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Pharmaceutical Cocrystals of Ribavirin with Reduced Release Rates Jia-Mei Chen,† Song Li,‡ and Tong-Bu Lu*,†,§ †

School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China § MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China ‡

S Supporting Information *

ABSTRACT: Cocrystals have been extensively utilized to improve drugs’ properties. Ribavirin is a water-soluble broad-spectrum antiviral drug and its application is severely limited by the peak-to-trough fluctuation in plasma drug concentrations and some undesirable side-effects. We show here that formation of cocrystals may be a useful approach to overcome this problem by reducing the release rate of ribavirin. Three cocrystals of ribavirin with 3,5-dihydroxybenzoic acid (1), gallic acid (2), and barbituric acid (3) were successfully prepared and characterized by powder and single crystal X-ray diffraction, infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis, as well as dynamic vapor sorption measurement. The dissolution process revealed that 1−3 showed a reduced release rate as compared to ribavirin in the buffer solution representing intestinal pH 6.8. This study indicates that the release rate of ribavirin can be manipulated over a wide range by the formation of cocrystals, which may subsequently help lower its peak-to-trough fluctuation in plasma concentrations.

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INTRODUCTION

retains their structural integrity and pharmacological activity.11−19 Ribavirin (RBV, Scheme 1) is a water-soluble (142 mg/mL, 25 °C) synthetic nucleoside with broad spectrum antiviral

The successful delivery of any drugs to patients requires the ability to produce safe and effective drug products. However, certain large groups of drugs are characterized by unfavorable physical, chemical, or biological properties that compromise their efficacy or safety and thus challenge their successful delivery. Crystal engineering is the study of intermolecular interactions in crystal packing and the utilization of such study to design new solids with desirable physicochemical properties.1 Recently, crystal engineering has played an important role in the context of drug delivery by means of exerting a control on the intermolecular interactions to optimize the properties of drugs.2−7 Cocrystallization is one of the crystal engineering approaches emerging in the pharmaceutical field. Pharmaceutical cocrystal is defined as a multiple crystalline molecular complex that often relies on hydrogen-bonded assemblies between API and neutral molecular coformers with well-defined stoichiometries.8−10 Numerous examples have well established the potential of cocrystals in enhancing the properties of drugs, such as hygroscopicity, mechanical properties, permeability, solubility, dissolution rate, and bioavailability, in a way that © XXXX American Chemical Society

Scheme 1. Molecular Structures of Ribavirin and Three Coformers and the Numbering of the Cocrystals Described in This Article

Received: August 22, 2014 Revised: October 5, 2014

A

dx.doi.org/10.1021/cg501247x | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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Table 1. Crystallographic Data and Refinement Parameters for Cocrystals 1−3 chemical formula formula wt temperature (K) crystal size (mm3) crystal system space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) volume (Å3) Z Z′ density (g cm−3) 2θ range F (000) index ranges

no. of reflns no. of unique reflns no. of params Rall, Robsa wR2,all, wR2,obsb flack GOF CCDC No. a

1

2

3

C8H12N4O5, 6(C7H6O4),7(H2O) 1295.04 150(2) 0.15 × 0.10 × 0.08 triclinic P1 9.44471(17) 10.38896(19) 15.6489(3) 77.4892(15) 72.8378(16) 77.6285(16) 1413.55(4) 1 1 1.521 3.00−66.91 678 −11 ≤ h ≤ 11 −12 ≤ k ≤ 12 −18 ≤ l ≤ 18 9726 9513 873 0.0398, 0.0389 0.1085, 0.1074 0.01(10) 1.055 1003744

C8H12N4O5, C7H6O5,H2O 432.35 150(2) 0.10 × 0.06 × 0.06 monoclinic P21 11.0567(2) 7.10570(10) 11.4404(2) 90 96.913(2) 90 892.29(3) 2 1 1.609 3.89−67.03 452 −13 ≤ h ≤ 13 −8 ≤ k ≤ 8 −13 ≤ l ≤ 13 3170 3064 286 0.0258, 0.0241 0.0612, 0.0598 0.04(13) 1.072 1003743

C8H12N4O5, 0.5(C4H4N2O3),H2O 326.28 150(2) 0.10 × 0.08 × 0.05 monoclinic C2 34.8962(7) 8.12494(16) 4.91985(11) 90 90.939(2) 90 1394.73(5) 4 1 1.554 5.07−65.38 684 −40 ≤ h ≤ 38 −9 ≤ k ≤ 9 −5 ≤ l ≤ 5 2228 2185 226 0.0310, 0.0291 0.0733, 0.0718 0.06(18) 1.076 1003745

R1 = Σ||Fo| − |Fc||/Σ||Fo|. bwR2 = [Σ[w(Fo2 − Fc2)2]/Σw(Fo2)2]1/2; w = 1/[σ2 (Fo)2 + (aP)2 + bP], where P = [(Fo2) + 2Fc2]/3. Aladdin Reagent, Inc. They were used without any further purification. All other reagents and solvents obtained from commercial suppliers were used as received. Preparation of RBV-35DHBA Cocrystal Hydrate (1:6:7) (1). A solution of RBV (244 mg, 1 mmol) in 2 mL of water was mixed with a solid of 35DHBA (924 mg, 6 mmol). The resulting suspension was allowed to stir at room temperature for 24 h, in which the solid of 35DHBA slowly transformed to cocrystal of 1 as white solid. The reaction mixture was filtered and the isolated solid was dried under ambient condition. Yield: 936 mg, 72%. Anal. (%) Calcd for C50H62N4O36: C, 46.37; H, 4.83; N, 4.33. Found: C, 46.32; H, 4.80; N, 4.35. IR (KBr, ν): 3283 (s), 3078 (m), 2999 (m), 2654 (m), 1695 (s), 1603 (s), 1516 (m), 1486 (s), 1419 (s), 1343 (s), 1307 (s), 1268 (s), 1225 (m), 1207 (m), 1167 (s), 1107 (m), 1083 (w), 1041 (w), 1005 (s), 946 (m), 911 (w), 864 (w), 839 (m), 769 (m), 734 (m), 666 (m), 615 (m), 558 (w), 528 (w), 476 (w) cm−1. The filtrate was left to evaporate slowly at RT. After about 1 week, block-shaped crystals of 1 were obtained. Preparation of RBV-35DHBA Cocrystal Hydrate (1:6:5) (1a). When drying a bulk batch of 1 under vacuum, another RBV-35DHBA cocrystal pentahydrate was obtained. Anal. (%) Calcd for C50H58N4O34: C, 47.70; H, 4.64; N, 4.45. Found: C, 47.00; H, 5.02; N, 4.77. IR (KBr, ν): 3271 (s), 3074 (m), 2998 (m), 2833 (m), 2654 (m), 2513 (m), 2003 (w), 1695 (s), 1645 (m), 1603 (s), 1487 (s), 1420 (s), 1343 (s), 1306 (s), 1270 (s), 1224 (m), 1206 (m), 1166 (s), 1105 (m), 1038 (w), 1005 (s), 945 (m), 867 (w), 838 (m), 768 (m), 734 (m), 695 (w), 666 (m), 613 (m), 558 (w), 528 (w), 476 (w) cm−1. Preparation of RBV-GA Cocrystal Hydrate (1:1:1) (2). A mixture of RBV (244 mg, 1 mmol) and GA (170 mg, 1 mmol) was added to 0.5 mL of water. The resulting suspension was allowed to stir at RT for 24 h in which the solid of the reactants slowly transformed to

properties and currently indicated for use as a combination therapeutic for Hepatitis C.20 As such, RBV is administered in dosages with 800−1200 mg per day, together with interferon injections.21 The large dose of RBV as well as its high dissolution rate causes the peak-to-trough fluctuation in plasma drug concentrations, which is closely related to undesirable side-effects for some patients.22,23 Herein we attempt to obtain cocrystals of RBV that may be advantageous to overcome this problem by decreasing its release rate. Formation of a cocrystal requires complementary hydrogen bonding between RBV and a second molecule. The most useful hydrogen bonding group of RBV is the amide functionality, which is known to form robust hydrogen bonding interactions with carboxylic acids and amide compounds.24,25 A group of nontoxic carboxylic acids, including 4-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, gallic acid, methyl gallate, and barbituric acid, were selected for cocrystal screening. Finally, three cocrystals of RBV with 3,5-dihydroxybenzoic acid, gallic acid, and barbituric acid were successfully obtained through a slurry method (Scheme 1). The resulting cocrystals were characterized by powder and single crystal X-ray diffraction, infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis, as well as dynamic vapor sorption measurement. The dissolution behavior was also investigated.

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EXPERIMENTAL SECTION

Reagents. Ribavirin (form II)26 was purchased from Suizhou Hongqi Chemical Co., Ltd. 3,5-Dihydroxybenzoic acid (35DHBA), gallic acid (GA), and barbituric acid (BA) were purchased from B



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Table 2. Hydrogen Bond Geometrical Parameters of Cocrystals 1−3 1

2

3

hydrogen bond

H···A (Å)

D···A (Å)

∠DH···A (deg)

symmetry

N4−H4A···O4 O3−H3A···O36 O4−H4···O19 O5−H5A···O33 O6−H6···O11 O8−H8···O31 O9−H9···O13 O10−H10···O7 O12−H12···O8 O13−H13A···O30 O14−H14···O19 O16−H16···O36 O17−H17···O21 O18−H18···O15 O20−H20···O16 O21−H21···O33 O22−H22···O27 O24−H24···O28 O25−H25···O35 O26−H26···O23 O28−H28···O34 O29−H29···O25 O30−H30B···O1 O30−H30A···O12 O31−H31B···O9 O31−H31A···O32 O32−H32A···N1 O32−H32B···O15 O33−H33B···O1 O33−H33A···O20 O34−H34A···O5 O34−H34B···O29 O35−H35B···O3 O35−H35A···O24 O36−H36B···O9 O36−H36A···O17 N4−H4B···O9 O3−H3A···O11 O4−H4···O9 O5−H5A···O11 O6−H6···O3 O7−H7A···O5 O8−H8···O1 O10−H10···N1 O11−H11A···O1 O11−H12A···O3 N4−H4A···O7 N4−H4B···O6 N5−H5B···O1 O3−H3A···O8 O4−H4···O3 O5−H5A···O4 O8−H8D···O1

2.29 1.97 2.01 2.07 1.82 1.77 1.85 1.84 1.91 1.79 1.80 1.90 1.87 1.83 1.89 1.88 1.82 1.90 1.85 1.82 1.80 1.92 2.05 2.06 2.08 1.74 1.88 1.82 1.85 1.93 2.00 2.07 1.86 2.00 2.20 1.97 2.13 1.93 2.23 1.96 2.01 2.14 1.93 1.89 2.00 2.05 2.14 2.09 1.87 1.94 1.96 1.75 1.90

3.082(3) 2.753(3) 2.860(2) 2.800(2) 2.631(2) 2.584(3) 2.660(2) 2.642(2) 2.728(2) 2.603(2) 2.618(2) 2.705(2) 2.679(2) 2.648(2) 2.698(2) 2.695(2) 2.632(2) 2.706(2) 2.669(2) 2.626(2) 2.607(2) 2.725(2) 2.831(2) 2.887(2) 2.772(3) 2.658(3) 2.787(3) 2.886(2) 2.686(2) 2.817(2) 2.878(3) 2.861(3) 2.773(4) 2.894(3) 2.955(3) 2.819(3) 2.9452(16) 2.7104(16) 2.9924(16) 2.7496(16) 2.8118(15) 2.8444(15) 2.6721(15) 2.7017(16) 2.7951(17) 2.8902(16) 2.9907(19) 2.911(2) 2.734(2) 2.755(2) 2.758(2) 2.690(2) 2.900(2)

154 159 169 148 171 171 170 165 171 170 176 168 171 178 170 172 170 168 174 168 168 167 152 165 164 158 156 177 163 171 174 169 174 170 149 174 158 158 156 160 166 145 150 171 156 167 171 159 179 175 160 169 170

x, y + 1, z x, y, z x, y + 1, z x + 1,y, z x, y, z x, y, z x − 1, y, z + 1 x, y, z x, y, z −1 x + 1, y, z x, y, z x, y, z x − 1, y, z + 1 x, y, z x, y, z − 1 x + 1, y, z x, y, z x − 1, y, z + 1 x, y, z x, y, z x, y, z x, y, z − 1 x − 1, y − 1, z x, y, z x + 1, y, z x, y, z x, y − 1, z x, y, z x − 1, y − 1, z x, y, z x, y, z x + 1, y, z x, y, z x +1, y, z x +1, y, z x +1, y, z x, y, z + 1 −x +2, y + 1/2, −z + 1 x + 1, y, z + 1 −x +2, y − 1/2, −z + 1 −x +1, y − 1/2, −z + 1 x −1, y, z x, y, z x, y, z − 1 x, y, z x, y, z x, y + 1, z + 1 x, y, z + 1 x, y − 1, z − 1 x, y, z −x + 3/2, y + 1/2, −z + 1 −x + 3/2, y + 1/2, −z + 2 x, y, z − 1

cocrystal of 2 as white solid. The reaction mixture was filtered, and the isolated solid was dried under vacuum for 24 h. Yield: 357 mg, 83%. Anal. (%) Calcd for C15H20N4O11: C, 41.67; H, 4.66; N, 12.96. Found: C, 41.68; H, 4.69; N, 12.95. IR (KBr, ν): 3414 (s), 3238 (s), 3195 (s), 3137 (s), 2987 (m), 2943 (m), 2917 (m), 2864 (m), 2756 (m), 2492 (m), 1881 (w), 1689 (s), 1667 (s), 1621 (s), 1532 (m), 1489 (m), 1477 (m), 1448 (m), 1387 (m), 1329 (s), 1297 (s), 1271 (m), 1249

(s), 1199 (s), 1138 (w), 1115 (s), 1086 (m), 1041 (s), 1028 (m), 997 (m), 961 (w), 941 (m), 889 (m), 869 (m), 847 (m), 801 (w), 791 (w), 766 (m), 748 (m), 714 (m), 655 (s), 571 (w), 558 (m), 534 (m), 447 (w), 443 (w) cm−1. The filtrate was left to evaporate slowly at RT. After about 1 week, rod-shaped crystals of 2 were obtained. Preparation of RBV-BA Cocrystal Hydrate (2:1:2) (3). A solution of BA (64 mg, 0.5 mmol) in 1 mL of water was mixed with a C



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Figure 1. (a) First, (b) second, and (c) third 2D sheet of 35DHBA and water molecules, and (d) 3D structure of 1 (the magenta, green, and yellow color are the first, second, and third sheets, respectively). solid of RBV (244 mg, 1 mmol). The resulting suspension was allowed to stir at room temperature for 24 h, in which the solid of RBV slowly transformed to cocrystal of 3 as white solid. The reaction mixture was filtered and the isolated solid was dried under vacuum for 24 h. Yield: 513 mg, 79%. Anal. (%) Calcd for C10H16N5O7.5: C, 36.81; H, 4.94; N, 21.47. Found: C, 36.78; H, 4.89; N, 21.45. IR (KBr, ν): 3605 (s), 3507 (s), 3336 (s), 3221 (s), 3154 (m), 2984 (w), 2962 (m), 2931 (m), 2878 (w), 2750 (s), 2535 (w), 2126 (w), 2056 (w), 1728 (s), 1703 (s), 1677 (s), 1630 (s), 1603 (m), 1528 (m), 1460 (m), 1439 (s), 1403 (s), 1354 (s), 1311 (s), 1284 (m), 1257 (s), 1228 (w), 1216 (w), 1181 (m), 1138 (m), 1101 (m), 1090 (w), 1065 (s), 1031 (s), 1006 (m), 969 (m), 943 (w), 911 (m), 898 (m), 876 (w), 837 (m), 803 (w), 763 (m), 725 (m), 705 (m), 660 (m), 646 (s), 574 (w), 550 (m), 520 (m), 501 (m), 419 (m), 411 (w) cm−1. The filtrate was left to evaporate slowly at RT. After about 1 week, rod-shaped crystals of 3 were obtained. Single Crystal X-ray Diffraction (SXRD). SXRD data of 1−3 were collected at 150 K on an Agilent Xcalubur Nova CCD diffractometer, with graphite monochromated Cu Kα radiation (λ = 1.5418 Å). Cell refinement and data reduction were applied using the program package CrysAlis PRO. The structures were solved by the direct methods and refined by the full-matrix least-squares method on F2. All the non-hydrogen atoms were refined anisotropically. Hydrogen positions on water were located in Fourier-difference electron density maps. Other hydrogen atoms were refined at geometrically constrained riding positions. All the calculations were performed using the SHELX-97 program.27 Molecular diagrams were generated using Mercury 3.3 (Cambridge crystallographic data center, U.K.).28 Crystallographic CIF files (CCDC Nos. 1003743, 1003744, and 1003745) are available at www.ccdc.cam.ac.uk/data_request/cif. The crystallographic data are summarized in Table 1, and selected hydrogen bond geometrical parameters are given in Table 2. Powder X-ray Diffraction (PXRD). PXRD analysis was performed on a Bruker D2 Advanced diffractometer (Bruker, PHASER) operated

with Cu Kα radiation (λ = 1.5418 Å) at 30 kV and 10 mA. The data were collected over an angular range of 5−40° (2θ) value in continuous scan mode using a step size of 0.014° (2θ) and a step time of 0.1 s. Typically, 30 mg of solid was used for analysis and pressed gently on a silicon slide to give a level surface. Calculated PXRD patterns were generated from the single crystal structure data using Mercury CSD 3.3. Infrared (IR) Spectroscopy. IR (KBr pellet) spectra were recorded on a Bruker EQUINOX 55 FT-IR spectrometer. A total of 64 scans were collected over a range of 4000−400 cm−1 with a resolution of 0.2 cm−1 for each sample. Differential Scanning Calorimetry (DSC). DSC was recorded in a nitrogen atmosphere using a Netzsch DSC-204 Instrument. The sample was placed in an aluminum pan and scanned from 30 to 200 °C at a heating rate of 10 °C/min. Thermogravimetric (TG) Analysis. TG analysis was performed in a nitrogen atmosphere using a Netzsch TG-209 instrument. The sample was placed in an aluminum sample pan and heated over the temperature range of 30−500 °C at a heating rate of 10 °C/min. Dynamic Vapor Sorption (DVS) Study. The DVS experiments were carried out on a SMS DVS instrument. Crystalline samples of 1− 3 were placed onto the DVS sample pan and pre-equilibrated at 0% RH and 25 °C under a stream of dry nitrogen for several hours. The relative humidity was then increased in 10% RH steps to 90% RH. Finally, the humidity was decreased in a similar fashion for the desorption phase. The temperature was maintained at a constant of 25 ± 0.1 °C. The equilibration criterion dm/dt (change in mass as a function of time) was set at 0.002%/min for all steps. The sorption isotherms were calculated from the equilibrium mass values. Dissolution Study. Dissolution study was performed using the paddle method29 on a ZQY-2 Dissolution Tester (Shanghai Huanghai Yaojian instrument distribution Co., Ltd.). Approximately 35 mg of RBV (the same mass of RBV for each cocrystal) was compressed in a hydraulic press at 0.5 t for 5 s in a die of 5 mm diameter disk. The disk D



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Figure 2. (a) Hydrogen bonding interactions of RBV with the first (magenta), (b) second (green), and (c) third (yellow) 35DHBA water sheet, and (d) 1D chain of RBV constructed in the channel framework in 1. was coated using paraffin wax, leaving only the surface under investigation free for dissolution. Then the disk was dipped into 150 mL of 0.02 M phosphate buffer saline (PBS, pH 6.8) at 37 °C, with the paddle rotating at 50 rpm. At each time interval, 2 mL of the dissolution medium was withdrawn and replaced by an equal volume of fresh medium to maintain a constant volume. After filtered through a 0.22 μm nylon filter, the quantity of dissolved RBV was determined by HPLC analysis of the filtrate. The peak area values were related to solution concentrations using a calibration curve. The dissolution profiles were represented as the cumulative percentages of the amount of RBV released at each sampling interval. Each profile is the average of three individual disks. HPLC Analysis. The contents of RBV were analyzed by a Shimadzu LC-20A HPLC system, with a C18 column (Inertsil ODS-3, 5 μm × 4.6 mm × 150 mm column, GL Sciences Inc., Japan) and a UV detection wavelength of 218 nm. The mobile phase consisted of methanol and 0.5% formic acid (30/70, v/v), with a flow rate of 1.0 mL/min.

and interactions of RBV and coformers in the crystal lattice. The crystal structure of 1 belongs to the triclinic, P1̅ space group (Table 1). The asymmetric unit contains one RBV, six 35DHBA, and seven water molecules. Two 35DHBA molecules are connected through a hydrogen-bonded R22(8) homosynthon to form a dimer, and the 35DHBA dimers are further connected with two water molecules through four O−H···O hydrogen bonds to form a one-dimensional (1D) tape (Figure 1a). The 1D tapes are held together to form a two-dimensional (2D) sheet via two O−H···O hydrogen bonds (Figure 1a). Another four 35DHBA and four water molecules, respectively, form two similar 2D sheet structures (Figure 1b,c). Three 2D sheets are packed together to generate a three-dimensional (3D) channel structure (Figure1d) via intersheet π···π interactions (∼3.4 Å) and hydrogen bonds. The seventh water molecule is linked to one water molecule of the first 35DHBA sheet through O−H···O hydrogen bonds (Figure 1a), and it is prone to escape from the crystal lattice due to the lack of direct interactions with 35DHBA framework. RBV molecules are captured in the channels through multiple hydrogen bonds with water molecules of every sheet (Figure 2a−c). RBV molecules assemble themselves into a 1D chain through N− H···O hydrogen bonds in each channel (Figure 2d). The molecular interactions of RBV in 1 is different from those in pure RBV (form II, refcode VIRAZL01)26 in which each RBV forms three hydrogen bonds between the carboxamide group of RBV and the base ring and hydroxyl of adjacent RBV molecules to form a one-dimensional chain (Figure S2, Supporting Information).

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RESULTS AND DISCUSSION Preparation of the Cocrystal. A series of pharmaceutically acceptable carboxylic acids were selected as potential cocrystal coformers. Grinding of RBV and guest acids resulted in poor crystalline products containing amorphous impurity. Thus, we choose to use a slurry method to screen cocrystals. Finally, three cocrystals of RBV with 35DHBA, GA, and BA were identified by the appearance of new reflections in their diffractograms (2θ) (Figure S1, Supporting Information), which do not exist in diffractograms of the starting materials. Crystal Structures Analysis. The availability of single crystal X-ray structures should help identify the arrangement E



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Figure 3. (a) Two-dimensional sheet and (b) top view and (c) side view of 3D structure (the green and magenta colors are the first and second sheets, respectively) of 2.

π···π interactions (∼3.5 Å) to form a 3D structure (Figure 3b,c). The crystal structure of 3 was solved in a monoclinic, C2 space group (Table 1). The asymmetric unit contains one RBV, one-half BA, and one water molecule. Two RBV molecules are simultaneously linked to one BA molecule through a hydrogenbonded R22(8) synthon (N4−H4A···O7 and N5−H5B···O1) to form a trimer, and the trimers are connected to each other via N4−H4B···O6 hydrogen bonds to form a 1D chain (Figure 4a). The adjacent 1D chains are connected by interchain O4− H4···O3 hydrogen bonds to generate a 2D framework (Figure 4b). The 2D frameworks are further held together with one water through O3−H3A···O8 and O8−H8D···O1 hydrogen

The crystal structure of 2 was solved in a monoclinic, P21 space group (Table 1). The asymmetric unit contains one RBV, one GA, and one water molecule. RBV molecules alternatively link GA molecules through O10−H10···N1, N4−H4B···O9, and O8−H8···O1 intermolecular hydrogen bonds to generate a 1D chain (Figure 3a). Water molecules are linked to RBV molecules via O11−H11A···O1 and O11−H12A···O3 hydrogen bonds (Figure 3a). The adjacent 1D chains are further connected by interchain O4−H4···O9 and O7−H7A···O5 hydrogen bonds to generate a 2D sheet (Figure 3a). The 2D sheets are further held together via intersheet O6−H6···O3, O5−H5A···O11, and O3−H3A···O11 hydrogen bonds and F



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Figure 4. (a) One-dimensional tape, (b) 2D sheet, and (c) side view of 3D structure (the magenta and green colors are the first and second sheets, respectively) of 3.

within it, while for 2 and 3, coformers alternatively connect to RBV with multiple hydrogen bonds to form sheet structures. PXRD Analysis. The PXRD patterns of the bulk batch of 1−3 showed the absence of characteristic peaks of the starting compounds and closely match the simulated patterns generated from the single crystal diffraction data (Figure S1, Supporting Information). It indicated that the structures obtained from single crystal X-ray diffraction data were representative of the bulk crystalline samples. The slight displacement of the peaks at the high angle regions between measured and simulated patterns is mainly caused by the different measuring temperature between the powder patterns (room temperature) and the single crystal X-ray diffraction data (150 K). When drying bulk batch of 1 under vacuum, the PXRD analysis of the

bonds as well as O5−H5A···O4 to form a 3D structure (Figure 4c). From crystal structure analysis, we can see that 1−3 are cocrystal hydrates with variable stoichiometry of coformers and water molecules in the crystal lattice due to diverse hydrogen bonding sites and conformations of RBV. The formation of a hydrate may be helpful to reduce the dissolution rate of RBV since the hydrate is typically considered to be less soluble in water than the corresponding anhydrate.30,31 The stoichiometry of RBV/coformer/water is diverse in these cocrystals, varying from 1:6:7 in 1, 1:1:1 in 2, to 2:1:2 in 3. For 1, six coformers and seven water molecules form an atypical supramolecular organic channel framework that tightly captures RBV molecules G



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Figure 5. DSC-TG curves for 1 (a), 2 (b), 3 (c), and 1a (d).

induced phase transition of 3. Furthermore, the third peak at 157 °C points to a process of melting. The melting point of 3 is also below those of RBV (174 °C) and BA (245 °C). DVS Study. The influence of humidity on the stability of 1− 3 was studied by a DVS experiment. The resulting vapor sorption isotherms are shown in Figure 6. The phase transitions during the DVS experiment were confirmed by PXRD detection (Figures S4 and S5, Supporting Information). During the initial drying stage, 1 lost two water molecules (approximately 2.9% in mass) to form pentahydrate species 1a (Figure S4d, Supporting Information) around 0% RH, and

resulting solids shows the position and intensity of the peaks have changed, suggesting that 1 may lose part of crystalline water and transform to new form 1a during the drying process (Figure S3, Supporting Information). Compound 1a is preliminarily identified as a pentahydrate species according to the elemental analysis. Thermal Analysis. The thermodynamic stability of these new crystalline forms was investigated by means of DSC and TG analysis (Figure 5). The TG curve of 1 shows a weight loss of 9.75% starting at 30 °C (Figure 5a), which is associated with the loss of seven H2O molecules (calcd 9.73%). In contrast, TG curve of 1a shows a weight loss of 7.46% over the 64 to 125 °C temperature range, which is consistent with the loss of five H2O molecules (calcd 7.15%) (Figure 5d). This result further confirms that 1a is a pentahydrate. Both 1 and 1a begin to decompose before melting, and their DSC curves just show the endothermic peak of the dehydration process (Figure 5a,d). The TG curve of 2 evidences a weight loss of 3.91% starting at 95 °C corresponding to the expected value for the 1:1 hydrate (calcd 4.16%). In the same temperature range, an endothermic peak due to dehydration is present in the DSC curve, followed by the second endothermic peak due to a process of melting from 150 to 170 °C (Figure 5b). The melting point of 2 is below those of RBV (174 °C) and GA (260 °C). The DSC trace of 3 shows three endothermic peaks between 70 and 180 °C (Figure 5c). The first peak at 109 °C corresponds to a mass loss of 5.23% in the TG curve, which is consistent with the loss of one H2O molecule (calcd 5.52%). The second peak at 116 °C may be related to a dehydration-

Figure 6. Water vapor sorption/desorption isotherm plots of 1−3 at 0−90% RH, 25 °C. H



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°C) of 35DHBA (84 mg/mL) and GA (12 mg/mL) is less than that of RBV (142 mg/mL), while the aqueous solubility of BA (142 mg/mL) is the same as that of RBV. (2) The formation of hydrate in cocrystals 1−3 will decrease their aqueous solubility and subsequently reduce their release rates. (3) The effective crystal packing arrangement in cocrystals with variable supramolecular interactions, such as hydrogen bonding and π···π interactions, will also contribute to their reduced release rates. (4) The lowest release rate for 1 can be attributed to the higher stoichiometric coformer and hydrate in 1 (1:6:7).

1a absorbed negligible water (0.41%) below 60% RH. Then it rapidly absorbed water above 60% RH and gradually transformed to heptahydrate species 1 as the humidity increased. The sample absorbed 3.87% of water at 90% RH and completely converted to 1, which was proven by PXRD measurement (Figure S4e, Supporting Information). During the desorption process, 1 can be stable above 10% RH, and then it lost two water molecules and converted to 1a below 10% RH. The above results display a humidity-induced transformation relationship between 1 and 1a at room temperature. In contrast, cocrystals 2 and 3 exhibit simple sorption/ desorption behavior (Figure 6). Their crystalline water molecules are not lost even at 0% RH (Figure S5b,e, Supporting Information). As the humidity is increased up to 90% RH, 2 and 3 only uptake little water on the surface, and no phase transition is observed (Figure S5c,f, Supporting Information). During the desorption process, 2 and 3 reversibly lose the absorbed water, and no hysteresis gap is observed in these two instances (Figure 6), demonstrating 2 and 3 are stable under ambient conditions. Dissolution Study. To verify whether formation of cocrystals could modify the dissolution behavior of RBV, the release of RBV from cocrystals 1−3 was investigated via dissolution experiments and compared with that of pure, original RBV. PBS (pH 6.8) was chosen as incubation media. The cumulative release profiles of RBV are shown in Figure 7.

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CONCLUSIONS In this study, three cocrystals of ribavirin with 3,5dihydroxybenzoic acid (1), gallic acid (2), and barbituric acid (3) were facilely synthesized by a solution crystallization method. The formation of cocrystals was verified by IR, XRD, DSC, TGA, and DVS measurements. The crystal structures reveal that all cocrystals are hydrates and the stoichiometry of ribavirin/coformer/water varies from 1:6:7 in 1, 1:1:1 in 2, to 2:1:2 in 3. For 1, six 3,5-dihydroxybenzoic acid and seven water molecules form an atypical supramolecular organic channel framework that tightly traps ribavirin molecules within it, while for 2 and 3, coformers alternatively connect to ribavirin with multiple hydrogen bonds to form sheet structures. Cocrystals 1−3 display reduced release rate when compared to original ribavirin. Compound 1 displays the slowest release rate due to its high water/coformer/ribavirin stoichiometry and the tight capture of ribavirin molecules of its channel framework structure. These results revealed that the release rate of the ribavirin can be manipulated over a wide range by the formation of cocrystals. In addition, gallic acid possesses potential antiviral activity, and this multi-API cocrystal (2) may find application toward developing a combination analgesic drug.

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ASSOCIATED CONTENT

S Supporting Information *

PXRD of 1−3, PXRD, DSC, and TG of 1a, and PXRD analysis with regard to DVS study. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +86-20-84112921. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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Figure 7. Release of RBV from 1−3 in contrast to raw RBV in PBS (pH 6.8). The ordinate represents dissolved RBV expressed as a percentage of the total dissolved and undissolved RBV.

ACKNOWLEDGMENTS This work was financially supported by NSFC (grant no. 21101173, 91127002, and 21331007), NSF of Guangdong Province (S2012030006240), and Guangzhou Pearl River New Star Fund Science and Technology Planning Project (2013J2200054).

It could be found that although 1−3 showed a similar release profile with raw drug, the RBV release rate from the cocrystal formulations is significantly reduced. Raw RBV was quickly released in PBS, and the cumulative released amount was up to 80% within 4 min. In contrast, RBV was slowly released from the cocrystals 1−3 and reached 80% dissolved RBV in PBS in about 64, 30, and 16 min, respectively. The rank order of the release rates is 1 < 2 < 3 < RBV. Several issues could be attributed to the observed release rate variations of the cocrystals. (1) The incorporation of less soluble coformers into the crystal lattice of cocrystals may reduce the release rate of RBV, as the aqueous solubility (20

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