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Cocrystals of Acyclovir with Promising Physicochemical Properties
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Cocrystals of Acyclovir with Promising Physicochemical Properties ANINDITA SARKAR, SOHRAB ROHANI Department of Chemical and Biochemical Engineering, The University of Western Ontario, London N6A 5B9, Ontario, Canada Received 1 September 2014; revised 30 September 2014; accepted 13 October 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24248 ABSTRACT: Cocrystal forming ability of antiviral drug acyclovir (ACV) with different coformers was studied. Three cocrystals containing ACV with fumaric acid, malonic acid, and DL-tartaric acid were isolated. Methods of cocrystallization included grinding with dropwise solvent addition and solvent evaporation. The cocrystals were characterized by powder X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis. The crystal structure of the cocrystal with fumaric acid as conformer was determined by single crystal X-ray diffraction. Formation of supramolecular synthon was observed in the cocrystal. Stability with respect to relative humidity for the three cocrystals was evaluated. The aqueous solubility of the ACV-cocrystal materials was significantly improved with a maximum of malonic acid cocrystal, which was about six times more soluble at 35◦ C compared with that of parent ACV. The dissolution profile indicates that at any particular dissolution time, the concentration of cocrystals in the solution was higher than that of the parent ACV, and malonic acid C 2014 Wiley Periodicals, Inc. and the American Pharmacists cocrystals had a maximum release of about twice than the hydrated ACV. Association J Pharm Sci Keywords: co-crystal; synthon; crystal engineering; single crystal; stability; dissolution rate; solubility
INTRODUCTION Acyclovir (2-amino-1,9-dihydro-9-((2-hydroxyethoxy)methyl)6H-purin-6-one, hereinafter abbreviated as ACV, Fig. 1) is an antiviral agent administered for the treatment of infections such as herpes simplex (HSV-1 and HSV-2), shingles, and varicella (chickenpox). ACV has been marketed under several brand names such as Cyclovir, Herpex, Acivir, Acivirax, Zovirax, Zoral, Xovir, and Imavir, and it is available as 200, 400, and 800 mg tablets as well as flavored liquid. The 1988 Nobel Prize in Medicine was awarded to G.B. Elion, partly for the invention and development of ACV. The commercial ACV exists as a hydrated form and the crystal structure revealed a 3:2 ACV:water hydrate with a monoclinic space group.1 The dihydrated form with a triclinic unit cell2 is obtained by dissolving commercial ACV in water at 60◦ C. A metastable anhydrous form was obtained by heating commercial ACV at a temperature between 130◦ C and 150◦ C.3 Two stable anhydrous forms were obtained by heating commercial ACV at a temperature above 150◦ C and 180◦ C, respectively.3 Sohn and Kim4 showed existence of one more anhydrous form of ACV when commercial ACV was dissolved in methanol at 60◦ C followed by cooling crystallization to room temperature. An acetic acid solvate form has been reported after dissolving ACV in acetic acid followed by slow evaporation at room temperature.4 However, the hydrated and anhydrous forms are poorly soluble in water. Acyclovir belongs to the Class IV drugs, according to the Biopharmaceutics Classification System, which have low solubility and low permeability. Hence, to explore various solid forms of ACV that can enhance its solubility, dissolution Correspondence to: Sohrab Rohani (Telephone: +519-661-4116; Fax: +519661-3498; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com. Journal of Pharmaceutical Sciences
C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
profile, and bioavailability is of significance. Various methods have been used to increase the solubility of ACV. For example, solubility and dissolution rate of ACV could be improved by forming complex with cytosine,5 self-microemusifying of drug delivery system,6 and inclusion compound with $-cyclodextrin.7 Pharmaceutical cocrystals are obtained by combining one or more solid components (conformers) together with an active pharmaceutical ingredient (API).8–12 There are only three cocrystals of ACV reported, but the stability of the cocrystals with respect to atmospheric humidity was not described.13,14 Moreover, ACV–fumaric acid cocrystal that was reported earlier differs from the fumaric acid cocrystal reported herein. Although there is no definite structure reported by the previous authors, but by observing the PXRD pattern it is clear that the cocrystal reported earlier is different from ours. Probably the synthon formation via H-bonding is completely different from what we reported. The aim of this study, therefore, was to rationally design and prepare a series of pharmaceutically acceptable ACV cocrystals, evaluate the stability of the cocrystals with respect to atmospheric humidity, and also to improve the physicochemical properties of ACV. Three dicarboxylic acids, viz. fumaric acid, malonic acid, and DL-tartaric acid, were selected as cocrystal formers. The dicarboxylic acids were selected as conformers from the view of crystal engineering. In the context of current crystal engineering experiment, the desired supramolecular interaction involved two hydrogen bonding viz. O–H···N and C–O···H forming ACV base–dicarboxylic acid heterodimer synthon (Scheme 1). Moreover, there was also possibility of formation of homo synthon between two ACV molecules via N– H···N hydrogen bonding interaction (Scheme 1). The following ACV cocrystals were prepared in this study: ACV/fumaric acid (cocrystal I), ACV/malonic acid (cocrystal II), and ACV/tartaric acid (cocrystal III). Although the formation of ACV/tartaric acid and ACV/fumaric acid was already reported,13,14 but there is a lack of stability tests with respect to the atmospheric relative humidity (RH) and a systematic crystal engineering study. Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
was dissolved in water (10 mL) and kept for crystallization at room temperature. Single crystals of hydrated form of ACV– fumaric acid were obtained after 7 days and were analyzed by single crystal X-ray diffraction. Cocrystal II (1:1 ACV/Malonic Acid)
Figure 1. Chemical structure of acyclovir.
The cocrystal of ACV/malonic acid was obtained only by solvent evaporation technique. Anhydrous ACV (225 mg, 1 mmol) and malonic acid (104 mg, 1 mmol) were dissolved in 10 mL of acetic acid and heated at 100◦ C on a hot plate with constant stirring using a magnetic stirrer for 2 h in a sealed tube. The filtered solution was then evaporated slowly at room temperature under a fume hood. Cocrystal III (1:1 ACV/Tartaric)
Scheme 1. Heteromeric synthon showing N–H···O and O–H···O hydrogen bonding interactions and homo-synthon showing N-H···O hydrogen bonding.
Two different approaches were employed to prepare cocrystals of ACV viz. solution evaporation and solvent drop grinding. Physical states of ACV cocrystals were characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Formation of heterodimer and homo synthon was confirmed by the crystal structure of ACV–fumaric acid, determined by single crystal Xray diffraction. For the three ACV cocrystals, the stability with respect to atmospheric RH was evaluated. Solubility and dissolution rates of the cocrystals were also evaluated and compared with those of parent ACV.
This material was prepared both by solution evaporation and grinding technique. Anhydrous ACV (225 mg, 1 mmol) and DLtartaric acid (116 mg, 1 mmol) after carefully weighing were dissolved in 10 mL of acetic acid in a sealed tube and heated at 100◦ C by a hot plate with constant stirring with a magnetic stirrer for 2 h. The solution was then filtered through 5 :m filter paper (VWR brand; VWR International Ltd.) to remove insolubles, and allowed to evaporate slowly at room temperature under a fume hood. The cocrystals were also obtained by grinding 1:1 stoichiometric amount of anhydrous ACV/DL-tartaric acid with a mortar and pestle for 20 min with the addition of 5 drops from a pipette (ca. 0.1 mL) of methanol. Powder X-Ray Diffraction The PXRD spectra were collected on a Rigaku-Miniflex benchtop X-ray powder diffractometer (Carlsbad, California) using ˚ radiation obtained at 30 kV and 15 mA. CuK" (8 = 1.54059 A) The scans were run from 5.0◦ to 40.0◦ 22, increasing at a step size of 0.05◦ 22 with a counting time of 2 s for each step. The diffractograms were processed using JADE 7.0 software. Calibration was performed using a silicon standard. Single Crystal X-Ray Diffraction
EXPERIMENTAL PROCEDURE Materials Apotex PharmaChem Inc. (Brantford, Ontario, Canada) donated 3:2 ACV: water hydrated form. Other chemicals were purchased from Sigma–Aldrich (London, Ontario) and were used as received. The anhydrous ACV was prepared by heating 3:2 ACV:water hydrated form at 180◦ C for an hour.2,4,13 Cocrystal I (1:1 ACV/Fumaric Acid) This material was prepared by solution evaporation and grinding technique. Anhydrous ACV (225 mg, 1 mmol) and fumaric acid (116 mg, 1 mmol) after carefully weighing were dissolved in 10 mL of acetic acid in a sealed tube and heated at 100◦ C by a hot plate with constant stirring with a magnetic stirrer for 2 h. The solution was then filtered through 5 :m filter paper (VWR brand; VWR International Ltd., London, Ontario) to remove insolubles, and allowed to evaporate slowly at room temperature under a fume hood. The cocrystals were also obtained by grinding 1:1 stoichiometric amount of anhydrous ACV/fumaric acid with a mortar and pestle for 20 min with the addition of 5 drops from a pipette (ca. 0.1 mL) of methanol. The ground material Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
Single crystals of ACV–fumaric acid were grown from a water solution at room temperature. The single crystal sample was mounted on a Mitegen polyimide micromount with a small amount of Paratone N oil. X-ray measurements were made on a Bruker Kappa Axis Apex2 diffractometer at a temperature of 110 K. The unit cell dimensions were determined from a symmetry constrained fit of 5682 reflections with 4.9◦ < 22 < 58.56◦ . The frame integration was performed using SAINT.15 The resulting raw data were scaled and absorption corrected, using a multiscan averaging of symmetry equivalent data using SADABS.16 The structure was solved by direct methods using the SIR2011 program.17 Most of the non-hydrogen atoms were obtained from the initial solution. The remaining atomic positions were obtained from a subsequent difference Fourier map. The hydrogen atoms were introduced at idealized positions and were allowed to ride on the parent atom. The structural model was fit to the data using full matrix least-squares based on F2 . The calculated structure factors included corrections for anomalous dispersion from the usual tabulation. The structure was refined using the SHELXL-2013 program from the SHELX suite of crystallographic software.18 DOI 10.1002/jps.24248
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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Differential Scanning Calorimeter The melting points were measured with a Mettler Toledo DSC 822e differential scanning calorimeter (Greifensee, Switzerland). Accurately weighed samples (∼3 mg) were prepared in a covered aluminum crucible having pierced lids to allow escape of volatiles. The sensors and samples were under nitrogen purge during the experiments. The temperature calibration was carried out using the melting point of highly pure indium in the medium temperature range. A heating rate of 5◦ C/min was used. Thermogravimetric Analysis Thermogravimetric analysis was performed on a MettlerToledo TGA/SDTA 851e instrument. Approximately 2 mg sample was heated from 25◦ C to 300◦ C at 10◦ C/min under nitrogen purge. Relative Humidity Relative humidity conditions were achieved at room temperature (ca. 20◦ C) within sealed glass desiccator jars containing P2 O5 for the 0% RH condition and the appropriate saturated aqueous salt solutions for other RH values (K2 CO3 for 43%; NaCl for 75%; and K2 SO4 for 98%). To compare the stability of ACV to the various cocrystals produced, cocrystals I–III were evaluated along with anhydrous ACV and commercial ACV for physical stability at the conditions of 0%, 43%, 75%, and 98% RH for time periods of 1 day, 3 days, 1 week, and 3 weeks. Cocrystal II that was formed from solution was at first ground gently with a mortar and pestle to homogenize the particle size before the experiments commenced. The cocrystal materials that were formed by solvent drop grinding (I and III) were obtained as microcrystalline powders and were used as isolated. Open glass vials containing 40–60 mg of powdered cocrystal materials were stored in the RH chambers at ambient temperature. A vial was removed for each cocrystal material at each time point. Upon removal from the desiccator, the samples were quickly evaluated for any form change by PXRD.
Figure 2. PXRD pattern of two third, dihydrated and anhydrous ACV.
RESULT AND DISCUSSION Cocrystal Characterization Powder X-Ray Diffraction The formation of cocrystals was confirmed by PXRD pattern. The PXRD patterns of anhydrous, 3:2 ACV:water hydrated, and dihydrated ACV are shown in Figure 2, those of fumaric acid, ACV–fumaric acid, simulated pattern of ACV–fumaric acid, malonic acid, ACV–malonic acid, DL-tartaric acid and ACV– tartaric acid are shown in Figure 3. The PXRD patterns of cocrystal materials were different from those of ACV and the corresponding conformer carboxylic acid. These unique PXRD patterns of ACV cocrytals indicate the formation of new crystalline phases.
Solubility and Dissolution Rate The solubility of hydrated ACV and cocrystal materials in water was measured at 35◦ C. To measure the saturation concentration of each sample, 20 g of the solvent mixture and an excess amount of solids were added to a flask at a given temperature and mixed using a magnetic stirrer plate (AGE Magnetic Stirrer; Newtec Inc., Hull, Iowa). These suspensions were then placed on a multiplate mechanical shaker and were left to equilibrate for 72 h in a temperature controlled water bath. Samples were filtered through 0.45 mm cellulose acetate syringe filters into volumetric flasks. Supernatant were then analyzed by UV spectrophotometric analysis at 8max 252 nm (Cary 100 Bio UV visible Spectrophotometer, Palo Alto, California). Subsequently, dissolution rate experiments were conducted on hydrated ACV and cocrystal materials. In order to evaluate the dissolution, 100 mg of 3:2 ACV:water hydrated form and the cocrystal materials were placed in 200 mL of phosphatebuffered saline (PBS) as dissolution medium at 37◦ C for 90 min at 100 rpm using a magnetic stirrer. Aliquots of 5.0 mL were withdrawn every 10 min. The dissolution medium was replaced after every sampling. Dissolution testing was performed in triplicate. The concentration of ACV and cocrystal material in the solution was measured by UV spectrophotometer Cary 100 Bio. DOI 10.1002/jps.24248
Cocrystal by Grinding It is generally possible to prepare cocrytals by dual method of solution growth and neat/solvent drop grinding as observed in cocrystals I and III in this study, but there are some exceptions too. As mentioned earlier, cocrystal II was easily prepared by evaporating acetic acid solution, but solvent drop grinding inhibited the formation of II. For the solvent drop grinding, for this particular cocrystal formation, solvent screening with numerous solvents viz. N,N -dimethyl formamide, dimethyl sulfoxide, methanol, ethanol, 1-propanol, isopropyl alcohol, acetone, acetonitrile, chloroform, n-hexane, acetic acid, and water was performed, but in all cases, anhydrous ACV was observed in PXRD. It must be pointed that not all the dicarboxylic acids that were attempted, successfully formed cocrystals with ACV, neither by solvent drop grinding nor by evaporation method. When screened for cocrystallization of ACV, the following three pharmaceutically acceptable dicarboxylic acids did not result in cocrystal formation: succinic acid, glutaric acid, and adipic acid. Differential scanning calorimetry results of ACV–fumaric acid after various grinding times are shown in Figure 4a. As the cogrinding of the anhydrous ACV and fumaric acid Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Figure 3. PXRD pattern of (a) Fumaric acid, (b) AVC-fumaric acid and (c) simulated pattern of AVC-fumaric acid, (d) Malonic acid, (e) ACV-malonic acid, (f) Tartaric acid, (g) ACV-tartaric acid.
proceeded, the endothermal peak at about 220◦ C decreased and finally disappeared. A melting endotherm at 185◦ C, which differs from the melting points of either anhydrous ACV (254.4◦ C) or fumaric acid (205.0◦ C), occurred all the time. This also indicated that during the solvent assisted grinding, a new phase was formed between anhydrous ACV and fumaric acid. Thermal Analysis The DSC and TGA of the cocrystallization product of ACV–fumaric acid from slow evaporation are presented in Figure 4b. The cocrystallization material from slow evaporation from acetic acid showed two endothermic peaks, one at 174.0◦ C and another small peak at 205.0◦ C. The small peak at 205.0◦ C corresponding to melting point of fumaric acid indicates that there was incomplete crystallization during the slow evaporation process. Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
The DSC thermogram of cocrystals II and III are depicted in Figure 5. The DSC thermogram of the cocrystals was distinguishable from parent ACV and the corresponding conformer dicarboxylic acids. The thermal stability of cocrystal materials was assessed by TGA. In case of ACV–fumaric acid·3H2 O, there was no weight loss until 150.0◦ C in TGA analysis (Fig. 4b). The thermal analysis of ACV–fumaric acid·3H2 O indicates that this cocrystal hydrate was stable up to 150.0◦ C. The retention of water molecules in the ACV–fumaric acid cocrystal hydrate up to 150.0◦ C indicates that all the three water molecules are located in the crystal lattice via strong O–H···O hydrogen bonds. For the cocrystals II and III, a weight loss at 135.0◦ C and 140.0◦ C occurred in TGA analysis. The TGA curves (Fig. 5) indicate the absence of any unbound or absorbed solvents. DOI 10.1002/jps.24248
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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Figure 4. (a) DSC analysis of cocrystal-I of hte resulting powders after various grining time and (b) TGA curve of cocrystal I and DSC thermogram of cocrystallization product from solvent evaporation.
Table 1.
Crystallographic Data of ACV–Fumaric Acid·3H2 O
ACV–Fumaric Acid·3H2 O Chemical formula Formula weight Crystal size (mm) Crystal system Space group T/K ˚ a (A) ˚ b (A) ˚ c (A) " (◦ ) $ (◦ ) ( (◦ ) Z ˚ 3) V (A Dcalcd. (g cm−3 ) : (mm−1 ) Reflections collected Unique reflections Reflections I ≥ 2FI R1 (I > 2F(I)) wR2 (all) Goodness-of-fit CCDC number
C24 H36 N10 O17 736.63 0.366 × 0.354 × 0.077 Triclinic P−1 110(2) 6.534 11.023 23.529 97.73 91.93 105.33 2 1615.3 1.197 0.075 59,837 5682 5333 0.0359 0.1164 1.110 1005729
Single Crystal Structure of ACV–Fumaric Acid The crystallographic data are summarized in Table 1. The hydrogen-bonding geometry is listed in Table 2. The 1:1 ACV/fumaric acid stoichiometry with three water molecules was observed for cocrystal I. As mentioned in the scheme 1, a heteromeric synthon was observed with only one of the two carboxylic groups of each dicarboxylic acid. The other carboxylic group forms a hydrogen bond with another fumaric acid. The second fumaric acid interacts with another ACV via O–H···N hydrogen bond. Again this ACV forms a heteromeric synthon with carboxylic acid of fumaric acid. As shown in the Figure 6, with two ACV and four fumaric acid molecules, a six membered ring (R1 in Fig. 6) is formed having two hetromeric synthons. Self assembly via O–H···O, N–H···N, and O–H···N interaction leads to a chain of six-membered rings running parallel to the a-axis in the crystal lattice of ACV– fumaric acid cocrystal. The ACV molecules of the abovementioned six-membered ring further form a homosynthon via DOI 10.1002/jps.24248
Table 2. Hydrogen-Bonding Geometry of ACV–Fumaric Acid·3H2 O ˚ and Angle in ◦ ) (d in A D–H···A
d(D–H)
d(H···A)
d(D···A)
D–H···A
O(3)–H(3)···O(15)§1 O(5)–H(5)···N(8)§2 N(5)–H(5A)···N(7)§3 N(6)–H(6)···O(14)§4 O(7)–H(7)···N(3)§5 O(10)–H(10)···O(16)§6 O(11)–H(11)···O(6)§7 O(13)–H(13)···O(8)§8 O(15)–H(15A)···O(14)§9 O(15)–H(15B)···O(21)§10 O(16)–H(16A)···O(10)§11 O(16)–H(16B)···O(10)§12 O(21)–H(21A)···O(3)§13 O(21)–H(21B)···O(3)§14
0.82 0.82 0.86(2) 0.87(2) 0.82 0.82 0.82 0.82 0.92(2) 0.99(5) 0.92(3) 0.83(3) 0.96(4) 0.96(4)
1.90 1.77 2.09(2) 2.03(2) 1.73 2.03 1.84 1.75 1.866(19) 1.78(5) 1.91(3) 2.02(3) 1.81(4) 1.81(4)
2.7123(18) 2.5881(18) 2.9466(19) 2.8743(18) 2.5522(17) 2.8027(18) 2.6551(17) 2.5648(16) 2.7130(17) 2.704(2) 2.8130(19) 2.8027(18) 2.7635(19) 2.774(2)
173.0 178.0 176.0(2) 165.0(2) 178.0 158.0 175.0 175.0 152.0(2) 153.0(5) 169.0(3) 159.0(3) 177.0(3) 174.0(3)
Symmetry transformations used to generate equivalent atoms: (§1) x,y,z; (§2) x,1+y,z; (§3) 1+x,y,z;(§4) −x,3−y,2−z;(§5) x,1+y,z; (§6) −x,3−y,2−z; (§7) x,1+y,z; (§8) −x,2−y,2−z; (§9) 1−x,4−y,2−z; (§10) 1−x,3−y,2−z; (§11) −1+x,y,z; (§12) −1+x,y,z; (§13) x,2+y,z; (§14) −x,3−y,2−z; (§15) −x,3−y,2−z.
weak N–H···N interaction. Another six-membered ring (R2 in Fig. 6) is formed via O–H···O supramolecular interaction involving O–H groups of two ACV molecules and O15–H3···O15 and O21–H21···O3 interactions of two different water molecules. Furthermore, the third water molecule (O16) forms a fourmembered ring (R3 in Fig. 6) with O–H groups of two ACV molecules via O16–H16A···O10 and O10–H10···O16 hydrogen bonding interaction.
COCRYSTAL PROPERTIES Relative Humidity The RH stability results of 3:2 ACV:water hydrated ACV, anhydrous ACV, and the cocrystals are described below and are summarized diagrammatically in Table 3. (a) The 3:2 ACV:water hydrated: The commercial ACV (3:2 ACV:water hydrated) rapidly rehydrated to dihydrated form at all RH condition except at 0% RH. The PXRD pattern of dihydrated form was evaluated by Sohn and Kim.4 At 0% RH, the 2:3 hydrated form lost water and Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
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Figure 5. DSC thermogram of (a) ACV-malonic acid and (b) ACV-tartatic acid, TGA curve of (c) of ACV-malonic acid and (d) ACV-tartatic acid.
Figure 6. Hydrogen bonding network of ACV-fumaric. 3H2 O showing formation of synthon and a six membered ring R1, a six membered ring R2 and a four membered ring R3.
as reported earlier it was converted to anhydrous form within 1 day (Sohn and Kim4 ). (b) Anhydrous ACV: The anhydrous ACV was converted to dihydrated form of ACV within 1 day at all RH conditions, but at 0% RH, it remained stable for 3 weeks. (c) Cocrystal I: This cocrystal material was found to be stable at all RH conditions for 3 weeks long study. This cocrystal clearly demonstrated enhanced stability over hydrated and anhydrous ACV. Its stability is additionally demonstrated by slurrying the cocrystal material in water for Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
2 days at ambient temperature without any observed physical form change. (d) Cocrystal II: This cocrystal material was also found to be stable at all RH conditions for our 3 weeks long study as that of cocrystal I. (e) Cocrystal III: This material was stable only at 0% RH for the total duration of our study. At 98% RH, the cocrystal III showed instability at just 1 day, forming a sticky substance with a color turning to light pink. We were not able to identify this substance. At the end of 1 week, this DOI 10.1002/jps.24248
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Table 3.
Stability of Cocrystals with Respect to RH
Table 4. Solubility of Anhydrous ACV and Cocrystal Materials in Water at 35◦ C
Stability with Respect to RH Material
% RH
Anhydrous ACV
0 43 75 98
2/3 Hydrated ACV
Cocrystal I
1 Day √
3 Days √
1 Week √
3 Weeks √
× × × √
× × × √
× × × √
× × × √
× × × √
× × × √
× × × √
× × × √
0 43 75 98 0 43 75 98
Cocrystal II
0 43 75 98
Cocrystal III
0 43 75 98
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
× ×
× ×
√ ×
2:3 hydrated ACV (moles/L) 1.28 × 10−1
Cocrystal I (moles/L)
Cocrystal II (moles/L)
Cocrystal III (moles/L)
7.01 × 10−1
7.25 × 10−1
6.78 × 10−1
× × ×
√ The symbol and × indicate that the material is stable and unstable respectively at that particular RH condition and time point.
unidentified sticky material started transforming to a liquid. The instability of cocrystal III at 75% RH started at day 3 and transformation into liquid at day 10. At 45% RH, the material was stable up to day 7. From the above discussion on relative stability of the ACV cocrystals, we can conclude that cocrystals I and II were most stable, whereas cocrystal III was the least stable. The order of cocrystal stability with respect to RH is thus: I ≈ II > III. The acid strength of the conformers are malonic acid (2.83) > fumaric acid (3.03) > DL-tartaric acid (3.22). The weakest acid among the four is DL-tartaric acid, which formed the least stable cocrystal. From this study, it is evident that acidity of the coformer is not the only issue affecting the stability of the cocrytals as both ACV–fumaric acid and ACV–malonic acid cocrystals had identical stability. The other driving force for the stabilization of the cocrystal is possibly due to the hydrogen bonds. Generally, introduction of dicarboxylic acids should prevent consumption of water into the cocrystal by formation of supramolecular synthon through hydrogen bonds created via donor and acceptor atoms and the cocrystals are expected to be more stable than the original drug molecule. Cocrystal I hydrate was stable at all test conditions, which indicates that water molecules form strong hydrogen bonds with the cocrystal components leading to a direct correlation between the structure and stability of the cocrystal hydrate. Solubility and Dissolution Rate The dissolution rate and solubility have a major impact on the bioavailability of pharmaceutical drugs. Solubility of the cocrystals in water at 35◦ C was compared with the 3:2 ACV:water DOI 10.1002/jps.24248
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Figure 7. Dissolution profile of 3:2 ACV:water hydrated and ACVcocrystal materials.
hydrated and the results are summarized in Table 4. The hydrated ACV is more soluble than the anhydrous forms (Kristl et al.3 ). The trend of solubility of cocrystals in water is as follows: cocrystal I ≈ cocrystal II > cocrystal III. The dissolution experiment was conducted on hydrated ACV and all the three cocrystals in PBS at 35◦ C. The highest concentration of 3:2 ACV:water hydrated was released approximately after 50 min, whereas the peak concentration of cocrystal I, II, and III in the solution appeared approximately 30, 30, and 40 min, respectively (Fig. 7). At the same dissolution time, the released concentrations of ACV cocrystals in the solution were consistently higher than that of ACV, as for example, at 20 min, the concentrations of ACV–fumaric acid and hydrated ACV were 1.69 and 1.22 mg cm−2 , respectively. Initially ACV–fumaric acid cocrystal dissolved rapidly and reached equilibrium after 20 min, but dissolution of ACV was slow at the beginning and reached the equilibrium point after 50 min. The remaining powder samples, after the dissolution experiment, were examined by PXRD. Surprisingly, it was observed that anhydrous ACV and the cocrystal III were converted to dihydrated ACV, whereas cocrystal I and II remain stable. The rapid dissolution of the cocrystal III at the early time point indicate that transformation of cocrystal III to dihydrated ACV occurred only after reaching the maximum concentration.
CONCLUSIONS Three cocrystals of a pharmaceutical compound ACV with dicarboxylic acids in 1:1 stoichiometric ratio have been described. The RH of the cocrystals differs from the hydrated and anhydrous ACV. In terms of physicochemical properties, such as solubility and dissolution rate, the cocrystals show remarkably higher solubility and faster dissolution compared with the hydrated ACV. Taking into account of improved physicochemical Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
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properties of cocrystals reported herein, novel formulations of ACV with dicarboxylic acids are possible.
ACKNOWLEDGMENT We are also thankful to the Natural Sciences and Engineering Council of Canada for its financial support of this project through the Discovery Grant.
REFERENCES 1. Birnbaum GI, Cygler M, Shugar D. 1984. Conformational features of acyclonucleosides—Structure of acyclovir, an anti-herpes agent. Can J Chem 62:2646–2652. 2. Lutker KM, Rosalynn Q, Xu J, Ramamoorthy A, Matzger AJ. 2011. Polymorphs and hydrates of acyclovir. J Pharm Sci 100:949– 963. 3. Kristl A, Srcic S, Vrecer F, Sustar B, Vojnovic D. 1996. Polymorphism and pseudopolymorphism: Influencing the dissolution properties of the guanine derivative acyclovir. Int J Pharm 139:231– 235. 4. Sohn YT, Kim SH. 2008. Polymorphism and pseudopolymorphism of acyclovir. Arch Pharm Res 31:231–234. 5. Tatughamiarso M, Guido W, Egert E. 2012. Cocrystals of 5fluorocytosine. I. Coformers with fixed hydrogen-bonding sites. Acta Cryst Sec B B68:431–443. ˜ JS, Baeza MR, Alderete JB. 2000. Inclusion com6. Rossel CP, Carreno plex of theantiviral drug acyclovir with cyclodextrin in aqueous solution and in solid phase. Quim Nova 23:749–752. 7. Sachan NK, Pushkar S, Solanki SS, Bhatere DS. 2010. Enhancement of solubility of acyclovir by solid dispersion and inclusion complexation methods. World Appl Sci J 11:857–864.
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8. Jones W. 1997. Organic molecular solids: Properties and applications; Jones W, Ed. New York: CRC Press, p. 177. 9. Bond AD, Jones W. 2002. Supramolecular organisation and materials design; Jones W, Rao CNR, Eds. Cambridge: Cambridge University Press, p. 391. 10. Atwood JL, Davies JED, MacNicol, DD, Vogtle F. 1996. Comprehensive supramolecular chemistry, vol. 9. Oxford: Pergamon. 11. Remenar JF, Morissette SL, Peterson ML, Moulton B, MacPhee ¨ 2003. Crystal engineering of novel ´ HR, Almarsson O. JM, Guzman cocrystals of a triazole drug with 1,4-dicarboxylic acids. J Am Chem Soc 125:8456. 12. Walsh RDB, Bradner MW, Fleischman S, Morales LA, Moulton B, Rodr´ıguez-Hornedo N, Zaworotko MJ. 2003. Crystal engineering of the composition of pharmaceutical phases. Chem Commun 186– 187. 13. Masuda T, Yoshihashi Y, Yonemochi E, Fujii K, Uekusa H, Terada K. 2012. Cocrystallization and amorphization induced drug–excipient interaction improves the physical properties of acyclovir. Int J Pharm 422:160–169. 14. Bruni G, Maietta M, Maggi L, Mustarelli P, Ferrara C, Berbenni V, Milanese C, Girella A, Marini A. 2013. Preperation and physicochemical characterization of acyclovir with improved dissolution properties. J Pharm Sci 102:4079–4086. 15. Bruker-Nonius. 2012. SAINT version 2012.12. Madison, Wisconsin: Bruker-Nonius. 16. Bruker-Nonius. 2012. SADABS version 2012.1. Madison, Wisconsin: Bruker-Nonius. 17. Burla MC, Caliandro R, Camalli M, Carrozzini B, Cascarano GL, Giacovazzo C, Mallamo M, Mazzone A, Polidori G, Spagna R. 2012. VLD algorithm and hybrid Fourier syntheses. J Appl Cryst 45:357– 361. 18. Sheldrick GM. 2008. A short history of SHELX. Acta Cryst A64:112–122.
DOI 10.1002/jps.24248
Cocrystals of Acyclovir with Promising Physicochemical Properties ANINDITA SARKAR, SOHRAB ROHANI Department of Chemical and Biochemical Engineering, The University of Western Ontario, London N6A 5B9, Ontario, Canada Received 1 September 2014; revised 30 September 2014; accepted 13 October 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24248 ABSTRACT: Cocrystal forming ability of antiviral drug acyclovir (ACV) with different coformers was studied. Three cocrystals containing ACV with fumaric acid, malonic acid, and DL-tartaric acid were isolated. Methods of cocrystallization included grinding with dropwise solvent addition and solvent evaporation. The cocrystals were characterized by powder X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis. The crystal structure of the cocrystal with fumaric acid as conformer was determined by single crystal X-ray diffraction. Formation of supramolecular synthon was observed in the cocrystal. Stability with respect to relative humidity for the three cocrystals was evaluated. The aqueous solubility of the ACV-cocrystal materials was significantly improved with a maximum of malonic acid cocrystal, which was about six times more soluble at 35◦ C compared with that of parent ACV. The dissolution profile indicates that at any particular dissolution time, the concentration of cocrystals in the solution was higher than that of the parent ACV, and malonic acid C 2014 Wiley Periodicals, Inc. and the American Pharmacists cocrystals had a maximum release of about twice than the hydrated ACV. Association J Pharm Sci Keywords: co-crystal; synthon; crystal engineering; single crystal; stability; dissolution rate; solubility
INTRODUCTION Acyclovir (2-amino-1,9-dihydro-9-((2-hydroxyethoxy)methyl)6H-purin-6-one, hereinafter abbreviated as ACV, Fig. 1) is an antiviral agent administered for the treatment of infections such as herpes simplex (HSV-1 and HSV-2), shingles, and varicella (chickenpox). ACV has been marketed under several brand names such as Cyclovir, Herpex, Acivir, Acivirax, Zovirax, Zoral, Xovir, and Imavir, and it is available as 200, 400, and 800 mg tablets as well as flavored liquid. The 1988 Nobel Prize in Medicine was awarded to G.B. Elion, partly for the invention and development of ACV. The commercial ACV exists as a hydrated form and the crystal structure revealed a 3:2 ACV:water hydrate with a monoclinic space group.1 The dihydrated form with a triclinic unit cell2 is obtained by dissolving commercial ACV in water at 60◦ C. A metastable anhydrous form was obtained by heating commercial ACV at a temperature between 130◦ C and 150◦ C.3 Two stable anhydrous forms were obtained by heating commercial ACV at a temperature above 150◦ C and 180◦ C, respectively.3 Sohn and Kim4 showed existence of one more anhydrous form of ACV when commercial ACV was dissolved in methanol at 60◦ C followed by cooling crystallization to room temperature. An acetic acid solvate form has been reported after dissolving ACV in acetic acid followed by slow evaporation at room temperature.4 However, the hydrated and anhydrous forms are poorly soluble in water. Acyclovir belongs to the Class IV drugs, according to the Biopharmaceutics Classification System, which have low solubility and low permeability. Hence, to explore various solid forms of ACV that can enhance its solubility, dissolution Correspondence to: Sohrab Rohani (Telephone: +519-661-4116; Fax: +519661-3498; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com. Journal of Pharmaceutical Sciences
C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
profile, and bioavailability is of significance. Various methods have been used to increase the solubility of ACV. For example, solubility and dissolution rate of ACV could be improved by forming complex with cytosine,5 self-microemusifying of drug delivery system,6 and inclusion compound with $-cyclodextrin.7 Pharmaceutical cocrystals are obtained by combining one or more solid components (conformers) together with an active pharmaceutical ingredient (API).8–12 There are only three cocrystals of ACV reported, but the stability of the cocrystals with respect to atmospheric humidity was not described.13,14 Moreover, ACV–fumaric acid cocrystal that was reported earlier differs from the fumaric acid cocrystal reported herein. Although there is no definite structure reported by the previous authors, but by observing the PXRD pattern it is clear that the cocrystal reported earlier is different from ours. Probably the synthon formation via H-bonding is completely different from what we reported. The aim of this study, therefore, was to rationally design and prepare a series of pharmaceutically acceptable ACV cocrystals, evaluate the stability of the cocrystals with respect to atmospheric humidity, and also to improve the physicochemical properties of ACV. Three dicarboxylic acids, viz. fumaric acid, malonic acid, and DL-tartaric acid, were selected as cocrystal formers. The dicarboxylic acids were selected as conformers from the view of crystal engineering. In the context of current crystal engineering experiment, the desired supramolecular interaction involved two hydrogen bonding viz. O–H···N and C–O···H forming ACV base–dicarboxylic acid heterodimer synthon (Scheme 1). Moreover, there was also possibility of formation of homo synthon between two ACV molecules via N– H···N hydrogen bonding interaction (Scheme 1). The following ACV cocrystals were prepared in this study: ACV/fumaric acid (cocrystal I), ACV/malonic acid (cocrystal II), and ACV/tartaric acid (cocrystal III). Although the formation of ACV/tartaric acid and ACV/fumaric acid was already reported,13,14 but there is a lack of stability tests with respect to the atmospheric relative humidity (RH) and a systematic crystal engineering study. Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
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was dissolved in water (10 mL) and kept for crystallization at room temperature. Single crystals of hydrated form of ACV– fumaric acid were obtained after 7 days and were analyzed by single crystal X-ray diffraction. Cocrystal II (1:1 ACV/Malonic Acid)
Figure 1. Chemical structure of acyclovir.
The cocrystal of ACV/malonic acid was obtained only by solvent evaporation technique. Anhydrous ACV (225 mg, 1 mmol) and malonic acid (104 mg, 1 mmol) were dissolved in 10 mL of acetic acid and heated at 100◦ C on a hot plate with constant stirring using a magnetic stirrer for 2 h in a sealed tube. The filtered solution was then evaporated slowly at room temperature under a fume hood. Cocrystal III (1:1 ACV/Tartaric)
Scheme 1. Heteromeric synthon showing N–H···O and O–H···O hydrogen bonding interactions and homo-synthon showing N-H···O hydrogen bonding.
Two different approaches were employed to prepare cocrystals of ACV viz. solution evaporation and solvent drop grinding. Physical states of ACV cocrystals were characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Formation of heterodimer and homo synthon was confirmed by the crystal structure of ACV–fumaric acid, determined by single crystal Xray diffraction. For the three ACV cocrystals, the stability with respect to atmospheric RH was evaluated. Solubility and dissolution rates of the cocrystals were also evaluated and compared with those of parent ACV.
This material was prepared both by solution evaporation and grinding technique. Anhydrous ACV (225 mg, 1 mmol) and DLtartaric acid (116 mg, 1 mmol) after carefully weighing were dissolved in 10 mL of acetic acid in a sealed tube and heated at 100◦ C by a hot plate with constant stirring with a magnetic stirrer for 2 h. The solution was then filtered through 5 :m filter paper (VWR brand; VWR International Ltd.) to remove insolubles, and allowed to evaporate slowly at room temperature under a fume hood. The cocrystals were also obtained by grinding 1:1 stoichiometric amount of anhydrous ACV/DL-tartaric acid with a mortar and pestle for 20 min with the addition of 5 drops from a pipette (ca. 0.1 mL) of methanol. Powder X-Ray Diffraction The PXRD spectra were collected on a Rigaku-Miniflex benchtop X-ray powder diffractometer (Carlsbad, California) using ˚ radiation obtained at 30 kV and 15 mA. CuK" (8 = 1.54059 A) The scans were run from 5.0◦ to 40.0◦ 22, increasing at a step size of 0.05◦ 22 with a counting time of 2 s for each step. The diffractograms were processed using JADE 7.0 software. Calibration was performed using a silicon standard. Single Crystal X-Ray Diffraction
EXPERIMENTAL PROCEDURE Materials Apotex PharmaChem Inc. (Brantford, Ontario, Canada) donated 3:2 ACV: water hydrated form. Other chemicals were purchased from Sigma–Aldrich (London, Ontario) and were used as received. The anhydrous ACV was prepared by heating 3:2 ACV:water hydrated form at 180◦ C for an hour.2,4,13 Cocrystal I (1:1 ACV/Fumaric Acid) This material was prepared by solution evaporation and grinding technique. Anhydrous ACV (225 mg, 1 mmol) and fumaric acid (116 mg, 1 mmol) after carefully weighing were dissolved in 10 mL of acetic acid in a sealed tube and heated at 100◦ C by a hot plate with constant stirring with a magnetic stirrer for 2 h. The solution was then filtered through 5 :m filter paper (VWR brand; VWR International Ltd., London, Ontario) to remove insolubles, and allowed to evaporate slowly at room temperature under a fume hood. The cocrystals were also obtained by grinding 1:1 stoichiometric amount of anhydrous ACV/fumaric acid with a mortar and pestle for 20 min with the addition of 5 drops from a pipette (ca. 0.1 mL) of methanol. The ground material Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
Single crystals of ACV–fumaric acid were grown from a water solution at room temperature. The single crystal sample was mounted on a Mitegen polyimide micromount with a small amount of Paratone N oil. X-ray measurements were made on a Bruker Kappa Axis Apex2 diffractometer at a temperature of 110 K. The unit cell dimensions were determined from a symmetry constrained fit of 5682 reflections with 4.9◦ < 22 < 58.56◦ . The frame integration was performed using SAINT.15 The resulting raw data were scaled and absorption corrected, using a multiscan averaging of symmetry equivalent data using SADABS.16 The structure was solved by direct methods using the SIR2011 program.17 Most of the non-hydrogen atoms were obtained from the initial solution. The remaining atomic positions were obtained from a subsequent difference Fourier map. The hydrogen atoms were introduced at idealized positions and were allowed to ride on the parent atom. The structural model was fit to the data using full matrix least-squares based on F2 . The calculated structure factors included corrections for anomalous dispersion from the usual tabulation. The structure was refined using the SHELXL-2013 program from the SHELX suite of crystallographic software.18 DOI 10.1002/jps.24248
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Differential Scanning Calorimeter The melting points were measured with a Mettler Toledo DSC 822e differential scanning calorimeter (Greifensee, Switzerland). Accurately weighed samples (∼3 mg) were prepared in a covered aluminum crucible having pierced lids to allow escape of volatiles. The sensors and samples were under nitrogen purge during the experiments. The temperature calibration was carried out using the melting point of highly pure indium in the medium temperature range. A heating rate of 5◦ C/min was used. Thermogravimetric Analysis Thermogravimetric analysis was performed on a MettlerToledo TGA/SDTA 851e instrument. Approximately 2 mg sample was heated from 25◦ C to 300◦ C at 10◦ C/min under nitrogen purge. Relative Humidity Relative humidity conditions were achieved at room temperature (ca. 20◦ C) within sealed glass desiccator jars containing P2 O5 for the 0% RH condition and the appropriate saturated aqueous salt solutions for other RH values (K2 CO3 for 43%; NaCl for 75%; and K2 SO4 for 98%). To compare the stability of ACV to the various cocrystals produced, cocrystals I–III were evaluated along with anhydrous ACV and commercial ACV for physical stability at the conditions of 0%, 43%, 75%, and 98% RH for time periods of 1 day, 3 days, 1 week, and 3 weeks. Cocrystal II that was formed from solution was at first ground gently with a mortar and pestle to homogenize the particle size before the experiments commenced. The cocrystal materials that were formed by solvent drop grinding (I and III) were obtained as microcrystalline powders and were used as isolated. Open glass vials containing 40–60 mg of powdered cocrystal materials were stored in the RH chambers at ambient temperature. A vial was removed for each cocrystal material at each time point. Upon removal from the desiccator, the samples were quickly evaluated for any form change by PXRD.
Figure 2. PXRD pattern of two third, dihydrated and anhydrous ACV.
RESULT AND DISCUSSION Cocrystal Characterization Powder X-Ray Diffraction The formation of cocrystals was confirmed by PXRD pattern. The PXRD patterns of anhydrous, 3:2 ACV:water hydrated, and dihydrated ACV are shown in Figure 2, those of fumaric acid, ACV–fumaric acid, simulated pattern of ACV–fumaric acid, malonic acid, ACV–malonic acid, DL-tartaric acid and ACV– tartaric acid are shown in Figure 3. The PXRD patterns of cocrystal materials were different from those of ACV and the corresponding conformer carboxylic acid. These unique PXRD patterns of ACV cocrytals indicate the formation of new crystalline phases.
Solubility and Dissolution Rate The solubility of hydrated ACV and cocrystal materials in water was measured at 35◦ C. To measure the saturation concentration of each sample, 20 g of the solvent mixture and an excess amount of solids were added to a flask at a given temperature and mixed using a magnetic stirrer plate (AGE Magnetic Stirrer; Newtec Inc., Hull, Iowa). These suspensions were then placed on a multiplate mechanical shaker and were left to equilibrate for 72 h in a temperature controlled water bath. Samples were filtered through 0.45 mm cellulose acetate syringe filters into volumetric flasks. Supernatant were then analyzed by UV spectrophotometric analysis at 8max 252 nm (Cary 100 Bio UV visible Spectrophotometer, Palo Alto, California). Subsequently, dissolution rate experiments were conducted on hydrated ACV and cocrystal materials. In order to evaluate the dissolution, 100 mg of 3:2 ACV:water hydrated form and the cocrystal materials were placed in 200 mL of phosphatebuffered saline (PBS) as dissolution medium at 37◦ C for 90 min at 100 rpm using a magnetic stirrer. Aliquots of 5.0 mL were withdrawn every 10 min. The dissolution medium was replaced after every sampling. Dissolution testing was performed in triplicate. The concentration of ACV and cocrystal material in the solution was measured by UV spectrophotometer Cary 100 Bio. DOI 10.1002/jps.24248
Cocrystal by Grinding It is generally possible to prepare cocrytals by dual method of solution growth and neat/solvent drop grinding as observed in cocrystals I and III in this study, but there are some exceptions too. As mentioned earlier, cocrystal II was easily prepared by evaporating acetic acid solution, but solvent drop grinding inhibited the formation of II. For the solvent drop grinding, for this particular cocrystal formation, solvent screening with numerous solvents viz. N,N -dimethyl formamide, dimethyl sulfoxide, methanol, ethanol, 1-propanol, isopropyl alcohol, acetone, acetonitrile, chloroform, n-hexane, acetic acid, and water was performed, but in all cases, anhydrous ACV was observed in PXRD. It must be pointed that not all the dicarboxylic acids that were attempted, successfully formed cocrystals with ACV, neither by solvent drop grinding nor by evaporation method. When screened for cocrystallization of ACV, the following three pharmaceutically acceptable dicarboxylic acids did not result in cocrystal formation: succinic acid, glutaric acid, and adipic acid. Differential scanning calorimetry results of ACV–fumaric acid after various grinding times are shown in Figure 4a. As the cogrinding of the anhydrous ACV and fumaric acid Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
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Figure 3. PXRD pattern of (a) Fumaric acid, (b) AVC-fumaric acid and (c) simulated pattern of AVC-fumaric acid, (d) Malonic acid, (e) ACV-malonic acid, (f) Tartaric acid, (g) ACV-tartaric acid.
proceeded, the endothermal peak at about 220◦ C decreased and finally disappeared. A melting endotherm at 185◦ C, which differs from the melting points of either anhydrous ACV (254.4◦ C) or fumaric acid (205.0◦ C), occurred all the time. This also indicated that during the solvent assisted grinding, a new phase was formed between anhydrous ACV and fumaric acid. Thermal Analysis The DSC and TGA of the cocrystallization product of ACV–fumaric acid from slow evaporation are presented in Figure 4b. The cocrystallization material from slow evaporation from acetic acid showed two endothermic peaks, one at 174.0◦ C and another small peak at 205.0◦ C. The small peak at 205.0◦ C corresponding to melting point of fumaric acid indicates that there was incomplete crystallization during the slow evaporation process. Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
The DSC thermogram of cocrystals II and III are depicted in Figure 5. The DSC thermogram of the cocrystals was distinguishable from parent ACV and the corresponding conformer dicarboxylic acids. The thermal stability of cocrystal materials was assessed by TGA. In case of ACV–fumaric acid·3H2 O, there was no weight loss until 150.0◦ C in TGA analysis (Fig. 4b). The thermal analysis of ACV–fumaric acid·3H2 O indicates that this cocrystal hydrate was stable up to 150.0◦ C. The retention of water molecules in the ACV–fumaric acid cocrystal hydrate up to 150.0◦ C indicates that all the three water molecules are located in the crystal lattice via strong O–H···O hydrogen bonds. For the cocrystals II and III, a weight loss at 135.0◦ C and 140.0◦ C occurred in TGA analysis. The TGA curves (Fig. 5) indicate the absence of any unbound or absorbed solvents. DOI 10.1002/jps.24248
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Figure 4. (a) DSC analysis of cocrystal-I of hte resulting powders after various grining time and (b) TGA curve of cocrystal I and DSC thermogram of cocrystallization product from solvent evaporation.
Table 1.
Crystallographic Data of ACV–Fumaric Acid·3H2 O
ACV–Fumaric Acid·3H2 O Chemical formula Formula weight Crystal size (mm) Crystal system Space group T/K ˚ a (A) ˚ b (A) ˚ c (A) " (◦ ) $ (◦ ) ( (◦ ) Z ˚ 3) V (A Dcalcd. (g cm−3 ) : (mm−1 ) Reflections collected Unique reflections Reflections I ≥ 2FI R1 (I > 2F(I)) wR2 (all) Goodness-of-fit CCDC number
C24 H36 N10 O17 736.63 0.366 × 0.354 × 0.077 Triclinic P−1 110(2) 6.534 11.023 23.529 97.73 91.93 105.33 2 1615.3 1.197 0.075 59,837 5682 5333 0.0359 0.1164 1.110 1005729
Single Crystal Structure of ACV–Fumaric Acid The crystallographic data are summarized in Table 1. The hydrogen-bonding geometry is listed in Table 2. The 1:1 ACV/fumaric acid stoichiometry with three water molecules was observed for cocrystal I. As mentioned in the scheme 1, a heteromeric synthon was observed with only one of the two carboxylic groups of each dicarboxylic acid. The other carboxylic group forms a hydrogen bond with another fumaric acid. The second fumaric acid interacts with another ACV via O–H···N hydrogen bond. Again this ACV forms a heteromeric synthon with carboxylic acid of fumaric acid. As shown in the Figure 6, with two ACV and four fumaric acid molecules, a six membered ring (R1 in Fig. 6) is formed having two hetromeric synthons. Self assembly via O–H···O, N–H···N, and O–H···N interaction leads to a chain of six-membered rings running parallel to the a-axis in the crystal lattice of ACV– fumaric acid cocrystal. The ACV molecules of the abovementioned six-membered ring further form a homosynthon via DOI 10.1002/jps.24248
Table 2. Hydrogen-Bonding Geometry of ACV–Fumaric Acid·3H2 O ˚ and Angle in ◦ ) (d in A D–H···A
d(D–H)
d(H···A)
d(D···A)
D–H···A
O(3)–H(3)···O(15)§1 O(5)–H(5)···N(8)§2 N(5)–H(5A)···N(7)§3 N(6)–H(6)···O(14)§4 O(7)–H(7)···N(3)§5 O(10)–H(10)···O(16)§6 O(11)–H(11)···O(6)§7 O(13)–H(13)···O(8)§8 O(15)–H(15A)···O(14)§9 O(15)–H(15B)···O(21)§10 O(16)–H(16A)···O(10)§11 O(16)–H(16B)···O(10)§12 O(21)–H(21A)···O(3)§13 O(21)–H(21B)···O(3)§14
0.82 0.82 0.86(2) 0.87(2) 0.82 0.82 0.82 0.82 0.92(2) 0.99(5) 0.92(3) 0.83(3) 0.96(4) 0.96(4)
1.90 1.77 2.09(2) 2.03(2) 1.73 2.03 1.84 1.75 1.866(19) 1.78(5) 1.91(3) 2.02(3) 1.81(4) 1.81(4)
2.7123(18) 2.5881(18) 2.9466(19) 2.8743(18) 2.5522(17) 2.8027(18) 2.6551(17) 2.5648(16) 2.7130(17) 2.704(2) 2.8130(19) 2.8027(18) 2.7635(19) 2.774(2)
173.0 178.0 176.0(2) 165.0(2) 178.0 158.0 175.0 175.0 152.0(2) 153.0(5) 169.0(3) 159.0(3) 177.0(3) 174.0(3)
Symmetry transformations used to generate equivalent atoms: (§1) x,y,z; (§2) x,1+y,z; (§3) 1+x,y,z;(§4) −x,3−y,2−z;(§5) x,1+y,z; (§6) −x,3−y,2−z; (§7) x,1+y,z; (§8) −x,2−y,2−z; (§9) 1−x,4−y,2−z; (§10) 1−x,3−y,2−z; (§11) −1+x,y,z; (§12) −1+x,y,z; (§13) x,2+y,z; (§14) −x,3−y,2−z; (§15) −x,3−y,2−z.
weak N–H···N interaction. Another six-membered ring (R2 in Fig. 6) is formed via O–H···O supramolecular interaction involving O–H groups of two ACV molecules and O15–H3···O15 and O21–H21···O3 interactions of two different water molecules. Furthermore, the third water molecule (O16) forms a fourmembered ring (R3 in Fig. 6) with O–H groups of two ACV molecules via O16–H16A···O10 and O10–H10···O16 hydrogen bonding interaction.
COCRYSTAL PROPERTIES Relative Humidity The RH stability results of 3:2 ACV:water hydrated ACV, anhydrous ACV, and the cocrystals are described below and are summarized diagrammatically in Table 3. (a) The 3:2 ACV:water hydrated: The commercial ACV (3:2 ACV:water hydrated) rapidly rehydrated to dihydrated form at all RH condition except at 0% RH. The PXRD pattern of dihydrated form was evaluated by Sohn and Kim.4 At 0% RH, the 2:3 hydrated form lost water and Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
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Figure 5. DSC thermogram of (a) ACV-malonic acid and (b) ACV-tartatic acid, TGA curve of (c) of ACV-malonic acid and (d) ACV-tartatic acid.
Figure 6. Hydrogen bonding network of ACV-fumaric. 3H2 O showing formation of synthon and a six membered ring R1, a six membered ring R2 and a four membered ring R3.
as reported earlier it was converted to anhydrous form within 1 day (Sohn and Kim4 ). (b) Anhydrous ACV: The anhydrous ACV was converted to dihydrated form of ACV within 1 day at all RH conditions, but at 0% RH, it remained stable for 3 weeks. (c) Cocrystal I: This cocrystal material was found to be stable at all RH conditions for 3 weeks long study. This cocrystal clearly demonstrated enhanced stability over hydrated and anhydrous ACV. Its stability is additionally demonstrated by slurrying the cocrystal material in water for Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
2 days at ambient temperature without any observed physical form change. (d) Cocrystal II: This cocrystal material was also found to be stable at all RH conditions for our 3 weeks long study as that of cocrystal I. (e) Cocrystal III: This material was stable only at 0% RH for the total duration of our study. At 98% RH, the cocrystal III showed instability at just 1 day, forming a sticky substance with a color turning to light pink. We were not able to identify this substance. At the end of 1 week, this DOI 10.1002/jps.24248
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Table 3.
Stability of Cocrystals with Respect to RH
Table 4. Solubility of Anhydrous ACV and Cocrystal Materials in Water at 35◦ C
Stability with Respect to RH Material
% RH
Anhydrous ACV
0 43 75 98
2/3 Hydrated ACV
Cocrystal I
1 Day √
3 Days √
1 Week √
3 Weeks √
× × × √
× × × √
× × × √
× × × √
× × × √
× × × √
× × × √
× × × √
0 43 75 98 0 43 75 98
Cocrystal II
0 43 75 98
Cocrystal III
0 43 75 98
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
× ×
× ×
√ ×
2:3 hydrated ACV (moles/L) 1.28 × 10−1
Cocrystal I (moles/L)
Cocrystal II (moles/L)
Cocrystal III (moles/L)
7.01 × 10−1
7.25 × 10−1
6.78 × 10−1
× × ×
√ The symbol and × indicate that the material is stable and unstable respectively at that particular RH condition and time point.
unidentified sticky material started transforming to a liquid. The instability of cocrystal III at 75% RH started at day 3 and transformation into liquid at day 10. At 45% RH, the material was stable up to day 7. From the above discussion on relative stability of the ACV cocrystals, we can conclude that cocrystals I and II were most stable, whereas cocrystal III was the least stable. The order of cocrystal stability with respect to RH is thus: I ≈ II > III. The acid strength of the conformers are malonic acid (2.83) > fumaric acid (3.03) > DL-tartaric acid (3.22). The weakest acid among the four is DL-tartaric acid, which formed the least stable cocrystal. From this study, it is evident that acidity of the coformer is not the only issue affecting the stability of the cocrytals as both ACV–fumaric acid and ACV–malonic acid cocrystals had identical stability. The other driving force for the stabilization of the cocrystal is possibly due to the hydrogen bonds. Generally, introduction of dicarboxylic acids should prevent consumption of water into the cocrystal by formation of supramolecular synthon through hydrogen bonds created via donor and acceptor atoms and the cocrystals are expected to be more stable than the original drug molecule. Cocrystal I hydrate was stable at all test conditions, which indicates that water molecules form strong hydrogen bonds with the cocrystal components leading to a direct correlation between the structure and stability of the cocrystal hydrate. Solubility and Dissolution Rate The dissolution rate and solubility have a major impact on the bioavailability of pharmaceutical drugs. Solubility of the cocrystals in water at 35◦ C was compared with the 3:2 ACV:water DOI 10.1002/jps.24248
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Figure 7. Dissolution profile of 3:2 ACV:water hydrated and ACVcocrystal materials.
hydrated and the results are summarized in Table 4. The hydrated ACV is more soluble than the anhydrous forms (Kristl et al.3 ). The trend of solubility of cocrystals in water is as follows: cocrystal I ≈ cocrystal II > cocrystal III. The dissolution experiment was conducted on hydrated ACV and all the three cocrystals in PBS at 35◦ C. The highest concentration of 3:2 ACV:water hydrated was released approximately after 50 min, whereas the peak concentration of cocrystal I, II, and III in the solution appeared approximately 30, 30, and 40 min, respectively (Fig. 7). At the same dissolution time, the released concentrations of ACV cocrystals in the solution were consistently higher than that of ACV, as for example, at 20 min, the concentrations of ACV–fumaric acid and hydrated ACV were 1.69 and 1.22 mg cm−2 , respectively. Initially ACV–fumaric acid cocrystal dissolved rapidly and reached equilibrium after 20 min, but dissolution of ACV was slow at the beginning and reached the equilibrium point after 50 min. The remaining powder samples, after the dissolution experiment, were examined by PXRD. Surprisingly, it was observed that anhydrous ACV and the cocrystal III were converted to dihydrated ACV, whereas cocrystal I and II remain stable. The rapid dissolution of the cocrystal III at the early time point indicate that transformation of cocrystal III to dihydrated ACV occurred only after reaching the maximum concentration.
CONCLUSIONS Three cocrystals of a pharmaceutical compound ACV with dicarboxylic acids in 1:1 stoichiometric ratio have been described. The RH of the cocrystals differs from the hydrated and anhydrous ACV. In terms of physicochemical properties, such as solubility and dissolution rate, the cocrystals show remarkably higher solubility and faster dissolution compared with the hydrated ACV. Taking into account of improved physicochemical Sarkar and Rohani, JOURNAL OF PHARMACEUTICAL SCIENCES
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
properties of cocrystals reported herein, novel formulations of ACV with dicarboxylic acids are possible.
ACKNOWLEDGMENT We are also thankful to the Natural Sciences and Engineering Council of Canada for its financial support of this project through the Discovery Grant.
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DOI 10.1002/jps.24248