Development of docetaxel-loaded solid self-nanoemulsifying drug delivery system (SNEDDS) for enhanced chemotherapeutic effect

International Journal of Pharmaceutics 452 (2013) 412–420

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Development of docetaxel-loaded solid self-nanoemulsifying drug delivery system (SNEDDS) for enhanced chemotherapeutic effect Youn Gee Seo a,1 , Dae Hwan Kim a,1 , Thiruganesh Ramasamy a , Jeong Hwan Kim a , Nirmal Marasini a , Yu-Kyoung Oh b , Dong-Wuk Kim c , Jin Ki Kim c , Chul Soon Yong a , Jong Oh Kim a,∗∗ , Han-Gon Choi c,∗ a

College of Pharmacy, Yeungnam University, 214-1, Dae-Dong, Gyongsan 712-749, South Korea College of Pharmacy, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Ku, Seoul 151-742, South Korea c College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 426-791, South Korea b

a r t i c l e

i n f o

Article history: Received 23 March 2013 Received in revised form 25 April 2013 Accepted 16 May 2013 Available online 22 May 2013 Keywords: Anti-tumor efficacy Bioavailability Docetaxel Self-nanoemulsifying drug delivery systems Toxicity

a b s t r a c t The main purpose of this study was to investigate the potential of self-nano-emulsifying drug delivery system (SNEDDS) in improving the bioavailability of docetaxel (DCT) and its chemotherapeutic effect. The DCT-loaded SNEDDS was prepared by employing rational blends of capryol 90, labrasol, and transcutol HP using ternary phase diagram. The liquid nano-emulsion was spray-dried into solid SNEDDS (D-SNEDDS) using an inert porous carrier, colloidal silica. The optimized formulation was characterized in terms of physico-chemical and pharmacokinetic parameters. Furthermore, anti-tumor efficacy of D-SNEDDS was compared with commercial marketed product, Taxotere® . The various compositions of SNEDDS were screened and found optimal at a volume ratio of 10/75/15 for capryol 90, labrasol, and transcutol HP, respectively. We observed a high oral bioavailability of 17% DCT for D-SNEDDS than compared to only 2.6% for pure DCT solution. Notably, D-SNEDDS exhibited an augmented anti-tumor efficacy with a reduced toxicity profile when compared with intravenously administered Taxotere® , the commercialized formulation of DCT. Taken together, D-SNEDDS could be a potential candidate for an oral dosage form of DCT with enhanced antitumor activity and reduced toxicity. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Docetaxel (DCT), a second generation taxoid, is considered one of the most potent chemotherapeutic agents in the clinical setting (Zhao and Astruc, 2012). DCT is effective against a wide spectrum of cancers such as ovarian cancer, breast cancer, head/neck cancer, and small and non-small cell lung cancers (Sanna et al., 2011). Despite such promising efficacy, severe systemic toxicity including bone marrow suppression, hypersensitivity reactions, peripheral neuropathy, musculoskeletal disorders, and fluid retention become major obstacles for successful treatment (Hu et al., 2012). Recently, a commercial injectable product ‘Taxotere® ’, in which DCT was solubilized in a polysorbate 80/ethanol system was launched in the market; however, it was reported to cause several adverse effects due to both the drug itself and the solvent system (Jun et al., 2012).

∗ Corresponding author. Tel.: +82 31 400 5802; fax: +82 31 400 5958. ∗∗ Co-corresponding author. Tel.: +82 53 810 2813; fax: +82 53 810 4654. E-mail addresses: [email protected] (J.O. Kim), [email protected] (H.-G. Choi). 1 Both authors contributed equally to this work. 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.05.034

Similar adverse effects have been reported for another commercial injectable product named Duopafei® (Liu et al., 2011). To overcome such side effects, efforts have focused on the development of the oral route as a viable alternative over the systemic delivery of DCT (Choi et al., 1999; Yong et al., 2005). However, the major problem associated with oral delivery is its low aqueous solubility (<5 ␮g/mL) and poor bioavailability (∼5%) (Hu et al., 2012; Jun et al., 2012). The low solubility is due to its highly lipophilic nature and bulky polycyclic structure. The poor oral bioavailability is attributed to its high hepatic first pass metabolism and P-glycoprotein-mediated multidrug efflux transporters (Yin et al., 2009; Gao et al., 2008). Several formulation approaches including liquid self-nanoemulsifying drug delivery systems (SNEDDS) (Yan et al., 2010; Wang et al., 2009; Elsabahy et al., 2007; Gao et al., 2008), prodrug (Du et al., 2007; Esmaeili et al., 2009), microcapsules including liposomes (Straubinger and Balasubramanian, 2005), solid dispersions (Chen et al., 2008a), nanoparticles (Esmaeili et al., 2010), and pre-treatment with P-glycoprotein inhibitors (Malingré et al., 2001; Engels et al., 2006; Ben Reguiga et al., 2007) have been attempted to improve its in vivo and physicochemical properties. Among these approaches, lipid-based SNEDDS has attracted significant attention due to its promising ability to

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improve the oral bioavailability of lipophilic drugs (Balakrishnan et al., 2009a, 2009b; Cui et al., 2009; Marasini et al., 2012). SNEDDS can improve the bioavailability by circumventing the hepatic portal route, protecting against drug degradation in the harsh GI environment, facilitating lymphatic transport of drugs, decreasing cytochrome P450-induced metabolism in the liver, and inhibiting the P-glycoprotein mediated efflux (Beg et al., 2012; Singh et al., 2013). Nevertheless, limited stability, low drug loading, and production issues often hinder its pharmaceutical application (Woo et al., 2008). Therefore, a solid-SNEDDS is highly sought-after due to its scalability and robustness, as well as its ability to avail all the benefits of a liquid system. Thus, this nanoparticulate system was anticipated to improve the bioavailability and therapeutic profile of DCT in the treatment of a broad spectrum of cancers. Toward this goal, a unique DCT-loaded solid-SNEDDS (DSNEDDS) was formulated to improve the antitumor properties of DCT. Herein, capryol 90 and polyglycolyzed glycerides were used to prepare the liquid SNEDDS. Silicon dioxide was employed as an inert solid carrier to prepare D-SNEDDS by a spray-drying method. The aim of this study was to investigate the potential of SNEDDS to improve the bioavailability of DCT as well as the anti-tumor efficacy/toxicity profile of the DCT-loaded nanoparticulate system. The D-SNEDDS was characterized in terms of physicochemical and pharmacokinetic parameters. In addition, the anti-tumor efficacy of the optimized formulation was compared with that of the commercially available product, Taxotere® . 2. Materials and methods 2.1. Materials Docetaxel was purchased from Taihua Co. (Xi’an, China). Polyglycolyzed glycerides (capryol 90, labrafac CC, labrasol, labrafil M 1944 CS, labrafil M 2125 CS, lauroglycol FCC and transcutol HP) were procured from Gattefosse (Saint-Priest Cedex, France). Polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), sorbitan monolaurate 20 (Span 20) and sorbitan monooleate 80 (Span 80) were purchased from DC Chemical Co. (Seoul, South Korea). Silicon dioxide was obtained from Degussa (Frankfurt, Germany). Taxotere® was purchased from Sanofi-Aventis Korea (Seoul, South Korea). All other chemicals were of reagent grade and were used as supplied. 2.2. Solubility studies The solubility of DCT in various vehicles was determined using shake flask method. An excess quantity of DCT powder (about 250 mg) was added to each vehicle (capryol 90, capryol PGMC, labrafac WL 1349, labrafil M 2125 CS, labrafil M 1944 CS, peceol, tween 20, span 80, span 20, transcutol HP, lauroglycol FCC, cremophore; 1 mL), vortexed and shaken in a water bath for 7 days at 25 ◦ C. The mixtures were centrifuged (Eppendorf, Hauppauge, NY, USA) at 3000 rpm for 15 min and the supernatant was filtered through a Millipore membrane filter (0.45 ␮m). The supernatant was suitably diluted with acetonitrile and the DCT concentration was quantified by the HPLC method described below.

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and co-surfactant varied between 70 and 95%. 300 ␮L of the formulation was introduced into 300 mL of water and gently stirred at 37 ◦ C for 2 h. The self-emulsification process in the water was visually inspected. Formation of milky white emulsion in the large quantity of water without any aggregation was considered good, while coalescence of oil droplets and breaking of nanoemulsion were considered bad. Phase diagrams were constructed using the ternary phase diagram software to identify the good or optimal self-emulsifying region. 2.4. Preparation of liquid SNEDDS formulations On the basis of the solubility study and phase diagram plot, capryol 90, labrasol, and transcutol HP were selected as oil, surfactant, and co-surfactant, respectively. The liquid SNEDDS was prepared as reported previously (Quan et al., 2013). Briefly, weighed quantity of DCT (100 mg) was dissolved in a mixture of 10% capryol 90, 75% labrasol and 15% transcutol HP. The resulting mixture was vortexed to obtain a homogenous solution. The prepared liquid SNEDDS was stored in sealed transparent glass bottles at room temperature until used. The formulations were recorded for any changes in turbidity or phase separation. The DCT concentration was maintained at 3.3% (w/w) throughout all the formulations. 2.5. Preparation of solid SNEDDS formulation A lab-scale mini-spray dryer, Büchi 190 nozzle-type (Flawil, Switzerland) was employed for the preparation of D-SNEDDS. Colloidal silicon dioxide was used as an inert carrier. 3 g of colloidal silica was dissolved in 500 mL of ethanol, followed by magnetic stirring: 3 mL of liquid SNEDDS (equivalent to 100 mg of DCT) was introduced to the above solution and stirred constantly at room temperature until a clear homogenous suspension was formed. The resulting suspension was spray-dried using a peristaltic pump (0.7 mm nozzle diameter) under the following operating conditions: feeding flow rate, 5 mL/min; inlet temperature, 62 ◦ C; outlet temperature, 32 ◦ C; air pressure, 4 kg/cm2 ; aspirator pressure, −25 mbar. The solid D-SNEDDS powder was collected and subsequently characterized. 2.6. Characterization of the solid SNEDDS 2.6.1. Solid state characterization Differential scanning calorimeter (DSC-Q200, TA Instruments, USA) was used to study the thermal behavior of DCT, colloidal silica, physical mixture (same as that of final formulation), and D-SNEDDS. The experiments were performed under a dynamic nitrogen atmosphere with a flow rate of 25 mL/min. DSC scans were recorded at a heating rate of 10 ◦ C/min from 50 to 200 ◦ C. The powder crystalline state properties of all the samples were analyzed using an XRD spectrometer (X’ Pert MPD diffractometer, PANalytical, Almelo, The Netherlands). The samples were analyzed using a copper anode operated at a voltage of 40 kV with a current of 30 mA radiation scattered in the crystalline regions of the sample. The patterns were obtained in the 2 angle range of 10–60◦ using a step width of 0.02 ◦ C at ambient temperature.

2.3. Pseudo-ternary phase diagram A ternary phase diagram was constructed to study the phase behavior of oil/surfactant/co-surfactant over the whole concentration range. The existence of self-emulsification region within this diagram was observed visually. The formulations were prepared using capryol 90 as oil phase, labrasol as surfactant, and Transcutol HP as a co-surfactant. The concentration of oil phase varied from 5 to 30%, while that of surfactant varied from 50 to 90%

2.6.2. Morphological analysis The morphologies of DCT, colloidal silica, and D-SNEDDS were examined using a scanning electron microscope (S-4100, Hitachi, Japan) with an image analysis system (Image Inside version 2.32). The samples were fixed/mounted on a brass stub using doublesided adhesive tape and vacuum coated with platinum (6 nm/min) for 120 s at 15 kV using the Hitachi Ion Sputter (E-1030, Tokyo, Japan).

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2.6.3. Dynamic light scattering measurements Hydrodynamic size and size distribution of both liquid and solid SNEDDS were determined at 25 ◦ C by dynamic light scattering (DLS) using Zetasizer Nano ZS (Malvern Instruments, U.K.) equipped with a He–Ne laser that operated at a wavelength of 635 nm. Measurements were taken at a fixed scattering angle of 90◦ . Software (version 6.34) that employs cumulants analysis was provided by the manufacturer and used to analyze the size, polydispersity index (PDI), and -surface charge. Each sample was analyzed in triplicate. 2.7. In vivo pharmacokinetic study 2.7.1. Animals The experimental protocols for the animal study were approved by the ethics committee of Seoul National University. Male Sprague-Dawley rats (approximately 250 ± 20 g body weight, Orient Bio. Inc., Seungnam, South Korea) were selected and fasted for 24 h before the commencement of experiments. 2.7.2. Administration and blood collection The rats were divided into three groups and fasted for 24 h prior to the experiments. The rats were anesthetized with diethylether and polyethylene tubing was inserted into the surgically bisected right femoral artery of each rat. The rats in first group (3 rats) were orally administered D-SNEDDS at a dose of 2 mL/kg (10 mg/kg as docetaxel). For comparison of bioavailability, the rats in the other groups received DCT solution orally at a dose of 10 mg/kg as DCT or intravenously at a dose of 5 mg/kg as DCT. Throughout the experiment, the DCT solution for oral and IV administration was prepared by diluting the commercial injectable product (Taxotere® ) with 13% ethanol in water prior to injection. Serial blood samples (0.3 mL) were collected from the right femoral artery at specified intervals. The blood samples were centrifuged at 3000 × g for 10 min, and the plasma was then separated. 2.7.3. Blood sample analysis 150 ␮L of plasma were mixed with acetonitrile (1.5 mL) and 50 ␮L of propyl paraben (5 ␮g/mL) were added as an internal standard. The mixture was centrifuged at 3000 × g for 10 min and the supernatant was separated and subjected to centrifugal evaporation (EYELA CVE-200D, Tokyo, Japan) at 40 ◦ C. The residue obtained after the evaporation was reconstituted with 100 ␮L of mobile phase and quantified by HPLC (Hitachi, Tokyo, Japan). The column was Intensil C8 (GL Science, 3.5 ␮m, 15 cm × 0.46 cm) with a UV/Vis detector (Model L-2420) set at a wavelength of 232 nm with a flow rate of 1.0 mL/min. Acetonitrile and phosphate buffer (pH 5) at a volume ratio of 49/51 was used as the mobile phase (Mu et al., 2010). The pharmacokinetic parameters were evaluated using WinNonlin software (CA, USA). The parameters included plasma concentration-time profile from area under the curve (AUC), halflife (t1/2 ), elimination rate constant (Kel ), absolute bioavailability (A), and volume of distribution (Vd ). 2.8. In vivo anti-tumor efficacy The antitumor effects of oral/IV DCT solution and D-SNEDDS were studied in 5-week-old female Cg-Foxnl-nu/CrljBgi nude mice (Orient Bio. Inc., Seungnam, South Korea). For tumor induction, 1 × 106 KB cells in 0.1 mL of PBS were subcutaneously injected into the right flank (Chang et al., 2011). The treatment of nude mice started when the tumor volume reached 100–150 mm3 and this day was designated as day 0. The mice were divided into four groups with five mice in each group. The first three groups respectively received phosphate-buffered saline solution (PBS, pH 7.4), DCT solution (10 mg/kg), and D-SNEDDS (10 mg/kg); the fourth group received DCT solution intravenously through the tail vein.

Fig. 1. Pseudo-ternary phase diagram depicting the nanoemulsion region.

The above formulations were administered on days 0, 3, and 6. The length and width of the tumor in each mouse were measured using calipers. The anti-tumor effect of the D-SNEDDS was compared to that of orally and intravenously administered DCT solution in the tumor-bearing mice by observing tumor volume reduction and measuring total body weight. Tumor volumes and body weights were normalized by the values at day 0 (before administration). The experimental protocols for the animal study were approved by the ethics committee of Seoul National University. 2.9. Statistical analysis All data were expressed as mean ± S.D. Data were statistically analyzed by ANOVA with Student–Newman–Keuls post hoc test. SigmaStat software (version 3.5, Systat Software, Richmond, CA, USA) was used for all analyses. A p-value of less than 0.05 was considered statistically significant. 3. Results 3.1. Formulation of liquid SNEDDS The DCT-loaded SNEDDS formulation was prepared to increase the solubility and bioavailability of drug via the oral route. Thus, each component used in the system should have high solubilizing capacity for the drug. In this context, we performed solubility studies to identify suitable oil, surfactant, and co-surfactant for the development of optimal D-SNEDDS. The study of these variables was important to achieve an optimum drug loading and emulsification efficiency. Although DCT is poorly soluble in water (Yin et al., 2009; Kintzel et al., 2006; Persohn et al., 2005), the drug showed good solubility in all the tested vehicles (Quan et al., 2013). Among the various oil phases screened, the commercially modified oil capryol 90 exhibited the highest solubilization capacity (142.43 ± 5.63 mg/mL) for the drug. Similarly, transcutol HP (255.84 ± 6.39 mg/mL) and labrasol (73.37 ± 4.83 mg/mL) showed the highest solubility among surfactants and co-surfactants, respectively. Therefore, aforementioned oil, surfactant and cosurfactant were selected for the development of the DCT-loaded SNEDDS formulation. 3.2. Ternary phase diagram A ternary phase diagram was constructed to determine the self nano-emulsifying region and to quantify the probable concentration of oil, surfactant and co-surfactant to develop a stable D-SNEDDS (Fig. 1). The phase diagram consists of oil, surfactant, and co-surfactant in each corner with 100% of each component.

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3.3. Effect of surfactant and co-surfactant on droplet size Droplet size plays a vital role in determining the rate and extent of drug release and bioavailability (Constantinides et al., 1994). For a stable and efficient SNEDDS formulation, the appropriate ratio of oil-surfactant-co-surfactant is greatly favored. Therefore, the system was optimized by observing the effect of various surfactant/oil and surfactant/oil/co-surfactant ratios on the droplet size of the emulsion. Fig. 2A clearly shows that an increase in surfactant concentration (from 60% to 75%, v/v) decreased the average size of the emulsion droplets. However, the trend reversed when the surfactant concentration rose beyond 75%, indicating an optimal concentration for the formulations. While keeping the surfactant concentration constant at 75%, the co-surfactant concentrations were increased from 5 to 20%. As can be seen from Fig. 2B, the average size of the emulsion droplets decreased up to two-fold with the increase in co-surfactant from 5 to 15%. However, further increase did not result in any size reduction. Furthermore, dilution factor had no effect on the droplet size or the self-nanoemulsifying ability of the emulsion system. Thus, capryol 90/labrasol/transcutol HP at a volume ratio of 10/75/15 was selected as the optimized liquid SNEDDS formulation for further experiments. 3.4. Formulation of solid SNEDDS Spray-drying was employed to prepare dry and solid DCTloaded SNEDDS with the aid of silicon dioxide as a carrier. Upon reconstitution in the aqueous environment, solid SNEDDS spontaneously produced fine droplets of oil-in-water (o/w) type nano-emulsions with a size of around 190 nm with acceptable PDI (0.250). Although the emulsion droplet size (after reconstitution) was smaller than that of liquid SNEDDS (215 nm), the difference was insignificant. These results suggest that the spray-drying of liquid SNEDDS solution does not have any effect on the droplet size. Furthermore, the solid SNEDDS preserved the self emulsifying property of liquid SNEDDS. This observation is consistent with our previous report in which solid SNEDDS prepared from silicon dioxide showed similar droplet size to liquid SNEDDS, while the hydrophilic dextran carrier significantly increased the size (Yi et al., 2008).

Emulsion droplet size (nm)

(A) 250 200

150

100

50 60/40 65/35 70/30 75/25 80/20 85/15 90/10 Ratio of surfactant/oil (v/v%)

(B) 350 Emulsion droplet size (nm)

The dark/shaded region depicts the nano-emulsion region and the wider part indicates the better nano-emulsifying region. A surfactant concentration of less than 50% resulted in turbid or crude emulsion with large particle size, suggesting that a minimum concentration is required to form a stable emulsion. Similarly, oil and co-surfactant were required to be 10–30% and 10–40%, respectively. In the present study, we selected two different surfactants with a blend of high and low HLB values, which is a prerequisite for the formation of stable nano-emulsion (Pouton, 2000; Villar et al., 2012). The thermodynamic stability of the nano-emulsion was improved by the preferential adsorption of surfactant and cosurfactant at the interface, thereby reducing the interfacial energy required for zeroing coalescence (Rao and Shao, 2008). In addition, transcutol HP (co-surfactant) with a HLB value of 4 acted as a amphiphile and was expected to increase the interfacial fluidity of the outer surface of surfactants in the micelles due to the entrapment of the former in the high HLB labrasol (HLB 14), enhancing the emulsification process upon dilution in aqueous medium (Taha et al., 2004). Generally, the longer the length of the hydrophobic alkyl chain, the higher will be the molecular volume of the oil phase affecting the emulsification ability of surfactant mixtures. Capryol 90 in this context is a C8 medium chain fatty acid of mono- and diester of caprylic acid that can be emulsified with ease (Rao and Shao, 2008).

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300 250 200 150 100 75/20/5

75/15/10

75/10/15

75/5/20

Ratio of surfactant/oil/co-surfactant (v/v/v%) Fig. 2. Effect of surfactant/oil ratio (A) and surfactant/oil/co-surfactant ratio (B) on the mean droplet size of liquid SNEDDS. These emulsions were composed of a 0.1 mL mixture of surfactant/oil and 100 mL of water.

3.4.1. Morphological analysis The surface morphology of DCT, colloidal silica, and DCT-loaded solid SNEDDS (D-SNEDDS) were studied with the aid of SEM (Fig. 3). As can be seen (Fig. 3A), the pure drug is present as rectangular irregular crystals ranging from 150 to 250 ␮m in size. The silicon dioxide (1–5 ␮m) had a rough and porous surface (Fig. 3B), which might have allowed the ingress of aqueous phase into the matrix. Although D-SNEDDS showed a satisfactory spherical shape with shallow dents, the particles were present as an aggregated mass (Fig. 3C). It is worth noting that none of the drug crystals were visible in the solid SNEDDS image, indicating complete incorporation of drug inside the matrix system. 3.4.2. Physical characterization The DSC technique was used to acquire qualitative information about the physicochemical status of drug in the formulations. The thermograms of DCT, silicon dioxide, physical mixture and D-SNEDDS are presented in Fig. 4A. DCT powder showed a sharp endothermic peak at 165 ◦ C that corresponded to the melting point of the drug in the crystalline form. No distinct peaks were observed for silicon dioxide over the entire range of temperatures tested. However, the physical mixture showed a slight peak of reduced intensity indicating the presence of crystalline objects. No such peaks were observed in the thermogram of D-SNEDDS, suggesting that the drug is either completely incorporated or molecularly dispersed in the amorphous state in the matrix (Umadevi et al., 2010).

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Fig. 3. Scanning electron microscopic images of (A) docetaxel, (B) silicon dioxide, and (C) D-SNEDDS.

The exothermic peak corresponds to crystallization of the unstable melt at higher temperature. The molecular dispersion of drug in the D-SNEDDS formulation was further verified by powder-XRD analysis. Typical diffraction patterns of DCT, silicon dioxide, physical mixture and D-SNEDDS are shown in Fig. 4B. The diffraction patterns of pure DCT revealed a highly crystalline structure with characteristic 2 peaks at 11.0◦ , 13.5◦ , 18.5◦ , 19.8◦ , 24.6◦ , and 28.0◦ . These typical patterns were absent in the D-SNEDDS, indicating the presence of drug in the amorphous form after its encapsulation (Oh et al., 2011). 3.5. In vivo pharmacokinetic study The plasma concentration–time profiles of DCT following oral administration of a single dose of 10 mg/kg of DCT and equivalent dose of D-SNEDDS are shown in Fig. 5. For comparison, DCT solution (Taxotere® ) in ethanol/water system was administered intravenously at a single dose of 5 mg/kg via tail vein. Only half the dose was used in IV administration versus the oral route due to its severe systemic toxicity. When commercial DCT solution was administered by IV route, the plasma concentration rapidly slumped to <10 ng/mL within 4 h and went below the quantification limit of the HPLC analytical technique by 5 h. No trace of DCT in systemic circulation was observed after 6 h of study period; oral administration of DCT solution produced a plasma concentration of 10 ng/mL at 1 h and gradually decreased until 8 h (sub-therapeutic concentration). These observations clearly demonstrate that free DCT by either the oral or IV route is rapidly cleared from the plasma circulation and attains sub-therapeutic level shortly after administration. In contrast, D-SNEDDS showed significantly higher drug levels throughout the study period. The concentration in the plasma was 40 ng/mL right after the 1 min of administration and attained a peak of approximately 130 ng/mL at 1 h. Furthermore, high plasma levels of 20 ng/mL were maintained until 8 h. The corresponding pharmacokinetic parameters are listed in Table 1. As reflected in Table 1, D-SNEDDS demonstrated a higher

capacity to promote the plasma circulation time of DCT. The DSNEDDS exhibited a significantly (p < 0.01) higher AUC (6 fold) and Cmax (12 fold) than oral DCT solution. Importantly, it also showed a 6.5-fold higher absolute bioavailability (17%) than the oral DCT solution. Thus, the results clearly suggest that the carrier system can increase the retention time and provide a sustained release profile for the drug. 3.6. In vivo anti-tumor efficacy of D-SNEDDS The anti-tumor efficacy of D-SNEDDS was compared with those of free DCT solutions in tumor-bearing nude mice (Fig. 6A). For this reason, D-SNEDDS and DCT solutions were given orally (10 mg/kg) along with DCT solution given intravenously at a single dose of 5 mg/kg. Phosphate-buffered saline (PBS) was used as a control. The respective formulations were given on days 0, 3, and 6, and the anti-tumor efficacy was calculated from the reduction in the ratio of tumor volume increase. PBS did not have any measurable effect on tumor growth, and tumor volumes increased rapidly and reached 2100 mm3 . A similar trend was observed for the orally administered DCT solution. However, D-SNEDDS showed a significant (p < 0.01) anti-tumor effect compared to oral DCT solution. To be specific, tumor volume increased relatively slowly and attained only 900 mm3 at the end of day 23. Interestingly, mice administered IV solution exhibited smaller tumor volumes (250 mm3 ) compared to those in the D-SNEDDS treated group. Still, the remarkable tumor regression property of D-SNEDDS is attributed to its ability to increase the bioavailability of drug and hence the maximum therapeutic concentrations in the systemic circulation. Changes in the body weights of tumor-bearing nude mice after oral/intravenous administration of formulations are presented in Fig. 6B. The same groups of mice that were used for anti-tumor studies were monitored for body weight change. As expected, PBS resulted in no loss of body weight. Mice treated with oral DCT solution and D-SNEDDS also showed no change in body weight throughout the study period. However, as opposed to the former

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Table 1 Pharmacokinetic parameters of oral DCT solution, oral D-SNEDDS, and IV DCT solution. Parameters

D-SNEDDS (oral, 10 mg/kg)

Solutiona (oral, 10 mg/kg)

AUC (h ng/mL) Tmax (h) Cmax (ng/mL) t1/2 (h) Kel (h−1 ) Absolute BA (%)

524.8 ± 44.7 0.85 ± 0.02 132.0 ± 5.9* 1.44 ± 0.14 0.49 ± 0.05 17.0

81.6 ± 15.6 0.87 ± 0.29 11.2 ± 3.8 2.11 ± 1.99 0.30 ± 0.09 2.6

*

Solutiona (i.v., 5 mg/kg) 1573.0 ± 481.6 – 11,162.9 ± 4118.1 0.6 ± 0.1 0.63 ± 0.11 –

Absolute bioavailability (%) = (AUCoral /Doseoral )/(AUViv /Doseiv ) × 100. Each value represents the mean ± S.D. (n = 6). * p < 0.05 compared with oral DCT solution. a Modified from Quan et al. (2013).

two groups, mice treated with IV DCT solution experienced a weight loss of 20% and were extremely lethargic from day 6 onwards. It is worth noting that while IV DCT solution had a remarkable effect on the tumor volume growth, the statistical difference (p < 0.05) in body weight between D-SNEDDS and DCT solution-treated groups clearly reflected the toxicity potential of the latter. 4. Discussion DCT is considered a potent chemotherapeutic agent against a wide spectrum of malignancies (Zhao and Astruc, 2012). However, the oral bioavailability of DCT has been reported to be either

Fig. 4. (a) Differential scanning calorimetry and (b) powder X-ray diffraction of docetaxel (A), silicon dioxide (B), physical mixture (C) and D-SNEDDS (D).

low or negligible which limits its clinical application. This may be explained by the fact that P-glycoprotein mediated multidrug efflux transporters and hepatic first pass metabolisms inhibit the transport of drug across the intestinal mucosal barrier (Jun et al., 2012). Other factors, including low aqueous solubility, also hamper the GI absorption of DCT. Although many alternatives have been proposed in the past to enhance the bioavailability of the drug, none has reached clinical practice (Yan et al., 2010). Considering the fact that oral treatment of anti-cancer drugs may have extraordinary pharmacodynamic benefits versus the intermittent intravenous administration, the present study was an effort to increase the oral bioavailability and therapeutic index of DCT by incorporating it into a lipid-based self-nanoemulsifying drug delivery system. In this study, an optimized and stable solid SNEDDS formulation was developed for the oral administration of DCT with enhanced bioavailability and anti-tumor activity. In order to evaluate the maximum soluble fraction of DCT in different vehicles, solubility studies were carried out. Accordingly, capryol 90, labrasol and transcutol HP were selected as oil, surfactant, and co-surfactant, respectively, to formulate a DCT-loaded SNEDDS formulation. Furthermore, a pseudo-ternary phase diagram was constructed to ensure the stability and spontaneity of nano-emulsions in aqueous phase at room temperature. In this context, the optimized liquid SNEDDS composed of capryol 90, labrasol and transcutol HP at a volume ratio of 10/75/15 produced the smallest and most stable nano-emulsion droplets. Such small droplets provide a large interfacial surface area and promote the absorption and lymphatic transport of drugs (Rao and Shao, 2008). The acceptability of liquid

Fig. 5. Plasma concentration–time profiles of docetaxel after intravenous and oral administration of docetaxel solution and D-SNEDDS to rats. Each value represents the mean ± S.D. (n = 6); *p < 0.05 compared to the oral docetaxel solution. Data for IV solution (5 mg/kg) and oral solution (10 mg/kg) were modified from Quan et al. (2013).

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Fig. 6. Antitumor efficacy of docetaxel (A) and body weight change of tumor-bearing Cg-Foxnl-nu/CrljBgi nude mice (B) after the intravenous and oral administration of docetaxel solution and the oral administration of D-SNEDDS formulation to tumorbearing mice according to a dose schedule regimen of three single administrations on days 0, 3 and 6; *p < 0.05 and **p < 0.01 compared to the phosphate-buffered saline and oral docetaxel solution; ## p < 0.01 compared to D-SNEDDS. Each value represents the mean ± S.D. (n = 5).

SNEDDS was further improved by solidification of the former formulation into solid SNEDDS by a spray-drying method. Such solid SNEDDS offers excellent stability, long storage time, and all of the advantages of a solid dosage form (Balakrishnan et al., 2009a; Woo et al., 2008). For the pharmacokinetic study, D-SNEDDS and DCT solution were given orally at a dose of 10 mg/kg and intravenously at a single dose of 5 mg/kg of DCT solution (Fig. 5). Immediately after the IV administration, the plasma concentration declined below 10 ng/mL, which is typical for this formulation (Taxotere® ). A similar plasma profile has been demonstrated by other authors (Jun et al., 2012; Lee et al., 2009). It was previously reported that DCT from Taxotere® was released quickly due to the breakdown of esterase sensitive polysorbate-80 micelles in the blood (Lee et al., 2011). In addition, after oral administration of DCT solution (in ethanol/water system), the plasma levels were too low to produce any therapeutic activity. The AUC and Cmax were very low due to the incomplete absorption of this poorly aqueous soluble BCS class II drug. On the other hand, D-SNEDDS produced a remarkable increase in the plasma concentration compared to free

DCT solutions. The AUC escalated from 81.6 ± 15.6 ng/mL for oral DCT solution to 524.8 ± 44.7 ng/mL for the D-SNEDDS formulation. Importantly, D-SNEDDS showed a 6.5-fold higher absolute bioavailability than the oral DCT solution. Recently, we reported a 12.5% BA from DCT-loaded SNEDDS (formulated from capryol 90, cremophore EL and transcutol HP at a volume ratio of 45/35/20) and showed a 5-fold increase than comparing to free drug suspension (Quan et al., 2013). The slight improvement in the pharmacokinetic parameters in the present study might be attributed to the presence of labrasol that acted as a better penetration enhancer and apparently changed the spontaneity of emulsion droplets (Oh et al., 2011). In another study, DCT-loaded SMEDDS was reported to improve the overall MRT and decreased the clearance of drug from the systemic circulation, consistent with our result (Yao et al., 2012). Such an unprecedented high bioavailability is attributed to a multitude of factors including its ability to disperse well in GI tract fluid, to cross intestinal epithelial layers, to transport the drug via lymphatic pathways, to bypass the P-glycoprotein efflux system, and to bypass cytochrome P-450 mediated drug metabolism (Rao and Shao, 2008; Sun et al., 2011; Craig et al., 1995). Furthermore, it was reported that the oil and surfactants used in the SNEDDS formulation played vital roles as absorption enhancers and P-glycoprotein inhibitors, which will in turn augment the GI permeability (Chang et al., 2011; Oh et al., 2011). This significant bioavailability result, which is highest among those previously reported, indisputably vouches for the distinct enhancement in the anti-tumor property of the drug. It has been well documented that p-gp mediated efflux transporters are primarily responsible for the low bioavailability of DCT (Yin et al., 2009; Gao et al., 2008). Augmentation in the drug transport across the intestine clearly suggests the inhibition of differential transport mechanism such as P-gp efflux. A marked 6-fold increase in the BA clearly demonstrates that SNEDDS were able to augment the drug absorption by inhibiting the p-gp efflux mechanism. Previously, SNEDDS has been reported to improve the intestinal absorption of drugs such as sirolimus, vimpocetine, and carvidelol by inhibiting the p-gp transport (Chen et al., 2008b; Singh et al., 2013). In addition, labrasol and transcutol were also reported to decrease the intestinal flux and drug biotransformation owing to their ability to inhibit p-gp and/or CYP450 enzymes (Chang et al., 2011; Oh et al., 2011; Wu et al., 2006). Generally, lymphatic pathway plays an important role in improving the bioavailability of lipid/oil-based delivery system. However, the extent to which lymphatic transport plays a role in improving the bioavailability in the present study is still uncertain and further studies are warranted to surface this possibility. After achieving satisfactory bioavailability of DCT, we evaluated the anti-tumor potential of D-SNEDDS. The tumor burden of orally administered D-SNEDDS treated mice was significantly smaller than in mice treated with free DCT solution and PBS. It is clear from Fig. 6A that there is no difference in the tumor volume until day 3. However, after the third dose (day 6), the tumor volume decreased remarkably in the D-SNEDDS treated group. The average tumor volume in the latter group increased very slowly and reached only 900 mm3 at the end of the study period. This means that the present dose of 10 mg/kg is not sufficient to completely eradicate the tumor and higher dose is required for a complete tumor regression. However, the tumor volume grew rapidly, resulting in severe ulceration in the case of free DCT solution. Such a poor performance of oral DCT solution was in accordance with the poor pharmacokinetic response, where only a small amount of drug was available to elicit its action. On the other hand, the enhanced tumor regression by D-SNEDDS was attributed to its ability to prolong the half-life of the drug and to reduce the elimination rate constant, which could increase drug accumulation at cancer sites. Such a high intracellular accumulation of drug prevents its removal from the cytosol and subsequently permits the tumor killing action (Kim et al., 2008). The

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enhanced permeability and retention (EPR) also contributed to the higher drug level inside tumor cells (Mendoza et al., 2011). Furthermore, DCT was well protected within the core of D-SNEDSS, which prevented its rapid clearance from the plasma circulation before targeting the cancer cells and allowed its subsequent release postinternalization (Singh et al., 2013). Interestingly, in the present study, the intravenous DCT solution (Taxotere® ) showed the best anti-tumor action among all the formulations despite its short circulation time in the rats. The toxicity profile of DCT was also assessed in terms of changes in the mean body weight. The oral DCT solution and D-SNEDDS did not show concomitant overt signs of toxicity and the mice maintained their body weights throughout the study period. This can be explained in two ways: first, incomplete absorption of drug in the former case may have led to a low plasma concentration and hence no noticeable side effects. Second, in the latter case, the drug from the core domain was released in a sustained manner into the blood stream and thus the drug did not reach a toxic level in the mice. Contrary to these two groups, mice treated with IV Taxotere® were lethargic and shed at least 20% of their body weight. This result is concordant with other authors’ reports (Lee et al., 2011; Xu et al., 2009). This body weight difference was significant (p < 0.05) when compared with the other two groups which indicates toxicity. It is worth noting that the same group exhibited the maximum tumor volume regression. Therefore, based on the tumor volume and body weight results, it can be presumed that D-SNEDDS is more efficacious and less toxic than either free drug or Taxotere® in tumor-induced mice. 5. Conclusion In the present study, a novel approach of combining the unique benefits of SNEDDS and anti-cancer drug was successful. DCTloaded SNEDDS was prepared to improve the bioavailability and anti-tumor potential of DCT. The composition of the SNEDDS formulation with capryol 90, labrasol, and transcutol HP at a volume ratio of 10/75/15 was judiciously selected to produce a stable nano-emulsion. D-SNEDDS remarkably improved the systemic circulation of DCT by augmenting the rate and extent of drug absorption and thereby bioavailability. However, the most important aspect of the present work is that Taxotere® -induced toxicity was overcome by D-SNEDDS with a plausible anti-tumor response. Therefore, D-SNEDDS offers an exciting mode of DCT delivery to improve its chemotherapeutic potential. The promising result of the present investigation can be extrapolated to other BCS class II anti-cancer drugs, which is the subject of ongoing research. Acknowledgement This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (Nos. 2012R1A2A2A01045658 and 2012R1A1A1039059). References Balakrishnan, P., Lee, B.J., Oh, D.H., Kim, J.O., Hong, M.J., Jee, J.P., Kim, J.A., Yoo, B.K., Woo, J.S., Yong, C.S., Choi, H.G., 2009a. Enhanced oral bioavailability of dexibuprofen by a novel solid self-nanoemulsifying drug delivery system (SEDDS). Eur. J. Pharm. Biopharm. 72, 539–545. Balakrishnan, P., Lee, B.J., Oh, D.H., Kim, J.O., Lee, Y.I., Kim, D.D., Jee, J.P., Lee, Y.B., Woo, J.S., Yong, C.S., Choi, H.G., 2009b. Enhanced oral bioavailability of Coenzyme Q10 by self-emulsifying drug delivery systems. Int. J. Pharm. 374, 66–72. Beg, S., Swain, S., Singh, H.P., Patra, C.N., Rao, M.E., 2012. Development, optimization, and characterization of solid self-nanoemulsifying drug delivery systems of valsartan using porous carriers. AAPS PharmSciTech 13, 1416–1427. Ben Reguiga, M., Bonhomme-Faivre, L., Farinotti, R., 2007. Bioavailability and tissular distribution of docetaxel, a P-glycoprotein substrate, are modified by interferonalpha in rats. J. Pharm. Pharmacol. 59, 401–408.

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