Insight into the Phase Transformation among Various Solid Forms of

Aug 27, 2015 - A new polymorph and three hydrates of baicalein, a widely prescribed anti-inflammatory TCM drug, were discovered through comprehensive ...
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Insight into the Phase Transformation among Various Solid Forms of Baicalein Bingqing Zhu, Jian-Rong Wang, and Xuefeng Mei* Pharmaceutical Analytical & Solid-State Chemistry Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China S Supporting Information *

ABSTRACT: A new polymorph and three hydrates of baicalein, a widely prescribed anti-inflammatory TCM drug, were discovered through comprehensive polymorph screening experiments. The forms were fully characterized by a range of analytical techniques, including PXRD, Raman spectra, FTIR, HSM, SEM, TGA, DSC, and DVS. Single crystal structures and the transformation pathways among different polymorphs and hydrates are discussed in detail. Single-crystal-to-single-crystal transformation behavior between monohydrate and hemihydrate was revealed. Thermodynamic stability, hygroscopicity, and powder dissolution behavior were investigated. The results show that the newly discovered form γ presents better dissolution behavior and remarkably greater apparent solubility compared with the current widely used marketed drug substance.

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INTRODUCTION A given molecule can exist in different crystalline forms, such as polymorphs, hydrates, and solvates.1 The various solid forms generally present distinguished physicochemical properties2,3 and are closely related to the bioavailability4,5 and manufacture of active pharmaceutical ingredients (APIs).6,7 The selection of a desired solid modification provides an opportunity to further optimize the performance of a drug candidate with predetermined chemical structures. Formation of hydrate is widely observed during drug development processes. APIs are usually exposed to water or water vapors in a variety of unit operations, such as precipitation, crystallization, formulations, and storage. The propensity to form hydrates needs to be fully understood.8 Precise knowledge of the thermostability of potential hydrates is a prerequisite for designing reliable industrial processes, where parameters, such as solvent composition, concentration, and drying temperature, are to be optimized.9−11 Therefore, during the development of a new drug product, studies are performed on the diversity of the solid forms via a thorough screening, aiming to develop a mechanistic understanding of the solid form landscape and corresponding phase transformation mechanism that controls the stability of a particular solid-state form. Baicalein (Scheme 1) is one of the most important bioactive flavonoids isolated from the well-known Traditional Chinese Medicine (TCM) called “Huang Qin”, which has been used for over 2000 years in China. In addition to its well-known pharmacological effects, such as anticancer,12 anti-inflammatory,13 antibacterial,14 anti-HIV,15 and anti-adipogenic activities,16 recent studies have indicated that baicalein is also a promising therapeutic agent for the treatment of devastating disorders, such as Alzheimer’s disease (AD) and Parkinson’s © 2015 American Chemical Society

Scheme 1. Chemical Structure of Baicalein

disease (PD).17−19 Baicalein usually exists as its glycoside form, baicalin, in natural resources. There has been much work concerning the metabolic pharmacokinetics of baicalein and its glycoside partner. Although baicalin exhibited better water solubility than baicalein, it was, however, much less well absorbed as its glycoside form due to its poor lipophilicity. Baicalin might be absorbed only after hydrolyzed to baicalein by enterobacteria in the colon, whereas baicalein was directly absorbed through the small intestine. It was believed that baicalin serves as a sustained-release prodrug of baicalein.20 Based on the AUC (area under curve) of total baicalein after enzymatic hydrolysis, the absolute bioavailability of baicalein (36.1% ± 4.4%) was much higher than that of its glycoside form, baicalin (28.0 ± 5.7%).21 The various pharmacologic effects and the relatively higher absolute bioavailability demonstrate that baicalein presents advantageous pharmacokiReceived: June 19, 2015 Revised: August 14, 2015 Published: August 27, 2015 4959

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After 12 h, yellow plate crystals were harvested for crystal structure identification. Preparation of MH1. To a 4 mL glass vial, 10 mg of baicalein was dissolved in 4 mL of a 1:1 (v/v) mixture of acetone and water. The resulting solution was filtered and placed at room temperature for evaporation. After 24 h, yellow needle crystals were obtained and identified as MH1. Preparation of HH. HH cannot be obtained directly from the anhydrous form. It was obtained upon maintaining MH1 under low humidity conditions (RH < 45%) at 25 °C for 24 h. HH can also be obtained by heating MH1 solid to 30 °C for 1 h or longer. Preparation of MH2. A different modification of monohydrate, MH2, was serendipitously discovered upon maintaining MH1 in the mother liquor under 4 °C for nearly 2 months. Powder X-ray Diffraction (PXRD). PXRD patterns were obtained using a Bruker D8 Advance X-ray diffractometer (Cu Kα radiation). Voltage and current of the generator was set to 40 kV and 40 mA, respectively. Data over the range 3−40° 2θ were collected with a scan rate of 5°/min at ambient temperature. The data were imaged and integrated with RINT Rapid and peak-analyzed with Jade 6.0 from Rigaku. Single Crystal X-ray Diffraction (SCXRD). X-ray diffractions of all single crystals were performed at 296(2) K on a Bruker Apex II CCD diffractometer using Mo Kα radiation (λ = 0.71073 Å). The integration and scaling of intensity data was performed using the SAINT program. The data were corrected for the effects of absorption using SADABS. The structures were solved by direct methods and refined with the full-matrix least-squares technique using SHELX-97 software. Non-hydrogen atoms were refined with anisotropic displacement parameters, and hydrogen atoms were placed in calculated positions and refined with a riding model. Crystallographic data in cif format have been deposited in the Cambridge Crystallographic Data Center, CCDC No. 1405657−1405660 for form γ, HH, MH1, and MH2, respectively. Thermogravimetric Analysis (TGA). Thermogravimetric analysis was carried out on Netzsch TG 209F3 equipment. The samples were placed in open aluminum oxide pans and heated at 10 °C min−1 to 400 °C. Nitrogen was used as the purge gas at 20 mL min−1. Differential Scanning Calorimetry (DSC). DSC experiments were performed on a PerkinElmer DSC 8500 instrument under a nitrogen gas flow of 20 mL/min purge. Ground samples weighing 3−5 mg were heated in sealed nonhermetic aluminum pans. To study phase transformation, the sample was sieved through 100-mesh sieves (mesh size 149 μm) before being analyzed at a heating rate from 30 °C/min to 250 °C/min. Two-point calibration using indium and tin was carried out to assess the temperature axis and heat flow of the equipment. Dynamic Vapor Sorption (DVS). The water sorption and desorption processes were measured on an Intrinsic DVS instrument from Surface Measurement Systems, Ltd. The samples were mounted on a balance and studied over a humidity range from 0% to 95% RH, and then decreased to 0% RH at 25 °C. Each humidity step was made if less than a 0.02% weight change occurred over 10 min. Fourier-Transform Infrared (FTIR) Spectroscopy. Fouriertransform-infrared (FTIR) spectra were collected by a NicoletMagna FT-IR 750 spectrometer in the range from 4000 to 350 cm−1, with a resolution of 4 cm−1 at ambient conditions. Confocal Raman Microscope. Raman spectra were recorded with the Thermo Scientific DXR Raman microscope equipped with a 532 nm laser. Raman scans range from 3500 to 50 cm−1. The samples were analyzed directly in a glass sheet using 10 mW laser power and 50 μm pinhole spectrograph aperture. Calibration of the instrument was performed using a polystyrene film standard. Hot-Stage Microscopy (HSM). All HSM examinations were performed on a XPV-400E polarizing microscope and a XPH-300 hot stage coupled with a JVC TK-C9201 EC digital video recorder (Shanghai Changfang Optical Instrument Company Ltd.). Crystals of anhydrous form γ and hydrates were focused under the microscope (10×) and set to capture images periodically over time during the heating program.

netic properties compared to baicalin in clinical applications. The development of baicalein as a new drug candidate has drawn particular interest in recent years.22 However, even with greater bioavailability compared with its glycoside counterpart, the therapeutic application of baicalein is still greatly limited by the low solubility (16 μg/mL in water).20 Therefore, improving its solubility and dissolution rate is a practical approach to better development of this drug. Although there have been endeavors aimed at improving the solubility of baicalein, such as formation of cyclodextrin (CD) complex,23 solid dispersion,24 and cocrystal with nicotinamide,25 studies concerning its polymorphs and hydrates have rarely been explored. Indeed, although baicalein has been used in TCM formulations for centuries, there has been little information about its solid state landscape. Currently, there is only one crystal structure reported as form α,26 which is the same as the currently available form on the market. Yang claimed the discovery of a new solid form, named form β, in his 2007 patent.27 However, a close analysis of the PXRD pattern provided in the patent revealed that the newly claimed form is actually identical to that of form α. Because there are no further solid-state characterizing data reported in the literature for form β, we believe the claimed form is most likely form α. Apparently, there is a need to comprehensively screen and fully characterize the various solid forms of this widely applied medicine. In this study, we focus on the solid form landscape of baicalein and report herein four novel solid forms, including a new pure form (γ), a hemihydrate (HH), and two polymorphic monohydrate modifications (MH1 and MH2). Their structures were elucidated by single-crystal X-ray diffraction (SXRD) and characterized by powder X-ray diffraction (PXRD), Fourier transform-infrared (FTIR) spectroscopy, and Raman microscopy. The physicochemical properties of these new forms were investigated by hot stage microscopy (HSM), scanning electron microscopy (SEM), thermal analysis (TGA and DSC), and dynamic vapor sorption (DVS) isotherms. The interconversion pathways of these forms were discussed in detail. Furthermore, the powder dissolution of the newly discovered pure form γ was evaluated and compared with those of the marketed form.

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EXPERIMENTAL SECTION

Materials. The sample of baicalein used in the present work was purchased from Shanghai Boylechem Co., Ltd. (Shanghai, China), with purity greater than 98%. The unprocessed material was confirmed by PXRD to be a monophasic sample (form α). All analytical-grade solvents were purchased from Sinopharm Chemical Reagent Company and were used without any further purification. Polymorph Screening. Many approaches, such as slurry, antisolvent, cooling, and evaporation, were applied to polymorph screening. Baicalein is soluble in most polar solvents, such as acetone, tetrahydrofuran, and alcohols, while it is practically insoluble in water. Slurry experiment was conducted in water, ethyl acetate, isopropanol, acetonitrile, nitromethane, toluene, chloroform, and hexane at both room temperature and 50 °C. All slurry experiments led to exclusive formation of α. Antisolvent experiments were conducted using a variety of solvent combinations, such as ethanol/hexane, ethanol/ chloroform, ethanol/ether, tetrahydrofuran/hexane, tetrahydrofuran/ chloroform, and tetrahydrofuran/ether. All antisolvent experiments also led to exclusive formation of α. Although the marketed form α appeared frequently using these methods, form γ and MH1 were unambiguously identified using evaporation method. Preparation of Form γ. In total, 10 mg of baicalein was dissolved in 2 mL of a 1:1 (v/v) mixture of tetrahydrofuran (THF) and toluene. The resulting solution was filtered and placed at 50 °C for evaporation. 4960

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Figure 1. Polarizing microscopy picture and SEM images for form α, form γ, MH1, and MH2. Scanning Electron Microscopy (SEM). The surface morphology of all of the samples was viewed using scanning electron microscopy (Agilent 8500) operated at a beam voltage of 1 kV. The samples were mounted onto a steel stage using double-sided adhesive tape before the analysis. Powder Dissolution. To minimize the size effect on dissolution results, form α and form γ were sieved through 100-mesh sieves. Accurately weighed powders of 5 mg baicalein (n = 3) were added to dissolution vessels containing 15 mL of pH 2.0 hydrochloric acid (HCl) buffer and pH 4.5 phosphate buffer. 0.5% Tween 80 was added to the buffer to improve the wettability of baicalein. The dissolution studies were conducted at a rotation speed of 75 rpm at 37 °C. Sampling was performed at 5, 10, 15, 20, 30, 40, 60, 80, 100, 120, 150, and 180 min. The withdrawn suspensions were filtered with 0.22 μm PTFE filters prior to HPLC analysis. The sample concentration was determined by an Agilent 1260 series HPLC (Agilent Technologies), equipped with a quaternary pump (G1311C), diode-array detector (G1315D) set at 276 nm, and a 4.6 × 100 mm, 2.5 μm Agilent Eclipse plus C18 column. Mobile phase consisting of a methanol and 0.05% (v/v) phosphoric acid solution (70/30, v/v) was run at 1.0 mL/min. The column temperature was set at 30 °C. After stirring for 3 h, the residual solids were examined using PXRD, indicating that a small amount of form γ had been transformed to form α.

to lose water and convert to anhydrous form. Notably, HH and MH2 cannot be prepared directly from a solution. The two hydrates can only be obtained through solid-to-solid transformation, e.g., by maintaining MH1 under a suitable temperature and relative humidity over a certain period of time (see Experimental Section). The various solid forms presented distinctive crystal habits, as illustrated in Figure 1. Form γ presented sheet-like morphology, while form α showed rod-like shape. MH1 crystallized as very long needles, while a triangular block-like habit can be observed for MH2. As shown in Figure 2, PXRD patterns of baicalein present distinguishable differences between four newly discovered solid

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RESULTS AND DISCUSSION To discover as many new solid forms as possible, comprehensive crystallization screening experiments were conducted with a variety of solvents and crystallization methods. The resulting crystallized samples were first analyzed by PXRD, and a selection of some successful results is listed in Table S1. MH1 was routinely obtained under 25 °C in the presence of water solvent. Form α can be expected if saturated baicalein solution was allowed to evaporated at room temperature. However, at higher temperatures, the resulting material was found to be a mixture of forms α and γ in screened solvent. The selection of solvent is also critical in dictating the resulting physical state of the material. Interestingly, when toluene was employed in the crystallization solvent mixture, thin sheet-like crystals of form γ were recovered after the crystallization solution was allowed to evaporate under 50 °C (Figure 1). However, a solvent mixture including MTBE (methyl tert-butyl ether) or MIBK (methyl isobutyl ketone) always resulted in a mixture of form α and form γ using the same protocol. Notably, under elevated temperatures, no hydrate can form even with water as the crystallization solvent, indicating that MH1 is thermodynamically unstable and prone

Figure 2. PXRD patterns of various solid forms of baicalein.

forms. Prominent peak positions for MH2 are at 2θ 9.9°, 11.2°, 13.0°, 17.6°, 9.8°, 26.4°, and 27.4°. The other polymorphic monohydrate, MH1, shows characteristic peaks at 2θ 8.8°, 10.3°, 11.5°, 13.8°, 14.4°, 16.5°, 20.8°, and 27.9°. The PXRD pattern of HH is quite similar to that of MH1, while differences can be observed at 2θ 7.0°, 8.8°, 10.4°, 11.8°, 14.1°, 14.4°, and 17.0°. All of the peaks displayed in the measured patterns of the bulk powder are closely matched with those in the simulated patterns generated from single crystal diffraction data (see Figure S1), confirming the formation of highly pure phases. The two pure polymorphs present distinctive PXRD patterns as well. Form α shows characteristic peaks at 2θ 10.2°, 11.4°, 13.3°, 15.4°, 23.9°, and 26.4°, which conforms to the reported 4961

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patent.27 It is worth mentioning that form γ was obtained with a remarkably high degree of crystallinity and showed sheet-like morphology. Therefore, the PXRD pattern presents only three distinctly strong peaks at 2θ 6.6°, 13.1°, and 19.7° due to the preferred orientation. To alleviate the orientation effects in PXRD, we ground the powder sample of form γ; however, this procedure only resulted in amorphous material. The simulated PXRD data are consistent with the experimental data. TGA analysis indicates a negligible mass loss prior to decomposition for both forms α and γ, confirming that there is no solvent or water molecule involved. However, a dehydration process is observed for MH1 after 30 °C (see Figure S2). The experimental weight loss from TGA of MH1 is determined to be only 3.1%, corresponding to 0.5 equiv of water (theory: 6.2%, with respect to the mass of the monohydrate). This may be attributed to the unstable nature of MH1. Half the amount of water was already lost during the equilibrium period before TGA measurement started. Characterization of the different solid forms by Raman spectra and FTIR are given in Figures 3

structures of baicalein obtained in this work and previous work,26 it is observed that a repeating intramolecular O−H···O H-bonding interaction between the carbonyl group and the adjacent phenolic substituent is present. However, the arrangement of the molecules in three dimensions in the crystal lattice reveals various salient features. Crystallographic data and refinement details are summarized in Table 1. The parameters of H-bonding in different solid forms are summarized in Table S2. The crystallization of baicalein from mixed solvents of THF and toluene gave plate-shaped crystals of form γ. The crystal structure reveals that form γ crystallizes in the Pc space group, with two molecules in the asymmetric unit, while form α crystallizes with only one molecule in the asymmetric unit. As shown in Figure 4, baicalein molecules in form γ are connected together via OH···OC H-bonding between the carbonyl group on the C ring and the 5-substitution of the phenol moiety on the A ring (O4′H···O2 distance of 2.674 Å and O4H···O2′ distance of 2.720 Å). The above-mentioned Hbonding network is extended along the c axis to give a onedimensional infinite chain structure. The adjacent chains are held together in parallel through weak H-bonding C12H7··· O5′ (distance of 3.390 Å), leading to a two-dimensional layer in the (010) plane. The layers are further stacked in three dimensions adopting a herringbone packing arrangement, stabilized by π···π interactions between adjacent layers along the b axis (π···π interaction parameters are listed in Table S3). However, the interaction mode in form α is completely different. Except for the π···π interaction, a three-dimensional H-bonding network is involved. Every baicalein molecule is involved in two different types of intermolecular H-bonding interactions: (1) two molecules of baicalein form hydrogen bonded dimers through a R22 (8) graph set motif (O5H···O4 distance of 2.806 Å) and 2) the carbonyl group forms additional H-bonding interactions with a phenol moiety through O4H···O2 (distance of 2.626 Å) to connect dimers. Viewed along the b axis, the dimers are aligned in parallel and stacked together through π···π contacts (π···π interaction parameters are also listed in Table S3). Single crystal of MH1 was obtained through evaporation under room temperature, while MH2 was serendipitously discovered when samples of MH1 were placed under 4 °C for 2 months. The water molecule of MH1 is in a DA (D: donor; A: acceptor) environment, i.e., it is involved in two distinctive intermolecular H-bonding interactions, while in MH2 the water molecules attain optimal stability by a DDAA H-bonding pattern, i.e., each water molecule acts as two H-bonding donors and two H-bonding acceptors to interact with four baicalein molecules.28 MH1 and MH2 display completely different packing arrangements (Figure 5). In MH1, baicalein molecules recognize each other via OH···OC H-bonding (distance of 2.716 Å) between the carbonyl group on the C ring and the phenolic group on the A ring, leading to a zigzag chain structure (colored in green and red). Two adjacent chains are held together by water molecules acting as a bridge, engaging in strong O1SH2S···O4 and O4H···O1S H-bonding (distance of 2.887 and 2.797 Å, respectively). Two water molecules and two baicalein molecules take turns to form a tetrameric R44 (8) supramolecular heterosython. However, there is no interaction between the adjacent water molecules. Looking along the a axis, MH1 exhibits rectangular channels occupied by water molecules (Figure 9). In MH2, the water molecules are isolated and engaged in four H-bonding involving four different

Figure 3. Raman patterns of baicalein (a) form α, (b) form γ, (c) MH1, (d) MH2, and (e) HH.

and S3, respectively. As shown in Raman spectra, form α and form γ present a distinguishable difference in the carbonyl C O stretching band, with form γ at 1673 cm−1 and form α at 1655 cm−1. Furthermore, the broad peak at 1386 cm−1 in form γ undertakes a peak splitting in form α, and obvious differences can be further identified from the peaks in the range of 1195− 1220 cm−1 corresponding to C−O stretching vibration. Similar differences have also been discovered in the polymorphic hydrate forms MH1 and MH2, e.g., a slight red shift of the carbonyl CO stretching band is observed for MH1 (at 1655 cm−1) when compared with MH2 (1663 cm−1). For the FTIR spectra, the most obvious difference for form α and form γ lies in the region of 3300−3500 cm−1 corresponding to hydroxyl O−H band stretching. MH1 and MH2 show significant differences in the region of 1200−1500 cm−1. However, the high similarity in both the FTIR spectra (Figure S3) and Raman spectra (Figure) indicates similar H-bonding interactions of MH1 and HH. Single Crystal Structures. Baicalein has three phenolic hydroxyl groups and a carbonyl group that can take part in Hbonding. The rotation of the single bond C1−C10 (Scheme 1) could provide conformational flexibility. From single crystal 4962

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Table 1. Crystallographic Data for Various Modifications of Baicalein Formula Crystal system Space group Temperature (K) a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) V (Å3) DCal (g/cm3) Z λ (Mo Kα) Independent reflns. S Rint R1 wR2 a

form αa

form γ

MH1

MH2

HH

C15H10O5 monoclinic P21/c 100(2) 7.7707 13.2373 11.5427 90 101.027 90 1167.2 1.538 4 0.71073 0.0230 0.0330 -

C15H10O5 monoclinic Pc 296(2) 13.562(2) 3.8415(7) 22.846(4) 90 93.241(7) 90 1188.3(4) 1.510 2 0.71073 5243 1.123 0.0536 0.0821 0.1045

C15H10O5·H2O monoclinic P21/n 296(2) 3.7722(18) 19.907(9) 16.748(9) 90 95.43(4) 90 1252.1(11) 1.529 4 0.71073 2112 1.035 0.0532 0.0709 0.2551

C15H10O5·H2O triclinic P1̅ 296(2) 7.8486(13) 9.7046(16) 10.4672(19) 104.171(10) 110.512(10) 107.425(10) 655.6(19) 1.460 2 0.71073 3006 1.040 0.0236 0.0418 0.1283

C15H10O5·0.5H2O monoclinic P21/c 296(2) 3.8097(2) 19.9030(11) 16.2375(8) 90 97.830(4) 90 1219.7(11) 1.515 4 0.71073 2165 0.976 0.1377 0.0602 0.1334

Data of form α are extracted from CCDC 155914.

Figure 4. (a) 2-D H-bonding packing of form γ on the ac plane (left) and bc plane (right). (b) H-bonding interactions of the baicalein molecule in form α (left) and packing on the ac plane (right).

subtle differences in the molecular packing between the two polymorphic monohydrates, Hirshfeld two-dimensional fingerprint plots are presented in Figure S4. Both MH1 and MH2 show spikes representative of strong H-bonding. Generally, more H-bonding interactions result in a closer packing mode. However, MH2, with more and stronger H-bonding interactions, has smaller packing coefficient and density (packing coefficient = 74.2%, density = 1.460 g/cm3) than

baicalein molecules (Figure 5): two as donor to the carbonyl oxygen (O1sH1s···O2 distance of 2.736 Å) and 5-substituent of phenol oxygen (O1sH2s···O4 distance of 2.841 Å), two as acceptor from the 5- and 6-substituent OH groups (O4H3··· O1s distance of 2.723 Å and O5H4···O1s distance of 2.647 Å). Unlike MH1, every baicalein molecule in MH2 is separated from the others by water molecules. Such an interaction mode results in a network structure in Figure 5 (right). To perceive 4963

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Figure 5. Channel waters in MH1 and isolated waters in MH2.

Figure 6. Solid-state transformation of form γ to form α upon heating, monitored by (a) VT-PXRD and (b) VT-Raman.

those of MH1 (packing coefficient = 77.8%, density = 1.529 g/ cm3). This fact may be explained by the observation in 2-D plot that MH2 has more dispersed H−H interactions, with (di + de) larger than that of MH1, thus leading to a relatively loose packing. Based on the crystal structures and the H-bonding formation between water and baicalein molecules, MH1

belongs to a characteristic channel hydrate, whereas MH2 is an isolated hydrate form. Polymorphic Transformation between Form α and Form γ. HSM can be used to directly observe real-time change in crystals during the heating process, such as morphology, transparency, and color.29 As shown in Figure S5, the flakeshaped crystal of form γ was found to transfer to form α at 4964

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rate, e.g., lower than 30 °C/min. As the heating ramp was increased to 100 °C/min or higher, the exothermic peak became increasingly more pronounced and slightly shifted to a higher temperature. The exothermic peak corresponding to polymorphic transformation was observed between 150 and 180 °C. As the sample was further heated, form α melted at onset T of 266.8 °C. Although the exothermic perk was particularly small, it showed a great reproducibility among different sample batches and different scan rates, confirming the exothermic event corresponding to a form transition signal rather than the fluctuation of baseline. Given that there are no other peaks between this exothermal transition point and the melting point, according to the Heat-of-Transition Rule,30 form γ and form α are monotropically related, with form α being the more stable form. This was further verified by slurry experiment, wherein a 1:1 mixture of form γ and form α was slurried in water at 25 °C for 12 h and only form α was recovered after reaching equilibrium. Preparation of a New Hemihydrate form HH through the Single Crystal to Single Crystal (SCTSC) Transformation of MH1. An outline of the hydration and dehydration pathways for MH1 is demonstrated by DVS measurement, whereby the mass uptake is monitored as a function of relative humidity (see Figure 8). The DVS result

approximately 114 °C. This transformation can be identified by the appearance of a much brighter domain emerging from the left corner of the crystal, and the new domain slowly expanded to the rest of the crystal with the increment of temperature. The original light-yellow color of the crystal turned to darkyellow, presumably attributed to changes in the degree of πconjugation in the molecule, resulting in a different optical property. The polymorphic solid-state transition was closely monitored by VT-PXRD and VT-Raman techniques. VTPXRD patterns collected in the range of RT to 140 °C are shown in Figure 6a. The sample was held for 5 min at each temperature step and then submitted for PXRD analysis. It is evident that the position of the peaks of form γ remains intact over the temperature range from 25 to 120 °C. After 120 °C, additional peaks at 2θ = 11.4°, 22.9°, 23.9°, and 26.4° start to appear. These new peaks were identified as the characteristic peaks of form α, indicating that the phase transformation was underway. As the temperature increased, characteristic peaks of form γ at 2θ = 6.6°, 13.1°, and 19.7° were gradually weakened while the new peaks were strengthened. At 150 °C, the characteristic peaks of form γ disappeared, indicating the complete transformation to the thermodynamically more stable form α. Raman spectroscopy has a prominent role in solid-state characterization as it provides spectra that can be considered a molecular structure fingerprint. As expected, clear changes can be observed during the heating process of pure form γ crystals (see Figure 6b). Specifically, the Raman signal corresponding to carbonyl CO stretching vibration was observed at 1673 cm−1 for form γ. When heated to 145 °C, an obvious red shifting for carbonyl CO vibration (1655 cm−1) was observed, which can be attributed to a stronger H-bonding formation of the carbonyl group in form α. From the single crystal structure data, a much shorter H-bonding length of the carbonyl group, illustrated by O4H···O2, was observed in form α (2.626 Å) compared with that of form γ (2.720 Å). To further explore the nature of phase transformation between the two pure polymorphic forms, hyper DSC was also employed. The DSC results obtained for form γ at different scan rates are summarized in Figure 7. There was almost no thermodynamic event that can be identified before the melting endotherm when the sample was scanned at a normal heating

Figure 8. DVS traces of MH1 and MH2 at 25 °C.

shows stoichiometric hydration and dehydration patterns for MH1. Initially, the powder MH1 sample was equilibrated at 0% RH for an extended amount of time, and then subjected to 0− 95−0−95% RH cycles. It is clear that the starting MH1 sample is totally dehydrated after being equilibrated under 0% RH, and then readily absorbs water upon increasing relative humidity. When RH is increased to 45%, the mass of absorbed water reaches a plateau and is determined to be approximately 3.1%, corresponding to ca. 0.5 molecules of water per molecule of baicalein. Then, a second stage of water absorption occurs, indicated by a steep increase in water uptake at approximately 50% RH. By approximately 55% RH, the amount of absorbed water (6.1%) is close to ca. 1 molecule of water per molecule of baicalein. As RH is increased further to reach 95%, there is essentially no pronounced increment of water content. The moisture uptake process can be reversed with hysteresis upon subsequently decreasing RH from 95% to 0%. The DVS result clearly verifies the existence and the interconversion pathway

Figure 7. DSC diagrams of form γ under different ramping rates. 4965

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Figure 9. Comparison of the channel waters in MH1 (left) and HH (right).

HH by PXRD. MH2 was found to be stable until 70 °C (see Figure S7). Upon further heating, the crystal of MH2 turned totally black due to the loss of hydrate and formation of amorphous material. The two polymorphic monohydrates showed different dehydration temperatures, wherein MH2 was less prone to dehydration compared with MH1. Ultimately, all the hydrates will result in form α upon complete dehydration. This was also supported by DSC results of MH1 and MH2 (see Figure S8). The two monohydrates have the same melting onset temperature which can be designated to the melting event of form α. The interconversion relationship of various solid forms is presented in Scheme 2, wherein the red

for MH1 and HH. According to the DVS results, a SCTSC transformation experiment was designed and performed by placing MH1 under 45% RH. The SCXRD data show great resemblance in the unit cell parameters of MH1 and HH (see Table 1), with only β angles differing by 2.4°. Both MH1 and HH belong to a characteristic channel hydrate (Figure 9). The framework of the crystal structures is almost unchanged during the dehydration and rehydration process; the only change lies in the content of the highly mobilized channel water. However, further observation of the crystal structure reveals notable conformational differences concerning the dihedral angle between ring B and C of the baicalein molecule. The dihedral angle measured by C2−C1−C10−C15 is determined to be 20.9° and 8.5° in MH1 and HH, respectively (the overlay diagram is presented in Figure 10). Of note, the dehydrated form at 0%

Scheme 2. Schematic Representation Showing Interconversion between These Forms

Figure 10. Overlay of molecules in MH1 (green) and HH (red).

RH may neither be form α nor form γ, because the DVS of both forms α and γ does not show any stages indicating water absorption (Figure S6). Interestingly, the DVS behavior of MH2 is completely different from the moisture uptake behavior of MH1. The DVS diagram of MH2 shows only approximately 1% moisture uptake at a humidity range up to 95% RH, which is much less hygroscopic than the polymorphic MH1. This distinctive moisture sensitivity may be directly attributed to the differences in the crystal structures and water properties between polymorphic hydrate forms MH1 and MH2 (channel and isolated hydrate). Interconversion between Different Forms of Baicalein. Both HSM and VT-PXRD were applied to investigate the dehydration process of the two polymorphic monohydrates during the heating process. HSM photography shows that MH1 started to dehydrate and turned partially opaque after being heated to 30 °C. The resulting material was confirmed to be

arrow is representative of the SCTSC transformation process from MH1 to HH. Relative humidity plays an important role in the conversion pathway of MH1 and HH, and the critical moisture content is found to be in the range of 45−55% RH. The newly discovered anhydrous form γ is in monotropic relationship with form α. Powder Dissolution of Form α and Form γ. A drug must be dissolved before it can be absorbed; therefore, improving the 4966

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elaborated in detail through comparison of their crystal structures. Moreover, bulk access to the pure anhydrous form γ was achieved, and thermodynamic stability studies show that form γ and form α are monotropically related, with form α being more stable. In addition, powder dissolution and stability experiments were performed and the results are compared with the marketed form α. The newly discovered form γ exhibits a larger apparent solubility and acceptable stability, suggesting a promising solid form for formulations of baicalein.

dissolution rate of poorly water-soluble drugs can be significant for pharmaceutical development. Because HH and MH1 are unstable forms under ambient temperature and are easily converted to form α, while MH2 cannot be prepared on a large scale, the powder dissolution rate was only conducted on form α and form γ. Figure 11 shows the dissolution profiles of both

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b00858. Additional data and figures (PDF) X-ray crystallographic data (CCDC 1405657) (CIF) X-ray crystallographic data (CCDC 1405658) (CIF) X-ray crystallographic data (CCDC 1405659) (CIF) X-ray crystallographic data (CCDC 1405660) (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; fax: +86-21-50807088; tel.: +86-21-50800934.

Figure 11. Powder dissolution profiles of baicalein polymorphs.

Notes

pure forms under pH 2.0 and pH 4.5 buffer solution with the presence of 0.5% Tween 80. The figure demonstrates that the apparent solubility of form γ is approximately 2.5 times larger than that of form α under the same dissolution condition. In particular, the higher concentration can be maintained for a considerable duration of time (at least 3 h). The amount dissolved for form γ seems to be reduced over time following a maximum at 60−80 min. This indicates that the undissolved solid in the sample was converted to the stable form α, which has lower solubility. This trend is also indicative of the relative thermodynamic stability of the two polymorphic forms. The higher solubility corresponds to lower stability. Commonly, the most stable polymorphic modification is used in a marketed formulation, because any other metastable polymorphs are likely to transform to the most stable one. However, there are cases where an unstable form was deliberately selected for better solubility if the metastable form fulfills the prerequisition of registration stability. In the present case, a stability study performed for form γ at 40 °C and 75% RH indicates no form change for form γ after 4 weeks under stressed conditions (see Figure S9). In summary, the superior solubility and relative stability of form γ encourage better bioavailability of baicalein and thus a new promising solid form for formulation.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Grants 81273479 and 81402898) and the Shanghai Institute of Materia Medica New-Star Plan B for funding.

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REFERENCES

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CONCLUSIONS Water is the most common solvent widely employed in the API’s manufacture process and formulation. Moisture is an almost unavoidable impurity in the finished drug product. The extra water content may lead to a solid-state form transformation during processing or the storage period. Fully investigating the polymorphic behavior of various hydrates and pure forms is very important to ensure the desired forms and drug quality. Four new solid forms of baicalein, including an anhydrous form (γ) and three hydrates (MH1, MH2, and HH), were obtained. The single crystal structures of these forms were reported in this work. The conditions for transformation among these phases were clearly revealed. Interestingly, SCTSC transformation occurs between MH1 and HH, which is 4967

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