Fast dissolving drug-drug eutectics with improved compressibility and synergistic effects

Accepted Manuscript Fast dissolving drug-drug eutectics with improved compressibility and synergistic effects

Rajesh Thipparaboina, Dinesh Thumuri, Rahul Chavan, V.G.M. Naidu, Nalini R. Shastri PII: DOI: Reference:

S0928-0987(17)30177-X doi: 10.1016/j.ejps.2017.03.042 PHASCI 3982

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

15 October 2016 23 December 2016 29 March 2017

Please cite this article as: Rajesh Thipparaboina, Dinesh Thumuri, Rahul Chavan, V.G.M. Naidu, Nalini R. Shastri , Fast dissolving drug-drug eutectics with improved compressibility and synergistic effects. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), doi: 10.1016/j.ejps.2017.03.042

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ACCEPTED MANUSCRIPT Fast Dissolving Drug-Drug Eutectics with Improved Compressibility and Synergistic Effects Rajesh Thipparaboina1, Dinesh Thumuri2, Rahul Chavan1, VGM Naidu2, Nalini R Shastri1,* 1

Solid State Pharmaceutical Research Group (SSPRG), Department of Pharmaceutics, National

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Institute of Pharmaceutical Education and Research, Hyderabad, India Department of Pharmacology, National Institute of Pharmaceutical Education and Research,

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Hyderabad, India

*Corresponding author. Nalini R Shastri

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Tel. +91-040-23423749 Fax. +91-040-23073751

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E-mail: [email protected], [email protected]

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Address: Department of Pharmaceutics, NIPER (National Institute of Pharmaceutical Education & Research), Balanagar, Hyderabad, India, Pin Code – 500037.

ACCEPTED MANUSCRIPT Abstract Combinational therapy has become increasingly popular in recent times due to various advantages like greater therapeutic effect, reduced number of prescriptions, lower administrative costs, and an increase in patient compliance. Drug–drug multicomponent adducts could help in combination of drugs at supramolecular level. Two drug-drug eutectics of etodolac with

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paracetamol (EP) and etodolac with propranolol hydrochloride (EPHC) were successfully

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designed and synthesized for the first time. These eutectics significantly improved dissolution and material properties. A 6 to 9 fold enhancement in % dissolution efficiency was found at 1

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min suggesting the fast dissolving capabilities of the eutectic mixtures when compared to plain drug. In addition, eutectic mixtures have shown improved hardness compared to plain drugs. EP

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and EPHC have shown around 5 fold and 3 fold improvements in hardness respectively at 10 MPa when compared to plain etodolac. Cell culture studies have shown improved effects of EP.

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Western blotting analysis revealed that the said combination successfully reduced various inflammatory mediators like TNF-α, COX-2 and IL-6. Whereas, the eutectic combination EPHC

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has shown enhanced cytotoxic effects with synergistic combination index and favorable dose reduction index. The generated multi-component systems EP and EPHC with fast dissolving

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capabilities, improved hardness at lower pressures and synergistic effects represent prospective

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combinations for effective treatment of osteoarthritis and cancer chemotherapy respectively.

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dissolution

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Keywords: Crystal engineering, eutectics, cocrystals, etodolac, paracetamol, compression,

ACCEPTED MANUSCRIPT 1.0 Introduction Multi-component adducts like cocrystals (Duggirala et al., 2016), eutectics (Cherukuvada and Nangia, 2014), and co-amorphous (Dengale et al., 2016) systems have diverse applications in pharmaceutical sector. Multi-component adducts with excipients are vastly explored. Current research is trending towards the development of combinational therapeutics using solid form

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screening. Literature available on drug-drug multicomponent adducts is limited and are relatively

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less explored due to many complications involved in their development (Cherukuvada and

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Nangia, 2012; Laitinen et al., 2013; Thipparaboina et al., 2016). Eutectics have been the outcomes during many cocrystals screening experimentations (Cherukuvada, 2016; Cherukuvada and Guru Row, 2014; Cherukuvada and Nangia, 2012; Ganduri et al., 2015; Kaur et al., 2015;

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Prasad et al., 2015; Prasad et al., 2014) due to the classic battle between “interactions” and “packing” for achieving a stable supramolecular assembly (Cherukuvada and Guru Row, 2014).

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They have found diverse applications in energy storage devices, in ceramics and glass industry, traditional refrigeration, snow removal, as an anti-freeze in vehicles along with few applications

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in drug delivery. Lidocaine and prilocaine combination marketed under trade name EMLA is a eutectic composition with enhanced transdermal permeation of lidocaine (Cherukuvada and

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Nangia, 2014) As per IUPAC Gold Book, eutectic formation is defined as “an isothermal, reversible reaction between two (or more) solid phases during the heating of a system, as a

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result of which a single liquid phase is produced” (Brito et al., 2015). Very few drug-drug

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eutectic compositions reported include lidocaine–prilocaine, and their combinations with tetracaine, bipuvacaine (Broberg and Evers, 1985), fenofibrate–acetylsalicylic acid (Gorniak et al., 2011), acetaminophen–propylphenazone (Bi et al., 2003), pyrazinamide–isoniazid

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(Cherukuvada and Nangia, 2012) and rifampicin–isoniazid (Lavor et al., 2011), curcumin– hydroquinone (Goud et al., 2012), simvastatin-aspirin (Gorniak et al., 2013), aspirin-paracetamol (Jain et al., 2014) and metronidazole-clarithromycin (Agafonova et al., 2014). Etodolac is chemically 2-(1,8-diethyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-yl)acetic acid with a molecular weight of 287.35 g/mol, melting point 146.5 0C, pKa 4.65 and LogP of 2.5. It is a poorly soluble drug (aqueous solubility 0.016 mg/mL) and belongs to BCS class II (O Neil, 2013). It exhibits bitter taste, poor flow and compressibility (Hayashi et al., 2013; Jitkar et al., 2016). It is an NSAID used in the treatment of inflammation and pain caused by osteoarthritis

ACCEPTED MANUSCRIPT and rheumatoid arthritis. Its activity is well documented in Alzheimer’s disease in combination with quercetin (Singh et al., 2014). Recent reports have revealed its activity in melanoma and carcinoma in combination with propranolol (Glasner et al., 2010). Patent has been filed for its activity against hyperplasia (Carson et al., 2007) and multiple myeloma (Carson et al., 2006). Recommended dose for analgesic effect is 100 to 450 mg and for anticancer effect it is 600 mg (Bhattacharyya et al., 2010). Various approaches like salt formation (David et al., 2012), ionic

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liquids (Cojocaru et al., 2013), hydrotalcite complexes for the suppression of bitter taste,

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cyclodextrin complexation (Brito et al., 2015; Hayashi et al., 2013), hydrotrophy (Kadam et al.,

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2016), nanosuspensions (Afifi et al., 2015) and spherical agglomeration (Jitkar et al., 2016) are reported for improving various physicochemical properties of etodolac.

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Current study was designed to develop drug-drug multi component systems of etodolac along with propranolol hydrochloride and paracetamol for improving their pharmaceutical

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properties and exploring their applications in prostate cancer (Bhattacharyya et al., 2010) and osteoarthritis (Pareek et al., 2010) respectively. Etodolac and propranolol is a unique chrono-

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modulated combination widely known for its utility in precision immunotherapy which acts through various pathways involving adrenergic beta receptors 1-2, nociception signaling

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cascades, COX-2, TRPA1, MAPK, PI3K, PKA and STAT3 (VT-122) (Bhattacharyya et al., 2010). Etodolac and paracetamol combination is well known for its application in osteoarthritis

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which acts through multiple pathways which are unexplored to date and has shown improved safety and efficacy (Pareek et al., 2010). Chemical structures of the compound studied are given

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in Fig. S1 (Supporting Information).

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2.0 Materials and methods

Etodolac (Racemic form) and paracetamol were obtained from Yarrow Chem laboratories, Mumbai and Nihal traders, Hyderabad respectively. Propranolol hydrochloride was obtained as a gift sample from Aurobindo Laboratories Pvt Ltd, Hyderabad. HPLC grade acetonitrile was purchased from Merck. Ammonium formate was obtained from SD Fine chemicals. Polyvinylidene difluoride membranes were obtained from Millipore (Bedford, MA, USA). Radioimmunoprecipitation assay buffer (RIPA) was obtained from Sigma Aldrich. ECL (enhanced luminol-based chemiluminescent substrate) detection kit was purchased from

ACCEPTED MANUSCRIPT Amersham Bioscience. Dulbecco's Modified Eagle Medium (DMEM) was obtained from Sigma Aldrich. In-house, ultra-pure water from Millipore® was used for all the experiments. 3.0 Prediction and synthesis Hydrogen bond formation of etodolac with paracetamol and propranolol hydrochloride was predicted using amorphous cell tools of Material Studio software® 7.0. Multicomponent

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forms were synthesized by grinding stoichiometric proportions of etodolac with paracetamol

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(EP) and etodolac with propranolol hydrochloride (EPHC) in presence of ethanol. Phase

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diagrams were constructed by grinding the pure materials in a mortar and pestle. Batches of 400600 mg were prepared from the pure materials in the entire range of said combinations in molar

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fractions of etodolac from 0 to 100 %. A small amount of volatile solvent diethyl ether was added to aid mixing between components, followed by gentle mixing in pestle for 5 min.

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Mixtures were stored in dessicator until further characterization. Phase diagrams were constructed by carrying out thermal analysis of various mixtures of said combinations in various

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mole fractions of etodolac like 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, (Diarce et al., 2015).

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4.0 Characterization

Thermal analysis was performed using a Mettler Toledo DSC with Stare module. Samples

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of about 10 mg were weighed in aluminium pans (40µL) using Sartorius balance (CAP22D, Germany) and crimped with pin holed aluminium lids. Heating experimentations were carried at a ramp of 5 °C min−1 in the ranges 35-175 °C and 35-185 °C for propranolol hydrochloride and

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paracetamol combinations respectively in the presence of dry nitrogen flow at 80 mL min −1. Powder diffractograms were recorded using Bruker D8 Advance diffractometer (Bruker-AXS,

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Karlsruhe, Germany) with Cu-Kα X-radiation (λ = 1.5406 Å) at 40 Kv S20 and 30 mA power. Data was collected over the 2θ range 5–50°, at a scan rate of 1°/min. FTIR spectrums of samples were recorded from 400 to 400 cm-1 by placing sample disc in suitable holder in PerkinElmer IR spectrophotometer. Disc was prepared by compressing the blend of sample (2 mg) with potassium bromide IR powder (100 mg) under vacuum at a pressure of 12 psi for 3 min. 5.0 Evaluation 5.1 HPLC

ACCEPTED MANUSCRIPT Drugs were quantified using HPLC system (e2695 Waters) consisting of a HPLC pump, an automated injector equipped with a UV detector (2998 PDA) and an auto sampler. Fortis C18 column (150 x 4.6 mm, 5 µ) was used for analysis. Acetonitrile and ammonium formate (5 mM) buffer in 80:20 ratio was used as a mobile phase at a flow rate of 1mL/min in isocratic mode with a run time of 4 min. Sample analysis was carried out by injecting 10 µL of the sample and absorbance of the elute was recorded at 226 nm for etodolac and propranolol hydrochloride, and

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at 248 nm for paracetamol, following suitable dilutions.

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5.2 In vitro dissolution studies

Powder dissolution studies were carried out in USP Apparatus II at 50 rpm by adding

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weighed quantities of solid forms equivalent to 200 mg of etodolac (BSS #sieve no 60 passed) in 900 mL pH 6.8 phosphate buffer, pre-warmed to 37±0.5 °C. Aliquots of 3 mL samples were withdrawn at predetermined time intervals of 1, 3, 5, 10, 15, 30, 45, 60 and 120 min substituting

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the same with equal quantity of fresh dissolution medium (Ibrahim et al., 2010). Samples were filtered using 0.22 µ filters. The aliquots following suitable dilution in the mobile phase were

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analyzed using a validated HPLC method as described above. Dissolution profiles were analyzed

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for the amount of drug released in 5 min (Q5) and the dissolution efficiency (DE5) at 5 min. All the calculations were performed using DDSolver (Zhang et al., 2010).

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5.3 Cell line studies

In vitro cell line studies were carried out for both alloys. The cytotoxic effects of EPHC

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combination were evaluated on PC-3 cells while the effect of EP combination on inflammatory

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markers in RAW 264.7 cells was determined using Western Blotting studies. 5.3.1 MTT assay

PC-3 (Prostrate cancer cell lines) and RAW 264.7 (mouse macrophage) cells were maintained in RPMI (Roswell Park Memorial Institute medium) and DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS and 1 % penicillin-streptomycin. Briefly, 1 × 103 RAW 264.7 cells and 5 × 103 PC-3 cells per well were seeded in 96 well plate and maintained at 37 °C and 5 % CO2. After 24 h PC-3 cells were treated with various concentrations etodolac and propranolol hydrochloride ranging from 25 to 1000 μM for 48 h. RAW 264.7 cells were treated with 100, 200 μM etodolac and paracetamol for 48 h to assess cell viability.

ACCEPTED MANUSCRIPT Further, cell viability was determined by MTT method. At the end of the experiment, 10 μL of MTT (5 mg/mL) in 100 μL medium was added and incubated at 37 °C for 4 h. Then the media with MTT was removed and the purple formazan crystals formed were dissolved in 200 μL of dimethyl sulphoxide and read at 570 nm using multidetection plate reader (Thummuri et al., 2015).

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5.3.2 Western blotting studies

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For western blotting, RAW 264.7 (mouse macrophage) cells were pre-treated with

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etodolac, paracetamol and EP eutectic at 25 and 50 µM concentrations for 2 h, and then stimulated with 100 ng/mL lipopolysacharide (LPS) for 12 h. Whole cell extracts were prepared

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using RIPA buffer, phosphate and protease inhibitor cocktails. Protein concentration was measured using Bradford protein assay. For western blotting, proteins were resolved by SDS-

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PAGE; then transferred to polyvinylidene difluoride membranes. Non-specific interactions were blocked with 5% bovine serum albumin for 1 h. Then membranes were probed with the indicated primary antibodies for 12 h at 4 °C followed by incubation with the appropriate secondary

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antibodies conjugated with horseradish peroxidase (HRP). The antigen-antibody complex was

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visualized with an ECL detection kit. For subsequent antibody treatment, membranes were stripped in stripping buffer and re-probed with another antibody. The immune blots were

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quantified by densitometry scanning with NIH Image J software. Combination effects observed from in vitro experiments were assessed using Chou-

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Talalay method, which is based on the median-effect equation. In this method, drug dose and the effect for each of the drugs and the eutectic mixtures were fed as input to the CompuSyn

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software. Based on the median-effect plot outputs generated from the software, different parameters such as median dose (Dm), m (slope or kinetic order) and r (linear correlation coefficient of the median-effect plot) for each drug and eutectic mixtures were obtained. From the Fa-CI plot (combination index plot or Chou-Talalay plot) the efficacy of combination was determined. Similarly from the Fa-DRI plot (dose-reduction index plot or Chou-Martin plot) possible dose reduction for each of the drugs used in the combination were successfully determined (Chou and Martin, 2005). 5.3.3 Compression properties

ACCEPTED MANUSCRIPT Compression properties were evaluated by Well’s method and compared with plain drugs. 500 mg of drug-drug multicomponent forms prepared as per Well’s protocol were compacted at 10 MPa using 13 mm die and punch in an IR pellet press and then equilibrated in dessicator for 24 hrs (Wells and Aulton, 2002). In separate experiments, 500 mg of etodolac, propranolol hydrochloride, paracetamol, EP, and EPHC were compressed at different pressures using 13 mm die and diametrical crushing strength of the compacts were determined using

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LABINDIA hardness tester to identify the best form compressible at lowest pressures.

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6.0 Results and discussion

Formation of co-crystal or a eutectic is dependent on the dominance of heteromeric and

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homomeric molecular interactions in a given combination of materials. Various factors like nature of intermolecular interactions and supramolecular synthons, functional group disposition and complementarity, interaction strength, and packing efficiency play an important role in the

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formation of eutectic or cocrystal (Cherukuvada, 2016). The functional groups that can potentially form bonds in etodolac (carboxylic acid and pyrazine –N-) and coformers (ketone,

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amine and hydroxyl groups) are shown in Fig. S2 and given in Table S1 (Supporting

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information). Etodolac has shown medium hydrogen bonding with paracetamol while no hydrogen bonding was observed with propranolol. Possible synthons available for hydrogen

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bondings were O-H....O and N-H....O. Formation of eutectics is due the lack of strong heteromeric interactions that are energetic enough to replace the homomeric interactions. Strong

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acid homodimers of etodolac would lead to eutectic, even if strong heterodimers (for eg. acidpyridine heterodimer competing acid homodimer) are present and the auxiliary interactions that

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can propagate them in the lattice are absent. 6.1 Phase Diagrams

Etodolac has shown melting endotherm in the region of 148-153 °C with a melting peak at 150.33 °C whereas paracetamol has shown melting in the region 168 to 172 °C with a melting peak at 169.6 °C. DSC thermograms of etodolac-paracetamol have shown liquidus point at 132.85 °C in 1:1 molar ratio (Fig. 1). Propranolol hydrochloride has shown melting in the region 163 to 168 °C with a melting peak at 164.82 °C. DSC thermograms of various mixtures of etodolac-propranolol hydrochloride

ACCEPTED MANUSCRIPT have shown liquidus point at 125.08 °C in 1:1 molar ratio (Fig. 2). Onset, peak and endset melting temperatures for both systems are given in Table S2 (Supporting Information). DSC thermograms of various stoichiometric mixtures of etodolac with paracetamol and propranolol hydrochloride are given in Fig. S3 and S4 respectively (Supporting Information). 6.2 p-XRD

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The powder X-ray diffraction (PXRD) technique, routinely employed for the

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characterization of cocrystals, is not found to be useful to diagnose a eutectic. This is because the

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formers are manifested by adhesive interactions that direct distinctive crystal packing, but in case of the latter, as the inclusion of a minor component happens substitutionally or interstitially in

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the major component, the cohesive interactions as well as the lattice structures of parent components remain largely unaffected. Even though, it was used to support the absence of cocrystal formation (Fig. 3). The diffraction patterns obtained for etodolac (Ozkan et al., 2000),

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paracetamol (Wang et al., 2011) and propranolol hydrochloride (Roberts and Rowe, 1994) were in agreement with the published literature. Eutectic mixtures did not show any new peaks and the

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diffraction patterns in eutectics were essentially were additive in nature indicative of absence of

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new crystal formation among the components. 6.3 FTIR

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Core functional groups in all the three drug molecules show secondary amine stretching in the region 3400-2800 cm-1, hence, significant changes in this region can confirm possible

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bond formation. However, no such change was observed in IR spectrum of both the eutectic

(Fig. S6).

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mixtures. The peaks were broad and no conclusive evidence was obtained from the IR studies

6.4 Invitro dissolution studies HPLC was used for sample analysis of etodolac in the linear range of 5-50 µg/mL (Fig. S5, Supporting Information). Powder dissolution studies revealed that about 50 % of drug was released from plain etodolac in 5 min (Q5) whereas around 80 % and 99 % of etodolac was released from EP and EPHC respectively (Fig. 4). This indicates fast dissolving capabilities of eutectic mixtures compared to pain drug. DE5 was found to be increased by 2.5 and 3.2 folds respectively for EP and EPHC. A 6 and 9 fold enhancement in % DE was found at 1 min

ACCEPTED MANUSCRIPT suggesting the fast dissolving capabilities of the eutectic mixtures when compared to plain drug. This enhancement might be due to local solubilization effects produced by paracetamol and propranolol hydrochloride in addition to altered thermodynamic properties such as high free energy, molecular mobility and intermolecular interactions (Cherukuvada and Nangia, 2012; Goud et al., 2012; Sekuguchi and Obi, 1961; Sekuguchi et al., 1964). Amount of drug released

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(Q) and % DE values at 1, 5 and 60 min are provided in Table 1.

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6.5 Cell culture studies

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6.5.1 Cytotoxic effects in PC-3 cells

PC-3 cells are human prostate cancer cell lines and are used in prostate cancer research.

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PC-3 cells have high metastatic potential compared to DU145 cells which have a moderate metastatic potential and to LNCaP cells which have low metastatic potential among all the

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prostate cancer cell lines. PC-3 cells were used for evaluating the efficacy of etodolacpropranolol eutectic combination considering their utility in prostate cancer. Combination effects

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of etodolac and propranolol hydrochloride were investigated by comparing the cytotoxic effects at fixed and varying concentration of etodolac. IC50 values of etodolac, propranolol

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hydrochloride and EPHC were found to be 887.8, 86.7 and 185.6 micrograms. About 4.8 fold decrease in IC50 was observed for EPHC when compared to etodolac. Dose effect and median

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effects of drugs and eutectic mixture are given in Fig. 5. Combination index (CI) for median effective dose was found to be 0.59. A CI value less than 1 indicates synergism, > 1 indicates

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antagonism and =1 indicates additive effects (Chou and Martin, 2005). A dose reduction index (DRI) of > 1 was observed favoring reduction of dose for desired effect in terms of IC 50 value

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resulting may be reduced side effects and toxicity. CI and DRI plots are given in Fig. 6. 6.5.2 Suppression of inflammatory markers RAW 264.7 cells are a macrophage-like cell line derived from BALB/c mice and were chosen to study effect of eutectics on inflammatory markers as etodolac-paracetamol combination is used for osteoarthritis. Cell viability studies revealed safety of the cells used. Western blotting studies have shown marked reduction in inflammatory mediators TNF-α, COX2 and IL-6 with EP eutectic system. Pareek et al. have shown that combination of etodolac and paracetamol was more effective as compared etodolac alone but the mechanisms and mediators

ACCEPTED MANUSCRIPT involved were unexplored (Pareek et al., 2010). An attempt was made to determine the regulation of inflammatory markers for EP system. Arthritis and osteolytic disorders are associated with increased inflammation characterized by up regulation of inflammatory cytokines such as TNF-α, IL-6 and COX-2. Eutectic system has shown significant reduction in inflammatory cytokines such as TNF-α, IL-6 and COX-2 (Fig. 7).

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6.6 Compressibility

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Three different blends A (Blending time (BT) 5 min and dwell time (DT) 2 sec), B (BT 5

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min and DT 30 sec) and C (BT 30 min and DT 2 sec) were used as per Wells protocol to understand compressibility of the materials. Results of compressibility studies are given in Table

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2. Wells compression studies revealed elastic nature of etodolac, paracetamol, propranolol hydrochloride as the blends have shown capping tendency (Fig. 8). EP eutectic has shown good

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hardness in both A and B blends with different dwell times (2 S and 30 S), and blend C was showing low hardness due to increased blending time leading to surface coating of magnesium

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stearate resulting poor binding tendency. EPHC eutectic has shown anomalous behaviour where in blend A was showing lower hardness and blend B and C were showing nearly similar hardness

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despite change in dwell time and blending times. Hence, it was concluded that the eutectics were possibly plastic although interpretations from the outcome of the study was not strongly

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conclusive.

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6.6.1 Crushing strength measurements Crushing strength measurements were carried out to understand the compressibility of the eutectics at lowest possible pressures. A crushing strength of 4-8 Kiloponds is generally

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recommended for tablets intended for oral use. Both etodolac and propranolol hydrochloride were showing very low hardness at 10 MPa. Paracetamol is well known for its compressibility issues and was showing capping tendency even at higher pressures. Eutectic mixtures have shown improved hardness compared to plain drugs (Fig. 9). Etodolac-paracetamol and etodolacpropranolol hydrochloride eutectic mixtures have shown around 5 fold and 3 fold improvement in hardness at 10 MPa compared to etodolac. Increased hardness might be due to altered microstructure in eutectic compositions and would help in development of directly compresisble tablets (Jain et al., 2014).

ACCEPTED MANUSCRIPT 6.7 CONCLUSIONS Novel drug-drug eutectics of etodolac with paracetamol and propranolol hydrochloride was successfully synthesized for possible applications in osteoarthritis and prostate cancer respectively.

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etodolac-propranolol

hydrochloride eutectics have shown fast dissolving capabilities with around 60 % and 92 % drug

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release in 5 minutes. Propranolol hydrochloride and etodolac eutectic combination has shown

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enhanced cytotoxic effects with synergistic combination index and favorable dose reduction index. Etodolac-paracetamol combination has shown good improvement in dissolution, hardness

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and suppression of inflammatory mediators. These prospective combinations could be helpful in

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developing formulations with improved manufacturability and reduced side effects. ACKNOWLEDGEMENTS

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The authors acknowledge financial support from Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India. The authors also acknowledge Dr. N. Satheesh

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Kumar (Assistant Professor) and Mr. David Paul (Research Scholar), Department of Pharmaceutical Analysis, NIPER, Hyderabad for analytical support. Dr. Suryanarayana

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Cherukuvada, Indian Institute of Science is acknowledged for useful discussions. We also thank the reviewers for helping us present the manuscript in the best shape.

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7.0 References

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Afifi, S.A., Hassan, M.A., Abdelhameed, A.S., Elkhodairy, K.A., 2015. Nanosuspension: An emerging trend for bioavailability enhancement of etodolac. Int J Polym Sci 2015, 1-16. Agafonova, E.V., Moshchenskiy, Y.V., Tkachenko, M.L., 2014. DSC study and calculation of metronidazole and clarithromycin thermodynamic melting parameters for individual substances and for eutectic mixture. Thermochim Acta 580, 1-6. Bhattacharyya, G., Julka, P., Bondarde, S., Naik, R., Ranade, A., Bascomb, N., Rao, N., 2010. Phase II study evaluating safety and efficacy of coadministering propranolol and etodolac for treating cancer cachexia, ASCO Annual Meeting Proceedings, p. 18059. Bi, M., Hwang, S.-J., Morris, K.R., 2003. Mechanism of eutectic formation upon compaction and its effects on tablet properties. Thermochimica acta 404, 213-226. Brito, R.G., Araújo, A.A., Quintans, J.S., Sluka, K.A., Quintans-Júnior, L.J., 2015. Enhanced analgesic activity by cyclodextrins–a systematic review and meta-analysis. Expert Opin Drug Deliv 12, 1677-1688. Broberg, B.F., Evers, H.C., 1985. Local anesthetic mixture for topical application and method for obtaining local anesthesia. US Patent 4562060. Carson, D.A., Cottam, H.B., Adachi, S., Leoni, L.M., 2006. Use of etodolac in the treatment of multiple myeloma. Carson, D.A., Leoni, L.M., Corr, M.P., 2007. Use of etodolac to treat hyperplasia. US7211599 B2.

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Figure Captions Fig. 1 Phase diagram depicting liquidus point in etodolac-paracetamol eutectic mixtures (EP) Fig. 2 Phase diagrams depicting liquidus point of etodolac-propranolol hydrochloride (EPHC) Fig. 3 p-XRD patterns of eutectics (EPHC and EP) along with plain drugs

ACCEPTED MANUSCRIPT Fig. 4 Dissolution profiles of etodolac and eutectic combinations Fig. 5 Dose effect (A) and median effects (B) (E-etodolac, PHC-propranolol hydrochloride, EPHC-Eutectic) Fig. 6 Combination index (CI) and dose reduction index (DRI) plots (E-etodolac and PHCpropranolol hydrochloride)

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Fig. 7 (A) Cell viability in control cells. (B) Western blotting analysis of TNF-α, COX-2 and IL6 in RAW 264.7 cells. (C) Graphical depiction of western blotting analysis of TNF-α, COX-2

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and IL-6. Data expressed as mean ± SEM (n = 3). *P < 0.05, p** < 0.01, ***P < 0.001 significant vs Lipopolysacharide control.

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Fig. 8 Pictorial compilation of tablets formed from compressibility studies

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Fig. 9 Crushing strength measurements at 10 MPa pressure (n=3, mean±SD) * Capping Table Captions

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Table 1 Inferences from dissolution profile analysis

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Table 2 Inference from Well’s compressibility studies (n=3, mean±SD)

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Phase diagram of EP

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Molar fraction of E

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Fig. 1 Phase diagram depicting liquidus point in etodolac-paracetamol eutectic mixtures (EP)

Phase diagram of EPHC

170.0

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Fig. 2 Phase diagrams depicting liquidus point of etodolac-propranolol hydrochloride (EPHC)

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Fig. 3 p-XRD patterns of eutectics (EPHC and EP) along with plain drugs

Fig. 4 Dissolution profiles of etodolac and eutectic combinations

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Fig. 5 Dose effect (A) and median effects (B) (E-etodolac, PHC-propranolol hydrochloride,

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Fig. 6 Combination index (CI) and dose reduction index (DRI) plots (E-etodolac and PHCpropranolol hydrochloride)

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Fig. 7 (A) Cell viability in control cells. (B) Western blotting analysis of TNF-α, COX-2 and IL6 in RAW 264.7 cells. (C) Graphical depiction of western blotting analysis of TNF-α, COX-2 and IL-6. Data expressed as mean ± SEM (n = 3). *P < 0.05, p** < 0.01, ***P < 0.001 significant vs Lipopolysacharide control.

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Fig. 8 Pictorial compilation of tablets formed from compressibility studies

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Hardness (kP)

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EPHC

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Fig. 9 Crushing strength measurements at 10 MPa pressure (n=3, mean±SD) * Capping

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Q (%)

EP

EPHC

DE (%)

Q (%)

DE (%)

Q (%) 91.66

45.82

99.68

84.05

10.56

5.28

60.46

30.22

5

50.91

25.5

79.13

62.07

60

99.83

80.54

100

91.67

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Time (min)

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DE (%)

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Inference

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2.9±0.35

3.8±0.49

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Observation

Capping

Capping

Elastic

P

3±0.92

Capping

Capping

Capping

Elastic

PHC

2.1±0.44

3.5±0.6

1.6±0.53

Capping

Elastic

EP

10.3±1.52

8.13±0.81

3.97±0.65

A~B>C

Possibly plastic

EPHC

2.43±0.25

9.53±1.06

9.16±0.26

B~C>A

Possibly plastic

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Table 2 Inference from Well’s compressibility studies (n=3, mean±SD)

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Graphical abstract