Solid self-nanoemulsifying drug delivery system (S-SNEDDS) of darunavir for improved dissolution and oral bioavailability: In vitro and in vivo evaluation

Accepted Manuscript Solid self-nanoemulsifying drug delivery system (S-SNEDDS) of darunavir for improved dissolution and oral bioavailability: in vitro and in vivo evaluation Spandana Inugala, Basanth Babu Eedara, Sharath Sunkavalli, Rajeshri Dhurke, Prabhakar Kandadi, Raju Jukanti, Suresh Bandari PII: DOI: Reference:

S0928-0987(15)00140-2 http://dx.doi.org/10.1016/j.ejps.2015.03.024 PHASCI 3226

To appear in:

European Journal of Pharmaceutical Sciences

Received Date: Revised Date: Accepted Date:

25 December 2014 30 March 2015 30 March 2015

Please cite this article as: Inugala, S., Eedara, B.B., Sunkavalli, S., Dhurke, R., Kandadi, P., Jukanti, R., Bandari, S., Solid self-nanoemulsifying drug delivery system (S-SNEDDS) of darunavir for improved dissolution and oral bioavailability: in vitro and in vivo evaluation, European Journal of Pharmaceutical Sciences (2015), doi: http:// dx.doi.org/10.1016/j.ejps.2015.03.024

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Solid self-nanoemulsifying drug delivery system (S-SNEDDS) of darunavir for improved dissolution and oral bioavailability: in vitro and in vivo evaluation Spandana Inugala, Basanth Babu Eedara, Sharath Sunkavalli, Rajeshri Dhurke, Prabhakar Kandadi, Raju Jukanti, Suresh Bandari* Department of Pharmaceutics, St. Peter’s Institute of Pharmaceutical Sciences, Vidyanagar, Hanamkonda, Warangal-506001, Andhra Pradesh, India.

Running Title: S-SNEDDS of darunavir for improved oral bioavailability.

*Corresponding author. Dr. Suresh Bandari, Principal, Professor, Department of Pharmaceutics, St. Peter’s Institute of Pharmaceutical Sciences, Hanamkonda, Warangal 506001, Andhra Pradesh, India. Tel: +91 8702567303; Fax: +91 8702567304; Email: [email protected]

ABSTRACT The current study was aimed to investigate the potential of solid self-nanoemulsifying drug delivery system (S-SNEDDS) composed of capmul MCM C8 (oil), tween 80 (surfactant) and transcutol P (co-surfactant) in improving the dissolution and oral bioavailability of darunavir. Liquid self-nanoemulsifying drug delivery systems (L-SNEDDS) were developed by using rational blends of components with good solubilizing ability for darunavir which were selected based on solubility studies, further ternary phase diagram was constructed to determine the self-emulsifying region. The prepared L-SNEDDS formulations were evaluated to determine the effect of composition on physicochemical parameters like rate of emulsification, clarity, phase separation, thermodynamic stability, cloud point temperature, globule size and zeta potential. In vitro drug release studies showed initial rapid release of about 13.3±1.4% within 30 min from L-SNEDDS followed by slow continuous release of entrapped drug and reached a maximum of 62.6±3.5% release at the end of 24h. The globule size analysis revealed the formation of nanoemulsion (144±2.3nm) from the optimized LSNEDDS formulation and was physically adsorbed onto neusilin US2. In vitro dissolution studies indicated faster dissolution of darunavir from the developed S-SNEDDS with 3 times greater mean dissolution rate (MDR) compared to pure darunavir. Solid state studies concluded the presence of drug in non-crystalline amorphous state without any significant interaction of drug with the components of S-SNEDDS. Furthermore, in vivo pharmacokinetic studies in Wistar rats resulted in enhanced values of peak drug concentration (Cmax) for L-SNEDDS ((2.98±0.19 µg/mL) and S-SNEDDS (3.7±0.28µg/mL) compared pure darunavir (1.57±0.17 µg/mL). Keywords: Darunavir; Solid self-nanoemulsifying drug delivery system; Dissolution; Globule size; Bioavailability.

1. Introduction Darunavir (TMC114) is a second generation synthetic peptidomimetic protease inhibitor (PI), designed for the treatment of human immunodeficiency virus (HIV-1) infection with resistance to other available protease inhibitors (Mitsuya et al., 2008). In contrast to other PIs, darunavir acts both by blocking cleavage of the natural peptide substrate and inhibiting dimerization of the HIV-1 protease enzyme (Koh et al., 2003). It has potent activity against wild-type HIV-1 and several multidrug resistant strains with a high genetic barrier to resistance (Koh et al., 2003; De Meyer et al., 2005). It develops resistance only at very low drug concentrations (< 200 nM) after a minimum of 3 to 4 mutations (De Meyer et al., 2005; Menendez-Arias, 2013). However oral delivery of darunavir is suffering with low oral bioavailability (37%) because of high hydrophobicity (log p=3.9) which is the main cause for the low water solubility (Van Gyseghem et al., 2009), extensive first pass metabolism by intestinal and hepatic cytochrome P450 (CYP) 3A4, P-glycoprotein (P-gp) efflux. Hence it is prescribed in combination with ritonavir, an older PI, which boosts the oral bioavailability of darunavir from 37 to 82% by inhibition of cytochrome P450 enzymes and P-glycoprotein (Youle, 2007). Lipid based formulations were chosen to overcome the above barriers and among them selfnanoemulsifying drug delivery systems (SNEDDS) have recently exhibited an intriguing role in oral delivery of highly lipophilic drugs due to ease of production, practical enhancement of drug solubility and oral bioavailability (Elsheikh et al., 2012). SNEDDS are preconcentrates composed of isotropic mixtures of oils, surfactants, co-surfactants which spontaneously form a fine oil in water (o/w) emulsion in situ upon contact with aqueous medium with a globule size in the range of 20–200 nm (Porter et al., 2008). Various other potential features of SNEDDS in enhancing oral bioavailability of lipophilic drugs consists of facilitating transcellular and paracellular absorption, reducing cytochrome-P450 metabolism in the gut

enterocytes, promoting lymphatic transport via peyer’s patches protects drug from hepatic first pass metabolism (Porter and Charman, 2001; Balakrishnan et al., 2009; Date et al., 2010; Balakumara et al., 2013). The major drawbacks of L-SNEDDS such as chemical instability, precipitation of drugs at storage temperature due to incompatibility of the volatile components of the formulation with gelatin capsule shell, leakage, portability, high production cost (Wilson and Mahony, 1997; Tuleu et al., 2004; Franceschinis et al., 2005) were overcome by adsorbing them on to highly porous carriers like neusilin US2, without affecting self-emulsifying properties (Beg et al., 2013). Extensive review of literature reveals lack of information about the bioavailability enhancement of poorly water soluble darunavir using self-nanoemulsifying drug delivery systems. Thus the current study was aimed to develop a solid self-nanoemulsifying drug delivery system (S-SNEDDS) for darunavir as a potential drug delivery system to enhance its oral bioavailability. To achieve this, darunavir solubility was tested in various vehicles and vehicles with highest solubility for darunavir were selected as components (oil, surfactant and co-surfactant) of L-SNEDDS. The developed darunavir loaded L-SNEDDS formulations were evaluated for self-emulsification time, robustness to dilution, thermodynamic stability, globule size of the emulsion and in vitro drug release. Optimized L-SNEDDS was adsorbed onto the nanostructured solid inert carrier such as neusilin US2 and evaluated for in vitro drug dissolution behaviour and oral bioavailability in Wistar rats. 2. Materials and methods 2.1. Materials Darunavir was supplied by Hetero Drugs Limited (Hyderabad, India). Among the vehicles, polyglycolyzed glycerides such as Capryol 90 (Propylene glycol monocaprylate), Labrafac CC (medium chain triglycerides), Labrafil M 1944 CS (Oleoyl macrogol-8 glycerides),

Labrafil M 2125 CS (Linoleoyl macrogolglycerides), Labrasol (Caprylocaproyl macrogol-8 glycerides EP), Lauroglycol FCC (Propylene glycol monolaurate-type-I EP) and Transcutol P (Diethylene glycol monoethyl) were obtained as gift samples from Gatteffose (Saint-Priest Cedex, France). Acconon-E (Polyoxypropylene 15 stearyl ether), Capmul MCM C8 (Glyceryl monocaprylate), Capmul MCM L8 (Glyceryl Mono-dicaprylate1,2,3-propanetriol decanoic acid monoester), Capmul PG8NF (Propylene glycol monocaprylate), Caproyl microexpress (a mixture of PEG-6 caprylic/capric triglyceride, glyceryl caprylate/caprate, polyglycerol-6 dioleate,), Captex 200 (Propylene glycol dicaprylocaprate), Captex 355 (Capric triglyceride), and Captex 8000 (Glyceryl tricaprylate) were provided by ABITEC Corporations (Cleveland, USA). Cremophore EL was procured as a generous gift sample from BASF Corp. (Ludwigshafen, Germany). Tween 80 was purchased from Merck (Mumbai, India). Neusilin US2 (Magnesium aluminometasilicate) was obtained as gift from Fuji Chemical Industry CO., Ltd. (Toyama, Japan). Dialysis membrane (DM-70; MWCO 10000) was purchased from Hi-media (Mumbai, India). All other chemicals used in this study and solvents were of analytical or HPLC grade respectively. Freshly collected double distilled water was used throughout the study. 2.2. HPLC analysis HPLC analysis of darunavir was determined using a reverse-phase isocratic Shimadzu HPLC system (Shimadzu, Kyoto, Japan) equipped with SPD-10 AVP UV/Vis detector (sensitivity of 0.005 absorbance units full scale, AUFS) and LC-10 AT solvent delivery unit. Chromatographic separation was accomplished by a Phenomenex® Luna C18 column (5µm, 4.6×250mm; Phenomenex, Torrence, California) maintained at 25oC. A mobile phase composed of 60% HPLC grade acetonitrile and 40% double distilled water was delivered at a flow rate of 1mL/min for the analysis of in vitro study samples. Samples of 20µL were injected through Rheodyne injector and analysed at a wavelength of 266 nm.

2.3. Solubility studies The saturation solubility of darunavir in various vehicles (oils, surfactants, co-surfactants) was assessed using shake flask method (Wang et al., 2009). Briefly an excess amount of drug was mixed with 1 gm of chosen vehicles (Acconon E, Capmul PG 8 NF, Capmul MCM L8, Capmul MCM C8, Captex 355, Captex 200, Captex 8000, Caproyl 90, Caproyl Microexpress, Cremophor EL, Labrafil M 1944 CS, Labrafil M 2125 CS, Labrasol, Lauroglycol, Labrafac CC, Transcutol-P and Tween 80) in 5 ml clean glass vials with vortexing to aid the proper mixing of darunavir with the vehicle. Then the stoppered vials were agitated for 48h at 37oC in a shaking water bath. After equilibration all the samples were centrifuged at 10000 rpm for 15 min to remove the un-dissolved darunavir from saturated solutions. Accurately measured quantities of supernatants were appropriately diluted with methanol and darunavir concentration was quantified by HPLC system. 2.4. Preparation of L-SNEDDS Based on the saturation solubility studies, vehicles with good solubilization capacity for darunavir were selected as components (oil, surfactant and co-surfactant) of the L-SNEDDS formulation. A series of L-SNEDDS (Table 1) were prepared by varying oil, surfactant and co-surfactant composition. Accurately weighed quantities of oil, surfactant and co-surfactant were vortex mixed in a glass vial for 30 s to get a clear homogenous mixture. To this mixture, 20 mg of darunavir was added in small increments with continuous vortex mixing to form a monophasic system. Then L-SNEDDS were stored in screw capped clean glass vials at room temperature until further evaluation. 2.5. Construction of ternary phase diagram Various ratios of selected oil, surfactant and co-surfactant were plotted on a ternary-phase diagram to establish the stable spontaneous self-emulsification zone. A visual test reported by Craig et al., 1995 with minor adaptation was conducted to assess the self-emulsification

properties of prepared L-SNEDDS and ternary phase diagram was constructed using Tri plot v1-4 software (David Graham and Nicholas Midgley, Loughborough, Leicestershire, UK) based on the tendency to form emulsion, clarity, phase separation, coalescence of droplets and drug precipitation. In brief, L-SNEDDS (200 μL) was dropped in small quantities into distilled water (37°C; 300 mL) in a glass beaker with continuous mixing on a magnetic stirrer (100 rpm). Then the stability of formed emulsions was determined by visual observations such as extemporary emulsification, phase separation, drug precipitation, cracking of the emulsion on storage (48h) at room temperature. Poor or no emulsion formation with immediate coalescence of droplets with phase separation and drug precipitaion indicates formation of unstable emulsion. Further L-SNEDDS which formed stable clear emulsions were subjected to increasing dilutions (10, 100 and 1000) using distilled water and 0.1 N hydrochloric acid as mediums to evaluate the effect of dilution on stability of formed emulsions, which mimics in vivo gastric condition. 2.6. Thermodynamic stability studies and cloud point measurement Stability of the prepared L-SNEDDS formulations at various stress conditions was evaluated by heating cooling cycles (4°C and 40°C) and freeze thaw cycles (-21°C and +25 °C) with storage at specified temperature for 48h. For centrifugation stress, the L-SNEDDS formulations were diluted with distilled water (1:100) and centrifuged at 3500 rpm for 15 min and visually observed for any phase separation (Kallakunta et al., 2012). The cloud point temperature of the diluted L-SNEDDS formulation (10 mL) was determined by gradual heating on a water bath and the temperature at which cloudiness appears was denoted using thermometer (Zhang et al., 2008). 2.7. Determination of globule size and zeta potential Mean globule size, polydispersibility index (PDI) and zeta potential of the emulsion formed from stable L-SNEDDS formulations (100 µL) on dilution with double distilled water (100

mL) were determined by photon correlation spectroscopy (PCS) using a Zetasizer (Nano ZS90; Malvern instruments Ltd., UK) with a 50 mV laser at a fixed angle of 90o at room temperature. The measurement time was 2 min and each run underwent 12 sub-runs. All the data obtained was the average of three determinations. 2.8. In vitro drug release studies In vitro drug release behaviour of drug from pure darunavir and L-SNEDDS dispersion was determined using modified dialysis method (Kang et al., 2004). L-SNEDDS upon oral administration, forms o/w emulsion on contact with gastro intestinal aqueous medium. At this stage, the drug is present in different complex states such as molecular state, entrapped in emulsion globules and micellar solution. For this purpose dialysis bag method was followed. Dialysis bag with molecular weight cut off of 10000 was used to circumscribe escape of emulsion into release medium and separate free drug from drug entrapped in emulsion globules and/or micelles. This helps in determination of real drug release pattern from LSNEDDS. Prior to experimentation, dialysis bag tubings (MWCO 10000, Hi-media Mumbai, India) were soaked in freshly prepared release medium for 12h at room temperature. L-SNEDDS formulation containing 20 mg of darunavir was diluted to 10 times with release medium and was placed inside the tubular regenerated cellulose dialysis bag. Both the ends of dialysis bags were sealed tightly without any leaks and were immersed in 100 mL release medium in a shaking water bath (100 rpm) maintained at 37oC. Samples (1 ml) were withdrawn at predetermined time intervals with fresh medium replacements, filtered, diluted with release medium and were analyzed using HPLC. 2.9. Preparation of S-SNEDDS formulations Optimized L-SNEDDS formulation was transformed into solid self-nanoemulsifying drug delivery system (S-SNEDDS) using solid inert carrier such as neusilin US2, which has high

surface and good adsorption capacity by physical adsorption method. In a glass mortar, neusilin US2 was admixed in small quantities with the optimized L-SNEDDS formulation (1 g) to get a non-sticky solid powder (Ramasahayam et al., 2014). Prepared S-SNEDDS were then passed through the sieve no. 60 (size 250 µm) and stored in a desiccator at room temperature until further evaluation. 2.10. Determination of liquid load factor and micromeritics Liquid adsorption capacity of neusilin US2 used in the preparation of S-SNEDDS was evaluated by computing the liquid load factor which is the ratio of the L-SNEDDS to the inert carrier (%w/w) used as adsorbent (Elkordy et al., 2012). The flowability of the developed SSNEDDS was determined using Carr’s compressibility index or Carr’s index (CI %) and Hausener’s ratio. Carr’s index (CI %) was calculated using tapped density (Pt) and poured bulk density (Pb) of S-SNEDDS formulation (Carr, 1965). 2.11. In vitro dissolution studies The relative in vitro dissolution behaviour of darunavir from pure darunavir and S-SNEDDS in 0.1N hydrochloric acid (500 mL; pH 1.2; 37±0.5°C) was assessed using USP type II apparatus (paddle type; Electrolab, TD L8, Mumbai, India) at a paddle rotation speed of 50 rpm. At predetermined time points, an aliquot of 5 mL was withdrawn with equal volumes of fresh dissolution medium replacements to maintain the medium volume constant. All the samples were filtered, diluted and the concentration of darunavir dissolved was assayed by HPLC. From the dissolution profiles, various dissolution parameters like dissolution efficiency (DE), mean dissolution time (MDT) and mean dissolution rate (MDR) were calculated out (Khan, 1975; Eedara et al., 2013). 2.12. Solid state characterization 2.12.1. Fourier transform infrared spectroscopy (FTIR)

FTIR spectra of pure darunavir, neusilin US2, and the S-SNEDDS formulation were recorded by KBr disc method using Perkin Elmer FT-IR Spectrometer (Paragon 1000, PerkinElmer, Waltham, Massachusetts, USA) to illustrate the promising interactions among the components used in the formulation. Powder sample (4 mg) and IR grade dry potassium bromide (KBr; 200 mg) were mixed gently in a glass mortar, compacted to form disk by applying a force of 5.5 metric tons using hydraulic press. The corresponding discs were scanned over the wave number range of 4000–400 cm-1 at a scanning speed of 4 scans /s with a resolution of 1 cm-1 for each spectrum. 2.12.2. Differential scanning calorimetry (DSC) DSC thermograms of pure darunavir, neusilin US2, and the S-SNEDDS formulation were carried out to determine the physical state of darunavir in the developed S-SNEDDS using differential scanning calorimetry (Mettler DSC 823e, Mettler-Toledo, Germany), calibrated with indium (calibration standard, purity > 99.9%). Accurately weighed sample (4 mg) was placed in a flat bottomed standard aluminium pan and scanned at a scanning speed of 10°C/min from 20○C to 300○C under nitrogen gas flow of 80 mL/min. 2.12.3. Powder X-ray diffraction studies (PXRD) Powder X-ray diffraction patterns of pure darunavir, neusilin US2, and the S-SNEDDS formulation were recorded at ambient conditions using X-ray diffractometer (X’Pert PRO Panalytical, Eindhoven, Netherlands) equipped with real time multistrip X’Celerator detector, 2θ compensating slit and Cu Kα source (1.54 Å) operating at a generator power of 40 kV and current 40 mA. Powder sample was loaded in the sample holder as a thin layer and scanned in a step scan mode over an angular range from 4-50° (2θ) at scan rate of 1 s/step). 2.12.4. Scanning electron microscopy (SEM) Scanning electron microscopic photographs of pure darunavir, neusilin US2, and the SSNEDDS formulation were captured using scanning electron microscope (SEM, S-410,

Hitachi Ltd., Tokyo, Japan). Each sample was mounted by light dusting on a sterilized metal stub with double sided adhesive carbon tape and gold coated in vacuum (3-5 nm/min; 100 s; 30 W; 4 Psi) to make them electrically conductive using ion sputter (E-1010, Hitachi Ltd., Tokyo, Japan). The SEM micrographs of samples were captured at an accelerating voltage of 15 kV. 2.13. Transmission electron microscopy Morphological evaluation of emulsion developed from selected S-SNEDDS formulation was examined using transmission electron microscopy (TEM; FEI TECNAI G2 200Kv D-2083, The Netherlands). Selected S-SNEDDS formulation (10 mg) was diluted with 10 mL of simulated gastric fluid (pH 1.2). For the negative-staining sample, a drop of emulsion was mounted on a copper grid coated with carbon film and excess sample was wiped-out immediately using a filter paper. After a while, a drop of phosphotungstic acid solution (2% w/v PTA) was dripped on the copper grid for about 60 seconds. Excess of solution was drained. Then the grid was dried in the air at room temperature before loading in the microscope. 2.14. In vivo oral bioavailability studies All animal experimental protocols were established and approved by the Institutional Animal Ethical Committee, St. Peter’s Institute of Pharmaceutical Sciences, Hanamkonda, Warangal, Andhra Pradesh, India. All animals were acclimatized to the standard laboratory environment with 25±2°C temperature, 55±5% relative humidity with free access to water and standard laboratory diet. In vivo oral bioavailability of darunavir from pure drug, L-SNEDDS, and S-SNEDDS was assessed in male albino Wistar rats (180-200 g). Prior to experimentation, all the animals were fasted overnight with free access to water and divided into three groups containing six animals in each group and were randomly administered with each sample. Pure drug, L-

SNEDDS, and S-SNEDDS were dispersed in 0.5% w/v of sodium carboxymethyl cellulose with gentle manual mixing. Control group animals were administered with pure darunavir dispersion and the test group animals were treated with L-SNEDDS, S-SNEDDS dispersion at a dose equivalent to 20 mg/kg body weight. At predetermined time points, blood samples (0.5 mL) were withdrawn from retro orbital plexus under mild ether anaesthesia into microcentrifuge tubes and allowed to clot. After coagulation of the blood samples, serum was separated by centrifugation at 10000 rpm for 10 min in a cooling-centrifuge (Remi equipments, India) and stored at -20°C until analysis using HPLC. All serum samples were equilibrated at room temperature before treatment and processed as follows. In brief, 200 µL of serum sample was treated with 100 µL of methanol, 100 µL of carbamazepine as internal standard (2.5µg/mL) and 1 mL of 0.5 M sodium carbonate and vortex mixed for 3 min. Darunavir was extracted from the above mixture using ethyl acetate (2 mL) with centrifugation at 4000 rpm and the separated organic layer was dried under vacuum. The residue was reconstituted in mobile phase (100 µL) and analysed chromatographically using 50mM phosphate buffer (pH 6.2): acetonitrile: methanol (39:39:22 v/v/v) as mobile phase. 2.14.1. Pharmacokinetic parameters The peak concentration (Cmax) and its time (Tmax) were obtained directly from serum concentration vs. time profile. Area under the curve (AUC0-t) was calculated by using trapezoidal rule method. AUCt-∞ was determined by dividing the serum concentration at last time point with elimination rate constant (K). Mean residence time (MRT) was calculated by dividing AUC0-t with AUMC0-t. Percentage relative bioavailability (%F) was estimated by dividing the AUC0-∞ of formulations with pure drug. 2.15. Statistical analysis

All the data expressed as mean±standard deviation (SD) was subjected to statistical analysis using Instat Graphpad prism software (version 4.00; GraphPad Software, San Diego California) by one way analysis of variance (ANOVA). The significance of difference between formulations was calculated by student–Newman-Keuls (compare all pairs). The differences were considered to be significant at P < 0.05. 3. Results and discussion 3.1. Solubility study In the present study, non-ionic surfactants which were reported (Subramanian et al., 2004) to be less toxic compared to ionic surfactants, greater compatibility with biological tissues, less affected by change in pH and ionic strength throughout the GI tract were selected as vehicles. The solubility of darunavir was determined in the screened vehicles to choose a suitable vehicle with maximum drug loading capacity and the results were shown in the Table 2. Apart from the drug solubility in the vehicles, mutual solubility of the selected vehicles is a crucial factor in the formation of stable L-SNEDDS formulation. Among the tested vehicles Capmul MCM C8 (>300 mg/ml), Transcutol P (>500 mg/ml) and Tween 80 (>500 mg/ml) showed the highest drug solubilization capacity for darunavir. Based on the solubility results, Capmul MCM C8 (Glyceryl monocaprylate [(68% MG, 27% DG, 3% TG; >95% C8, 3% C10)]) was selected as the oil phase, Tween 80 (a water-soluble surfactant; Polyethylene glycol sorbitan monooleate) as surfactant and Transcutol-P (Diethylene glycol monoethyl ether) as co-surfactant. Previous reports demonstrate that medium chain monoglycerides (polar lipids) like Capmul MCM C8 shows good solvent capacity for hydrophobic drugs and also promote water penetration and self dispersibility of lipid formulations upon hydration. Further, Capmul is likely to increase the interfacial fluidity of surfactant boundaries in the micelles because of the entrapment of Capmul in high HLB surfactant enhances the emulsification process upon

dilution with aqueous medium (Taha et al., 2004). The combination of surfactant and cosurfactant with high and low hydrophilic lipophilic balance (HLB) values results in the rapid formation of stable emulsion with fine emulsion globule size upon dispersion in water (Craig et al., 1995). Hence Tween 80 (HLB 15) and Transcutol-P (HLB 4) were chosen as surfactant mixture in this study. Tween 80 is a partial fatty acid ester of sorbitol and its anhydride contain 20 units of oxyethylene. The fatty acid composition is 70% oleic acid with several other fatty acids such as palmitic acid (Shah et al., 1994). Transcutol-P used as co-surfactant forms more stable interfacial film with surfactants. It also decreases the fluidity of hydrocarbon region of the interfacial film and modify the film curvature, which promotes drug loading into the LSNEDDS, self-dispersibility properties and possesses penetration enhancement effect (Cui et al., 2005; Ghosh and Murthy, 2006; Shen and Zhong, 2006; Hong et al., 2006). 3.2. Construction of pseudo ternary phase diagrams Based on the results of solubility studies, ternary phase diagram (Fig. 1) of the Capmul MCM C8 (oil), Tween 80 (surfactant) and Transcutol-P (co-surfactant) was constructed to evaluate the self-emulsifying properties of the compositions and to determine the concentration range of components for formation of a clear nanoemulsion. In ternary phase diagram, the concentration of components was expressed as percent weight/weight (%w/w). The enclosed area in the phase diagram represents the region of self-emulsification. All the L-SNEDDS compositions exhibited good spontaneity of emulsification with emulsification time less than 60sec. The coloured region in the enclosed area indicates the formation of clear translucent fine oil in water emulsion upon gentle agitation. However L-SNEDDS compositions F1, F2, F6, F7 and F12 upon dispersion produced milky emulsions without any signs of drug or excipient precipitation. A higher concentration of surfactant mixture (Tween 80/Transcutol-P; >75%) or lower concentration of oil (Capmul MCM C8; >25%) resulted in formation of clear

translucent emulsions with nanosized globules. This may be due to higher HLB value of Tween 80 (HLB 15) and solubilizing effect of Transcutol P. Right mixture of surfactants favourably adsorbed at interface and produces thermodynamically stable nanoemulsion by reducing the interfacial energy as well as providing a mechanical barrier to coalescence (Reiss, 1975). In addition, co-surfactants increase interfacial fluidity by penetrating into the surfactant film creating void space between surfactant molecules (Constantinides and Scalart, 1997). The translucent emulsions formed were visually evaluated for clarity and stability after 48h at room conditions. All tested emulsions remained clear transparent even at the end of 48h. LSNEDDS which produced stable clear transparent emulsions spontaneously were diluted with distilled water and 0.1 N HCl to 10, 100 and 1000 times. The resultant emulsions were also clear transparent without any phase separation and precipitation with both the media indicating stability of formed emulsions at various dilutions and pH conditions which mimics in vivo situation. 3.3. Thermodynamic stability studies and cloud point measurement Thermodynamic stability study was conducted to identify and avoid the metastable LSNEDDS formulation. The L-SNEDDS formulations which produced translucent emulsions upon dispersion in distilled water and their emulsions were tested for stability at different temperatures and centrifugal stress conditions. All the tested L-SNEDDS formulations passed the thermodynamic stability studies without any signs of phase separation and precipitation during alternative temperature cycles (4°C and 40°C), freeze thaw cycles (-21°C and +25 °C) and centrifugation at 3500 indicating good stability of formulations and their emulsions. The cloud point is an essential parameter in the selection of a stable L-SNEDDS particularly when composed with non-ionic surfactants (Itoh et al., 2002). The cloud point temperature (lower consolute temperature) indicates the temperature at which the transparent monophasic

system was transformed into cloudy biphasic system as dehydrated surfactant molecules associated together as precipitate, which can affect the formulation adversely (Chen et al., 2000; Warisnoicharoen et al., 2000). Hence, the cloud point for SNEDDS should be higher than body temperature (37°C), which will avoid phase separation occurring in the gastrointestinal tract. The cloud point temperature of the tested L-SNEDDS was found to be in the range of 87-95°C. Therefore, it would suggest that the developed formulation do not requires a precise storage temperature and it develops a stable emulsion upon administration at physiological temperature in vivo (Zhang et al., 2008). 3.4. Globule size analysis and zeta potential Globule size and size distribution of the emulsion are key parameters which influences in vivo stability of emulsion developed from L-SNEDDS after oral administration. Globule size of the emulsion also affects rate of drug release and absorption, as drug diffusion is faster from smaller globules with large surface area (Tarr and Yalkowsky, 1989). Globule sizes and poly dispersity index values of the L-SNEDDS formulations which produced translucent emulsions upon hydration with double distilled water are summarized in Table 1. Globule sizes were found to be in the range of 25-207 nm, which indicated that globules are in nanometric size range. In conventional self-emulsifying drug delivery systems, the amount of free energy required to form an emulsion is very low, thereby allowing the spontaneous formation of an interface between emulsion globules and the water (Balakrishnan et al., 2009). The variation in fatty acid carbon chain lengths of oil, surfactant and their degree of un-saturation plays a significant role in rapid self-emulsification with small globule size and stability of formed emulsion (Shah et al., 1994). Capmul MCM C8 (Glyceryl monocaprylate [(68% MG, 27% DG, 3% TG; >95% C8, 3% C10)]; HLB-3.5) consists of >95% of medium chain fatty acid such as caprylic acid (C8). Tween 80 (Polyethylene glycol sorbitan monooleate) is a highly water-soluble surfactant with greater surface activity because of the

presence of a carbon unsaturated bond in its tail portion. The presence of fatty acids with varying carbon chain length and combination of hydrophilic surfactant (Tween 80; HLB 15) and hydrophobic co-surfactant (Transcutol P; HLB 4) might be responsible for the formation of smaller globules with compact interfacial mixed film at oil water interface (Craig et al., 1995). Among the tested formulations, globule size of the emulsion developed from F5 formulation was 144±2.3 nm with significantly very low PDI value (0.14 ± 0.021), indicating the narrow size distribution of the globules in the developed emulsion. Hence, L-SNEDDS (F5) was selected as optimized formulation for further evaluation and development of SSNEDDS. Zeta potential values of the emulsions produced upon dilution with double distilled water were found to be in the range of −5 to -8.6 mV. Both the surfactant (Tween 80) and cosurfactant (Transcutol-P) used in this present study are non-ionic in nature and didn’t contribute any charge to emulsion globules (Lu et al., 2008, Cui et al., 2009). However, the small negative zeta potential values of L-SNEDDS could be due to the ionization of free fatty acids and glycols present in the oil and surfactants which improves stability by preventing globule coalescence. 3.5. In-vitro drug release studies In vitro drug release profile from pure drug dispersion and L-SNEDDS was observed using modified dialysis method. Fig. 2 shows the highest drug release form L-SNEDDS formulation compared to pure darunavir over 24h. The release of drug from drug dispersion was significantly lower and showed only 12.4± 1.9% drug release in 24h. Whereas LSNEDDS showed initial rapid release of about 13.3±1.4% within 30 min which might be owing to the release of free molecular form of the drug followed by slow continuous release of entrapped drug and reached a maximum of 62.6±3.5% release at the end of 24h. Such a pattern of drug release from L-SNEDDS by carrying entrapped drug in the form of fine

emulsion droplets to the site of absorption is advantageous in increasing bioavailability, by enhancing release of poorly water soluble drug (Nielsen et al., 2008). 3.6. Preparation of S-SNEDDS and its flow properties L-SNEDDS were transformed into solid SNEDDS using neusilin US2 as adsorbent carrier. Neusilin US2 showed adsorption capacity of 0.82 %w/w with 55.5% load of L-SNEDDS. Flow properties of S-SNEDDS were determined from the Carr’s index and Hausner’s ratios. S-SNEDDS prepared using neusilin US2 as adsorbent carrier has shown good flow behaviour with Carr’s index value of 18.6±0.1 and Hausner’s ratio 1.18±0.15, respectively. The SSNEDDS formulation containing neusilin US2 as it possess very small particles with large specific surface area leads to high oil adsorbing capacity. Furthermore silica derivatives are more frequently used to adsorb lipid formulations by forming a three dimensional network with hydrogen bonds between silanol groups (Takeuchi et al., 2005). Simultaneously high compression characteristic of neusilin US2 favours easy transformation into tablets and also thermo stable and does not vary its quality on storage. 3.7. In vitro dissolution studies The comparative dissolution profiles of pure darunavir and S-SNEDDS formulation as carried out in 0.1 N HCl (pH 1.2) as dissolution medium is presented in Fig. 3. The dissolution profile shows that the S-SNEDDS showed faster drug release (61.7±2.5% within 5 min) vis-à-vis pure drug with maximum drug release in 60 min as 35.6%, respectively. The S-SNEDDS (DE60=73.1±3.1%) showed better dissolution performance with higher mean dissolution rate (MDR=5.23±0.25) and lower mean dissolution time values (MDT=8.6±1.2) vis-à-vis pure drug (DE60= 28.3±3.9%; MDR=1.76±0.27; MDT=12.3±4.9) signifies rapid release of drug from the prepared S-SNEDDS. Rapid drug dissolution from the S-SNEDDS may be due to low surface free energy of the self-emulsifying systems which favours rapid emulsification by quick establishment of interface between dissolution medium and oil (Craig

et al., 1995). Further improved dissolution also credited by the greater surface area of the neusilin US2 with high porosity which allows quick entrance of release medium into the pores and rapid emulsification, small size of the globules, transformation of darunavir physical state from low water soluble crystalline form to a non-crystalline amorphous or disordered crystalline phase of a molecular dispersion in S-SNEDDS. 3.8. Solid state characterization 3.8.1. Fourier transform infrared spectroscopic studies To characterize possible interactions between the components of the S-SNEDDS, infrared spectra of pure darunavir, neusilin US2, S-SNEDDS were recorded and shown in Fig. 4. The pure darunvir (Fig 4A) exhibits characteristic absorption peaks at 3363, 2965, 1704, 1596, 1551, 1310, 1266, 1151, 769, 671 and 554 cm−1. Whereas neusilin US2 (Fig 4B) used as inert carrier showed broad absorption peaks at 3457, 1638, 1021, 680, and 449 cm−1. The characteristic absorption peaks of the darunavir at 2965, 1310, 1266, 671, cm−1 were retained with broad neusilin peaks in S-SNEDDS formulation [Fig 4C]. However the remaining drug peaks were disappeared may be due to the overlapping of drug peaks with broad neusilin peaks. The FTIR spectrum of S-SNEDDS with no new additional peaks signifies the presence of only physical interaction between the drug and other components. 3.8.2. Differential scanning calorimetric studies The thermotropic behavior and the physical state of the darunavir in S-SNEDDS were evaluated by performing DSC analysis. The DSC thermograms of darunavir, neusilin US2 and S-SNEDDS were recorded and shown in Fig. 5. Pure darunavir (Fig. 5A) showed an endothermic peak at 77.5°C corresponding to its melting point. The neusilin US2 (Fig 5B) used as a carrier exhibited a diffused peak at 226.94°C. The absence of conspicuous drug peak in S-SNEDDS (Fig 5C) formulation over the melting range of darunavir unravels the

transformation of the physical state of the drug (crystalline to amorphous) which was further confirmed from powder X-ray diffraction studies. 3.8.3. Powder X-ray diffraction studies The PXRD patterns of darunavir, neusilin US2 and S-SNEDDS were represented in Fig. 6. The pure darunavir (Fig 6A) showed several high intense characteristic diffraction peaks at different diffraction angles of 12.5, 9.5, 6.4, 5.3, 5.0, 4.3 and 4.1 Å signifying the crystalline state of the drug. Whereas the neusilin US2 (Fig 6B) showed absence of intrinsic peaks suggesting its amorphous nature. The absence of characteristic darunavir peaks in SSNEDDS (Fig 6C) formulation indicates the change in physical state to a non-crystalline amorphous or disordered crystalline phase of a molecular dispersion. 3.8.4. Scanning electron microscopy The surface morphology of the pure darunavir, neusilin US2 and S-SNEDDS were examined by SEM and the images were presented in Fig. 7. SEM image of pure darunavir (Fig. 7A) showed mixture of irregularly shaped crystals of various sizes. While neusilin US2 (Fig. 7B) existed as granular fine porous powder. The absence of typical crystalline structures of darunavir in S-SNEDDS (Fig. 7C) indicates the transformation of drug to amorphous or molecular state. Further, the granular and porous structure of neusilin US2 was illegible in SSNEDDS because of the deposition of liquid formulation (L-SNEDDS) on the surface of neusilin US2. 3.9. Transmission Electron Microscopy (TEM) Fig. 7D illustrates the transmission electron microscopic image of reconstituted S-SNEDDS in distilled water. From the Fig. 7D it is evident that all the emulsion globules were of spherical in shape. The globules were also found to be of uniform size with no signs of coalescence, signifying the physical stability of formed nano emulsion. 3.10. In vivo pharmacokinetic study

In vivo oral bioavailability studies were conducted in Wistar rats and comparative pharmacokinetic profiles of darunavir from pure darunavir, L-SNEDDS, and S-SNEDDS were shown in Fig. 8. The noncompartmental pharmacokinetic parameters were calculated (Table 3) to evaluate the absorption behaviour of darunavir from L-SNEDDS, S-SNEDDS. In comparison to pure darunavir, both L-SNEDDS and S-SNEDDS showed significantly higher serum drug profiles. The peak drug concentration (Cmax) of L-SNEDDS and S-SNEDDS (2.98±0.19 and 3.7±0.28µg/mL) was approximately 2 and 2.5 fold higher than pure drug (1.57±0.17 µg/mL) respectively. However the time to reach the peak drug concentration (Tmax) remained constant which clearly indicate that the transformation of emulsion from LSNEDDS and S-SNEDDS was spontaneous. Similarly, other parameters such as biological half-life (t1/2), mean residence time (MRT0−t) and area under curve (AUC0-∞) were also found to significantly higher from both L-SNEDDS and S-SNEDDS with respect to pure darunavir. However, L-SNEDDS showed a greater initial rate of absorption compared to pure darunavir and S-SNEDDS, which might be due to the presence of L-SNEDDS in the liquid solution state. Overall, these results corroborate the improved rate and extent of the drug absorption from developed S-SNEDDS. Improvement in oral bioavailability of darunavir from S-SNEDDS can be attributable to many factors which, either in combination or alone contribute for favoured magnitude of absorption, like presence of lipophilic drug in solution or in small emulsion globules eliminates the dissolution step and keeps drug in a dissolved state during transport to the unstirred water layer of the GI membrane, lymphatic transport through intestinal transcellular pathways. In addition, the vehicles used in the formulation modulate the P-gp efflux pumps and/or CYP450 enzymes function at intestine region and improve the absorption of drug. Earlier reports suggest that the majority of the lipid based systems comprising of long chain and medium fatty acids gain admittance to intestinal lymph and bypass the portal circulation,

whereas a larger portion of shorter chain lipids get absorbed into the systemic circulation (Caliph et al., 2000). Our results also envisage favoured absorption of darunavir from SSNEDDS formulation because of the medium chain triglycerides in capmul MCM C8 enhances lipoprotein synthesis and subsequent lymphatic absorption (Porter 2007). 4. Conclusions Liquid self-emulsifying drug delivery systems (L-SNEDDS) composed of Capmul MCM C8 (16.6%), Tween 80 (41.7%) and Transcutol-P (41.7%) was selected as optimized formulation as it has produced clear translucent nanoemulsion (144 nm) upon dispersion with water. The optimized L-SNEDDS formulation was physically adsorbed onto the neusilin US2. The significant increase in drug dissolution rate of darunavir from S-SNEDDS suggests that the developed S-SNEDDS could be promising system for the oral bioavailability enhancement of low water soluble drug. Solid state studies conclude the presence of non-crystalline amorphous drug in the S-SNEDDS. In conclusion, in vivo pharmacokinetic studies showed improved oral bioavailability which might be due to the collective mechanism of nano emulsion formation with greater surface area, improved drug dissolution and release, transcellular and paracellular absorption, P-gp modulation potential of excipients, reduced cytochrome-P450 metabolism in the gut enterocytes, lymphatic bypass via peyer’s patches protects drug from hepatic first pass metabolism. Acknowledgements The authors acknowledge the help of Hetero Drugs Limited (Hyderabad, India) for providing the generous gift sample of darunavir. The authors are also acknowledging the ABITEC Corporations (Cleveland, USA), BASF Corp. (Ludwigshafen, Germany), Gatteffose (SaintPriest Cedex, France), Fuji Chemical Industry CO., Ltd. (Toyama, Japan) for providing the gift samples. Authors are thankful to Prof. TP Radhakrishna, School of Chemistry, University

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Table 1. Composition of the darunavir loaded liquid self-nanoemulsifying drug delivery systems (L-SNEDDS; % w/w) and evaluation parameters. Liquid self-nanoemulsifying drug delivery systems (L-SNEDDS) Ratio F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

F12

F13

F14

F15

S/CS

1:1 1:1

1:1

1:1 1:1

3:1

3:1

3:1

3:1

3:1

5:1

5:1

5:1

5:1

5:1

O/Smix

1:1 1:2

1:3

1:4 1:5

1:1

1:2

1:3

1:4

1:5

1:1

1:2

1:3

1:4

1:5

O(%)

50

33.3 25

33.3 25

20

16.6 50

33.3 25

20

16.6

S(%)

25

33.3 37.5 40

41.7 37.5 50

CS(%)

25

33.3 37.5 40

41.7 12.5 16.7 18.7 20

20.8 8.3

11.1 12.5 13.3 13.9

M

T

Appearance M

T

20

T

16.6 50

56.3 60

62.6 41.7 55.6 62.5 66.6 69.5

M

M

T

T

T

M

T

T

T

T

Evaluation Parameters

F3

F4

F5

F8

F9

F10

F12

F13

F14

F15

Cloud point (oC)

90

94

95

90

89

87

94

92

93

94

Globule size (nm)

207

198

144

155

98

47

199

144

86

25

PDI

0.39 0.3

0.14 0.25 0.4

0.45 0.31 0.24 0.32 0.35

Zeta potential (mV) in water

-6.8

-8.4

-8.6

-12

-8.2

-7.6

-7.9

-5

-8.5

-7.3

Zeta potential (mV) in SGF

5.8

4.8

4.9

3.4

3.9

4.9

1.9

6.4

3.8

4.8

O: Oil, S: Surfactant, CS: Co-surfactant, Smix: Mixture of surfactant and co-surfactant; Mmilky; T- Translucent.

Table 2. Darunavir solubility in various vehicles (Mean±SD, n=3). Vehicle Acconon E

Solubility(mg/ml) Vehicle 1.95± 0.12

Solubility(mg/ml)

Cremophor EL

31.58±0.25

Capmul PG 8 NF

28.5±0.15

Labrafil M 1944 CS

4.26± 0.24

Capmul MCM L8

30.42±0.2

Labrafil M 2125 CS

6.31±0.16

Capmul MCM C8

>300

Labrasol

5.42± 0.38

Captex 355

1.42±0.26

Lauroglycol

6.42± 0.36

Captex 200

12.12±0.19

Labrafac CC

1.20± 0.37

Captex 8000

10.53±0.5

Transcutol-P

>500

Caproyl 90

9.01± 0.28

Tween 80

>500

Caproyl Microexpress

6.38± 0.28

Table 3. Pharmacokinetic parameters of darunavir (20 mg/kg) in Wistar rats following oral administration of pure darunavir, L-SNEDDS, and S-SNEDDS (mean±SD, n=6). Pharmacokinetic Parameters Pure darunavir L-SNEDDS

S-SNEDDS

Cmax (µg/mL)

1.57 ± 0.17

2.98 ± 0.19‡1 3.7 ± 0.28‡1,‡2

Tmax (h)

1.0

1.0

1.0

AUC 0-∞ (µg h mL-1)

11.3±3.54

22.9±2.1‡1

32.28±2.5‡1, ‡2

MRT (h)

6.81 ±0.84

7.7± 0.27*1

8.66± 0.21‡1, †2

T1/2 (h)

6.41 ± 1.33

8.4 ± 0.59†1

9.91 ± 0.5‡1,*2

%F

-

202.7

285

Cmax- peak drug concentration; Tmax- time to reach the peak concentration; AUC – area under the curve; MRT – mean residence time; T1/2 –half-life; %F-percentage relative bioavailability. 1, 2 indicates pure darunavir, L-SNEDDS respectively. *, †, ‡ indicates significant difference at p<0.05, p<0.01, and p<0.001 respectively.

FIGURE LEGENDS Fig. 1. Ternary phase diagram of liquid self-nanoemulsifying drug delivery systems (LSNEDDS) composed with Capmul MCM C8 (oil), Tween 80 (surfactant) and Transcutol P (co-surfactant). Fig. 2.

In vitro drug release profiles of pure darunavir and optimized L-SNEDDS formulation in 0.1 N hydrochloric acid (pH 1.2; mean±SD; n=3).

Fig. 3.

In vitro dissolution profile of pure darunavir and S-SNEDDS in 0.1 N hydrochloric acid (pH 1.2; mean ± SD; n=3).

Fig. 4.

Fourier transform infrared (FTIR) spectra of (A) pure darunavir, (B) neusilin US2, and (C) S-SNEDDS.

Fig. 5.

Differential scanning calorimetric thermograms (A) pure darunavir, (B) neusilin US2, and (C) S-SNEDDS..

Fig. 6.

Powder X-ray diffractrograms of (A) pure darunavir, (B) neusilin US2, and (C) S-SNEDDS.

Fig. 7.

Scanning electron microscopy (SEM) images of (A) pure darunavir, (B) neusilin US2, (C) S-SNEDDS, and (D) transmission electron microscopy (TEM) image of the reconstituted nanoemulsions from S-SNEDDS.

Fig. 8.

Pharmacokinetic profiles of darunavir in serum following oral administration of pure darunavir, L-SNEDDS, and S-SNEDDS (mean ± SD; n=6).

TABLE LEGENDS Table 1. Composition of the darunavir loaded liquid self-nanoemulsifying drug delivery systems (L-SNEDDS; % w/w) and evaluation parameters. Table 2. Darunavir solubility in various vehicles. All the data are presented as mean±SD; n=3. Table 3. Pharmacokinetic parameters of darunavir (20 mg/kg) in Wistar rats following oral administration of pure darunavir, L-SNEDDS and S-SNEDDS (mean±SD, n=6).

Graphical abstract