Development and characterization of fast dissolving tablets of oxaprozin based on hybrid systems of the drug with cyclodextrins and nanoclays

Accepted Manuscript Title: Development and characterization of fast dissolving tablets of oxaprozin based on hybrid systems of the drug with cyclodextrins and nanoclays Authors: Francesca Maestrelli, Paola Mura, Marzia Cirri, Natascia Mennini, Carla Ghelardini, Lorenzo Di Cesare Mannelli PII: DOI: Reference:

S0378-5173(17)30445-3 http://dx.doi.org/doi:10.1016/j.ijpharm.2017.05.033 IJP 16681

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

11-2-2017 12-5-2017 14-5-2017

Please cite this article as: Maestrelli, Francesca, Mura, Paola, Cirri, Marzia, Mennini, Natascia, Ghelardini, Carla, Di Cesare Mannelli, Lorenzo, Development and characterization of fast dissolving tablets of oxaprozin based on hybrid systems of the drug with cyclodextrins and nanoclays.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.05.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development and characterization of fast dissolving tablets of oxaprozin based on hybrid systems of the drug with cyclodextrins and nanoclays Francesca Maestrellia, Paola Muraa,*, Marzia Cirria, Natascia Menninia, Carla Ghelardinib, Lorenzo Di Cesare Mannellib a

Department of Chemistry, School of Human Health Sciences, University of Florence, Via Schiff 6, Sesto Fiorentino I-50019, Florence, Italy b Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Pharmacology and Toxicology Section, University of Florence, Florence, Italy

*Corresponding author: Paola Mura, tel. 0039 055 4573672; e-mail address: [email protected]

Graphical abstract

1

ABSTRACT Previous studies highlighted an increase of the randomly-methylated-ß-cyclodextrin (RAMEB) solubilizing power towards oxaprozin when used in combination with Larginine (ARG) or sepiolite nanoclay (SV). Therefore, the aim of this work was to investigate the possibility of maximising the RAMEB solubilizing efficacy by a joined approach based on the entrapment in SV of the drug-RAMEB-ARG complex. The quaternary nanocomposite was prepared by different techniques and characterized for solid state and dissolution properties, compared to ternary drug combinations with RAMEB-ARG, RAMEB-SV or ARG-SV. The dissolution rank order was drug-RAMEB-ARG-SV>>drug-RAMEB-ARG≈drug-RAMEB-SV>>drugARG-SV. The new hybrid nanocomposite enabled an increase from 60 up to 90 % of oxaprozin dissolution parameters compared to the ternary systems with RAMEBARG and RAMEB-SV. Moreover, the lowest solubilizing efficacy of ternary systems with ARG-SV evidenced the specific synergic effect of both ARG and SV with RAMEB in enhancing oxaprozin dissolution properties. The superior performance of the quaternary nanocomposite was maintained after incorporation in a tablet formulation. In vivo studies on rats proved that the developed fast-dissolving tablet formulation, containing oxaprozin as cofused system with RAMEB, ARG and SV was more effective than the marketed tablet in terms of faster and more intense pain relieving effect in the treatment of adjuvant-induced arthritis.

Keywords oxaprozin; randomly methylated-ß-cyclodextrin; arginine; nanoclay; fast dissolving tablets; in vivo pain relieving effect. Chemical compounds studied in this article Randomly-methylated-beta-cyclodextrin (PubChem CID 51051622); oxaprozin (PubChem CID 4614); L-arginine (PubChem CID 6322); sepiolite (PubChem CID 61797).

2

1. Introduction The low dissolution rate in gastrointestinal fluids of poorly-soluble drugs is a crucial factor limiting their absorption rate and bioavailability (Vemula et al., 2010; Sharma et al., 2009). Therefore, the search for suitable approaches able to overcome such a critical drawback remains as one of the most challenging aspects in the development of new oral drug delivery systems (Yellela, 2012; Kumar et al., 2011). Oxaprozin, a non-steroidal anti-inflammatory drug mainly utilized to treat inflammatory conditions, including rheumatoid arthritis, is currently available on the market as 600 mg oral conventional tablets, with an usual daily dosage of 1.2 g. It is labelled in Class II of BCS (Biopharmaceutics Classification System), due to its high permeability but very poor water-solubility (Yazdanian et al., 2004). In particular, despite its acidic character, it fulfils the BCS low solubility criteria over the entire gastrointestinal pH range from 1.2 to 7.4 (Yazdanian et al., 2004), indicating that its oral absorption is most likely limited by its poor solubility and dissolution in the entire gastrointestinal region. Therefore, the development of a new tablet formulation, with improved oxaprozin solubility and dissolution rate would allow to enhance its bioavailability and reduce its dosage and dose-related side-effects (Rothstein 1998). Cyclodextrins have been widely and successfully used as carriers to improve the solubility and bioavailability of several poorly soluble drugs (Uekama et al., 1998; Loftsson and Duchêne, 2007). Moreover, cyclodextrin complexation can provide additional advantages, including taste masking, dose lowering and side effects reduction, particularly beneficial in the case of anti-inflammatory drugs (Otero Espinar et al., 1991; Ann et al., 1997; Elkeshen et al., 2002; Muraoka et al, 2004). Based on these premises, in a previous study we investigated the performance of different cyclodextrins in improving oxaprozin dissolution properties, and we

3

individuated the randomly methylated ß-cyclodextrin (RAMEB) as the most effective one (Maestrelli et al, 2009). However, the amount of methylated cyclodextrins actually usable in pharmaceutical formulations is restricted by problems of potential dose-related toxicity (Bouldemarat et al., 2005; Ulloth et al., 2007; Kiss et al., 2010). For this reason, it would be very important to find proper ways to increase the solubilizing efficacy of such cyclodextrin derivative, and then reduce the necessary amount to use. Ternary complexation involving salt formation with suitable cations, including basic aminoacids, has been suggested as a possible approach to improve the cyclodextrin solubilizing power towards acidic drugs (Redenti et al., 2001; Mura et al., 2005; Figueiras et al., 2010). In particular, we have found recently that the equimolar ternary system of oxaprozin with RAMEB and arginine exhibited a very marked increase in dissolution efficiency with respect to the corresponding binary system without the basic aminoacid (Mennini et al., 2016). Alternatively, combined strategies based on drug-cyclodextrin complexation and complex entrapment into various carriers such as solid-lipid (Cavalli et al., 1999) or polymeric (Vega et al., 2013) nanoparticles, liposomes (Bragagni et al., 2010; Maestrelli et al., 2010), niosomes (Marianecci et al., 2015), micelles (Li et al., 2010), microemulsions (Mura et al., 2014) or nanostructured lipid carriers (Cirri et al., 2012) proved to be able to enhance the performance of both kinds of nano-carriers, overcoming or reducing the problems related to their use. In this regard, we have experimented recently a new combined approach, by developing a hybrid system, based on loading the oxaprozin-RAMEB complex into nanoclays, with the aim of joining and, possibly, potentiating the relative benefits of both carriers in a single drug delivery system (Mura et al., 2016). The particular interest in the use of inorganic

4

matrices as drug carriers was due to their biocompatibility and high entrapment power (Aguzzi et al., 2007; Viseras et al., 2010), together with their ability to provide increased drug dissolution properties, sustained release, improved drug stability (Ambrogi et al., 2001; Ambrogi et al., 2003; Zheng et al., 2007; Rojtanatanya and Pongjanyakul, 2008; Perioli et al., 2011). The developed "oxaprozin in RAMEB in sepiolite" ternary system allowed to double the % of drug dissolved with respect to the complex as such (Mura et al., 2016). Therefore, considering the interesting results obtained either by ternary complexation of oxaprozin with RAMEB and arginine (ARG) (Mennini et al., 2016) or by the combined use of RAMEB and sepiolite (SV) (Mura et al., 2016), in the present work we considered it worthy of interest to join these different strategies in an only system, to maximise the RAMEB performance. Moreover, in order to evaluate the influence of the preparation method on the performance of the final product, ternary and quaternary combinations of the drug with RAMEB, ARG and SV were prepared by cofusion, cogrinding or coevaporation and characterized for solid state (by Differential Scanning Calorimetry and X-ray powder diffractometry analyses) and dissolution properties. The best systems were selected for the development of tablets, which were evaluated for technological properties and dissolution behaviour. The most effective tablet formulation was finally selected for in vivo studies on rats, to evaluate its therapeutic efficacy in the treatment of induced rheumatoid arthritis compared to the marketed OXA tablet formulation.

2. Materials and Methods

5

2.1. Materials Oxaprozin (OXA) (4,5-diphenyl-2oxazole propionic acid) was kindly provided by S.I.M.S. (Firenze, I) and used as received. Amorphous randomly substituted methyl-ß-cyclodextrin (RAMEB), average MS 1.8, was donated by Wacker-Chemie GmbH (München, Germany). Sepiolite (SV) was from Vicalvaro (Spain). L-arginine (ARG) was from Sigma Chemical Company (St Louis, MO, USA). Microcrystalline cellulose (Emcocel® 90M) was purchased from Penwest Pharmaceuticals Oy (New York, U.S.A.). Magnesium stearate was obtained from Aldrich Chemie GmbH (Steinhelm, Germany). Sodium starch glycolate (Explotab®) was from JRS Pharma (Rosenberg, Germany). Tablets of oxaprozin commercially available in Italy (Walix ®) were from Fidia Farmaceutici, S.p.A, All other chemicals and solvents were of reagent grade and used without further purification.

2.2. Phase-solubility studies Phase-solubility studies of OXA with RAMEB alone (Maestrelli et al., 2009), or in the presence of ARG (1:1 mol:mol) (20 Mennini et al., 2016) were previously performed. Therefore, in order to evaluate the effect of the presence of SV on such systems, excess amounts of OXA (100 mg) combined with SV (1:4 w/w) or ARG (1:1 mol:mol) and SV (1:4 w/w) together, were added to 10 mL of pH 5.5 phosphate buffer solutions, containing increasing concentrations of RAMEB (0-25 mM) in sealed glass containers preserved from the light and electromagnetically stirred (500 rpm) at 25 ± 0.5 ◦C. Aliquots were withdrawn every 24 h with a filter syringe (0.45 µm pore size) until equilibrium (72 h), and spectrometrically assayed for drug content at 285.2 nm (UV-Vis 1600 Shimadzu spectrophotometer, Tokyo, Japan). Each test was performed in triplicate (C.V. < 4%). The apparent stability constants (K1:1) of the

6

complexes were calculated from the slope of the straight line portions of the phasesolubility diagrams (Higuchi and Connors, 1966).

2.3. Preparation of drug-carrier systems OXA-RAMEB-SV, OXA-RAMEB-ARG, OXA-ARG-SV and OXA-RAMEBARG-SV interaction products were obtained by different methods: -Physical mixing: physical mixtures (PM) were obtained by 15 min blending in a turbula mixer the single components previously sieved (75–150 µm sieve fraction); -Cofusion: PMs were heated 20 min at 200 °C, and then let solidify to room temperature in a desiccator (cofused products, COF); -Co-grinding: PMs were ball-milled in a high-energy vibrational micro-mill (Mixer Mill MM 200, Retsch GmbH, Düsseldolf, Germany) at 24 Hz for 30 min (cogroundproducts, GR); -Coevaporation: PMs were dissolved/dispersed in a 60:40 v/v ethanol-water mixture and then the solvent was removed in a rotary evaporator (Laborota 4000, Heidolph, Milan, Italy) (coevaporated products, (COE). Based on previous studies, regardless of the preparation method used, RAMEB and ARG were always added in equimolar ratio with the drug (Mennini et al., 2016), while SV was added at 1:4 w/w ratio (Mura et al., 2016).

2.4. Solid state characterization of drug-carrier systems The solid state properties of the pure components and of their different combinations were characterized by: -Differential scanning calorimetry (DSC): Mettler TA4000 Stare system (Mettler Toledo, Greifensee, Switzerland) equipped with a DSC 25 cell. Accurately weighed

7

samples (5-10 mg, Mettler M3 Microbalance) were scanned in pierced Al pans at 10 °C/min between 30 and 300 °C under static air. The residual crystallinity degree (RCD%) of OXA in the samples was calculated by the following equation: 𝑅𝐶𝐷% =

ΔH𝑠𝑎𝑚𝑝𝑙𝑒 ΔH 𝑑𝑟𝑢𝑔

x 100

where ΔHsample is the sample heat of fusion (normalized to the drug content in the sample) and ΔHdrug the pure OXA heat of fusion. -X-ray powder diffractometry (XRPD): Bruker D8-advance instrument (thetatheta geometry) (Silberstreifen, Germany), using Cu K radiation and graphite monochromator, at 40 mV voltage and 55 mA current. Samples were examined at room temperature in the 5-40° 2θ range, at a scan rate of 0.05°/s. -Environmental Scanning Electron Microscopy (ESEM): the micromorphology of the individual components and of selected interaction products was investigated by FEI Quanta 200 ESEM apparatus, equipped with an EDS-X-ray Microanalysis System (EDAX, software EDAX Genesis).

2.5. Dissolution studies Dissolution studies were carried out according to the dispersed amount method. A sample of 100 mg of drug or drug-equivalent, previously sieved (75-150 µm granulometric fraction), was added to 100 mL of pH 5.5 phosphate buffer solution thermostated at 37±0.5 °C, in a 150 mL beaker. A three-blade paddle (9.5 mm radius) was centrally put in the beaker and rotated at 100 rpm. Aliquots (2 mL) periodically withdrawn with a syringe-filter (pore size 0.45 µm) were spectrometrically assayed for drug content as described above, and replaced with an equal volume of fresh medium. A correction for the cumulative dilution was made. The results are the mean of four experiments. The parameters considered to compare the OXA performance

8

from the different systems were: the relative dissolution rate at 5 min and % drug dissolved at 15 min and the Dissolution Efficiency at 60 min as index, respectively, of the rate and of the totality of the process. All data were analysed by ANOVA (oneway analysis of variance) (GraphPad Prism version 4.0 program, Inc. San Diego CA). Differences were considered statistically significant when P <0.05.

2.6. Preparation and characterization of tablets Tablets containing 100 mg of drug were prepared from binary (OXA-SV or OXA-RAMEB),

ternary

(OXA-RAMEB-SV

and

OXA-RAMEB-ARG),

and

quaternary (OXA-RAMEB-ARG-SV) systems. In order to obtain mixtures with proper flowability, disintegration and compactability properties, 200 mg of microcrystalline cellulose as binder, 4 % Explotab® as superdisintegrant and 1% Mg stearate as lubricant were added to the drug or drug-carrier systems. The powders were mixed 15 min in a turbula mixer and then the mixture compressed in a hydraulic press at 2.5 tons for 1 min. Characterization of tablets was accomplished by uniformity of mass and weight, diameter and thickness, friability, and disintegration tests. All the tests were performed according to European Pharmacopoeia 8th Ed. Dissolution studies of OXA from tablets (mean of four experiments) were performed according to the same experimental conditions above described for powder samples (par. 2.5), in order to be able to compare the results.

2.7. In vivo studies Male Sprague-Dawley rats (Harlan, Varese, Italy) weighing 220-250 g at the beginning of the experimental procedure, were used for all the experiments. Animals were housed in the Centro Stabulazione Animali da Laboratorio, University of

9

Florence, and used no earlier than one week after their arrival. Four rats were housed per cage (size 26 × 41 cm); animals were fed a standard laboratory diet and tap water ad libitum, and kept at 23±1 °C with a 12 h light/dark cycle, light at 7 a.m. All animal manipulations were carried out according to the Directive 2010/63/EU of the European parliament and of the European Union council (22 September 2010) on the protection of animals used for scientific purposes. The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85-23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the experiments described was obtained from the Animal Subjects Review Board of the University of Florence. Experiments involving animals have been reported according to ARRIVE guidelines (McGrath and Lilley, 2015). All efforts were made to minimize animal suffering and reduce the number of animals used. Adjuvant-induced arthritis, triggered by injection of complete Freund’s adjuvant (CFA, Sigma-Aldrich) in the right tibio-tarsal joint of the rats, was used ads model of rheumatoid arthritis (Butler et al., 1992; Di Cesare Mannelli et al., 2016). Briefly, the rats were slightly anesthetized by 2% isoflurane, the left leg skin was sterilized with 75% ethyl alcohol, and the lateral malleolus located by palpation; then, a 28-gauge needle was inserted vertically to penetrate the skin and turned distally for insertion into the articular cavity at the gap between the tibio-fibular and tarsal bone until a distinct loss of resistance was felt. A volume of 50 μL of CFA was then injected (day 0). The control animals were treated with the same volume of physiological saline. The anti-nociceptive effect of the developed OXA tablet formulation, compared with that of the marketed OXA tablet formulation, was evaluated according to the Von Frey test (mechanical allodynia) (Carter and Shieh, 2010). Each tablet

10

formulation, after crushing and dispersion in 1% carboxy-methylcellulose aqueous solution, was orally administered by gavage to the rats, at three different doses (10, 30 and 100 mg kg-1). Control animals received the same volume of blank vehicle. The animals were placed in 20×20 cm Plexiglas boxes equipped with a metallic meshy floor, 20 cm above the bench. A habituation of 15 min was allowed before the test. An electronic Von Frey hair unit (Ugo Basile, Varese, Italy) was used: the withdrawal threshold was evaluated by applying a force ranging from 0 to 50 g with a 0.2 g accuracy. Punctuate stimulus was delivered to the mid-plantar area of each anterior paw from below the meshy floor through a plastic tip and the withdrawal threshold was automatically displayed on the screen. Paw sensitivity threshold was defined as the minimum pressure required to elicit a robust and immediate withdrawal reflex of the paw. Voluntary movements associated with locomotion were not taken as a withdrawal response. Stimuli were applied on each anterior paw with an interval of 5 s. The measure was repeated 5 times and the final value of sensitivity threshold was obtained by averaging the 5 measures (Sakurai et al., 2009). The results were expressed as mean (±S.E.M.) with one-way analysis of variance. A Bonferroni’s significant difference procedure was used as a post hoc comparison. P-values <0.05 or <0.01 were considered significant. Data were analysed using the Origin 9 software (OriginLab, Northampton, MA, USA).

3. Results and Discussion

3.1. Phase-solubility studies Previous phase-solubility studies indicated that the presence of ARG (added at 1:1 mol:mol ratio with the drug) strongly increased the solubilizing efficiency of RAMEB towards OXA, even though it give rise to a decrease of the complex stability

11

constant from 2340 M-1 (binary complex) to 1230 M-1 (ternary complex) (Mennini et al., 2016). On the other hand, dissolution studies performed on ternary OXA-RAMEB-SV cofused systems showed an about 2 times increase in both % drug dissolved at 15 min and Dissolution Efficiency at 60 min, suggesting a synergistic effect of CD complexation and nano-entrapment in clay in improving the OXA dissolution performance (Mura et al., 2016). Therefore, in the present work further phase-solubility studies were initially performed to evaluate the possible role of SV on the solubilizing and complexing properties of RAMEB towards OXA. As in the previous studies (Mennini et al., 2016), also in this case AL type phase-solubility diagrams were obtained, showing a linear increase of drug solubility with increasing the RAMEB concentration, indicative of the formation of soluble complexes (Higuchi and Connors, 1966) (please see the Supplement, Fig. S1). The 1:1 apparent stability constants of OXA-RAMEB and OXA-ARG-RAMEB complexes in the presence of SV were 2380 M-1 and 1210 M-1, respectively. These values were in good agreement with those previously obtained in the absence of SV (2340 M-1 and 1230 M-1, respectively, Mennini et al., 2016), proving that the addition of SV to the complexation medium did not significantly change either the complexing or the solubilizing efficiency of RAMEB towards OXA. Therefore, these results indicated that SV had only a favourable effect on the dissolution rate properties of the drug, both as such and, even more, as complex with RAMEB (Mura et al., 2016), but it did not enhance its equilibrium solubility, and did not affect the binary OXA-RAMEB or ternary OXA-RAMEBARG complex formation.

12

3.2. Preparation and characterization of solid systems of OXA in combination with RAMEB, ARG and SV Taking into account that the technique used for solid systems preparation can strongly affect the performance of the final product (Mura et al., 1999; Maestrelli et al., 2009; Mennini et al., 2014), ternary (OXA-RAMEB-SV, OXA-RAMEB-ARG, OXA-ARG-SV) and quaternary (OXA-RAMEB-ARG-SV) systems were prepared by different methods, i.e. cogrinding (GR), coevaporation (COE) and cofusion (COF), and the obtained interaction products were characterized for solid state and dissolution properties, in comparison with the corresponding physical mixtures (PM) taken as reference. Solid-state characterization was performed by DSC and XRPD analyses. As a first step, to evaluate the possible effect of the preparation techniques on the drug properties, samples of OXA alone were treated by these same methods and compared with untreated OXA. The thermal profile of OXA, characterized by a sharp endothermal peak at 162.6 °C (Hfus 136.8 J.g-1) remained almost unchanged in terms of melting temperature and enthalpy after both grinding (Tfus 162.2, Hfus 129.6 J.g-1), fusion (Tfus 162.0, Hfus 126.6 J.g-1), and solvent evaporation (Tfus 162.1, Hfus 127.3 J.g-1) processes, indicating the absence of relevant amorphization phenomena (RCD% ranged between 97.9 and 94.6). No appreciable variations were detected also in XRPD spectra (data not shown) confirming that the different treatments did not change the crystal structure of the drug. The thermal profile of OXA was still well detectable in its ternary PMs with RAMEB-SV, ARG-SV and RAMEB-ARG, even though shifted to lower temperatures and decreased in intensity (RCD 23, 19 and 16 %, respectively), while a complete disappearance of the drug melting peak was observed in the quaternary PM

13

(Fig. 1). Any significant difference was instead noted among ternary and quaternary products obtained by the different preparation methods, since in all cases the complete absence of the drug melting peak was found, as shown, for example for the series of OXA-RAMEB-SV products. This finding is indicative of solid state interactions between the components, brought about by the different treatments, leading to complete drug amorphization and/or inclusion complexation XRPD studies showed that the typical drug diffraction peaks were clearly evident in all ternary PMs, while a marked, even if not complete, loss of drug crystallinity was detected in the case of the quaternary PM (Fig. 2). The complete disappearance of the drug melting peak observed in the DSC curve of such sample can be reasonably attributable to the easy amorphization of the residual crystalline drug by the thermal energy supplied to the sample during the run. In agreement with DSC results, no crystalline OXA was detected in GR, COF and COE products of all ternary systems, as shown for example for the series of OXA-RAMEB-SV products, where only the diffraction peaks of SV were still detectable. Interestingly, some loss of SV crystallinity was observed in the ternary COF system, probably due to a more intimate interaction of the clay with the amorphous drug-CD complex achieved during the cofusion process. This effect was even more marked in the case of the quaternary COF system, which showed an almost completely diffuse pattern, where only a residual peak of SV at 7.35° 2was still detectable, indicating the nearly full amorphization of all the sample components, as a consequence of a deeper and complete interaction among them. The morphological characterization of the quaternary system, performed by ESEM analysis, substantially confirmed the XRPD findings. In fact, some typical oblong drug crystals were still recognizable in the PM, mixed with the amorphous,

14

almost spherical particles of RAMEB and the irregularly sized crystals of ARG, dispersed on the surface of the lamellar SV structure of (Fig. 3 A). On the contrary, an evident modification of morphology was observed in the corresponding cofused product (Fig. 3 B), where it was no longer possible to distinguish the single components, and only an amorphous mixture appeared, where the EDS-X-ray microanalysis evidenced the highly homogeneous distribution of SV (indicated by the presence of Mg, Si and Al) throughout the sample surface. These findings further confirmed that the individual components, clearly separated in the physical mixture, became closely amalgamated by co-fusion, which enabled an effective adsorption of the ternary complex into the lamellar structure of the clay .

3.3. Dissolution rate studies Previous studies showed that solid systems obtained by the combined use of RAMEB and ARG (Mennini et al., 2016), as well as of RAMEB and SV (Mura et al., 2016) allowed a marked improvement of OXA dissolution properties with respect to those containing RAMEB alone. However, the above systems were prepared, respectively, only by cogrinding (Mennini et al., 2016) or cofusion (Mura et al., 2016). Therefore, in the present work we considered worth of interest to investigate both the effect of different preparation techniques of such ternary systems, as well as of the use of RAMEB, ARG and SV all together. Moreover, to better evaluate the role of ARG in improving the drug dissolution properties when used in combination with RAMEB, also ternary systems based on the combined use of ARG and SV were examined. The dissolution profiles of OXA from its different combinations with RAMEB, ARG, and SV, prepared by physical mixing (as reference), co-grinding, coevaporation

15

and cofusion of the components, are shown in Fig. 4, compared with that of pure drug, while the relative main dissolution parameters are presented in Table 1. By comparing the results obtained with the different ternary combinations, the dissolution performance rank order was OXA-RAMEB-ARG≈OXA-RAMEBSV>OXA-ARG-SV. The limited efficacy exhibited by the ternary systems containing the ARG-SV combination confirmed that the role of ARG in enhancing the drug dissolution properties is mainly related to its synergic action with RAMEB, and not simply due to salt formation with the drug (Mennini et al., 2016), and, at the same time, highlighted the specific synergic effect between SV and RAMEB in enhancing the drug dissolution properties. Interestingly, the combined use of ARG, SV and RAMEB, achieved in the quaternary systems, gave rise to a significant improvement of the drug dissolution properties in terms of both relative dissolution rate at 5 min, % dissolved at 15 min and Dissolution Efficiency at 60 min (P<<0.05) and enabled to reach almost 100% of dissolved drug at the end of the test. Evidently, the simultaneous presence of the three components allowed to join and potentiate the favourable effects shown by RAMEBARG and RAMEB-SV combinations, maximising the RAMEB solubilizing power. As for the effect of the preparation method on the performance of the final product, it was found to be dependent on the kind of system. In fact, in the case of OXA-RAMEB-ARG ternary systems, the rank order in terms of both % dissolved at 15 min and Dissolution Efficiency at 60 min was GR>COF>COE>PM, while for all systems containing SV (i.e. OXA-RAMEB-SV, OXA-ARG-SV and OXA-RAMEBARG-SV) it was COF>GR>COE>PM. Such findings indicated that the cofusion process was the most effective technique in the presence of SV, probably by favouring a more efficient loading of the OXA-RAMEB or OXA-RAMEB-ARG-

16

complexes in the clay lamellar structure. It is reasonably assumable that the adsorption of the binary drug-CD or ternary drug-ARG-CD complex onto the wide surface offered by the nanoclay structure, enhanced the effective surface area, and, at the same time, enabled its fast release from the nanocomposite in the dissolution medium, owing to the weak interaction forces between them.

3.4. Tablet preparation and characterization Based on the results of dissolution studies, OXA-RAMEB-ARG GR, OXARAMEB-SV COF and OXA-RAMEB-ARG-SV COF products were selected as the best systems for preparing fast dissolving tablets of OXA. Tablets containing the corresponding simple binary systems (OXA-RAMEB GR and OXA-SV COF) and the drug alone were also prepared for comparison purposes. All the prepared tablets passed the tests of uniformity of mass and weight (RSD <3%), diameter (RSD<0.3%) and thickness (RSD<1%), friability (weight loss <0.2%) and disintegration time (< 5 min). The dissolution profiles of the drug from the different tablets are presented in Fig. 5 and the related dissolution parameters are collected in Table 2. Dissolution studies were performed under the same experimental conditions previously used, in order to be able to compare the results and evaluate the effect of tablet formulation on the OXA dissolution performance. As can be seen, also after tablet formulation, OXA-RAMEB-ARG GR and OXARAMEB-SV COF ternary systems showed significantly (P<<0.05) better drug dissolution performance than the corresponding OXA-RAMEB GR and OXA SV COF binary systems, as observed in previous studies for the respective powders compounds (Mennini et al., 2016; Mura et al., 2016). Moreover, the same trend was found as in dissolution rate studies from the corresponding powder ternary samples (Table 1). In fact, the two ternary systems exhibited analogous drug dissolution 17

parameters (P>0.05), both allowing an increase of more than 15 times of the drug dissolution efficiency at 60 min, with respect to the plain drug. Furthermore, the OXA-RAMEB-ARG-SV COF system was significantly more effective of both ternary systems (P<<0.05) in terms of dissolution rate and dissolution efficiency, enabling an additional increase of about 1.5 folds of all the considered drug dissolution parameters. These findings indicated that the superior performance of the quaternary nanocomposite system was maintained also after its incorporation into the tablet formulation. On the other hand, the OXA dissolution curve from the commercial tablet (Walix®) was similar (P>0.05) to that from the tablet containing the binary OXA SV cofused system, and clearly lower compared to those from the ternary and even more from the quaternary OXA-RAMEB-ARG-SV cofused product. Based on these results, the new tablet formulation containing the drug as cofused system with RAMEB, ARG and SV was selected for in vivo studies aimed to evaluate its actual therapeutic efficacy in comparison with the commercial OXA tablet formulation (Walix®).

3.5. In vivo studies Previous studies showed that the painful articular inflammation induced by intraarticular injection of CFA generally reaches the peak 14 days after the joint treatment (Di Cesare Mannelli et al., 2016). Therefore, on day 14 after CFA injection, the effect of acute administration of OXA at three different doses, both as the new tablet formulation, in the form of cofused system with RAMEB, ARG and SV, and as commercial tablet (Walix®) was evaluated by stimulation of the rat damaged paw with a non noxious mechanical stimulus according to the Von Frey test (Carter and Shieh, 2010). The results of these studies are shown in Figure 6. The withdrawal threshold of CFA-treated rats decreased to 18.0 ± 0.5 g in comparison to the normal 18

value of 26.9 ± 1.1 g shown by control rats (saline + vehicle). The novel OXA tablet formulation was able to induce a dose-dependent pain reliever effect after a single administration per os. Starting from 10 mg kg-1, the developed OXA formulation significantly increased the pain threshold after only 15 min post-administration. Furthermore, the higher dose (100 mg kg-1) fully reverted the CFA-induced hypersensitivity. On the contrary, the acute administration per os of the OXA marketed tablet (Walix®) was ineffective till the dosage of 30 mg kg-1 and its effect appeared only after 30 min post-administration, even with the higher dose (100 mg kg-1) which, moreover, did not fully revert the CFA-induced rat hypersensitivity. In all cases the pain relieving effect vanished at 60 min. The better therapeutic efficacy of the new OXA tablet formulation can be ascribed to the very faster dissolution of OXA compared to the commercial tablet formulation (see Fig. 5), obtained in virtue of its incorporation as "drug-ARG-RAMEB in SV" hybrid nano-composite. The higher drug dissolution rate as quaternary system allowed a more rapid absorption and a consequent more rapid and powerful action, being effective also at the lower administered dose.

4. Conclusions A hybrid system consisting in the ternary OXA-RAMEB-ARG complex loaded into the SV nanoclay was successfully prepared by a cofusion process. The new system joined the favourable properties previously shown by RAMEB-ARG and RAMEB-SV combinations, enabling to maximize the RAMEB solubilizing effect towards the drug. In fact the new hybrid nanocomposite was clearly more effective than the respective OXA-RAMEB-ARG and OXA-RAMEB-SV systems in improving the

19

drug dissolution properties, enabling a more than 90 % increase of the relative dissolution rate at 5 min and of about 60 % of dissolution efficiency at 60 min. It can be hypothesized a nano-encapsulation of the ternary OXA-RAMEB-ARG complex in the finely divided clay lamellar structure. Such a phenomenon both strongly enhanced the effective surface area, hindering possible aggregation phenomena, and also enabled a very fast release of the complex from the clay surface, when in contact with the dissolution medium, owing to the weak interaction forces between them. In vivo studies on rats proved that the new developed fast-dissolving tablet formulation of OXA, containing the drug as cofused system with RAMEB, ARG and SV had a beneficial effect on CFA-induced arthritis and was more effective than the marketed OXA tablet in terms of both faster and more intense pain relieving effect. In conclusion, the proposed approach proved to be a useful way for enhancing the therapeutic effectiveness of OXA, and also reducing the amount of RAMEB to use for achieving the required drug solubility value. Acknowledgments The authors gratefully acknowledge the valuable scientific advise of prof. César Viseras (Department of Pharmacy and Pharmaceutical Technology, University of Granada, Spain) in the field of nanoclays pharmaceutical applications.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

20

References Aguzzi, C., Cerezo, P., Viseras, C., Caramella, C., 2007. Use of clays as drug delivery systems: possibilities and limitations. Appl. Clay Sci. 36, 22-36. Ambrogi, V., Fardella, G., Grandolini, G., Perioli, L., 2001. Intercalation compounds of hydrotalcite-like anionic clays with antiinflammatory agents - I. Intercalation and in vitro release of ibuprofen. Int. J. Pharm. 220, 23–32. Ambrogi, V., Fardella, G., Grandolini, G., Nocchetti M., Perioli, L., 2003. Effect of hydrotalcite-like compound on the aqueous solubility of poorly-ware-soluble drugs. J. Pharm. Sci. 92, 1407-1418. Ann, H.J., Kim, K.M., Choi, J.S., Kim, C.K., 1997. Effects of cyclodextrin derivatives on bioavailability of ketoprofen. Drug Dev. Ind. Pharm. 23, 397-401. Bouldemarat, L., Bochot, A., Lesieur S., Fattal, E., 2005. Evaluation of Buccal Methyl-β-Cyclodextrin Toxicity on Human Oral Epithelial Cell Culture Model. J. Pharm. Sci. 94, 1300-1309. Bragagni, M., Maestrelli, F., Mennini, N., Ghelardini, C., Mura, P., 2010. Liposomal formulations of prilocaine: effect of complexation with hydroxypropyl- bcyclodextrin on drug anesthetic effect. J. Lipos. Res. 20, 315-322. Butler, S.H., Godefroy, F., Besson, J.M., Weil-Fugazza, J., 1992. A limited arthritic model for chronic pain studies in the rat. Pain 48, 73-81. Carter, M., Shieh, J.C., 2010. "Nociception". Guide to Research Techniques in Neuroscience. Burlington, MA: Academic Press. pp. 51-52. Cavalli, R., Peira, E., Caputo, O., Gasco, M.R., 1999. Solid lipid nanoparticles as carriers of hydrocortisone and progesterone complexes with -cyclodextrins. Int. J. Pharm. 182, 59-69. Cirri, M., Bragagni, M., Mennini, N., Mura, P., 2012. Development of a new delivery system consisting in drug – in cyclodextrin – in nanostructured lipid carriers for ketoprofen topical delivery. Eur. J. Pharm. Biopharm. 80, 46-53. Di Cesare Mannelli, L., Micheli, L., Cinci, L., Maresca, M., Vergelli, C., Pacini, A., Quinn, M.T., Giovannoni, M.P., Ghelardini, C., 2016. Effects of the neutrophil elastase inhibitor EL-17 in rat adjuvant-induced arthritis, Rheumatology (Oxford) 55, 1285-1294. Elkheshen, S.A., Ahmed, S.M., Al-Quadeib, B.T., 2002. Inclusion complexes of piroxicam with -cyclodextrin derivatives in comparison with the natural -

21

cyclodextrin: in vitro and in vivo drug availability. Pharm. Ind. 64, 708-715. Figueiras, A., Sarraguca, J.M., Pais, A.A., Carvalho, R.A., Veiga, J.F., 2010. The role of L-arginine in inclusion complexes of omeprazole with cyclodextrins, AAPS Pharm. Sci. Tech. 11, 233-240. Higuchi, T., Connors, K.A., 1965. Phase-solubility techniques. Adv. Anal. Chem. Instrum. 4, 117-212. Kiss, T., Fenyvesi, F., Bacskay, I., Varadi, J., Fenyvesi, E., Ivanyi, R., Szente, L., Tosaki, A. Vecsernyes, M., 2010. Evaluation of cytotoxicity of -cyclodextrin derivatives: evidence for the role of cholesterol extraction. Eur. J. Pharm. Sci. 40, 376-380. Kumar, A., Sahoo, S.K., Padhee, K., Kochar, P.S., Sathapathy, A., Pathak, N., 2011. Review on solubility enhancement techniques for hydrophobic drugs. Pharmacie Globale 3, 1-7. Li, D.X., Han, M.J., Balakrishnan, P., Yan, Y.D., Oh, D.H., Joe, J.H., Seo, Y., Kim, J.O., Park, S.M., Yong, C.S., Choi, H.G., 2010. Enhanced oral bioavailability of flurbiprofen by combined use of micelle solution and inclusion compound. Arch. Pharm. Res. 33, 95-101.
 Loftsson,T., Duchêne, D., 2007. :Cyclodextrins and their therapeutic applications. Int. J. 
 Pharm. 329, 1-11. Maestrelli, F., Cecchi, M., Cirri, M., Capasso, G., Mennini, N., Mura, P., 2009. Comparative study of oxaprozin complexation with natural and chemically modified cyclodextrin in solution and in the solid state. J. Incl. Phenom. Macrocycl. Chem. 63, 17-25. Maestrelli, F., Gonzalez-Rodriguez, M.L., Rabasco, A.M., Ghelardini, C., Mura, P., 2010. New drug-in cyclodextrin-in deformable liposomes formulations to improve the therapeutic efficacy of local anaesthetics. Int. J. Pharm. 395, 222-231. Marianecci, C., Rinaldi, F., Esposito, S., Di Marzio, L., Carafa, M., 2015. Niosomes encapsulating ibuprofen-cyclodextrin complexes: preparation and characterization. Curr. Drug Targets 14, 1070-1078. Mennini, N., Bragagni, M., Maestrelli, F., Mura, P., 2014. Physicochemical characterization in solution and in the solid state of clonazepam complexes with native and chemically-modified cyclodextrins. J. Pharm. Biomed. Anal. 89, 142149.

22

Mennini, N., Maestrelli, F., Cirri, M., Mura, P., 2016. Analysis of physicochemical properties of ternary systems of oxaprozin with randomly methylated-cyclodextrin and L-arginine aimed to improve the drug solubility. J. Pharm. Biomed. Anal. 129, 350-358. Mura, P., Faucci, M.T., Manderioli, A., Bramanti, G., 1999. Influence of the preparation method on the physicochemical properties of binary systems of econazole with cyclodextrins. Int. J. Pharm. 193, 85-95. Mura, P., Bettinetti, G.P,. Cirri, M., Maestrelli, F., Sorrenti, M., Catenacci, L., 2005. Solid-state characterization and dissolution properties of Naproxen-ArginineHydroxypropyl--cyclodextrin ternary system. Eur. J. Pharm. Biopharm. 59, 99106. Mura, P., Bragagni, M., Mennini, N., Cirri, M., Maestrelli, F., 2014. Development of liposomal and microemulsion formulations for transdermal delivery of clonazepam: Effect of randomly-methylated--cyclodextrin, Int. J. Pharm 475, 306-314. Mura, P., Maestrelli, F., Aguzzi, C., Viseras, C. 2016. Hybrid systems based on “drug – in cyclodextrin – in nanoclays” for improving oxaprozin dissolution properties Int. J. Pharm. 509, 8-15. Muraoka, A., Tokumura, T., Machida, Y., 2004. Evaluation of the bioavailability of flurbiprofen and its -cyclodextrin inclusion complex in four different doses upon oral administration to rats. Eur. J. Pharm. Biopharm. 58, 667-671. Otero Espinar, F.J., Anguiano Igea, S., Blanco Mendez, J., Vila Jato, J.L., 1991. Reduction in the ulcerogenicity of naproxen by com- plexation with cyclodextrin. Int. J. Pharm. 70, 35-41. Perioli, L., Ambrogi, V., Nocchetti, M., Sisani, M., Pagano, C., 2011. Preformulation studies on host–guest composites for oral administration of BCS class IV drugs: hTlc and furosemide. Appl. Clay Sci. 53, 696-703. Redenti, E., Szente, L., Szejtli, J., 2001. Cyclodextrin complexes of salts of acidic drugs. Thermodynamic properties, structural features, and pharmaceutical applications, J. Pharm. Sci. 90, 979-986. Rothstein, R., 1998. Safety profiles of leading nonsteroidal anti-inflammatory drugs, Am. J. Med. 105, 39-43.

23

Rojtanatanya, S., Pongjanyakul, T., 2008. Propranolol–magnesium aluminum silicate complex dispersions and particles: characterization and factors influencing drug release. Int. J. Pharm. 383, 106-115. Sakurai, M., Egashira, N., Kawashiri, T., Yano, T., Ikesue, H., Oishi, R., 2009. Oxaliplatin-induced neuropathy in the rat: involvement of oxalate in cold hyperalgesia but not mechanical allodynia. Pain 147, 165-174. Sharma, D., Soni M., Kumar, S., Gupta, G.D., 2009. Solubility enhancement eminent role in poorly soluble drugs. Res. J. Pharm. Tech. 2, 220-224. Uekama, K., Hirayama, F., Tetsumi, I., 1998. Cyclodextrin drug carrier systems. Chem. Rev. 98, 2045-2076. Ulloth, J.E., Almaguel, F.G., Padilla, A., Bu, L., De Leon, M., 2007. Characterization of methyl--cyclodextrin toxicity in NGF-differentiated PC12 cell death. Neurotoxicology 28, 613-621. Vega, E., Egea, M.A., Garduño-Ramírez, M.L., García, M.L., Sánchez, E., Espina, M., Calpena, A.C., 2013. Flurbiprofen PLGA-PEG nanospheres: role of hydroxypropyl--cyclodextrin on ex vivo human skin permeation and in vivo topical anti-inflammatory efficacy. Colloids Surf. B Biointerfaces 110, 339-346. Vemula, V.R., Lagishetty, V., Lingala, S., 2010. Solubility enhancement techniques. Int. J. Pharm. Sci. Rev. Res. 5, 41–51. Viseras, C., Cerezo, P., Sanchez, R., Salcedo, I., Aguzzi, C., 2010. Current challenges in clay minerals for drug delivery. Appl. Clay Sci. 48, 291-295. Yazdanian, M., Briggs, K., Jankovsky, C., Hawi, A., 2004. The high solubility definition of the current FDA guidance on biopharmaceutical classification system may be too strict for acidic drugs. Pharm. Res. 21, 293-299. Yellela, S.R.K., 2010. Pharmaceutical technologies for enhancing oral bioavailability of poorly soluble drugs. J. Bioequiv. Availab. 2, 28-36. Zheng, J.P., Luan, L., Wang, H.Y., Xi, L.F., Yao, K.D., 2007. Study on ibuprofen/ montmorillonite intercalation composites as drug release system. Appl. Clay Sci. 36, 297-301.

24

Legend for Figures Fig. 1. DSC curves of pure oxaprozin (OXA) and its physical mixtures (PM), coground (GR) and coevaporated (COE) products with RAMEB, L-arginine (ARG) and sepiolite (SV).

Fig. 2. Powder X-ray diffraction patterns of: (A) raw materials; (B) oxaprozin (OXA) physical mixtures (PM) with ARG-SV, RAMEB-SV, RAMEB-ARG and RAMEBARG-SV combinations; (C) OXA coevaporated (COE), coground (GR) and cofused (COF) systems with RAMEB-SV, and RAMEB-ARG-SV.

Fig. 3. ESEM micrographs of oxaprozin physical mixture (A) and cofused system (B) with RAMEB-ARG-SV.

Fig. 4. Dissolution profiles of oxaprozin (OXA) alone () or from its physical mixtures (),

coevaporated (), coground () and cofused () systems with

RAMEB-SV (A), RAMEB-ARG (B), ARG-SV (C) and RAMEB-ARG-SV combinations (D).

Fig. 5. Percent of oxaprozin (OXA) dissolved from tablets containing the drug alone or as coground system (GR) with RAMEB or RAMEB-ARG or cofused system (COF) with SV or RAMEB-SV or RAMEB-ARG-SV.

Fig. 6. Effect of acute oral administration in rats of oxaprozin (OXA) as OXARAMEB-ARG-SV tablet formulation (new tab) in comparison to its commercial tablet (Walix) on CFA-induced rheumatoid arthritis model. Both tablets were crushed, suspended in 1% carboxy-methyl cellulose aqueous solution and administered per os. Pain relieving properties were evaluated by Von Frey test using a non noxious mechanical stimulus (allodynia-like measurement) Key: Withdrawal threshold (g) after 0 (

), 15 (☐), 30 ( ), 45 (

) and 60 (

) min after each

treatment (mean ±S.E.M., n=5).

25

26

27

28

29

30

31

Table 1 Relative dissolution rate (rdr), percent dissolved at 15 min (PD.15) and Dissolution Efficiency at 60 min (D.E.60) of oxaprozin (OXA) alone and from its physical mixtures (PM), co-ground (GR), cofused (COF) or coevaporated (COE) products with RAMEB, ARG, SV (mean ±S.D., n=4). sample preparation r.d.r. P.D.15 D.E.60b method 5 mina OXA ----3.5±0.1 4.4±0.2 OXA-RAMEB-ARG

PM

17.9

15.0±0.7

15.4±0.8

OXA-RAMEB-ARG

GR

100.1

55.8±2.8

56.0±2.7

OXA-RAMEB-ARG

COF

71.5

34.5±1.7

34.7±1.5

OXA-RAMEB-ARG

COE

50.9

27.1±1.4

26.9±1.3

OXA-RAMEB-SV

PM

12.7

13.0±0.5

13.3±0.6

OXA-RAMEB-SV

GR

75.8

36.0±1.8

35.0±1.7

OXA-RAMEB-SV

COF

101.3

52.6±2.5

53.0±2.6

OXA-RAMEB-SV

COE

55.1

28.7±1.4

27.5±1.3

OXA-SV-ARG

PM

10.1

9.1±0.5

9.2±0.4

OXA-SV-ARG

GR

42.2

19.8±1.0

19.0±0.9

OXA-SV-ARG

COF

65.0

32.6±1.6

31.7±1.5

OXA-SV-ARG

COE

27.5

14.0±0.7

14.2±0.6

OXA-RAMEB-ARG-SV

PM

25.3

17.1±0.8

16.9±0.7

OXA-RAMEB-ARG-SV

GR

136.4

68.6±3.2

67.7±3.1

OXA-RAMEB-ARG-SV

COF

194.9

89.1±4.2

89.3±4.0

OXA-RAMEB-ARG-SV

COE

100.2

46.9±2.3

45.7±2.2

a Ratio between amount of OXA dissolved from a sample and that from plain drug at 5 min. b Calculated from the area under the dissolution curve at 60 min; expressed as % of the rectangle area described by 100% dissolution in the same time .

32

Table 2 Relative dissolution rate (rdr), percent dissolved at 15 min (P.D.15) and Dissolution Efficiency at 60 min (D.E.60) of OXA from tablets containing the drug alone, or as co-ground (GR) or cofused (COF) or product with RAMEB, ARG, SV (mean ±S.D., n=4). sample rdr P.D.15 D.E.60b 5 mina OXA --2.0±0.1 2.6±0.2 OXA-RAMEB GR

13.8

16.7±1.0

19.3±1.2

OXA-RAMEB-ARG GR

28.7

36.5±2.2

43.5±2.6

OXA-SV COF

7.8

9.9±0.6

10.6±0.7

OXA-RAMEB-SV COF

24.9

32.8±1.8

40.3±2.4

OXA-RAMEB-ARG-SV COF

43.3

52.3±3.1

63.0±3.6

a Ratio between amount of OXA dissolved from a sample and that from plain drug at 5 min. b Calculated from the area under the dissolution curve at 60 min; expressed as % of the rectangle area described by 100% dissolution in the same time

33