Aprepitant loaded solid preconcentrated microemulsion for enhanced bioavailability: A comparison with micronized Aprepitant

Accepted Manuscript Aprepitant loaded solid preconcentrated microemulsion for enhanced bioavailability: a comparison with micronized Aprepitant Sunil Kamboj, Radhika Sharma, Kuldeep Singh, Vikas Rana PII: DOI: Reference:

S0928-0987(15)00329-2 http://dx.doi.org/10.1016/j.ejps.2015.07.008 PHASCI 3318

To appear in:

European Journal of Pharmaceutical Sciences

Received Date: Revised Date: Accepted Date:

31 March 2015 6 July 2015 8 July 2015

Please cite this article as: Kamboj, S., Sharma, R., Singh, K., Rana, V., Aprepitant loaded solid preconcentrated microemulsion for enhanced bioavailability: a comparison with micronized Aprepitant, European Journal of Pharmaceutical Sciences (2015), doi: http://dx.doi.org/10.1016/j.ejps.2015.07.008

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Aprepitant loaded solid preconcentrated microemulsion for enhanced bioavailability: a comparison with micronized Aprepitant Sunil Kamboj, Radhika Sharma, Kuldeep Singh and Vikas Rana* Pharmaceutics Division Dept. of Pharmaceutical Sciences and Drug Research Punjabi University Patiala-147002

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

1

Abstract Aprepitant (APT) is a lipophilic, poorly water soluble drug with moderate permeability characteristic. Therefore, we aimed to improve solubility as well as permeability that could possibly improve oral bioavailability of APT. For this purpose, Quality by design (QbD) approach employing simplex lattice mixture design was used to prepare solid preconcentrated microemulsion (S-PCM). Further, the software generated numerically optimized S-PCM formulations were developed by utilizing desirability function. The spectral attributes (powder X-ray diffraction, ATR-FTIR, and differential scanning calorimetry) of S-PCM formulations suggested that APT was present in amorphous form. The results of droplet size (150 to 180 nm), zeta potential (-13 to -15 mV), poly dispersity index (PDI) (0.211 to 0.238) and emulsification time (<1 min), of these S-PCM formulations (SP1, SP2 and SP3) suggested spherical shape morphology (Transmission electron microscopy) with thermodynamic stability. The comparison of in vitro/ex vivo behavior of S-PCM (SP1) with micronized and non-micronized formulations of APT suggested 2 fold and 5 fold enhancement in solubility and permeability, respectively. This was further evident from pharmacokinetic studies in rabbits that showed 1.5 fold enhancement in bioavailability of S-PCM with respect to micronized APT. Thus, it could be envisaged that development of S-PCM formulation of APT is the best alternative to micronization technology based APT formulations reported earlier.

Keywords: Preconcentrated microemulsion (PCM); Simplex lattice design; Spray drying; Oral bioavailability; Apparent permeability 2

1.0 Introduction Aprepitant (APT) is an antiemetic agent that mediates its effect by blocking neurokinin 1 (NK1) receptors [1]. Previous studies have shown that the projected efficacious human dose for APT is relatively high due to low solubility of nanoformulated APT in simulated intestinal fluids [2–4]. Thus, the development of enhanced bioavailability formulation of APT could potentially reduced the required dose [3,5]. Currently, APT is available in capsule formulation with different dose (40 mg, 80 mg and 125 mg) manufactured by Merck and Company, Inc., and commercially marketed in the United States of America [5]. APT is a lipophilic compound [log P (pH 7)=4.8], weakly basic in nature with a low water solubility (3-7 µg/mL, pH 2-10) [1,6]. The first pass metabolism by CYP3A4 leads to low pharmacokinetic profile [7,8]. Further, an intermediate permeability of APT (7.85×10-6 cm/s) across Caco2 model indicated that APT did not have “high permeability”. Therefore APT was categorized as a BCS class IV drug, being “low soluble” and “low permeable” [2,3]. Various attempts have been made to enhance bioavailability of APT. The Merck and Company, Inc. utilized size reduction principle to developed micronized/nanosized APT, that was found to enhance solubilization rate of the drug [5]. Wu et al., [9] investigated APT bioavailability in beagle dogs that exhibited 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 5.49µm to 0.12 µm. Although, the Emend ® (nanoparticulate formulation of APT available in market) improved bioavailability of APT, but the efforts to enhance the solubility and dissolution rate of APT are still on pursuit, probably due to high cost of this drug. Several methods have been introduced to enhance the solubility of APT, which include, the use of surfactants [10], solid dispersion [11,12], hot melt extrusion technique [13], cyclodextrin complexes [5,14,15] and nanoparticles [16]. But, a most effective alternative approach to increase solubility as well as permeability of BCS class IV drugs could be the development of microemulsion based drug delivery systems [17]. In this system, the drug exists in lipid phase that is dispersed in highly solubilized form which has a capability to easily cross permeability barriers and transport the drug via lymphatic route [18]. Thus, solubility as well as permeability problem of APT could be resolved. In addition, Emulsion-based delivery systems are convenient means of delivering poorly water soluble drugs via the oral route, protecting and encapsulating 3

drugs for pharmaceutical applications [19,20]. Microemulsion are considered to be an ideal liquid vehicles for drug delivery as they possess additional merits like very low interfacial tension with spontaneous formation, high solubilization, thermodynamic stability (long shelflife), low viscosity (with Newtonian behavior) and high surface area (high solubilization capacity), droplet size (5–200 nm) [21–23]. Preconcentrated microemulsion (PCM) formulation design approach has been used for many poorly water soluble and poorly permeable drugs like talinolol [24], amphotericin B [25], carvidilol [17],

ezetimibe [26],

etc. to improve their

solubility as well as permeability that leads to enhanced oral bioavailability. Solid preconcentrated microemulsion delivery system (S-PCM) is one of the lipid-based drug delivery systems prepared by incorporation of liquid excipients into powders by using various solidification techniques [27]. It is a beneficial drug delivery system for low water-soluble drugs as it possesses the advantages of solid dosage forms (high stability with various dosage form options) with those of liquid preconcentrated microemulsion drug delivery system (L-PCM) (solubility and bioavailability enhancement). S-PCM produce oil-in-water microemulsion upon mild agitation in aqueous media (such as gastrointestinal fluids) [28,29]. The micro/nano sized droplets generated in this process bears the advantage of carrying the drug in a solubilized form with a high interfacial surface area for an enhanced, more uniform and reproducible bioavailability [30]. In the present study, an attempt was made to enhance the solubility, in vitro dissolution and permeability that could possibly enhance the oral bioavailability of APT. For this purpose LPCM formulations of APT were prepared using a medium chain oil, surfactant and co-surfactant combination as per simplex lattice mixture design and evaluated using surface response methodology. The L-PCM formulations developed were characterized for its ability to form microemulsion based on droplet size, dissolution characteristics and zeta potential. These LPCM formulations were then converted into S-PCM using Aerosil 200 employing spray drying technique. The S-PCM formulations were then evaluated for in vitro, ex vivo and in vivo performance in comparison to micronized form of APT. Therefore, the present investigation could be an effective alternative to enhance oral bioavailability of APT as compare to already available techniques. 2.0 Materials and methods 2.1 Materials 4

Micronized Aprepitant (particle size 3.21±0.9 µm) and non-micronized Aprepitant (particle size 25.91±3.2 µm) were provided ex gratia by Dr. Reddy’s Laboratories Ltd., Andhra Pradesh, India and Ranbaxy laboratory, Gurgaon, India, respectively. Labrafil M 1944CS (Oleoyl macrogol-6 glycerides), Labrafil M 2125CS (Linoleoyl macrogol glycerides), Lauroglycol 90 (Propylene glycol monolaurate), Plurol olique (Polyglyceryl-3 dioleate) and Transcutol (Diethylene glycol monoethyl etherol) were received as gift samples from M/s Gattefosse, Saint-Priest, France. Capmul MCM C10 (Glyceryl mono and dicaprate), Captex 200 (Propylene glycol dicaprylate/dicaprate), Captex 355 (Caprylic/Capric triglyceride) were received as gift samples from M/s Abitec Corp., Wisconsin, USA. Aerosil 200 ex-gratia was a gift by Panacea Biotech Pvt. Ltd., Derabassi, Punjab, India. The HPLC grade solvents like methanol, acetonitrile etc. were used for liquid chromatographic studies. All the reagents were of analytical grade and used as received. 2.2 Methods 2.2.1 Equilibrium Solubility studies for Aprepitant Solubility of APT was determined in various oils (Labrafil M1944CS, Labrafil M212CS, Captex 355, Lauroglycol 90, Capmul MCM C10, Labrafac lipophile, Captex 200, Plurol oleique), surfactants (Tween 80) and co-surfactant (Transcutol). For this purpose, an excess amount of APT was transferred to each of the glass vial previously containing 1mL of oil phase and/or surfactant phase and/or co-surfactant phase and vortexed for 5 min after every 2 h for 24 h. In between these intervals, these vials were kept at a constant temperature in shaking incubator at 50 rpm and 25± 0.5 °C. All the vials were centrifuged (3000 rpm for 10 min) after keeping them aside for 24h to attain equilibrium. The obtained supernatant was filtered through a membrane filter having pore size of 0.45 µm (Millipore, Darmstadt, Germany), 0.1 mL of solution was taken after filtration and diluted with mobile phase and analyzed employing HPLC method. Based upon solubility studies Capmul MCM C10, Tween 80 and Transcutol were selected as oil, surfactant and co-surfactant, respectively. 2.2.2 Analytical method The analytical profile of APT was validated for its quantification on high-performance liquid chromatography (HPLC) system. The samples obtained from solubility, dissolution tests, ex vivo permeation and in vivo pharmacokinetic analysis were quantitatively analyzed for APT concentration using an isocratic HPLC system. The HPLC system consists of 515 HPLC pump 5

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 acetonitrile and 0.1% orthophosphoric acid (60:40) at 1 mL min-1 flow rate and 20 µL sample volume. The detection wavelength was set at 210 nm. The limit of detection and limit of quantification were found to be 0.035 µg/mL and 0.113 µg/mL, respectively. The method was found to be linear in the range of 0.1-50 μg/mL with regression coefficient (r2=0.999). The analysis was performed under ambient conditions. 2.2.3 Construction of ternary phase diagrams The ternary phase diagrams of the system Tween 80, Transcutol and Capmul MCM C10 in the absence and presence of model drug APT (80 mg/mL), were constructed by carefully measuring each component mixture (total 10 mL) into a glass vial. Various compositions of Capmul MCM C10 (10-90 %), Tween 80 (10-90 %) and Transcutol (10-40 %) were used to prepare ternary mixtures. After complete solubilization of all the ternary components, a clear and transparent solution was obtained. These clear mixtures were then diluted 100 times with distilled water at room temperature (25±1°C) for the evaluation of droplet size, PDI and self-emulsification time. 2.2.4 Optimization of L-PCM Experimental design The design of experiment (DoE) technique provides efficient means for estimating the optimal formulation of a mixture and optimizing their processes. The goal of optimizing pharmaceutical formulations is to explore the variable/position that influenced the selected response variables and find the process or formulation variable levels from which a robust product with enhanced quality characteristics may be generated. The influence of the oil, surfactant and co-surfactant on the APT loaded L-PCM properties was studied using simplex lattice design (mixture design) [31–33]. Preliminary experiments were performed to select the different variables of the formulation, which were chosen on the basis of the ability of a mixture to form a selfemulsifying system and to achieve lowest droplet size. Thus, the levels of design were selected on the basis of ternary phase diagram results, where the area obtained using maximum oil (upto 40% v/v), minimum surfactant (40-60 % v/v) and co-surfactant (upto 30% v/v) was further used. The three independent variables are; Capmul MCM C10 as oil phase (X1), Tween 80 (X2), and Transcutol (X3). For three component system, the simplex lattice experimental design was a most 6

appropriable design that is shown by an equilateral triangle exist in a two-dimensional space [31–33]. On the basis of ternary phase diagrams and preliminary experiments, the range of each component was selected as follows: 300µL ≤ X1 ≥ 600 µL, where, X1 is Capmul MCM C10 (Lipid phase) 300µL ≤ X2 ≥ 600 µL, where, X2 is Tween 80 (Surfactant) 100 µL ≤ X3 ≥ 300 µL, where, X3 is Transcutol (Co-surfactant) The Design-Expert® software (Version 8.0.4; Stat-Ease, Inc., MN, USA) was used to analyze correlation between dependent and independent variables. The experimental design (simplex lattice mixture design) that contains 13 runs was utilized in generating suitable mathematical models (linear, cubic, quadratic, quadratic and/or simple cubic) and statistical significance (r2, predicted r2, adjusted r2, lack of fit) employing Design-Expert® software. The best model was predicted by the software on the basis of maximum magnitude of r2, predicted r2 and insignificant lack of fit. Moreover, adjusted r2 and predicted r2 should lie within 0.2 for ensuring the validity of the model. For the best model the PRESS value should be small. The excellence of the model was determined using PRESS. The droplet size (in nanometers; Y1), polydispersity index (PDI) (Y2) and self emulsification time (in seconds; Y3) were taken as the dependent variables (responses). The detailed description of independent variables and dependent variables is shown in table 1. After the fitting of the mathematical model, the desirability function was studied for the optimization of independent variables for desirable responses. A numerical optimization criterion for PCM is summarized in table 1. 2.2.5 Preparation of solid-PCM The numerically optimized L-PCM formulations were used for the preparation of S-PCM formulations. APT loaded S-PCM was prepared by using spray drying technology. Briefly, Aerosil 200 was suspended in ethanol (1 g/100 mL) to produce suspension. 1 mL of L-PCM containing APT (80 mg/mL) was then added into the Aerosil 200 suspension with constant stirring (15 min at room temperature) and homogenous suspension was obtained. The resultant suspension was then spray dried in a laboratory scale mini spray dryer apparatus (Labultima, India) utilizing inlet aspirator flow rate = 40 Nm3/h, temperature of 80 °C and feeding rate was 5

7

mL/min. The powder appeared in cyclone was collected and measured for the final drug content using a validated HPLC method. 2.2.6 Solid state characterization of S-PCM Scanning electron microscopy (SEM) The surface morphological changes in Aerosil 200 powder in presence or absence of APT loaded L-PCM was investigated by JSM-6610LV (Jeol, Tokyo, Japan) scanning electron microscope at accelerating voltage of 15 KV. Each sample was mounted on SEM-stub using double-sided adhesive tape and was gold coated in the presence of argon gas at 25 mA to make them electrically conductive. The SEM images were taken and analyzed for any physical differences. Differential Scanning Calorimetry The thermal characteristics of pure APT powder (both micronized and non-micronized), S-PCM formulations, the solid carrier (Aerosil 200) and physical mixture (APT+Aerosil 200) were investigated using a differential scanning calorimeter (DSC Q10, TA Instruments, USA). For the test, about 3 mg of the samples was placed in sealed aluminum pans before heating under dry nitrogen which was used as effluent gas at a flow rate of (25 mL/min) and heating rate of 10 °C/min from 40 ºC to 400 ºC. X-ray Powder Diffractometry (XpRD) The amorphous or crystalline nature of APT before or after adsorption into S-PCM was explored by studying diffraction patterns generated through X'pert-PRO High Resolution Powder Diffractometer (PANalyticals, Almelo, Netherlands), with a divergent beam (Cu Kα radiation source with wavelength of λ 1.5418) and scintillation counter detector. The scanning rate was maintained fixed over a range of 10-90° 2θ at 25°C, with a step size of 0.01° 2θ. FTIR- ATR Spectroscopy A pure sample of APT (micronized), APT (non-micronized), Aerosil 200, physical mixture (APT + Aerosil 200) and S-PCM were analyzed by FTIR-ATR analysis (RXI-JR, Alpha-E, Bruker, Germany) in the spectral region of 500–4000 cm−1. 2.2.7 Properties of L-PCM and S-PCM after reconstitution Emulsification time determination Emulsification time was determined according to the method reported by Parmar et al.[34] and Venkatesh et al. [35]. The study was conducted in a dissolution apparatus II paddle (Electrolab india, Mumbai, India). For the study, either L-PCM (1 mL) or S-PCM (500 mg) was introduced 8

into 500 mL of distilled water at 37°C under constant stirring by a standard stainless steel dissolution paddle rotating at 100 rpm. The time taken by each sample to completely disperse in a dissolution basket was taken as end point. Emulsion droplet size and charge determination Droplet size of the microemulsion formed by the addition of 0.1 mL of L-PCM, or 100 mg of SPCM into 50 mL of distilled water was stabilized for 2 h and then centrifuged at 3000 rpm for 10 min and clear supernatant was examined by Zetasizer Nano ZS90 (Instruments, UK) with dynamic light scattering droplet size analyzer at a wavelength of 635 nm and at a scattering angle of 90° at 25 °C. All studies were conducted three times, and the z-average diameters were reported. Transmission Electron Microscopy S-PCM formulations (100 mg) were dispersed in 50 mL deionized water. After 2h stabilization each sample was centrifuged (3000 rpm for 10 min). A clear supernatant was examined for morphological and structural behavior using transmission electron microscopy (TEM; Hitachi, Tokyo, Japan) working at 100 kV. Each sample was prepared by withdrawing droplet (0.5 mL) of the reconstituted S-PCM (clear centrifuged solution) formulation. This droplet was directly positioned on the copper electron microscopy grids supported by formvar films. The excess was siphoned off using filter paper. The grids were stained with phosphotungstic acid (0.5% w/v aqueous solution) for 30 s, and the excess was siphon off. The grids were then viewed under TEM. 2.2.8 Stability testing The stability of reconstituted microemulsion was assured by determining cloud point levels and thermodynamic behavior of optimized samples (L-PCM and S-PCM) according to the method reported by Bali et al. [36], Elnaggar et al. [37] and Beg et al. [38]. 2.2.9 In vitro dissolution of S-PCM APT cumulative release from pure APT (micronized suspension), APT (non-micronized suspension), L-PCM and S-PCM was evaluated by using dissolution apparatus-paddle II USP (Electrolab, Mumbai, India) at 37 ± 0.5 °C using 900 mL of 2.2% SLS as dissolution media with stirring speed of 100 rpm [5]. Samples were withdrawn at an interval of 5, 15, 30, 45, 60 minutes, respectively. At different time intervals, 5 mL sample was withdrawn and replaced by fresh dissolution medium. After filtration (using 0.22 µm size filter paper, Millipore, Darmstadt, 9

Germany), each sample was suitably diluted with respective dissolution medium. Amount of drug released was determined by HPLC method already validated for this purpose. Further, dissolution data was analyzed using similarity factor (f2) and dissimilarity factor (f1) approach [39]. 2.2.10 Ex vivo permeation studies An everted rat gut sac method was used as per method reported by Ghai and Sinha, [24], Tan et al. [40] and Singh et al. [25]. The protocol was approved by the Institutional Animal Ethical Committee (IAEC), Punjabi University, Patiala, India (107/99/CPCSEA-2011-23) guidelines for the use and care of experimental animals. For this study male Wistar rats (200-240 g) were anesthetized using excessive ether inhalation after keeping them fasted for 24 h with an exception to drinking water. The incision was made in the abdomen portion of rat to locate small intestine region. The Jejunum region was separated (~7 cm; area 9.14 cm2), washed with cold Kreb’s Ringer phosphate buffer (KRPB) and placed inside aerated KRPB solution [17,24,25,40]. After washing and removing adhered muscle layer, one end of tissue segment was ligated with thread and everted on thin glass rod. A sample equivalent to 80 mg of APT present in different formulations (L-PCM, S-PCM, APT micronized and APT non-micronized) was transferred to the donor compartment. The everted gut sac was filled with 1 mL of KRPB solution, was submerged inside the aerated (15 bubbles/min) bath (50 mL; 37±0.5 °C) containing L-PCM/S-PCM/ APT micronized/APT nonmicronized. An aliquot of drug solution was withdrawn from the serosal compartment (inside portion of everted rat sac) at predetermined time intervals up to 2 h and immediately replaced with fresh KRPB solution. The amount of APT diffused across everted rat gut sac was determined by HPLC. The percentage cumulative amount per unit area permeated with respect to time was used to determined flux, relative permeability and apparent permeability (Papp) as per method reported by Ghai and Sinha, [24]. Papp of APT was calculated from mucosal to serosal direction according to the equation: Papp (cm/sec) = (dQ/dt)/(A×Co) Where, the dQ/dt is the drug permeation rate from the tissue, A is the cross-sectional area of the tissue, and Co is the initial APT concentration in the donor compartment at t = 0. Statistical analysis of ex vivo permeation data (i.e., permeation flux) and Papp of formulations was performed using one-way ANOVA followed by post hoc Tukey’s multiple-comparisons test 10

(SigmaStat software 3.5, Systat Software, Inc., Richmond, CA) with p value of <0.001 was considered as significant. 2.2.11 Pharmacokinetic studies in rabbits In- vivo studies were performed using New Zealand male rabbits. All animal experiments were carried out after approval of the protocol by the Institutional Animal Ethical Committee (IAEC), Panjabi University, Patiala, India, (107/99/CPCSEA-2011-23) guidelines for the use and care of experimental animals. New Zealand male rabbits, weighting 2.4–2.9 kg were fasted for 14 h before drug administration but were allowed to free access of water. Animals were divided into different groups, each containing four rabbits and receive the following treatments. Group A

:

Oral administration of suspension of non-micronized APT

Group B

:

Oral administration of suspension of micronized APT

Group C

:

Oral administration of SP1 batch

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 micro-centrifugation tubes containing 40 µL of disodium EDTA. The collected blood was centrifuged at 4000 rpm (15 min). The upper plasma layer was separated carefully using micropipette and transferred to micro-centrifugation 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. Standard non-compartmental pharmacokinetic parameters (±SD) were calculated using the pharmacokinetic program Phoenix Win-Nonlin 6.4 (Pharsight Corporation, Mountain View, CA). Statistical analysis of in vivo pharmacokinetic data was conducted using one-way ANOVA followed by post hoc Tukey’s multiple-comparisons test, with P values of <0.05 were considered as significant. 3.0 Results and discussion 3.1 Equilibrium Solubility studies for APT The solubility of APT was determined in various oils (Labrafil M1944CS, Labrafil M212CS, Captex 355, Capmul MCM C10, Labrafac lipophile, Captex 200P and Soybean oil) and in various surfactants (Plurol oleique and Tween 80). The results of solubility of APT in various 11

carriers are summarized in Fig. 1. The solubility of APT in Capmul MCM C10 was found to be maximum (69.19±3.0 mg/mL) amongst oil phase chosen for solubility study, i.e. 5 to 6 fold higher solubility as compared to Labrafil M1944CS, Labrafil M212CS, Captex 355, Labrafac lipophile, Captex 200 P and Soybean oil. The solubility of APT in Tween 80, Plurol oleique and Transcutol was found to be 77.34±1.8 mg/mL, 22.5±0.9 mg/mL and 105±3.1 mg/mL, respectively. The solubility of APT in Tween 80, Transcutol and Capmul MCM C10 was highest. Hence, Capmul MCM C10, Tween 80 and Transcutol were selected amongst various oils and surfactants for the preparation of ternary phase diagrams. This combination of Capmul MCM C10 with Tween 80 and Transcutol were expected to provide maximum solubility/ entrapment efficiency of APT with microemulsion region in lowest possible concentration of Tween 80. This combination was further evaluated using ternary phase diagram. 3.2 Ternary phase diagrams For the purpose of selecting proportions of components that can provide maximum microemulsion region, ternary phase diagrams with various levels of components were constructed. It was reported that a large amount of surfactant causes GI irritation [41]. Therefore, it is beneficial to develop microemulsion formulation that contains minimum amount of surfactant. Thus, ternary mixtures with less amount of surfactant for a fixed concentration of oil in microemulsion were selected from the phase diagrams. Ternary phase diagrams were also examined to determine the concentration range of each component that could provide stable and acceptable microemulsion [24,26]. The ternary phase diagrams were prepared using Capmul MCM C10, Tween 80 and Transcutol (Fig. 2). Visual experiments were conducted to assess the self emulsification efficiency. The microemulsion region was further screened for the area with droplet size less than 200 nm (Fig. 2b). The developed formulations from the ternary phase diagram were subjected to dilution with distilled water (1 in 100 mL of D.H2O). The systems were examined visually for a capability to emulsify spontaneously and the end point appearance of the microemulsion. The microemulsion area was plotted according to the visual inspection depending upon the ability of the mixture to form clear microemulsion with small droplet size and high dispersibility. The results suggested 5-40 %v/v of Capmul MCM C10, 50-80 %v/v Tween 80 and 10-40 %v/v Transcutol as acceptable microemulsion area. Thus, on this basis the levels used in simplex lattice design were chosen for the formulation optimization of L-PCM. 3.3 Formulation optimization of APT-loaded L-PCM 12

The optimization of APT loaded L-PCM formulation using selected Tween 80, Capmul MCM C10 and Transcutol was studied employing a simplex lattice design. The total amount of cosurfactant, surfactant and oil phase in the formulation was kept constant while the ratio of the three was varied according to table 1. The levels of the three independent variables selected were: Capmul MCM C10 (X1), 300–500 µL; Tween 80 (X2), 300–600 µL; Transcutol (X3), 100– 300 µL. According to simplex lattice model and the selected concentration levels of surfactant, co-surfactant and oil, thirteen different formulations of L-PCM carrying APT were prepared. The results of their mean droplet size, PDI and emulsification time are given in Table 1. The model fitting of each response was conducted for getting highest r2 value generated for various polynomial models. Based on the adjusted and predicted multiple correlation coefficient (r2), lack of fit and model p value; cubic model was expected to be the best fit models for interpreting data. The equations generated after correlating droplet size, PDI and emulsification time with independent variables are: Droplet size (Y1) =+275.55X1+157.44X2+177.55X3-147.10X1X2-191.07X1X3-86.45X2X3 -82.04 X1X2X3 -137.86X1X2(X1-X2) -296.22X1X3(X1-X3) +17.24X2X3(X2-X3) (Equation 1, r2=0.9982) PDI

(Y2)=+0.33X1+0.24X2+0.54X3-0.13X1X2-0.28X1X3-0.10X2X3-0.18X1X2X3-0.37X1X2(X1-

X2) -1.34 X1X3(X1-X3) -0.79X2X3(X2-X3) (Equation 2, r2=0.9970) Emulsification time (Y3)=+72.21X1+63.60X2+24.70X3-52.40X1X2-77.85X1X3-37.71X2X3-148.4 2X1X2X3-19.45X1X2(X1-X2)-40.94X1X3(X1-X3)-14.37X2X3(X2-X3)(Equation 3, r2=0.9985) Polynomial equations obtained above were used to draw conclusions by correlating the magnitude of coefficient and the mathematical sign it carries. The positive sign in the polynomial equations is an indicative of direct correlation between responses (like Droplet size, polydispersity index and self emulsifying time) and independent variables. In addition, a positive value represents a synergistic effect, while a negative value indicates an antagonistic effect [42]. Equation 1 shows highest positive magnitude of X1 suggesting increase in amount of Capmul MCM C10 increased the droplet size. Thus, revealing additive influence of Capmul MCM C10 in increasing the droplet size. Interestingly, the polynomial coefficients of cubic model reflect joint effort of Capmul MCM C10, Tween 80 and transcutol. Further, negative magnitude of interaction variables [(X1X2, X2X3, X1X3, X1X2X3, X1X2(X1-X2) and X1X3(X1-X3)] revealed the 13

reduction in droplet size is associated with interaction variables rather than individual independent variables (X1, X2 and X3). Further, the response surface plot and the corresponding contour plot (Fig. 3a) showed crucial role of Transcutol in decreasing droplet size. Thus, the goal was to achieve minimum PDI. The PDI of all the L-PCM formulations after reconstitution was in range 0.19 to 0.56, suggesting all the L-PCM formulations to have uniform droplet size distribution. The equatation generated between independent variables and PDI suggested that a combination of Capmul MCM C10, Tween 80 and Transcutol strongly contributed in preparation of PCM bearing uniform droplet size distribution. The response surface plots and corresponding contour plots also revealed a joint effect of influence of X1 (Capmul MCM C10), X2 (Tween 80) and X3 (Transcutol) in creating uniform droplet size and PDI (Fig. 3b, Table 1). The magnitude of X2 (Tween 80) was minimum thus, owing to its role in decreasing PDI as compared to X1 (Capmul MCM C10) and X3 (Transcutol) which is why increasing the proportion of both increased the PDI. Whereas, the interaction of Tween 80 and Transcutol provide sufficient capability of reconstituted L-PCM to decrease their PDI (Equation 2). The equation 3 revealed that Transcutol predominantly influences self emulsification time as compared to Capmul MCM C10 and Tween 80. Interestingly, the interaction between Transcutolwith either Capmul MCM C10 or Tween 80 in L-PCM has higher influence in decreasing self emulsification time, suggesting overwhelming influence of Transcutol in the depression of self emulsification time (Fig. 3c, Table 1). To obtain compositions of optimized formulation, a numerical optimization technique with desirability function was used. For this purpose, independent variables were set within the range and constraints were set to all the responses. Y1, Y2 and Y3 were minimized. Equal weightage and importance was given to all the responses. The global desirability value was obtained using Design Expert 8 software® shown in Fig. 4 and the values are given in Table 1. Numerical optimization technique of desirability approach using Design Expert 8 software® was employed to develop a new formulation with desired responses. The desirability functions combine all responses in one measurement and provide a possibility to predict the optimum levels of independent variables. Some constrains for selection of desired response were added to design expert 8 software® to obtain optimized formulation with maximum desirability of respective responses (e.g. PS, PDI and SET were set to be minimized). Upon comprehensive 14

evaluation of feasibility search and subsequent exhaustive grid search, the formulation compositions Capmul MCM C10 (30-50%), Tween 80 (30-50%) and Transcutol (14.5-18.5%) fulfilled the maximum requirement of an optimum formulation (Table 1). The predicted desirability of the optimized L-PCM prepared with different compositions of Capmul MCM C10, Tween 80 and Transcutol was found to be 0.991-0.847 (Fig. 4). The value of desirability close to 1 indicates that the response variables are consequently nearer to the largest value [43,44]. With the application of design expert 8 software® three optimized formulation compositions were developed and final formulations were prepared on the basis of these compositions (Table 2). The results showed not more than 5% bias in the predicted results. Hence, it can be concluded that the simplex lattice model equation accurately predicts the experimental results. All the three numerically optimized L-PCM formulations were converted into solid form using spray drying technique [28,45–47] and aerosil 200 was used as a hydrophobic solid carrier. Three S-PCM formulations (SP1, SP2 and SP3) were prepared and used for further evaluation and characterization (Table 2). 3.4 Solid State evaluation of numerically optimized APT loaded S-PCM 3.4.1 Surface characterization The surface morphological changes on solid carrier (Aerosil 200) before and after spray drying of APT loaded L-PCM was ascertained using scanning electron microscope JSM-6610LV (Jeol, Tokyo, Japan). SEM image of Aerosil 200 that appears to be irregular shaped loose aggregate of particles is shown in Fig. 5a. The SEM images of S-PCM (SP1) formulation obtained after adsorption using spray drying of APT loaded L-PCM showed aggregated and larger particles. The S-PCM particles did not show any crystalline shape of APT, indicating complete adsorption of APT loaded L-PCM on to Aerosil 200 (Fig. 5b). 3.4.2 Spectral attributes The FTIR-ATR analysis of Aerosil 200 was performed to rule out any possibilities of molecular level interaction or chemical interaction among the excipients of L-PCM with Aerosil 200. The FTIR-ATR spectra of pure APT (micronized) or APT (non-micronized) indicated the most characteristic peaks at 1603 cm-1 N-H bending, 1704 cm-1 C=O stretching, 1506 cm-1 Ar C=C stretching, 1132 cm-1 C-F stretching [48] (Fig. 6a,b). The FTIR spectra of pure Aerosil 200 presented a broad band at 1085 cm-1 and 801.51 cm-1 indicating the presence of Si-OH moieties. 15

The peaks in the range of 3000-3800 cm-1 indicating OH (bridged) and 3750 cm-1 indicating O-H (isolated) (Fig. 6d) was reported by Nagpal et al. [49]. The physical mixture of Aerosil 200 and APT showed broadening of peak at 1097 cm-1. This peak was attributed to the merger of characteristic peak of APT at 1132 cm-1 (C-F stretching with characteristic peak of Aerosil 200 at 1085 cm-1 (Si-O-H stretching) (Fig. 6e). FTIR-ATR spectra of APT loaded L-PCM showed peak at 1132 cm-1 as observed in pure APT (Fig. 6c). This indicated the presence of APT in solubilized form in L-PCM. The control sample of S-PCM (without APT) showed no additional peaks indicating absence of any interaction of Capmul MCM C10/Tween 80/Transcutol with particles of Aerosil 200 (Fig. 6f). A broadening of band at 1096 cm-1 was observed in FTIR-ATR spectra of S-PCM (SP1, SP2 and SP3) prepared with Aerosil 200 (Fig. 6g). This peak was found to same as observed in FTIR-ATR spectra of physical mixture between APT and Aerosil 200 indicating merger of C-F stretching peak of APT at 1132 cm-1 and Si-O-Si stretching at 1085 cm1

. However, origin of prominent peak at 805 cm-1 in SP1 samples pointed towards entrapment of

Capmul MCM C10/Tween 80/Transcutol and APT mixture between the Si-O-Si plains. This could probably enhance the availability of APT in solution form when it comes in contact with water or GIT solvents. The same observations were made in the SP2 and SP3 formulations. 3.4.3 X-ray powder diffraction studies The X-ray diffraction pattern of APT (non-micronized) and APT (micronized) samples is shown in Fig. 7a,b. The sample of APT (non-micronized) showed maximum relative intensities at 21.11°, 24.95 and 25.05° (2θ). Whereas, APT (micronized) showed maximum relative intensities at 17.35°, 17.77°, 20.82°, 21.34°, 22.13°, 23.70° and 25.98° (2θ). Braun et al. [50] reported that APT exists in two forms (form I and II). Form I shows X-ray diffraction pattern with key reflection at approximately 15.6°, 17.7° and 22.21° (2θ). Form II shows the key reflections at 18.3° and 21.11° (2θ). Thus, the sample of APT (non-micronized) was form II and that of APT (micronized) was form I. The results were in consonance with the findings of Helmey et al. [48] that showed 21.1° (2θ) diffraction patterns as characteristic of form II. Absence of any diffraction peaks in X-ray diffraction of Aerosil 200 suggested amorphous nature (Fig. 7c). The S-PCM formulations (SP1, SP2 and SP3) prepared from Aerosil 200, Capmul MCM C10, Tween 80, Transcutol and APT showed no significant difference in diffraction patterns as compared to pure Aerosil 200 sample (Fig. 7d-f) suggesting conversion of crystalline state of APT to amorphous state of APT in S-PCM (SP1, SP2 and SP3) formulations. 16

3.4.4 Thermal attributes Thermal attributes of APT, Aerosil 200, physical mixture of APT and Aerosil 200, S-PCM without drug and S-PCM with drug were identified using DSC analysis (Fig. 8). The DSC analysis of APT (non-micronized) and APT (micronized) suggested that drug melts at 248°C and 253°C, respectively (Fig. 8a,b). The increase in melting point of APT (micronized) could be due to micronized particles. The DSC of APT showed sharp endothermic transition which indicated crystalline nature of APT. The results were in consonance with the thermal attributes reported by Braun et al. [50], suggesting high purity of both APT non-micronized and APT micronized samples. The DSC thermogram of Aerosil 200 did not show any characteristic peak in range of 40-400°C (Fig. 8c). This was also observed in S-PCM (SP1, SP2 and SP3) sample prepared from Aerosil 200 (Fig. 8f-h). Therefore, it could be concluded that APT in the S-PCM (SP1, SP2 and SP3) was present in amorphous form after fabrication. It is known that transformation of physical state of drug to amorphous or partially amorphous state leads to high energy state and high disorder, resulting in enhancement of solubility [46,51]. This marked increase in solubility of amorphous APT leads to strike the optimal solubility-permeability interplay that results in enhancement of permeability [53–55]. 3.5 Properties of numerically optimized L-PCM and S-PCM formulations after reconstitution 3.5.1 Droplet size, PDI, zeta potential and emulsification time The droplet size as well as zeta potential of reconstituted solution of L-PCM and S-PCM formulations (L1, L2, L3, SP1, SP2 and SP3) in water was evaluated. The results suggested no significance difference (p<0.05) in particle size before and after conversion of L-PCM to SPCM. This was evident from Sb/Sa ratio [47]. The ratio near to 1 is an indicator of lowest deviation (Table 3). The PDI of all the formulations (L1, L2, L3, SP1, SP2 and SP3) was in range of 0.2-0.25 reflecting uniform particle size distribution (Table 3). The zeta potential of all the PCM formulations was found to be negative. However, the magnitude of zeta potential was higher for L-PCM formulations in comparison to S-PCM formulations. The emulsification time is the reflector of quick emulsifying ability of PCM formulations. The conversion from L-PCM to S-PCM showed enhancement in emulsification time and this enhancement in emulsification time could be associated with time required to redisperse adsorbed L-PCM over S-PCM. The emulsification time of S-PCM formulations was less than 1 min. 3.5.2 TEM analysis 17

The TEM analysis of reconstituted S-PCM formulations (SP1, SP2 and SP3) was conducted and the images are shown in Fig. 9. The spherical shaped oil globules were observed in all the SPCM formulations. In addition, black colored droplets indicated the encapsulated APT. No signs of drug precipitation suggested solubilized and stable APT microemulsion. Further, the TEM analysis also showed that droplets maintained their morphology and uniformity in shape or size after reconstitution. These findings pointed towards stability of microemulsion formulation after reconstitution of S-PCM. 3.6 Stability testing The liquid and solid PCM formulations (L1, L2, L3, SP1, SP2 and SP3) were easily dispersible in water. The PCM formulations upon centrifugation test (after reconstitutions) did not show any sign of phase separation and precipitation. Upon freeze thaw cycles, all the formulations were found to be stable. These results suggested that liquid and solid PCM formulations have inherent capacity to successfully generate microemulsion when comes in contact with aqueous environment. The results indicated that all the liquid and solid PCM formulations (L1, L2, L3, SP1, SP2 and SP3) were found to be thermodynamically stable and the results are summarized in Table 4. 3.7 In vitro dissolution studies The high in-vitro dissolution profile indicated high solubility of drug in the simulated gastric environment. The data pertaining to in vitro performance of selected batches L-PCM (L1, L2, and L3) and S-PCM (SP1, SP2 and SP3) is shown in Fig. 10. The dissolution study results of L-PCM formulations (L1, L2 and L3) and S-PCM formulations (SP1, SP2 and SP3) showed more than 80% APT release indicating enhancement in dissolution performance. However, the dissolution performance of APT (non-micronized) showed 60.5 ± 4.2 % cumulative APT released in 180 min whereas, APT (micronized) showed 58.6 ± 1.9 % cumulative APT released in 15 min and maximum 78.2 ± 3.5 % cumulative APT release in 180 min. The results showed 4 fold reduction in time to release maximum APT which was incorporated into PCM formulations. However, Shono et al. [3] observed 35 % APT was released from micronized APT (7 µm average particle size) in 60 min employing biorelevent dissolution media. This could be correlated with low free energy requirement of PCM formulations as compared to particle size reduction technique. Due to this low free energy requirement, APT loaded in highly solubilized admixtures immediately disperse into small droplets (less than 180 nm) that leads to enhance the dissolution [56]. Further, 18

a similarity/dissimilarity statistics was applied to determine point to point correlation between different PCM formulations of APT and pure APT (non-micronized or micronized). The results suggested that all the PCM formulations L-PCM and S-PCM (L1, L2, L3, SP1, SP2 and SP3) were statistically dissimilar to APT (micronized) (f2 value less than 31 and f1 values above 35) and APT (non-micronized) (f2 value less than 20 and f1 value above 45) (Fig.9). However, a similar point to point correlation of L-PCM formulations (L1, L2 and L3) with S-PCM (SP1, SP2 and SP3) (f2 value above 60 and f1 values below 10) was evident. This could be due to uniformity in particle size distribution (PDI 0.2-0.3) and particle size less than 180 nm of all the PCM formulations (Table 3). In the light of above findings SP1 was selected for pharmacokinetic investigations as it showed more than 80% APT release in 10 min, 148±7.1 nm droplet size and uniform particle size distribution (PDI 0.211±0.019) amongst all the S-PCM formulations. Further, the results also indicated development of preconcentrated microemulsion formulations of APT were found to be a good alternative to already available micronization technique for APT. 3.8 Ex vivo performance The ex vivo performance of S-PCM and L-PCM formulations was determined employing everted rat gut sac method [17,24,25]. The study was conducted to explore the permeation behavior of APT before and after loading into PCM system. For this purpose flux (µg/s/cm2), relative permeability (µg*ml/cm2) and apparent permeability (cm/s) were determined and results are summarized in table 5. The flux provided the amount of drug permeated per unit area per unit time across a biological membrane and the rise in flux indicated increase in drug transport across membrane. The relative permeability determined the rate with which APT travelled across serosal side of the intestine [25] and the apparent permeability (Papp) is the indicator of mass permeated by area of tissue over time. The results suggested that the value of flux, relative permeability and Papp of APT when loaded in L1 and SP1 formulation was significantly higher (p>0.05) as compared to drug suspensions (non-micronized APT or micronized APT). Overall, the

flux

associated

with

respective

PCM

formulations

follows

an

order

as

L1=SP1>L2>SP2>L3>SP3 >APT micronized>APT non-micronized. Thus, indicated L1 and SP1 were better option than other formulations. Interestingly, a similar order was observed with relative permeability and apparent permeability for L1 and SP1. A comparison of Papp of APT loaded in S-PCM formulations SP1, SP2 and SP3, respectively showed 2.43 fold, 2.12 fold and 19

1.78 fold enhancement, respectively as compared to APT micronized. In addition, the increase in the Papp of APT from micronized APT suspension could be due to smaller particle size of micronized APT. Similarly, a reduced particle size of droplets containing solubilized APT in flexible small droplets could also be the possible reason for enhancement of apparent permeability, flux and relative permeability of APT from PCM formulations. From the above understanding, it can be attributed that small droplet size of the microemulsion increased the adherence to intestinal membrane which in turn enhanced the transport of the drug and optimized intestinal absorption and permeation [57]. Overall, the findings pointed towards new possibilities for reducing required dose and lowering adverse effects of APT in PCM system. Further, it could be envisaged that large surface area provided by the microemulsion formulations of APT for partitioning of APT across the intestinal membrane could be the cause of enhanced transportation of APT [24]. Furthermore, presence of APT in amorphous form in PCM formulations as evident from DSC studies leads to enhancement in apparent solubility of APT. This enhances supersaturation level and provides trigger force to APT for the transportation across biological tissues. Similar findings were obtained by Dhan et al. [52], Miller et al. [53] and Frank et al. [54,55] for the enhancement in solubility and permeability of amorphous forms of progesterone, nifedipine and ABT-102. Thus, on the basis of in vitro dissolution performance and ex vivo investigations SP1 formulation was selected for pharmacokinetic study. 3.9 In vivo bioavailability studies After obtaining clearance from in vitro and ex vivo performance of S-PCM formulation (SP1), it was essential to challenge the developed formulation in rabbit models. For this purpose, SP1 formulation, micronized APT and non-micronized APT (containing APT equivalent to 1 mg/kg for 2.5 kg rabbit) were orally administered to rabbits and APT reached in the blood plasma was estimated at different time intervals. The results of pharmacokinetic parameters calculated from plasma concentration vs. time plot are summarized in Table 6 and Fig. 11. The Cmax of SP1 formulation was 1.53 fold and 1.97 fold enhanced, respectively from APT (micronized) and APT (non-micronized), respectively. However, 2 fold reduction in T max in comparison to APT (micronized) or APT (non-micronized) suggested reduction in lag time with faster transportation of APT. The AUC of different formulations followed the order SP1>APT (micronized)>APT (non-micronized), indicating entrapment of APT in microemulsion formulation system was beneficial. Further, the oral relative bioavailability from SP1 was 2 fold increased as compared to 20

orally administered APT non-micronized suspension and 1.5 times increased as compared to APT micronized suspension. This may be due to the enhancement in solubility as well as permeability of APT when incorporated into PCM system. Thus, the developed S-PCM formulation showed a significant increase in oral bioavailability of the poorly water-soluble and poorly permeable drug “Aprepitant”. 4.0 Conclusion In the present research, an attempt was made to improve solubility and permeability of APT. For this purpose, the formulation were prepared as per simplex lattice mixture design, showing quadratic correlation of Capmul MCM C10, Tween 80 and Transcutol with droplet size, PDI and self-emulsification time. The main outcome of QbD based procedure was advantageous in achieving three best numerically optimized formulations containing Capmul MCM C10 (3050%), Tween 80 (30-55%) and Transcutol (14.5-18.5%). These three numerically optimized formulations (SP1, SP2 and SP3) containing APT in solubilized form, provide <1 min selfemulsification time and 150-180 nm particle size with low particle size distribution. Further, the developed S-PCM formulations were thermodynamically stabile with spherical shape morphology (SEM/TEM analysis). Furthermore, 2.34 fold, 2.12 fold and 1.78 fold enhancement in Papp of APT was observed when incorporated in SP 1, SP2 and SP3, respectively. The pharmacokinetic study also revealed 1.5 times and 2 times increase in oral bioavailability of APT as compared to APT (micronized) and APT (non-micronized), respectively. Overall, the developed S-PCM formulation (SP1) with increased solubility and permeability of APT was found to be a better alternative to micronized APT. Acknowledgements The authors would like to acknowledge the financial assistance provided by CSIR, New Delhi (Project no. 02/(0064)/12/EMR-II). The authors would also like to thanks IIT Ropar for extending the facility of SEM and XRD. References [1]

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26

Legends to figures Fig. 1: Solubility study of Aprepitant in various oils, surfactant and co-surfactant Fig. 2: Ternary phase diagrams of Capmul MCM C10, Tween 80 and Transcutol showing a) self emulsification region b) microemulsion compositions with droplet size equal to and less than 200 nm Fig. 3: Response surface and corresponding contour plot of different APT loaded L-PCM formulations prepared as per simplex lattice mixture design with dependent variables: a) Droplet size, b) PDI and c) self emulsification time Fig. 4: Software generated contour plots of APT loaded L-PCM as a function of a) Desirability function and b) Graphical optimization Fig. 5: SEM images of a) Aerosil 200, B) SP1 Fig. 6: FTIR-ATR spectra of different APT compositions Fig. 7: XpRD diffractogram of a) APT non-micronized, b) APT micronized, c) Aerosil 200, d) SP1, e) SP2, f) SP3 Fig. 8: Thermal attributes of a) APT non-miconized, b) APT micronized, c) Aerosil 200, d) APT and Aerosil 200 physical mixture, e) Aerosil-200 S-PCM (without APT), APT loaded S-PCM f) SP1, g) SP2 and h) SP3 Fig. 9: TEM images of SP1 formulation at magnification of 100,000X after reconstitution Fig. 10: In vitro dissolution performance of optimized L-PCM and S-PCM formulations Fig. 11: The pharmacokinetic profile of SP1 formulation, APT micronized and APT nonmicronized after oral administration (1mg/kg APT) to New Zealand strain Rabbits

27

Table 1: Simplex lattice mixture design for the preparation and evaluation of L-PCM of APT Simplex lattice mixture design using Cubic model Independent variables Response Formulation no. Point Type Oil surfactant Co-surfactant Droplet Size Emulsification PDI (µl) (µl) (µl) (nm) time (s) 1. Axial CB 500 350 150 171±8.3 0.2467±0.0081 39±4 2. 3.

Third Edge Vertex

400 600

500 300

100 100

4.

Center

400

400

200

5.

Vertex

300

600

100

6. 7.

Third Edge Vertex

300 300

500 300

200 400

8.

Third Edge

300

400

300

9.

Axial CB

350

500

150

10. 11.

Third Edge Third Edge

500 500

400 300

100 200

12.

Axial CB

350

350

300

13.

Third Edge

400

300

300

170±9.7 270±12.3 156±10.9 160±6.4 181±14.1 155±6.3 151±8.7 155±3.9 188±6.2 170±4.8 160±9.5 193±10.4 Cubic 441.45 101.92 0.9982 0.9960 0.9686 70.18 692.27 1.31

0.3071±0.0102 0.3645±0.0067 0.3213±0.0052 0.2421±0.0034 0.5642±0.0294 0.2848±0.0091 0.4628±0.0262 0.2132±0.0057 0.2054±0.0039 0.1932±0.0024 0.4774±0.0173 0.5072±0.0125 Cubic 254.75 538.56 0.9970 0.9930 0.9377 42.327 1.52 3.14

Model Model F value Lack of fit F value Statistical analysis of R2 various responses i.e. Adjusted R2 particle size, PDI and Predicted R2 emulsification time Adequate precision PRESS % C.V Numerical optimization Formulation Droplet Size Desirability Oil Surfactant Co-surfactant no. (nm) Criteria used for response numerical optimization minimized 300 555 145 L1 0.991 151 457 390 153 L2 0.924 160 515 300 185 L3 0.847 182

Data expressed as mean±SD (n=3)

28

55±5 72±8 32±7 65±9 26±5 42±6 28±5 41±5 55±7 36±8 25±4 27±5 Cubic 510.04 76.24 0.9985 0.9965 0.9606 59.43 173.24 2.26

minimized

Emulsification time (s) in range

0.199 0.222 0.216

51 37 39

PDI

Table 2: Composition, drug loading and yield of various optimized PCM formulations

Formulation

Capmul MCM C8 (µl)

Tween 80 (µl)

300 555 L1 457 390 L2 515 300 L3 300 555 SP1 457 390 SP2 515 300 SP3 Data expressed as mean±SD (n=3)

Transcutol (µl)

Aprepitant (mg/ml)

Aerosil 200 (g)

Drug content (%)

Yield (%)

145 153 185 145 153 185

80 80 80 80 80 80

1 1 1

98±1 95±2 96±2 85±4 82±4 80±3

77±3 75±4 75±3

29

Table 3: Characterization of optimized L-PCM and S-PCM formulations after reconstitution Formulation

Droplet size (nm)

PDI

L1 L2 L3 SP1 SP2 SP3

143±4.9a 154±5.4b 175±7.6c 148±7.1a 161±8.3b 181±8.9c

0.201±0.026 0.235±0.018 0.226±0.035 0.211±0.019 0.229±0.027 0.238±0.031

a, b, c, d

Zeta potential (mV) -18.8±0.21a -20.6±0.83b -23.6±0.56c -13.8±0.54d -16.4±0.32e -15.3±0.63e

Emulsification time (sec) 56±5a 42±8b 44±7b 64±9c 53±6a 55±7ac

Sb/Sa 1.05 1.01 1.04 1.03 1.04 1.03

Means within the same column, labeled with the same letter, do not statistically differ from each other (p > 0.05).

30

Table 4: Thermodynamic stability of optimized L-PCM and S-PCM formulations Formulatio n code

L1 L2 L3 SP1 SP2 SP3

Physical appearance without reconstitution Clear, no drug precipitation Clear, no drug precipitation Clear, no drug precipitation Free flowing powder Free flowing powder Free flowing powder

Centrifugation Phase Precipitatio separatio n n

Freeze thaw cycle 4°C

40°C

Cloud point (°C)

×

×

stable

stable

73±2

×

×

stable

stable

69±2

×

×

stable

stable

65±1

×

×

stable

stable

71±2

×

×

stable

stable

68±2

×

×

stable

stable

63±2

31

Table 5: Ex vivo permeation studies of various optimized L-PCM and S-PCM formulations Parameters

L₁

L₂

L₃

SP₁

SP₂

SP₃

Flux (×10-1) (µg/sec/cm²) Relative Permeability (µg*ml/cm²) Apparent Permeability (×10⁶) (cm/sec)

4.09±0. 14a 4.05±0. 19a 5.12±0. 2a

3.69±0. 09b 3.53±0. 2b 4.62±0. 12 b

3.21±0. 11b 2.33±0. 05b 4.01±0. 12 c

3.89±0. 12a 3.83±0. 17a 4.82±0. 13ab

3.37±0. 17bc 3.23±0. 12ab 4.22±0. 11c

2.82±0. 11c 2.15±0. 19c 3.52±0. 20c

APT micronized

APT nonmicronized

1.54±0.16d

0.64±0.14e

2.04±0.12c

1.21±0.16d

1.93±0.09d

0.796±0.21e

(a,b,c,d,e means labeled with the same letter, within group do not statistically differ from each other ; p > 0.05, n=3)

32

Table 6: Pharmacokinetic parameters of S-PCM (SP1) and APT suspension (micronized and non-micronized) after 1mg/kg single oral dose administration to New Zealand strain rabbits

Parameters

APT nonmicronized suspension

APT micronized suspension

S-PCM (SP1) formulation

Ka (hr-1)*

0.1504±0.003a

0.1642±0.005a

0.1881±0.007b

K (hr-1)

0.0612±0.001

0.0632±0.003

0.0592±0.002

AUC0-24h (ng/ml/h)

4094±314.5a

5287±451.2b

8080±421.7c

T1/2 (hr)

11.32±0.5a

10.46±0.7a

9.31±0.4b

Cmax (ng/ml)

308±35a

451±28b

601±48c

Tmax (h)

4.15±0.6a

4.10±0.7a

2.03±0.3b

% Fr

100

130.3

197.36

(n=3) Cmax, peak concentration; Tmax, time to peak concentration; AUC0-24h, area under the concentration-time curve from time 0 to 24 hours; Fr, relative oral bioavailability; a, b, c, Means within the same row, labeled with the same letter, do not statistically differ from each other (p > 0.05).

33

Transcutol Plurol oleique Tween 80 Lauroglycol 90 Soybean oil Captex 200P Labrafil lipophile

Captex 355 Labrafil M 212CS Labrafil M 1944CS Capmul MCM C10 0

20

40

60 Solubility (mg/ml)

Fig. 1

34

80

100

120

Fig. 2

Fig. 3 35

Fig. 4

Fig. 5

36

Fig. 6 37

Fig. 7 38

Fig. 8

39

Fig. 9

40

120

% Cumulative drug release

100

80

60 L₁ L₂

40

L₃ SP₁ SP₂

20

SP₃ APT micro APT non-micronized

0 0

10

20

30 Time (minutes)

Fig. 10

41

40

50

60

Plasma concentration of APT (ng/mL)

700 600 APT non-micronized

500

APT micronized

400

SP₁

300 200 100 0 0

5

10

15

20 Time (h)

Fig. 11

42

25

30

35

Graphical Abstract)