Cocrystal Hydrate of an Antifungal Drug, Griseofulvin, with Promising

Nov 9, 2012 - (4) GF has been one of the highly prescribed antifungal drugs, with an annual worldwide market value of USD 63.7 M, and its ..... 1968, ...
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Cocrystal Hydrate of an Antifungal Drug, Griseofulvin, with Promising Physicochemical Properties Published as part of the Crystal Growth & Design virtual special issue In Honor of Prof. G. R. Desiraju. Srinivasulu Aitipamula,*,† Venu R. Vangala,† Pui Shan Chow,† and Reginald B. H. Tan*,†,‡ †

Crystallization and Particle Science, Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833 ‡ Department of Chemical & Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576 S Supporting Information *

ABSTRACT: Cocrystallization of an antifungal drug, griseofulvin (GF), with an artificial sweetener, acesulfame (Ace-H), resulted in a cocrystal monohydrate with GF and Ace-H in a 2:1 stoichiometric ratio. The cocrystal was characterized by differential scanning calorimetry and thermogravimetric analysis, and its crystal structure was determined by singlecrystal X-ray diffraction. The Ace-H molecule is in its enol form in the crystal structure. The cocrystal hydrate shows remarkable thermal stability, which was traced to strong hydrogen bonds in the crystal structure. The dissolution rate of the (GF)2·Ace-H hydrate is significantly improved compared to that of the parent GF, and the aqueous solubility of the cocrystal hydrate is greater than the solubility of GF at 37 °C. Furthermore, the cocrystal hydrate is found to be stable at different relative humidity conditions for up to 13 weeks. The remarkable thermal stability, improved solubility and dissolution rate, and pharmaceutical acceptability of Ace-H make the (GF)2·Ace-H hydrate a preferable solid form for development of GF formulations (Singapore patent application 201107310-3 (ref 1)).

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crystal structure of GF has been reported in 19776a and later revisited in 1982.6b A recent study by Willart and co-workers revealed two new crystalline polymorphs of GF which originate from the melt crystallization.7 GF is known to form several solvates. For example, Grant and Abougela reported solvates with a series of fatty acids.8 GF is also known to form solvates with alkyl halides, alkyl dihalides, chloroform, benzene, and 1,4dioxane.9 Differences in the amorphous phases of GF prepared by cryogenic milling and melt-quenching have been investigated recently.10 GF belongs to Class II11 drugs according to the Biopharmaceutics Classification System, which have low solubility and high permeability. Hence, GF is an ideal active pharmaceutical ingredient (API) to explore various solid forms that can enhance solubility and directly impact dissolution profile and bioavailability. However, the nonionizable nature of GF hampers application of the most-common solubility increasing method, such as salt formation, and poses challenges for solid form screening. Various alternate methods are used for improving the solubility and dissolution rate of GF. For example, it has been reported that the solubility and dissolution rate of GF could be improved by micronization of GF into fine particles,12a preparation of GF nanoparticles from water-

riseofulvin ((2S,6′R)-7-chloro-2′,4,6-trimethoxy-6′-methyl-3H,4′H-spiro[1-benzofuran-2,1′-cyclohex[2]ene]-3,4′dione, hereinafter abbreviated as GF, Figure 1) is a classic

Figure 1. Chemical diagram of GF and Ace-H.

antifungal drug administered orally for the treatment of dermatomycoses including ringworm, athlete’s foot, and infections of the scalp and nails.2 GF has been marketed under several brand names, such as Fulvicin, Grifulvin V, GrisPeg, and Grisactin, and it is available as tablets containing 125 mg or 250 mg of the active ingredient. Since the isolation of GF in 1939, more than 400 analogues have been synthesized and tested for their antifungal activity.3 More recently, GF has been found to be active toward a range of mammalian cancer cell lines.4 GF has been one of the highly prescribed antifungal drugs, with an annual worldwide market value of USD 63.7 M, and its annual consumption is nearly 85,000 kg.5 The first © 2012 American Chemical Society

Received: August 21, 2012 Revised: November 6, 2012 Published: November 9, 2012 5858

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dilutable microemulsions,12b cogrinding of GF with a pharmaceutical excipient, crospovidone,12c etc. Most recently, inclusion complexes of GF with β-cyclodextrin at the surface of silica particles have been suggested for topical formulations.13 Amorphization of crystalline GF is another way to achieve higher dissolution rate; however, it has been noted that GF crystallizes during dissolution of amorphous solid, making it a rapidly crystallizing compound in solution.14 Furthermore, it has been recently found that the experimental solubility of amorphous GF is only 1.4 times the solubility of crystalline GF.14 Therefore, there is a need to find a stable solid form which can enhance the solubility and dissolution rate of GF. Pharmaceutical cocrystals are multicomponent crystals which are actively being explored due to their ability to fine-tune the physicochemical properties of APIs.15 Pharmaceutical cocrystals contain one or more of the solid components (coformers) together with an API. It is a prerequisite that the coformer should be pharmaceutically acceptable if the cocrystal is to be considered as a suitable solid form for drug development. Hence, the coformers are generally selected from the list of United States Food and Drug Administration (US FDA) generally recognized as safe (GRAS) chemicals.16 The impact of cocrystal formation in the pharmaceutical development has been well documented.17 For example, a cocrystal of an anticonvulsant drug, carbamazepine, involving saccharin showed a comparable bioavailability with the marketed drug (Tegretol tablets).18 The importance of pharmaceutical cocrystals in drug development has been recently recognized by the US FDA, which released a draft guidance on the subject of the regulatory classification of pharmaceutical cocrystals.19 In this Communication, we report a cocrystal hydrate of GF with an artificial sweetener, acesulfame (Ace-H, Figure 1). In order to assess the stability and structural features of the reported solid form, a variety of analytical techniques were applied; these include differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and single crystal and powder X-ray diffraction (PXRD). Physicochemical properties such as stability, solubility, and dissolution rate of the cocrystal hydrate are also evaluated. Design of cocrystals is difficult for molecules such as GF that lack potential hydrogen-bonding functionalities (e.g., carboxylic acid, pyridyl, amide). Therefore, our cocrystallization trials included a variety of coformers that consisted of carboxylic acids, phenols, amides, and pyridines (Table S1 of the Supporting Information). Thus, a total of 40 cocrystal formers were chosen, and a screening for cocrystals was conducted using solution crystallization and solid-state grinding methods including neat grinding and solvent-drop grinding (see Table S1 of the Supporting Information). Most of these experiments were found to be unsuccessful, except that a grinding experiment on a 2:1 stoichiometric ratio of GF and Ace-H using a ball mill hinted at the formation of a cocrystal (see Figure S1 of the Supporting Information), and cocrystallization of GF and Ace-H in a 2:1 stoichiometric ratio from methanol (10 mL) at ambient conditions resulted in a monohydrate of a 2:1 (GF/Ace-H) cocrystal. Ace-H is an aliphatic calorie-free sweetener, and its potassium salt is widely used in food products and pharmaceutical formulations.20 Potential pharmaceutical applications of Ace-H as a taste masker have been highlighted recently in a patent application that revealed a pleasant sweet taste for the hydrogen bonded adducts (or cocrystals) of Ace-H with xanthines such as propentofyllin, pentoxifyllin and caffeine,

and phenazone.21 These adducts have been claimed as suitable candidates for preparation of chewing gums, chewable tablets, etc. The X-ray structural analysis of these adducts revealed that the Ace-H molecule is in its enol form (Figure 2). The mechanical behavior of two polymorphs of Ace-H has been investigated recently.22

Figure 2. Keto−enol tautomerism in Ace-H.

Single crystal X-ray diffraction revealed that the cocrystal hydrate belongs to the monoclinic P21 space group.23 The asymmetric unit contains two molecules of GF and one molecule each of Ace-H and water, and the Ace-H is present in its enol form (see Figure S2 of the Supporting Information for an overlay of different conformations of GF and Ace-H). As shown in Figure 3, the water molecule plays an important role in the cocrystal formation, such that it interacts with all other components of the asymmetric unit via O−H···O (2.529 Å, 173°; 2.789 Å, 174°; 2.753 Å, 165°; Table S2 of the Supporting Information) hydrogen bonds that involve hydroxyl groups of water and Ace-H, and carbonyl groups of the symmetry independent molecules of GF. The resulting four-component supramolecular units are connected to each other via Cl···O (3.01 and 3.18 Å) interactions to generate columns along the crystallographic a-axis. The crystal structure features several C− H···O (2.30−2.54 Å, 114−170°) and C−H···π (2.80 Å, 125°; 2.91 Å, 105°) interactions from the adjacent columns. A comparison of the hydrogen bonds in the crystal structure of (GF)2·Ace-H hydrate revealed that the O−H···O hydrogen bond between the enol OH group of Ace-H and the water molecule is the strongest hydrogen bond among all other O− H···O hydrogen bonds present in the crystal structure. Therefore, we surmise that the ability of the enol OH group to be involved in a strong hydrogen bond facilitates the formation of the enol form. A similar observation of keto−enol tautomerism has been observed by Etter and co-workers in a molecule that is structurally related to Ace-H.24 Acylsulfonamide exists in the enol form in a cocrystal with triphenylphosphine oxide. On the basis of the structural and spectroscopic analysis, it has been confirmed that a strong intermolecular O−H···O hydrogen bonding between the enol OH and phosphoryl oxygen stabilizes the enol tautomer in the solid-state. The thermal stability of the cocrystal hydrate was assessed by DSC and TGA. Crystals obtained from the crystallization batches were air-dried before they were subjected to DSC or TGA analysis. The DSC thermogram shows a major endotherm at 150 °C, followed by exotherms (Figure 4). TGA analysis did not show any weight loss until 150 °C (Figure 4). The thermal behavior of (GF)2·Ace-H hydrate was also analyzed by hotstage microscopy (HSM, Figure 5). Water release and partial melting of the crystal was observed in the temperature range 150−185 °C. Subsequent heating of the crystal resulted in complete melting at 200 °C. Our attempts to obtain an anhydrous cocrystal by dehydration experiments were not successful. Samples of cocrystal hydrate stored in a vacuum oven at 150 °C for a day resulted in a powder for which the 5859

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Figure 3. Crystal structure of the (GF)2·Ace-H hydrate: (a) a four-component supramolecular unit; (b) arrangement of columns formed by the fourcomponent supramolecular units. Symmetry independent molecules of GF in part b are shaded with different colors.

water molecules in the crystal lattice of (GF)2·Ace-H hydrate to a temperature of 150 °C is most likely due to the fact that the water molecule is located in the crystal lattice via strong O− H···O hydrogen bonds. Furthermore, as the water plays a decisive role in the cocrystal formation, removal of the water molecule led to complete collapse of the crystal lattice, and hence, decomposition of the free Ace-H was observed above 150 °C. In a recent study, Zaworotko and co-workers analyzed several cocrystal hydrates and studied the correlation between their crystal structure and thermal stability.25 It has been found that cocrystal hydrates that consist of isolated water molecules show a greater thermal stability compared to the hydrates that contain water molecules in channels. The high thermal stability of (GF)2·Ace-H hydrate up to a temperature of 150 °C can be reasoned by the ability of the water molecule to form three strong hydrogen bonds with the components of the cocrystal, leading to a direct correlation between the structure and thermal stability of the cocrystal hydrate. The powder samples of crystalline GF and cocrystal hydrate were found to be stable at ambient conditions for over one year (Figure S4 of the Supporting Information). The remarkable thermal stability of (GF)2·Ace-H hydrate and its ambient stability prompted us to evaluate the physical stability of the (GF)2·Ace-H hydrate at various relative humidity (RH) and temperature conditions recommended by the ICH guidelines for pharmaceutical stability testing.26 Incubated samples were analyzed by PXRD at designated time points (1, 3 days, 1, 2, 4, 7, and 13 weeks) that are shown in Figure 6. Some of these samples were also analyzed by DSC/TGA (Figure S5 of the Supporting Information). Results of the stability studies suggest that the cocrystal hydrate is stable at all test conditions that are evidenced by a perfect match of the PXRD pattern of the incubated samples with that of the PXRD pattern of the cocrystal hydrate at its initial stage. Among the different known solid forms of GF (crystalline GF, an amorphous form, and various solvates), the amorphous form has been thoroughly investigated for its physicochemical properties.10,12,14 Therefore, it is important to compare the physicochemical properties of (GF)2·Ace-H hydrate with the physicochemical properties of the amorphous GF. Amorphous GF was prepared by melt quenching. In a typical experiment, crystalline GF was melted at 225 °C in an oven and quenched

Figure 4. DSC and TGA profiles of the (GF)2·Ace-H hydrate.

Figure 5. Photomicrographs from HSM experiment showing the thermal behavior of (GF)2·Ace-H hydrate at different temperatures.

PXRD pattern is identical to the PXRD pattern of GF (see Figure S3 of the Supporting Information). This observation confirms that the weight loss in TGA analysis is due to concomitant events of water loss and decomposition of the Ace-H (reported melting point of Ace-H: 122 °C22). Thermal analysis and dehydration experiments suggest that the cocrystal hydrate is stable up to a temperature of 150 °C. Retention of 5860

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The physical stability of the cocrystal hydrate in the presence of water was also compared with the stability of the amorphous form by solution mediated transformation experiments. Excess solids of the amorphous GF and cocrystal hydrate (approximately 1 g in 4 mL of deionized (DI) water) were stirred in DI water at 37 °C for 24 h. PXRD patterns of the filtered solids are compared in Figure 8 with those of the respective solids at their

Figure 6. PXRD patterns of pure and incubated samples of (GF)2·AceH hydrate. Notice that the cocrystal remained intact in all the tested storage conditions.

with liquid nitrogen. PXRD analysis of the melt quenched sample shows broad humps centered at 2θ =13.26° and 27.35° which are characteristic of the amorphous nature of GF (Figure S6 of the Supporting Information).10 DSC analysis of the melt quenched sample showed a glass transition around 85 °C and a recrystallization exotherm at 127 °C (Figure S7 of the Supporting Information). The physical stability of the amorphous GF was tested at various RH conditions (Figure 7). Notably, amorphous GF converts into crystalline GF at

Figure 8. Comparison of PXRD patterns of (a) crystalline GF, (b) amorphous GF, (c) cocrystal hydrate as prepared by solution crystallization, (d) powder from slurry experiment on amorphous GF, and (e) powder from slurry experiment on cocrystal hydrate for 24 h. Notice that the cocrystal hydrate is intact in the slurry experiments.

initial stage. It was observed that while the amorphous GF completely transforms into crystalline GF, cocrystal hydrate was intact for 24 h in the aqueous medium, and this confirms that there was no solution mediated transformation to the parent GF. The rate of dissolution has a major impact on the performance and bioavailability of APIs that are poorly watersoluble. In the present study, encouraged by the remarkable thermal and physical stability of (GF)2·Ace-H hydrate, solubility and dissolution experiments were conducted to gauge whether the cocrystal hydrate shows any effect on the GF solubility and dissolution rate. Since amorphous GF converts into crystalline GF in slurry experiments (Figure 8), solubility experiments were not conducted on this material. Bulk samples of the GF and (GF)2·Ace-H hydrate were prepared by solution crystallization experiments at a 10 g scale. Prior to the solubility experiments, crystals were ground to a fine powder and sieved to achieve an approximate particle size of 75−106 μm. Saturated solutions of each of the samples were obtained by stirring an excess of the sample in DI water at 37 °C for 48 h. The resulting samples were filtered and quantified upon appropriate dilution using high performance liquid chromatography (see Supporting Information for experimental details). Thermodynamic solubility values thus obtained revealed that the solubility of (GF)2·Ace-H hydrate is three times the solubility of GF (Table 1). Subsequently, powder dissolution experiments were conducted on crystalline GF, amorphous GF, and cocrystal hydrate in pH 7.5 phosphate buffer at 37 °C. As shown in Figure 9, the (GF)2·Ace-H hydrate dissolves significantly faster initially and reaches equilibrium within approximately 20 min. On the other

Figure 7. PXRD patterns of the powder samples from stability experiments on amorphous GF. Notice that the amorphous GF converts to crystalline GF at higher RH conditions.

higher RH (>75%) within a day. Partial conversion to crystalline GF was observed at 57% and 33% RH after 2 days and 1 week, respectively. Amorphous GF was found to be stable even after storage at 2σ(I)). The final wR(F2) values were 0.1216 (I > 2σ(I)). The final R1 values were 0.0483 (all data). The final wR(F2) values were 0.1275 (all data). (24) Etter, M. C.; Gillard, R. D.; Gleason, W. B.; Rasmussen, J. K.; Duerst, R. W.; Johnson, R. B. J. Org. Chem. 1986, 51, 5405. (25) Clarke, H. D.; Arora, K. K.; Bass, H.; Kavuru, P.; Ong, T. T.; Pujari, T.; Wojtas, L.; Zaworotko, M. J. Cryst. Growth Des. 2010, 10, 2152. (26) (a) ICH harmonized guideline, 2003. Q1A (R2) stability testing of new drug substances and products. (b) Vangala, V. R.; Chow, P. S.; Tan, R. B. H. CrystEngComm 2011, 13, 759. (27) Chen, J.; Sarma, B.; Evans, J. M. B.; Myerson, A. S. Cryst. Growth Des. 2011, 11, 887.

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