Oral Bioavailability and Pharmacodynamic Activity of Hesperetin

Oral Bioavailability and Pharmacodynamic Activity of Hesperetin...

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Oral Bioavailability and Pharmacodynamic Activity of Hesperetin Nanocrystals Generated Using a Novel Bottom-up Technology Ganesh Shete, Yogesh B. Pawar,† Kaushik Thanki, Sanyog Jain, and Arvind Kumar Bansal* Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Punjab 160062, India S Supporting Information *

ABSTRACT: In the present study, nanocrystalline solid dispersion (NSD) was developed to enhance the release rate and oral bioavailability of hesperetin (HRN). NSD of HRN was prepared using a novel bottomup technology platform. It is a spray drying based technology to generate solid particles, containing drug nanocrystals dispersed in small molecule excipients. HRN and mannitol were used in a 5:5 ratio, and an average crystallite size of HRN in NSD with mannitol was found to be 137.3 ± 90.0 nm. An in vitro release study revealed a statistically significant release rate enhancement for HRN nanocrystals (46.3 μg/mL/min) as compared to that of the control (29.5 μg/mL/min). Further, a comparative oral bioavailability study of NSD and control in Sprague− Dawley rats established significant improvement in Cmax and oral bioavailability (AUC0−∞) by 1.79- and 2.25-fold, respectively, for HRN nanocrystals. The findings of oral bioavailability were corroborated by intestinal uptake and Caco-2 cell uptake studies, wherein HRN, when administered in nanocrystalline form, showed higher penetration in intestinal mucosa and higher uptake in Caco-2 cells. Finally, the therapeutic efficacy of HRN nanocrystals was tested by a reactive oxygen species (ROS) generation assay and carrageenan induced anti-inflammatory model. HRN nanocrystals markedly inhibited ROS generation in MCF-7 cells, and carrageenan induced inflammation in rats. The process of NSD formation was found to be based on classical nucleation theory wherein mannitol contributed to NSD formation by acting as a plasticizer and crystallization inducer, and by providing sites for heterogeneous nucleation/crystallization. KEYWORDS: hesperetin, nanocrystals, nanocrystalline solid dispersion, release rate improvement, bioavailability enhancement

INTRODUCTION Flavonoids are polyphenolic plant secondary metabolites ubiquitously found in citrus fruits.1 They occur naturally as glycosides and consist of flavones, flavonols, flavanones, and isoflavones.2 Hesperidin is one of the important flavanone glycoside whose aglycone, hesperetin (HRN), is linked to glucose and rhamnose sugars at position 7. Its rutinose (6-O-αL-rhamnosyl-D-glucose) group is hydrolyzed in the distal part of the intestine and the colon by colonic microflora with the help of enzymes such as α- and β-rhamnosidases. After this hydrolysis, HRN is released and absorbed in the lower part of the gut. Hydrolysis of the rutinoside to release the active compound is a rate limiting step in the availability and absorption of HRN.3 HRN is reported to exert multiple beneficial effects on the human body such as antioxidant,4 antiaging,5 anti-inflammatory,6,7 blood lipid- and cholesterollowering,8 anticarcinogenic,9 and estrogenic activities.10 HRN helps to maintain the health of the liver,11,12 prevents bone loss,13 and regulates the expression of cancer gene transcription.14 HRN suffers from poor aqueous solubility leading to its poor oral bioavailability.15−17 Various formulation approaches have been tried to enhance the dissolution performance and oral © XXXX American Chemical Society

bioavailability of this potentially useful antioxidant. Among these are particle size reduction,18 delivery as amorphous solid dispersion with povidone,15,16 and complexation with cyclodextrins.19 HRN is a Biopharmacetics Classification System (BCS) class II compound with an aqueous solubility of 1.4 μg/ mL18 and permeability of 4 × 10−4 cm/s.20 It is reported to be absorbed through active and passive routes. The dissolution time (Tdisso) is the minimum time required to dissolve a single drug particle under sink conditions. The Tdisso values for 50 μm and 500 nm HRN particles were found to be 1428.5 and 142.8 min, respectively (aqueous solubility, 1.4 μg/mL; dose, 135 mg).18,20−22 This indicated the “dissolution-rate limited” oral bioavailability for HRN. This also indicated the possible benefits of nanonization on the oral bioavailability of HRN. Nanonization involves the use of submicron or nanosized particles and has been commercially exploited in the pharmaceutical industry for the improvement of the bioavailability of poorly water-soluble drugs. Nanocrystal generation Received: October 13, 2014 Revised: February 16, 2015 Accepted: March 18, 2015


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Molecular Pharmaceutics

Figure 1. Chemical structures of HRN (left) and mannitol (right).

techniques are classified as “bottom-up” and “top-down” approaches. The “bottom-up” approaches involve the generation of nanocrystals by molecular aggregation using techniques like solvent precipitation. The top-down approaches rely on reducing the size of coarser particles to achieve nanocrystals using techniques like pearl milling, microfluidization, and high pressure homogenization.23 Formulating poorly water-soluble drugs as nanocrystals can solve their biopharmaceutical delivery problems of low oral bioavailability after oral administration, low penetration into the skin (low dermal bioavailability), large injection volume for intravenous administration, and undesired side effects after intravenous injection when using high concentrations of solubilizers. Oral bioavailability advantage of nanocrystals are due to mechanisms like (i) increased dissolution rate, (ii) increased apparent solubility, and (iii) increased adhesiveness to surfaces/cell membranes.24 Recently, our laboratory has developed a novel spray drying based process for the generation of nanocrystalline solid dispersion (NSD) of drugs. Nanocrystals are achieved in the presence of nonpolymeric excipients that act as crystallization inducers of the drug. Spray drying is used to generate a solid powder wherein nanocrystals of the drug are dispersed in the small molecule excipient.25 In the current work, we discuss the development of NSD of HRN in crystalline mannitol, wherein the latter acts as a crystallization-inducing-excipient. NSD was characterized by microscopic, thermal, and crystallographic techniques. Performance evaluation of NSD was carried out by in vitro release testing in relevant media and bioavailability assessment in female Sprague−Dawley rats. Finally, pharmacodynamic studies, i.e., anti-inflammatory activity, intestinal uptake, cellular uptake, and antioxidant activity, of HRN nanocrystals were conducted using suitable animal models and cell culture studies. Chemical structures of HRN and mannitol are shown in Figure 1.

3 mL/min, air atomization pressure of 0.95 kg/cm2, and vacuum of 100 mm of the water column. NSD of the HRNmannitol system was labeled as HRN-M, while the HRNmannitol physical mixture was labeled as HRN-M-PM. Furthermore, NSD of the HRN-mannitol system was also generated by adding 0.005% w/w coumarin 6 into the methanolic solution of HRN prior to spray drying. The rest of the NSD generation procedure remained the same. Coumarin 6, being chemically similar to HRN, preferentially tagged HRN nanocrystals in the NSD. This NSD was labeled as C6-HRN-M. Additionally, HRN was recrystallized from its methanolic solution in the presence of 0.005% w/w coumarin 6. The generated crystals were mixed with mannitol to obtain a physical mixture of 5:5 proportion. The physical mixture was labeled as C6-HRN-M-PM. Differential Scanning Calorimetry (DSC). Conventional DSC experiments were conducted on a DSC Q2000 (TA Instruments, Delware, USA) equipped with a refrigerated cooling system operating with Universal Analysis 2000 software version 4.5A. The sample cell was purged with dry nitrogen at a flow rate of 50 mL/min. DSC instrument was calibrated for temperature and heat flow using a high purity indium standard. Accurately weighed samples (3−5 mg) were scanned at a heating rate of 20 °C/min in crimped aluminum pans from 25 to 300 °C. All of the measurements were performed in duplicate. DSC Screening Studies. Amorphous HRN was generated in situ by heating the crystalline sample up to 245 °C at a heating rate of 20 °C/min and quenching to 25 °C at 20 °C/min. This sample was then scanned in DSC at a rate of 20 °C/min up to 300 °C. HRN-M-PM was prepared in 5:5 proportion. The sample was heated in a DSC pan up to 245 °C at a heating rate of 20 °C/min and quenched to 25 °C at 20 °C/min. This procedure generated amorphous HRN in the presence of mannitol. This sample was then scanned in DSC at a rate of 20 °C/min up to 300 °C. Thermogravimetric Analysis (TGA). TGA was performed using Mettler Toledo 851e TGA/SDTA (Mettler Toledo, Switzerland) operating with STARe software version Solaris 2.5.1. Drug samples (5−7 mg) were weighed and analyzed under nitrogen purge (20 mL/min) in alumina crucibles at a heating rate of 20 °C/min over a temperature range of 25−300 °C. All of the measurements were performed in duplicate. Powder X-ray Diffraction (PXRD). Powder X-ray diffraction (PXRD) patterns of samples were recorded at room temperature on a Bruker’s D8 Advance diffractometer (Karlsruhe, West Germany) with Cu Kα radiation (1.54 A), at 40 kV, 40 mA passing through a nickel filter with a divergence slit (0.5°), antiscattering slit (0.5°), and receiving slit (1 mm). The diffractometer was equipped with a 2θ compensating slit and was calibrated for accuracy of peak positions with corundum.

EXPERIMENTAL SECTION Materials. HRN, coumarin-6, Triton X-100, and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich, USA. Mannitol was supplied by Lobachemie Pvt. Ltd., India. Dulbecco’s modified Eagle’s medium, fetal bovine serum, and Hanks’s balanced salt solution (HBSS) were purchased from PAA Laboratories, Austria. Tissue culture plates were purchased from Tarsons, India, and 8-well culture slides were procured from BD Falcon, USA. All other reagents, materials, and solvents used were of analytical grade. Methods. Preparation of NSD. NSD of the HRN-mannitol system was prepared using a laboratory scale spray dryer (U228 Model, Labultima Ltd., Mumbai, India). A mixture of HRN and mannitol in 5:5 weight proportion was dissolved in methanol and water (7:3) to achieve a final composition of 2.0% (w/v) in a solvent mixture. The solution was spray dried at an inlet temperature of 70 °C, outlet temperature of 60 °C, feed rate of B

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plotting the cumulative percent drug released against time. Data were evaluated by application of Student’s t test for assessing statistical significance of differences. Cell Culture Experiments. Cells. MCF-7 (human breast cancer cells) were obtained from National Centre for Cell Sciences, Pune, India, and Caco-2 cells were obtained from ATCC, Manassas, VA, USA. The cells were maintained in complete medium containing Dulbecco’s modified Eagle’s medium (DMEM; PAA, Austria), 10% fetal bovine serum (FBS; PAA, Austria), and antibiotics (antibiotic−antimycotic solution; PAA, Austria). Stock cultures were maintained in the exponential growth phase by passaging them every 3 days with their growth medium in a 25 cm2 plastic flask (Tarson, Kolkata, India) at 37 °C in 5% CO2/95% air. Caco-2 and MCF-7 Cell Uptake Study. The cellular uptake of HRN was studied using C6-HRN-M and C6-HRN-M-PM. Caco-2 and MCF-7 cells were seeded in 96-well tissue culture plates (1 × 105 cells/mL) and incubated overnight for cell attachment. The culture medium was replaced with fresh medium containing C6-HRN-M and C6-HRN-M-PM and incubated with cells for 4 h. Following incubation, cells were washed 5 times with Hank’s balanced salt solution (HBSS) to remove noninternalized material, and the fluorescence images were acquired using CLSM. Antioxidant Activity in MCF-7 Cells. Oxidative stress in the form of intracellular reactive oxygen species (ROS) was determined via the H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate, also known as dichlorofluorescin diacetate) assay. There were four groups in this study: (1) control group, MCF-7 cells were untreated; (2) H2O2 group, MCF-7 cells were exposed to freshly prepared 50 μM H2O2 for 30 min; (3) HRN-M-PM treated group, MCF-7 cells were treated with HRN-M-PM formulation for 4 h and then exposed to 50 μM H2O2 for 30 min; and (4) HRN-M treated group, MCF-7 cells were treated with HRN-M formulation for 4 h and then exposed to 50 μM H2O2 for 30 min. For groups 3 and 4, MCF-7 cells (1 × 105 cells/well) were incubated with 0.1, 0.2, 0.5, 1, 2, and 5 μg/mL concentrations of HRN-M-PM and HRN-M for 4 h. Following incubation, cells were washed 5 times with HBSS. Then, cells of group 2, 3, and 4 were incubated with 50 μM H2DCFDA (dissolved in DMSO and filtered through a 0.22 μm filter) for 30 min. The cells of groups 2, 3, and 4 were further incubated with H2O2 [50 μM solution in physiological buffered saline (PBS)] for 30 min, and cells were again washed to remove H2O2 and lyzed with 0.1% Triton X-100, and the fluorescence intensity of the cell lysate was determined at 488 nm excitation and 530 nm emission (PerkinElmer LS50B, USA). HRN concentrations were plotted against fold-change in DCF (2′,7′-dichlorofluorescein) fluorescence with respect to the control. Additionally, the fluorescence images of cells were acquired using CLSM before 0.1% Triton X-100 treatment for qualitative evaluation of the antioxidant activity of samples. Ex Vivo Intestinal Uptake Study. CLSM was performed to access the uptake and localization of HRN into the intestinal region. Briefly, C6-HRN-M-PM and C6-HRN-M were administered orally in overnight fasted female Sprague−Dawley rats at a dose of 80 mg/kg as a suspension in double distilled water containing 0.5% w/v Na CMC. After 1 h of administration, rats were sacrificed, and the duodenal region was dissected and washed thoroughly with Ringer’s solution. Tissue samples were embedded in Tissue Freezing Medium (Leica Biosystems, Nussloch, GmBH, Germany) and frozen at

Samples were subjected to X-ray powder diffraction analysis in continuous mode with a step size of 0.01° and a step time of 1 s over an angular range of 3−40° 2θ. Three hundred milligrams of a powder mixture was loaded in a 25 mm holder made of poly methyl methacrylate (PMMA) and pressed by a clean glass slide to ensure coplanarity of the powder surface with the surface of the holder. The sample holder was rotated in a plane parallel to its surface at 30 rpm during the measurements. Obtained diffractograms were analyzed with DIFFRACplus EVA (ver. 9.0) diffraction software. The measurements were performed in triplicate. Particle Size Analysis. NSD samples were evaluated for their mean particle size and polydispersity index (PDI) by using Zeta Sizer (Nano ZS, Malvern Instruments, UK). Powder samples were added to water. The dispersion was sonicated for 3 min and analyzed for particle size. All of the values were taken as the average of 6 measurements. Microscopy. Powder samples were observed under a Leica DMLP polarized microscope (Leica Microsystems, GmbH, Wetzlar, Germany) equipped with a Linkam LTS 350 Hot stage. Photomicrographs were acquired using a JVC color video camera and analyzed using Linksys32 (version 1.8.9) software. The distribution of HRN particle size, taken as the length along the longest axis of the individual crystal, was plotted using 100 particles. Average size of the particles was determined from the size distribution plot. The surface morphology of powder samples was viewed under a scanning electron microscope (SEM) (S-3400, Hitachi Ltd., Tokyo, Japan) operated at an excitation voltage of 25 kV. The powder samples were mounted onto a steel stage using double sided adhesive tape and sputter coated with gold using ion sputter (E-1010, Hitachi Ltd., Tokyo, Japan) before analysis. Powder samples were also analyzed under transmission electron microscopy (TEM) (FEI TF-20; FEI, Hillsboro, Oregon). Briefly, HRN-M was added to water to form 1% w/v dispersion and was sonicated for 5 min. This dissolved the mannitol and dispersed HRN nanocrystals in water. Two drops of the dispersion were added onto carbon coated copper grids and allowed to dry under ambient conditions. The dried sample was analyzed under a microscope at 200 kV. Alternatively, powder samples were directly mounted on the carbon coated copper grids and analyzed. Fluorescence images were acquired using a confocal laser scanning microscope (CLSM) using the Olympus FV1000 confocal microscope. The parameters, namely, pinhole size, electron gain, neutral density filters, and background levels, were set up before the confocal experiment and were not changed throughout the measurements. In Vitro Release Profile. HRN-M-PM and HRN-M equivalent to 1.5 mg of HRN were introduced into a dialysis tube [Spectra/Por Float-A-Lyzer, dialysis tubes; molecular weight cut off (MWCO), 25000 g/mol; and diameter, 10 mm]. The dialysis tube contained 2 mL of pH 6.8 phosphate buffer (50 mM) containing 0.125% w/v sodium carboxy methyl cellulose (Na CMC). Dialysis tubes were introduced into the release medium containing 38 mL of pH 6.8 phosphate buffer (50 mM). The entire assembly was equilibrated to 37.0 ± 0.5 °C before the release study and covered by parafilm to avoid water evaporation. The amount of drug released was calculated at each sampling point by spectrophotometry measurement at λmax = 288 nm. The drug concentration was linearly related to the measured absorbance. Release profiles were constructed by C

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Figure 2. PXRD patterns of HRN and HRN-M. Peaks used for particle size measurement are shown by stars. The inset depicts peak broadening.

−30 °C in a Semiautomatic Cryostat (Medimeas Instruments, Ambala, Haryana, India). Circular tissue sections were prepared, mounted on glass slides using phosphate buffered glycerol, and visualized using CLSM. In Vivo Studies. Oral Bioavailability Study. The animal experiment was performed in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC/12/27). Female Sprague−Dawley rats ranging from 230 to 270 g were kept on fasting for 12 h before the start of the experiment and were allowed free access to water before and during the experiment. HRN-M-PM and HRN-M were suspended in double distilled water containing 0.5% w/v Na CMC and administered at a dose of 80 mg/kg of rat body weight via an oral gavage. Blood samples were collected from the retro-orbital plexus after 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h in heparinized microcentrifuge tubes. Plasma was separated immediately by centrifugation at 13,000 rpm for 10 min at 4 °C and stored at −20 °C until processed and analyzed. Plasma samples were extracted with methanol and quantified by a validated LC-MS/ MS method. Bioanalytical Method. The LC-MS/MS system consisted of an Accela Thermo Scientific HPLC system and a linear ion trap LTQ XL (Thermo Scientific, USA) mass spectrometer equipped with an electrospray ionization (ESI) source (Thermo Scientific, USA). Data acquisition was performed with Xcalibur 2.0.7 SP1 software. Chromatographic analysis was performed at 25 °C on a Thermo Electron Corporation Hypersil Silica column (50 mm × 4.6 mm). The mobile phase consisted of 0.1% formic acid in methanol. The flow rate was 0.5 mL/min, and the injection volume was 25 μL. The mass spectrometer was operated in negative mode. Quantification was obtained using single reaction monitoring (SRM) mode. Segment I: HRN was monitored at an m/z transition of 301 → 286, with collision energy of 30 eV. The MS parameters were as

follows: spray voltage, 4.6 kV; heated capillary temperature, 250 °C; sheath gas (nitrogen), 40 psi; auxiliary gas (nitrogen), 20 psi; and collision gas (helium) pressure, 4 kg/cm2. MS/MS operating conditions were optimized by infusion of the standard solution (1 μg/mL) of HRN into the ESI source via a syringe pump. Pharmacokinetic and Statistical Analysis. Various pharmacokinetic parameters were calculated from the mean plasma HRN concentration−time profiles using the Thermo Kinetica, software version 245 5.0 (Thermo Fischer Scientific). Statistical significance for pharmacokinetic parameters was compared using the paired t test assuming equal variances. The test was considered to be statistically significant if P < 0.05. Anti-inflammatory Activity after Oral Administration. The animal experiment was performed in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC/14/58). The anti-inflammatory activity of HRN-M and HRN-M-PM was evaluated using the carrageenan-induced paw edema method described by Winter et al.26 In brief, female Sprague−Dawley rats were fasted overnight before the start of the experiment. The animals were divided into three groups having six animals each (n = 6). HRN-M-PM and HRN-M were suspended in double distilled water containing 0.5% w/v Na CMC and administered at a dose of 80 mg/kg of rat body weight via an oral gavage to two groups. The control group was treated with vehicle only (0.5% w/v Na CMC solution via oral route). Edema was induced by subcutaneous injection of 100 μL of 1% carrageenan in normal saline solution into the left hind paw of rats 1 h after drug/ vehicle administration to all three groups. The paw volume was measured immediately before (basal) and after carrageenan injection up to 24 h using UGO Basile Plethysmometer Model 7140 (Ugo Basile Srl, Varese, Italy). Edema rate was calculated by subtracting initial paw volume from paw volume measured at a particular time point; and % inhibition was calculated from D

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Figure 3. DSC traces of crystalline HRN, amorphous HRN, and HRN-M.

edema rate (E) =

% inhibition =

Vt − V0 V0

Ec − Et × 100 Ec

RESULTS Solid State Characterization. The solid form of crystalline ̈ material) was confirmed by polarized light HRN (naive microscopy, PXRD, DSC, and TGA. HRN was found to be birefringent irregular shaped crystalline material with a particle size range of 4.82 ± 1.92 μm when analyzed by microscopy (data not shown). PXRD revealed the crystalline nature of HRN with characteristic peaks at 2θ values of 7.19°, 14.41°, 16.94°, 17.65°, and 20.89° in the diffractogram (Figure 2). TGA analysis revealed moisture content below 0.1% w/w (data not shown). Figure 3 shows DSC heating curves of crystalline and amorphous forms of HRN. HRN exhibited a single melting endotherm at a temperature (Tm onset) of 228.7 °C (157.1 J/ g) in the DSC scan. Amorphous HRN, generated by an in situ method exhibited a Tg at 72.7 °C. DSC Screening Studies for the Selection of Spray Drying Parameters. Quench cooling of HRN generated its amorphous form. A subsequent heating run revealed a glass transition (Tg) of 72.7 °C. No recrystallization (Tc) exotherm was evident for amorphous HRN (Figure 3). A heat−cool− heat scan of HRN was then carried out in the presence of mannitol (Figure 4). The first heating run showed melting of βD-mannitol at 164.4 °C followed by melting of HRN at 213.9 °C. The cooling run revealed only crystallization of mannitol at about 97 °C. In the second heating run, HRN showed a Tg at 59.2 °C associated with enthalpy relaxation followed by recrystallization at 103.4 °C. This was followed by melting of δ-D-mannitol at about 153 °C and then melting of β-D-mannitol at 164.2 °C. Melting of HRN in the second heating run was not observed. The heat−cool−heat cycle of mannitol alone indicated a melting at 164.3 °C in the first heating run. Mannitol recrystallized in the cooling run, and in the subsequent heating run, it showed a melting at 164.9 °C

this edema rate of the control and treated groups using the following equations:



Here, Vt = mean paw volume after carrageenan injection, V0 = mean paw volume before carrageenan injection, Ec = edema rate of the control group, and Et = edema rate of the treated group. Estimation of Tumor Necrosis Factor-α (TNF-α) in Plasma. TNF-α levels were estimated in rats’ plasma after 24 h of the carrageenan-induced paw edema study. Quantification was performed using the rat specific TNF-α enzyme linked immune sorbent assay (ELISA) method (RayBiotech Inc., Norcross, Georgia) according to the manufacturer’s protocol. Briefly, a 96-well plate coated with an antibody specific to rat TNF-α was used. The plate was washed with wash buffer and rat TNF-α standard, and study samples were added to plate, and biotinconjugate was added to all of the wells. The plate was incubated for 2 h at room temperature, on a shaker. The wells were washed with buffer, and horseradish peroxidase conjugated streptavidin was added to the plate. The plate was then incubated for 1 h at room temperature, on a shaker. The wells were washed with buffer, 3,3′,5,5′-tetramethylbenzidine substrate solution was added to the wells, and the plate was incubated in the dark for 10 min at room temperature. The samples were analyzed at 450 nm using a plate reader (BioTek Power Wave XS2, Vermont, USA) for quantification of TNF-α levels. E

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quantified by gas chromatography and was found to be about 400 ppm, which was below the recommended limits (less than 3000 ppm) mentioned in the International Conference of Harmonization (ICH) Q3C guideline.27 HRN-M showed a moisture content of about 0.3% w/w when measured by Karl Fischer titrimetry (716 DMS Titrino, Metrohm Limited, Switzerland). The entrapment efficiency (or the drug content) of HRN-M was determined using spectrophotometry measurement at λmax = 288 nm. The drug concentration was linearly related to the measured absorbance. The amount of HRN per unit gram of HRN-M was 0.512 ± 0.015 g (n = 3). This confirmed the uniformity of HRN and mannitol in HRN-M. HRN-M was stored over phosphorus pentoxide in a desiccator at ambient conditions. Characterization of NSD. Microscopy. Surface morphology of HRN-M was viewed using SEM (Figure 5a and b). HRN-M showed discrete particles of NSD powder with average size ranging from 2 to 50 μm. A single discrete NSD particle contained nanocrystals of HRN and micron sized crystals of mannitol at a ratio of 5:5. It may be seen that there are no distinct regions of HRN and mannitol and that the texture is rough. Figure 5c and d shows TEM images of HRN-M. To acquire Figure 5c, HRN-M was added to water and sonicated for 5 min. This dissolved the mannitol and dispersed HRN nanocrystals in water. Thus, Figure 5c shows HRN nanocrystals in the aggregated state. Figure 5d was acquired by directly mounting the HRN-M powder on carbon coated copper grids. Figure 5d shows HRN nanocrystals (darker spots) dispersed in the crystalline mannitol phase (lighter spots). The differences in the darkness of HRN and mannitol were observed due to their different molecular electron density. HRN has a flavonoidal ring structure and possesses more numbers of electrons than mannitol in a single molecule (Figure 1). This provides high electron density to the HRN molecule. However, mannitol lacks a ring structure and has lower electron density. Because of differences in electron density, HRN results in darker spots than mannitol in the TEM image. This formed the basis of differentiation between HRN and mannitol in their TEM analysis. DSC. NSD particles were confirmed to be crystalline by DSC analysis (Figure 3). No glass transition or recrystallization event was observed between 25 to 300 °C. DSC trace of HRN-M showed three well-separated and defined endothermic peaks corresponding to the melting of δ-D-mannitol (152.9 °C), β-Dmannitol (163.0 °C), and HRN (215.4 °C). The melting point depression in the HRN was attributed to the miscibility of HRN with mannitol, which is discussed in detail later. Generally, particle size reduction to a significant extent reduces heat of fusion as less energy is required for melting. Hence, HRN which was present in nanocrystalline form in HRN-M exhibited less heat of fusion (130.4 J/g) as compared to pure HRN (157.1 J/g). Particle Size Analysis. PXRD analysis of HRN-M showed peaks at 2θ values corresponding to that of initial pure HRN confirming the formation of the same crystalline form after spray drying. Additionally, characteristic peaks for mannitol were also observed. DSC and PXRD analysis suggested the absence of any amorphous impurity of either HRN or mannitol in HRN-M. As compared to the sharp lines in the diffraction pattern of crystalline HRN, corresponding diffraction peaks for NSDs were significantly wider. PXRD peak broadening could be

Figure 4. Heat−cool−heat DSC traces of HRN-M-PM. The inset depicts a zoomed-in version of the second heating cycle from 40 to 140 °C.

(data not shown). HPLC analysis confirmed that no degradation of HRN happened during the DSC experiments. Generation of NSD by Spray Drying. DSC screening studies for HRN and mannitol were carried out using ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. Details of DSC screening studies are provided in the Supporting Information. On the basis of DSC screening studies, the 5:5 ratio was selected for formulation development. Further, the HRN and mannitol batch in 5:5 proportion was spray dried by varying the spray drying parameters. Inlet temperature was found to have a significant effect on the crystallinity and crystallite size of HRN in NSD powder. This was in accordance with classical nucleation theory (CNT), which is discussed in detail later. HRN and mannitol NSD in 5:5 proportion were generated at four different inlet temperatures selected on the basis of DSC screening studies and CNT (60, 70, 80, and 90 °C). An inlet temperature of 60 °C resulted in the generation of partially amorphous HRN, while the completely crystalline phase was obtained above 70 °C. The crystallite size increased with increase in temperature. Thus, the 70 °C inlet temperature value provided the smallest size of crystallites with complete crystallinity, and hence, this temperature was selected for the generation of HRN-M, which was used for further studies. HRN-M was obtained as a powder with a yield of about 74%. Residual solvent content (methanol) in spray dried powder was F

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Figure 5. SEM (a and b) and TEM (c and d) images of HRN-M.

Zeta sizer than that calculated by PXRD data was attributed to nanocrystal aggregation. When HRN-M was added to water for analysis, the mannitol, being the hydrophilic component, dissolved rapidly in water. This led to dispersion of HRN nanocrystals in water, wherein nanocrystals aggregated owing to their high surface free energy.30 Experimental evidence for this phenomenon can be seen in Figure 5c, wherein aggregation of HRN nanocrystals was observed. Zeta sizer analysis is based on dynamic light scattering, which measured the aggregated nanocrystals as a single particle. Thus, it showed a higher size of HRN nanocrystals than that measured by PXRD. In Vitro Release Profile. Figure 6 shows in vitro release profiles of HRN-M-PM and HRN-M, respectively. Comparison

caused by multiple factors which include mechanical strain in the crystal lattice and crystal defects. These factors often occur for preferred orientations leading to different extents of peak broadening in different PXRD scans.28 The size of HRN crystals obtained from three PXRD scans, and the results from characteristic peaks of HRN were in close agreement with each other. This indicated that factors like crystal lattice strain and defects were not affecting peak broadening significantly. Further, the PXRD pattern of HRN-M-PM did not show any line broadening (data not shown). This confirmed that the peak width of HRN remained unaffected by the presence of mannitol. The possibility of polycrystallinity in HRN nanocrystals cannot be ruled out, though such evidence was not present in the TEM photograph. The peak broadening of HRN in HRN-M was attributed to the loss in the original degree of perfection of the crystallites and significant reduction in their size. Assuming that size reduction is the main reason for PXRD line broadening, as suggested by the TEM analysis, the Scherrer equation was used to estimate average crystallite size of HRN in HRN-M which is given as follows:29 τ=

Kλ Bt cos θ


where, τ is the crystallite dimension, K is the shape factor (0.9), λ is the wavelength, and βt is line broadening due to the effect of small crystallites, as measured by the full width of the PXRD peak at half-height.29 Specific diffraction peaks of HRN at 2θ values of 7.19°, 14.41°, 16.94°, 17.65°, and 20.89° were used for crystallite size analysis. Peak width at half maxima (PWHM) values for these peaks were determined. Particle size was calculated as the average of these five peaks. HRN in HRN-M exhibited an average crystallite size of 137.3 ± 90.0 nm. Zeta sizer analysis revealed an average size of 756.1 nm for HRN nanocrystals in HRN-M. The higher size observed in the

Figure 6. In vitro release profiles of HRN-M-PM and HRN-M. Solid lines represent the Korsmeyer−Peppas model fit to the experimental data. G

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Table 1. Pharmacokinetic Parameters of HRN Observed after Administration of HRN-M-PM and HRN-M after Single Oral Dose (80 mg/kg) to Female Sprague−Dawley Rats (n = 6) formulation HRN-M-PM HRN-M a

pharmacokinetic parameters (mean ± SEM) Cmax (ng/mL)

Tmax (h)

t1/2 (h)

AUC0−∞ (ng·h/mL)

297.3 ± 44.2 534.4 ± 51.5a

0.6 ± 0.1 0.8 ± 0.1

3.36 ± 0.48 3.6 ± 0.21

2074.9 ± 525.4 4680.1 ± 705.3a

P < 0.05, statistically significant difference in comparison with HRN-M-PM.

an average value was determined. Average dc/dt for HRN-MPM was found to be 29.5 μg/mL/min, while that of HRN-M was found to be 46.3 μg/mL/min. Tdisso of HRN reduced from 503.3 min (particle size, 4.82 ± 1.92 μm; apparent solubility in release study medium, 3.83 μg/mL) to 4.5 min (particle size, 137.3 ± 90.0 nm; apparent solubility in release study medium, 12.05 μg/mL). Oral Bioavailability Study. HRN-M showed a significant advantage in in vitro release rate over HRN-M-PM. To confirm whether this in vitro performance of nanocrystals was able to convert to in vivo oral bioavailability advantage, HRN-M was tested for the oral bioavailability along with a control of HRMM-PM. Table 1 shows pharmacokinetic parameters, and Figure 7 shows mean plasma concentration−time profile for HRN-M-

of the profiles showed the drug release rate enhancement obtained with HRN nanocrystals, as almost 29% of the drug was dissolved following 240 min as compared to 7% release for HRN-M-PM. Yet, complete release for HRN-M was not achieved even after 240 min. The difference in the extent of release between HRN nanocrystals in HRN-M and microcrystalline HRN in HRN-M-PM was found to be statistically significant by employing Student’s t test (p < 0.05). The experimental data were evaluated for well-known release kinetics models such as zero order, first order, and Higuchi and Korsmeyer−Peppas models.31,32 The release profile in both cases followed the Korsmeyer−Peppas model.32 The correlation coefficient (R2) for HRN-M-PM and HRN-M was found to be 0.967 and 0.992, respectively for the Korsmeyer−Peppas model. The Korsmeyer−Peppas model is used to describe the release of the solute when the prevailing mechanism is a combination of drug diffusion by Fickian as well as non-Fickian transport. The model is described by the following equation:32

Mt = Kt n M∞


where Mt/M∞ is the fraction of drug released at time t, K is the rate constant, and n is the diffusion exponent. According to this model, the value of n identifies the release mechanism of the drug. Values of n between 0.5 and 1.0 indicate anomalous transport kinetics, n approximately 0.5 indicates the pure diffusion controlled mechanism (Fickian diffusion). The smaller n values below 0.5 may be due to drug diffusion partially through a swollen matrix and water filled pores in the formulations.32 The value of n was found to be 0.505 for HRN-M-PM, which indicated the pure diffusion controlled mechanism (Fickian diffusion). However, n was found to be 0.949 for HRN-M. The closeness of the n value to 1 indicated zero order (concentration independent) release kinetics of HRN nanocrystals.32 The microcrystalline powder of HRN achieved a concentration of 3.83 μg/mL 24 h, at 37 °C. The increase in apparent solubility can be calculated from the Ostwald−Freundlich equation as follows:23


Cs 2σM = 2.303RTρr Cα

Figure 7. Mean plasma concentration−time profile for HRN-M-PM and HRN-M.

PM and HRN-M. The Cmax and oral bioavailability (AUC0‑∞) of HRN-M showed statistically significant improvement of 1.79- and 2.25-fold, over the control (HRN-M-PM). Against the expectation, a left shift in the Tmax for HRN-M was not observed, and similar Tmax values were observed for HRN-M and HRN-M-PM. This may be attributed to both active as well passive absorption of HRN from the gastrointestinal tract.33 Caco-2 and Intestinal Uptake of HRN Nanocrystals. The insights of higher oral bioavailability of HRN nanocrystals were obtained by Caco-2 and intestinal uptake studies. HRN internalization in Caco-2 cells was studied using CLSM. C6HRN-M and C6-HRN-M-PM equivalent to 10 μg/mL HRN were added to exponentially growing cells for 4 h. As shown in Figure 8a, intracellular concentration of HRN in the case of C6HRN-M was quite higher than that of C6-HRN-M-PM. After 4 h of incubation, higher extent of fluorescence was observed in the case of C6-HRN-M as compared to that of C6-HRN-MPM. The higher uptake of HRN was attributed to the better apparent solubility of HRN nanocrystals. Further, the higher


where Cs is the apparent solubility, Cα is the solubility of the solid consisting of large particles, σ is the interfacial tension of the substance with the medium, M is the molecular weight, R is the gas constant, T is the absolute temperature, ρ is the density of the solid, and r is the radius. As per the Ostwald−Freundlich equation, HRN nanocrystals were expected to provide an apparent solubility of 4.28 μg/mL. However, an experimental value of 12.05 μg/mL was achieved after 24 h in the case of HRN-M. Further, the in vitro release rate (dc/dt) was calculated by the amount of HRN released per unit time, for all time points, and H

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Figure 8. CLSM images of HRN uptake in (a) Caco-2 cells, (b) rat intestine, and (c) MCF-7 cells.

Figure 9. (a) Comparative concentration dependent antioxidant activity of HRN-M and HRN-M-PM in MCF-7 cells and (b) CLSM images of study samples for HRN-M-PM and HRN-M equivalent to 5 μg/mL HRN. I

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Figure 10. In vivo anti-inflammatory activity of HRN-M and HRN-M-PM, (a) % inhibition of paw edema and (b) post-anti-inflammatory TNF-α level in plasma after 24 h. Each data point is expressed as the mean ± SEM (n = 6).

apparent solubility of HRN nanocrystals followed by the greater extent of penetration of HRN in MCF-7 cells. Figure 8c demonstrates the penetration of HRN exposed as C6-HRN-MPM and C6-HRN-M in MCF-7 cells. Anti-inflammatory Activity. It has been reported that carrageenan produces inflammation as a consequence of free radical generation.37 In the present study, this effect was found to be suppressed by HRN. HRN-M showed a higher extent of inhibition than HRN-M-PM, and the difference in % inhibition was found to be statistically significant at 0.5, 1, 2, 4, and 6 h by employing Student’s t test. The inflammatory cascade associated with tissue injury results in the up-regulation of TNF-α and other cytokines.38 The TNF-α level in the plasma was measured after 24 h to endorse the anti-inflammatory activity of HRN. Carrageenan markedly increased the TNF-α level as compared to that of the negative control. Both HRNM-PM and HRN-M decreased the TNF-α level. The decrease in TNF-α level was more in the case of HRN-M and was statistically significant from HRN-M-PM. Figure 10 shows the effect of HRN-M-PM and HRN-M on carrageenan induced paw edema and TNF-α levels in the plasma.

oral bioavailability of HRN nanocrystals was corroborated by intestinal uptake studies (Figure 8b). HRN nanocrystals showed higher penetration into the mucosa and higher uptake in intestinal cells in solubilized form as compared to those of the HRN microcrystalline material. This was visually distinguished by higher fluorescence in the case of C6-HRNM as compared to that of C6-HRN-M-PM. Oral bioavailability and uptake studies proved the efficacy and superiority of HRNM over HRN-M-PM in terms of oral bioavailability advantage. Antioxidant Activity. ROS are chemically reactive molecules containing oxygen. They are formed as a natural byproduct of the normal metabolism of oxygen and play a vital role in cell signaling and homeostasis. However, during times of environmental stress, ROS levels can increase dramatically, which results in significant damage to cell structures. Cumulatively, this is known as oxidative stress.34 HRN is known to possess antioxidant activity, which was tested using the H2DCFDA assay.35 The cell-permeant H2DCFDA is a chemically reduced form of fluorescein. It is used as an indicator of ROS in cells. Upon cleavage of the acetate groups by oxidative stress causing agents like UV light, temperature, or chemicals such as H2O2, the nonfluorescent H2DCFDA is converted to the highly fluorescent DCF. Pretreatment of cells with the antioxidant drug lowers the conversion of H2DCFDA to DCF by reducing oxidative stress, thereby inhibiting the fluorescence. The extent of inhibition of fluorescence is the quantitative measure of antioxidant activity of the drug.36 As mentioned in the Experimental Section, the control group was untreated MCF-7 cells, the H2O2 group was MCF-7 cells exposed to H2O2, the HRN-M-PM treated group was MCF-7 cells pretreated with HRN-M-PM and then exposed to H2O2, and the HRN-M treated group was MCF-7 cells pretreated with HRN-M and then exposed to H2O2. The plot for the antioxidant activity of HRN-M-PM and HRN-M in MCF-7 cells is shown in Figure 9a. MCF-7 cells showed HRN concentration dependent reduction in H2O2-stimulated ROS generation. The difference between reduction in DCF fluorescence for HRN-M-PM and HRN-M was found to be statistically significant by employing Student’s t test. CLSM images of the antioxidant activity study are shown in Figure 9b. The H2O2 group exhibited DCF fluorescence as compared to that of the control group. The DCF fluorescence was reduced by both HRN-M-PM and HRN-M, indicating their antioxidant activity. However, the reduction in DCF fluorescence was more prominent in the case of HRN-M than HRN-M-PM, indicating the superior antioxidant activity of HRN nanocrystals. This effect was attributed to the higher in vitro release rate and

DISCUSSION Process of NSD Formation. HRN and mannitol were dissolved in a methanol−water (7:3) solvent mixture and spray dried; the solvents evaporate in the drying chamber of a spray dryer leaving behind HRN and mannitol. HRN is expected to sequentially attain amorphous and nanocrystalline states as per the Ostwald’s rule.39 This sequence of events was confirmed, as spray drying of HRN alone in a mixture of methanol and water (7:3) produced its amorphous form. The NSD formulation contains nanocrystals of HRN dispersed in the crystalline mannitol phase. Mannitol acts as a crystallization-inducingexcipient and encourages crystallization of HRN.25 As per the classical nucleation theory (CNT), nucleation is thermodynamically favored at lower temperatures close to glass transition, Tg, and crystal growth is favored at higher temperatures close to melting, Tm.40 Thus, the nanocrystals can be generated at conditions that encourage attenuated crystal growth with high nucleation rate. Such crystallization conditions can be achieved near the Tg of the system. DSC screening studies had revealed a Tg and Tc of HRN as 59.7 and 103.4 °C, respectively, in the presence of mannitol. The inlet temperature of 70 °C used for spray drying was expected to generate NSD because of its proximity to Tg (59.2 °C). In the NSD formation process, mannitol acted as a plasticizer by decreasing the Tg of HRN by 13.5 °C (72.7−59.7), thereby J

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CONCLUSIONS The spray drying based novel technology successfully generated nanocrystalline solid dispersion of HRN in the crystalline mannitol. Solid state characterization of HRN-M established an average crystallite size of 137.3 ± 90.0 nm of HRN. Mannitol acted as a crystallization-inducing-excipient by plasticization of HRN and providing sites for heterogeneous nucleation. HRN nanocrystals showed enhanced oral bioavailability due to higher release rate, apparent solubility, and penetration into gastric mucosa and intestinal cells as compared to those of the control. HRN nanocrystals resulted in higher antioxidant and antiinflammatory activities as compared to those of the control. The novel bottom-up technology discussed in this article provides a simple and robust platform for bottom-up generation of nanocrystals. Further investigations on this technology such as the role of excipients in the generation of nanocrystals, scale-up, and wider applications for enhancement of oral bioavailability of BCS class II and IV drugs are ongoing in our research group.

leading to enhanced molecular mobility of HRN and exerting a crystallization inducing effect. The plasticization effect was a result of miscibility between HRN and mannitol, as established by their close solubility parameter (δt) values.41−44 δt values for HRN and mannitol, as calculated by the Hildebrand method, were found to be 35.7 and 43.4, respectively. This miscibility encourages interaction between HRN and mannitol, wherein the latter induces crystallization in the amorphous phase of HRN. Mannitol is reported to possess high crystallization tendency.45 This further encouraged the crystallization of HRN during spray drying, as mannitol provided sites for heterogeneous nucleation. Heterogeneous nucleation is the spontaneous formation of nuclei of the drug on the solid particles of the crystallization-inducing-excipient, i.e., mannitol.25 Hence, mannitol exerted a crystallization inducing effect by lowering the Tg of HRN and by facilitating heterogeneous nucleation. Spray drying of HRN alone under similar conditions had produced amorphous HRN. In Vitro Release Rate and Apparent Solubility of HRN Nanocrystals. Increase in surface area for nanocrystals was one of the prominent reasons for increase in release rate and apparent solubility of HRN-M. Additionally, mannitol being the carrier for HRN nanocrystals, provided a highly hydrophilic environment around HRN nanocrystals that encouraged dissolution. This effect of mannitol has been reported earlier in the literature.45 The presence of Na CMC, a hydrophilic polymer, effectively prevented aggregation of nanocrystals during dissolution studies. In order to verify that the release rate/solubility enhancement was not due to the presence of the HRN amorphous form, evaluation of HRN-M was carried out using DSC and PXRD. A shift in the melting point of HRN in the DSC scan toward lower temperature (228.7 to 215.4 °C) was observed due to the miscibility of HRN and mannitol. No glass transition event was evident in the DSC scan (Figure 3). PXRD analysis confirmed that the spray drying operation retained the crystalline state as the diffraction pattern for HRN was conserved in the nanocrystals (Figure 2). Further, PXRD analysis revealed broadening of the diffraction peaks for HRN nanocrystals due to the size of crystals in the nanometer range. These results confirmed that the increase in release rate and apparent solubility was not due to the formation of the amorphous phase or the polymorphic transition of HRN. Pharmacokinetics and Pharmacodynamics of Formulations. The enhanced apparent solubility and release rate of HRN nanocrystals transformed into improved oral bioavailability. The improved oral bioavailability due to particle size reduction was in accordance with previous reports.46 The pharmacokinetic parameters after a single oral administration of HRN-M showed significant enhancement in Cmax and AUC0−∞. The findings of in vivo pharmacokinetics were further corroborated by intestinal and cell uptake studies. HRN, when administered in nanocrystalline form, showed higher uptake in intestinal mucosa and Caco-2 cells. Finally, the therapeutic efficacy of HRN nanocrystals was tested by antioxidant assay and the carrageenan induced anti-inflammatory model. HRN-M indicated distinctly superior pharmacodynamic activity in these studies. HRN nanocrystals markedly inhibited ROS generation in MCF-7 cells and carragenan induced inflammation in Sprague−Dawley rats.


S Supporting Information *

Additional methods and results; DSC scans of HRN-M-PM samples; thermal events of HRN and mannitol in the second heating run; crystallite size of HRN particles in HRN-M batches; dissolution profiles of HRN-M and HRN-M-PM; and CLSM images of the uptake study in Caco-2 and MCF-7 cells. This material is available free of charge via the Internet at http://pubs.acs.org.


Corresponding Author

*Tel: +91-172-2214682 ext. 2126. Fax: +91-172-2214692. Email: [email protected]; [email protected] Present Address †

(Y.B.P.) Proprietary Products, R&D, Innovation Plaza, Bachupally, Qutubullapur, R.R. District 500090, India.


The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the Department of Science and Technology, Government of India for providing financial support. G.S. and K.T. are grateful to the Council of Scientific and Industrial Research, Government of India for providing research fellowships. We thank Mr. Vikas Grover for LC-MS studies and Guru Raghavendra Valicherla for discussions in the initial phase of this work.


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