecules of chloroform (Figure 9). Crystallographic data and hydrogen bond parameters for compound 1 are given in Tables 3 and 4 respectively. The C−O and CO bond distances of the carboxyl group of SA are 1.302(3) and 1.208(4) Å respectively and thus ΔDc‑o > 0.08 Å (0.094 Å) suggesting that SA is present as a neutral molecule in the crystal lattice of 1.61 In the basic supramolecular unit, both the -COOH groups of each SA molecule are forming a carboxylic acid amide heterosynthon with the -CONH2 group of OXCBZ molecules through O−H···O (O4−H4B···O2, O···O 2.564 (3) Å, O···H 1.760 Å, O−H···O 4218



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dissolution studies, but the PXRD analysis of solids obtained at the end of experiment matched exactly with that of pure OXCBZ indicating that 1a reverted back to the free base form of OXCBZ upon slurrying in aqueous medium (post dissolution PXRD pattern shown in Figure 5e). No significant pH change was observed in the dissolution media indicating that the enhancement of the dissolution profile of 1a is not due to any pH change. In the case of compound 3, not much change was observed in the concentration achieved in comparison with pure OXCBZ. The post dissolution PXRD analysis proved that 3 also converted to pure OXCBZ during slurrying in aqueous medium (Figure 5l). An examination of dissolution profile of 2 indicated that this cocrystal sustains a higher concentration of OXCBZ throughout the 6 h study achieving a maximum concentration of 0.430 mg/ mL at a time period of 10 min. Thus, the study showed that the cocrystallization of OXCBZ with SAC improved its maximum solubility by ∼3.5 fold. As the dissolution proceeded, compound 2 showed a decrease in OXCBZ concentration indicating that the cocrystal has started dissociating slowly in acidic aqueous medium. However, the PXRD pattern of the leftover powder of 2 after the 6 h dissolution exhibited an almost similar spectrum as that of the original cocrystal, except for the appearance of a few small peaks of pure OXCBZ (indicated by ● in Figure 5i) confirming that 2 slowly dissociates during the slurrying but to a lesser extent. No significant change in pH was observed after the slurry experiment of 2, suggesting that the dissolution profile so obtained was not affected by the pH change. To further investigate the overall increase in solubility of OXCBZ upon cocrystallization with SAC, the concentration of cocrystal (measured in terms of concentration of OXCBZ) was plotted against total SAC concentration achieved during the dissolution study (Figure 14). The solubility of cocrystal 2 was found to decrease nonlinearly as the total SAC concentration increased over the remainder of the study. To understand this behavior, the solubility product and complexation constant of cocrystal 2 was anticipated by utilizing mathematical models developed by Rodriguez-Hornedo et al.64 The dissolution of 2 in 0.1 N HCl can be explained by following equilibrium reaction

Table 3. Crystal Data and Structure Refinement for Compound 1 empirical formula formula weight temperature diffractometer used radiation used, wavelength crystal system, space group unit cell dimensions

volume Z, calculated density absorption coefficient F(000) crystal size theta range for data collection scan type scan speed scan range (ω) background measurement index ranges reflections collected independent reflections absorption correction max and min transmission refinement method data/restraints/ parameters goodness-of-fit on F2 weighting scheme data to parameter ratio final R indices, 1934 reflections [I > 2σ(I)] R indices (all data) extinction coefficient largest diff peak and hole

C35H31Cl3N4O8 741.99 293(2) K Siemens P4 MoKα, 0.71073 Å monoclinic, C2/c a = 37.41(4) Å, α = 90° b = 5.355(1) Å, β = 109.97(1)° c = 18.535(3) Å, γ = 90° 3489.9(9) Å3 4, 1.412 Mg/m3 0.320 mm−1 1536 0.24 × 0.17 × 0.10 mm 2.23 to 25.00°. 2θ−θ variable, 2° to a maximum of 60°/min in ω 0.82° plus Kα separation stationary crystal and stationary counter at the beginning and end of scan, each for 25.0% of total scan time −44 ≤ h ≤ 0, 0 ≤ k ≤ 6, −20 ≤ l ≤ 22 3106 3055 [R(int) = 0.0370] Psi-Scan 0.83 and 0.73 full-matrix least-squares on F2 3055/48/281 1.048 1/[σ2(Fo2) + (0.1052*P)2 + 0.67*P], where P = (max(Fo2,0) + 2*Fc2)/3 10.9:1 R1 = 0.0641, wR2 = 0.1651

K sp

OXCBZ ·SACsolid ← → OXCBZsoln + SACsoln R1 = 0.1061, wR2 = 0.1867 0.0012(6) 0.266 and −0.397 e·Å−3

Thus, the solubility product Ksp is given by equation K sp = [OXCBZ][SAC]

[OXCBZ] =

Table 4. Hydrogen Bond Parameters for Compound 1

O4− H4B···O2 N2− H2A···O3 N2− H2B···O3 C18A− H18A···O1

symmetry

−x, −y + 1, −z

R (D− H) (Å)

r (H···A) (Å)

r (D···A) (Å)

∠D−H···A (deg)

0.820

1.760

2.564(3)

166.16

0.860

2.149

2.952(4)

155.18

0.860

2.404

2.963(3)

123.18

0.980

2.409

3.341(16)

158.71

(2)

Solubility product (Ksp) was then calculated from the slope of plot of total OXCBZ concentration in the solution versus the inverse of total SAC concentration (Figure 15) according to equation

maximum concentration of 0.195 mg/mL achieved in 25 min as compared to OXCBZ alone which exhibited a peak solubility of 0.128 mg/mL at a similar time point. Although from its dissolution profile, 1a seems to remain stable throughout the

D−H···A

(1)

K sp [SAC]

(3)

A linear dependence was observed and its regression analysis resulted in the following equation of line (±standard error of slope and y-intercept). y = 0.00000587( ±0.42 × 10−7)x + 0.00013 ( ±0.12 × 10−4)

This equation clearly reveals that y-intercept has a significant value relative to slope, which suggested that a 1:1 solution complex is in equilibrium with the dissolved single components and the cocrystal according to equation 4219



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Figure 10. The layered structure of compound 1 in which OXCBZ molecules are connected to an SA molecule by O−H···O and N−H···O hydrogen bonds. Interaction between CHCl3 molecule hydrogen and keto oxygen of the OXCBZ molecule of adjacent layers can also be observed.

Figure 11. Projections of crystal packing in compound 1 illustrating the corrugated layers that extend along the c-axis. K11

OXCBZsoln + SACsoln ↔ OXCBZ ·SACsoln

The complexation constant K11 was then calculated from the yintercept based on equation derived by Rodriguez-Hornedo et al.64

(4)

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Figure 13. Dissolution profiles for pure commercial sample of OXCBZ in comparison with compounds 1a, 2, and 3 in 0.1 N HCl at 37 °C.

Figure 14. Solubility of cocrystal 2 in terms of OXCBZ concentration as a function of SAC concentration in 0.1 N HCl at 37 °C.

of cocrystal degradation or dissociation was observed even after 4 weeks. Thus, the cocrystal 2 appeared to be physically stable under ambient as well as accelerated stability conditions studied (Figure S2 available in Supporting Information). Figure 12. (a) Packing in space filling mode along the crystallographic caxis showing CHCl3 molecules fitted in voids between layers (green = OXCBZ, blue = SA, red = CHCl3). (b) Space filling mode representing the void in which the CHCl3 molecule fits in.

[OXCBZ] =

K sp [SAC]

+ K11K sp

(5)

The calculated values for Ksp and K11 are given in Table 5. A higher value of K11 (22.64 M−1) for the cocrystal suggest a significant complexation between cocrystal components in the solution which is thus responsible for overall increase in the solubility of 2 as compared to OXCBZ alone. Solid-State Physical Stability. The samples 1a, 2, and 3 were kept under ambient and stability conditions of 25 °C/60% RH and 40 °C/75% RH and analyzed by PXRD after 4 weeks. Compounds 1a and 3 were found to be stable under ambient and 25 °C/60% RH conditions, but converted to pure OXCBZ at higher humidity conditions. In the case of compound 2, no sign

Figure 15. Total OXCBZ concentration in equilibrium with cocrystal 2, as a function of the inverse total SAC concentration in 0.1 N HCl at 37 °C. 4221



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Table 5. Solubility Values of Reactants and Ksp, K11, and ΔG° for Cocrystal OXCBZ·SAC in 0.1 N HCl at 37 °Ca parameter

value

SOXCBZ (M) SSAC (M) Ksp (M2) K11 (M−1) ΔG° (kJ·mol−1)

5.07× 10−4 ± (6.05 × 10−6) 141.39 × 10−4 ± (163.77 × 10−6) 5.87 × 10−6 ± (0.42 × 10−7) 22.64 ± 2.36 −0.58 ± 0.01

a

SOXCBZ and SSAC are the solubilities of OXCBZ and SAC respectively in 0.1 N HCl at 37 °C. All the values are mean ± standard deviation of n = 3.

Further, any transformation of equimolar mixtures of solid reactants (OXCBZ and SAC) to cocrystal 2 was investigated by FT-IR spectroscopy and it was found that this mixture of the unmilled reactants did not form cocrystal even after 90 days of storage at 40 °C−75% RH. (Figure S3 available in Supporting Information). This is in contrast with the physical mixture of unmilled CBZ and SAC, which resulted in cocrystal formation within 60 days.65 This can be explained on the basis of the magnitude of free energy of cocrystal formation for 2, calculated from solubility and Ksp values according to the following chemical equilibrium.65 OXCBZ(solid) + SAC(solid) ↔ OXCBZ ·SAC(solid)

Figure 16. Dose−response graph for cocrystal 2 in comparison with pure commercial sample of OXCBZ in mice.

Table 6. ED50 Values of OXCBZ in Pure Drug and Its Cocrystal Form along with the 95% Confidence Limits

(6)

compound

ED50 mg/kg (95% CL)

OXCBZ OXCBZ·SAC

15.67 (10.61−22.39) 9.02 (7.61−19.39)

The free energy of the reaction becomes ΔG° = ‐RT ln

SOXCBZSSAC K sp

solvated cocrystal 1 of OXCBZ/SA/CHCl3 by a solution method. Compound 1, identified in the present work is the earliest example of OXCBZ cocrystal with complete structural characterization to our knowledge. Compounds 2 and 3 have been characterized by DSC/TGA, PXRD, FT-IR, and ss-NMR spectroscopy. The desolvated form of compound 1, i.e., compound 1a, along with compounds 2 and 3 were selected for solubility and stability measurements. Out of these three multicomponent crystals, the one formed with saccharin, 2, showed improved pharmaceutical properties and efficacy compared with single component OXCBZ crystals. Further, an attempt has been made to describe the solubility of this cocrystal by determining the solubility product and complexation constant. The free energy of cocrystal formation for 2 from the reactant mixtures in solid state has also been determined and has been found to be negative but very reduced in magnitude which accounts for its low spontaneity of formation. In conclusion, the study demonstrates the formation of cocrystals in search for exploring better formulations of OXCBZ. Crystal Structure Prediction calculations for compounds 2 and 3 are under process by utilizing polymorph predictor and Reflex module of Material Studio 6.0 (Accelerys).

(7)

where SOXCBZ and SSAC are the solubilities of pure OXCBZ and SAC in 0.1 N HCl at 37 °C and the values are given in Table 5. From eq 7, ΔG° was calculated to be −0.58 kJ mol−1 as compared to −5.1 kJ mol−1 for CBZ−SAC cocrystal.65 This negative free energy change indicates that although cocrystal 2 is the thermodynamically stable phase, the magnitude of its ΔG° is very small which explains the low spontaneity of its formation. ED50 Values in Mice. Upon the basis of a significant improvement in the initial rate of dissolution of OXCBZ from the cocrystal 2, we hypothesized that this improved solubility of drug would translate into enhanced exposure and hence improved oral absorption of the drug. To support this, we examined the dose-dependent effect of 2 by performing MES test in mice. Equivalent amounts of both OXCBZ and cocrystal 2 were given orally to a sufficient number of animals in various doses and the abolition of hind limb extension was observed. Both the pure drug as well as cocrystal 2 produced abolition of hind limb extension depending upon the dose. Thus, a dose− response curve was subsequently calculated on the basis of percentage of animals protected against the HLE and the ED50 with 95% confidence limits (CL) was calculated from this curve (Figure 16). The ED50 value for 2 following oral administration was found to be lower as compared to pure OXCBZ (Table 6). Thus, a smaller dose of cocrystal produces an effect similar to that produced by larger dose of the pure drug. This lowering of the dose provides an evidence that cocrystal based enhanced solubility of the drug resulted in its better oral absorption in mice.

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ASSOCIATED CONTENT

S Supporting Information *

PXRD and FT-IR plots for physical stability studies, 1H MAS ssNMR spectra, and crystallographic .cif file. This information is available free of charge via the Internet at http://pubs.acs.org/.

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CONCLUSION The present study illustrates the formation and characterization of three novel multicomponent crystalline forms of OXCBZ. A crystal engineering approach was utilized to prepare a 2:1:1

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +91-9316015096. Notes

The authors declare no competing financial interest. 4222



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ACKNOWLEDGMENTS We gratefully acknowledge the financial support provided by University Grants Commission (UGC), New Delhi, India, for accomplishing this work. We also acknowledge NMR Research Centre, Indian Institute of Science, Bangalore (India) for their support in conducting solid state NMR analysis on our samples.

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