Cocrystals of Hesperetin: Structural, Pharmacokinetic, and

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Co-crystals of Hesperitin: Structural, Pharmacokinetic and Pharmacodynamic Evaluation Karan Vasisht, Kunal Chadha, Maninder Karan, Yashika Bhalla, Renu Chadha, Sadhika Khullar, and Sanjay Mandal Cryst. Growth Des., Just Accepted Manuscript • Publication Date (Web): 23 Mar 2017 Downloaded from http://pubs.acs.org on March 24, 2017

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Crystal Growth & Design

Co-crystals of Hesperetin: Structural, Pharmacokinetic and Pharmacodynamic Evaluation 1

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Karan Vasisht* , Kunal Chadha , Maninder Karan ,Yashika Bhalla , Renu Chadha , Sadhika Khullar , 3 Sanjay Mandal 1. University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, INDIA 2. Department of Chemistry, D.A.V. University, Jalandhar - Pathankot National Highway, Jalandhar 144012, Punjab, India 3. Department of Chemical Sciences, Indian Institute of Science Education and Research Mohali, Sector 81, Manauli PO, S.A.S. Nagar, Mohali (Punjab) 140306, INDIA Co-crystallization by the solvent drop grinding technique has been employed successfully to generate highly water soluble co-crystals of a poorly soluble nutraceutical hesperetin with different coformers, picolinic acid, nicotinamide and caffeine. The miniscule amount of solvent, (ethanol here), added during grinding, expectedly imparts high molecular mobility and efficiency to the method. Based on preliminary indication of the phase transformation by DSC, these co-crystals were further characterized by FT-IR and SSNMR spectroscopy. However, the final structural confirmation of these distinct cocrystalline forms was provided by either single crystal XRD for HESP-PICO or powder XRD data in Material Studio® software to generate the crystal structure of HESP-NICO and HESP-CAFF. The data revealed the existence of supramolecular synthons established by novel hydrogen bonds between hydroxyl groups of hesperetin with acid or amide carbonyl (C=O), and/or amidic NH2, and/or pyridine/aromatic nitrogen (Naromatic) of coformers. Dissolution studies of co-crystals in aqueous buffer showed maximum concentration of hesperetin to be nearly 4-5 times higher than the pure substance. This has led to optimized pharmacokinetics as exhibited by improved relative bioavailability (HESP-PICO:1.36, HESP-NICO:1.57, HESP-CAFF:1.60). Furthermore, the enhanced antioxidant and antihaemolytic effect, coupled with the protective action against inflammation signify the development of a clinically useful and a pharmaceutically acceptable form of hesperetin.

Hydrogen bonding between hesperetin and picolinic acid in HESP-PICO * Corresponding Author: Professor Karan Vasisht University Institute of Pharmaceutical Sciences Faculty of Pharmaceutical Sciences. Panjab University, Chandigarh 160014, INDIA Email: [email protected] M: +91 9876067171

ACS Paragon Plus Environment


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Co-crystals of Hesperetin: Structural, Pharmacokinetic and Pharmacodynamic Evaluation

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Karan Vasisht* , Kunal Chadha , Maninder Karan ,Yashika Bhalla , Renu Chadha , Sadhika Khullar , 3 Sanjay Mandal 1. University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, INDIA 2. Department of Chemistry, D.A.V. University, Jalandhar - Pathankot National Highway, Jalandhar 144012, Punjab, India 3. Department of Chemical Sciences, Indian Institute of Science Education and Research Mohali, Sector 81, Manauli PO, S.A.S. Nagar, Mohali (Punjab) 140306, INDIA

* Corresponding Author: Professor Karan Vasisht University Institute of Pharmaceutical Sciences Faculty of Pharmaceutical Sciences. Panjab University, Chandigarh 160014, INDIA Email: [email protected] M: +91 9876067171


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ABSTRACT Co-crystallization by the solvent drop grinding technique has been employed successfully to generate highly water soluble co-crystals of a poorly soluble nutraceutical hesperetin with different coformers, picolinic acid, nicotinamide and caffeine. The miniscule amount of solvent, (ethanol here), added during grinding, expectedly imparts high molecular mobility and efficiency to the method. Based on preliminary indication of the phase transformation by DSC, these

co-crystals

were

further

characterized

by

FT-IR

and

SSNMR

spectroscopy. However, the final structural confirmation of these distinct cocrystalline forms was provided by either single crystal XRD for HESP-PICO or powder XRD data in Material Studio® software to generate the crystal structure of HESP-NICO and HESP-CAFF. The data revealed the existence of supramolecular synthons established by novel hydrogen bonds between hydroxyl groups of hesperetin with acid or amide carbonyl (C=O), and/or amidic NH2, and/or pyridine/aromatic nitrogen (Naromatic) of coformers. Dissolution studies of co-crystals in aqueous buffer showed maximum concentration of hesperetin to be nearly 4-5 times higher than the pure substance. This has led to optimized pharmacokinetics as exhibited by improved relative bioavailability (HESP-PICO:1.36, HESP-NICO:1.57, HESP-CAFF:1.60). Furthermore, the enhanced antioxidant and antihaemolytic effect, coupled with the protective action against inflammation signify the development of a clinically useful and a pharmaceutically acceptable form of hesperetin. keywords:

hesperetin,

co-crystallization,

structure

pharmacokinetics, pharmacodynamics


elucidation,


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1. INTRODUCTION The comprehensive knowledge about the biopharmaceutical properties and other performance parameters of a drug molecule is very important in its developmental stages, with solubility, dissolution rate and thermal stability being of prime importance, albeit in conjunction with permeability.1-2 Since an increased emphasis is being placed on economically and environmentally sustainable techniques, the pharmaceutical industry during this decade has embraced crystal engineering as a premier technology to modulate these performance parameters to optimum values and in turn improve their overall clinical application. The non covalent derivatization at the core of every crystal engineering product, including co-crystals, polymorphs, solvates and eutectics, effect a desired change in the physical properties without any impact on the pharmacological profile. Cocrystal is a homogenous crystalline entity that contains in a definite stoichiometric amounts, two or more co existing molecular and/or ionic compounds, in the same crystal lattice stabilized by non covalent interactions like hydrogen bonds, π- π interactions, ionic interactions and van der Waals forces.3 The technique of cocrystallization itself involves the breaking of existing cohesive non covalent interactions especially hydrogen bonding, to establish new adhesive interactions between different components of the cocrystal.4 The non-covalent derivatization involves the replacement of interactions between moieties that establish weak associations with more robust ones.5 These resultant interactions form what has been described by Desiraju as supramolecular synthon complexes.5,6 Crystal engineering, with significant departure from traditional organic solvent based synthetic derivatization technologies, offers a solvent less, clean, uncomplicated, environment friendly and a cost effective method that is known for its versatile ability to tune the process to generate products with required parameters like better solubility, dissolution, stability, tableting and/or compressibility and dispersion properties.6-10 Hesperetin,

[2,3-dihydro-5,7,-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)

benzopyran-4-one], commonly found in citrus fruits11 is a powerful

-

4H-1-

antioxidant

molecule.12 This free radical scavenger, apart from reducing atherosclerosis, hypercholesterolemia and lipid peroxidation also exhibits, antiplatelet effect, therby


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manifesting a potent prophylactic action against ischemic stroke.13-16 It also offers protection against oxidative stress induced neurological,12 hepatic,17

and testicular

dysfunction.18 Antimicrobial properties in form of antiviral and antibacterial action11 as well as a prominent protective effect against lung, breast, colon and carcinoid cancers1921

has further drawn attention to this molecule. Despite the exemplary therapeutic

profile, and negligible mammalian toxicity,22-25 hesperetin is still not approved for use as a pharmaceutical agent. This is primarily due to critically low aqueous solubility (reported to be in the range of 16 µg/mL - 40 µg/mL by various researchers)25 which leads to very frugal absorption from the gastrointestinal tracts after oral dose and consequently resulting in sub-therapeutic concentrations in the systemic circulation.21-24 In this predicament, the manipulation of the solubility parameter has by far been the easiest and most cost effective way of improving bioavailability.21-24 There have been previous attempts to improve upon the solubility of hesperetin like development of nanoparticles and beta cyclodextrin inclusion complexes.26-28 But, due to the inherent disadvantages of these techniques including uncertain kinetics and neurotoxicity for nanoparticles,29 gastric toxicity and bulkiness for cyclodextrins,30 these have not really been that successful. Although, co-crystals of hesperetin with two coformers, isonicotinamide and nicotinic acid are reported but this study is not supported by any biological evaluation.31 The present attempt to crystal engineer a nutraceutical hesperetin into clinically significant molecule is an encouragingly successful journey to prepare non covalent derivatives with desired properties, which on account of its neutral nature and no ionizable groups do not lend itself easily to salt formation. Moreover sensitive moieties in these natural compounds are quite prone to racemization or decomposition in harsh pH conditions of salt formation.6,32-37 The three non toxic coformers, picolinic acid, nicotinamide and caffeine, used in the present study possess inherent useful therapeutic profile and have satisfactorily high aqueous solubility and physical stability.35 Whereas, picolinic acid (endogenous catabolite of tryptophan amino acid, via kynurenine pathway in liver and kidneys) when secreted into the intestine via pancreas acts as body's prime natural chelator for elements like chromium, zinc, manganese, copper, iron and molybednum,42 the nicotinamide, being a vitamin B2 derivative is converted to the same in body,43 and



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caffeine (purine alkaloid) is a powerful CNS stimulant with strong diuretic and smooth muscle relaxing properties.44 Hesperetin, with three hydroxyl groups, one keto and two ether groups is greatly expected to form strong adhesive forces with amide group on nicotinamide, carboxylic acid on picolinic acid and hydroxyl in caffeine apart from the Naromatic in all three. (Scheme 1). The three new co-crystals prepared by us with, picolinic acid, nicotinamide and caffeine, apart from covering the characterization by DSC, FT-IR and PXRD in conjunction with SSNMR and structure determination also reports a detailed and encouraging experiments on dissolution profile, equilibrium solubility, pharmacokinetic analysis, in vivo pharmacodynamic studies by evaluation of antiinflammatory response and finally the in vitro study of antioxidant and antihaemolytic effects.

Scheme 1: Co-crystallization of hesperetin with picolinic acid, nicotinamide and caffeine providing three new co-crystals with unique structural and physical properties.

2. EXPERIMENTAL SECTION: 2.1. Materials Hesperetin, picolinic acid, nicotinamide, caffeine and carrageenan, (λ form and type IV) were purchased as anhydrous crystalline powders with >99% purity from Sigma Aldrich, India and other solvents from Merck India Ltd.


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2.2. Preparation Liquid assisted grinding for preparation of co-crystals was done using catalytic amounts of solvent. Stoichiometric amount of hesperetin (302 mg) was triturated for 30 min in a glass pestle mortar with each of the coformers, viz. picolinic acid (123.11 mg), nicotinamide (122.12 mg) and caffeine (195 mg) individually, with the addition of a drop or two of ethanol to accelerate molecular mobility and promote high atomic efficiency. The final products with picolinic acid, nicotinamide and caffeine were coded as HESPPICO, HESP-NICO and HESP-CAFF, respectively and stored in an air tight dessicator under controlled humidity. Furthermore, these were recrystallized from minimum amount of warm ethanol to obtain single crystals. However, only HESP-PICO was crystallized out in form of pale white, transparent needle shaped crystals which were robust enough and suitable for detailed structure analysis by single crystal X-Ray diffraction. To obtain uniform particle size of the powders for further analysis, the bulk cocrystal powders were sieved through mesh #44 and particle size was measured by Mastersizer 2000 (Malvern Instruments Inc.,U.K.) as 8.5 µm. (Particle size distribution graph is presented in Supporting information as Figure S1) 'The method of solvent drop grinding used here, involves the addition of a suitable solvent, like ethanol, in miniscule or catalytic amounts during the grinding of the two cocrystal components. The addition of a solvent like ethanol in this case imparts significantly enhanced efficiency, higher yield and higher crystallinity of the product along with an increased ability to control polymorphic transitions.10,37 This is in addition to the comprehensive conversion of the reactants into the desired products with no intermediate and waste products. Also the ethanol added evaporates during the process itself with no leftover solvent that requires waste management.7,9,10,36,37 Besides the economization of energy, this procedure is an integral part of the "green chemistry" initiative with features like, almost trace amounts of solvent use, no waste or adduct formation and no subsequent requirement for recovery, storage and disposal of solvents and waste materials..6,9,10,45,46



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2.3. Identification and Characterization Differential Scanning Calorimetry The DSC thermogram of prepared co-crystals were obtained on a calibrated DSC Q20 differential scanning calorimeter (TA Instruments). A precise amount of sample (1-2 mg) sealed in aluminium pans using an empty pan as reference were heated in DSC cell from 50 oC to 350 oC at 5 oC/min rate under inert conditions maintained by dry nitrogen flow (50 cc/min) Powder X-Ray Diffraction (PXRD) The PXRD analysis performed on X'Pert PRO diffractometer (PANalytical, Netherlands) used Cu Kα radiation at 1.54060 oA with tube voltage and power at 45 kV and 40 mA, respectively, while divergence slit and antiscattering slit were kept at 0.48o. A dry sample (about 100 mg), in an aluminium sample holder, was analyzed by a continuous scan between 3o and 50o of 2θ values with step size of 0.017o and step time as 25 sec/step. Fourier Transform Infrared Spectroscopy (FT-IR) The FT-IR studies were done on calibrated RX I FT-IR spectrometer (Perkin Elmer, United Kingdom), in diffuse-reflectance mode. The dataset of the samples dispersed in a dry KBR pellet, was collected over the range of 4000-400 cm-1 and processed by Spectrum software (Perkin-Elmer). Solid State NMR analysis The solid state

13

C NMR analysis of hesperetin, coformers and HESP-PICO, HESP-

NICO and HESP-CAFF was carried out on Jeol Resonance JNM-ECX400II Instrument at Indian Institute of Science, Bangalore, India. Crystal structure determination a. Single crystal X-ray diffraction The single crystal X-ray diffraction datasets for HESP-PICO were collected on Bruker AXS Kappa APEX II diffractometer with CCD detector (crystal-to-detector distance as 60 mm) and sealed-tube monochromated Mo-Kα radiation (0.71073 Å) at room temperature (296 K) The computer program APEX2 controlled the crystal centering, unit


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cell determination, refinement of the cell parameters and data collection. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. Data were also corrected for absorption effects using the multi-scan method (SADABS).47 The structure was solved by direct methods using SHELXS-97 and refined against F2 using SHELXL-97.48-50 All calculations were performed using the SHELXTL V 11.0 suite of programs. There were no residual peaks >1e/Å3. All hydrogen atoms were placed in ideal positions and refined as riding atoms with individual isotropic displacement parameters. Crystallographic parameters and basic information pertaining to data collection and structure refinement for all compounds are summarized in Table 1. All figures were drawn using Mercury V 3.251 and hydrogen bonding parameters were generated using PLATON.52 The final positional and thermal parameters for HESPPICO are listed in the CIF file. b. Structure by utilizing the powder XRD pattern The structure of co-crystals HESP-NICO and HESP-CAFF was determined by utilizing the PXRD pattern of cocrystal in Powder Solve module of Materials Studio® (by BIOVIA) The structure determination was initiated separately for both the co-crystals. Beginning with a search and identification of the useful peaks of the experimental PXRD pattern, the software then indexes these peaks in XCELL module to generate the relevant unit cell with the appropriate spacegroup. The subsequent unit cell is refined by Pawley refinement that uses experimental PXRD pattern as the reference to obtain a structure with minimum Rwp. Then, molecular structures of hesperetin and the coformers were sketched using the drawing tool and optimized by DMOL3 module calculations to produce optimized minimum energy structures. These optimized structures were imported into the unit cell with lowest Rwp and this conglomerate was subjected to POWDER SOLVE module calculations which took considerable time and used the 'direct space MonteCarlo simulatedannealing approach' with full-profile comparison method. This process was run for 10 cycles consisting of 510000 steps in each cycle. The solution structure thrown up by these calculations had the maximum possible concurrence between



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calculated and the experimental PXRD patterns (as denoted by a lowest possible value of Rwp). Finally, to further reduce the Rwp and increase the agreement with the experimental data, the solution of Powder Solve module was optimized by Reitveld Refinement calculations. All the crystallographic data (except the structure factors) for HESP-PICO, HESP-CAFF and HESP-NICO have been deposited with the Cambridge Crystallographic Data Centre. 2.4. Evaluation of equilibrium solubility and dissolution profile Since hesperetin is majorly absorbed from the intestine therefore, the co-crystals were evaluated for solubility and dissolution in aqueous phosphate buffer pH 7.2 (I.P.). However, in view of the extremely poor aqueous solubility of hesperetin, and to avoid using a cosolvent like ethanol, for dissolution carried out in large amounts of medium, a modified shake flask method was used. This involved shaking an excess of sample (25 mg) in 5 mL of the buffer contained in stoppered conical flasks in a mechanical water bath shaker at constant rate of 100 rpm and bath temperature at 37oC. Sampling intervals were fixed at 10, 30, 60, 90, 120, 150, 180 and 240 mins and 24 hours with 0.1 mL aliquots withdrawn and replaced with equal amount of buffer. After suitable dilution with HPLC grade methanol, the samples were filtered through 2 mm nylon filters and then assayed for hesperetin content by HPLC. After 24 hours, the residual material in each flask was filtered and analyzed by FT-IR.6,53 2.5. Biological Studies The protocols of animal experiments were approved by the Institutional Animal Ethics Committee of Panjab University, Chandigarh, INDIA (vide ref. no: PU/IAEC/S/14/85). A. DPPH Free radical-scavenging activity Antioxidant molecules hydrogenate purple coloured 1,1-diphenyl-2-picryl hydroxyl (DPPH) free radical to form stable, yellow coloured 1,1-diphenyl-2-picrylhydrazine. This forms the basis of antioxidant potential evaluation. Herein, modified version of the Blois (1958) method was followed.54-57 A mixture of 1 mL of DPPH methanolic solution (10mM) and 1 mL of sample (predetermined concentrations of 10, 6, 5, 4, 2 and 1


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µg/mL in 30:70 methanol:water),

was shaken and

incubated for 30 min at room

temperature in a dark place. Subsequently, volume was made up to 5 ml with 50:50 methanol:water,

and

absorbance

noted

at

517

nm

in

UV–Visible

EZ201

Spectrophotometer (Perkin Elmer) against a blank with no sample. The percentage inhibition of free radical scavenging capacity is calculated as: % Free radical scavenging activity= (Absorbance of control – Absorbance of sample) X 100 (Absorbance of control)

The degree of discolouration depends on the scavenging or the antioxidant potential of the sample which increases proportionately with the solubility of antioxidant compound. B. Antihaemolytic activity Solutions: Retro-orbitally drawn rat blood (about 2 mL) into sterilized centrifuge tubes containing equal amount of sterilized Alsevar's solution was centrifuged at 3000 rpm for 15 min at 4oC. After discarding the supernatant, the packed RBCs at the bottom of the tube were thrice washed with isotonic phosphate buffer pH 7 PBS and finally suspended in the same buffer as 10 % v/v suspension. Procedure: The reaction mixture containing 0.5 mL of RBC suspension and 4.5 mL of the hypotonic phosphate buffer pH 7 containing the appropriate concentrations (50, 100, 150 µg/mL) of hesperetin or its co-crystals, was shaken and incubated at 37°C for 30 minutes. This was centrifuged at 3000 rpm for 15 min and supernatant was measured by UV-Visible spectrophotometer at 540 nm to determine the extent of haemolysis. Maximum control haemolysis was achieved by pure hypotonic PBS and percentage inhibition of

hesperetin and co-crystals was calculated as the extent to which the

haemolysis is prevented as compared to pure hypotonic PBS.57 C. Pharmacokinetic studies The pharmacokinetic parameters were evaluated in orally dosed, food but not water deprived (for 18h) male Sprague Dawley rats, each weighing 200-250 g (n = 5 per group). The hesperetin, HESP-PICO, HESP-CAFF and HESP-NICO were delivered via oral gavage at a dose equivalent 100 mg of hesperetin/kg body weight of animal as a suspension in aqueous 0.5 % sodium CMC. Subsequently, the blood withdrawn (approx 300µL) at intervals of 10, 30, 60, 90, 120, 180 and 240 min in heparinized centrifuge



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tubes was centrifuged at 10000 rpm for 20 min to separate the plasma. The plasma extraction was done three times with acetonitrile (3X50 µL) followed by methanol (1 X 50 µL). The commercially available computer program, Thermo Scientific Kinetica® (Thermo Fischer Scientific Inc.) along with Pk solver (add in software for Microsoft Excel) were used to determine the pharmacokinetic parameters including CMAX, TMAX, AUCtot, AUClast, T1/2 and the mean retention time (MRT).58 D. Anti-inflammatory activity The anti-inflammatory estimation was done by the 'carragenan induced paw edema model on male Sprague-Dawley rats (200-250g). Animals were divided into 5 groups (n=3), viz. negative control, hesperetin and three co-crystal groups with one for each form. The animals were deprived of only food and not water for the night before the experiment. The animals were administered an oral dose equivalent to 100 mg of hesperetin per kg body weight. After 30 mins of the oral dose, subplantar injection of carrageenan (0.1 ml of a 1% suspension in 0.85% saline) was given into the right hind paw. Paw volume was measured with a plethysmometer (Ugo-Basile, Varese, Italy) immediately prior to the injection of carrageenan and thereafter at time points 0, 60, 120, 180 and 240 mins. Edema was calculated as the percentage increase in paw volume after carrageenan injection relative to the pre-injection value for each animal.59 Percent inhibition = (1 - ∆Vtreatment / ∆Vcontrol ) × 100 where: ∆V= Vt - V0 Vt is the volume displaced by paw at the specific time V0 is the volume displaced at 0 hr ∆Vcontrol is the change in displacement of control group 2.6. HPLC analysis The

quantification

of

hesperetin

in

both

the

dissolution

samples

and

the

pharmacokinetic samples was done using the Waters Alliance® HPLC Systems (Waters Corp., Millford, MA., USA) equipped with a quaternary pump system and a photo diodearray detector. The separation was carried out on Sunfire® C18 column (3.5 µm, 4.6 mm X 150 mm) by Waters Corporation (Milford, MA., USA) and used in conjunction with Phenomenex RP-C18 guard column (California, U.S.A.). Isocratic Solvent system


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comprising methanol: acetonitrile: 0.1% (v/v) aqueous ortho-phosphoric acid in ratio 40:10:50 respectively was pumped at flow rate of 0.8 ml/min with sample injection volume kept at 20 µL and hesperetin detected at 280 nm. The final chromatograms were interpreted using the EMPOWER® software. Fixed concentrations of 10, 20, 30, 50, 75 and 100 µg/ml of hesperetin in methanol provided the calibration curve (AUC vs/ fixed conc.)

to measure solubility samples whereas hesperetin spiked rat plasma

(concentrations of 0.1, 0.2, 0.4, 0.8, 1, 2, 4, 8 and 10 µg/mL of hesperetin) gave the calibration curve to measure pharmacokinetics 3. RESULTS AND DISCUSSION In a non covalent derivatization process here, the adhesive interactions have prevailed over the size/shape disparity of hesperetin and the coformers to generate co-crystals. The distinct melting point, unique and signature peaks in X-ray diffraction pattern and spectroscopic techniques are characteristics of the cocrystals. Hydrogen bonding interactions, between the complimentary functional groups of constituents, generates supramolecular synthons that presents a distinct, but ordered and homogenous crystal lattice to the cocrystals.

4,36,37,45

This is in stark contrast to the random arrangement of

molecules in a physical mixture which is heterogeneous in nature with individual crystal latttices retaining their identity.7,9,45 The new lattice arrangement of cocrystals is responsible for the change in the thermodynamic functions like free energy, entropy and enthalpy that leads to a unique and specific melting point among other characteristic physical properties.7-9,37,45 The clear advantage of preparing these co-crystals was ascertained by manifold increase in solubility, the improved values of various biopharmaceutical

parameters

including

bioavailability

and

the

enhanced

pharmacological response over pure hesperetin. 3.1. Differential scanning calorimetry The thermal behaviour of the co-crystals examined by DSC was represented by a definite sharp melting point that are distinct from hesperetin and the respective coformers. DSC endotherms of hesperetin (Fig. 1a, 2a and 3a), picolinic acid Fig. 1(b), nicotinamide (Fig. 2b), and caffeine Fig. 3(b) show the melting points to be 231.71 oC, 138.16 oC, 130.65 oC and 236.06 oC, respectively. The co-crystals HESP-PICO (Fig.



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1c), HESP-NICO (Fig. 2c), and HESP-CAFF (Fig. 3c) exhibit formation of sharp specific melting endotherms at 197.84 oC, 143.98 oC and 157.79 oC respectively. The position, nature and shape of the melting endotherms, speaks volumes about the nature of the product formed. Firstly the distinctive and specific temperature at which these endotherms appear point towards the formation of a new product. Secondly the sharp and narrow endotherm indicates a high degree of crystallinity and purity of the prospective non covalent derivative. Thirdly the existence of single peaks (except in HESP-NICO) is representative of the homogenous, single phasic entity with no impurity that could include extraneous matter, the leftover starting materials and adducts or waste materials formed during the process. The HESP-CAFF cocrystal shows a melting point that is lower than that of the constituents. This is although a little less common but not unheard off. There are quite a few reports detailing cocrystals with lower melting points.31 Furthermore, another endothermic event in HESP-NICO cocrystal, at around 275oC is accompanied by the weight loss (nearly 30 %) in thermogravimetric analysis (TGA) of this cocrystal (Supporting information S2). This points towards the decomposition of the cocrystal at this high temperature and negates the polymorphic transition. The final identification and confirmation of cocrystal forms was however determined by the appearance of signature and characteristic peaks in spectroscopic methods.


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Fig. 1: DSC patterns of (a) Hesperetin, (b) Picolinic acid and (c) HESP-PICO

Fig.2: DSC Patterns of (a) Hesperetin, (b) Nicotinamide and (c) HESP-NICO



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Fig. 3: DSC patterns of (a) Hesperetin, (b) Caffeine and (c) HESP-CAFF

3.2. PXRD analysis PXRD is kind of a fingerprint characterization method for solid phases like co-crystals and salts since a different pattern of products implies the formation of a new phase. Existence of new phases as suggested by DSC were confirmed by the powder X-ray diffraction, on the basis of their novel patterns in contrast to hesperetin and the coformers. The powder XRD analysis of HESP-PICO (Fig. 4e) exhibits characteristic reflections at 14.54o, 16.27 o, 16.39o, 19.55

o

positions (2θ) which are absent in

hesperetin (Fig. 4a) and picolinic acid (Fig. 4b). Similarly, HESP-NICO co-crystals (in Fig. 4f) shows characteristic new reflections at 18.64o, 20.08o, 20.55o and 27.69o positions (2θ) that were different from string molecules. Similarly the hesperetin-caffeine cocrystal in Fig. 4g shows distinct diffraction reflections at 11.89o, 13.14o and 13.54o positions (2θ) which were absent in the hesperetin and caffeine. 3.3. FT-IR analysis Since PXRD data is not quite enough to differentiate between hydrates, solvates, and co-crystals, therefore methods like FT-IR and solid state NMR become relevant in these


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situations. The vibrational characteristics of

various atoms in the cocrystal

microenvironment due to changes in physical state as well as the formation of novel and new hydrogen bonding interactions are unique to every new cocrystal, and measured by the infrared spectroscopy.6 The supramolecular synthon formed in the cocrystal and stabilized by these hydrogen bonds involve functional groups on both hesperetin and the respective coformers. Now, these functional groups in the pure sample of single substance throw up infrared absorption patterns at a specific value which undergo a considerable alteration when hydrogen bonding starts acting on them to form supramolecular synthons that are bedrock of co-crystallization. Thus a distinct characteristic infrared pattern, apart from indicating towards new crystalline forms also confirms and reveals about the functional groups and the position of the hydrogen bonding. In hesperetin (Fig. 5a). two bands in the alcoholic region points to possibility of both intermolecular and intramolecular hydrogen bonding at 3497 and 3117 cm-1 respectively The positions of vibrational frequencies due to supposed hydrogen bonding in HESPPICO (Fig. 5e) are distinct and different from their original positions in hesperetin (Fig. 5a), and picolinic acid ( Fig. 5b). The hydroxyl (-OH) stretch at 3117 cm-1 and carbonyl (C=O) stretch at 1640 cm-1 in hesperetin rearrange to 2973 and 1635 cm-1 respectively in HESP-PICO. In case of HESP-NICO, the OH hydroxyl stretch at 3117 cm-1 in hesperetin with amidic nitrogen (-NH2) stretch at 3162 cm-1 in nicotinamide (Fig. 5c) throw up a combined new band at 3141 cm-1 in HESP-NICO (Fig 5f). Also, the phenolic OH stretch at 3497 cm-1 in hesperetin along with amidic carbonyl (-C=O) stretch at 1688cm-1 and C=Naromatic stretch at 1613 cm-1 in nicotinamide show shifts to 3414, 1686 and 1632 cm-1 respectively in HESP-NICO, thus establishing a hydrogen bond between phenolic OH on one side and amidic C=O and Naromatic of nicotinamide on another. The existence of hydrogen bonding in HESP-CAFF (Fig. 5g) is indicated to be between phenolic OH in hesperetin and Naromatic of imidazole moiety of caffeine as the maxima due to OH stretch at 3117 cm-1 and C=N stretch in caffeine at 1547 cm-1 (Fig. 5d) have shifted to 3291 and 1553 cm-1 respectively in HESP-CAFF.



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Page 18 of 50

3.4. Solid State NMR analysis The solid state

13

C NMR spectral analysis in conjunction with crystal structure

determination by PXRD acts as conclusive proof of formation of co-crystals in those cases where a single crystal suitable for single crystal XRD analysis are not obtained. The structural information interpreted from the unique and characteristic peaks in solid state

13

C

NMR spectroscopy provide much relevant information on realignment in

hydrogen bonding, molecular conformation, flexibility and mobility as well as the short range order in produced crystalline solids.6 The solid state NMR analysis patterns of hesperetin, nicotinamide, caffeine and the two co-crystals, HESP-NICO and HESPCAFF are represented in Fig. 6a, Fig. 6b,Fig. 6c, Fig. 6d and Fig. 6e respectively. Perturbations at 163.8, 170.6, 146.7 ppm corresponding to carbon atoms on positions 5,7 and 3' of hesperetin (Fig 6a) and the peaks at 152.5, 149.2 ppm corresponding to carbons adjacent to the pyridine N (Naromatic) in nicotinamide (Fig. 6b) show shifts to 164.4, 169.0, 145.3 and 154.6, 148.7 ppm, respectively in HESP-NICO (Fig. 6d). These novel peaks raise the prospect of hydrogen bonding between hydroxyl (OH) groups on hesperetin at C5, C7 and C3' with (Naromatic) in nicotinamide forming a O-H....Naromatic heterosynthon. Similarly, the peaks at 143.4 and 148.4 ppm corresponding to carbons adjacent to the Naromatic in the imidazole ring of purine caffeine (Fig. 6c) and the peak at 146.7 ppm representing C3' of hesperetin (Fig. 6e) result in two peaks at 142.1 and 147.3 ppm (broad bifurcated peak with merger of two peaks at 148.4 and 146.7 ppm) respectively in pattern of HESP-CAFF (Fig. 6e) thus lending credibility to existence of hydrogen bonding between hydroxyl group (-OH) on C3' of hesperetin and Naromatic of imidazole ring of caffeine to form [-O-H...Naromatic] heterosynthon. Evidence of fresh hydrogen bonding by carbonyl group (C=O) on C4 and the hydroxyl group (-OH) on C7 in hesperetin is provided by the shifts in peaks due to these carbon atoms from 198.4 and 170.6 ppm to 196.4 and 165.4 ppm respectively in HESP-CAFF. However, no shift in the position of C5, even though the hydroxyl group on it is involved in hydrogen bonding can be explained by the maintenance of existing intramolecular interactions.


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Fig. 4: PXRD pattern of (a) Hesperetin, (b) Picolinic acid, (c) Nicotinamide, (d) Caffeine, (e) HESPPICO, (f) HESP-NICO and (g) HESP-CAFF



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 5: FT-IR pattern of (a) Hesperetin, (b) Picolinic acid, (c) Nicotinamide, (d) Caffeine, (e) HESP-PICO, (f) HESP-NICO, (g) HESP-CAFF


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13

Fig. 6: Solid State C NMR spectra of (a) Hesperetin, (b) Nicotinamide, (c) Caffeine, (d) HESPNICO and (e) HESP-CAFF

3.5. Crystal structure determination The determination and analysis of the crystal structures of the prepared new non covalent derivatives has confirmed these to be co-crystals in definite stoichiometry and stabilized by hydrogen bonding interactions. The hydrogen bonding involves a hydrogen donor moiety like OH- present as hydroxyl (or phenolic group as in hesperetin) alone or as part of acid, (-COOH) (as in picolinic acid) and NH2 present as amine alone (as in



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Page 22 of 50

caffeine) or as a part of amide (-CONH2) (as in nicotinamide). These interact through a hydrogen bond with hydrogen acceptors primarily represented by ketone (C=O), carboxyl (COO- ) and -Naromatic (as in picolinic acid and nicotinamide and caffeine). This replacement is done according to the Etter's rule of best donor-best acceptor pairing of hydrogen bonds.6,37,45 O

H

O

N

O

H

O

carboxylic acid - pyridine heterosynthon

'A

successful

recrystallization

of

H H

O N

carboxylic acid -carboxamide heterosynthon

HESP-PICO

involves

the

solubilization

of

mechanochemical product in a warm ethanol to generate a supersaturated solution. The crystal of HESP-PICO that was isolated was large and robust enough to be analyzed by single crystal X-ray diffraction. However, many times, a suitable single crystal is not obtained from the bulk product due to the mismatch in the solubilities of the cocrystal components in the appropriate solvent. These situations demand the radical approach of directly determination of structure from powder X Ray diffraction data sets. It imparts basically the same information as single crystal diffraction data, but is compressed into one dimension against the usual three in latter. Therefore, it has potential to elucidate the structure of microcrystalline HESP-CAFF and HESP-NICO obtained from liquid assisted grinding. The structure determined by Powder solve calculations is further supported with the data from FT-IR and 13C solid state NMR. Hesperetin structure (Fig. 7a and b) elucidated by Fuji et al. in 1994 crystallized in the monoclinic space group P21/c with cell dimensions: a = 12.464(2)Å, b = 16.226(3)Å, c = 7.102(1)Å, α = 90°, β = 104.24(2)°, γ = 90°. Intermolecular hydrogen bonding between OH on C7 of one molecule and the OH on C5 of other is exhibited.60


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Fig. 7(a): Hesperetin molecule showing hydrogen bonding interactions

Fig. 7(b): Hesperetin molecule as present in the monoclinic unit cell

The single crystal X-ray analysis of HESP-PICO (Fig. 8a) reveals it to exist in triclinic unit cell with P -1 spacegroup. (Fig. 8a)



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Fig. 8(a): Hesperetin and picolinic acid in the unit cell of HESP-PICO

Herein, interestingly the picolinic acid existing in its zwitterionic state has the carboxylate group as hydrogen acceptor and protonated pyridine N (Naromatic)

as

hydrogen donor. The hydroxyl group (OH) on C3' of hesperetin forms a bifurcated hydrogen bond to bind to both oxygen atoms in the carboxylate ion (-CO...O--) of zwitterionic picolinic acid. One of this carboxylate oxygen is further hydrogen bonded to protonated pyridine moiety of another picolinic acid which in turn has its corresponding carboxylate oxygen, bonded to protonated pyridine of first picolinic acid. The carboxylate ion of zwitterionic picolinic acid is also attached to hydroxyl (OH) on C7 of another hesperetin molecule.(Fig. 8b)


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Fig. 8(b): Hydrogen bonding between hesperetin and picolinic acid in HESP-PICO

Moreover, the supramolecular homosynthon (OH...OH) between hydroxyl groups on C5 of one and C7 of another in the structure of pure hesperetin, rearranges in cocrystal HESP-PICO to give rise to a new supramolecular heterosynthon (C=O....OH-) due to interactions between C4 of one and C7 of another hesperetin molecules. This synthon formation creates a head to tail bonding and creating hesperetin chains that are connected by picolinic acid bridges. (Fig. 8c)



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Fig. 8 (c): Hydrogen bonding of hesperetin with picolinic acid in HESP-PICO.


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The structure of HESP-NICO (Fig. 9a) crystallized in monoclinic space group P-21. (Fig. 9b)

Fig 9(a). Asymmetric unit in HESP-NICO

Fig. 9(b). Unit cell of HESP-NICO

Both the unit cell and the final structure showed excellent agreement with the experimental data, with Rwp of unit cell after Pawley refinement at 9.33 and the value of the final structure after Reitveld refinement at 28.53. The atomic arrangement of HESP-NICO is represented by the Figures 9 (c to e). Herein, primarily, Naromatic and amide carbonyl (-C=O) of nicotinamide as principal hydrogen acceptors whereas the amidic -NH2 and hydroxyl (OH) of hesperetin function as hydrogen donors. The hydroxyl group on C3' of hesperetin forms a bifurcated hydrogen bond with amidic carbonyl of nicotinamide to form [O-H....O=C] heterosynthon on one hand and another with amidic amino (NH2) of yet another nicotinamide molecule to form [O...H-NH] heterosynthon. Further, this amidic amine also pairs with pyridine nitrogen (Naromatic) of yet another nicotinamide molecule to establish [N-H....Naromatic] heterosynthon. The Fig. 9c explains these new heterosynthons of hesperetin with 3 separate nicotinamide molecules. The hesperetin molecules are attached in a head to tail fashion by maintaining the pre-existing intermolecular hydrogen bonding between hydroxyl groups (-OH) on C5 of one and C7 of another hesperetin molecule. (Fig. 9d) Nicotinamide molecules act as bridges between the horizontal long chains of hesperetin in HESP-NICO. (Fig. 9e)



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Fig. 9(c). Hydrogen bonding between hesperetin and 3 separate nicotinamide molecules giving rise to three new heterosynthons.

Fig. 9(d). Stacking of hesperetin molecules in the HESP-NICO structure


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Fig 9 (e). Chains of hesperetin bridged by nicotinamide in HESP-NICO

The PXRD pattern was in turn simulated from this determined structure. This pattern is quite similar to the experimental pattern as well as the powder pattern obtained by stripping away of kα2 peaks followed by smoothening of Comparison is shown in Fig.9f.


experimental PXRD.


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Fig. 9(f). The comparison of the experimental, processed and simulated from the determined structure PXRD patterns of HESP-NICO.


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The simulated pattern and the experimental PXRD pattern of HESP-NICO along with the difference is shown in Fig. 9g. The similarity between the two is explained by the low values of Rp and Rwp.

Fig. 9(g). The simulated and experimental PXRD patterns of HESP-NICO along with their difference.

The cocrystal, HESP-CAFF (Fig 10.a) existing in the triclinic unit cell with P-1 space group had the final Rwp value of 13.16 after Reitveld refinement. (Fig. 10b).

Fig. 10(a): Assymmetric unit representation of hesperetin and caffeine in HESP-CAFF



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Fig. 10 (b): Unit cell of HESP-CAFF

HESP-CAFF crystal structure consists of alternating parallel chains of hesperetin molecules bridged by caffeine. The linear chain arrangement of hesperetin molecules arranged in head to tail fashion has hydrogen bond interactions between hydroxyl group on C7 and the ketone group on C4 of hesperetin.(Fig. 10c)

Fig. 10(c) The chain of hesperetin molecules in HESP-CAFF


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The aromatic nitrogen (-N) of imidazole moiety in caffeine is hydrogen bonded with hydroxyl (-OH) on C3' of one hesperetin whereas the keto(=C) on C6 is hydrogen bonded to phenolic hydrogen on C5 of second hesperetin molecule. (Fig. 10d). The two hesperetin molecules bridged by two caffeine molecules are in turn connected to another hesperetin molecules forming a kind of ladder like structure. Herein, hesperetin chains form the support columns of the ladder and the bridging caffeine molecules forms rungs of the ladder. (Fig. 10e)

Fig. 10(d). Hydrogen bonding between hesperetin and caffeine



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 10 (e). Layered packing arrangement of HESP-CAFF showing chains of hesperetin bridged by caffeine molecules

The simulated PXRD generated from the determined structure of HESP-CAFF is quite similar to the experimental PXRD and the pattern obtained after stripping away of the kα2 peaks followed by smoothening. (Fig. 10f)


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Fig. 10(f). The comparison of the experimental, processed and the simulated from determined structure, PXRD patterns of HESP-CAFF.



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Page 36 of 50

The Fig 10(g) represents the simulated and the experimental PXRD along with the difference between them.

Fig 10(g). The simulated PXRD and the experimental PXRD of HESP-CAFF along with the difference

Table 1: Crystallographic Parameters of hesperetin, HESP-PICO, HESP-NICO and HESPCAFF. Form Parameter

CCDC Number Formula

Hesperetin

HESP-PICO

HESP-NICO

HESP-CAFF

1300986

1503858

1503935

1503932

C16 H14 O6 C22 H19 N O8 C22 H20 N2 O7

C24 H24 N4 O8

formula weight

302.07

425.38

424.4

496.47

Stoichiometry

-

1:1

1:1

1:1

296

296

296

Temperature (K) wavelength (Å)

-

0.71073

1.5046

1.5046

crystal system

Monoclinic

triclinic

monoclinic

triclinic

Spacegroup

P 21 / c

P -1

P -21

P -1

a (Å)

12.46

7.438 (9)

16.98

14.00

b (Å)

16.22

8.527(11)

13.36

12.39

c (Å)

7.10

15.007(19)

6.07

7.94


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


α (deg)

90

85.390(17)

90

97.11

β (deg)

104.24

77.245(14)

95.16

100.90

γ (deg)

90

87.120(16)

90

100.58

Volume (Å3)

1392.18

925(2)

1372.81

1312.17

D g/cm3

1.3754

1.528

1.027

1.257

Z

4

2

2

2

µ (mm−1 )

0.118

0.581

0.722

θ range (deg)

1.39-25.00

3.5-49.98

3.5-49.98

444

444

520

F (000)

-

range h

-8 to 8

8 to 0

0 to 6

range k

-10 to 10

6 to 0

-6 to 6

range l

-18 to 17

-3 to 3 -

-3 to 3

reflections collected

-

10306

-

3202

-

-

Observed reflections

-

2857

-

-

No. of parameters

-

288

-

-

-

0.0527/0.0583

-

-

-

0.1438/0.1510

-

-

Goodness of Fit

-

0.996

1.26

1.34

Rp

-

-

22.54

9.78

-

28.53

Independent reflections

R1 (I > 2σ(I)) / R1 (all data) wR2 (I > 2σ(I)) / wR2 (all data)

Rwp a

b

2

2 2

2 2 1/2

2

2

-

13.16 2

R1 = Σ||Fo| − |Fc||/Σ|Fo|. wR2 = [Σw(Fo − Fc ) /Σw(Fo ) ] , where w = 1/[σ (Fo ) + (aP) + bP], P = (Fo 2 + 2Fc )/3.

2

3.6. Evaluation studies The relevance of the triumph of co-crystallization as technique to make hesperetin more pharmaceutically acceptable is truly felt only when the co-crystals while overcoming the obstacles faced by hesperetin, do not show any decline in the biological efficacy as well. Fulfilling these crucial criteria, the co-crystals of hesperetin possess vastly superior



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solubility and dissolution rate that manifests into exceptionally valuable increase in all the biopharmaceutical parameters with bioavailability being the most essential, and the therapeutic effect measure by a slew of in vitro and invivo biological studies that measure the antioxidant and anti-inflammatory effect of hesperetin. A. Dissolution profile and equilibrium solubility studies The dissolution profile represents the rate at which the final concentration of a substance at chemical equilibrium, with an excess of undissolved substance, known as equilibrium solubility is achieved. The chemical equilibrium for practical purposes is taken at 24 hours. The dissolution profile measured upto 4 h (in Fig 11) depicts the increase in maximum solubility of HESP-NICO to 4.5 times, HESP-CAFF to 4 times and HESP-PICO to be 4 times the solubility of pure hesperetin in distilled water (literature value 23.78 µg/mL). The maximum concentrations achieved for HESP-NICO (94.02 µg/mL) and HESP-CAFF (90.2 µg/mL ) at 180 mins as well as for HESP-PICO (68.54 µg/mL ) at 150 mins started dropping down slowly but steadily to near about the levels shown by pure hesperetin : 23.78 µg/mL (Fig.12). This change in solubility is explained by the break down of co-crystals to its starting molecules on extended exposure to aqueous medium as is confirmed by the FT-IR analysis of residue. This peculiar effect known as parachute effect offers a comfortable time period window considered fair enough for the cocrystal to be absorbed into the systemic circulation before it releases the active constituent.8


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Fig. 11. Dissolution profile of hesperetin, HESP-PICO, HESP-NICO and HESP-CAFF

Fig 12. Equilibrium solubility (24h) of hesperetin, HESP-PICO, HESP-NICO and HESP-CAFF

B. Antioxidant activity One of the main pharmacological effects of hesperetin as an antioxidant and a free radical scavenger makes it useful in neuro-, cardio- and hepatoprotection. The biological free radicals are ably and effectively removed from the system by hesperetin only if it is able to reach the distant and proximal sites of their occurrence inside the body which to a large extent depends, apart from permeation, on solubility and



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dispersability in aqueous medium. The increase in antioxidant activity of hesperetin and the co-crystals measured as percentage inhibition of oxidation of DPPH free radical shows nearly 50 % enhancement in activity of HESP-CAFF followed by around 30% for HESP-NICO and 20% for HESP-PICO over that shown by pure hesperetin. (Fig. 13)

Fig. 13. Percentage inhibition of oxidation of DPPH radical by hesperetin and the co-crystals.

C. Antihaemolytic activity There are a range of free radical mediated reactions out of which the cell membrane destruction of red blood cells leading to haemolysis is quite common due to reactive oxidative species (ROS). The prevention of this damage manifested by hesperetin increases with the increase in solubility and is thus a quite helpful barometer to study the improved characteristics of its co-crystals. The Fig 14. lucidly depicts that the inhibition of haemolysis of rat RBC's by hesperetin is much lower as compared to the co-crystals. On an average for all tested concentrations, there was seen a significant decrease in haemolysis of rat RBC's with maximum of 60 % shown by HESP-CAFF, followed by a nearly 40 % decrease by HESP-NICO and about 30 % by HESP-PICO, over that for pure hesperetin. The decrease was further proportional to the increase in the concentration of the samples.


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Fig. 14: Antihaemolytic activity represented as the percentage inhibition of haemolysis by hesperetin and the co-crystals.

D. Pharmacokinetic studies The increase in solubility makes available more of hesperetin in the solution form from where it is much easily absorbed and thus directly has an impact on the amount entering

into

systemic

circulation.

This

further

translates

in

to

improved

biopharmaceutical parameters including maximum concentration (Cmax), time taken to reach this conc. (Tmax), and most importantly impacting the bioavailability. The relative bioavailabilities (calculated as: Frel = AUCtot of cocrystal / AUCtot of hesperetin) were found to be at 1.36, 1.57 and 1.6 for HESP-PICO, HESP-NICO and HESP-CAFF. The results are elucidated by concentration against time graph in Fig.16 and the pharmacokinetic

parameters

tabulated

in

Table

2.

Whereas,

the

maximum

concentration (Cmax) achieved was nearly 3 times for HESP-CAFF and HESP-NICO, it was 1.5 times for HESP-PICO as compared to pure hesperetin. The time taken to achieve this maximum concentration (Tmax) was reduced to 90 minutes for HESP-CAFF and HESP-NICO and to 120 mins for HESP-PICO as compared to 180 mins for hesperetin. The early arrival of a higher peak concentration is significantly better for a quick and an effective therapeutic effect.



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Fig. 15. Pharmacokinetic profile of hesperetin, HESP-PICO, HESP-NICO and HESP-CAFF. Table 2: Pharmacokinetic parameters of hesperetin, HESP-PICO, HESP-NICO and HESPCAFF with comparison of relative bioavailability of co-crystals compared to hesperetin. Parameters

Hesperetin

HESP-PICO

HESP-NICO

Tmax (min)

180

120

90

90

Cmax (µg/ml)

0.47

0.63

1.15

1.27

AUC last (µg/ml*min)

97.98

119.37

169.54

172.61

AUC tot (µg/ml*min)

126.65

173.44

199.75

203.10

264.24

179.07

175.29

1.36

1.57

1.60

MRT (min) 222.64 Relative bioavailability(Frel) = AUC tot(cocrystal) / AUCtot(hesperetin)

HESP-CAFF

E. Pharmacodynamic anti-inflammatory activity The implicated direct role of reactive nitric oxide free radicals (NO2/NO3),

oxygen-

derived free radicals and prostaglandins (PG) was investigated in carrageenan-induced model of acute hind paw inflammation. The inflammatory response elicited by the intraplantar injection of carrageenan is generally characterized by a time-dependent increase in paw oedema, neutrophil infiltration, and increased levels of NO2/NO3 and prostaglandin E2(PGE2) in the paw exudate. As an anti-inflammatory agent, hesperetin not only reduces the effect of free radicals but also reduces the edema. At all time points, the effect of pure hesperetin was overshadowed by the response shown by the co-crystals. As shown in Fig. 16 and Table 3, the maximum percentage inhibition of inflammation was seen generally after 240 minutes of carragenan injection and which reached an astounding 87 % for HESP-CAFF, 79 % for HESP-NICO and 72 % for HESP-PICO as compared the maximum inhibition of 60 % seen for pure hesperetin.


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These impressive values of inhibition of inflammation show a clear advantage of cocrystals as compared to pure hesperetin in achieving the desired pharmacological response. Table 3. The percentage inhibition of inflammation by hesperetin and co-crystals..

Time (min)

60 120 180 240

Hesperetin 36.95 53.84 60.15 61.19

Percentage inhibition of inflammation HESP-PICO HESP-NICO 51.63 65.21 56.83 69.23 65.23 75.78 72.01 78.73

HESP-CAFF 59.78 75.64 84.37 87.68

Fig. 16. Percent inhibition of inflammation of hesperetin, HESP-PICO, HESP-NICO and HESPCAFF

4. CONCLUSION The usefulness of hesperetin keeps the research scientists deeply interested to develop it as a clinically useful molecule utilizing newer and better technologies to overcome the limitations on use imposed by its critically poor solubility and bioavailability. This has been overcome with aplomb by successfully preparing the non covalently derived cocrystals of hesperetin using highly soluble non toxic therapeutically active coformers: picolinic acid, nicotinamide and caffeine. The crystal structures of the co-crystals determined either by single crystal XRD or from powder XRD are distinct and different from the parent molecules and clearly show establishment of hydrogen bond interactions between the OH (hydroxyl groups) on the C7, C5 and C3' of hesepritin with carbonyl(C=O), aromatic N(Naromatic) and amidic amino (CO-NH2) of the coformers. The



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success of this approach of co-crystallization to improve the solubility and optimize the bioavailability of hesperetin can be gauged by the massive improvements seen in solubility studies and further corroborated by the in vitro activities like anti-oxidant evaluation and antihaemolytic studies as well as the in vivo estimation of antiinflammatory activity. The pharmacological benefits attributed to the conformers (caffeine, nicotinamide and picolinic acid) as well as their low inherent toxicity have made these co-crystals of hesperetin particularly attractive for further work up. Further research can directly take up these co-crystals for disease or tissue targeted estimation in cell lines or organ studies leading to a clearer and better clinical evaluation. ACKNOWLEDGEMENTS The UGC-RFMS fellowship awarded to Kunal Chadha and financial support by DST, New Delhi (File number: SR/SI/OC-90/2012) are gratefully acknowledged. The instrumentation facilities provided under DST-FIST and UGC-CAS to the University Institute of Pharmaceutical Sciences, Panjab University, are also acknowledged. Special thanks for the use of X-ray facility at IISER Mohali. CONFLICT OF INTEREST None REFERENCES

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Figure S1, that is the graph of the powder size determination of powders passed through seive before use.

•

Figure S2, The thermogravimetric analysis (TGA) graph of HESP-NICO cocrystal



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For Table of contents page only: Co-crystals of Hesperetin: Structural, Pharmacokinetic and Pharmacodynamic Evaluation. Karan Vasisht*, Kunal Chadha, Maninder Karan,Yashika Bhalla, Renu Chadha, Sadhika Khullar, Sanjay Mandal.

Synopsis: Solvent drop grinding of hesperetin with picolinic acid, nicotinamide and caffeine using ethanol has generated co-crystals. After characterization by DSC, FT-IR, PXRD, and SSNMR, the crystal structure is also determined through SCXRD or from PXRD. An enhancement of nearly 4-5 times in aqueous solubility coupled with optimized pharamcokinetics shows a manifold increase in pharamcological action as well.


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Cocrystals of Hesperetin: Structural, Pharmacokinetic, and

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