Enhanced Biopharmaceutical Performance of Rivaroxaban through

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Enhanced Biopharmaceutical Performance of Rivaroxaban through Polymeric Amorphous Solid Dispersion Sunita Metre, Sumit Mukesh, Sanjaya K. Samal, Mahesh Chand, and Abhay T. Sangamwar Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01027 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Molecular Pharmaceutics Page 1 of 43

Enhanced Biopharmaceutical Performance of Rivaroxaban through Polymeric Amorphous Solid Dispersion Sunita Metreψ, Sumit Mukeshψ, Sanjaya K. Samal, Mahesh Chand, Abhay T Sangamwar* Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, 160062, Punjab, India.

Ψ = Authors with equal contribution

*

Corresponding author

Abhay T. Sangamwar Tel: +91-0172 2214682; Fax: +91-0172 2214692 E-mail: [email protected], [email protected]

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

Page 2 of 43 Page 2 of 43

Abstract Rivaroxaban (RXB) is an orally active direct inhibitor of the activated serine protease Factor Xa, given as monotherapy in the treatment of venous thromboembolism (VTE). It has been characterized in-vitro as a substrate for the active, non-saturable efflux via P-gp transporter, limiting its high permeability. Therefore, role of P-gp inhibiting polymers in enhancing the biopharmaceutical performance of RXB by preparing polymeric amorphous solid dispersion and subsequent improvement in solubility and permeability was investigated. Initially, solubility parameter and Flory-Huggins interaction parameter were determined for miscibility studies between drug and polymers. Binary dispersions were prepared by dissolving drug with polymers eudragit S100, eudragit L100 and soluplus in common solvent (5% v/v water in tetrahydrofuran) using spray dryer. Prepared binary dispersions were analyzed by differential scanning calorimetry (DSC), microscopy, powder X-ray diffractometry (PXRD), Fourier transform infrared spectroscopy (FTIR), dynamic vapor sorption (DVS) and solution nuclear magnetic resonance (NMR) spectroscopy. Superior performance of binary dispersions was observed upon dissolution and solubility studies over micronized API. Amorphous solid dispersion (ASD) prepared with soluplus showed 10 fold increase in apparent solubility and maintenance of supersaturation for 24 hrs than the crystalline RXB. Further, pharmacokinetic study performed in animals was in good correlation with the solubility data. 5.7 and 6.7 fold increase was observed in AUC and Cmax, respectively for ASD prepared with soluplus than crystalline RXB. FTIR and NMR spectroscopy unveiled the involvement of N-H group of RXB with C=O group of polymers in intermolecular interactions. The decreased drug efflux ratio was observed for ASDs prepared with eudragit S100 and soluplus in Caco-2 transport study suggesting improvement in the absorption of RXB. Hence, the present study demonstrates ASD using soluplus as a promising formulation strategy for enhancing the biopharmaceutical performance of RXB by increasing the solubility and circumventing the P-gp activity.

Keywords: Rivaroxaban, Polymeric Amorphous solid dispersion, Apparent solubility, P-gp inhibition, Pharmacokinetic, Caco-2 permeability.


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Graphical Abstract




INTRODUCTION Rivaroxaban

(RXB),

chemically designated

as

5-Chloro-N-({(5S)-2-oxo-3-[4-(3-oxo-4-

morpholinyl) phenyl]-1, 3 oxazolidin-5-yl} methyl)-2-thiophene-carboxamide is an orally active direct inhibitor of the activated serine protease Factor Xa, given as monotherapy in the treatment of venous thromboembolism (VTE).1-3 It is marketed as oral film coated immediate release tablet under the brand name of Xarelto® in 10, 15 and 20 mg dose by Bayer Healthcare AG.4 RXB exists in 3 different polymorphic forms and form I is thermodynamically most stable polymorph and hence used in commercial formulation.3 It is non-ionizable and, practically insoluble in water with pH independent solubility of 5-7 mg/L at 25° C. RXB is lipophilic in nature, exhibiting high permeability across gastrointestinal tract (GIT) and hence classified as a BCS class II drug.2 However, it has been characterized in-vitro as a substrate for the active, nonsaturable efflux via P-glycoprotein (P-gp) transporter which limits its high permeability.5, 6 The presence of food results in the improved rate and extent of absorption of RXB (implying positive food effect), whereas drug exposure is reduced if released in the proximal small intestine and, even further reduced if released in the distal small intestine or ascending colon (site specific absorption).5, 7 Various formulation strategies such as amorphous co-precipitates,8 co-crystals,9 lipid solid dispersion,10 self microemulsifying drug delivery systems11 and, mesoporous dosage form12 have been explored to improve the limited solubility issues of RXB. Among the various techniques available to improve the solubility, amorphous solid dispersion (ASD) is a well known and most widely employed strategy for enhancing solubility/dissolution of poorly water soluble active pharmaceutical ingredients (APIs).13 Amorphous form exists in disordered structure and possesses higher free energy, enthalpy, and entropy compared to the corresponding crystalline state resulting in higher apparent solubility, dissolution rate and oral bioavailability.14 However, the excess thermodynamic properties of amorphous forms are associated with inherent physical instabilities, negating any solubility advantage.15 This necessitates the stabilization of amorphous form of an API by preparing molecular level dispersion with crystallization inhibiting polymers which are capable of lengthening the physical stability and maintenance of supersaturation in the aqueous environment.16 The matrix polymer traps the drug in metastable form through intermolecular interaction to prevent recrystallization from the supersaturated state, either through the formation of drug-polymer assemblies or by impeding nucleation and crystal growth.14, 17, 18


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To take the maximum advantage from the solid dispersion strategy, understanding of the drug solubility and drug-polymer miscibility in the matrix of choice is mandatory.19 Thermodynamic miscibility of drug and polymer governs the stabilization of amorphous drug through strong adhesive interactions during storage and processing. Therefore, an assessment of miscibility is essential in terms of polymer type, drug loading and recommended storage conditions, in order to maximize the physical stability.20,

21

Interaction parameter and solubility parameters are often

used to predict the miscibility of a drug-polymer system, and is well established in polymersolvent/polymer-polymer solution studies.19, 22, 23 Oral drug absorption and the fraction of drug absorbed in the intestine are a function of drug’s solubility and permeability. Oral drug absorption processes occur mainly in intestinal regions and P-gp is widely expressed in the membrane of endothelial cells in the intestine. Many clinically used agents are the substrates of P-gp and therefore, P-gp plays an important role in intestinal absorption of such drugs.24 Broad substrate recognition by P-glycoprotein (P-gp) receptors and clinical implications due to its inhibition has been explored for improving peroral drug delivery. P-gp is gaining importance in absorption enhancement due to its selective and extensive distribution at the site of drug absorption.25 Its presence on the villus tip of the apical brush border membrane of gut enterocytes causes the efflux of drugs from gut epithelial cells back into the intestinal lumen.24 Thus, by understanding the physiological and biochemical role of P-gp in efflux of drugs can be explored to improve the bioavailability of drugs restricted by Pgp.25 Various formulation strategies such as co-administration of another P-gp substrate/specific inhibitor and/or incorporation of a nonspecific lipid and/or polymeric excipients have been used to counter the P-gp mediated drug efflux.26, 27 The ideal P-gp inhibitor must be non-toxic and devoid of any pharmacological activity of its own with the advantages of being safe, not being absorbed from the gut and pharmaceutically acceptable.27 Few studies are reported in literature wherein inhibition of P-gp has been exploited for improving the intestinal permeability. Xiang Jin et al. developed soluplus micelles as the delivery system for doxorubicin (DOX). In-vitro, soluplus micelles have significantly enhanced the cellular accumulation of DOX in MCF-7/DOX cells by inhibition of P-gp mediated drug efflux confirmed via membrane fluidity study. In-vivo, both verapamil (the P-gp inhibitor) and soluplus improved the cytotoxicity of DOX in MCF-7/DOX tumor-bearing mice.28 In an another study, conducted by Ramin Mohammadzadeh et al., examined the potential effects of three




grades of Eudragit namely RL100, S100 and L100 on the intestinal epithelial membrane transport of rhodammine-123 (Rho-123), using a monolayer of 24 human colon cancer cell line (Caco-2). Treatment of cells with eudragit RL100 and L100 led to a very slight change while eudragit S100 showed 61% increase in Rho-123 accumulation with reduced transporter expression.29 The interplay of solubility, dissolution rate and permeability of the drug through the gastrointestinal membrane substantiate for limited oral drug absorption.30, 31 When both the drug solubility and permeability are being targeted and enhanced, there will be a subsequent increase in the rate and extent of oral absorption.24 The use of excipients may serve to improve the bioavailability of orally administered BCS class II/IV drugs having low solubility and limited permeability due to their efflux by P-gp.32 Therefore, the objective of the present investigation was to develop amorphous solid dispersion of RXB with the polymers capable of inhibiting the P-gp mediated efflux of RXB to enhance the solubility, intestinal permeability and the overall biopharmaceutical performance of RXB. With best of our knowledge, no ASD have been reported so far for rivaroxaban. EXPERIMENTAL SECTION Materials Rivaroxaban (RXB) form I was obtained as a gift sample from Lupin Pharmaceutical INC (Lupin research park, Pune, India). Eudragit S100 (ES100) and Eudragit L100 (EL100) were received from Vikram Thermo Limited (Ahmedabad, India). Soluplus was obtained as a gift sample from BASF India Limited (Navi Mumbai, India). Paclitaxel was provided as a gift sample from Fresenius Kabi Oncology Limited (Gurgaon, India). Cyclosporine A was obtained as a gift sample from Panacea Biotec (Delhi, India). Verapamil, Tetrahydrofuran (THF), Hanks' balanced

salt

solution

(HBSS),

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-

tetrazoliumbromide (MTT), 2-N morpholinoethanesulfonic acid monohydrate (MES), 2-(4-(2hydroxyethyl)-1-piperazinyl) ethanesulphonic acid (HEPES) and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich, Steinheim, Germany. Dulbecco's modified Eagle's medium (DMEM), non-essential amino acids, trypsin-EDTA, penicillin (100 IU/ml), streptomycin (100 µg/ml) and fetal bovine serum (FBS) were obtained from Biological Industries, Israel Beit-


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Haemek Ltd. (Beit-Haemek, Israel). All other chemicals/ reagents were of analytical grade and used without further purification.

Figure 1. Molecular structures of (a) RXB,2 (b) EL100 (x=y) and ES100 (x=2y), and (c) Soluplus33

Methods Biopharmaceutical Characterization of RXB The dissolution number (Dn) is the ratio of the residence time to the dissolution time (Tdisso) (Equation 1), which includes solubility (Cs), diffusivity (D), density (ρ), and the initial particle radius (r) of a compound and the intestinal transit time (Tsi). Do (dose number) is the ratio of dose concentration to drug solubility given by Equation 2, where Cs is the solubility, M is the dose, and V0 is the volume of water taken along with the dose (i.e. 250 ml.) Dabs and Tdisso is calculated by Equation 3 and Equation 4 respectively, where Peff is effective permeability, S is solubility, IA is an intestinal area, ITT is intestinal transit time, DLT is diffusion layer thickness, and Dcoeff is diffusion coefficient.30, 31

3D C

D T

T

/T

r ρ D M/V /C

D P S IA ITT


Equation 1 Equation 2 Equation 3



T

DLTρr 3D S

Equation 4

Drug-Polymer Miscibility Using Solubility Parameters (δ) Fedors and Hoftyzer-Van Krevelen group contribution methods were used to calculate the solubility parameter. Hildebrand solubility parameter was calculated using Fedors method from the cohesive energy density (CED) using Equation 5, where Ev is the energy of vaporization, Vm the molar volume and CED is the cohesive energy per unit volume. Hoftyzer-Van Krevelen method considers the dispersive forces, interactions between polar groups, and hydrogen bonding groups as shown in Equation 6, where δd, δp and, δh are the dispersive, polar, and hydrogen bonding solubility parameter components, respectively. These components can be further calculated using Equation 7, where Fdi, Fpi, and Ehi are the group contributions for different components of structural groups and V is the group contribution to molar volume.19, 22, 23

∆E% .# δ CED.# V& δ' δ + δ) + δ'

-∑ F) ∑ F ∑ E' δ , δ , δ . V V V

Equation 5 Equation 6

Equation 7

Flory-Huggins Interaction Parameter (χ) The lattice-based Flory–Huggins (F–H) theory is a well-known theory for describing polymer– solvent or polymer–polymer miscibility owing to change in Gibbs free energy before and after mixing.19, 34, 35 The F−H interaction parameter, χ can be estimated by using solubility parameters and/or melting point depression method using physical mixtures of drug and polymer.19, 23 A) Estimation of χ using solubility parameter Difference in solubility parameter values of drug and polymer has frequently been used as a means for predicting the miscibility in pharmaceutical systems. χ can be estimated from solubility parameters, where χ refers to the square of the difference in solubility parameter (δ) of drug and polymer, calculated from group contribution methods at 25°C. Equation 8 is used for


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this purpose, where R is the gas constant; T is the absolute temperature, and Vsite is the volume per lattice site.23, 34

V

/ 0δ123 − δ)56&1 7 χ RT

Equation 8

B) Estimation of χ using melting point depression

The melting point depression of the drug is related to the interaction parameter as shown in & : Equation 9, where T9 is the melting point of the drug-polymer mixture, T9

)21

is the melting

point of the pure drug, φ123 and φ)56&1 are the corresponding volume fraction of the drug and

polymer respectively, ∆H2 is the heat of fusion of drug, and m is the ratio of the volume of the

polymer to that of drug lattice site. The densities of drug and polymers were calculated using helium pycnometry which was further used to determine value of m. Estimation of the interaction parameters from melting point depression data requires rearrangement of Equation 9. Specifically, a plot of =

>

?ABC @

>

− ?DEFG H @

∆IJEK LM

>

NO − lnφ123 − H1 − &N φ)56&1 vs. φ)56&1 yields

a linear relationship at low polymer weight fractions with a slope equal to χ.19, 23

S

1

& : T9

−

1

)21 T T9

−

R 1 =lnφ123 + 1 − φ)56&1 + χφ)56&1 O ∆H2

m

Equation 9

In-Silico Studies to Estimate Drug-Polymer Miscibility RXB and polymers (monomeric units of ES100, EL100, and Soluplus) were optimized by using the Smart algorithm with ultra-fine quality (Materials Studio 6.0, Forcite Module, Accelrys Inc. San Diego, CA). The molecular structures were then subjected to the UNIVERSAL force-field with the assigned charges. The atom based summation method was employed for both electrostatic and van der Waals interactions, with cubic spline truncation with cut-off distance of 18.5Å, spline width of 1Å and buffer width of 0.5Å. Further, the solubility parameter (at 298K) was calculated for optimized structures of drug and polymers by using Synthia module for the given molecular weight. Interaction parameters were calculated for combination of drug (as a base) and individual polymers (as a screen) using Blends module. Blend calculations were performed at ultra-fine quality for the task. Each combination was subjected to Universal forcefield with atom based summation method without truncation cut-off limits. The obtained results were then analyzed by using Blend Analysis Toolbox.36




Preparation of Amorphous Solid Dispersions Amorphous solid dispersions were prepared by spray drying technique. RXB and polymers were dissolved in 5% v/v water in tetrahydrofuran (THF), resulting in total solution concentration of 2% w/v. RXB solubility in 5% (v/v) water in THF was determined to be >20 mg/mL. The prepared solutions were spray dried with following process parameters; inlet temperature of 90 ºC, feed rate of 10% , air atomization pressure of 500 L/h and aspiration 90% using a lab scale spray-dryer (B191, Buchi Labortechnik AG, Flawil, Switzerland). All the products were stored in vacuum desiccator over phosphorous pentoxide at RT and 0 % RH. Differential Scanning Calorimetry (DSC) The calorimetric responses of samples were recorded on a DSC 821e (Mettler-Toledo GmbH, Schwerzenbach, Switzerland) equipped with a refrigerated cooling system and operating with STARe software version 5.1. The instrument was calibrated for temperature and heat flow using high purity indium standard. The sample cell was purged with dry nitrogen at a flow rate of 40 ml/min. Accurately weighed samples (3-5 mg) in crimped aluminum pans were scanned at a heating rate of 20 ºC/min upto 250ºC. Powder X-Ray Diffraction (PXRD) PXRD patterns of different samples were recorded at room temperature using Bruker’s model D8 Advance Diffractometer (Karlsruhe, West Germany) equipped with a 2θ compensating slit, using Cu Kα radiation (1.54 Å) at 40 kV and 40 mA passing through nickel filter with divergence slit (0.5º), antiscattering slit (0.5º) and receiving slit (0.1 mm). Samples were mounted on zero-background sample holder and subjected to a continuous scan over an angular range of 5–40º 2θ at a step size of 0.01º and scan rate of 1 s/step. Obtained diffractograms were analyzed with DIFFRACplus EVA (ver. 9.0) diffraction software. Microscopy The morphology of powder samples were observed under Leica DMLP polarized microscope (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) and photomicrographs were acquired in both optical and birefringence mode. These photomicrographs were acquired using Leica JVC digital camera and analyzed using Leica IM 50 (version 1.20) software under the resolution of 50X. Additionally, the particle size distribution (PSD) of RXB was carried out and the diameter (i.e. length along the longest axis of individual particles) of 300 particles was determined. The


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cumulative particle size distribution curves were plotted to determine the diameters corresponding to 10, 50 and 90% of cumulative undersize particles, i.e., d (0.1), d (0.5) and d (0.9), respectively. Fourier Transform Infra-red (FTIR) Spectroscopy The FTIR spectra were recorded on an FTIR multiscope spectrophotometer (Perkin Elmer, Buckinghamshire, UK) equipped with spectrum v3.02 software by a conventional KBr pellet method. FTIR study was performed for crystalline RXB and ASDs to determine the intermolecular interaction between drug and polymers. Nuclear Magnetic Resonance (NMR) Spectroscopy Solution 1H NMR spectra were recorded on Bruker 400 UltraShieldTM (Germany) operating at 400 MHz using a 5 mm BBO 400 S1 with Z gradient. The measurements were performed at room temperature in deuterated dimethyl sulphoxide (DMSO-d6). The NMR spectrums were recorded for pure RXB and all the ASDs in solution. Dynamic Vapor Sorption (DVS) The moisture uptake studies were performed with a dynamic vapor sorption instrument (Q5000SA, TA instruments, New Castle, Delaware, USA). The temperature was maintained at 25.0 ± 0.1°C. Approximately 15 mg of sample was added to the sample basket and placed in the instrument. The sample was pre-dried at 25ºC/0% RH for 60 min and the instrument was programmed for moisture sorption from 0-90% RH at 10% RH steps with a maximum dwell time of 60 min at 25ºC. The moisture sorption profile was generated for ASDs and crystalline RXB. Scanning Electron Microscopy (SEM) The surface morphology of powder samples was viewed under a scanning electron microscope (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. In-vitro Dissolution Studies Dissolution studies were performed in USP 37 type II automated dissolution test apparatus (ElectrolabTDT-08L, Mumbai, India). The dissolution profiles of ASDs and crystalline RXB




were determined in triplicate using 500 mL of fasted state simulated intestinal fluid (FaSSIF media) of pH 6.5 previously maintained at 37±0.5°C and stirred at 75 rpm. Powder samples equivalent to 10 mg of RXB were weighed and then added into the dissolution vessel. 5 mL samples were withdrawn at predetermined time points, and filtered using 0.22 µm syringe filter. Equal volume of fresh dissolution medium was compensated each time immediately after the withdrawal of the aliquots to maintain the sink condition. The concentration of RXB in samples was determined by HPLC. Apparent Solubility Studies Apparent solubility was determined by adding excess quantity of samples (equivalent to 15 mg of RXB) in 30 ml screw-capped glass vials containing 15 ml of water as media, pre-equilibrated at 37±0.5°C. The vials were mechanically shaken at 100 rpm in a shaking water bath (Julabo SW 23, Seelbach, Germany) maintained at 37±0.5°C. The samples were withdrawn at predetermined time intervals upto 24 hrs, filtered, centrifuged and, analyzed for drug content by HPLC. High Performance Liquid Chromatography The RXB samples from the in-vitro studies were analyzed using a reversed-phase Shimadzu HPLC system equipped with a model series SPD-10A UV-Visible detector, a pump with degassing device DGU-20A5, an auto sampler SIL-10AD, a column heater/cooler CTO-10AS and a system controller CBM-20A. Separations were performed by isocratic elution at 40ºC

using a C18 (5 µm, 250 mm 4.6 mm) stationary phase. Data were acquired via LC solutions

software. The mobile phase (55 % v/v acetonitrile and 45 % v/v water) was pumped isocratically at 1 mL/min for run time of 8 min at the detection wavelength of 249 nm. In-Vitro Permeability Study Cell Culture Caco-2 cells were obtained from National Centre for Cell Science (NCCS), Pune, India and cultured in DMEM supplemented with 15% fetal bovine serum (FBS), 1% non-essential amino acids, 100 IU/ml penicillin and 100 µg/ml streptomycin at humidified atmosphere of 5% CO2 at 37°C. MTT Cytotoxicity Assay


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The cells were harvested and seeded in 96-well plates at a seeding density of 1.5104 cells per well. RXB solutions were prepared at a concentration range of 10–300µM and incubated for 2, 12 and 24 hrs. Cell culture media without RXB was used as a control. 50µL of MTT solution (0.5mg/mL in PBS) was then added to each well and incubated for 4 hrs (37ºC, 5% CO2) to allow MTT to be metabolized. The media was removed and formazan (metabolic product of MTT) precipitate was dissolved in 200µL of DMSO. Optical density was read at 570 nm using ELISA Plate Reader (Bio-Tek Instruments, Inc., Vermont, USA), and background was subtracted at 630 nm. The percent cell viability was measured from Equation 10.37-39

Cell Viability%

Signal − background 100 Blank − Background

Equation 10

Drug Permeability Studies Cells were harvested by trypsin-EDTA, seeded at a density of 75,000 cells per well apically on 0.4 µm pore size polycarbonate membrane with 6.5 mm diameter inserts polystyrene plate (Transwell® Permeable support 24 well plate, tissue culture treated, Corning Incorporated Costor®, Kennebunk, USA) and then grown as monolayer by incubating at 37°C/5% CO2 for 21 days. The growth medium was changed every 2–3 days. The trans-epithelial electrical resistance (TEER) value was measured after every 2nd day and calculated using Equation 11, where RM is the resistance of the Caco-2 monolayer, RB is the resistance of the polycarbonate membrane filter and A is the surface area of the membrane filter. Drug permeability assay was performed once the TEER value reached ≥ 400 Ω·cm2.

TEER R 9 − R e A

Equation 11

Prior to permeability assay, the growth medium was replaced with HBSS and gently rinsed thrice with the same solution. Thereafter, the cells were equilibrated in HBSS for 30 min at 37°C. For apical to basolateral transport (A → B), RXB and ASDs (equivalent to 50 µM of RXB) in HBSS buffer with MES 2.5 mM, pH 6.5 were added apically (250 µl) and 600 µl blank HBSS buffer containing HEPES 5 mM, pH 7.4 was added basolaterally. For basolateral to apical transport (B → A), samples were added basolaterally. The permeability assay was also performed in the presence of verapamil (100 µM) and cyclosporine A (10 µM), the well-known P-gp inhibitors. The Caco-2 plate was then incubated for a predetermined time at 37 °C in 5% CO2. Samples were taken from both apical and basolateral compartments at time points 15, 30, 60, 90 and 120




min and quantified using HPLC system. The apparent permeability coefficients (Papp) were calculated according to Equation 12.37-39

P)) dQ/dT/C A

Equation 12

where dQ/dt is the cumulative transport rate (µM/min) defined as the slope obtained by linear regression of cumulative transported amount as a function of time (min), A is the surface area of the filters or inserts (0.7 cm2 in 24-wells), C0 is the initial concentration of the compounds on the donor side (µM). The concentration of the transported drug was measured from A → B and B → A i.e., Papp (AB) and Papp (BA) respectively. The efflux ratio (ER) was calculated using the

following Equation 13.37-39

ER P)) BA/P)) AB

Equation 13

In-Vivo Pharmacokinetic Study Animals: Male Wistar rats weighing 180-200 g, were obtained from the central animal facility (CAF), National Institute of Pharmaceutical Education and Research (NIPER), S.A.S Nagar, India. The animals were housed at 22±2 °C and 50-60% relative humidity (RH) under 12 hrs light/dark conditions for one week before the commencement of experiment. Standard pellet diet (Ashirwad Industries, Kharar, Punjab, India) and water was given ad libitum. The study protocol was duly approved by the Institutional Animal Ethics Committee (IAEC), NIPER, S.A.S Nagar, India (IAEC/17/19; Dated 17-05-2017). Pharmacokinetic Study: All the animals were randomly distributed into four groups, each containing five animals, as described below. Before the commencement of study, animals of each group were fasted overnight with free access to water for 12 hrs. Group I: Administered crystalline RXB suspension in double distilled water containing 0.25% w/v sodium carboxymethyl cellulose as suspending agent, at a dose equivalent to 10 mg/kg body weight of RXB, peroral (p.o.). Group II: Administered RXB:ES100 solid dispersion (1:4) in double distilled water containing 0.25% w/v sodium carboxymethylcellulose as suspending agent, at a dose equivalent to 10 mg/kg body weight of RXB, p.o.


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Group III: Administered RXB:EL100 solid dispersion (3:7) in double distilled water containing 0.25% w/v sodium carboxymethyl cellulose as suspending agent, at a dose equivalent to 10 mg/kg body weight of RXB, p.o. Group IV: Administered RXB:Soluplus solid dispersion (1:5) in double distilled water containing 0.25% w/v sodium carboxymethyl cellulose as suspending agent, at a dose equivalent to 10 mg/kg body weight of RXB, p.o. Tail vein was selected for the collection of blood samples. The blood samples (approximately 0.2 ml) were collected into the micro centrifuge tubes containing 50 µL of EDTA solution (4 % w/v) upto 12 hrs. Blood samples were centrifuged at 10,000 rpm for 10 min at 4°C and stored at 20°C prior to analysis. The pharmacokinetic analysis of plasma drug concentration-time data was analyzed by biphasic clearance kinetics using Kinetica Software (Version 5.0, Thermo scientific) and required pharmacokinetics parameters were determined. Quantification of RXB in Plasma Samples: The in-vivo study samples were treated (to remove blood cells and proteins) and diluted with acetonitrile:methanol (1:1) mixture and analyzed using the same HPLC system mentioned previously using paclitaxel as the internal standard (IS). Separations were performed by isocratic elution at 35ºC using a C18 (250 mm 4.6 mm, 5µm) stationary phase. The mobile phase (55% v/v acetonitrile and 45% v/v water) was pumped isocratically at 0.8 mL/min for of 25 min at detection wavelengths of 249 nm (RXB) and 227 nm (IS), respectively. Statistical Data Analysis The mean, standard deviation (SD) and standard error mean (SEM) of all values were calculated. The statistical comparisons were performed by one-way analysis of variance (ANOVA) using GraphPad Prism 5 software, version 5.04 (GraphPad Software, Inc San Diego, CA, USA). The results were considered statistically significant when p < 0.05. RESULTS AND DISCUSSION Biopharmaceutical characterization of RXB Calculated Tdisso and Dabs values showed that unmicronized RXB exhibits both solubility and dissolution rate limited absorption owing to the higher Tdisso value than small intestinal transit




time (Tsi) and lower Dabs than the maximum daily dose of RXB. Table I listed the biopharmaceutical parameters which helped in estimating the absorption rate limiting factors for the drug. The Tdisso value was decreased considerably for the micronized RXB indicating that the microcrystalline nature of RXB do not exhibit dissolution rate limited absorption. Therefore, the theoretical calculations of Dn and Do of micronized (based on PSD analysis, discussed later) RXB indicates that it belongs to BCS class II (Dn > 1, Do > 1) having only solubility limited oral bioavailability. Table I. Biopharmaceutical characterization of RXB

Parameters

Values

Comments

Tdisso unmicronized (min)

583.09

Tdisso≫199

Tdisso micronized (min)

46.65

Tdisso≪50

0.317

Dabs≪dose (20 mg)

Dose number (Do)

11.43

Solubility limited bioavailability (Do > 1)

Dissolution number (Dn)

4.27

Bioavailability is not dissolution rate limited (Dn > 1)

Dose absorbable (Dabs) (mg)

Drug-Polymer Miscibility Using Solubility Parameter (δ) Compounds with similar δ values were considered to be miscible, as the energy required for mixing is compensated by the energy released (exothermic energy of mixing) in interactions between the components. For such miscibility evaluation, if the difference in solubility parameter (∆δ) between the components to be mixed is smaller than 7 MPa1/2, then the components are likely to be miscible. If ∆δ is smaller than 2 MPa1/2, the components may form a solid solution, while ∆δ value greater than 10 MPa1/2 indicates immiscibility.34,

40

Solubility parameters

estimated using the Fedors, Hoftyzer and Van Krevelen method are given in Table II. The difference between the solubility parameters of RXB and each polymer were small i.e. < 7 MPa1/2, indicating the good miscibility between RXB and all the polymers. RXB and soluplus system exhibited the ∆δ of 2.051 MPa1/2, which suggests the higher miscibility of RXB with soluplus than that of other two polymers. Flory-Huggins interaction parameter (χ)


Page 17 of 43 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


(A) Estimation of χ using solubility parameter The values of interaction parameters for RXB with each polymer system at 25°C (298K) were calculated using solubility parameter method (using Equation 8) are shown in Table III. The drug-polymer interaction parameter χ, for each system was found to be positive. The positive value of χ indicates the unfavorable Gibb's free energy of mixing (∆Gmix) resulting in limited miscibility between RXB and all the polymers at 25°C.34 (B) Estimation of χ using melting point depression The DSC thermograms of physical mixtures of the drug with EL100, ES100 and soluplus system with varying ratios are given in Figure 2a, 2b and, 2c, respectively. With increasing weight fraction of all the polymers, the melting endotherms were shifted to a lower value indicating depression in the melting point of drug. For RXB-EL100 and RXB-ES100, the melting point depression of about 7.32ºC and 6.84°C (Figure 2a and Figure 2b) were observed at drug-polymer ratio 55:45, respectively, whereas for the RXB-soluplus system (at ratio 60:40) melting point depression of 9.7ºC was obtained (as shown in Figure 2c). The presence of a polymer altered the melting behavior of a drug depending on the drug miscibility with the polymer. Thus, presence of miscible system was quite evident due to reduction in the melting temperature of the crystalline drug. The melting point depression method has been widely applied for the evaluation of the miscibility for several drug–polymers, wherein miscible systems exhibited melting point depression with varying polymer concentrations. On other hand, immiscible or partially miscible system led to slight or no melting point depression.34, 40, 41 A graph of =

>

?ABC @

>

− ?DEFG H @

∆IJEK LM

>

NO − lnφ123 − H1 − &N φ)56&1 vs. φ)56&1 plotted using the

melting point depression data yielded a linear relationship at lower polymer weight fractions

with a slope equal to χ as shown in Figure 3. χ value (near the melting temperature of drug) obtained from the slope of the plotted graphs using melting point depression data were -0.3752, 0.7076 and, -0.7395 for RXB-soluplus, RXB-EL100 and RXB-ES100, respectively. The decrease in the melting point with increasing polymer fraction and the negative values of χ near the melting temperature of RXB were representatives of negative free energy and an exothermic heat of mixing with all the polymers, suggesting miscibility with the drug.34




(a)

(b)


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(c) Figure 2. DSC thermograms of physical mixtures of (a) RXB and EL100, (b) RXB and ES100 and, (c) RXB and Soluplus

Figure 3. Graphs of =

>

lmn jk

−

>

opqr

jk

H

∆stpu Lv

>

NO − wxyz{|} − H1 − ~N y~{ vs y~{ to determine

the χ values for system - (a) RXB-ES100, (b) RXB-EL100 and (c) RXB-soluplus




In-silico studies for determination of solubility and interaction parameter Solubility parameters estimated by Fedors, Hoftyzer and Van Krevelen method at 298K using Synthia module are given in Table II. The mean δ values obtained for RXB and EL100 were 25.119 and 21.344 MPa1/2 respectively (i.e. ∆δ of 3.776 MPa1/2), whereas ES100 have a least solubility parameter value i.e. 20.201 MPa1/2. The difference between the solubility parameters of RXB and each polymer were found to be < 7 MPa1/2, this suggests that RXB and all the polymers have a good miscibility. There was a good correlation observed between the Synthia module generated results and theoretically calculated solubility parameter values. Interaction parameters calculated for combination of drug (as a base) and individual polymers (as a screen) using Blends module are given in Table III. The drug-polymer interaction parameter χ, for each system was found to be positive. This suggests limited miscibility existed between RXB and polymers at a temperature of 25°C (298 K), which is congruent with theoretically calculated χ values using solubility parameters. Table II. Solubility parameter value calculation

δ (MPa1/2)

Method Name of Compound

used for

Fedors

Hoftyzer and Van

calculation

method

Krevelen method

Theoretical

26.064

In-silico

EL100

∆ MPa1/2)

(MPa1/2)

(δdrug-δpolymer)

24.875

25.47

-

25.001

25.238

25.119

-

Theoretical

21.86

18.31

20.09

5.38

In-silico

21.007

19.394

20.201

4.92

Theoretical

22.7

21.36

22.03

3.44

In-silico

21.980

20.707

21.344

3.78

Theoretical

23.998

22.84

23.42

2.05

In-silico

21.128

20.576

20.852

4.27

RXB

ES100

Mean δ

Soluplus


Page 21 of 43 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


Table III. χ values calculated at room temperature

System

χ at 25ºC Using δ

In-silico

RXB-ES100

3.464

11.499

RXB-EL100

1.416

20.211

RXB-Soluplus

0.503

1.562

Solid State Characterization Physical nature of starting materials and prepared spray dried ASDs were confirmed by DSC, PXRD and polarized light microscopy. When observed under polarized light microscopy RXB showed birefringence, indicating its crystalline nature. Conventional DSC thermogram as shown in figure 4a also showed sharp melting endotherm at temperature (Tm onset) of 230.59°C. Analysis of powder diffraction patterns (Figure 5a) of RXB showed a distinct crystalline phase with characteristic sharp peaks, confirming the polymorphic form I of RXB. All the polymers showed small and broad endotherm of moisture in DSC and halo pattern in PXRD. All the ASDs prepared by spray drying were found to be stable and showed absence of crystallinity as evident by DSC and PXRD. The spray dried ASDs gave a halo PXRD pattern (Figure 5b) and were found to be nonbirefringent when analyzed under polarized light microscopy. A single glass transition peak was observed for all the prepared ASDs in DSC thermogram, representing the single phase and uniformly mixed drug and carrier system (Figure 4b). Based on the presence of single glass transition peak and complete absence of melting endotherm, the drug to polymer ratios of 3:7, 1:4 and 1:5 were finalized with polymers EL100, ES100 and soluplus, respectively and were designated as ASD-L100, ASD-S100 and ASDSLPS, respectively, for further discussions. Higher glass transition temperatures (Tg midpoint) of 126°C and 122°C were observed for ASD-L100 and ASD-S100, respectively suggesting the antiplasticization effect of eudragit polymers on the amorphous form of the drug. ASD-SLPS showed the glass transition temperature of 71°C, lower than that of reported Tg of RXB. Tg of soluplus is 70°C42 which is less than the Tg of pure amorphous RXB 83°C3, hence as per the theory of antiplasticization, intermediate Tg was observed for ASD-SLPS.14




(a)

(b) Figure 4. (a) The DSC thermogram of RXB and polymers, (b) DSC results of optimized spray dried samples indicating Tg.


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(a)

(b) Figure 5. (a) PXRD diffractograms of RXB and polymers, (b) PXRD diffractograms of RXB and ASDs

Fourier Transform Infra-red (FTIR) Spectroscopy FTIR analysis was conducted for the evaluation of possible molecular interactions between RXB and polymers in the ASD formulations. Infra-red (IR) spectroscopy is extensively used to identify drug–polymer interactions in ASD through alteration in peak shape and/or position that can be linked to molecular interactions. The hydrogen bonding for specific functional groups, particularly the vibrational frequencies of O-H, N-H, and C=O bonds in the mid-IR region serves




to be imperative. The presence of hydrogen bonding between the drug and polymer is important in order to inhibit drug crystallization (upon storage) and precipitation (during solvation process). In particular, the stretching frequencies of N-H, O-H group act as hydrogen bond (Hbond) donor thus their bands tend to broaden, decrease or disappear depending on the H-bond donor–acceptor distances. The changes in frequency of carbonyl bond (H-bond acceptor) in the dispersion can also be related to intermolecular interactions.43 The obtained FTIR spectra showed the characteristic peak at 1011 cm-1 confirming the polymorphic form I of the RXB.44 The crystalline RXB shows the secondary amide N-H stretching vibration band at 3354 cm-1 and bending mode (scissoring) appears as a medium intensity bands at 1547 and 1518 cm-1. Strong band of C=O stretching was seen at 1738 cm-1 for ester group while, amide group exhibited strong stretching frequencies at 1668 and 1644 cm-1. The C-O-C-functions of ethers and esters are typically found as strong peaks in the range between 1000 and 1300 cm-1. The bands occurring in the range 850-550 cm−1 were assigned for C-Cl stretching. The presence of more than two bands might be due to the different possible conformations. FTIR spectra of Eudragit S-100 and Eudragit L-100 showed the C-H stretching vibrational peak at 2998 cm-1 and 2953 cm-1 due to the presence of O-H (carboxylic acid). The Eudragit L100 and Eudragit S100 polymers contain both carboxylic acid and ester groups. The spectra of Eudragit L100 and Eudragit S100 showed the carbonyl vibrations of the ester group at 1731 cm−1 and 1732 cm-1, respectively.45-47 From the chemical structures, hydrogen bonding could be expected between the hydroxyl groups of soluplus® and the carbonyl function of RXB.48 Spectra of soluplus showed inter-molecularly hydrogen bonded O-H stretching in the 3350–3600 cm1

range specifically at 3461 cm-1. Ester carbonyl stretching was observed at 1738 cm-1 and C=O

stretching for tertiary amide at 1639 cm-1.49 The bands corresponding to N-H stretching (at 3354 cm-1) of secondary amide group got broadened and disappeared in case of all the ASDs. The stretching frequency of -NH groups that act as hydrogen bond donors decreases in amorphous solid dispersion and their bands tend to broaden as hydrogen bond donor–acceptor distances get shorter. Broadening of peak can also be attributed to alteration of solid state form of drug from crystalline to amorphous in solid dispersion. This suggested that all the prepared spray dried products were amorphous in nature. C=O ester stretching vibrations at 1738 cm-1 in RXB shifted to 1733 cm-1 in ASDs prepared with


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both eudragit polymers, while remain unaffected in ASD prepared with soluplus. The apparent shift in both C=O and O-H band frequencies, imply the participation of these moieties in hydrogen bond interaction in the ASD. The first C=O amide stretching band at 1668 cm-1 in RXB got disappeared in ASDs prepared with eudragit L-100 and eudragit S-100 whereas second C=O amide stretching band present at 1644 cm-1 in RXB shifted to 1648 cm-1 in ASDs prepared with eudragit S-100 and eudragit L-100 while it got down shifted to 1639 cm-1 in ASD prepared with soluplus. This clearly indicates that, N-H bond of drug is interacting molecularly with C=O bond of polymers that resulted in the formation of strong intermolecular hydrogen bond between drug and polymers. Also, changes in carbonyl band structure in the amorphous solid dispersion can be related to hydrogen bonding between drug and polymers. Analysis of the fingerprint region in the FTIR spectra revealed that characteristic peak that appeared at 1011 cm-1 in crystalline RXB got disappeared in all the ASDs suggesting the complete conversion of crystalline to amorphous form. Significant down-shift and up-shift of the wave numbers in the ASDs, signifies a higher degree of interaction between the molecules.50

Figure 6. Overlay showing FTIR spectra of RXB and ASDs

Nuclear Magnetic Resonance (NMR) Spectroscopy Proton (1H) NMR spectroscopic studies were performed in solution state to further explore the evidences for interactions between drug and polymers. Figure 7a and Figure 7b shows the proton (1H) NMR spectra of RXB and ASDs, respectively. Changes in chemical shift values suggest the




presence of interactions between drug and polymer. One possible site of interaction on the drug is situated at its amide function for interaction with polymers. Therefore, to study the interaction between RXB and polymers, the focus was kept on the protons present on nitrogen in the amide group of RXB as shown in Figure 7a. The change in the electron density at the interacting atoms is denoted by variations in chemical shift values which suggested the possible molecular interactions between drug and polymers.51 The maximum peak shift (∆δ) of 0.0238 ppm was observed in RXB and ASD-L100, compared to other ASDs. The change in chemical shift of 0.0226 and 0.0172 ppm was observed for ASDS100 and ASD-SLPS, respectively. The chemical shift due to the proton at amide group shifted upfield to a lower value in case of ASD-L100 and ASD-S100; this suggests the shielding of same proton. While increase in δ value in ASD-SLPS denotes the deshielding of the proton as it has shifted downfield to higher value.

(a)


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(b) Figure 7. (a) Solution 1H NMR spectrum of RXB, (b) Chemical shift peaks for proton at amide group of RXB in ASDs

Dynamic Vapor Sorption (DVS) Analysis Data obtained from DVS analysis showed that RXB in crystalline form was non-hygroscopic in nature with moisture content of 0.155% at 90 % RH, while amorphous solid dispersions have shown higher moisture uptake than crystalline RXB. ASD-SLPS has shown the highest hygroscopicity with 14.17 % moisture gain at 90% RH, whereas ASD-S100 and ASD-L100 showed moisture uptake of 4.135% and 4.546% at 90% RH, respectively, as shown in Figure 8. This data suggests that all ASDs comprising RXB in amorphous form have higher propensity to gain moisture. Amorphous solids show higher moisture sorption as compared to their crystalline counterpart because both adsorption (onto the surface) as well as absorption (into the bulk) takes place in amorphous solids.52 Eudragit S100 and L100 both are anionic, water insoluble polymers while soluplus is hydrophilic hence shows higher inherent hygroscopicity.53




Figure 8. Sorption isotherms of RXB and ASDs generated by DVS

Scanning Electron Microscopy (SEM) SEM analysis of amorphous solid dispersions is widely used to probe the particle morphology after the formation of amorphous dispersions. SEM photomicrographs were acquired for pure drug, polymers as well as for prepared ASDs. RXB consisted of mixture of large and small size particles with microparticles deposited on them as shown in Figure 9 a, which could be due to the micronization or any other size reduction process at the time of manufacturing. The polymers ES100 and EL100 have shown the spherical particles with smooth surface (Figure 9 b and c), whereas soluplus showed spherical particles with rough surface (Figure 9 d). The ASD-SLPS system has shown spherical surface without any imperfections (Figure 9 g). All the samples showed no aggregation. The possible reason for such different surface morphologies can be the nature of polymers being used and their drying mechanisms. The RXB-soluplus system dissolved in solvent completely and subsequently formed a more homogeneous emulsified system due to amphiphilic nature of soluplus.54 Therefore, soluplus may have plasticizing effect on the porous and permeable microspheres before the drying which demonstrated small particle size, regular shape and smooth surface.54, 55 Conversely, the powder samples of ASD-S100 and ASD-L100 showed the collapsed and corrugated surface morphology (Figure 9 e and f).


Page 29 of 43 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


Figure 9. SEM images of samples (a) RXB, (b) EL100, (c) ES100, (d) Soluplus, (e) ASD-L100, (f) ASDS100 and, (g) ASD-SLPS

In-Vitro Dissolution Studies In-vitro dissolution was carried out for pure RXB and prepared ASDs in FaSSIF media, pH 6.5. As illustrated in figure 19, ASD prepared with eudragit S100 showed highest drug release of 65% at the end of 120 min whereas ASD-L100 and ASD SLPS showed 48% and 56%, respectively. Crystalline RXB exhibited release of 37%, which was more than expected. The reason for this may be accounted for micronization of API during manufacturing which increased the surface area of API and according to the modified Noyes-Whitney equation; dissolution rate of the API got increased.56 We observed overlapping release behavior for RXB and ASD-S100 for initial 30 min, and then release of ASD-S100 got enhanced after 60 min. A similar release pattern was observed for ASD-L100 and ASD SLPS for initial 30 min. The reason for this may be limited solubility of drug in this media or formation of complex with components of FaSSIF which subsequently resulted in slow and incomplete release of drug from the formulated ASDs.57,

58

Another reason for slow release of API may be accounted to poor

ionization of eudragit polymers during dissolution.59 This may be the case with eudragit S100 since it dissolves at pH 7 and above, and is used for delivery of drugs in colon. When the pH value reaches the soluble threshold of eudragit S-100, a constant and slow drug release is




observed. This resulted in slow diffusion of drug from the poor ionization of polymeric matrix of eudragit S100. However, for eudragit L100, the targeted drug release area is jejunum and it dissolves at pH above 6. It is used for effective and stable coating with fast dissolution in upper gastrointestinal tract.60 Hence, assumption of formation of complex of eudragit ASD with FaSSIF components is more prominent. This is also quite evident from the dissolution profile of physical mixtures in FaSSIF media. The release of drug from the physical mixture got hampered and was even reduced than the release pattern of pure crystalline RXB alone. This clearly indicate that drug polymer physical mixture did not dissociate in FaSSIF media and resulted in poor release profile during dissolution studies. The same reason may be accounted for soluplus polymer also. The release of RXB from the physical mixture did not show significant improvement over that of pure RXB. It has also been reported previously that presence of hydrophilic polymer did not improve the wettability of cilostazol significantly.61 However, improved drug release profile was observed for all the ASDs than pure drug alone. ASD-S100, ASD-L100, and ASD-SLPS showed 1.77, 1.31, and 1.52 fold increases in drug release, respectively after 120 min.

Figure 10. Dissolution studies performed in FaSSIF. Values are represented as % amount of drug release ± SD for RXB (n = 3)

Apparent Solubility Study


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Solubility study was conducted for crystalline RXB and generated ASDs in water for 24 hrs, data of which have shown in figure 11. All the ASDs solubilized quickly and reached highest concentration exhibiting spring effect followed by maintenance of supersaturation during the period of study exhibiting parachute effect. Significant enhancement in solubility was observed for all the ASDs as compared to crystalline RXB. The maximum concentration was achieved with ASD-SLPS, showed solubility enhancement of ≈10-fold compared to the crystalline form. The ASD-SLPS reached a maximum soluble concentration of 98 µg/ml after 12 hrs and exhibited supersaturation up to 24 hrs. Soluplus is a highly water soluble graft copolymer. It combines the feature of water solubility and amphiphilicity and thus functions as a polymeric solubilizing agent. Soluplus is miscible with water in any ratio, hence, ASD-SLPS showed maximum solubility in water.62 Also, soluplus has been reported for forming micelles and maintaining a high supersaturation of drug by inhibiting both drug nucleation and crystal growth.63 The concentration of crystalline RXB in water was found to be 9 µg/ml after 24 hrs. ASDs with eudragit S100 and L100 exhibited similar type of solubility profile in water. In comparison, the maximal soluble concentration of ASD-S100 and ASD-L100 was around 45 µg/ml and 39 µg/ml after 1 hr. At the end of 24 hrs solution concentration reduced to 22.73 µg/ml and 29 µg/ml which were nearly 2-fold and 3-fold higher than that of the crystalline form, respectively. The drastic enhancement in solubility for ASD-SLPS did not translate during dissolution study in FaSSIF. In FaSSIF media, release of ASD-SLPS was lower than that of ASD-S100. The underlying mechanism for slow release of ASD-SLPS may be attributed to extensive swelling of soluplus polymer in FaSSIF media which hindered the diffusion and dissolution of RXB.64 Hence, ASD-S100 exhibited better dissolution profile as compared to ASD-SLPS.




Figure 11. Solubility profiles of RXB and ASDs in water. Values are represented as concentration ± SD for RXB (n = 3)

In-Vitro Permeability Studies MTT Cytotoxicity Assay Viability of cells was directly measured using the MTT test to evaluate the cytotoxicity of RXB on Caco-2 cells. Cell viability value of less than 50% indicates reduced mitochondrial activity. A higher cell viability of > 80% as shown in Figure 12 ensured that RXB concentrations were nontoxic to the cells. The cytotoxic concentration at 50 % cell viability or IC50 value was observed at 124.9 µM, 88.58µM, and 68.71µM after 2, 12 and 24 hrs incubation, respectively. The proposed working concentration of 50 µM displayed approximately 83.98% cell viability after 24 hrs incubation.


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(a)

(b)

(b) Figure 12. Cytotoxicity results of RXB at different concentrations in Caco-2 cells using MTT assay. Incubation was done at (a) 2 hrs, (b) 12 hrs and (c) 24 hrs. Values are represented as % cell viability ± SD for rivaroxaban (n = 3)

Drug Permeability Studies The Caco-2 permeability model was used to determine the permeability of crystalline RXB and ASDs. According to the Caco-2 cell assay, the drugs having experimental Papp values of 14.010-6 cm/s were considered highly permeable.65 Based on this fact, it can be concluded that RXB is highly permeable through the Caco-2 cell monolayer. The Papp values of the samples from A→B and B→A as well as in the presence of verapamil and cyclosporine A are shown in Table IV. The Papp values of the samples from A→B direction did not show significant difference (p ˃ 0.05). Contribution of active transport was investigated by performing the transport studies in the reverse direction, i.e., B→A. Papp values for the samples from B→A were significantly higher than the values obtained from A→B studies (p < 0.01). RXB showed the




significant increase in permeability in the presence of cyclosporine A and verapamil, the P-gp efflux inhibitors. These findings supported the possibility of efflux mechanism by involvement of P-gp in transport of RXB. The ASD-S100 showed the significant decrease in efflux ratio (ER) of RXB by 3-fold whereas the ASD-L100 did not alter the ER of RXB. The efficacy of ES100 had already been demonstrated in decreasing the P-gp expression and consequently its possible inhibitory role on the efflux process of P-gp substrate (e.g. Rho-123) whereas EL100 failed to do so.29 Conversely, soluplus neither affected the binding of substrate to P-gp nor altered the expression of P-gp located in membrane. The P-gp inhibition was through indirect interaction with P-gp and changing the cell membrane fluidity.28 Therefore, ASD-SLPS sample resulted in slightly allayed ER of RXB (1.24-fold). Table IV. Permeation studies result of RXB (50 µM) showing the values of Papp ± SD (n = 3) and mean efflux ratio

Formulation

Papp (± SEM) 10-4 (cm/s)

Mean Efflux

A→B

B→A

Ratio

RXB

15.2±3.5

174.8±11.7

11.53

RXB + Cyclosporine A

39.6±9.5

82.4±7.4

2.08

RXB + Verapamil

26.9±6.7

98.3±16.2

3.66

ASD-S100

13.6±3.2

51.2±9.7

3.76

ASD-SLPS

17.1±4.3

158.4±7.0

9.27

ASD-L100

12.9±3.4

164.9±31.9

12.78

In-Vivo Pharmacokinetic Study The pharmacokinetic studies of developed formulations were performed in Wistar rats by oral administration and sampling done via tail vein method. All evaluated parameters e.g. maximum serum concentration (Cmax), area under curve (AUC), half-life (t1/2) and mean residence time (MRT) are enumerated in Table V. Graph of plasma conc. vs. time profile is depicted in figure 13. It was observed that values of all the parameters were increased in case of ASDs compared to


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pure crystalline drug. After 12 hrs, drug concentration was not detected in RXB and ASD-L100 samples. Compared to RXB, the solid dispersions ASD-L100, ASD-S100, and ASD-SLPS exhibited a significant (p < 0.01) enhancements in oral bioavailability with 1.7, 4.1 and, 5.7-fold increase in AUC0-12hrs, respectively. Similarly, maximum plasma concentration (Cmax) was increased by 1.7, 2.9 and, 6.7-fold for ASD-L100, ASD-S100 and, ASD-SLPS, respectively. The enhanced bioavailability of ASD with soluplus may be attributed to the increase in apparent solubility and in-vivo interaction of the RXB and/or soluplus with the intestinal fluids, which contain bile salts and phospholipids. This might have led to the formation of mixed micelles which provided synergistic effect on drug absorption in-vivo.66 Table V. Pharmacokinetic parameters evaluated for drug and ASDs

Parameters

RXB

ASD-SLPS

ASD-S100

ASD-L100

Cmax (ng/ml)

752.636

5045.152

2192.134

1293.027

AUC0-12hr (ng/ml*hour)

2700.408

15291.178

11051.623

4560.092

t1/2 (hour)

2.046

3.803

3.377

3.139

MRT (hour)

3.547

5.289

5.398

4.773




Figure 13. Pharmacokinetic profiles of RXB and ASDs

CONCLUSIONS In the present study, ASDs were successfully generated using spray drying technique. The drug and polymer miscibility was estimated theoretically as well as by in-silico methodology, where all the three polymers were most likely to be miscible with drug. The drug to polymer ratio of 3:7, 1:4 and 1:5 were optimized for RXB and polymers EL100, ES100 and soluplus respectively. The generated ASDs were characterized for its microscopic, thermal, diffractometry and spectroscopic properties. Comparative assessment of thermal and spectroscopic data confirmed the amorphous nature of the drug in ASDs and confirmed their stability. All the ASDs and crystalline RXB were evaluated for their biopharmaceutical performances in terms of dissolution in FaSSIF media and solubility studies in water. There was a significant improvement observed in apparent solubility and dissolution of ASD-SLPS. This might be due to formation of micellar solution during the solvation process and slight surface active properties of soluplus. The permeability studies highlighted and supported the underlying and well reported P-gp inhibiting mechanisms of all the polymers. FTIR and NMR studies revealed the involvement of amide group of drug in the intermolecular interaction with carbonyl group of polymers, which can be the reason for stabilization of drug in amorphous form and during the solvation process. The pharmacokinetic study established a correlation with the in-vitro evaluation of ASDs and RXB. ASD-SLPS has shown better biopharmaceutical performance than all ASDs and crystalline drug.


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Therefore, ASD of RXB with soluplus enhanced the overall biopharmaceutical performance through improved solubility and enhanced intestinal uptake of RXB. DECLARATIONS The Authors declares that they have no conflicts of interest to disclose.

ACKNOWLEDGEMENT We are thankful to Prof. Arvind K. Bansal, Department of Pharmaceutics, NIPER, S.A.S Nagar for providing DSC facility. We also thank Director, NIPER, S.A.S Nagar for providing financial support and facilities. We thank Deepika Daksh and Shamandeep Kaur for their constant help. REFERENCES 1.

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Remko, M. Molecular structure, lipophilicity, solubility, absorption, and polar surface area of novel anticoagulant agents. J. Mol. Struc: THEOCHEM. 2009, 916, (1), 76-85.

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Grunenberg, A.; Lenz, J.; Braun, G. A.; Keil, B.; Thomas, C. R., Polymorphous form of 5-chloro-N-({(5S)-2-oxo-3

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