Inclusion Complexation of Etodolac with ... - ACS Publications

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Inclusion Complexation of Etodolac with Hydroxypropyl-betacyclodextrin and Auxiliary Agents: Formulation Characterization and Molecular Modeling Studies Atul P. Sherje,*,† Vaidehi Kulkarni,† Manikanta Murahari,‡ Usha Y. Nayak,§ Pritesh Bhat,∥ Vasanti Suvarna,† and Bhushan Dravyakar† †

Department of Quality Assurance, SVKM’s Dr. Bhanuben Nanavati College of Pharmacy, Gate No. 1, SVKM Campus, V. M. Road, Vile Parle (W), Mumbai 400 056, India ‡ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, M. S. Ramaiah University of Applied Sciences, Bangalore 560 064, India § Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576 104, India ∥ Schrodinger, Bengaluru 560 086, Karnataka India S Supporting Information *

ABSTRACT: The present investigation was aimed to prepare inclusion complexes of a therapeutically important nonsteroidal anti-inflammatory drug, etodolac (ETD) with hydroxypropyl-beta-cyclodextrin (HP-β-CD) and to study the effect of L-arginine (L-Arg) as an auxiliary agent on the complexation efficiency of HP-β-CD to improve aqueous solubility and the dissolution property of ETD. The binary and ternary complexes were prepared by physical mixing, coevaporation, and spray drying methods. The complexes were characterized using differential scanning colorimetry (DSC), Fourier transform-infrared spectroscopy (FT-IR), and powder X-ray diffraction (PXRD) studies. The mechanism of inclusion interaction of guest and host was established through 1 H NMR, molecular docking, and molecular dynamics studies. On the basis of preliminary screening studies, L-Arg was found to be the most efficient auxiliary agent for the present research problem. The change in crystallinity of ETD was evident from DSC and PXRD studies which indicated the formation of new solid forms. A remarkable increase in apparent stability constant (Kc) and complexation efficiency (CE) of HP-β-CD was observed in the presence of L-Arg in ternary complexes with improvement in solubility and dissolution of ETD than binary complexes. However, inclusion complexes of ETD obtained by computational studies is in good correlation with the results obtained through experimental methods. More stable complex formation with L-Arg was confirmed by molecular simulation studies too. Thus, the present study led to the conclusion that the ternary complex of ETD-HP-β-CD-L-Arg could be an innovative approach to augment the solubility and dissolution behavior of ETD. KEYWORDS: etodolac, ternary inclusion complex, arginine, solubility enhancement, binary inclusion complex, dissolution improvement, computational modeling

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therapeutic outcome.1 Hence, enhancing solubility and bioavailability of the drug is one of the most important milestones in the drug development process, which decides the fate of drugs in the market.2 Cyclodextrins (CD) are used as a promising solubility enhancement tool for water insoluble drugs over many years. These are cyclic oligosaccharides consisting of D-glucopyranoside units connected by glycosidic bonds. CDs have the ability

INTRODUCTION

In the pharmaceutical industry, screening programs conducted to detect the water insoluble drugs led to the fact that about 40% of new chemical entities are poorly water-soluble. The major problem associated with formulation development of new chemical entities is its low aqueous solubility, necessitating usage of a high dose to reach therapeutic plasma concentration levels. Most of the drugs available in the market are orally administered due to associated advantages like ease of administration and improved patient compliance. A drug with poor aqueous solubility will exhibit rate limited absorption. Thus, aqueous solubility of drugs is one of the critical factor affecting their bioavailability, which inturn influences their © XXXX American Chemical Society

Received: December 13, 2016 Revised: February 18, 2017 Accepted: February 21, 2017

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DOI: 10.1021/acs.molpharmaceut.6b01115 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

methods to elucidate the interaction of drug with HP-β-CD and

to form inclusion complexes with the drug and alter the physicochemical and biological characteristics of the drug molecule, leading to enhanced solubility in water.3,4 The structure of CDs (which resemble a truncated cone), is open at both ends, facilitating the incorporation of various organic molecules (apolar drugs) in their cavities.5 A guest molecule replaces the water moiety from the apolar cavity of CDs to form the complex. Naturally occurring cyclodextrins reported in the literature are of three types, namely, α-, β-, and γcyclodextrin, which are comprised of six, seven, and eight glucopyranose units, respectively. β-CD is the most widely used natural cyclodextrin in the pharmaceutical field. These naturally occurring cyclodextrins exhibit limited aqueous solubility and low complexation efficiency.6 Many chemically modified derivatives of natural cyclodextrins are commercially available. These are obtained by substitution of some hydroxyl groups on the external surface of CD with other functional groups like methyl, hydroxypropyl, sulfobutyl ether, etc., which result in improved aqueous solubility.7 Hydroxypropyl-β-cyclodextrin (HP-β-CD) is a modified form of naturally occurring β-CD containing a hydroxypropyl group, to be used widely as a water solubility improvement aid. Extensive studies by various research groups have established the fact that the solubilization and complexation efficiency of CDs can also be increased by addition of auxiliary or ternary agents. Some of the reported ternary agents include polymers, amino acids, and hydroxyl acids.8 The effect of various polymers like hydroxypropyl methyl cellulose (HPMC) K4,12 polyvinylpyrrolidone (PVP) K30,13 and polyethylene glycol (PEG) 400014 have been studied in the recent literature for enhancing the solubility of some poorly water-soluble dugs. The effect of L-Arg and other amino acids has also been demonstrated in the literature for enhancing the solubility of poorly water-soluble drugs.8,15,16 Etodolac (ETD) was selected as a model drug for the study (Figure 1). It is a nonsteroidal anti-inflammatory drug approved

L-Arg.

Molecular docking and dynamics studies were carried out to understand the significance of L-Arg in the complexation of ETD in both the R and S configurations with HP-β-CD. The inclusion complexes obtained by molecular docking studies were compared with the results obtained through experimental methods. Binding affinity parameters were calculated to investigate the stability of binary and ternary complexes.

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METHODS Reagents and Supplies. ETD was obtained as a gift sample from IPCA Laboratories, Mumbai, India. HP-β-CD was kindly provided as a gift sample from Gangwal Chemicals Pvt. Ltd., Mumbai, India. L-Arg was purchased from SD Fine Chemicals Ltd., Mumbai. All other chemicals used for the study were of analytical reagent grade. Determination of the Aqueous Solubility of ETD. The solubility of ETD in water, HCl buffer (pH 1.2), and phosphate buffer (pH 6.8) was determined by the classical saturation solubility method. Briefly, ETD was added in excess amount to the vials containing different solvents and shaken using thermostatic orbital shaker (Orbitek- Scigenics Biotech, India) for 48 h at 25 °C and 100 rpm. The solutions were filtered through a syringe filter (0.45 μm) and suitably diluted, and ETD was quantified using a UV−visible spectrophotometer (Shimadzu UV 1800, Japan) at 275 nm. Selection of Auxiliary Agents. Various polymers and amino acids were screened with ETD, in the presence and absence of cyclodextrin to select the suitable auxiliary agent. The auxiliary agents, viz., HPMC K4, PVP K30, PEG 4000, LArg, and glycine, were evaluated for their solubility enhancement property toward ETD. ETD in excess was added to the vials containing various concentrations of aqueous solutions of auxiliary agents. These dispersions were shaken on a thermostatic orbital shaker at a temperature of 25 °C for 48 h and 100 rpm. The amount of ETD solubilized in solutions of polymer and amino acids was estimated by using a spectrophotometer at 275 nm. Phase Solubility Study. Auxiliary substances are known to interact with the outer surface of CD or the drug−CD complex, forming aggregates or cocomplexes which exhibit higher stability constants (Kc) and complexation efficiency (CE) values than the binary drug−CD system. The improved Kc and CE of CD associated with the ternary system results in a lesser amount of CD required for preparation of the complex.17,18 The phase solubility study for both binary and ternary complex systems was performed according to the Higuchi-Connors method.19 In all complexation processes including the CDs and drug, the measurement of the stability constant (Kc) is vital since it provides information about the change of physicochemical properties due to host−guest interaction. An excess amount of ETD was added to the vials containing 10 mL of increasing concentrations of HP-β-CD solutions (0− 24 mM) prepared in water (binary systems). The influence of selected amino acid on the drug solubility was evaluated by adding an accurately weighed amount (to obtain 3 mM concentration) of L-Arg into 10 mL of HP-β-CD solutions containing excess of ETD (ternary system). The prepared samples were kept in sealed vials for stirring in a thermostatic orbital shaker at 25 °C for 48 h. Aliquots of the samples were filtered through a 0.45 μm syringe filter and the drug concentration was analyzed by a UV−visible spectrophotom-

Figure 1. Molecular structure of etodolac.

by the U.S. FDA in January 1991 and is classified as a BCS class II drug due to its low solubility and high permeability in the gastrointestinal (GI) tract. It reduces pain, swelling, and joint stiffness in arthritis, which is one of the most chronic inflammatory diseases. However, the use of ETD is restricted due to its low solubility, high dose, twice a day administration, and GI side effects. Various research investigations from the literature focusing on solubility enhancement of ETD include complexation of ETD with CD, HP-β-CD,9 solid dispersions,10 and self-emulsifying systems.11 In the present study, an attempt was made to investigate the effect of L-Arg, an auxiliary agent, on the solubilizing and complexation abilities of HP-β-CD with ETD through the ternary complexation technique. The binary and ternary complexes were prepared by techniques including physical mixing, coevaporation, and spray drying methods. The complexes were characterized using saturation solubility studies, in vitro dissolution studies, and various instrumental B


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dissolution study was performed. Dissolution studies on the complexes were performed using a USP type II paddle type dissolution apparatus. Complexes equivalent to 100 mg of ETD was weighed and placed into 900 mL of pH 1.2 HCl buffer. The dissolution medium was maintained at 37 ± 0.5 °C and stirred at 75 rpm. Aliquots (5 mL) were withdrawn at predetermined time intervals and filtered through Whatmann filter paper no. 41. Sink conditions were maintained by the addition of an equal volume of fresh dissolution medium. The filtered sample solutions were analyzed for the amount of drug by using a UV spectrophotometer (Shimadzu, UV 1800) at 275 nm, and the percent release of drug was calculated. Powder X-ray Diffraction (PXRD) Studies. The PXRD spectra of the samples were recorded using a high-power powder X-ray diffractometer (PANalytical) with Cu as the target filter having a voltage/current of 40 kV/40 mA at a scan speed of 4°/min. The samples were analyzed at a 2θ angle range of 5° to 40°. The step time was 0.5 s, and 1 h was the time of acquisition. Observations were made for changes in the characteristic peaks of pure ETD and ICs. Crystallinity was determined by comparing representative peak heights in the diffraction patterns of the ICs with that of ETD. Fourier Transform-Infrared Spectroscopy (FT-IR). The spectra were recorded on a FT-IR spectrometer (PerkinElmer, Inc.) by the KBr disc method in the range of 4000−400 cm−1 to study the interaction of ETD with CD and L-Arg. Differential Scanning Calorimetry (DSC) Studies. It is a widely used technique for quality control purposes, such as purity of samples. Differential scanning calorimeter was used to record the thermograms. The samples were examined on a differential scanning calorimeter (Mettler Toledo DSC 822) using Stare SW 10.00 software. The samples were scanned in a nitrogen atmosphere with a flow rate of 0.2 kg/m2 using aluminum pans. The scanning was performed in the temperature range of 50−300 °C at a heating rate of 10 °C min−1. Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR). 1H NMR study was carried out to understand the mechanism of interaction of protons of ETD with the hydrophobic cavity of HP-β-CD. The NMR spectra of ETD and ETD−HP-β-CD binary complexes and ETD−HP-β-CD− L-Arg ternary complexes were recorded in deuterated dimethyl sulfoxide (DMSO-d6) on a Bruker FT-NMR spectrophotometer operating at 500 MHz using tetramethyl silane (TMS) as an internal standard. Molecular Modeling Studies of ETD Inclusion Complexes. The molecular modeling studies of ETD with HP-βCD in the presence and absence of L-Arg was carried out using the Schrödinger software suite (Schrödinger, LLC, New York) in the Maetsro module (version 10.7). Structure Collection. Using the Maestro structure builder, ETD and L-Arg structures were drawn, and the obtained structures were optimized using the LigPrep (version 3.9, Schrödinger) module. To generate the proper ionization state, LigPrep was run with the Epik (version 3.7, Schrödinger) option set to generate a possible state at target pH 7.4. Since ETD is a racemic mixture, both R and S geometry were generated. Finally the geometry optimization was carried out using the OPLS2005 force field.21−23 The β-CD crystal structure was obtained from the Protein Data Bank PDB ID 1BFN. The crystal structure was processed using Protein Preparation Wizard, to add missing hydrogen, and then β-CD crystal structure was separated from β-amylase. HP-β-CD was generated by manually attaching isopropyl group on the 6-OH

eter (Shimadzu, UV 1800). Phase solubility curves for the binary and ternary systems were obtained by plotting the concentration of ETD against HP-β-CD concentration in the sample. The Kc was determined using the saturation solubility of ETD (S0) in water in the absence of HP-β-CD (for binary system) or HP-β-CD−L-Arg (for ternary system) and slope of the phase solubility curve. Kc =

slope S0(1 − slope)

The complexation efficiency (CE) value is defined as the ability of the CD to form a complex with the hydrophobic molecule through inclusion of drug in a hydrophobic cavity of CD. It is more convenient to compare the CE than the Kc values, since CE is less sensitive to errors related to estimation of intrinsic drug solubility and is useful to choose appropriate conditions for complexation of the drug with CD.6,7 Hence, CE was calculated using the following equation complexation efficiency (CE) =

slope (1 − slope)

Preparation of Inclusion Complexes (ICs). Binary inclusion complexes containing ETD−HP-β-CD (1:1 mol ratio) and ternary complexes containing ETD−HP-β-CD−LArg (1:1:1 mol ratio) were prepared by methods as described below.20 Physical Mixing (PM) Method. The physical mixture (PM) of binary system was prepared using 1:1 molar ratio of ETD and HP-β-CD. Sieving of this mixture was done using sieve no. 80. The ternary PM containing ETD−HP-β-CD−L-Arg was prepared in a similar manner with addition of an equimolar amount of L-Arg. Coevaporation (CE) Method. For preparation of binary complex, a solution of HP-β-CD in water and a solution of ETD in ethanol were prepared by maintaining a 1:1 molar ratio of ETD and CD. The aqueous phase was added gradually to the organic phase with stirring on a magnetic stirrer. After thorough mixing, the solvent was evaporated by heating the mixture at an elevated temperature. The ternary complex was prepared analogous to the binary complex with addition of an equimolar amount of L-Arg to the solution of HP-β-CD. The products were dried and passed through an 80 mesh sieve and stored in a desiccator. Spray Drying (SD) Method. The equimolar quantities of HP-β-CD and ETD were dissolved in water and ethanol, respectively. The aqueous phase was added to the organic phase stirred on a magnetic stirrer in a dropwise manner. The resultant solution was fed into the spray drying machine (LV222 advanced SD-1000) and sprayed in the chamber from the spray drying nozzle. The spray dried product obtained was collected. The ternary complex was prepared in a similar manner to the binary complex with addition of an equimolar concentration of L-Arg to the solution of HP-β-CD. Characterization of ICs. Saturation Solubility of ETD in IC. Solubility of ETD in IC was determined in distilled water and HCl buffer (pH 1.2) by the shake flask method. ICs were added in excess amount to the vials containing solvents and stirred on an orbital shaker for 48 h at 25 °C. The resulting dispersions were filtered through a syringe filter (0.45 μm), and ETD was quantified spectrophotometrically at 275 nm. In Vitro Dissolution Study. To elucidate the effect of complexation of ETD on its release behavior, an in vitro C


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Figure 2. Solubility of ETD in various auxiliary agents.

Figure 3. Phase solubility curves of binary system (ETD−HP-β-CD) and ternary system (ETD−L-Arg−HP-β-CD).

individual contributions of various types were calculated; the binding energy was calculated according to the equation:

group of glucopyranose. HP-β-CD geometry was optimized using the Macro Model (version 11.3, Schrödinger) suite using the OPLS2005 force field.24,25 Generation of Supramolecular Inclusion Complex Models. The Glide (version 7.2, Schrödinger) module was used for generating HP-β-CD inclusion complexes.26 The grid was generated using the Glide Grid Generation panel in Glide. For generating HP-β-CD binary supramolecular inclusion complex, ETD was docked with standard precision (SP) mode (separately with each “R” and “S” geometry) on HP-β-CD. The ternary supramolecular inclusion complex was generated by docking the binary inclusion complex with L-Arg in SP mode. Binding Affinity Calculation. The binding affinity “ΔG” was calculated using the Prime MM-GBSA module (version 4.5, Schrödinger), which calculates the free energy change upon formation of the complex in comparison to total individual energy based on change in the solvent accessible surface area.27 The properties including columbic energy, van der Waals energy, lipophilic energy, π−π packing energy, and generalized borne electrostatic solvation energy were calculated for complexes, ligand, and receptor. Strain energies were calculated for the ligand and receptor. For each of these properties,

ΔG _bind = E _complex(minimized) − E _ligand(minimized) − E _receptor(minimized)

The Embrace (Macromodel, version 11.3, Schrödinger) minimization panel was used to calculate the free energy change upon formation of complex in comparison to total individual potential energy. The Embrace gives a molecular mechanics estimate for binding free energy of a ligand. The potential energy calculations were run in two modes; the first mode was “energy difference mode”, where the calculation was first performed on the receptor, followed by ligand and finally on complex. This mode includes relaxation of the ligand and receptor on binding. The energy difference is Del E = Ecomplex − E ligand − Eprotein

The second mode used for potential energy calculation was “interaction energy mode”, where interaction energy between the receptor and ligand was calculated. This energy is calculated from interactions between ligand atoms and receptor atoms but D


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Molecular Pharmaceutics excludes the contributions coming from ligand only and receptor only. Molecular Dynamics Simulation. The Desmond (version 4.7, Schrödinger) module from Schrödinger was used for running MD simulations with periodic boundary conditions. The supramolecular inclusion complex was immersed in an orthorhombic simulation box, with the TIP4P explicit water model using the System Builder Panel with the minimum thickness of a solvent layer, 10 Å. In order to neutralize the system, counterions were added. The system was minimized and finally subjected to 5 ns MD simulation with the NPT ensemble at 300 K temperature and 1.013 bar pressure with default settings. The temperature and pressure was maintained during simulation using a Nose-Hoover Chain thermostat and a Matrtyna-Tobias-Klein barostate, respectively. A time step of 2 fs was used. Saving energy and structure enumerated for every 5 ps during simulation, the MD trajectory was generated. Finally to analyze trajectory simulation, an even analysis tool was used.

Table 2. Saturation Solubility of ICs solubility (mg/mL)

R2a

ETD−HP-β-CD ETD−HP-β-CD−L-Arg

162.11 ± 6.24 2573.31 ± 11.31

0.078 1.209

0.9813 0.9859

0.137 ± 0.0025 0.268 ± 0.0015 0.592 ± 0.0265 0.311 ± 0.0063 3.821 ± 0.0577 15.123 ± 0.4500 22.256 ± 0.1000

0.039 0.584 1.439 1.967 0.873 2.854 2.578

± ± ± ± ± ± ±

0.0003 0.0052 0.0613 0.1112 0.0669 0.0288 0.1258

enhancements of ETD in ternary complexes prepared by coevaporation and spray drying methods were found to be 163and 100-fold, respectively, in distilled water with respect to saturation solubility of pure ETD. The solubility enhancements in pH 1.2 HCl buffer were observed to be 65- and 73-fold, respectively. The increased solubility of ETD observed in the ternary system compared to binary complexes can be attributed to improved complexation efficiency of HP-β-CD due to the addition of L-Arg. Complexes prepared by the coevaporation method revealed the highest solubility of drug compared to complexes of the spray drying method and the physical mixing method. Hence, the complexes of coevaporation method were selected for further characterization studies. It has been reported earlier that auxiliary substance like hydrophilic polymer increases the aqueous solubility of drug by interacting with the external surface of the drug: CD complexes. In addition, van der Waal forces, formation of hydrogen bonds, hydrophobic interactions, and dipole−dipole electrostatic bonds between the drug and polymer has a crucial role in solubility enhancement.26,27 It has been studied in the literature that the basic amino acids like L-Arg form the ternary complex with drug and CD by concurrently interacting with CD via hydrogen bonding, electrostatic interactions, and salt formation.28,29 L-Arg is an amino acid with amphiphilic nature involving its hydrophobic region interacting with the hydrophobic portion of HP-β-CD to form a supramolecular ternary complex. The hydrophilic portion is orientated toward the CD complex as a surfactant and reduces the surface tension, thereby enhancing the aqueous solubility.30,31 In Vitro Dissolution Study. The in vitro dissolution study was performed to investigate the influence of enhanced solubility of ETD on release behavior of complexes. The results of in vitro dissolution study are showed in Figure 4. The dissolution study performed in pH 1.2 HCl buffer, showed 3.82 ± 0.73% release of ETD in initial 10 min. A maximum of 13.12 ± 1.40% drug release was obtained at 60 min. This poor release of pure ETD can be attributed to the less solubility of ETD in pH 1.2 HCl buffer. The binary complex with HP-β-CD showed limited improvement in the dissolution rate. The improvement in drug release was in the order of pure ETD < PM < CE or SD. Complex prepared by physical mixing, coevaporation, and the spray drying method exhibited 23.2 ± 3.12 (BPM), 29.45 ± 3.14 (BCE), and 32.5 ± 2.91 (BSD) % release of drug, respectively, at 10 min. The slightly higher drug release from PM compared to pure ETD can be attributed to formation of in situ soluble

Table 1. Phase Solubility Study of Binary and Ternary Systema CEa

ETD BPM BSD BCE TPM TSD TCE

pH 1.2 HCl buffer

BPM, binary physical mixture; BCE, binary inclusion complex by the coevaporation method; BSD, binary inclusion complex by the spray drying method; TPM, ternary physical mixture; TCE, ternary inclusion complex by the coevaporation method; TSD, ternary inclusion complex by the spray drying method.

RESULTS AND DISCUSSION Selection of Auxiliary Agents. As depicted in Figure 2, it was found that among five auxiliary agents used for the study (HPMC K4, PVP K30, PEG 4000, L-Arg, and glycine), the highest solubility of ETD was observed in L-Arg. Hence, L-Arg was used as an auxiliary agent for phase solubility study of a ternary system and synthesis of ternary complexes. Phase Solubility Studies. The phase solubility curves of binary and ternary systems are shown in Figure3, whereas, Kc, CE, and R2 values are depicted in Table 1. The solubility of

Kc (M−1)b

distilled water

a

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system

producta

a Kc indicates stability constant; CE, complexation efficiency; R2, correlation coefficient. bMean of three determinations ± standard deviation.

ETD increased linearly with respect to HP-β-CD concentration indicating an AL type curve. An improvement in ETD solubility was observed due to the addition of L-Arg. The higher Kc (2573.31 M−1) and CE (1.209) values for the ternary system than the binary system (Kc −162.11 and CE −0.078) can be attributed to addition of L-Arg and therefore suggests a more stable complex formation with improved complexation of CD with ETD. The AL shape of the curve and slope values of curves less than unity suggest existence of 1:1 stoichiometry between HP-β-CD and CD in the presence and absence of auxiliary substances. Saturation Solubility. ETD exhibited low solubility in water (0.137 ± 0.0025 mg/mL). The solubilities of ETD in pH 1.2 HCl buffer and pH 6.8 phosphate buffer were found to be 0.0392 ± 0.0231 mg/mL and 10.5 ± 0.0756 mg/mL, respectively. ETD showed a pH dependent solubility with maximum solubility in alkaline pH and reduced solubility at lower pH. The results of saturation solubility of ETD and complexes are depicted in Table 2. The binary and ternary systems of ETD showed improved solubility as compared to pure ETD. The solubility increment of ETD was higher in ternary complexes compared to binary complexes. The solubility E


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Figure 4. Dissolution profiles of various complexes (BPM, binary physical mixture; TPM, ternary physical mixture; BCE, binary inclusion complex by the coevaporation method; TCE, ternary inclusion complex by the coevaporation method; BSD, binary inclusion complex by the spray drying method; TSD, ternary inclusion complex by the spray drying method; and ETD, etodolac).

Table 3. FT-IR Spectral Data and Spectral Changes of ETD, HP-β-CD, and L-Arg in Binary and Ternary Complexes wavenumber (cm−1) and corresponding functional group samples

NH stretch

CH stretch

CO stretch

CH3 bend

CN stretch

ETD

3345.40

2971.92

1746.44

1412.04

1034.58

binary PM

3345.99

2931.58

1746.81

1411.20 slight splitting

1034.53

ternary PM

3345.95

2923.37

1746.38

1415.59

1034.45

binary CE

3346.24 broad

2931.57 slight broadening

1747.14

1412.10 split

1033.98 broadening

ternary CE

3378.93 highly broad

2929.83 flattening

1653.52 flattening

disappeared

1032.42 flattening

complex with HP-β-CD in the dissolution medium.29 The graphs clearly indicated a significant improvement in the dissolution profile of ETD. The binary complexes of the coevaporation and spray drying methods showed an increase in percent drug release by 7.70- and 8.50-fold, respectively, with respect to that of pure ETD. Ternary complexes of the coevaporation and spray drying methods showed an increase of 19.96- and 20.73-fold, respectively, with respect to that of pure ETD. Ternary complexes prepared with the addition of L-Arg showed remarkable improvement in the dissolution profile of ETD than the binary complexes. The complexes prepared by the coevaporation and spray drying methods showed greater dissolution as compared to physical mixtures. However, because of low yield of product by the spray drying technique, the coevaporation complexes were selected as the optimized complexes for further characterization studies. The improved drug release from complex prepared by CE and SD can be ascribed to reduced crystallinity of ETD due to formation of inclusion complexes, which is demonstrated by PXRD studies. Powder X-ray Diffraction (PXRD) Studies. PXRD is a useful technique to detect crystallinity of sample based on its diffraction pattern containing characteristic peaks. Diffraction pattern of pure ETD showed its highly crystalline nature,

indicated by numerous distinctive peaks at diffraction angles of 2θ (9.4°, 13.2°, 14.24°, 17.2°, 18.3°, 20.1°, 22.9°, and 27.3°). A typical hollow-pattern was recorded for HP-β-CD indicating its amorphous nature (Supporting Information). The crystallinity of ETD in IC was determined by comparing representative peaks in the diffractograms of IC with those of ETD. Differences in ETD diffractogram was seen when it is in complex form with HP-β-CD in the presence or absence of LArg. The binary physical mixtures showed relatively less intense peaks, but the crystallinity is evident. The reduction in intensity of characteristic peaks of ETD in binary IC was relatively more compared to physical mixtures. This suggests partially bound ETD in the HP-β-CD cavity leading to partial loss of the crystalline nature of the drug. However, the complete amorphous state was achieved in the case of ternary complexes with L-Arg as indicated by a broad halo in the diffraction pattern. These results were in compliance to the solubility enhancement observations of ETD in binary and ternary IC. Fourier Transform-Infrared Spectroscopy (FT-IR). The FT-IR technique is a valuable analytical tool to analyze the possible interaction of drug with cyclodextrin molecules in the solid state. The FT-IR spectral data of ETD and IC are depicted in Table 3, and FT-IR spectra of samples are F


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This confirms the hypothesis of complex formation. The change in the endothermic peaks is attributed to loss of water molecule, complex formation, reduction in crystallinity, and an increase in the amorphous nature of the sample. The binary IC prepared by CE method showed a small endothermic peak at 151 °C; however, the DSC endotherm of ETD in ternary IC completely disappeared, indicating improved interaction of ETD with HP-β-CD in the presence of L-Arg. Thus, on the basis of the changes and shifts in the various endothermic peaks, it can be concluded that there is a strong interaction between pure ETD and cyclodextrins during the complex formation process. 1 H NMR Spectroscopy. The formation of ETD, HP-β-CD, and L-Arg ternary complex was evidenced by comparing the 1H NMR spectra of ETD and binary and ternary complexes under the same experimental conditions. Chemical shift values of binary and ternary complexes were observed and compared with that of the pure drug. The NMR spectra of ETD, HP-βCD, L-Arg, and IC are depicted in Figure 5. ETD showed

presented in the Supporting Information. The FT-IR spectrum of pure ETD showed N−H stretch, C−H stretch, C−O stretch, −CH3 bend, and C−N stretch at 3345.40, 2971.92, 1746.44, 1412.04, and 1034.58 cm−1, respectively. The FT-IR spectrum of HP-β-CD showed absorption bands at 3397.65 cm−1 (O−H, stretch), 2926.32 cm−1 (C−H, stretch), and 1024.41 cm−1 (C− O−C stretch). The absorption bands for L-Arg were observed at 3293.0 and 3071.71 cm−1 (broad O−H carboxylic or N−H guanidine due to intramolecular hydrogen bonding), 2859.89 cm−1 (C−H stretch), 1681.25 cm−1 (CO, amide), 1322.63 cm−1 (C−N stretch), and 1618.40 cm−1 (CO carbonyl COOH). FT-IR spectra of binary and ternary complexes showed changes in the characteristic absorption bands of ETD. The intensity of the characteristic bands of ETD was strongly reduced in the complexed form. The N−H stretch of ETD was found to completely flatten in the FT-IR spectrum of HP-β-CD ternary complexes prepared by the CE method in contrast to that of binary IC, wherein a prominent peak with reduced intensity was observed. These results reveal the amorphization of ETD with HP-β-CD due to presence of L-Arg in ternary IC than that observed for ETD and HP-β-CD in binary IC. The absorption bands of L-Arg were observed to disappear in IR spectra of complexes indicating interaction of L-Arg with ETD and HP-β-CD, confirming formation of the ternary complex. The IC prepared by the physical mixing method showed less interaction of ETD with HP-β-CD and HP-β-CD−L-Arg in binary and ternary IC, respectively, compared to that of CE method as evidenced by prominent peaks with less reduced intensity seen in complexes prepared by the physical mixing method. Thus, the broadening, flattening, and disappearance of the peaks are indicative of formation of a new solid system between ETD−HP-β-CD and ETD−HP-β-CD−L-Arg resulting in increased solubility. Differential Scanning Calorimetry (DSC) Studies. The DSC technique is of significantly important to understand the compatibility between the drug and cyclodextrin or ternary agents in its complexes. When guest molecules are included in cyclodextrin cavities, their melting, boiling, and sublimation points shift to different temperatures or disappear. The DSC thermograms of ETD and IC are shown in the Supporting Information. The DSC thermogram of ETD displayed a sharp peak (endothermic in nature) at 151 °C, which corresponds to the melting point of ETD. For HP-β-CD, the endothermic peak at 122 °C was observed, which might be attributed to loss of a water molecule. L-Arg exhibited endothermic peaks at 102.7 and 178.4 °C, which could be attributed to loss of water of crystallization from small portions of L-Arginine·2H2O and a melting endotherm at 231.3 °C.32−34 In binary complexes (ETD−HP-β-CD), a shift in characteristic peaks was found in comparison to that of pure ETD. This indicates loss of crystallinity and increase in amorphous nature within the HP-β-CD matrix. The DSC profile of HP-β-CD binary and ternary physical mixture samples exhibited an endothermic peak at 154.4 and 137.7 °C. The ternary PM thermogram did not show any sharp endothermic peak. The DSC graph of the HP-β-CD binary complex of coevaporation method exhibited three endothermic peaks at 114.8, 147.5, and 274.8 °C. The DSC profile of the HP-β-CD ternary complex of the coevaporation method showed an endothermic peak at 212.4 °C. When all the peaks of various complexes were compared with that of pure drug, it was observed that there was a large difference in the melting points and the intensity of the peaks.

Figure 5. NMR spectrum of (a) ETD, (b) HP-β-CD, (c) L-Arg, (d) HP-β-CD binary CE, and (e) HP-β-CD ternary CE.

distinct signals at δ values of 10−12 ppm and 6−7.5 ppm, which correspond to the protons of the −COOH group and benzene ring with the −NH moiety, respectively. It also showed various minor signals at δ values of around 2−6 ppm which may correspond to various −CH moieties in the drug. The HP-β-CD NMR spectrum showed a characteristic signal of the −OH moiety attached to the aromatic ring at the δ value of about 4.5 ppm. The L-Arg NMR spectrum showed two distinct peaks between 2 and 4 ppm. NMR analysis of complexes G


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Molecular Pharmaceutics exhibited a CD induced chemical shift in the various peaks of ETD. A very prominent shift in the δ value (Δδ) was observed for the −COOH group. The δ value was found to change from about 11.9 ppm to 10.8 ppm indicating that the −COOH group of ETD has undergone inclusion in the HP-β-CD hydrophobic cavity to a major extent, leading to an increase in solubility of ETD. All the signals observed in the NMR spectrum of HP-β-CD exhibited a remarkable change in their positions, and the NMR spectrum of complexes could be ascribed to change in the cavity of HP-β-CD molecule to accommodate the guest drug molecule. The benzenic ring attached to −NH group showed a slight change in the number of protons. The number of protons at 5.9 ppm in HP-β-CD has increased from 2 to 5 indicating interactions in this benzene ring moiety. Numerous proton additions were noted in the δ range of 2−6 ppm, which may be attributed to the minor changes in the −CH3, −CH groups of the drug molecule, contributing to the change in the solubility. The NMR spectrum of ternary complex revealed that the LArg signals have encountered tremendous alterations indicating its major role in the solubility enhancement of ETD. There was a slight change in the −COOH moiety signal of ETD. A lot of new signals were seen along with slight shifts in the original signals in the δ range of 6−8 ppm. These evidence lead to the conclusion that the benzene ring substituted with a −NH group plays a major role in alteration of the solubility properties of ETD. It also supports interaction of L-Arg with this particular group to exhibit its effect as a solubility enhancer. Molecular Modeling Studies of ETD Inclusion Complexes. Supramolecular Inclusion Complex Models. Since ETD is commercially available as a racemic mixture, both the R and S forms of ETD were considered for supramolecular inclusion complex model generation. On docking the active S enantiomer of ETD into the HP-β-CD cavity, the docked pose exhibited combination of electrostatic and hydrophobic nonbonded interactions stabilizing the complex. The −NH group from ETD formed a strong hydrogen bond with the 6-OH group of glucopyranose of HP-β-CD with a bond distance of 1.8 Å. The ethyl phenyl moiety from ETD was placed toward 6OH group of glucopyranose of HP-β-CD; the hydroxyl propyl ether moiety of HP-β-CD formed at least three hydrophobic interactions. The pyran ring of ETD substituted with methyl carboxylate and ethyl group on chiral carbon was oriented toward the 2-OH and 3-OH group of glucopyranose of HP-βCD. The binding affinity expressed in the form of Glide docking score for the S enantiomer of ETD with HP-β-CD was found to be −5.88 kcal/mol. As depicted in Figure 6, the S enantiomer of ETD was oriented toward the hydroxyl propyl ether moiety of HP-β-CD. The R form of ETD also acquired the same head to tail orientation as that of the S-enantiomer. The docking score for R form was −5.31 kcal/mol, which was comparable to that of the S-enantiomer of ETD. As depicted in Figure 6, the R-enantiomer was oriented toward the 2-OH and 3-OH groups of glucopyranose of HP-β-CD. The −NH group of ETD formed a hydrogen bond with the oxygen atom forming the ether bridge between two glucopyranose with a bond length of 3.0 Å. The ethyl phenyl moiety from ETD formed hydrophobic interactions with the hydroxyl propyl ether moiety of HP-β-CD. From the docking of binary complexes, both the R and S isomers of ETD have shown a similar kind of binding interactions with the HP-β-CD. The ternary supramolecular inclusion complex was modeled by docking L-Arg on to the binary complex of ETD and HP-β-

Figure 6. Docking binding pose of the inclusion complex.

CD. The R and S enantiomeric ternary complexes were studied separately. Molecular docking studies identified that L-Arg was positioned freely facing toward the 2-OH and 3-OH groups of the glucopyranose units of HP-β-CD in both the enantiomers of ETD. The carboxyl group from ETD formed a strong salt bridge type of interaction with the guanido moiety of L-Arg. The guanido moiety also formed a hydrogen bond with the 2OH and 3-OH groups of glucopyranose. The carboxyl and amino group from the backbone of L-Arg formed several hydrogen bonds with the OH group from glucopyranose. In other words, the L-Arg further increased the stability of the complex by bridging between the ETD and the HP-β-CD. With this molecular docking study, binary and ternary complexes of ETD were further taken to calculate the binding energies to observe the stability of complexes and their interactions. Binding Energy Calculations with Embrace. Using the OPLS force field, the potential energy of the supramolecular inclusion complexes were measured and compared. As described in the Methods, the Embrace module was used for the calculation of the binding energy. Table 4 shows the results of Embrace energy calculations carried out on the dimer and ternary inclusion complexes of different enantiomers of ETD. The S-ETD exhibited the highest binding affinity with HP-βCD and L-Arg in the ternary supramolecular inclusion complex with the “energy difference” mode of Embrace calculations. The differential (Del) total energy was −175.33 kcal/mol, the Del van der Waals energy was −119.93 kcal/mol, and the Del electrostatic energy was −616.60 kcal/mol. The binary complex with the S-ETD had higher binding affinity among the two enantiomers. Embrace energy calculations in “interaction energy” mode estimates the amplitude of interaction between ETD with that of the host in the complex as shown in Table 4. The inclusion complexes with the S-enantiomer exhibited higher “total binding energy” for binary as well as ternary supramolecular inclusion complexes, which was in line with the observations of “energy difference mode”. For complex stabilization, contribution from electrostatic interaction was higher compared to that of Del van der Waals interactions for both the enantiomers in the ternary complex, whereas for the binary complex, contribution from Del van der Waals interactions was higher compared to that of electrostatic interaction for both the enantiomers. H


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Molecular Pharmaceutics Table 4. Embrace Energy Calculationsa energy difference mode kcal/mol

interaction energy mode kcal/mol

complex

Del total energy

Del vdW

Del electro

total energy

vdW

electrostat

R-ETD binary S-ETD binary R-ETD ternary S-ETD ternary

−3.202 −81.483 −91.626 −175.33

−141.043 −115.212 −141.843 −119.939

−2.149 −51.431 −481.639 −616.605

−348.82 −352.55 −789.58 −813.115

−110.322 −66.53 −79.924 −85.701

−238.507 −286.020 −709.656 −727.414

a Del total energy, differential total energy; Del vdW, differential van der Waals energy; Del electro, differential electrostatic energy; total energy, total binding energy; vdW, energy for van der Waals interactions; electrostat, energy for electrostatic interactions.

Binding Affinity Calculations with Prime MM-GBSA. The Prime MM-GBSA method estimates the energy based on the difference in solvent accessible surface area. The “ΔG Bind” is a measure of binding affinity, calculated considering strain energy terms of the ligand and the host. The ΔG binding energy exhibited a similar trend as observed with that of Embrace calculations, where the S isomer of ETD exhibited higher ΔG (−37.128 kcal/mol for the binary inclusion complex, −51.464 kcal/mol for ternary inclusion complex) compared to that of the R enantiomer (−27.326 kcal/mol for binary complex and −43.061 kcal/mol for ternary complex) for both binary and ternary supramolecular inclusion complexes (Table 5). Table 5. Prime MM-GBSA Calculationsa ΔG values in kcal/mol complex R-ETD binary S-ETD binary R-ETD ternary S-ETD ternary

ΔG bind

ΔG bind Colomb

ΔG bind Hbond

ΔG bind Lipo

ΔG bind vdW

−27.326

−1.300

−0.121

−20.728

−21.84

−37.128

−13.287

−0.545

−20.115

−16.493

−43.061

−40.304

−2.084

−21.964

−25.761

−51.464

−60.612

−2.973

−20.346

−16.024

Figure 7. Hydrophobic (brown) and hydrophilic (blue) surface area of ETD, binary complex, and ternary inclusion complexes (CÅ, cubic Angstroms).

ΔG bind, free energy of binding; ΔG bind Colomb, free energy of binding from Coulomb energy; ΔG bind Hbond, free energy of binding from hydrogen bonding; ΔG bind Lipo, free energy of binding from lipophilic binding; ΔG bind vdW, free energy of binding from van der Waals energy. a

upon addition of L-Arg which could be the reason for the observed better complexation efficiency (CE). The predicted binding pose by computational modeling was in agreement with the observed IR and NMR spectral changes. The docking pose showed formation of a hydrogen bond between the −NH group of ETD and HP-β-CD, which was also evident in the IR spectral changes, where a sharp band −NH stretch was noted to broaden or completely flatten. Similarly for −CN stretch, a sharp band observed for pure ETD became broadened or flattened. Similar evidence was observed in the NMR spectrum where the −NH signal disappeared for the ternary complex. The docking pose exhibited hydrophobic interactions between the CH2 and CH3 moieties attached on the chiral carbon of ETD with HP-β-CD, which was also apparent in the IR spectra showing flattening effects on sharp bands. The ternary inclusion complex with L-Arg formed a strong electrostatic interaction with the COOH group of ETD as evident in both the IR and NMR spectral changes. The NMR spectra exhibited no major changes in signals of the COOH moiety for a binary complex, but ternary complex interaction with L-Arg resulted in a large shift of the NMR signal, and a further IR spectral band for the COOH moiety was also observed to flatten for the ternary complex. Molecular Dynamics (MD) Simulations. To analyze the stability of ETD predicted binding mode in the binary and ternary supramolecular inclusion complexes, molecular dynamics (MD) simulation was run separately for each isomer on

Both the Embrace and the MM-GBSA binding energy calculations manifested observed improvement in the stability constant (Kc) value. It is evident that the Del van der Waals interaction energy remained the same with no significant changes for both binary and ternary inclusion complexes. The increase in the binding energy for the ternary complex was effectively due to an increase in the electrostatic interactions. The complex stabilizing agent L-Arg improved the complex stability by introduction of hydrogen bond and electrostatic interactions. The docking results confirmed the same, where in the binary complex the ETD was stabilized by combination of hydrogen bonding and electrostatic interactions. L-Arg introduced bridged interactions between ETD and HP-β-CD to increase the stability of the complex by occupying the outer surface of the inclusion complex, forming a lid like entity. Further, L-Arg increased solubility, by increasing the polar surface area of the supramolecular inclusion complex. Figure 7 shows details of the hydrophobic and hydrophilic surface areas of ETD, the binary complex, and ternary inclusion complexes. It is evident from Figure 7 that the hydrophilic area increased upon formation of the binary complex, and it further improved I


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Figure 8. Shows the RMSD plotted between the original structure and the structures enumerated during MD simulation of HP-β-CD (red), R and S isomers of ETD (green) and L-Arg (blue).

Figure 9. Shows the H-bond plotted between the original structure and the structures enumerated during MD simulation.

inclusion complex with S-ETD exhibited RMSD within the range of 1.31 Å for HP-β-CD, whereas for S-ETD, RMSD fluctuation was in the range of 2.31 Å. The inclusion complex with R enantiomer showed RMSD within the range of 0.84 Å for HP-β-CD, whereas for R-ETD, RMSD fluctuation was in the range of 1.32 Å. The RMSD deviation between structural frames for ETD was found to be less for ternary inclusion complex than compared to binary inclusion complex, which indicated better stability of the ternary inclusion complex. The ternary inclusion complex with S-ETD exhibited the RMSD within the range of 0.88 Å for HP-β-CD, whereas for S-ETD, the RMSD fluctuation was in the range of 1.08 Å. There was an initial structural drift observed for L-Arg until about 1700 ps, which latter stabilized and was within the range of 1.41 Å. The inclusion complex with R enantiomer showed RMSD within the range of 1.02 Å for HP-β-CD, whereas for the R-ETD the

binary and ternary supramolecular inclusion complexes. The inclusion complex was soaked in explicit solvent water, and MD simulation was run at a temperature of 300 K for a total time period of 5000 ps, during which 1000 structural frames of inclusion complex were enumerated, was saved in the trajectory. To access the stability of inclusion complex structures computed during MD simulation, the structures from the trajectory aligned to HP-β-CD atoms of the first frame and root mean square deviation (RMSD) was calculated for HP-β-CD and ETD with respect to the initial frame. The inclusion complex gets stabilized in the initial phase of MD simulation. So the analysis of the structural stability was carried out on the final phase of 2000 ps. RMSD value indicates the stability of complex and a value within 2.0 Å implies the formation of a stable system. Figure 8 shows the RMSD plot of structures computed through MD simulation to the original structure. The binary J



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RMSD fluctuation was in the range of 0.58 Å, and RMSD of LArg was within the range of 1.05 Å. L-Arg was found to retain interactions observed in docking pose during MD simulations. To find the reason for improved stability of ternary complex in the presence of L-Arg, the intermolecular interactions were observed. From interaction energy analysis, we could make out clearly that polar interaction contributions were higher compared to that of nonpolar interactions in stabilizing the ternary supramolecular inclusion complex. Figure 9A−D shows hydrogen bond interactions between ETD with other residues, and Figure 9E,F depicts the hydrogen bond interaction of L-Arg with other residues in ternary inclusion complex. From the figure, it can be clearly seen that both isomers of the ETD ternary complex have a higher number of hydrogen bonds compared to the binary complex. L-Arg in the ternary inclusion complex acted as a bridging agent and formed multiple hydrogen bonds and electrostatic interactions between ETD and HP-β-CD, which further stabilized the inclusion complexes. L-Arg was observed to position on the outer surface of the complex that further improved the polar surface of the inclusion complex resulting in increased solubility of the complex.

ACKNOWLEDGMENTS The authors are thankful to IPCA Laboratories and Gangwal Chemicals Pvt. Ltd., Mumbai, India, for providing the gift samples of etodolac (ETD) and HP-β-CD, respectively. They are also thankful to Tata Institute of Fundamental Research, Mumbai, India, for facilitating XRD analysis of the samples.

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ABBREVIATIONS ETD, etodolac; HP-β-CD, hydroxypropyl-beta-cyclodextrin; Kc, stability constant; CE, complexation efficiency; L-Arg, Larginine; CD, cyclodextrin; IC, inclusion complex; PXRD, powder X-ray diffraction; FT-IR, Fourier transform-infrared spectroscopy; DSC, differential scanning colorimetry; 1H NMR, proton nuclear magnetic resonance spectroscopy; SP, standard precision; MD, molecular dynamics

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CONCLUSIONS The present study successfully demonstrated the formation of a ternary complex between ETD, HP-β-CD, and L-Arg, proving L-Arg as an effective auxiliary substance in solubility enhancement of ETD using a strategy of inclusion phenomena. Improved apparent stability constant (Kc) and complexation efficiency (CE) of HP-β-CD on addition of L-Arg indicated the formation of stable complex. Change in the crystalline nature of ETD to an amorphous nature was clearly evident by characterization techniques such as DSC and PXRD. Computational modeling revealed that L-Arg increased the stability of the complex by bridging between the ETD and the HP-β-CD through hydrogen bonding and electrostatic interactions. Further molecular dynamics confirmed that L-Arg was positioned on the outer surface of complex and improved polar surface of inclusion complex resulting in increased solubility of the complex. Thus, it can be concluded that the inclusion complex of ETD with HP-β-CD and L-Arg can be used as a novel approach to improve solubility and dissolution performance of ETD for better oral bioavailability. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b01115. P-XRD graphs, FT-IR spectra, and DSC thermograms (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +91-22-42332052. Fax: +91-22-26132905. E-mail: [email protected]. ORCID

Atul P. Sherje: 0000-0001-7306-1646 Notes

The authors declare no competing financial interest. K


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