Zwitterionic Cocrystals of Flavonoids and Proline: Solid-State

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Zwitterionic Cocrystals of Flavonoids and Proline: Solid-State Characterization, Pharmaceutical Properties and Pharmacokinetic Performance Hongyan He, Ying Huang, Qi Zhang, Jian-Rong Wang, and Xuefeng Mei Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00142 • Publication Date (Web): 09 Mar 2016 Downloaded from http://pubs.acs.org on March 14, 2016

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Crystal Growth & Design

Zwitterionic Cocrystals of Flavonoids and Proline: Solid-State Characterization,

Pharmaceutical

Properties

Pharmacokinetic Performance Hongyan He, Ying Huang, 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 E-mail address: [email protected] (X. F. Mei); fax: +86-21-50807088; tel.: +86-21-50800934

ACS Paragon Plus Environment

and


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ABSTRACT: We utilized the concepts of crystal engineering to acquire cocrystals of D/L-proline with a variety of flavonoids and further evaluated the impact on solubility and pharmacokinetic of flavonoids. We have synthesized and characterized six D/L-proline cocrystals with flavonoids and their single crystal structures were revealed. The powder dissolution profiles were determined and the pharmacokinetic properties for Kae-L-Pro were analyzed and compared with corresponding physical mixture. D/L-proline was found to be a suitable coformer for a variety of structural similar flavonoids. This study may offer an alternative approach for the development of these widely used flavonoids.

KEYWORDS: flavonoid, cocrystals, dissolution, pharmacokinetic.


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INTRODUCTION Flavonoids are naturally occurring polyphenolic compounds that are abundant in vegetables, fruits and products of that origin1 and a large part of attention that flavonoids have attracted is due mainly to their antioxidant, antitumor and anti-inflammatory pharmacological properties.2 Though flavonoid components exhibit a quantity of beneficial health effects in vitro, their promising efficiency is impeded by low solubility, poor bioavailability and first pass metabolism,3 resulting in the decreased potential of possible application in pharmaceutical formulations or nutritional supplements. The dietary flavonoids involving weakly acidic phenolic hydroxyl groups are unlikely to give salt formation with bases at physiological pH, accordingly the strategy of cocrystallization tends to be an encouraging approach to modulate physicochemical properties and the feasibility of this approach has been proved by many cases.4-9 For instance, the bioavailability of quercetin cocrystals is sharply increased compared with pure quercetin.10 Yanting Huang and co-workers have explored baicalein-nicotinamide cocrystal with improved solubility and oral bioavailability.11 Recently cocrystallization has attracted expanding interest in a number of fields because the multicomponent materials can be rationally designed through crystal engineering principle with supramolecular synthon approach. Depending on the ionic state of the components, cocrystals can be divided into molecular cocrystals, ionic cocrystals and zwitterionic cocrystals.12-14 All the time molecular cocrystals are the research spotlight in pharmaceutical cocrystallization field, while zwitterionic



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cocrystals are less explored. In this scenario, amino acids, naturally zwitterions under physiological conditions, could be of interest in formation of zwitterionic cocrystals with dietary flavonoids under the guidance of crystal engineering concept. Proline appears to be a popular cocrystal former because the 5-membered ring “lateral chain” render the molecular structure restricted and rigid.15 The application of proline in cocrystallization has been reported in quite a few cases.6, team-workers

have

revealed

the

representative

of

13, 16-18

proline

Martin and

application

in

semiconductor and suggested the perspective for development of biomaterials with semiconducting properties.17 Tilborg et al. have investigated in detail the structural characteristic of proline cocrystals including naproxen-proline cocrystals and fumaric acid-proline cocrystals, proving out the proline molecules contribute general geometrical and organizational behavior for structures involving zwitterionic proline according to the survey of existing crystal structures in Cambridge Structural Database (CSD).13, 14, 16, 17, 19-25 In this presentation, we utilized the crystal engineering principle to investigate the cocrystallization behavior of zwitterionic proline with three sub-classes of dietary flavonoids, namely flavonols (Quercetin and Kaempferol), flavones (Luteolin, Baicalein and Chrysin) and isoflavones (Genistein) (Scheme 1). Flavonoids are polyphenolic compounds comprising 15 carbons with two aromatic rings connected by a three carbon bridge, hence C6-C3-C6 skeleton, and flavonols, flavones and isoflavones are the main sub-classes of dietary flavonoids. Quercetin and kaempferol are the most widespread distributed dietary flavonols. In fact the cocrystals of


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quercetin have been scrutinized,26 27 where Que-Pro cocrystal has been reported yet the single crystal structure has not been determined. Similarly, the cocrystals of luteolin, baicalein and genistein have also been explored.11,

28-31

In contrast, the

cocrystalline form of kaempferol and chrysin has not been exploited to date and previous research mainly focus on their broad biological properties.32-34 Herein, we have utilized the basic concept of crystal engineering to explore the novel cocrystallization behavior of a series of flavonoids with both D-proline and L-proline. We wish to investigate the influence of APIs’ subtle structural difference on the change of crystal packing and interactions, and hence on the changes of physicochemical properties and pharmacokinetic performance.

Flavonoid skeleton

Quercetin (Que)

Luteolin (Lut)

Baicalein (Bai)

Kaempferol (Kae)

Chrysin (Chr)

Genistein (Gen)



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L-proline (L-Pro)

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D-proline (D-Pro)

Scheme 1. Chemical structures of Que, Kae, Bai, Chr, Lut, Gen, L-Pro and D-Pro. EXPERIMENT SECTION Materials. Quercetin (Que), Kaempferol (Kae), Luteolin (Lut), Chrysin (Chr), Genistein

(Gen),

Baicalein

(Bai),

D/L-proline

(D/L-Pro,

99%),

sodium

carboxymethylcellulose and phosphoric acid were purchased from Aladdin Chemistry Company Limited. Kaempferol-3-O-glucosylside (99.19%, reference standard) was purchased from Shanghai Boylechem Company Limited. All analytical grade solvents were purchased from the Sinopharm Chemical Reagent Company and were used without further purification. Cocrystal preparation methods Quercetin-D/L-proline (1:2) (Que-D/L-Pro). Que and D/L-Pro in a 1:2 molar ratio were dissolved in 95% ethanol at 50 °C and evaporated slowly at that temperature. Yellow thin plate-like crystals suitable for X-ray diffraction were harvested after a few days (yield: 85.5%). Bulk Que-D/L-Pro powder was synthetized by slurrying a stoichiometric amount of Que and D/L-proline in ethyl acetate and ethanol (1:1, v/v). The resulting material was centrifuged and allowed to dry at 50 °C overnight. The identity and purity of the cocrystal were analysed by means of PXRD. Kaempferol-D/L-proline (1:2) (Kae-D/L-Pro). Kae and L-Pro in a 1:2 molar ratio were dissolved in methanol and tetrahydrofuran (1:1, v/v) at 50 °C and evaporated at that temperature. Yellow plate crystals available to X-ray diffraction were acquired


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over a few days (yield: 89.6%). Bulk Kae-D/L-Pro material were produced by slurrying a stoichiometric amount of Kae and D/L-Pro in ethyl acetate and ethanol (1:1, v/v). The resulting material was centrifuged and allowed to dry at 50 °C overnight. The identity and purity of the cocrystal were analysed by means of PXRD. Luteolin-D/L-proline (1:1) (Lut-D/L-Pro). Equimolar amounts of Lut and D/L-Pro were dissolved in 95% ethanol and acetonitrile (1:1, v/v) at 50 °C and evaporated at that temperature. Block-like crystals suitable for X-ray diffraction were obtained (yield: 86.3%). A stoichiometric amount of Lut and D/L-Pro was suspended at 50 °C in ethyl acetate and ethanol (1:1, v/v) and stirred on a stir plate over 24 h. The resulting material was centrifuged and allowed to dry at 50 °C overnight. The identity and purity of the cocrystal were analysed by means of PXRD. Chrysin-D/L-proline (1:1) (Chr-D/L-Pro). The equal molar ratio of Chr and D/L-Pro were dissolved in 95% ethanol, tetrahydrofuran and acetonitrile (1:1:1, v/v/v) at 50 °C and evaporated slowly at that temperature. Rod-like crystals suitable for X-ray diffraction were harvested (yield: 83.7%). Solution-mediated cocrystallization and liquid-assisted grinding (a few drops of ethanol) method were applied to obtain bulk Chr-D/L-Pro cocrystal powders. A stoichiometric amount of Chr and D/L-Pro was suspended in ethyl acetate and ethanol (1:1, v/v) at 25 °C and stirred on a stir plate over 72 h. The resulting material was centrifuged and allowed to dry at 50 °C overnight. The identity and purity of the cocrystal were analysed by means of PXRD. Genistein-D/L-proline (1:2) (Gen-D/L-Pro). Gen and D/L-Pro in the 1:2 molar ratio were dissolved in the mixture of 95% ethanol, acetonitrile and ethyl acetate (1:1:1,



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v/v/v) at 50 °C and evaporated slowly at that temperature. Plate-shaped crystals suitable for X-ray diffraction were harvested (yield: 84.5%). A stoichiometric amount of Gen and D/L-Pro were suspended in ethyl acetate and ethanol (1:1, v/v) at room temperature and stirred on a stir plate over 3 days. The resulting material was centrifuged and allowed to dry at 50 °C overnight. The identity and purity of the cocrystal were analysed by means of PXRD. Baicalein-D/L-proline (1:1) (Bai-D/L-Pro). Bai and D/L-Pro were suspended in ethyl acetate and ethanol (1:1, v/v) at room temperature and stirred on a stir plate over 72 h. The resulting material was centrifuged and allowed to dry at 50 °C overnight. Needle shape crystals were acquired by evaporation at 50 °C (yield: 82.6%). A stoichiometric ratio of Bai and D/L-Pro was determined to be 1:1 by NMR. However, diffraction quality crystals could not been obtained through solution evaporation, cooling method or anti-solvent experiment in various solvents. Single Crystal X-ray Diffraction. The single crystal X-ray diffraction measurements for Que-L/D-Pro, Kae-L/D-Pro, Chr-L-Pro, Lut-L/D-Pro and Gen-L-/D-Pro were conducted on a Bruker Smart Apex II diffractometer using Mo-Kα radiation (λ = 0.71073 Å) with a graphite monochromator at 296(2) K. The X-ray diffraction data for Chr-D-Pro was collected on a Bruker Smart Apex II CCD diffractometer using Cu-Kα radiation (λ = 1.54184 Å) at 296(2) K. Integration and scaling of intensity data was accomplished using the SAINT program. The structures were solved by Direct Methods using SHELXS97 and refinement was carried out by a full-matrix least-squares technique using SHELXL97. The hydrogen atoms were refined


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isotropically, and the heavy atoms were refined anisotropically. N-H and O-H hydrogens were located from different electron density maps, and C-H hydrogens were placed in calculated positions and refind with a riding model. Data were corrected for the effects of absorption using SADABS. Powder X-ray Diffraction. 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. Samples were measured in reflection mode in the 2θ range 3-40 ° with a scan speed of 15 °/min (step size 0.025 °, step time 0.1 s ) using a LynxEye detector. Data were imaged and integrated with RINT Rapid and the peaks are analyzed with Jade 6.0 software from Rigaku. Thermogravimetric Analysis (TGA). Thermogravimetric analysis was carried out on a Netzsch TG209 F3 equipment, using N2 as dry air with a flow of 20 mL/min and a scan rate of 10 °C/min. Differential Scanning Calorimetry (DSC). Differential scanning calorimetry (DSC) was performed with a PerkinElmer DSC 8500 instrument. Samples weighing 3-5 mg were heated in standard aluminum pans at scan rates of 10 °C/min under a nitrogen gas flow of 20 mL/min. 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). Dynamic vapor sorption experiments were performed on an Intrinsic DVS instrument from Surface Measurement Systems, Limited. Samples were studied over a humidity range from 0 to 95% RH at 25 °C.



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Each humidity step was made if less than a 0.02% weight change occurred over 10 min, with a maximum hold time of 3 h. Fourier Transform Infrared (FT-IR) Spectroscopy. Fourier transform infrared (FT-IR) spectra were collected by a Nicolet-Magna FT-IR 750 spectrometer in the range from 4000 to 350 cm-1, with a resolution of 4 cm-1 at ambient conditions. The IR spectra were recorded on samples dispersed in KBr pellet. Confocal Raman Microscope. Raman spectra were recorded with the Thermo Scientific DXR Raman microscope equipped with a 780 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. Polarized Light Microscopy (PLM). All PLM 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). Selective PLM photos for flavonoids and L-Pro cocrystals are presented in Figure 1 (magnified 50 times). Powder Dissolution. To minimize the particle size influence on dissolution results, samples were sieved through 100-mesh sieves prior to experiment. Accurately weighed powders of approximately 15 mg of samples (n = 3) were added to dissolution vessels containing 15 mL of 0.5% tween 80 water solution. The dissolution studies were conducted at a rotation speed of 75 rpm at 37 °C. Samples were performed at 5, 10, 15, 20, 25, 30, 40, 50, 60, 120, 180, 240, 300 and 360min.


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And the withdraw suspensions were filtered with 0.45 µm PTFE filters prior to HPLC analysis. The sample concentration was determined by an Agilent 1260 series Infinite HPLC, equipped with an Agilent Eclipse Plus C18 column (4.6 x 150 mm, 5 µm). Pharmacokinetic experiments. To evaluate the efficacy of cocrystallization for modulating the pharmacokinetics of flavonoids, Kae-L-Pro bulk samples were utilized to

conduct

the

pharmacokinetics

experiments.

0.5%

CMC-Na

(sodium

carboxymethylcellulose) aqueous solution (the crystal form was administered as a suspension in CMC-Na) was selected as the gavage vehicle according to a previously reported reference.35 18 male Sprague-Dawley rats weighing 220-250 mg were randomly allocated into three groups of 6 animals each and administered orally at a dose of 150 mg/kg body weight Kae raw, Kae-L-Pro and physical mixture (PM) of Kae and L-Pro (expressed as Kae equivalents). The rats were allowed water ad libitum and fasted overnight before drug administration. After administration, about 200 µL of blood sample was collected from the eyeball into heparinized tubes at 0, 15 min, 30 min, 1 h, 2 h, 4 h, 6,h, 8 h and 24 h. Plasma was immediately separated by centrifugation (10 °C, 10,000 g, 5 min) using a refrigerated table top centrifuge and kept frozen at – 20 °C until analysis. To accurately quantify the concentration of Kae in the plasma, a modified method based on previously reported work was employed using high performance liquid chromatography.36

37

In brief, frozen plasma samples were thawed at room

temperature before sample preparation. 100 µL of rat plasma was mixed with 15 µL of genistein solution (internal standard, 20.33 µg/ml in metanol) and 50 µL of 20%



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phosphoric acid solution.. After vortexing for 2 min, the plasma samples were extracted with 200 µL acetonitrile by vortex-mixing for 5 min and centrifuged for 10 min (10 °C, 10,000 g). Because kaempferol-3-O-glucosylside is the main active metabolite of Kae and mainly existing form in plasma, the total concentration of Kae and kaempferol-3-O-glucosylside in plasma was considered as the concentration of Kae. The concentrations of Kae and the kaempferol-3-O-glucosylside were simultaneously determined by a Agilent Eclipse Plus C18 column (150 × 4.6 mm, 5 µm) equipped in Agilent 1260 HPLC and detected at 266 nm. The mobile phase containing methanol and 0.1% phosphoric acid solution was run at 1.0 ml/min with methanol altering from 50% to 90% in 10 min. An injection of 50 µL was performed and the column temperature was set at 30 °C. The concentration range of the standard curve was 0.5 ug/mL to 10.0 ug/mL of Kae and kaempferol-3-O-glucosylside. 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. RESULTS AND DISCUSSION According to previous studies and a survey of Cambridge Structural Database (CSD)38

39

analysis, phenolic hydroxyl groups are preferable to form

charge-assisted hydrogen bonding with carboxylate moieties (57% in CSD, version 5.31).40 Flavonoids are rich in phenolic hydroxyl groups and proline is zwitterions under physiological conditions. Therefore the supramolecular


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heterosynthon between phenolic hydroxyl group and carboxylate moiety was expected (Figure 1).

Figure 1. Supramolecular heterosynthon between D/L-proline and flavonoids (R = H or OH) Cocrystallization of D/L-proline with six flavonoid compounds were employed in slurry experiments using ethyl acetate and ethanol (v/v, 1:1) as solvent. The single crystals were obtained by solution evaporation experiments. In addition, liquid-assisted grinding (LAG) method was also applied as a comparison. The LAG experiments herein used methanol and ethanol as assisting reagents and three cocrystals were prepared by grinding (Table S1). All the cocrystals can be obtained by slurry experiments in the same solvent mixture (ethyl acetate / ethanol, v:v/1:1). This crystallization conditions are also suitable for scale-up to 500 mg. The experimental powder X-ray diffraction of bulk cocrystal samples demonstrate good agreement with their single crystals simulated patterns (Figure S1-S6), which suggest that the high quality and purity of the cocrsytals in a single phase (Figure 2). Although a variety of methods have been undertaken, the single crystals of Bai-D/L-Pro suitable for diffraction could not been acquired. The stoichiometric ratio and purity of Bai-L-Pro was confirmed by 1H-NMR analysis (Figure S7). All of the cocrystals have been



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systematically investigated through thermal properties, spectroscopic analysis and dissolution rate behaviors.

Figure 2. Polarized light microscopies (5 × 10 times) for cocrystal (a) Que-L-Pro (b) Kae-L-Pro (c) Lut-L-Pro (d) Chr-L-Pro (e) Gen-L-Pro and (f) Bai-L-Pro Crystal structures analysis The cocrystals have been refined and solved by single crystal X-ray diffraction and the crystal structures revealed that Que-L-Pro, Kae-L-Pro, Lut-L-Pro, Chr-L-Pro and Gen-L-Pro are mainly driven by N-H…O and O-H…O hydrogen bonding interaction. In the cocrystal structures, L-Pro presents as zwitterions indicated by the comparable C-O bond lengths of the carboxylate in each crystal structure. It was interesting to notice that the ionic L-proline molecules are joined together into double column-like structures regardless of the change of non-polar flavonoids coformers (Figure 3 and S8). Subtle changes of H-bond length and angles are observed with very similar interaction patterns. Detailed H-bonding parameters will be discussed individually in the following sections. The formation of double 1-D chain structure of L-proline is a representative packing pattern according to the analysis of CSD as well.13, 16


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(a)

(b)

Figure 3. Illustration of the double column-like organization of L-proline moieties in (a) Que-L-Pro and (b) Lut-L-Pro A 6-membered ring structure build by intra-molecular hydrogen bonding interactions between 5-OH and carbonyl group of ring C was also commonly observed in all of the cocrystal structures. The six flavonoid compounds present the same flavonoid skeleton and a close structural similarity. Therefore in the following sections we mainly discuss on how the distinction of subtle geometric parameters and substitution groups of flavonoid compounds influence the crystal structure. Crystallographic data for the L-proline cocrystals are summarized in Table 1 and those with D-proline are summarized in Table S2, whereas hydrogen bond parameters are listed in Table S3. Que-L-Pro and Kae-L-Pro crystal structure On account of the structural resemblance between Que and Kae molecules, Que-L-Pro and Kae-L-Pro present iso-structural characters. Both cocrystals exhibited long plate-like morphology (Figure 2) and crystallized in P21 space group of monoclinic system with approximate cell parameters. The crystal structures revealed that Que or Kae and L-Pro are in a 1:2 stoichiometry ratio. Que-L-Pro and Kae-L-Pro



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also present identical crystal packing and H-bonding interactions. Subtle differences can be found in the bond length and H-bonding interaction on ring B. As discussed before, segregated L-Pro molecules present 1-D chain structure connected by charge-assisted hydrogen bonding: in Que-L-Pro, N1-H1B…O8, 2.918 Å; N2-H2A…O10, 2.855 Å; and in Kae-L-Pro, N1-H1A…O7, 2.827 Å; N2-H2B…O9, 2.839 Å (Figure 3a and S8a). The H-bonding interactions between flavonoids and L-Pro are also very similar. Slight differences can be found in the hydrogen bonding parameters (Figure 4a and 4b). It was found that adjacent hydroxyl groups of ring B in Que molecule interact with ionic L-Pro molecules on the same oxygen atom of the carboxylate moiety, leading to the motif of R21(7) (Figure 4a). The ring motif was absent in Kae-L-Pro which was ascribed to the lack of 3’-OH substituent on ring B of Kae molecule. The packing networks of Que-L-Pro and Kae-L-Pro both resemble sandwich-like assembly (Figure 5a and 5b), where both Que and Kae molecules were anchored between two sets of L-Pro columns and mild π…π interactions occurred between the offset stacking of Que and Kae molecules (centroid to centroid distance of π…π: 3.698 Å in Que-L-Pro and 3.971 Å in Kae-L-Pro). Overall, the hydrogen bonding organization and packing arrangement of Que-L-Pro and Kae-L-Pro are similar.


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Figure 4. Illustration of hydrogen bonding interaction in (a) Que-L-Pro (b) Kae-L-Pro (c) Lut-L-Pro (d) Chr-L-Pro and (e) Gen-L-Pro



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Figure 5. Illustration of the crystal packing network within (a) Que-L-Pro (b) Kae-L-Pro (c) Lut-L-Pro (d) Chr-L-Pro and (e) Gen-L-Pro (red and blue: L-Pro, green: corresponding flavonoid molecule) Lut-L-Pro crystal structure Cocrystallization of Lut-L-Pro in 95% ethanol and acetonitrile leads to block-like crystals. The crystal structure revealed that Lut-L-Pro crystallized in P21 space group


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of the monoclinic system and contains one Lut and one L-Pro molecules in its asymmetric unit. Similar to that of Que-L-Pro and Kae-L-Pro, L-Pro molecules generate 1-D chain structure. However, the adjacent L-Pro columns were connected by hydrogen bonding N1-H1B…O8 (2.817 Å, ∠N-H…O = 144.5°) of motif R33(11) (Figure 3b). Contrary to Que-L-Pro and Kae-L-Pro, which only one Que or Kae molecule inserts between columnar L-Pro patterns, there are two Lut molecules connecting by O6-H6A…O6 (3.016 Å) and O6-H6A…O5 (2.851 Å) of motif R12(5) adjusted between columnar networks (Figure 4c). The dihedral angle of two Lut molecule linked by motif R12(5) was 66.7° (Figure 4c). The twisted Lut molecules are aligned in parallel between ionic L-Pro 1-D chains as shown in Figure 5c. The parallel Lut sheet structures were reinforced by weak π…π interactions with centroid to centroid distance of 3.962 Å. Chr-L-Pro crystal structure Cocrystallization of Chr-L-Pro in 95% ethanol, tetrahydrofuran and acetonitrile gives rod-like shape crystals, which crystallizes in the P212121 space group of the orthorhombic system, with one Chr and one L-Pro molecules in the asymmetric unit. The Chr molecules were anchored to the L-Pro columns through hydrogen bonding O2…H1B-N1 (2.996 Å) and O4-H4…O6 (2.643 Å) as shown in Figure 4d, which is the only case that protonated nitrogen moieties of L-Pro molecules provide hydrogen bonding donors to flavonoids in all the cocrystals reported in this work. It was found that the Chr molecules are separated by the L-Pro columns along the b axis as shown



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in Figure 5d. In addition, moderate π…π interactions (centroid to centroid distance of 3.563 Å) were observed among the stacking Chr molecules. Gen-L-Pro crystal structure Cocrystallization of Gen-L-Pro in 95% ethanol, acetonitrile and ethyl acetate leads to plate-like crystals. Gen-L-Pro crystallizes in the P1 space group of the triclinic system, with one Gen molecule and two L-Pro molecules in the asymmetric unit. Akin to Chr-L-Pro, hydrogen bonding interaction was absent between the adjacent L-Pro columns. The Gen molecules are connected to the L-Pro columns through hydrogen bonding O4-H4…O9 (2.666 Å) and O5-H5…O7 (2.750 Å) as described in Figure 4e. Both sides of each Gen molecule were linked by L-Pro molecule, leading to the V-model organization, and the V-model organizations further arrange into rows in bc plane. Along the direction of b axis, the packing structures were adapted into “ladder” style and Gen molecules play the role of rung between two L-Pro columns (Figure 5e). Table 1. Crystallographic data for refinement of the cocrystals Que-L-Pro

Kae-L-Pro

Lut-L-Pro

Chr-L-Pro

Gen-L-Pro

(1:2)

(1:2)

(1:1)

(1:1)

(1:2)

1444393

1444361

1444362

1444363

1444364

C25H28N2O11

C25H28N2O10

C20H19NO8

C20H19NO6

C25H28N2O9

T (K)

296 (2)

296 (2)

296 (2)

296 (2)

296(2)

Crystal systetm

monoclinic

monoclinic

monoclinic

orthorhombic

triclinic

Space group

P21

P21

P21

P212121

P1

a (Å)

5.4354

5.3597

6.5107

5.3011

5.29670

b (Å)

18.8955

18.850

5.7951

18.145

6.2757

c (Å)

12.0279

12.177

23.939

18.386

17.5868

CCDC Empirical formula


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a (°)

90

90

90

90.00

84.456

β(°)

94.275

95.712

92.728

90.00

82.571

γ (°)

90

90

90

90.00

89.979

Volume (Å3)

1231.9

1224.2

902.21

1768.5

576.93

Z

2

2

2

4

1/

1.436

1.401

1.477

1.387

1.441

0.114

0.109

0.116

0.103

0.110

560

544

420

776

264

0.3×0.1×0.1

0.2×0.15×0.05

0.4×0.3×0.2

0.4×0.2×0.1

0.1×0.1×0.05

Rint

0.0570

0.0775

0.0186

0.0603

0.0261

R1 [I>2σ(I)]

0.0809

0.0535

0.0367

0.0451

0.0554

wR2 (all data)

0.1973

0.1143

0.0983

0.0948

0.1442

GOF

0.975

1.012

1.019

0.932

1.079

4 (2)

-0.6 (16)

-0.5 (9)

-0.6 (16)

-0.4 (11)

Calculated density (g/cm3) Absorption coeff. (mm-1) F(000) Crystal

size

(mm)

Flack parameters

Thermal analysis Thermal behaviors of the cocrystals and the corresponding pure components were investigated by TGA and DSC analysis. TGA profiles exhibit the absence of solvent molecules and the decomposition temperature of cocrystals were lower than corresponding flavonoids, indicating the thermal stability of each cocrystal is lower than the corresponding flavonoids (Figure S9). The DSC diagrams are presented in Figure S10 and the melting point (indicated by onset temperature) results are listed in Table S4. The melting point of Kae-L-Pro and Lut-L-Pro are between the values of



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the individual components, in agreement with most cocrystal cases.41 Notably, the melting point difference between Que and Kae was nearly 34 degree, while after forming cocrystal the melting point difference between Que-L-Pro and Kae-L-Pro decreased to 14 degree. This is probably ascribed to the overall impact of the same coformer and isostructural properties of Que-L-Pro and Kae-L-Pro. Among the cocrystals, the calculated density for Lut-L-Pro is the highest (1.477 g/cm3). In addition, the moisture sorption analysis for the cocrystals are performed at 25 °C, and the results are summarized in Figure S11. Lut-L-Pro was found to have very low hygroscopic sensitivity and the moisture content change was less than 2.5% over the 0-95% RH. In contrast, the other cocrystals were highly hygroscopic especially in the high humidity range of 80-95% RH, where the moisture content was found to increase up to 60% in most of the cases (Figure S11). Cocrystals of flavonoids with D-Pro Originally the cocrystallization experiments were conducted with L-Pro. We are wondering if cocrystals can be expected if using D-Pro as coformer. Since all the flavonoids were non-chiral compounds, it would be reasonable to assume that the D-Pro would form cocrystal iso-structural to the L-Pro cocrystals. The cocrystallization experiments were performed under the same conditions and successfully we able to synthesized six pairs of flavonoids with both D/L-Pro cocrystals utilizing the same crystallization conditions. Previous investigation suggest that the cocrystal stabilization energy was low (hardly surpassing 10 kJ mol-1),42-45 implying that subtle changes in structure such as chirality could change cocrystal


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formation.46 In our presentation, structural similarity flavonoids present difference in geometric parameters and substituent groups, and all the flavonoids could form cocrystals with L-Pro and D-Pro. Notably, the enantiomeric cocrystals exhibit similar crystal structures (Figure S12) and thermal properties (TGA and DSC). Since L-Pro is natural occurred enantiomer and readily available, further dissolution behavior and pharmacokinetic performance were conducted only employing L-Pro cocrystals. In vitro dissolution Dissolution rate and apparent solubility of solids are important properties to be considered in pharmaceutical development and quality control.47 Flavonoids were restricted to application in pharmaceutical fields for exhibiting low solubility and bioavailability. The powder dissolution profiles for all the cocrystals and corresponding flavonoids materials in 0.5% tween 80 solutions are illustrated in Figures 6. All of the cocrystal profiles display a type of “spring and parachute” effect, which is a general phenomenon in pharmaceutical cocrystals.4,

48

Results

demonstrated that the apparent solubility present significant differences amoung various cocrystals. In particular, the dissolution behavior of Chr-L-Pro, Bai-L-Pro and Kae-L-Pro exhibit approximate 70%, 50% and 270% higher of maximum solubility than corresponding starting material under the same dissolution condition. In general the high apparent solubility can only be maintained for a short time in the supersaturation state and subsequently decrease rapidly. However the higher concentration of Kae-L-Pro decrease to a constant value, which is still 1.8 times larger than raw material, and can sustain for a considerable duration of time (at least 6 h).



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Nevertheless, Chr-L-Pro and Bai-L-Pro decrease rapidly to the equilibrium concentration after 60 minutes, which is close to the equilibrium concentration of starting compounds. It can be found that the dissolution profiles of Que-L-Pro, Lut-L-Pro and Gen-L-Pro show comparable even reduced dissolution behaviors compared to starting materials. The results of PXRD analysis for all the residue solids after dissolution experiment indicate that Lut-L-Pro, Chr-L-Pro, Gen-L-Pro and Bai-L-Pro have transformed to corresponding starting materials, while Que-L-Pro and Kae-L-Pro has converted into amorphous state (Figure S13-18). In general, cocrystallization with L-proline have modulated the in vitro dissolution behaviors of flavonoids especially promoted the dissolution rate of Kae.


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Figure 6. Powder dissolution profiles of Que-L-Pro, Kae-L-Pro, Lut-L-Pro, Chr-L-Pro, Gen-L-Pro, Bai-L-Pro and their corresponding starting materials in 0.5% tween system (data are expressed as means ± SD, n = 3), dash refer to flavonoid materials, solid refer to corresponding cocrystals) In vivo pharmacokinetic To evaluate whether the bioavailability of flavonoids have been improved through crystal engineering principle, the pharmacokinetic experiment of Kae-L-Pro was carried out for its improved in vitro dissolution. Previous studies had reported that glycosylate metabolites were the major form existed in the blood concerning the pharmacokinetics of Kae.37,

49

In order to make a meaningful comparison, the

pharmacokinetic parameters of Kae-3-O-glucosylside, which was identified as the major

metabolite

in

this compound,

were

provided


and

analyzed.

The


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pharmacokinetic curves for Kae, Kae-L-Pro cocrystal and physical mixture (PM) of Kae and L-Pro are presented in Figure 7. The Kae-L-Pro cocrystal exhibited an improved pharmacokinetic profile compared with Kae pure component and corresponding physical mixture. The pharmacokinetic parameters are shown in Table 2. Surprisingly, Kae-L-Pro cocrystal has changed the overall shape of the pharmacokinetic curve with shorter Tmax, higher Cmax and AUC0-24 h in comparison to Kae component. It is notable that the Cmax and AUC0-24 h of Kae-L-Pro were 369% and 351% higher than that of Kae pure form respectively. This increasing in AUC of Kae-L-Pro is suspected to be correlated with its faster dissolution rate and higher solubility.50 Based on this, crystal engineering approach with L-proline is proved to be a feasible technique to ameliorate the performance of Kae component and progress the development of pharmaceutic cocrystals.


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Figure 7. Pharmacokinetic profiles of Kae-3-O-glucosylside (mean plasma concentration versus time). (data are expressed as means ± SD, n = 6) Table 2. Pharmacokinetic parameters of Kae-3-O-glucosylside solid forms

Tmax (h)

Cmax (ug/mL)

AUC0-24 (ug.h/mL)

Kae

6.00 ± 0.00

7.83 ± 1.26

39.55 ± 8.49

PM of Kae and L-Pro

4.00 ± 0.00

6.54 ± 3.45

29.86 ± 15.38

Kae-L-Pro

4.66 ± 3.05

36.79 ± 20.52

178.68 ± 79.96

CONCLUSION We successfully synthesized and characterized twelve pairs of D/L-proline and flavonoids cocrystals. The crystal structures reveal that D/L-proline moieties create the typical column-like patterns. The difference in structure network can lead to the distinction in dissolution behavior and bioavailability. All of the cocrystals dissolution profiles exhibit a common “spring and parachute” effect. Notably, Chr-L-Pro,



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Bai-L-Pro and Kae-L-Pro display promotion in dissolution behavior compared with their starting compounds. It is worth to notice that the equilibrium concentration of Kae-L-Pro is 1.8 times higher than that of Kae compounds, and this solubility advantage can be maintained for a considerable duration of time (at least 6 h). Kae-L-Pro presents a significantly improved performance with a sharply increased Cmax and AUC0-24 h with respect to Kae pure component and corresponding physical mixture. Supporting Information. PXRD, IR, Raman, TGA, DSC and DVS profiles for the cocrystals. X-ray crystallographic data (CCDC 1444393, 1444361-364, 1446360-1446364) in CIF format and CIF check. This information is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Grants 81273479 and 81402898), CAS Key Technology Talent Program, and Youth Innovation Promotion Association CAS for funding. REFERENCES (1) Crozier, I. B. J. a. A., Dietary flavonoids and phenolic compounds. In ed.; Fraga, C. G., Ed. John Wiley & Sons: Plant Phenolics and Human Health: Biochemistry, Nutrition, and Pharmacology, 2010. (2) Crozier, A.; Jaganath, I. B.; Clifford, M. N. Nat. Prod. Rep. 2009, 26, 1001-1043.


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For Table of Contents Use Only

Zwitterionic Cocrystals of Flavonoids and Proline: Solid-State Characterization, Pharmaceutical Properties and Pharmacokinetic Performance Hongyan He, Ying Huang, 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

Twelve pairs of D/L-proline and flavonoids cocrystals were synthesized and the single structures revealed that the ionic proline molecules joined together into double-column packing. The physicochemical properties characterization were performed and the pharmacokinetic properties for Kae-L-Pro were analyzed and exhibited an improved performance with respect to Kae pure component.


Zwitterionic Cocrystals of Flavonoids and Proline: Solid-State

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