Development of aprepitant loaded orally disintegrating films for enhanced pharmacokinetic performance

    Development of aprepitant loaded orally disintegrating films for enhanced pharmacokinetic performance Radhika Sharma, Sunil Kamboj, Gursharan Singh, Vikas Rana PII: DOI: Reference:

S0928-0987(16)30007-0 doi: 10.1016/j.ejps.2016.01.006 PHASCI 3454

To appear in: Received date: Revised date: Accepted date:

13 October 2015 11 December 2015 6 January 2016

Please cite this article as: Sharma, Radhika, Kamboj, Sunil, Singh, Gursharan, Rana, Vikas, Development of aprepitant loaded orally disintegrating films for enhanced pharmacokinetic performance, (2016), doi: 10.1016/j.ejps.2016.01.006

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ACCEPTED MANUSCRIPT Development of aprepitant loaded

orally disintegrating films for enhanced

pharmacokinetic performance

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Radhika Sharma, Sunil Kamboj, Gursharan Singh, Vikas Rana*

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Pharmaceutics Division, Dept. of Pharmaceutical Sciences and Drug Research, Punjabi

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University, Patiala-147002

*Address for correspondence:Dr. Vikas Rana, Dept. of Pharmaceutical Sciences and Drug Sesearch, Punjabi university Patiala (India), E mail: [email protected], Phone no.: +91-9872023038

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ACCEPTED MANUSCRIPT Abstract The present investigation was aimed to prepare orally disintegrating films (ODFs) containing

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aprepitant (APT), an antiemetic drug employing pullulan as film forming agent, tamarind

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pectin as wetting agent and liquid glucose as plasticizer and solubilizer. The ODFs were

design considering

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prepared using solvent casting method. The method was optimized employing 32 full factorial proportion of pullulan: tamarind pectin and concentration of liquid

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glucose as independent variables and disintegration time, wetting time, folding endurance, tensile strength and extensibility as dependent variables. The optimized ODF was evaluated

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for various physicochemical, mechanical, drug release kinetics and bioavailability studies. The results suggested prepared film has uniform film surface, non-sticky and disintegrated

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within 18 s. The in-vitro release kinetics revealed more than 87% aprepitant was released

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from optimized ODF as compared to 85%, 49%, and 12% aprepitant release from marketed formulation aprecap, micronized aprepitant and non micronized aprepitant, respectively. The

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results of animal preference study indicated that developed aprepitant loaded ODFs are accepted by rabbits as food material. Animal pharmacokinetic (PK) study showed 1.80, 1.56

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and 1.36 fold enhancement in relative bioavailability for aprepitant loaded ODF, aprecap and micronized aprepitant respectively, in comparison with non micronized aprepitant. Overall, the solubilised aprepitant when incorporated in the form of aprepitant loaded ODF showed enhanced bioavailability as compared to micronized /non micronized aprepitant based oral formulations. These findings suggest that aprepitant loaded ODF is likely to become one of the choices of aprepitant preparations for antiemesis during cancer chemotherapy. Keywords Orally Disintegrating films, Tamarind pectin, Pullulan, Tensile strength, Dissolution, Bioavailability

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ACCEPTED MANUSCRIPT 1. Introduction Orally disintegrating films (ODFs) are one of the most popular dosage forms. The key

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advantage of such dosage forms is its quick disintegration, when placed on the tongue

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without the need of water, releasing the drug which dissolves in saliva. This usually results in

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enhanced bioavailability with faster onset of action compared to conventional oral dosage forms (Puttalingaiah et al., 2011). The presence of larger surface area of ODF is the cause of quick disintegration and dissolution in the oral cavity. ODFs are flexible so they are not as

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fragile as tablet and need not any kind of special package for protection during transportation

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and storage as compared to ODT (Siddiqui et al., 2011). In addition, the orally disintegrating tablets are prepared at higher crushing strength for preventing breakage during transportation.

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The increase in hardness leads to compromise with disintegration time even with enhanced

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concentration of superdisintegrants (Fukami et al., 2005). Because of convenience and ease of use over other dosage forms, ODFs have been introduced in the market (Bhyan et al.,

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2011). ODF formulations containing cetirizine hydrochloride (Mishra and Amin, 2011), ropinirole (Panchal et al., 2012), anastrozole (Satyanarayana and Keshavarao, 2012), or

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miconazole (Murata et al., 2013) were developed that disintegrate in less than 30 s and showed more than 90 % of in-vitro drug release. However, no attempts have been made so far to load water insoluble drugs like aprepitant (APT). This may be associated with blockage of aqueous channels and interference in disintegration of films (Goel et al., 2009). Aprepitant (APT); is selected due to its selective high affinity neurokinin-1 receptor (NK-1R) antagonist activity against chemotherapy induced nausea and vomiting (Olver et al., 2007). However, the limiting factor for the use of APT is its low solubility in water (about 3-7 µg/ml over the pH range 2-10), weak basic and lipophilic nature (log P at pH 7 = 4.8). This is the reason that APT is categorized into BCS class IV, being “low permeable” and “low soluble” (Kesisoglou and Wu, 2008; Olver et al., 2007). Previous studies have shown that the

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ACCEPTED MANUSCRIPT projected efficacious human dose for APT is relatively high due to low solubility of nanoformulated APT in simulated intestinal fluids (Kesisoglou and Mitra, 2012; Kesisoglou

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et al., 2007; Shono et al., 2010). Thus, the development of formulation of APT with enhanced

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bioavailability potentially reduced its required dose (Ren et al., 2014; Shono et al., 2010).

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Presently APT capsules in different dose (40 mg, 80 mg and 125 mg) are manufactured by Merck & Co., Inc., and are commercially available in the USA (Ren et al., 2014). Further, the unformulated APT exhibit limited oral bioavailability in fasted state and showed marked

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enhanced food effects (Angi et al., 2014; Wu et al., 2004). Interestingly, an enhanced oral

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bioavailability was observed following oral administration of APT solution indicating negligible first pass metabolism effect (Angi et al., 2014). Therefore, entrapment of APT in

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solubilised form in ODF formulation is expected to enhance oral bioavailability of APT.

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Various attempts have been made to enhance bioavailability of APT. The Merck & Co., Inc utilized size reduction principle to develop micronized/nanosized APT that was found to

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enhance solubilisation rate of drug (Ren et al., 2014). The studies on beagle dogs conducted under fed and fasted condition showed that administration of micronized APT and nanosized

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(milled) crystals of APT results in increased bioavailability in the fasted state as well as reduced the food effect, both in animal models and clinical studies (Majumdar et al., 2006; Shono et al., 2010; Wu et al., 2004). Wu et al. (2004) investigated APT bioavailability in humans that exhibit dependency on solubility as well as on particle size. An increase in bioavailability (AUC 5.88 ± 1.86 µg/ml to 25.3 ± 3.29 µg/ml) was evident with decrease in particle size from 5.49 µm to 0.12 µm. Based on these findings a nanosized APT in capsule form is available in market. Although, the Emend® provide APT with enhanced bioavailability, but the efforts to enhance the solubility and dissolution rate of APT have never been stopped, probably due to high cost of this drug.

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ACCEPTED MANUSCRIPT Several methods have been introduced to enhance the solubility of APT. These include the use of surfactants (Niederquell and Kuentz, 2013), solid dispersion (Liu et al., 2006), hot melt

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extrusion technique (Breitenbach, 2002) and cyclodextrin complexes (Hiremath and Godge,

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2013; Torne and Vavia, 2006). Angi et al. (2014) attempted novel continuous flow

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technology for the development of nanostructured APT formulations in which the generation of the nanosized particles takes place at molecular level. The method produces stable amorphous solid form comprising nanostructured particles (less than 100 nm) with improved

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apparent solubility and permeability. This leads to improved pharmacokinetic properties

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(Huskey et al., 2003). Thus, suggested reduction in particle size enhances solubility as well as permeability of APT. Another feasible and commercially viable approach is cyclodextrin

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inclusion complexation. This approach has been extensively used to enhance drug solubility,

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convert liquid drugs in microcrystalline powders, prevent drug-drug or drug-additive interactions, reduce or eliminate unpleasant taste and odour in most of the formulations

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(Loftsson and Brewster, 1996; Ren et al., 2014). These benefits were utilized to develop APT-sulfobutyl ether-β-cyclodextrin complex to enhance solubility of APT (Ren et al.,

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2014). The results suggested enhancement in dissolution rates and extend of release of the test formulation as compared to Emend®. However, no significant effect on permeability of APT was evident from these investigations. Shono et al. (2010) investigated in silico simulation technology to forecast in-vivo oral absorption of micronized and nanonized APT formulations in pre and postprandial states. They suggested dissolution is the primary limitation to the rate of absorption for micronized APT. However, permeability issue was still evident for nanonized formulation. The present investigation is intended to formulate and evaluate aprepitant (APT) loaded orally disintegrating films that could enhance its oral bioavailability as compared to micronized APT, non micronized APT and marketed formulation (Aprecap).

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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Materials

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Aprepitant (non micronized) and aprepitant (micronized) were gifted by Ranbaxy

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Laboratories Ltd. Gurgaon, India and Dr. Reddy’s Pvt. Ltd., Hyderabad, India, respectively.

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Commercially available aprepitant capsules (Aprecap 80 mg, Glenmark Pharmaceuticals Ltd., India) were procured from local market. Pullulan was obtained as gift sample from Gangwal chemicals Ltd., Mumbai, India. Liquid glucose (DE = 38-44, Gujarat Ambuja

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exports Ltd.) was kindly gifted by Nayan Pharmaceuticals Pvt. Ltd., Patiala, India. Tamarind

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fruits were procured from local market, Patiala, India. HPLC grade acetonitrile and orthophosphoric acid were purchased from Thermo Fischer Scientific Pvt. Ltd., Mumbai,

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India. All other materials and chemical used were of analytical grade.

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2.2 Methods

2.2.1. Extraction of tamarind pectin (TP)

al. (2015).

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The extraction of tamarind pectin (TP) was carried out as per method reported by Sharma et

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2.2.2. Analytical method

The analytical profile of aprepitant (APT) was validated for its quantification on highperformance liquid chromatography (HPLC) system. The samples obtained from in-vitro drug release and pharmacokinetic studies were quantitatively analyzed for APT concentration using an isocratic HPLC system. For analysing blood plasma samples obtained during animal pharmacokinetic study, the method was validated in presence of blood plasma. The HPLC system consists of 515 HPLC pump and 2489 UV detector (Waters Ges.m.b.H. Wien/Austria). The chromatograms were evaluated with Empower 3 Software (Waters Ges.m.b.H. Wien/Austria). The analytical column used was a Discovery® C8 column 15cm × 4.6mm, 5µm particles (Supelco, Sigma-Adlrich, UK). The mobile phase was a mixture of

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ACCEPTED MANUSCRIPT acetonitrile and 0.1% orthophosphoric acid (60:40) at a flow rate of 1 ml min-1. The injection volume was 20 µl. The detection wavelength was set at 210 nm. The limit of detection and

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limit of quantification were found to be 0.035 µg/mL and 0.113 µg/mL, respectively. The

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0.999). The analysis was performed under ambient conditions.

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method was found to be linear in the range of 0.1-50 μg/mL with regression coefficient (r2 =

2.2.3. Preparation of blank ODFs

The blank ODFs were prepared by dissolving pullulan, (PU ;10-90%), tamarind pectin, (TP

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;90-10%) and different plasticizers sorbitol (0.1-1%), glycerine (0.1-1%) or liquid glucose

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(LG) (0.01-0.1%) in water, while maintaining total polymer concentration to 2.5% w/v. For the preparation of film formulation, PU was dissolved in minimum amount of purified water.

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Separately, TP was dissolved in purified water containing plasticizer. This TP - plasticizer

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solution was added drop wise to PU solution with stirring and make up the volume to 25 ml. A clear homogenous solution obtained was poured in to polypropylene petriplate (7cm

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diameter) and dried at 50˚C for 24 h. The dried films were stored in polyethylene bags till further use. The different films prepared were physically examined for their integrity, surface

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behaviour, smoothness, etc. Table 1 summarizes different film combinations. 2.2.4. Preparation of APT loaded ODFs For the preparation of APT loaded ODF, solution was prepared by dissolving APT (92.4 mg) in 0.5 ml of 0.1 M NaOH followed by addition of plasticizer LG ( 0.1% v/v of total polymer). This LG - APT solution was mixed with PU solution. Separately, TP was dissolved in purified water, added dropwise to PU- APT - LG mixture with stirring and make up the volume upto 25 ml. The clear homogenous solution obtained was poured in to polypropylene petriplate (7cm diameter) and dried at 50˚C for 24 h. The dried films were stored in polyethylene bags till further use. 2.2.5. Experimental design

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ACCEPTED MANUSCRIPT A 32 full factorial design was employed for optimization of ODF formulation. The two factors, each at three levels (-1, 0 and +1) were taken as independent variables (proportion of

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PU: TP (X1) and concentration of LG; X2). The dependent variables selected were

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disintegration time (DT; Y1), wetting time (WT; Y2), folding endurance (Y3), tensile strength

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(Y4) and extensibility (Y5). Table 2 summarizes nine different film formulations of PU: TP plasticized with LG per 32 factorial design. Design expert software 8.0.7.1 was used for obtaining correlation between independent variables with selected dependent variables.

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2.2.6. Evaluation of APT loaded ODF

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In vitro disintegration time (Y1)

The in-vitro disintegration time was estimated by placing 2 × 2 cm ODF in 6 ml of phosphate

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buffer (pH 6.8) kept at 37˚C. The time taken by the film to disintegrate completely into its

In vitro wetting time (Y2)

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components was taken as disintegration time of ODF (Panchal et al., 2012).

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Circular tissue paper of 7 cm diameter was placed in a petriplate. 10 ml of 0.05% w/v eosin dye solution in water was added to the petriplate. Film 2 × 2 cm was placed on the surface of

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tissue paper. The time required for the dye solution to appear on the surface of film was noted as wetting time.

Folding endurance (Y3) This test ensures the tensile strength of the film. Folding endurance was determined by repeatedly folding the film at the same point till it breaks or folding for 150 times which ever is less (Boateng et al., 2013). Tensile strength (Y4) and extensibility (Y5) The tensile strength and extensibility of ODF was determined using texture analyzer (TAXT plus, Stable Microsystems, Godalming Surrey, UK) employing probe A/TG; Tensile GRIPS, pretest speed 1.0 mm/s; test speed 1.0 mm/s; post test speed 10.0 mm/s; trigger force 5.0 g.

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ACCEPTED MANUSCRIPT Mechanical characterization The mechanical properties (Burst Strength, relaxation, resilience) of APT loaded ODFs were

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studied using texture analyzer (TAXT plus, Stable Microsystems, Godalming Surrey, UK).

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By employing probe: P/0.25 S; 0.25 inches spherical stainless steel, pretest speed 2.0 mm/s;

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test speed 1.0 mm/s; post test speed 10.0 mm/s; trigger force 5.0 g. Drug content

A 4 cm2 film (equivalent 9.6 mg APT) sample was dissolved in 500 ml of 2% w/v SLS

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solution with continuous stirring. This solution was kept aside for 3 h for stabilization. After

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3 h, 20 μL sample from flask was filtered through 0.45μm membrane filter and injected into HPLC (515 pump, 210nm, acetonitrile: 0.1% orthophosphoric acid (60:40) mobile phase, C8

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HPLC column and 1.0 ml min-1 flow rate). The data represented is the mean of six

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determinations. Film thickness

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The thickness of the ODF was evaluated using micrometer (3109-25, Insize Precision Measurement, Vienna, Austria) with range 0-25 mm and resolution 0.001 mm. The ODF

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sample (4 cm2) was taken and kept between the anvil and spindle of the micrometer. The average of three readings was taken as mean thickness (Kamboj et al., 2015). 2.2.7. In vitro drug release The APT release from ODF was determined as per method reported by Mishra and Amin, (2011). The USP dissolution apparatus– paddle I (Electrolab, Mumbai, India) at 37 ± 0.5°C using 500 ml of 2.2 % SLS as dissolution media with stirring speed of 100 rpm was employed for the study (Ren et al., 2014). The in vitro release performance was conducted on APT loaded ODF (approx. 5.8 × 5.8 cm equivalent to 80 mg APT), marketed formulation (Aprecap, 80 mg), micronized APT (80 mg), and non micronized (80 mg). Samples were withdrawn at different time intervals of 5, 10, 15, 20 and 30 minutes, respectively. At each

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ACCEPTED MANUSCRIPT time interval, aliquots of 5 ml was withdrawn, replaced by fresh dissolution medium, filtered and 1 ml of this filtrate was diluted suitably whenever required with respective dissolution

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medium. Amount of drug released was determined by HPLC method (20 µl injection volume;

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210 nm; acetonitrile: 0.1% orthophosphoric acid solution (60:40) mobile phase; 1ml min-1

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flow rate; Discovery® C8 column (15cm× 4.6mm, 5µm particles). Further, dissolution data was compared using similarity factor (f2) and dissimilarity factor (f1) approach. 2.2.8. Optimized film surface-buffer interaction

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Film surface contact angle

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Phosphate buffer of pH 6.8 (10 µl) was gently placed on film surface by a microsyringe, and the images of drop were obtained instantaneously by using a digital camera (SONY, 14.1

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mega pixel, 10x zoom, in mega mode). The contact angle between drop of the solvent and

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PU-TP film surface was calculated using software (MB - ruler 3.5). The results reported were mean of three determinations (Kamboj et al., 2015).

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Contact angle depression rate

With the help of microsyringe, 10 μl of phosphate buffer (pH 6.8) was placed on film surface

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and the images of drop were obtained after every second till the hemispherical drop became flat by using a digital camera (SONY, 14.1 mega pixel, 10x zoom, in mega mode). A negative slope of the graph between contact angle and time was taken as contact angle depression rate (Kamboj et al., 2015). 2.2.9. Drug excipient interaction studies Fourier transform infrared spectroscopy (FTIR) The excipient - excipient, excipient - drug interaction in ODF was investigated by FTIR-ATR analysis in the spectral region 500 - 4000cm-1 employing FTIR-ATR spectrometer (ALPHAe, Bruker IR, Germany). Differential scanning calorimetry (DSC)

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ACCEPTED MANUSCRIPT The thermal properties of samples were evaluated using differential scanning calorimeter (EVO 131, SETARAM Instrumentations France) with heating rate of 10˚C/min in the

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temperature range of 30 to 300˚C.

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X-ray powder diffraction (XpRD)

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The X-ray diffraction patterns of samples were obtained by using X'Pert Pro XRD instrument. The instrument consists of vertical theta - theta goniometer having range of 0°160° 2θ. Sample was glass coated and then x - ray was passed through it. The radiation used

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was Cu K-alpha-1 (45kV, 40mA) at range 5-50° (2θ). ODF samples were scanned at

Scanning electron microscopy (SEM)

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temperature 25°C, 0.030 (2θ) step size and scan time was 1.5 s at each step.

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Scanning electron microscopy (SEM) was used to characterize the morphology and

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transverse section of PU-TP films. Scanning electron micrographs of surface and transverse section of PU-TP film were taken using a SEM (Jeol JSM-6510 LV) machine. The samples

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were coated with gold and mounted in a sample holder. The photomicrographs of sample were taken at an accelerating voltage at 15 kV at different magnifications.

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2.2.10. Stability testing of optimized APT loaded ODF The stability study of the APT loaded ODF was carried out according to Nishimura et al. (2009). A 2 × 2 cm square piece of film preparation was stored in an aluminium package in a chamber controlled at 40°C and 75% humidity for 4–8 weeks. The films were characterized for the drug content and other parameters during the stability study period. 2.2.11. Taste assessment of optimized APT loaded ODF Taste assessment of the formulation was carried out by modifying the animal preference test reported by Laska et al. (2002). For this test, rabbits were kept indoors under natural light conditions at 18–22°C. They were housed individually and were provided with enough food and water. To determine whether formulation taste is acceptable or unacceptable by rabbits,

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ACCEPTED MANUSCRIPT each rabbit was given 20 ml solution in bottle containing pure APT suspension in water and solubilised APT in buffer pH 7.4. APT loaded ODF (1 × 1 cm) was replaced with food in the

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cage. After 24 h, the volume of liquid or amount of ODF consumed was recorded. Each trial

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summed for 10 times.

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2.2.11. Animal Pharmacokinetic performance

Animal pharmacokinetic (PK) study was performed using New Zealand male rabbits. All animal experiments were carried out after approval of the protocol by the Institutional Ethical

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committee

(IAEC),

Punjabi

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Animal

University,

Patiala,

India,

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(107/99/CPCSEA-2011-23) guidelines for the use and care of experimental animals. New Zealand male rabbits, weighting 2.5-3.0 kg were fasted for 12 h before APT

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administration (APT equivalent to 1 mg/kg) but were allowed free access to water (Kamboj et

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al. 2015). Animals were divided into 4 groups, each containing three rabbits. The study was four period four treatment crossover design with one week washout period. Each group was

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orally administered with APT loaded ODF (2.4 mg/cm2 - 3 mg/1.25cm2), aprecap (2.4 -3.0 mg), APTN (2.4 -3.0 mg) and APTM (2.4 -3.0 mg) suspension that was happily accepted.

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Table 3 shows the protocol in which each group received formulations. 0.5 ml of blood was withdrawn from alternate peripheral ear vein of each rabbit with the help of 26 gauge needle in the vacuum microcentrifugation tubes containing 40 µl of disodium EDTA. The collected blood was centrifuged at 4000 rpm (15 min). The upper layer of plasma was separated carefully with micropipette and transferred to microcentrifugation tube. Plasma proteins were precipitated by the addition of acetonitrile (0.9 ml) to plasma sample (0.1 ml). This mixture was centrifuged at 4000 rpm for 10 min. The supernatant layer was collected and evaporated. The residue obtained was reconstituted with mobile phase to analyze APT in blood plasma. The amount of APT in the blood plasma was determined by validated HPLC method. Statistical analysis of animal pharmacokinetic data was performed using one-way ANOVA

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ACCEPTED MANUSCRIPT followed by post hoc Tukey’s multiple-comparisons test, with P values of <0.05 were considered as significant.

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3. Results and Discussion

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3.1. Preparation of APT loaded ODFs

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Different combinations of film containing pullulan (PU) with tamarind pectin (TP) were prepared and results were shown in Table 1. The results suggested 10 - 20 % PU and 90 - 80 % TP causes stickiness in film. The films prepared with 70 - 90% PU and 30 - 10 % TP

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composition were non uniform. However, films prepared by taking composition PU: TP

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30:70 to 60: 40 were non sticky, uniform and clear.

Different plasticizers i.e. sorbitol (0.1 to 1%), glycerin (0.1 to 1%) and liquid glucose (LG)

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(0.01 to 0.1%) were examined for their compatibility and plasticizing effect. From the results,

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it was evident that 30: 70 (PU: TP) films were brittle. However, addition of plasticizers enhanced folding endurance. The folding endurance follows the order LG > sorbitol >

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glycerin indicating high plasticizing effect of LG. Interestingly, the disintegration time of plasticized film follows the order LG < control film (non - plasticized) < sorbitol < glycerin,

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suggesting incorporation of LG plays a role in enhancing flexibility and promotes disintegration of film (decreases disintegration time). Thus, on this basis, LG was considered as good plasticizer for PU: TP orally disintegrating films. Therefore, all the films prepared were plasticized with LG. These studies suggested blending of PU and TP plasticized with LG gives water soluble films. This may be due to high solubility of PU and TP in water. In addition, PU bears good film forming and swelling potential (Pathare et al., 2013). It has poor water absorbing property. However, TP was reported to have higher water absorbing capacity with lower swelling index as compared to citrus pectin, apple pectin etc (Sharma et al., 2015). This could be associated with enhanced equilibrium moisture content. Further, the wetting time of

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ACCEPTED MANUSCRIPT commercial pectin was found to be higher as compared to TP (Basu et al., 2013). The lowest wetting time is needed for good wetting agent. Therefore, TP was selected as wetting agent

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and could be used in combination with PU for preparing ODFs. The addition of LG in ODFs

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was compatible combination and showed enhanced flexibility. The incorporation of APT into

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the film seems to be a tedious task. This was due to its solubility. APT belongs to BCS class IV drug and insoluble in water. The incorporation of APT in PU: TP blends lead to nonuniform distribution of drug. Thus, solubilization of APT into PU: TP blend was conducted.

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Various attempts were made to uniformly disperse APT into film. The lipid phase dispersion,

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surfactant addition, complexation with β - cyclodextrin and pH change were the methods used (Table 4). Among these methods, pH change was found to be the best method to

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incorporate APT in to films. APT exhibits pH- dependent solubility with a solubility of 13

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µg/ml at pH 1.2, less than 7µg/ml between pH 2.5 and 6, 120 µgm/ml at pH 7.2 (Kesisoglou and Wu, 2008). Therefore, during preparation APT was initially dissolved in a 0.1 M NaOH

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and pH of film solution was adjusted to pH 7.0, so that APT remained in solubilised form even in films after drying.

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3.2. Experimental design

A 32 factorial design enables us to examine the joint (or interaction) effect of the independent variables on the dependent variables. Therefore, 32 full factorial design was used to prepare APT loaded ODF. The results are summarized in Table 2. The results suggested all the prepared films have uniform film surface, non-sticky and clear. The % in-vitro release of APT was found to be more than 87 % indicating the formulation excipients did not retain the drug which was present in solubilised form in the film. Thus, APT although belongs to BCS class IV was made available for immediate release when incorporated into novel film formulation. Further, the results were analysed employing Design expert software 8.0.7.1 and with the application of multiple linear regression, different equations showing behaviour of

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ACCEPTED MANUSCRIPT linear, quadratic, cubic were analyzed and fitted simultaneously into the software to generate optimized formulations. Adequacy and good fit of the models were tested using analysis of

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variance (ANOVA). Mathematical relationships generated for the studied response variables

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are expressed as equations.

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Disintegration time (Y1) and wetting time (Y2)

Following equations were generated when disintegration time was correlated with independent variables (X1 and X2).

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Y1 = 24.88 + 6.67X1 - 2.83X2 + 0.00X1X2 - 1.53X12 - 0.03X22------------(1)

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(R2= 0.99, Quadratic model).

In the above equation (1), positive sign signifies synergistic influence of coefficients on

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response variables. The negative sign in the above equation reflect inverse correlation of that

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coefficient/variable with response. From the above equation it was evident that polymer proportion had a high pronounced influence as compared to plasticizer concentration.

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Interestingly, the magnitude of X1 and X2 are significantly higher than coefficients of interaction variables. Further, the depression in disintegration time was associated with

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increase in concentration of LG and decrease in proportion of PU: TP to 30:70. Furthermore, the influence of independent variables on the in-vitro disintegration time of films was elucidated using response surface plots. The results suggested increase in concentration of PU in the films increases disintegration time. However, increase in concentration of TP in the films decreased disintegration time. In addition, the increase in concentration of LG decreased disintegration time. To further evaluate disintegration behaviour of films, wetting time was estimated. It is the time required to transport water throughout the film such that film immediately disintegrated into its constituents. The equation generated that correlates wetting time with independent variables are Y2 = 18.03 + 7.33X1 - 1.33X2 + 0.25X1X2 - 0.62X12 - 0.62X22 ------------(2)

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ACCEPTED MANUSCRIPT (R2= 0.99, Quadratic model) The equation (2) showed a similar correlation of X1 and X2 with wetting time suggesting

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disintegration time was associated with wetting time of films. In addition, the response

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surface plots also revealed a direct correlation of disintegration time and wetting time.

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Further, the enhancement in the concentration of TP in a film leads to decrease in wetting time. Thus, the property of wetting in a film could be associated with combined effect of properties of TP and LG.

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Folding endurance (Y3)

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The equation generated by equating folding endurance and independent variables is: Y3 = 48.45 - 76.33X1 + 16.67X2 – 19.00X1X2 + 29.93X12 + 2.93X22 ---------------(3)

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(R2=0.99, Quadratic model)

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The response surface plots (Fig. 1) generated showed significant influence of LG in enhancing folding endurance. The enhancement in concentration of LG in films enhanced

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folding endurance of ODFs. However, with decrease in proportion of PU: TP i.e. from 60: 40 to 30: 70 folding endurance was increased.

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Tensile strength (Y4) and extensibility (Y5) Tensile strength and extensibility were determined employing texture analyzer (Stable Micro Systems Ltd., Godalming, U.K.). Tensile strength is a descriptor denotes mechanical strength of films. The extensibility is related to the flexibility of films i.e. distance upto which the film could be stretched on application of force (5kg load cell). The constants and regression coefficients for tensile strength (Y4) and extensibility (Y5) are: Y4 = 2.93 + 2.29X1 + 0.96X2 + 0.13X1X2 + 0.49X12 + 0.60X22--------------(4) (R2 = 0.95, Quadratic model) Y5 = 26.11 - 6.46X1 + 2.09X2 - 2.60X1X2 - 1.23X12 - 0.58X22--------------(5) (R2= 0.94, Quadratic model)

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ACCEPTED MANUSCRIPT The results suggested increase in proportion of PU in the films and decrease in concentration of TP in films enhanced tensile strength of films (Fig. 1). This suggested PU contributed to

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tensile strength of films. The role of LG was also to enhance tensile strength. Thus, LG and

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PU could contribute more towards enhancement in tensile strength as compared to TP alone.

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Further, the extensibility was found to be directly related to concentration of LG (X2). The decrease in proportion of PU and increase in proportion of TP in the films, increased extensibility. This behaviour of film components was also observed in the negative

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magnitude of interaction coefficients (X1X2), suggesting film extensibility could be increased

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by decreasing the PU: TP (30:70) proportion (X1) in the films. The software showed 30: 70 (PU: TP) proportion and 0.1% of LG containing solubilised APT was the film compositions

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having desired constrains. On this basis, ODF was prepared and desired responses were

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evaluated. The values of disintegrating time and wetting time of the optimized batch were not significantly different, when compared to the ODF formulations batches prepared as per 3 2

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full factorial design. The response surface plots showing the effect of different combination of X1 and X2 on desirability are shown in Fig 1. The desirability function combines all

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responses in one measurement and provides a possibility to predict the optimum levels of the independent variables. The predicted desirability of the optimized formulation was found to be 0.750. The value of desirability closer to 1 indicates that the response values are consequently nearer to the target values. The optimized formulation was evaluated for various dependent variables. The response values were calculated and compared to corresponding predicted value. The prediction error for the response parameters ranged between 1.06 % and 3.55 %. 3.3. Evaluation of optimized APT loaded ODF

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ACCEPTED MANUSCRIPT The optimized ODF [30:70 (PU:TP) + 0.1% v/v LG + 92.4 mg APT] prepared showed 14 s disintegration time with sufficient tensile strength, extensibility, folding endurance, burst

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strength, relaxation, resilience and 87.67 % in-vitro release (Table 2).

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3.4. Mechanism of disintegration

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To determine the mechanism of disintegration, it was essential to explore the role played by each of the excipient of optimized film formulations. For this purpose each ingredient either alone or its combination was subjected to water sorption time, swelling index, solubility

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studies and solubilization rate. Water sorption time is the indicator of wicking action i.e.

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solvent sucking nature of excipient. Swelling index depicts water retaining/ holding behaviour of excipient. Solubilisation rate revealed speed with which excipient turned down

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into solution.

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Fukami et al. (2005) suggested a mixture of glycine with swelling agent could be used as a superdisintegrant. The role of glycine as wicking agent was further utilized by Goel et al.

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(2009) and Vora and Rana, (2008) for developing superdisintegrants in orally disintegrating tablets. However, glycine once gains water, unable to gain its wicking action after drying in

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oven (45°C). This probably due to its water of crystallization that holds the water into its crystal lattice. Hence, the pharmaceutical processes in which solvent evaporation followed by drying process involves wicking property of glycine could not be utilized. Thus, a process like formulation of orally disintegrating film that involves solvent evaporation followed by drying process, an alternative wetting agent is required. Therefore, pectins are hygroscopic in nature with negligible water holding capacity (Malviya and Kulkarni, 2012). Hence, Tamarind pectin was selected for this purpose as their extraction process involves final oven drying of pectin and no lyophilisation that produces hygroscopic materials by removing bound water (Srivastava and Malviya, 2011).The results of water sorption time, swelling index, solubilisation rate are shown in Table 5. The results suggested

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ACCEPTED MANUSCRIPT glycine loses its wicking property while TP regains this when both samples pretreated with water followed by oven drying (45°C). In addition, PU did not have wetting property and

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swelling index, although it acts as good film former. The physical mixture of PU with TP in

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different proportion did not reduce the water sorption time. However, the swelling index of

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physical mixture was 1.8 fold increased as compared to a pure PU or pure TP. Further, these mixtures were found to be sparingly soluble and this leads to enhancement in the solubilisation rate. To examine the possibility of interaction between PU and TP that leads to

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reduced disintegration time or wetting time, PU and TP was pretreated with water followed

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by oven drying (45°C). A significant reduction in WST and enhancement in solubility of pretreated PU: TP mixture indicated occurrence of interaction between PU and TP which

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influenced disintegration time and wetting time. Further, the increase in concentration of TP

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in the pretreated or physical mixture samples decreased water sorption time suggesting TP acts as a wetting agent. Interestingly, the pretreatment of PU: TP increased solubilisation rate,

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suggesting interaction of PU with TP enhanced solubility as well as reduced water sorption time. The PU: TP samples prepared by mixing LG during pretreatment were also subjected to

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evaluation. The results suggested inclusion of LG into PU: TP during pretreatment reduced wetting time and enhanced solubilisation rate. However, significant reduction in swelling index suggested disintegration was not swelling controlled. Overall, the interaction of PU with TP and addition of LG reduced water sorption time as well as increased solubility of mixture. Therefore, it could be envisaged that the mechanism of disintegration of optimized ODF formulation was associated with reduction in wetting time and enhancement in the solubility of polymer blend. Further, the addition of APT in solubilised or unsolubilised form did not influence water sorption time, swelling index, solubility and solubility rate. Thus, revealed mechanism behind disintegration was not influenced by APT.

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ACCEPTED MANUSCRIPT The contact angle is the parameter that reflects solvent loving or disliking nature of surface. However, the optimized film formulations were disintegrated in less than 30 s. Therefore,

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contact angle depression rate was estimated. The contact angle depression rate depicts the

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rate with which film surface solubilised when comes in contact with phosphate buffer pH 6.8

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(i.e. solvent loving nature or hydrophilic solvent). The images taken for total 10-13 s at the rate of one image/sec and plot of contact angle vs. time to estimate contact angle depression rate are shown in Fig. 2. The results suggested film surface was initially 70˚- 65˚. However,

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the contact angle decreases spontaneously to 30˚- 33˚. Further, a correlation between contact

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angle depression rate and disintegration time was linear with R2 = 0.971 (P > 0.05) (Fig. 2) indicating disintegration time of ODF was due to rapid contact angle depression. Thus,

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suggesting overwhelming influence of PU, TP, and LG on contact angle depression rate.

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Overall, it could be envisaged that interaction of PU with TP produces blend films that have potential of rapid solubilization when comes in contact with aqueous medium. This rapid

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solubilization is associated with reduction in contact angle that leads to reduced disintegration time and wetting time. The summary of mechanism of disintegration is shown in Fig. 3.

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3.5. Drug excipient interaction studies FTIR-ATR analysis of pure samples and blend films was obtained to explore the possibilities of any interaction that leads to decreased water sorption time, disintegration time and enhanced solubilisation rate. The FTIR - ATR spectra of pure PU sample showed strong absorption peaks at 3243.65 cm-1 indicated that PU had some repeating units of –OH. The additional strong peak at 2910.72 cm-1 indicated sp3 C–H bond of alkane compounds. The occurrence of strong peaks at 844.61 cm-1, 752.33 cm-1 and 987.75 corresponds to α-D– glucopyranoside units, α-(1→4)-D-glucosidic bonds and presence of α-(1→6)-D-glucosidic bonds, respectively (Prasad et al., 2012) (Fig. 4A). The FTIR-ATR analysis of pure TP exhibited peaks at 3238.67 cm-1, 2882.35 cm-1, 1705.60 cm-1 and 1009.83cm-1 corresponding

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ACCEPTED MANUSCRIPT respectively to –OH, –CH, -C=0 of ester and C–O–C stretching of galacturonic acid (Fig. 4B). There was additional strong peak at 1220.66 cm-1 and weak peak at 1589.37 cm-1

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indicating undissociated carbonyl moieties. In addition, the peaks exhibiting anionic

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carboxylic acid characteristic of strong intensity bonds between 1610 cm-1 and 1550 cm-1 of

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asymmetric stretching (1610.73 cm-1) and 1410 cm-1 and 1300 cm-1 of symmetrical stretching vibration (1380.88 cm-1) of carboxylic group were present (Tong et al., 2008). These peaks were absent for PU which is in accordance with its polymer structure. Therefore, the

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interaction of PU with TP was elucidated by shift in wavenumber of symmetric and

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asymmetric peaks as well as peaks correspond to H–bonding. Fig. 4C showed spectra of PU: TP (30: 70) blend film. Addition of PU caused the COO- bond

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(of TP) to shift from 1610 cm-1 to 1626.94 cm-1 for TP. This provided an evidence that the

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asymmetric and symmetric vibration of C=O and C–O bonds were enhanced, probably due to the disruption of intermolecular hydrogen bonds present originally between the carboxylic

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groups caused by added PU. The symmetric COO- stretching around 1380.86 cm-1 shifted to 1372.83 cm-1 in a blend films. A similar interaction occurred due to shifting in wavenumber

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between PU and alginate or CMC (Tong et al., 2008). Further, the peaks at 3277.82 cm-1 and appearance of strong peaks at 1005.48 cm-1 followed by peak at 803.76 cm-1suggested the hydrogen bond formation between the –OH moieties of PU/TP and –OH moieties of TP/PU ( i.e. intermolecular/ intramolecular H-bonding) either among themselves or with each other (Khurana et al., 2014). Further, incorporation of LG into PU: TP (30: 70) films for plasticization increased the intensity of peaks at 3263.74 cm-1, 998.06cm-1 followed by 801.41 cm-1 indicated for enhancement in H- bonding with LG (Fig. 4D). Thus, suggested the optimized blend film contains H- bonding between carboxyl group of TP and –OH groups of TP or PU or LG. Further, the increase in H- bonding was associated with hydrophilic character of the film that

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ACCEPTED MANUSCRIPT further correlated with enhancement in solubilisation of blend. Thus, this could be envisaged that enhancement in H- bonding in optimized film formulation was the cause of decreased

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water sorption time, disintegration time and enhanced solubilisation rate. Further, Fig. 4F

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showed FTIR-ATR spectra of optimized APT-ODF indicating additional peaks

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corresponding to APT along with peaks represented for PU-TP-LG films, suggesting no significant interaction of APT with excipients (i.e. PU, TP or LG).

DSC thermogram of pure PU showed endothermic transition at 320.96˚C indicating PU melts

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at 320.96˚C temperature (Fig. 5A). The pure TP showed one endothermic transition at

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142.15˚C and one exothermic transition at 291.22˚C (Fig. 5B). The first endothermic transition could be assigned to be due to presence of moisture and hydrogen bonding among the O- moieties and H+ moieties of galacturonic acid units (Hiorth et al., 2005). The

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exothermic transition at 291.22˚C could be due to the degradation of TP at this temperature. A similar peak was observed by Einhorn-Stoll and Kunzek (2009). DSC thermogram of PU:

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TP (30:70) showed three endothermic transitions at 162.018˚C, 241.019˚C and 298.161˚C. The first endothermic transition could be due to loss of water molecules (bound or unbound)

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in the films. However, second endothermic transition onset at 213.616˚C and offset at 264.14˚C could be ascribed to the loss of hydroxyl groups as water molecules or due to breakage of H-bonding either between COOH groups of TP and OH groups of PU or TP and/ or OH groups of PU with OH groups of PU or TP (Fig.5C). Soppirnath and Aminabhavi, (2002) reported the origin of DTA thermal transitions between 250-350˚C have appeared due to 50-60 % loss of hydroxyl groups of guar gum in the form of water molecules. Thus, the second endothermic transition could be attributed due to occurrence of interaction between PU and TP. The third endothermic transition at 298.161˚C could be due to melting of PU: TP (30:70) blend. A similar three endothermic transitions were observed in films prepared by the addition of liquid glucose in PU: TP (30:70) film. However, the transition temperature of

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ACCEPTED MANUSCRIPT these peaks decreased by 10-15˚C depicting decrease in melting point of film or enhancement in flexibility in films.

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Fig.5E showed six endothermic transition at 134.773˚C (due to water loss), 196.39˚C

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(interaction peak), 242.082˚C (melting of solubilised APT), 260.934˚C (insoluble APT),

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279.354˚C (melting of PU: TP blend), 320.042˚C (melting of unreacted PU) and one endothermic transition at 371.754˚C arises due to degradation of PU. However, the physical mixture of APT + PU + TP showed peaks as observed in their pure thermograms (Fig. 5F)

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and absence of interaction peaks at 196.39˚C, 242.082˚C and 279.354˚C corresponds to PU-

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TU interaction, solubilised ATP and melting of PU-TP blend, respectively. This suggested absence of any interaction when these excipients present in its physical form and occurrence

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of interaction (H–bonding) between PU and TP. Overall, the DSC investigation suggested

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occurrence of H–bonding interactions between PU and TP that had enhanced water sorption capacity as well as solubilisation rate of optimized ODF containing solubilised APT that had

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lead to decreased disintegration time.

The XRD of ODF was conducted to know the nature of drug when incorporated in soluble

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film formulations. The XRD of pure PU showed absence of peaks indicating amorphous nature of PU (Fig. 6b). The pure spectra of TP showed peaks at 12.4015°, 13.3442°, 18.4633°, 21.3787°, 29.2845°, 35.1987°, 43.8192° and 72.6661° (2θ) suggesting crystalline nature. However, absence of peaks in PU: TP (30: 70) film plasticized with LG (Fig. 6d) revealed conversion of crystalline TP into amorphous film formulations. Thus, reflected interaction of PU with TP changed physical nature of respective pure polysaccharides. Hence, it could be envisaged that interaction of PU with TP enhanced solubility of blend as well as solubilisation rate such that it disintegrate quickly. Interestingly, the incorporation of APT that showed peaks at 16.98°, 17.35°, 20.38°and 20.82° (2θ) into PU: TP (30: 70) plasticized with LG showed amorphous nature of APT in film blend. This was evident from absence of

23

ACCEPTED MANUSCRIPT peak in the optimized ODF spectra (Fig. 6e). This suggested APT exists in the film in its solubilised form that immediately disintegrated in oral cavity; APT solution immediately

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made shall be absorbed into GIT for antiemetic action (Chaudhary et al., 2012).

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The SEM of optimized ODF was performed to understand the surface behaviour of films

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(Fig. 7). The SEM of pure PU showed porous structured with small fragments of film. However, the crystalline nature of TP was evident from pure TP sample. The blending of PU with TP (30: 70) showed rough surfaced, porous and non-uniformity in the SEM images of

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film transverse section or at film surface. However, blending of LG with PU: TP (30: 70)

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form a film with smooth surface, non- porous and uniformity in the SEM images of transverse section or at film surface. Similar results were observed in the SEM images of

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optimized ODF indicating APT was present in solubilised form. Thus, the results were in

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consonance with these observed in XRD investigations. 3.6. Stability study of optimized APT loaded ODF

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The stability test of optimized ODF packed in polypropylene packs under 40˚C/ 75 % RH was performed for 60 days. The results are summarized in Table 6. There was no significant

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change in drug content, tensile strength, appearance, folding endurance and disintegration time suggesting APT-ODF was stable for 60 days. 3.7. In vitro drug release The in-vitro dissolution performance of optimized ODF (5.8 × 5.8 cm equivalent to 80 mg APT), marketed formulation (Aprecap, 80 mg), micronized APT (80 mg), non micronized APT (80 mg) was conducted at 37˚

0.5˚C using 500 ml of 2% SLS (Ren et al., 2014). The

results suggested more than 87%, 85%, 49%, and 12% APT was released respectively from optimized ODF, aprecap (ACAP), micronized aprepitant (APTM) and non micronized aprepitant (APTN) (Fig. 8a). The finding revealed enhanced solubilisation of APT when formulated in ODF formulations.

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ACCEPTED MANUSCRIPT Further, the point to point correlation between optimized ODF and ACAP employing f1 and f2 was investigated as they look closest to each other. The results showed 12.86 magnitude of f1

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and 41.03 magnitude of f2 indicating both formulations were dissimilar.

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3.8. Taste assessment of optimized APT loaded ODF

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From animal preference test it was observed that solubilised APT (1mg/kg) in buffer 7.4 was not consumed by rabbits. However, 4 ml APT suspension (1mg/kg) in water was accepted by rabbits. The findings pointed towards bitter nature of APT when present in solubilised form.

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An interesting finding was observed when rabbit accepted APT loaded ODF (1 × 1 cm). This

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indicated good acceptance of ODF by rabbits as preferred food. 3.9. Animal Pharmacokinetic performance

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For animal pharmacokinetic performance different formulations of APT (1mg/kg) entrapped

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in optimized ODF (1 × 1 cm), ACAP, APTM, APTN treatments were given to four groups of rabbits (n=3). The study was four period four treatment crossover design with one week

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washout period. For this purpose the rabbits were divided into four groups, each group contains three rabbits. The plasma concentration time profile obtained was shown in Fig 8b.

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The different pharmacokinetic parameters (AUC, AUMC, Tmax, Cmax, Ka, Ke, F and MRT) were estimated from the data obtained. The pharmacokinetic parameters of all the different formulations were compared employing ANOVA followed by Tukey`s method. The results suggested enhancement in AUC when APT was administered in ODF. The AUC of all the four dosage was found to be significantly different. Further, there was no significant difference in Tmax and MRT suggesting different forms of APT in a dosage did not influenced residence (stay) time and maximum time to attain maximum concentration of APT. However, comparison of relative bioavailability with respect to APTN suggested 1.80, 1.56 and 1.36 fold enhanced respectively for APT - ODF, marketed formulation and APTM (Table 7). Interestingly the absorption rate constant (Ka) of APT-ODF and ACAP were significantly

25

ACCEPTED MANUSCRIPT different from APTM and APTN. However, the elimination rate constant (Ke) of ACAP and APTM were significantly different from APT-ODF and APTN. This indicated overwhelming

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influence of solubilised APT (APT-ODF) or APTM on the dosage form and particle size of

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drug molecule. Overall, the solubilised APT when incorporated in the form of APT-ODF

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showed enhanced absorption and then enhances bioavailability as compared to APTM / APTN based oral formulations. 4. Conclusion

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The optimized orally disintegrating films prepared using PU:TP::30:70 and LG as plasticizer

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(0.1 % v/v) and loaded with solubilised APT by solvent casting method showed satisfactory drug dissolution rate (87%), in vitro disintegration time (18s) and acceptable The ODF formulation prepared by employing 3 2 full

D

physicomechanical characteristics.

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factorial design was found to be successful in providing numerically optimized formulation with desirability function. The significant difference in in vitro dissolution performance was

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evident when compared with APTM and APTN. Although, taste of APT is bitter but use of tamarind pectin as film forming agent had masked the taste of APT which was further

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happily accepted by the rabbits. Further, a significant difference in the AUC and AUMC of APT when loaded in a ODF formulation indicated significant improvement in oral bioavailability of APT. Therefore, the APT loaded ODF is considered to be potentially useful for cancer patients who receive radiotherapy and/or high- to moderate emetogenic anticancer drugs. Acknowledgments The authors would like to acknowledge the financial assistance provided by AICTE, New Delhi (Project No. 8-188/RIFD/RPS/Policy-1/2014-15). References

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Fast Dissolving Drug Delivery System and Their Patents. Advances in Biological Research. 5, 291-303.

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release application. European Journal of Pharmaceutics and Biopharmaceutics. 53, 87-

Srivastava, P., Malviya, R., 2011. Sources of pectin, extraction and its applications in pharmaceutical industry-An overview. Indian Journal of Natural Products and Resources. 2, 10-18. Tong, Q., Xiao, Q., Lim, L.-T., 2008. Preparation and properties of pullulan–alginate– carboxymethylcellulose blend films. Food Research International. 41, 1007-1014. Torne, J.S., Vavia, P.R., 2006. Inclusion complexation of anti-HIV drug with β-cyclodextrin. Journal of Inclusion Phenomena and Macrocyclic Chemistry. 56, 253-259.

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ACCEPTED MANUSCRIPT Vora, N., Rana, V., 2008. Preparation and optimization of mouth/orally dissolving tablets using a combination of glycine, carboxymethyl cellulose and sodium alginate: a

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comparison with superdisintegrants. Pharmaceutical Development and Technology. 13,

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233-243.

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Wu, Y., Loper, A., Landis, E., Hettrick, L., Novak, L., Lynn, K., Chen, C., Thompson, K., Higgins, R., Batra, U., 2004. The role of biopharmaceutics in the development of a clinical nanoparticle formulation of MK-0869: a Beagle dog model predicts improved

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bioavailability and diminished food effect on absorption in human. International

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Journal of Pharmaceutics. 285, 135-146.

Fig. 1: Response surface plots correlating dependent variables (Y1, Y2) with independent

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variables (X1, X2, X3, X4 and X5). Fig. 2: (a) The images of drop of buffer (pH 6.8; when placed over optimized film surface) taken after every second till 10th sec; (b) Effect of contact angle made by buffer (pH 6.8) on optimised ODF surface with respect to time (sec); (c) Correlation of disintegration time (sec) with contact angle depression rate (degrees/sec) Fig. 3: Flow diagram showing mechanism of disintegration of ODF Fig. 4: FTIR- ATR of (A) Pullulan; (B) Tamarind Pectin; (C) PU: TP (30:70) film; (D) PU:TP (30:70) + Liquid glucose film; (E) Aprepitant; (F) PU:TP (30:70) + LG + APT film

32

ACCEPTED MANUSCRIPT Fig. 5: DSC thermograms of (A) Pullulan; (B) Tamarind Pectin; (C) PU:TP (30:70) film; (D) PU:TP (30:70) + Liquid glucose film; (E) PU:TP (30:70) + LG + APT film; (F)

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PU:TP (30:70) + LG + APT physical mixture

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film; (E) PU:TP::30:70 +LG+APT film

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Fig. 6: XRD of (A) Aprepitant; (B) Pullulan; (C) Tamarind pectin; (D) PU:TP::30:70 + LG

Fig. 7: SEM images of (a) Aprepitant; (b) Pullulan; (c) Tamarind Pectin; (d), (e), (f)

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Transverse sections; (g), (h), (i) Surface images of PU:TP::3070 film; PU:TP::3070

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+ LG film; PU:TP::3070 + LG + APT film, respectively. Fig. 8: (a) In-vitro dissolution profile; (b) Pharmacokinetic profile of APT loaded optimized

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formulation, aprecap (ACAP), micronized APT (APTM) and non micronized APT

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(APTN)

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ACCEPTED MANUSCRIPT Table 1 Preliminary studies for choosing levels of factors for 32 factorial design

BF8 BF9

Film code PF1 PF2 PF3 PF4 PF5 PF6 PF7 PF8 PF9

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BF7

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BF6

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BF5

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BF4

Film formulations (30:70:: PU:TP) prepared for screening of plasticizers Plasticizer Stickiness Surface Film In vitro Folding (v/v) appearance clarity disintegration endurance time (sec) (folds) Glycerin NonUniform Clear 32 ± 1.1 81 ± 1.0 (0.1%) sticky Glycerin NonUniform Clear 36 ± 1.4 87 ± 1.0 (0.5%) sticky Glycerin (1%) NonUniform Clear 40 ± 1.4 96 ± 2.0 sticky Sorbitol (0.1%) NonUniform Clear 34 ± 1.3 88 ± 2.0 sticky Sorbitol (0.5%) NonUniform Clear 36 ± 1.1 96 ± 2.0 sticky Sorbitol (1%) NonUniform Clear 38 ± 1.0 104 ± 3.0 sticky Liquid glucose NonUniform Clear 20 ± 1.5 120 ± 3.0 (0.01%) sticky Liquid glucose NonUniform Clear 16 ± 1.0 No cracks (0.05%) sticky Liquid glucose NonUniform Clear 14 ± 1.2 No cracks (0.1%) sticky

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BF1 BF2 BF3

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Film code

Blank films prepared with PU and TP combination Composition Stickiness Surface Film In vitro Folding (PU: TP) appearance clarity disintegration endurance time (sec) (folds) 10:90 Sticky Film did not formed 20:80 Sticky Film did not formed 30:70 NonUniform Clear 22 ± 1.2 48 ± 2.0 sticky 40:60 NonUniform Clear 30 ± 1.5 55 ± 2.0 sticky 50:50 NonUniform Clear 35 ± 1.3 69 ± 3.0 sticky 60:40 NonUniform Clear 38 ± 1.2 32 ± 2.0 sticky 70:30 NonNonTurbid 20 ± 2.0 sticky uniform 80:20 NonNonTurbid 11 ± 1.0 sticky uniform 90:10 NonNonTurbid 8 ± 1.0 sticky uniform

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ACCEPTED MANUSCRIPT Table 2 32 full factorial experimental design for the fabrication of APT loaded ODFs

F6

F7

F8

F9

-1 (0.01 %) 0 (0.05 %) 1 (0.1% ) -1 (0.01 %) 0 (0.05 %) 1 (0.1% ) -1 (0.01 %) 0 (0.05 %) 1 (0.1% )

20 ± 0.2

11 ± 0.4

120 ± 3.0

16 ± 0.1

10 ± 0.2

150 ± 4.0

0.74 ± 0.01

14 ± 0.1

8± 0.1

200 ± 5.0

27 ± 0.4

19 ± 0.5

25 ± 0.3

18 ± 0.4

Y5 (Extensi bility (mm)

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-1 (30:7 0) -1 (30:7 0) -1 (30:7 0) 0 (45:5 5) 0 (45:5 5) 0 (45:5 5) 1 (60:4 0) 1 (60:4 0) 1 (60:4 0)

(Tensi le streng th) (N) 0.88 ± 0.01

22 ± 0.1

Y4

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Y3 (Foldi ng endur ance) (folds)

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Y2 (Wet ting time ) (sec)

Unifo rm

Nonsticky

Cle ar

0.10 ± 0.01

85.42 ± 1.1

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F5

Y1 (Disin tegrat ion time) (sec)

34.33 ± 1.5

Unifo rm

Nonsticky

Cle ar

0.11 ± 0.01

86.31 ± 1.3

2.93 ± 0.07

38.32 ± 1.7

Unifo rm

Nonsticky

Cle ar

0.10 ± 0.02

87.67 ± 1.1

42 ± 2.0

2.44 ± 0.04

24.80 ± 1.3

Unifo rm

Nonsticky

Cle ar

0.09 ± 0.01

86.16 ± 1.0

3.13 ± 0.08

26.90 ± 1.4

Unifo rm

Nonsticky

Cle ar

0.13 ± 0.03

86.63 ± 1.2

58 ± 2.0

3.59 ± 0.08

26.28 ± 1.4

Unifo rm

Nonsticky

Cle ar

0.12 ± 0.02

87.15 ± 1.3

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F4

X2 (% of LG v/v of total polym er)

Physical evaluation of APT loaded ODFs prepared by 32 full factorial experimental design Surfa Sticki Fil Thick In vitro ce ness m ness drug appea clar (mm) release rance ity (%)

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F3

X1 (PU:T P)

49 ± 3.0

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F2

Dependent variables

16 ± 0.5

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F1

Independent variables

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Bat ch No.

27.58 ± 1.3

33 ± 0.7

25 ± 0.9

2± 1.0

5.35 ± 0.09

20.38 ± 1.1

Unifo rm

Nonsticky

Cle ar

0.12 ± 0.01

85.98 ± 1.5

30 ± 0.5

25 ± 0.7

4± 1.0

5.06 ± 0.09

20.36 ± 1.1

Unifo rm

Nonsticky

Cle ar

0.09 ± 0.01

86.46 ± 1.1

27 ± 0.4

23 ± 0.6

6± 1.0

7.91 ± 1.1

20.73 ± 1.2

Unifo rm

Nonsticky

Cle ar

0.11 ± 0.02

85.59 ± 1.0

Variance analysis coefficients with magnitude

Film composition

Respo nse

Inter cept

X1

X2

X1 X2

X1 2

X22

Tamarind pectin

437.5 mg/film

Y1

24.88

-2.83

0.00

-1.53

-0.03

Pullulan

Y2

18.03

-1.33

0.25

-0.62

-0.62

48.45

16.67

-19.00

29.93

2.93

Liquid glucose 0.1 M NaOH

187.5 mg/film 0.1 % v/v

Y3 Y4

2.93

0.96

0.13

0.49

0.60

Purified H2O

q.s. 25 ml

Y5

26.11

6.6 7 7.3 3 76. 33 2.2 9 6.4

2.09

-2.60

-1.23

-0.58

Drying Time

24 h

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0.5 ml

Evaluation of optimized APT loaded ODF Smooth, Physical Nonappearanc sticky e and clear Evaluation parameters Disintegrati 14 ± 0.1s on time Wetting 8 ± 0.1s time Tensile strength Extensibilit y

2.93 ± 0.07N 38.32 ± 1.7mm

ACCEPTED MANUSCRIPT

Y3

Y4

Y5

Constrains

minim um 13.824

minimu m 7.879

maxi mum 2.553

maxi mum 37.908

maxim um 193.31

14

8

2.933

38.316

200

1.29

1.5

1.17

1.06

3.55

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Predicted values Observed values % error

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92.4 mg

Films were prepared in petriplates with diameter 7 cm (38.5 cm2 area) employing solvent casting method

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Y2

50°C

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Y1

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Response

Drying Temperature Aprepitant

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6 Numerical optimization

Folding endurance Drug content Thickness Burst strength Relaxation Resilience

200 ± 5.0folds 9.04 ± 0.3mg 0.10 ± 0.02mm 7.09 ± 0.2g 30.34 ± 1.5% 25.68 ± 2.4%

ACCEPTED MANUSCRIPT Table 3 Four period, Four treatment crossover design for evaluating animal pharmacokinetic performance Periods of giving formulations 1 2 3 4 1 ACAP APT-ODF APTN APTM 2 APT-ODF APTN APTM ACAP 3 APTN APTM ACAP APT-ODF 4 APTM ACAP APT-ODF APTN Each group contains three rabbits and total trials were 48. APT-ODF is optimized APT loaded ODF; APTN is non micronized drug; APTM is micronized drug and ACAP is marketed formulation (Aprecap). (Dose: 1 mg/kg APT equivalent dose)

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ACCEPTED MANUSCRIPT Table 4 Different trails conducted to incorporate APT into ODF Observation

Oil phase dispersion

9.6 mg APT was dissolved in 0.1 ml Capmul: Tween 80 (70:30). This solution was transferred to polymeric blend.

Film was rough, non- clear and disintegration property of film was lost

Niederquell and Kuentz, 2013

Solubility enhancement using complexation

9.6 mg of APT was dispersed in 10 ml of water: methanol (50:50) solution containing 19.2 mg of cyclodextrin. This dispersion was shaken for 24 hr in shaking incubator at 50°C. A clear solution obtained was evaporated on water bath at 60°C to obtain a solid mass. This solid mass was redispersed in minimum quantity of water and mixed with polymeric solution.

APT- βcyclodextrin complex broken and free βcyclodextrin present in the complex caused interaction with TP leading to increase in disintegration time of film

Hiremath and Godge, 2013

9.6 mg APT dissolved in 0.5 ml of 0.1 M NaOH solution

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9.6 mg APT was dissolved in 0.1 ml of Tween 80. This solution was transferred to polymeric blend.

pH change

9.6 mg of APT was dissolved in 0.1 M NaOH solution. This solution was transferred to polymeric solution and pH was adjusted to 7.

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9.6 mg APT was dissolved in 0.1 ml of Tween 80

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Preparation

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28.8 mg of APT- βcyclodextrin complex (1:2)

Principle

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Trials for incorporating APT 9.6 mg APT dissolved in 0.1 ml Capmul: Tween 80 (70:30) SMEDDs

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References

Pectin present in Niederquell the film and precipitated Kuentz, during drying 2013 process leading to a non- uniform film and disintegration property of film was lost Film formed was Kesisoglou smooth, nonand Wu, sticky, clear and 2008 retained its disintegration time of 14 sec

ACCEPTED MANUSCRIPT Table 5 Water sorption time, swelling index, solubility and solubilization rate of various compositions of excipients

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1

Glycine (Pretreatment)

20

1

TP (Pretreatment)

9

1

Pullulan

120

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1

D 70

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PU:TP::30:70 (Physical mixture) PU:TP::45:55 (Physical mixture) PU:TP::60:40 (Physical mixture) PU:TP::30:70 (Pretreatment) PU:TP::45:55 (Pretreatment) PU:TP::60:40 (Pretreatment) PU:TP::30:70+ LG (0.11%) (Pretreatment) PU:TP::30:70+ LG (0.05%) (Pretreatment) PU:TP::30:70+ LG (0.01%) (Pretreatment) PU:TP::30:70+ LG (0.11%) (Pretreatment) PU:TP::30:70+ LG (0.11%) (Pretreatment) + APT ( physical mix)

3

84

6

96

5

50 62 78 7

2 3 5 1

7

1

8

1

7

1

7

1

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Solubility (mg/ml)

IP 1.8 0.25 2.10 0.11 0.92 0.12 2.2 0.12 2.25 0.23 3.90 0.11 3.55 0.12 3.00 0.11 2.6 0.1 2.3 0.1 2.4 0.1 0.91 0.1 0.92 0.1 0.91 0.1 0.92 0.1 0.92 0.1

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Tamarind pectin (TP)

Swelling index

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Glycine

Water sorption time (WST) (sec) 7 1

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Composition of sample

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Solubilization rate (mg sec/ml)

200 (freely soluble)

28.6

100 (freely soluble)

12.5

50 (soluble)

2.5

200 (freely soluble)

22.2

10 (sparingly soluble) 10 (sparingly soluble) 10 (sparingly soluble) 10 (sparingly soluble) 50 (soluble) 50 (soluble) 50 (soluble) 1000 (very soluble)

0.08

1.00 0.81 0.64 142.85

1000 (very soluble)

142.85

1000 (very soluble)

125.00

1000 (very soluble)

142.85

1000 (very soluble)

142.85

0.14 0.11 0.10

ACCEPTED MANUSCRIPT Table 6 Evaluation of optimized APT loaded ODF during stability studies at 40˚C/ 75% RH

D

60

TE

45

CE P

30

AC

5

T

Smooth, nonsticky and clear Smooth, nonsticky and clear Smooth, nonsticky and clear Smooth, nonsticky and clear Smooth, nonsticky and clear

Folding endurance (folds) 200 ± 5.0

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9.4 ± 0.3 (94.22%) 9.4 ± 0.3 (94.11%) 9.3 ± 0.2 (93.01%) 9.3 ± 0.3 (93.69%) 9.2 ± 0.1 (92.38%)

Appearance

200 ± 5.0

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Tensile strength (N) 2.93 ± 0.07 2.92 ± 0.06 2.91 ± 0.06 2.90 ± 0.04 2.86 ± 0.03

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Drug content (mg/4 cm2)

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Day (s)

48

Disintegration time (sec) 14 ± 0.2 14 ± 0.1

195 ± 3.0

14 ± 0.3

192 ± 1.0

14.5 ± 0.2

190 ±1.0

15 ± 0.3

ACCEPTED MANUSCRIPT Table 7 Mean of all pharmacokinetic parameters of different formulations and their significance level

Tmax (hr)

6.00 ± 0.24 0.055 ± 0.002

6647.8 5760.62 ± 1± 230.42 280. 35 6.00 ± 6.00 ± 0.24 0.25 0.055 0.0495 ± ± 0.002 0.002

0.0235 ± 0.001

Cmax (ng.ml1 )

686.54 ± 27.46

614.52 ± 24.58

AUMC (ng.hr2.ml-1)

78469. 32 ± 3138.7 7 1.80 ± 0.07

10.26 ± 0.41

MRT (hr)

563.41 ± 22.54

No significant difference.

0.01875 ± 0.001

No significant difference between APT- ODF and Aprecap. Significant difference between APTODF & APTM, APT-ODF & APTN, Aprecap & APTM, Aprecap & APTN, APTM & APTN No significant difference between Aprecap and APTM. Significant difference between APTODF and Aprecap, APT- ODF & APTN, Aprecap & APTN, APTM & APTN

-0.0165 ± 0.001

470.84 ± 18.83

No significant difference between Aprecap and APTM. Significant difference between APTODF and Aprecap, APT- ODF & APTN, Aprecap & APTM, Aprecap & APTN, APTM & APTN.

67126. 57568.85 51 ± ± 2302.75 2685.0 6 1.56 ± 1.36 ± 0.06 0.05

43701.46 ± 1748.05

Significant difference (P< 0.05).

1.00 ± 0.04

No significant difference between Aprecap and APTM. Significant difference between APTODF and Aprecap, APT- ODF & APTM, APT- ODF & APTN, Aprecap & APTN, APTM & APTN

10.09 ± 0.40

10.26 ± 0.41

No significant difference.

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F

-0.0235 ± 0.001

Significant difference (P< 0.05).

6.00 ± 0.25

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-0.03 ± 0.001

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Ke (μg.hr-1)

Significance level

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7647.6 5± 305.91

Non micronized APT 4254.85 ± 170.19

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AUC (ng.hr.ml-1)

Ka (μg.hr-1)

Microniz ed APT

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Aprec ap

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APTODF

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PKParameters

9.99 ± 0.39

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Graphical abstract

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