Cocrystals of Baicalein with Higher Solubility and Enhanced

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Cocrystals of Baicalein with Higher Solubility and Enhanced Bioavailability Bingqing Zhu,†,‡ Qi Zhang,† 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 ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China S Supporting Information *

ABSTRACT: Baicalein (Bai) is one of the most important bioactive flavonoids isolated from the well-known traditional Chinese medicine called “Huang Qin”. Though it has broad therapeutic capability, the bioavailability is limited due to its poor solubility. In this study, we aimed to modulate its solubility through cocrystallization. Cocrystals of Bai with isoniazide (Inia), isonicotinamide (Inam), caffeine (Caf), and theophylline (Tph) were obtained. And different cocrystallization methods lead to different cocrystal phases for Inam and Tph. These cocrystals were characterized using powder X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, dynamic vapor sorption, and Fourier transform infrared spectroscopy. Among all the cocrystals studied, BaiCaf is found to be superior in powder dissolution and pharmacokinetic behavior. Area under the curve values of BaiCaf is improved by a factor of 4.1, and the bioavailability of baicalein is thus expected to be accordingly increased. Given that Caf is a central nervous system stimulant available in many prescription and nonprescription medications, BaiCaf can be a promising alternative solid form of Bai to be developed.

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INTRODUCTION Baicalein (5,6,7-trihydroxyflavone, Bai) (Scheme 1), one of the most important bioactive flavonoids isolated from the root of

vivo administration studies suggest that Bai is directly absorbed through small intestine and circulates in the bloodstream as its conjugated metabolites, baicalin.13 However, its poor water solubility (16 μg/mL) results in its low oral bioavailability (absolute bioavailability: 36.1% ± 4.4%) and limits its use in the pharmaceutical field.13 Considering its notable pharmacological activity, researchers have been motivated to improve its solubility and dissolution rate by all means to better develop this drug, such as formation of cyclodextrin (CD) complex,14 solid dispersion,15 and cocrystal with nicotinamide.16,17 The Bai solubility of cocrystal can be increased by 50−100% from pH 3.6 to 6.8 by forming a cocrystal with nicotinamide. In our previous study, the solid form landscape has been comprehensively studied, and four novel solid forms including a new pure form (γ), a hemihydrate (HH), and two polymorphic monohydrate modifications (MH1 and MH2) were discovered.18 However, a large number of literature reports on solubility or dissolution of polymorphs reveal that the solubility ratio of different polymorphs is typically less than 2,19 which is also the case for Bai polymorphs in our previous study. Cocrystals, defined as “crystalline single phase materials composed of two or more molecular and/or ionic compounds generally in a stoichiometric ratio which are neither solvates nor simple salts”,20 hold tremendous potential to modulate solubility to a larger extent. There have been literally hundreds

Scheme 1. Chemical Structures of Bai and Coformers

Sccutellaria baicalensis, has been demonstrated to exhibit diverse pharmacological activities, such as anticancer,1−3 anti-inflammatory,4 antibacterial,5 anti-HIV,6 and antiadipogenic activities.7 Apart from this, recent studies indicate that Bai is also a promising therapeutic agent for the treatment of devastating disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).8−10 Moreover, in the Chinese Pharmacopoeia, it has been described as a medicine for the treatment of fever, sore throat, and upper respiratory tract infection.11,12 The in © XXXX American Chemical Society

Received: December 19, 2016 Revised: February 20, 2017 Published: February 27, 2017 A

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Table 1. Crystallographic Data for Five Cocrystals of Bai 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

BaiInam·MeOH

BaiInam

BaiCaf

BaiTph·MeOH

BaiTph·H2O

C43H36O13N4 orthorhombic Pna21 100(2) 30.007(3) 16.7630(17) 3.7377(5) 90 90 90 1880.1(4) 1.443 2 0.71073 3305 1.044 0.1139 0.0761 0.2118

C27H22O7N4 monoclinic P21/n 100(2) 6.1137(3) 19.6757(9) 19.9136(9) 90 93.638(3) 90 2390.61(19) 1.429 4 0.71073 5371 1.018 0.0276 0.0439 0.1190

C38H30O12N4 monoclinic P21/c 100(2) 24.669(3) 4.5593(5) 28.267(3) 90 90.320(8) 90 3179.2(6) 1.535 4 0.71073 5590 0.954 0.1624 0.0586 0.1444

C31.5H34O11.5N8 monoclinic P21/c 100(2) 7.1154(6) 30.411(3) 15.5733(12) 90 102.708(5) 90 3287.3(5) 1.432 4 0.71073 5763 0.975 0.0550 0.0748 0.2135

C29H32O12N8 monoclinic P21 100(2) 6.7290(5) 14.9229(9) 15.1048(10) 90 97.145(4) 90 1504.99(18) 1.511 2 0.71073 6514 0.974 0.0489 0.0625 0.1660

special emphasis on improving its solubility is presented. Most notably, powder dissolution profiles show that BaiCaf exhibits a higher solubility than other cocrystals and crystalline Bai, which leads further to greatly improved bioavailability.

of prominent cocrystal cases on the improvement of dissolution and even bioavailability of active pharmaceutical ingredients (API).21−28 Because the physiochemical enhancement of API is largely dependent on the available coformer, it becomes possible to optimize the solubility of API by deliberate selection of the coformer. This approach is specifically suitable for those APIs which lack ionizable functional groups. The API and coformers are generally held together via noncovalent interactions such as hydrogen bonds, π−π interactions, and halogen bonds, in a crystalline lattice. The principle of crystal engineering and supramolecular synthon serves as a guide to cocrystal design. The key functional group commonly seen in flavonoids, i.e., phenols, underpin robust supramolecular synthons in crystal engineering.29 More than one cocrystals were commonly observed in various flavonoids, such as fisetin, myricetin, quercetin, and luteolin.25,29−31 Moreover, many of these cocrystals presented superior dissolution behavior and even bioavailability compared with the flavonoids. These previous works inspire us to design and prepare cocrystals of Bai, aiming to give better solubility and bioavailability. In addition, according to the FDA’s draft guidance on August 16, 2016, a pharmaceutical cocrystal has a regulatory classification similar to that of a polymorph of the API. It will be treated as a fixed-dose combination product and not a new API. This reclassification offers great opportunity for advancing the development of a pharmaceutical cocrystal.32 On the basis of a myriad of cocrystal structures of other flavonoids,25,29−31 we can see that the O−H···Narom heterosynthon can be the most competitive synthon in cocrystallizing phenolic compounds. Therefore, some specific coformers with aromatic N atoms were chosen for cocrystal construction (Scheme 1), and various cocrystallization methods were utilized for preparation. Ultimately, crystallization attempts resulted in six new Bai cocrystals, namely, baicalein−isoniazide (1:1) (BaiInia), baicalein−isonicotinamide (1:1) methanol solvate (BaiInam·MeOH), baicalein−isonicotinamide (1:2) (BaiInam), baicalein−cafffeine (2:1) (BaiCaf), baicalein−theophylline (1:2) methanol solvate (BaiTph·MeOH), and baicalein− theophylline (1:2) trihydrate (BaiTph·H2O). Comprehensive analysis of their structures and physicochemical properties with

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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 a purity greater than 98%. All analytical-grade solvents were purchased from Sinopharm Chemical Reagent Company and were used without any further purification. Preparation of BaiInia. Bulk powder sample of BaiInia was synthesized using the reaction crystallization method. Inia was added to 4 mL of methanol solvent to give a saturated solution, and the excess solid was removed by filtration. Then, an excess amount of Bai was added to the saturated solution. The resulting suspension was allowed to react 48 h at room temperature and was then subjected to centrifugation. The cocrystal solid was harvested and dried in a vacuum overnight. Unfortunately, the single crystal of BaiInia could not be obtained. Preparation of BaiInam·MeOH. The preparation method is same as that of BaiInia. Single crystal of BaiInam·MeOH was successfully obtained by slowly evaporating the final filtrate. Preparation of BaiInam. Both bulk sample and single crystal of BaiInam were prepared by a liquid diffusion method. A 1:2 stoichiometric ratio of Bai (0.2 mmol, 54.0 mg) and Inam (0.4 mmol, 48.8 mg) was dissolved in 2 mL of THF solution. The solution was transferred to the bottom of a tube and 10 mL of hexane was slowly added along the tube wall. After 5 days, yellow prismatic crystals with good quality for single crystal structure determination were grown on the tube wall. Preparation of BaiCaf. The preparation method is same as for BaiInia. A single crystal of BaiCaf was successfully obtained by slowly evaporating the final filtrate. Furthermore, the BaiCaf cocrystal can be easily prepared on a large scale. 38.8 g of Caf and 107.4 g of Bai (molar ratio in 1:2) were slurried in a 1 L saturated methanol solution of BaiCaf for 24 h. After filtration and drying in a vacuum for 48 h, 142 g of BaiCaf was obtained with a yield up to 97.1%. Furthermore, the saturated filtrate can be used repeatedly in the next batch. Preparation of BaiTph·MeOH. The preparation method is same as for BaiInia. A single crystal of BaiTph·MeOH was successfully obtained by slowly evaporating the final filtrate. Preparation of BaiTph·H2O. The preparation method is same as for BaiInia, except for using the mixture solvent of acetone and H2O B

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(1/1, v/v) instead of methanol. A single crystal of BaiTph·H2O was successfully obtained by slowly evaporating the final filtrate. 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 were set to 40 kV and 40 mA, respectively. Data over the range 3−40° 2θ were collected with a scan rate of 0.1 s/step at ambient temperature. 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 carried out at 100(2) K on a Bruker Apex II CCD diffractometer using Mo−Kα radiation (λ = 0.71073 Å). Integration and scaling of intensity data were performed using the SAINT program. Data were corrected for the effects of absorption using SADABS. The structures were solved by direct method and refined with full-matrix least-squares technique using SHELX-2014 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. 1522877−1522881 for BaiTph·H2O, BaiCaf, BaiInam·MeOH, BaiInam, and BaiTph·MeOH, respectively. Crystallographic data and refinement details are summarized in Table 1. Thermogravimetric Analysis (TGA). TGA was carried out on Netzsch TG 209F3 equipment. 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 nitrogen gas flow of 20 mL min−1 purge. Ground samples weighing 3− 5 mg were heated in sealed nonhermetic aluminum pans at a heating rate of 10 °C min−1. Two-point calibration using indium and tin was carried out to check 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 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 (FT-IR) 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. 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 BaiInam·MeOH, BaiInam, BaiCaf, BaiTph·MeOH, and BaiTph· H2O were focused under the microscope (10×). Powder Dissolution. To minimize the size effect on dissolution results, Bai and four edible cocrystals, BaiInia, BaiInam, BaiCaf, and BaiTph·H2O were sieved through 100-mesh sieves. Accurately weighed powders of (or corresponding to, for cocrystals) 5 mg of Bai (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 100 rpm at 37 °C. Sampling was performed at 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 120, 150, 180, 240, 300, and 360 min. The withdraw suspensions were filtered with 0.22 μm PTFE filters prior to HPLC analysis. Pharmacokinetic Experiments. To evaluate the efficacy of cocrystallization for modulating the pharmacokinetics of Bai, BaiCaf bulk samples were utilized to conduct the pharmacokinetics experiments. Bai, BaiCaf cocrystal and physical mixture (PM) of Bai and Caf were sieved through 100-mesh sieves. The obtained samples were then dispersed homogeneously in 0.1% CMC-Na (sodium carboxymethylcellulose) aqueous solution to get the suspension of 12.10 mg/mL of Bai. The experimental protocol was

approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Materia Medica for the use of experimental animals and conformed to the Guide for Care and Use of Laboratory Animals. Eighteen male Sprague−Dawley rats weighing 220−250 mg were randomly allocated into three groups (6 rats in each group). Each received gavage administration at a dose of 121 mg/kg body (expressed as Bai equivalents). Taking the caffeine toxicity into account, it will amount to a dose of 43.5 mg/kg in rates and 7 mg/kg in human. At this dose, it will not raise safety concerns either in animals or in the general population, according to FDA’s Select Committee on GRAS Substances (SCOGS) Opinion.33 The rats were allowed water and libitum and fasted overnight before drug administration. After administration, about 200 μL of blood sample was collected from orbital sinus into heparinized tubes at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 h from each rat. Plasma was immediately separated by centrifugation (10 °C, 10000g, 5 min) using a refrigerated table top centrifuge and kept frozen at −20 °C until analysis. To accurately quantify the concentration of Bai in the plasma, a modified method based on previously reported work was employed using high performance liquid chromatography. In brief, frozen plasma samples were thawed at room temperature before sample preparation. 100 μL of rat plasma was mixed with 15 μL of luteolin solution (internal standard, 48.86 μg/mL in methanol) and 50 μL of potassium dihydrogen phosphate buffer (0.5 M, containing 1% sodium ascorbate). After vortexing for 2 min, the plasma samples were extracted with 150 μL of acetonitrile by vortex-mixing for 5 min and centrifuged for 10 min (10 °C, 10000g). Because baicalein-7-Oglucuronide (BG) is the main active metabolite of Bai and the mainly existing form in plasma, BG was provided for statistical comparison and bioavailability calculation. The concentrations of BG were simultaneously determined by Agilent Eclipse Plus C18 column (250 × 4.6 mm, 5 μm) equipped in Agilent 1260 HPLC and detected at 276 nm. The mobile phase containing methanol and 0.2% phosphoric acid solution (63/37, v/v) was run at 1.0 mL/min. An injection of 20 μL was performed, and the column temperature was set at 30 °C. The concentration range of the standard curve was 0.05 μg/mL to 8.0 μg/ mL of BG. The results indicated that the standard curve performance was within acceptable range for bioanalytical method acceptance (R2 > 0.99). Pharmacokinetic analysis was performed by means of a model independent method using a DAS 2.0 computer program. All results were expressed as mean ± SD.

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RESULTS AND DISCUSSION On the basis of the hydrogen bonding possibilities of Bai and the competitiveness of O−H···Narom heterosynthon, appropriate coformers (Scheme 1) were selected for cocrystallization. Various crystallization methods were used, including slurry, evaporation, and antisolvent. And a wide set of solvents were applied in each crystallization method. As a result, cocrystals with Inia, Inam, Tph, and Caf were all formed. Interestingly, two cocrystallization phases for the combination of Bai and Inam were obtained using different crystallization methods. The 1:2 cocrystallization phase (BaiInam) can only be isolated by an antisolvent method (THF as good solvent and Hex as poor solvent), while other methods using methanol as solvent exclusively led to a 1:1 methanolic solvate cocrystal (BaiInam· MeOH). In addition, two different solvated cocrystals, BaiTph· MeOH and BaiTph·H2O, were obtained using methanol and water as crystallization solvents, respectively. PXRD analysis was applied to identify the cocrystalline phases of Bai (Figure S1). The patterns of the products are different from that of Bai and those of the corresponding coformers. And 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, confirming the formation of highly pure cocrystal phases. Single crystals suitable for X-ray C

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Figure 1. (a) Two independent Inam molecules form a hydrogen bonded homodimer. (b) View of a sheet motif in BaiInam.

Figure 2. (a) Side view of Inam molecules forming an infinitely right-hand helical chain structure. (b) Packing diagram viewed along the c axis, with channel methanol molecules in a space-filled mode.

Experimental Section). Crystallographic data are summarized in Table 1, and hydrogen bond parameters are listed in Table S1. To make information about some structural parameters clearer and understandable, asymmetric units and atom numbering schemes of all five cocrystals are presented in Figure S4. Bai has three hydroxyl substituents on the benzopyran ring, wherein hydroxyl atom O3 of each Bai molecule exclusively acts as a Hbond donor in an intramolecular H-bonding interaction (O3− H...O2). The remaining two H-bond donors are involved in intermolecular H-bonding interactions with either coformers or solvents in the cocrystal structures. Except for BaiTph·MeOH and BaiTph·H2O, the robust heterosynthon O−H···Narom is embedded in the other three cocrystal structures as we have expected. Bai and Inam cocrystallized in two different phases, namely, BaiInam and BaiInam·MeOH. The asymmetric unit of

diffraction were all obtained except for BaiInia, whose stoichiometric ratio of Bai and Inia (1:1) can only be confirmed by 1H NMR analysis (Figure S2). Interestingly, except for BaiCaf, single crystals of the other four cocrystals presented similar crystal habits and mechanical behavior upon application of stress. As shown in Figure S3, crystals of BaiInam, BaiInam·MeOH, BaiTph·MeOH, and BaiTph·H2O show prismatic morphology. When stress was applied on them, the crystals broke into pieces showing their brittle nature, with the exception of BaiCaf. It crystallized as extremely long needle crystals having about 1 cm length, which is remarkably flexible and shows elastic bending when applying mechanical stress. Single Crystal Structures. Single crystals of BaiInam· MeOH, BaiCaf, BaiTph·MeOH, and BaiTph·H2O were obtained by means of solution evaporation approach, while BaiInam was isolated by the liquid diffusion method (see D

DOI: 10.1021/acs.cgd.6b01863 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Figure 3. (a) Top view of right-/left-hand helical chains, R is representative of right-handed and L is representative of left-handed. (b) Packing diagram viewed along the b axis, with the basic building block hexamer highlighted in green.

Figure 4. (a) Intermolecular interaction environment of the water trimer. (b) Side view of columnar assembly, where water molecules serve as a bridge to connect Bai and Tph dimers together. (c) Packing diagram viewed along the a axis, with the columnar assembly highlighted in green.

of 2.994 Å) to form a sheet motif (Figure 1b). The driving force for a three-dimensional network is dominated by π···π interactions. BaiInam·MeOH crystallizes in the Pna21 space group and contains in the asymmetric unit one Bai, one Inam, and one methanol molecule. Inam molecule adopts an almost planar conformation, with the torsional angle O6−C21−C18− C19 being only 4.24°. Because of the inclusion of methanol molecules in the crystal lattice, the crystal structure and packing motif are obviously different from that of BaiInam. Inam molecules in BaiInam·MeOH are connected through N2− H2A···O6 (distance of 2.885 Å), giving an infinitely right-hand helical chain structure instead of a homodimer in BaiInam (Figure 2a). The remaining H-bond donor in the secondary amine of Inam is engaged in intermolecular H-bonding with Bai molecule via N2−H2B···O2 (distance of 2.885 Å). Besides, the pyridine N1 acts as a H-bond acceptor with vicinal diols of Bai molecule via O5−H5···N1 (distance of 2.668 Å) and O4−

BaiInam contains one Bai and two independent Inam molecules. The conformation of Inam molecule is expressed in the torsion angle of amide group and its pyridyl ring plane. The two independent Inam molecules adopt quite different conformations, wherein the O6−C21−C18−C17 torsion angle in InamA is 23.74° while in InamB it is 47.22° (O7−C27−C24− C23 torsion angle). The two independent Inam molecules form a dimeric R2 2(6) homosynthon via N4−H4A···O6 (distance of 2.918 Å) and N2−H2A···O7 (distance of 2.851 Å) hydrogen bonds (Figure 1a). Two available hydroxyl substituents on the benzopyran ring in the Bai molecule act as H-bond donors to connect Inam dimers through O4−H4···N1 (distance of 2.660 Å) and O5−H5···N3 (distance of 2.687 Å) hydrogen bonds, extending into zigzag chains running along the b axis (highlighted in green and blue in Figure 1b). Adjacent chains are further interconnected in an antiparallel fashion through N2−H2B···O2 (distance of 2.946 Å) and N4−H4B···O5 (distance E

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Figure 5. (a) Side view of the columnar assembly, where methanol molecule O10 serves as a bridge to connect Bai and Tph dimers together. (b) Packing diagram viewed along the a axis, with the columnar assembly highlighted in green and the channel methanol molecules in space-filled mode.

H4···N1 (distance of 3.027 Å). Thus, adjacent Inam helical chains are held together mediated by Bai molecules (Figure 2b). On the whole, the BaiInam·MeOH cocrystal belongs to a characteristic channel solvate along the c axis, wherein methanol molecule does not participate in sustaining the three-dimensional H-bonding architecture. BaiCaf crystallizes in P21/c space group with one molecule of Caf and two molecules of Bai in the asymmetric unit. Noticeably, the basic building block is a hexamer involving four Bai molecules and two Caf molecules, which is constructed via three distinctive types of hydrogen bonds, O9−H9···O2 (distance of 2.644 Å), O4−H4···O11 (distance of 2.823 Å), and O10−H10···N2 (distance of 2.786 Å). These hexamers are arranged in a head-to-tail (head: atom N2 in Caf molecule, tail: hydroxyl group O10−H10 in Bai molecule) fashion, forming an extraordinarily large left-/right-handed helical chain structures (Figure 3a). On the other hand, the adjacent opposite helical chains are inversion-related by means of O5−H5···O12 (distance of 2.709 Å) hydrogen bond, giving a two-dimensional H-bonding network, which is additionally extended into three-dimensional architecture throughout the crystal structure via relatively weak interactions (Figure 3b). Two different solvated cocrystals, BaiTph·MeOH and BaiTph·H2O, were isolated using methanol and water as crystallization solvents, respectively. The asymmetric unit of both contains two Tph molecules, one Bai molecule, and three water/2.5 methanol molecules. Unlike the three cocrystal structures discussed above, the Bai molecule does not directly interact with coformer molecules. On the contrary, the water/ methanol molecules in BaiTph·MeOH and BaiTph·H2O are indispensable in generating three-dimensional networks. In the case of BaiTph·H2O, three independent water molecules (namely, water O1S, water O2S, and water O3S) are engaged in multiple hydrogen bonds. Water O1S is in a DDAA environment; i.e., it is involved in four distinctive intermolecular Hbonding interactions. The other two water molecules take a DDA H-bond pattern; i.e., each water molecule acts as two Hbond donors and one H-bond acceptor. Specifically, the three independent water molecules form a trimer through O2S− H2SB···O3S (distance of 2.764 Å) and O3S−H3SA···O1S (distance of 2.836 Å) hydrogen bonds (Figure 4a). The remaining potential donor/acceptor sites in water molecules are engaged in intermolecular H-bonding with four Tph molecules and two

Bai molecules (Figure 4a). Analysis of the crystal packing reveals that two Tph molecules form a H-bonded dimer through a R2 2(8) motif (N1−H1···O8 distance of 2.818 Å and N5−H5A···O6 distance of 2.764 Å). Tph dimer and Bai molecule are alternatively stacked mediated by water molecules, wherein on one side water O2S links Tph dimer and Bai molecule (O2S−H2SA···O7 distance of 2.777 Å and O4−H4···O2S distance of 2.678 Å), and on the other side water O1S and water O3S stitch the Tph dimers together (O1S−H1SB···O9 distance of 2.869 Å, O3S−H3SA···O1S distance of 2.836 Å and O3S−H3SB··· O9 distance of 2.964 Å). Thus, a columnar assembly along the a axis is constructed (Figure 4b). Such columns are further extended into three-dimensional fabrication by means of O2S− H2SB···O3S (distance of 2.764 Å), O1S−H1SA···N6 (distance of 2.866 Å), and O5−H5···O1S (distance of 2.782 Å) hydrogen bonds (Figure 4c). The packing arrangement of BaiTph·MeOH is isostructural to BaiTph·H2O, which can be reflected in similar PXRD patterns and cell parameters. Relative to BaiTph·H2O, the a and b axes in the crystal of BaiTph·MeOH are longer for +5.74% and 3.01%, respectively, whereas the c axis is doubled. Thp molecules in BaiTph·MeOH give a dimer through only one hydrogen bond N3A−H3A···N1A (distance of 2.849 Å) instead of a R2 2(8) motif in BaiTph·H2O. Bai molecules and Tph dimers are alternatively stacked together into a columnar assembly (Figure 5a). Similar to the role of water molecule O2S in BaiTph·H2O, methanol molecule O10 serves as a bridge, linking Tph dimer and Bai molecule together on one side. However, unlike BaiTph·H2O, there are no hydrogen bonds between Tph dimers on the other side. The difference between BaiTph·MeOH and BaiTph·H2O may be attributed to the capability of H-bond formation of the solvent molecule, wherein water molecule is more abundant in H-donors/ acceptors compared with methanol molecule. Adjacent columns are further fabricated into three-dimensional network via N 2A −H 2A ···N 4A (distance of 2.823 Å), O5 −H5 ···O 11 (distance of 2.716 Å) and O11−H11A···O6 (distance of 2.766 Å) wherein the methanol molecule O11 plays an vital role (Figure 5b). Unlike BaiTph·H2O wherein the three independent water molecules are all involved in H-bonding, the methanol molecule O12 (in space-filled mode) in BaiTph· MeOH serves as a channel solvent and does not participate in sustaining the H-bonded network. F

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Figure 6. Powder dissolution profiles of Bai and its four cocrystals in (a) pH 2.0 and (b) pH 4.5 buffer solution.

FT-IR Spectroscopy. FT-IR spectra are used to identify the noncovalent interactions within the crystal structures. The FTIR spectra of cocrystals give many characteristic peaks that are different from neither Bai nor the coformers (Figure S5). The signal corresponding to carbonyl CO stretching vibration in the Inia and Inam were blue-shifted to 1693, 1707, and 1692 cm−1 in BaiInia, BaiInam·MeOH, and BaiInam respectively. However, in BaiTph·H2O and BaiTph·MeOH, it was redshifted to 1694 and 1695 cm−1, compared with the signal at 1717 cm−1 in Tph. This can be attributed to the extra intermolecular H-bond interactions of CO with H2O or methanol. Thermal Analysis. Thermal behavior of the six cocrystals was assessed by means of TG, DSC, and VT-PXRD. Analysis of TG plot of BaiInia, BaiInam, and BaiCaf reveals no significant weight loss before the melting event, confirming their unsolvated characteristics (Figure S6). No phase transitions were observed in the course of heating for BaiInam and BaiCaf based on the DSC results (Figure S7). In the case of BaiInia, an additional endothermic peak with the onset temperature of 164.2 °C can be observed before the melting point. Since there is no inclusion solvate, this endothermic peak is attributed to a phase transformation, which can be further verified by VTPXRD results (Figure S8). The sample was held for 10 min at each temperature step and then submitted for PXRD analysis. When heated to 175 °C, additional peaks at 2θ = 5.9°, 10.2°, 11.5°, 11.7°, 15.4°, and 23.9° start to appear. These new peaks are identified as the characteristic peaks of Bai and another solid form of BaiInia (named BaiInia II herein). In BaiInam· MeOH, a gradual weight loss of 7.9% was observed, corresponding to the loss of 1 equiv of methanol (calculated 7.5%). Accordingly, in DSC, an endothermic peak with the onset temperature of 110.4 °C was presented before the melting peak. Furthermore, VT-PXRD was utilized to determine the phase after desolvation (Figure S8). When heated to 90 °C, characteristic peaks of BaiInam at 2θ = 6.3° and peaks of Bai at 2θ = 11.5° appear, indicating that BaiInam· MeOH converts to a physical mixture of BaiInam and Bai after desolvation. The thermal behavior of BaiTph·H2O and BaiTph·MeOH is quite similar. A weight loss of 7.6% corresponding to 3 equiv of water (calculated 7.9%) was shown for BaiTph·H2O, while for BaiTph·MeOH, a weight loss of 10.7% was observed, corresponding to 2.5 equiv of methanol (calculated 11.5%). These desolvation processes are also reflected in DSC results (Figure S7). Both are desolvated at about 100 °C, resulting in a physical mixture of Bai and Tph.

However, during heating of BaiTph·H2O, new peaks at 2θ = 6.3° and 6.5°, which are neither characteristic peaks of Bai nor Tph, appear. It is assumed that the BaiTph·H2O dehydration process occurs in two separate steps, and an intermediate form (named BaiTph·H2O II herein) is detected during dehydration of BaiTph·H2O. This is also indicated in its DSC pattern wherein a small endothermic peak can be observed following the dehydration signal. Hygroscopicity. Outline of hydration and dehydration pathways for six cocrystals were demonstrated by DVS measurement, whereby the mass uptake is monitored as a function of relative humidity (see Figure S9). The analysis of solvated cocrystals, including BaiInam·MeOH, BaiTph·H2O, and BaiTph·MeOH, are initiated at ambient RH, so as to avoid initial drying, which can change the crystal form of the sample. Among six cocrystals, BaiCaf was found to be the least hygroscopic form, the water content of which is only 0.65% at 80% RH, showing the rather optimum hygroscopicity property. BaiInia, BaiInam·MeOH, and BaiInam have essentially no moisture increase between 40−80% RH. The absorption behavior of isostructural BaiTph·H2O and BaiTph·MeOH is quite similar. A distinct desolvation step is shown below 10% RH and 30% RH for BaiTph·H2O and BaiTph·MeOH, respectively. However, the absorption curve of BaiTph·MeOH is irreversible, while BaiTph·H2O can rehydrate and shows reversible absorption curves. In Vitro Powder Dissolution. After delivery, pharmaceutical drugs must dissolve in the intestinal fluid in order to be absorbed into circulation. Poor dissolution behavior will limit the amount of API that is available for absorption. Therefore, improvement of dissolution rate and apparent solubility of Bai is of great value for its application in pharmaceutical field. In this study, BaiInam·MeOH and BaiTph·MeOH are excluded from dissolution experiment since they are inedible. Powder dissolution profiles for Bai and the rest of four cocrystals determined in pH 2.0 and pH 4.5 buffer solution with the presence of 0.5% Tween 80 are shown in Figure 6. Compared to pure Bai, all the cocrystals exhibited faster dissolution rate in the early phase. In particular, BaiInia and BaiCaf exhibited a much larger apparent solubility (Smax), wherein BaiCaf gives the most significant improvement by approximately 2.5-fold and 1.5-fold in pH 2.0 and pH 4.5 buffer solution, respectively. All the cocrystals except for BaiCaf (in pH 2.0 buffer) display a “spring and parachute” effect34 which can be attributed to the dissociation of cocrystals into less soluble Bai in solution. Maintaining supersaturation levels achieved during cocrystal G

DOI: 10.1021/acs.cgd.6b01863 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

dissolution is very important for API absorption. Although the API can also provide the “spring” in a noncrystalline solid, the physical instability to crystallization is a big concern. Cocrystals offer the advantage over noncrystalline solid that this problem may be avoided to an extent. It is interesting to note that the higher concentration of BaiCaf can remain constant for at least 6 h in pH 2.0 buffer, and no “rapid parachute” effect was observed. The remaining solid after dissolution experiment was collected, dried, and analyzed by PXRD. The results of PXRD indicate that the undissolved solid of BaiCaf remains cocrystal phase without dissociation, while others have transformed to Bai (Figure S10). However, the extended supersaturation level of BaiCaf is not observed in pH 4.5 buffer. This may be attributed to the higher supersaturation level delivered by the spring in pH 4.5 buffer, giving more driving force for Bai to crystallization. In Vivo Bioavailability. It is hypothesized that the ability to achieve and sustain a high transient concentration of the drug in aqueous medium has immense potential for improving drug bioavailability. Therefore, an in vivo bioavailability study was performed on BaiCaf. Bai and physical mixtures of Bai and Caf (PM) were also tested for comparison. After oral dosing of Bai, traces of baicalein can be detected in a few samples. Therefore, the pharmacokinetic parameters of baicalein-7-O-glucuronide (BG), which was identified as the main form existed in blood,35 was provided for statistical comparison and bioavailability calculation. The mean plasma concentrations of BG versus time profiles for Bai, PM, and BaiCaf cocrystal are shown in Figure 7. It can be observed that plasma concentration of BG

Table 2. Pharmacokinetic Parameters from the Plasma Concentration of the Crystalline Bai, PM, and BaiCaf Cocrystala Baicalin solid form

Tmax (h)

Cmax (μg/mL)

AUC0−24 (μg h/mL)

Bai PM BaiCaf

0.42 ± 0.14 0.50 ± 0.00 0.67 ± 0.29

4.54 ± 1.12 5.19 ± 1.15 15.21 ± 0.95

17.75 ± 1.17 33.69 ± 2.03 73.48 ± 1.11

a

AUC0−24: the area under the curve (computed starting at the time the drug is administered and ending at 24 h) in a plot of concentration of drug in blood plasma against time.

absorption to occur. It can be found that PM has a modest improvement of Cmax and AUC compared with pure Bai. This indicates that the concomitant Caf may have a synergic effect on the absorption amount of Bai. Additionally, caffeine is classified by FDA as “generally recognized as safe” (GRAS). It is a central nervous system stimulant and is used to reduce physical fatigue and to treat drowsiness. So it is frequently found in some headache and migraine medications, and in many popular energy drinks. And it will not raise safety concerns either in animals or in the general population at that dose, according to FDA’s Select Committee on GRAS Substances (SCOGS) Opinion.33 Therefore, cocrystal BaiCaf can be a promising alternative to pure Bai to improve its efficacy and for further drug development.

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CONCLUSION With the aim of improving solubility of Bai, a natural polyphenolic compound of pharmaceutical interest, cocrystals with isoniazide (Inia), isonicotinamide (Inam), caffeine (Caf), and theophylline (Tph) were prepared. Different cocrystal phases were obtained for Inam and Tph by means of different cocrystallization methods. Except for BaiInia, single crystal structures of all the cocrystals were successfully determined by SXRD. BaiInam·MeOH, BaiInam, and BaiCaf share a common supramolecular motif O−H···Narom, which is not involved in BaiTph·H2O and BaiTph·MeOH due to the presence of solvent in the crystal structure. Physicochemical properties, such as mechanical behavior, thermal stability and hygroscopicity, were characterized by TG, DSC and DVS. Furthermore, powder dissolution studies were conducted for pure Bai and four edible cocrystals, wherein BaiCaf shows approximately 2.5-fold and 1.5-fold enhancement in pH 2.0 and pH 4.5 buffer solution, respectively. And this superior in vitro dissolution behavior is further reflected in its great enhancement in in vivo bioavailability. The AUC of BaiCaf was about 4.1 times that of pure Bai. In conclusion, BaiCaf can be easily prepared on a large scale. It shows great thermostability and low hygroscopicity, and most importantly, superior dissolution property and oral bioavailability. And Caf is considered as “GRAS” for human consumption. The combination of these advantages makes it a promising alternative to pure Bai to be developed in the future.

Figure 7. Plasma BG concentration−time curves of the crystalline Bai, PM, and BaiCaf cocrystal (data are expressed as means ± SD, n = 6).

displayed a second peak over the 4−8 h time interval following the initial absorption. This can be attributed to enterohepatic circulation, which contributes significantly to the exposure of BG in rats. The pharmacokinetic parameters listed in Table 2 are calculated from the concentration of BG. At a single administered dose of 121 mg/kg, BaiCaf cocrystal has a significantly higher Cmax and AUC compared with Bai and PM. On the basis of the AUC0−24h, the relative bioavailability of BaiCaf cocrystal is about 4.1 times that of crystalline Bai. This enhanced bioavailability of cocrystal is correlated with its higher dissolution profile. We can see that the time of supersaturation level in the BaiCaf dissolution profile is long enough for Bai

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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.6b01863. Figure S1. Comparison between experimental and simulated PXPD of cocrystals; Table S1. List of HH

DOI: 10.1021/acs.cgd.6b01863 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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bond lengths and angles for the cocrystals; Figure S2. Comparison of 1H-NMR ((CD3)2SO, 400 MHZ) of BaiInia, Bai, and Inia; Figure S3. (a−d) Brittle deformation of the crystal upon application of a mechanical stress (e) elastic deformation of the crystal upon application of a mechanical stress; Figure S4. Asymmetric units and atom numbering schemes of (a) BaiInam·MeOH, (b) BaiInam, (c) BaiCaf, (d) BaiTph· H2O, and (e) BaiTph·MeOH; Figure S5. FT-IR spectra of six cocrystals and the corresponding coformers; Figure S6. TG profiles of six cocrystals; Figure S7. DSC profiles of six cocrystals and the corresponding coformers; Figure S8. PXRD profiles of (a) BaiInia, (b) BaiInam·MeOH, (c) BaiTph·H2O, and (d) BaiTph·MeOH upon heating; Figure S9. DVS profiles of six cocrystals of Bai; Figure S10. PXRD results of the undissolved solids after dissolution experiment (PDF) Accession Codes

CCDC 1522877−1522881 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.R.W.). *E-mail: [email protected] (X.M.). ORCID

Jian-Rong Wang: 0000-0002-0853-7537 Xuefeng Mei: 0000-0002-8945-5794 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Grant Nos. 81273479 and 81402898), Youth Innovation Promotion Association CAS (Grant No. 2016257), CAS Key Technology Talent Program, and SANOFI-SIBS Scholarship for funding.

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DOI: 10.1021/acs.cgd.6b01863 Cryst. Growth Des. XXXX, XXX, XXX−XXX