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The University of Bradford Institutional Repository This work is made available online in accordance with publisher policies. Please refer to the repository record for this item and our Policy Document available from the repository home page for further information. To see the final version of this work please visit the publisher s website. Access to the published online version may require a subscription. Link to publisher s version: /acs.cgd.6b00759 Citation: Ketkar S, Pagire SK, Goud RN et al. (2016) Tracing the architecture of caffeic acid phenethyl ester cocrystals: studies on crystal structure, solubility, and bioavailability implications. Crystal Growth and Design. 16(10): Copyright statement: This document is the Accepted Manuscript version of a Published Work that appeared in final form in Crystal Growth and Design, copyright American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see /acs.cgd.6b00759 Tracing the architecture of Caffeic acid phenethyl ester cocrystals: Studies on crystal structure, solubility and bioavailability implications Sameer Ketkar, Sudhir K.Pagire, N. Rajesh Goud ǂ, KakasahebMahadik, AshwiniNangia ǂ, * and AnantParadkar * Centre for Advanced Research in Pharmaceutical Sciences, Poona College of Pharmacy, BharatiVidyapeeth University, Pune , India Centre for Pharmaceutical Engineering Science, University of Bradford, Bradford, BD7 1DP, UK ǂ School of Chemistry, University of Hyderabad, Prof. C. R. Rao Road, Gachibowli, Hyderabad, , India. CSIR-National Chemical Laboratory, Dr. HomiBhabha Road, Pune , India. 1 ABSTRACT Caffeic acid phenethyl ester (CAPE) is a polyphenolic active compound present in popular apiproduct, propolis obtained from beehives. Though it has broad therapeutic capability, the bioavailability of CAPE is limited due to poor solubility. In this study, we report novel cocrystals of CAPE engineered using coformers such as caffeine (CAF), isonicotinamide (INIC), nicotinamide (NIC) with enhanced solubility and bioavailability of CAPE. The cocrystals were prepared by microwave-assisted cocrystallization and characterized using PXRD, DSC and Raman spectroscopy. PXRD and DSC confirm the successful formation and phase purity of CAPE-CAF, CAPE-INIC and CAPE-NIC cocrystals. Raman spectra of CAPE cocrystals complement these results in confirming the formation of novel crystalline phases. CAPE-NIC cocrystal was further subjected to X-ray crystallography to understand its molecular arrangement and hydrogen bonding in the crystal structure. The CAPE-NIC cocrystal structure is found to be stabilized by a rare 1,2-benzenediol-amide heterosynthon. Cocrystallization of CAPE with NIC improved its aqueous solubility and pharmacokinetic profile thereby demonstrating 2.76 folds escalation in bioavailability. 1. Introduction Polyphenolic compounds either synthetic or obtained from natural sources play an important role in therapeutic and nutritional applications. 1,2,3 They are known to exhibit diverse pharmacological effects such as antioxidant, antimicrobial, anti-inflammatory, anticancer, antiviral, immunomodulatory, etc. 4,5,6,7 Despite broad therapeutic utility, the poor aqueous solubility of polyphenols limits their bioavailability and makes their clinical efficacy questionable. 1,2,8 Therefore, improving the physicochemical properties of polyphenols forms a 2 major focus of research worldwide. Pharmaceutical cocrystallization is a process by which a drug molecule and a coformer are brought together into the same crystal lattice to prepare novel solid forms of APIs with tailored physicochemical properties. This method has been successfully adapted to modulate the solubility of various polyphenolics. For example, the solubility and dissolution rate of curcumin and myricetin were improved by cocrystallizing with suitable 9, 10 coformers. Further, cocrystallization was found to be advantageous in altering the 11, 12 pharmacokinetic profile of polyphenolics such as quercetin and epigallocatechin-3-gallate. Binary solid composites, e.g. amorphous solid dispersions and eutectics are complementary 13, 14, 15 strategies to improve the solubility of polyphenolics. Caffeic acid phenethyl ester (CAPE) is a polyphenolic active present in popular apiproductpropolis, obtained from bee hives. CAPE is known to possess a wide array of pharmacological activities such as antioxidant, anti-inflammatory, antitumor, antibacterial, antiviral, neuroprotective, hepatoprotective, cardioprotective and immunostimulant Although numerous preclinical studies demonstrate the effectiveness of CAPE, its therapeutic utility is limited on account of its poor bioavailability which is ascribed to its poor aqueous solubility. 24, 25 Most of the documented scientific reports focus on chemical synthesis 26, 27, 28 and biological evaluation of CAPE or its derivatives. 29,16 However, a systematic analysis of its crystal structures directed towards improving its physicochemical properties is not known in the literature. A benzene solvate of CAPE is reported, 30 but its structural details were not found in the Cambridge Structural Database (CSD). Thus, till date there are no X-ray crystal structures on crystal forms of CAPE, either as single or multicomponent forms. The current study explores novel cocrystals of CAPE directed towards modulating its physicochemical properties. The microwave assisted cocrystallization 31 technique was used to screen CAPE with various GRAS 3 (Generally Regarded As Safe) listed coformers. In this study, we have found three novel cocrystals of CAPE with caffeine (CAF), isonicotinamide (INIC) and nicotinamide (NIC). The generation of novel crystalline forms of CAPE and understanding their structure-property relationships with special emphasis on improving its poor solubility and bioavailability forms the major focus of this article. Scheme 1.Chemical structures of CAPE and coformers. 2. Experimental 2.1 Materials and Methods CAPE, 97% pure, CAF, NIC, INIC, gallic acid, p-coumaric acid, curcumin, isoniazide, theophylline, urea, ibuprofen (purity 98%) and taxifolin analytical standard were purchased from Sigma-Aldrich Company Ltd., UK. Ethanol( 99% pure) and acetonitrile ( 99% pure) were also procured from Sigma-Aldrich Company Ltd., UK. 4 2.2. Synthesis of CAPE Cocrystals A common procedure was used to screen all coformers listed above (SI1,ESI ) using microwave assisted cocrystallization technique for generation of cocrystals with CAPE. Equimolar amount of CAPE and the coformer were mixed with ethanol to obtain 40 % w/w slurry in a 30 ml glass tube. The slurry was subjected to microwave irradiation in microwave reactor (Monowave 300, Anton Paar, Gmbh, Austria) to induce cocrystallization. The operating conditions were target temperature (80 C), hold time (60 sec) followed by cooling to 40 C, the solid product was dried and subjected to further characterization Powder X-ray diffraction (PXRD) The microwave processed materials were screened for cocrystal formation on Bruker D8 advanced diffractometer (X-ray wavelength λ = nm, source Cu-Kα, voltage 40kV) with filament emission of 40 ma current. The samples were scanned from 2 to 30 2θ at a scan speed of 0.01 step width Differential scanning Calorimetry (DSC) The microwave processed materials were subjected to thermal analysis using TA Instruments DSC 2000 differential scanning calorimeter. The samples were subjected to heating from 20 C to their respective melting temperature at rate of 10 C/ min in DSC cell under inert nitrogen environment. Standard aluminum pans were used. 5 2.5. Raman spectroscopy Raman spectra were recorded using Renishaw In via Reflex bench top spectrometer coupled with 683 nm stabilized diode (Renishaw Plc., UK). A laser spot of diameter (footprint) 2 μm was obtained at specimen using a 100X objective lens. The spectra were collected using extended scanning mode over the wave number region of cm scans were collected for each sample. Data analysis was performed using Galactic Grams AI 8.0 spectroscopy software (Thermo Electron Corporation) X-Ray Crystallography Single crystal X-ray data on CAPE-NIC cocrystal was collected on the Xcalibur Gemini EOS CCD diffractometer (Oxford Diffraction, Yarnton, UK) using Mo-Kα, radiation at 298 K. Data reduction were performed using CrysAlisPro (version ). OLEX2-1.0 and SHELX-TL 97 were used to solve and refine the reflections data. Non-hydrogen atoms were refined anisotropically. Hydrogen atoms on O and N were experimentally located in difference electron density maps. All C H atoms were fixed geometrically using HFIX command in SHELX-TL. A check of the final CIF file using PLATON did not show any missed symmetry. X-Seed was used to prepare packing diagrams. The X-ray data are deposited with the Cambridge Crystallographic Data Centre (www.ccdc.cam.ac.uk) (CCDC No ) Equilibrium Solubility measurements CAPE and the cocrystal samples were individually added in small portions to 1 ml distilled water at room temperature (25 ± 0.5 C) and stirred at 600 rpm. The addition of solids was stopped once they showed precipitation in the solvent. The systems were stirred for further 6 24 hrs to ensure equilibration of the solids with the solvent. The samples were filtered using 0.45 µm nylon filter. The filtrate was suitably diluted and subjected to High Performance Liquid Chromatographic (HPLC) analysis on Waters e-2695 system integrated with a PDA 2998 detector, Empower 3 software and Waters symmetry C18 column ( mm, 5 µm) maintained at 25 ± 0.5 C throughout study, see SI2ESI. Amount of CAPE solubilized was calculated from calibration curve by plotting the known concentration at each dilution vs. the corresponding peak area In vivo pharmacokinetics Male Wistar rats (n = 3 per group) weighing g were purchased from National Toxicology Centre, Pune, India. CAPE and CAPE-NIC cocrystal samples were administered per oral at a dose of 100 mg CAPE per kg body weight. Blood was collected from retro-orbital plexus of rats at different time points as: 0, 5, 10, 15, 30, 45, 60, 120, 240, and 360 min. and analyzed for CAPE plasma concentration, see SI3,ESI for more details. 3. Results and Discussion Data mining of the crystal structure database (CSD) in conjunction with the crystal engineering principles comprise the basic design elements for fabrication of multi-component cocrystals Considering the nature of functional groups in CAPE molecule, coformers with complementary functional moieties were selected so as to form stable cocrystals. The primary functional groups in CAPE are 1,2-benzenediol and ester moieties. In literature, COOH, CONH 2 and pyridine 33, 37 functional groups have been reported to assemble with hydroxyl containing molecules. Accordingly, CAPE was cocrystallized with GRAS listed coformers containing these functional groups. A complete list of all the coformers are listed in Table SI1, ESI. 7 3.1. Powder X-ray Diffraction PXRD was employed as a fingerprint tool to examine the crystallinity and novelty of CAPE cocrystals. The experimental powder patterns of bulk materials of CAPE cocrystals and their respective individual components are shown in Figure 1a and Figure 1b. The new and unique diffraction peaks in the powder XRD patterns of cocrystal samples different from that of pure CAPE and coformers confirm cocrystallization. Figure 1a. Experimental powder diffraction patterns of A) pure CAPE; B) CAPE-CAF; C) CAPE-INIC and D) CAPE-NIC 8 Figure 1b.Calculatedpowder diffraction patterns of pure A) caffeine; B) INIC and C) NIC 3.2. Differential Scanning Calorimetry Differential scanning calorimetric analysis displayed unique thermal behavior and the phase purity of CAPE cocrystals. Interestingly, none of the cocrystals exhibited a melting point in between that of the corresponding individual components as observed in certain cocrystal systems 35 (Figure 2a and 2b). Instead, all the cocrystal phases exhibited lower melting point as compared to CAPE and the respective coformers. The cocrystal melting points showed a direct dependence on the coformer melting point, i.e. higher melting cocrystal was formed by a higher melting coformer (Table 1). Further, heat-cool-heat experiments on these cocrystals indicated that they did not undergo recrystallization on cooling, possibly the recrystallization event 9 followed a different cooling profile than the method adapted during our DSC analysis. (SI4, ESI ) Figure 2a. DSC Heating curves for a) pure CAPE; b) CAPE-CAF; c) CAPE-INIC; d) CAPE- NIC; e) CAF f) INIC and g) NIC 10 Figure 2b. DSC Heating curves for a) pure CAPE; b) CAF; c) INIC and d) NIC. Table 1. DSC melting points of CAPE cocrystals and their conformers. S. No. CAPE cocrystal Cocrystal melting Point ( C) Coformer Coformer melting Point ( C) 1 CAPE-NIC Nicotinamide CAPE-INIC Isonicotinamide CAPE-CAF Caffeine Melting point of CAPE is 129 C 3.3. Raman spectroscopy CAPE and its cocrystals were characterized by Raman spectroscopy in order to ascertain the influence of solid state modification on the vibrational states of participating atoms and differentiate the novel crystalline phases from the starting materials. In the Raman spectrum of CAPE, the ester C=O stretch occurs at cm -1, the asymmetric and symmetric C=C stretch occurs at and cm -1 and the C-O stretch occurs at cm -1. Similarly in the Raman spectrum of NIC, the N-H stretch occurs at cm -1, the C=O at cm -1 and C- O at cm -1. On forming cocrystal, the amide C=O of NIC undergoes a red shift to cm -1 indicating the enhanced single bond character of the C=O group on forming a hydrogen bond with the phenolic O-H of CAPE. The other cocrystals where crystal structures are not available currently, changes in their vibrational patterns may be explained through Raman spectrum. In pure INIC, the N-H stretch occurs at cm -1 and the C=O at cm -1. In the cocrystal, the amide C=O undergoes a blue shift to cm -1 and C-O to cm -1. In pure CAF, the C=O appears at 1695 cm -1 which undergoes a red shift to cm -1 in the 11 cocrystal. Thus, the Raman spectroscopy complement with other characterization techniques in establishing the formation of novel crystalline phases. Figure 3. Raman spectrum of CAPE-NIC in comparison to its starting materials 12 Figure 4. Raman spectrum of CAPE-INIC in comparison to its starting materials Figure 5. Raman spectrum of CAPE-CAF in comparison to its starting materials 3.4. Crystal Structure Analysis CAPE-NIC cocrystal Among the novel crystalline phases, crystal structure of CAPE-NIC was determined by SC X-ray crystallography. The structural parameters of CAPE-NIC are shown in Table 2 and the hydrogen bond parameters are listed in Table 3. 13 Table 2. Crystallographic Parameters of CAPE-NIC cocrystal. Emp. Form. C 23 H 22 N 2 O 5 Form. Wt Cryst. Syst. Monoclinic Sp. Gr. P2 1 /c T (K) 298(2) a (Å) (15) b (Å ) (4) c (Å ) (16) α (º) 90 β (º) (8) γ (º) 90 Z 4 V (Å3) (3) Total No. of Reflns Unique Reflns Obsd. Reflns Parameters 287 R wr GOF Table 3. Hydrogen bond distances and angles in CAPE-NIC cocrystal (neutron-normalized D H A D A (Å) H A (Å) D H A ( ) Symmetry code CAPE NIC (1:1) O1 H1 O x+1,+y 1/2, z+1/2+1 N2 H2B O x+1,+y 1/2, z+1/2+1 N2 H2A O x, 1+y, z O2 H2 N x,+y-1,+z+1 C16 H16 O x+2,+y 1/2, z+1/2+1 N H Å, O H Å, and C H Å distances). This 1:1 stoichiometric cocrystal crystallized in monoclinic P2 1 /c space group. A two-point heteromeric interaction between the 1,2-benzenediol moiety of CAPE and the amide of NIC (O1 H1 O5, 1.96Å, 148 ; N2 H2B O2, 2.26Å, 137 ) is the primary H-bonding synthon in this cocrystal (Figure 6). Further, these heterodimers are connected through N H O (N2-H2B O1 (phenolic oxygen), 2.58 Å, 114 ; N2-H2A O5 (amide oxygen), 2.29Å, 150 ) H-bonds, extending them into the 2-dimensional space. The phenolic OH (O2H2) further propagates this molecular assembly into the third dimension through O-H N (O2-H2 N3 (pyridyl N), 1.74Å, 164 ) H-bond with the adjacent pyridyl nitrogen. Apart from these strong hydrogen bonding synthons, weak C-H O (C16-H16 O4, 2.67Å, 167 ) and C-H π interactions ably assist in stabilizing the 3D packing in this cocrystal (Figure 7). The benzenediol-amide heterosynthon between CAPE and NIC is relatively rare interaction as compared to their respective homosynthons and competing heterosynthons. A CSD search for cocrystals with 1, 2- benzenediol and primary amide functional group lends credence to this statement (Table SI5, 15 ESI ). A CSD search (version 5.37, March 2016) with these functional groups on chemically different molecules resulted in 28 hits. Of these 24 crystal structures were stabilized by competing homosynthons (OH OH and amide-amide) heterosynthons such as acid-amide, OHpyridine etc. Only four structures form the 1,2-benzenediol and amide heterosynthon. The predominance of crystal structures stabilized by competing heterosynthons indicates the diolamide heterosynthon as a much weaker interaction. In the CAPE-NIC structure, the diol-amide heterosynthon was favored due to the absence of other functional groups and preferred over the competing homosynthons. Figure 6. CAPE and NIC molecules connected through heteromeric two point synthon which are further held through N-H O H-bonds. The O-H N (pyridine) interaction is highlighted through a blue circle. 16 Figure 7. Weak C-H O and C-H π interactions further stabilize the CAPE-NIC crystal structure Equilibrium Solubility The primary objective behind developing cocrystals of CAPE was to address its poor solubility, which significantly lowers its bioavailability limiting its efficacy. Our objective was based on the hypothesis that the high solubility coformers would alter the hydrogen bond patterns in the CAPE, which would improve its aqueous solubility envisaging enhancement in bioavailability. Equilibrium solubility experiments on CAPE and its cocrystals were performed in distilled water and the samples were analyzed by HPLC. The HPLC chromatograms demonstrated retention time (Rt) for standard solutions of CAPE, CAF, INIC and NIC to be ± 0.02, ± 0.03, ± 0.02 and ± 0.01 min, respectively and the same were observed for respective cocrystals (Figure SI6, ESI ). Solubility experiments, apart from confirming the poor aqueous solubility of CAPE (0.021± mg/ml), highlighted the advantage in forming cocrystals where the CAPE-CAF, CAPE-INIC and CAPE-NIC exhibited about 5.5, 7.5 and 17.7 times higher solubility as compared to pure CAPE (Table 4). Interestingly, the solubility profiles of 17 CAPE cocrystals are inversely related to their melting point. The lower melting CAPE-NIC exhibited higher solubility followed by CAPE-INIC and CAPE-CAF. Significant improvement in the aqueous solubility of CAPE-NIC may be ascribed to the higher solubility documented for nicotinamide in water. 34,35. Further, the BFDH (Bravais, Friedel, Donnay and Harker) model hints (since this method is based purely on geometric considerations and completely neglects energetics, the result is mostly a qualitative view) at the morphology of CAPE-NIC cocrystal (Figure SI7, ESI ) where the hydrophilic -diol groups of CAPE project out of the major (100) face of the CAPE-NIC cocrystal structure burying the hydrophobic skeleton inside, enabling favorable interaction with the water molecules and thus increasing its solubility. 40 Table 4. Equilibrium solubility values of CAPE and its cocrystals. Compound Equilibrium Solubility (mg/ml) X fold increment compared to CAPE Melting point of cocrystal ( C) CAPE ± CAPE-CAF ± CAPE-INIC ± CAPE-NIC ± In vivo pharmacokinetics The poor water solubility of CAPE hampers its pharmacokinetic profile especially in terms of absorption upon oral administration. The present study was performed to trace the influence of cocrystallization of CAPE on its bioavailability. The 17 times improved solubility of CAPE-NIC 18 cocrystal, made it a lead entity to screen for its pharmacokinetic
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