http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.878003

ORIGINAL ARTICLE

Self-nanoemulsifying drug delivery system (SNEDDS) of the poorly water-soluble grapefruit flavonoid Naringenin: design, characterization, in vitro and in vivo evaluation Abdul Wadood Khan1, Sabna Kotta1, Shahid Husain Ansari2, Rakesh Kumar Sharma3, and Javed Ali1

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1

Department of Pharmaceutics, 2Department of Pharmacognosy & Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India, and 3Division of CBRN Defense, Institute of Nuclear Medicine and Allied Sciences, New Delhi, India

Abstract

Keywords

Naringenin (NRG), predominant flavanone in grapefruits, possesses anti-inflammatory, anti-carcinogenic, hepato-protective and anti-lipid peroxidation effects. Slow dissolution after oral ingestion due to its poor solubility in water, as well as low bioavailability following oral administration, restricts its therapeutic application. The study is an attempt to improve the solubility and bioavailability of NRG by employing self-nanoemulsifying drug delivery technique. Preliminary screening was carried out to select oil, surfactant and co-surfactant, based on solubilization and emulsification efficiency of the components. Pseudo ternary phase diagrams were constructed to identify the area of nanoemulsification. The developed selfnanoemulsifying drug delivery systems (SNEDDS) were evaluated in term of goluble size, globule size distribution, zeta potential, and surface morphology of nanoemulsions so obtained. The TEM analysis proves that nanoemulsion shows a droplet size less than 50 nm. Freeze thaw cycling and centrifugation studies were carried out to confirm the stability of the developed SNEDDS. In vitro drug release from SNEDDS was significantly higher (p50.005) than pure drug. Furthermore, area under the drug concentration time-curve (AUC0–24) of NRG from SNEDDS formulation revealed a significant increase (p50.005) in NRG absorption compared to NRG alone. The increase in drug release and bioavailability as compared to drug suspension from SNEDDS formulation may be attributed to the nanosized droplets and enhanced solubility of NRG in the SNEDDS.

Bioavailability, oil phase, pharmacokinetics, pseudoternary phase diagram, solubility

Introduction Epidemiological studies and nutritional experiments have suggested that increased consumption of fruits and vegetables rich in antioxidants protect against chronic diseases such as cardiovascular diseases and cancer (La Vecchia & Tavani, 1998). Besides variety of potentially beneficial nutrients such as vitamins C and E, fruit and vegetables also contain so called non-nutrients such as flavonoids. Flavonoids are a widely distributed group of polyphenolic compounds characterized by a common benzo-g-pyrone structure. Over 4000 different flavonoids have been described, and they are categorized into flavonols, flavones, flavanones, isoflavones, catechins and anthocyanidins. Plants containing flavonoids have been reported to possess strong antioxidant properties (Raj & Shalini, 1999). In recent years flavonoids have attracted much attention due to their antioxidant, anti-inflammatory, anticancer and antiviral activity (Hollman & Katan, 1996; O’Reilly et al., 2000; Scalbert & Williamson, 2000). Because of such Address for correspondence: Dr Javed Ali, Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110 062, India. Tel: +91 9811312247. Fax: +91 11 2605 9663. Email: [email protected]; [email protected]

History Received 26 November 2013 Revised 13 December 2013 Accepted 13 December 2013

beneficial properties, the use of flavonoids as possible therapeutic agents to protect from free radical-mediated disease has become a focal point in recent research (Middleton et al., 2000). Among the various types of flavonoids, the flavanone Naringenin (NRG) found in grapefruit and oranges are associated primarily in cancer prevention (Liu, 2004). NRG (40 ,5,7-trihydroxyflavanone), a citrus flavanone occurs abundantly in fruits such as grapes, grapefruit, blood orange, lemons, pummelo and tangerines (Jayaraman et al., 2009). NRG has been reported to posses several effects on biological system such as antioxidant (Rice-Evans et al., 1996; Cao et al., 1997), anti-inflammatory (Manthey 2000), anticancer (Goldwasser, 2010), antifibrogenic (Lee et al., 2004) and antiatherogenic (Lee et al., 2001). The beneficial effects on human health are mainly due to their antioxidant activity and ability to chelate metals, to scavenge oxygen free-radical, to inhibit enzymes and to prevent oxidation of low-density lipoproteins (LDL) (Madsen et al., 2000; Yu et al., 2005). Even though NRG possesses a wide range of activities, clinical studies exploring different schedules of administration of this drug have been hampered by its extreme water insolubility and poor bioavailability. To overcome these

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problems, delivery of NRG using novel lipid-based drug delivery systems will likely yield more promising clinical applications of this compound. Various strategies have been used to overcome the problems associated with oral absorption and bioavailability issues of poorly soluble drugs, such as particle size reduction (Aungst, 1993), complexation with cyclodextrins (Miyake et al., 2000), salt formation, solid dispersions (Sinha et al., 2010), use of surfactant, nanoparticles, nanocarriers (Bali et al., 2011), Nanoemulsions (Kotta et al., 2012), self-emulsifying drug delivery system (Khan et al., 2012), prodrug formation, etc. Approaches used in past to improve the dissolution and bioavailability of NRG include complexation with b-cyclodextrin (Tommasini et al., 2004), phospholipid complexes (Semalty et al., 2010), solid dispersion with PVP (Kanaze et al., 2006), etc. However, there is no published work for enhancing the bioavailability of NRG using selfnanoemulsifying drug delivery system (SNEDDS). In the current study SNEDDS have been used to enhance the dissolution and hence the bioavailability of NRG. Lipid-based drug delivery systems such as nanoemulsions and SNEDDS have a great potential to improve oral bioavailability of poor water soluble drugs by presenting the drug in a solubilized state in colloidal dispersion. SNEDDS are defined as isotropic mixtures of natural or synthetic oils, solid or liquid surfactants, or alternatively, one or more hydrophilic solvents and co-solvents/surfactants that have a unique ability of forming fine oil-in-water (o/w) emulsions upon mild agitation followed by dilution in aqueous media, such as gastrointestinal (GI) fluids (Singh et al., 2009). SNEDDS have been proved to improve the bioavailability of poorly soluble drugs like Talinolol (Ghai & Sinha, 2012), Coenzyme Q10 (Nepal, 2010), Zedoary turmeric oil (ZTO) (Zhao, 2010), biphenyl dimethyl dicarboxylate (BDD) (El-Laithy, 2008). An increase of 1.7- and 2.5-fold in AUC and Cmax, respectively was obtained when Zedoary turmeric oil (ZTO), an essential oil extracted from the dry rhizome of Curcuma zedoaria was administered as SNEDDS orally to rats. Unlike polymeric system, lipid-based systems are easily taken up by the body. The digestion of these formulation involve dispersion of fat globules into a coarse emulsion of high surface area, enzymatic hydrolysis of fatty acid glyceryl esters (primarily triglyceride lipid) at the oil/water interface and dispersion of the products of lipid digestion into an absorbable form. The resemblance of their degradation product with end product of intestinal degradation has contributed in their wide acceptance for SNEDDS (Khan et al., 2012). The objectives of the present study were to develop and characterize NRG-loaded SNEDDS formulation with an aim to increase the solubility and bioavailability. The in vivo behavior of the optimized formulations was also investigated in Wistar Albino rats.

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caproyl macrogol-8-glyceride), Labrafac (propylene glycol dicaprylocaprylate), Maisine (Glyceryl monolinoleate), Peceol (Glyceryl monooleate), and TranscutolÕ HP (Diethylene glycol monoethyl ether) were gifted by Gattefosse India (Mumbai, India), Capmul (Propylene Glycol Monolaurate) was gifted by Abitec Corporation (Columbus, OH, USA), Triacetin (Glycerol triacetate), Tween 80 (polyoxyethylene sorbitan monooleate), Tween 20 (polyoxyethylene sorbitan monolaurate), PEG (Polyethylene glycol) 400, PEG 200 and ethanol were purchased from Merck India Limited (Whitehouse Station, NJ, USA); Span 20 was purchased from Thomas Baker (Mumbai India); arachis oil, oleic acid, peanut oil, soyabean oil, caster oil, corn oil, jaloba oil, fish oil, linseed oil were purchased from Loba Chemie (Mumbai, India). Water was obtained from Milli-Qwater purification system (Millipore, MA). All other chemicals and reagents were of analytical reagent grade. Selection of oil Various oils were screened for their ability to dissolve maximum amount of NRG by shake flask method. Excess amount of NRG was added to each of the eppendorf tubes containing 1 ml of different oils. The mixture was vortexed on a vortex mixer (Nirmal International, Delhi, India) for 10 min to facilitate proper mixing and solubilization of NRG in the oil. The mixture was then transferred to incubator shaker (Shel Lab, Avenue Cornelius, USA) maintained at 25 ± 2  C for 72 h. The samples were then centrifuged at 5000 rpm for 20 min to separate the undissolved drug. Supernatant was filtered through 0.22 m membrane filter and then suitably diluted with methanol. NRG dissolved in various oils was quantified using high-pressure liquid chromatography (HPLC) method at 289 nm. Selection of surfactant and co-surfactant Similarly the solubility of NRG was also determined in different surfactants and co-surfactants also. Thereafter, the surfactants were screened based on their solubilization behaviour towards the oil phase. For the solubilization study, 2.5 ml of 15 weight % surfactant solution was prepared in water and 5 ml of oil was added with vigorous vortexing. If clear solution was obtained, the addition of the oil was continued until the solution became cloudy. The selected surfactant was combined with different co-surfactants, namely, PEG 200, PEG 400, Transcutol HP and Ethanol. The co-surfactants were selected based on their ability to maximize the nanoemusion area with the selected surfactant. At fixed Smix ratio of 1:1 (surfactant and co-surfactant), the pseudoternary phase diagrams were constructed with different weight ratios of oil and Smix, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3.5, 1:3, 3:7, 1:2, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1, respectively, in order to get maximum possible nanoemulsion formulations.

Material and methods NRG (Mw. 272.25; purity495%) was purchased from SigmaAldrich (St. Louis, MO, USA); Cremophor EL (polyethoxylated castor oil) and Cremophor RH(Polyoxyl 40 Hydrogenated Castor Oil) were kind gifts from BASF Corporation (Ludwigshafen, Germany); Labrasol (Caprylo

Construction of pseudo-ternary phase diagrams The pseudo-ternary phase diagrams were constructed by titration of homogenous liquid mixtures of lipid, surfactant and co-surfactant with water. Smix were prepared in different volume ratios (1:1, 1:2, 2:1, 3:1 and 4:1). For each phase

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diagram, oil and specific Smix ratio were mixed in different volume ratios ranging from 1:9 to 9:1 to obtain 16 different combinations, i.e., 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3.5, 1:3, 3:7, 1:2, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1. The monophasic mixtures were slowly titrated with aliquots of water (37  C) and vortexed. After equilibrium was reached, the mixture was further titrated with aliquots of water until they showed the turbidity. The tendency to emulsify spontaneously and also the progress of emulsion droplets were observed visually, and the pseudo-ternary phase diagrams were constructed. The experiments were performed in triplicate.

using Zetasizer 1000 HS (Malvern Instruments, Worcestershire, UK), wherein light scattering was monitored at 25  C at a 90 angle.

Formulation of NRG loaded SNEDDS

Time for self-nanoemulsification

NRG was added in the oily phase in small increment with continues stirring and added drop wise to vial containing calculated amount of mixture of surfactant and co-surfactant (Smix). The surfactant system was prepared by mixing the chosen surfactant and co-surfactant in their determined ratios. The components were mixed by gentle stirring and vortex mixing. Mixing was continued for 0.5 h to ensure uniformity. The formulation was equilibrated at ambient temperature for at least 48 h and examined for signs of turbidity or phase separation prior to dilution, self emulsification and particle size studies.

The time required for self-nanoemulsification of selected formulations was assessed on USP-II dissolution apparatus (Veego, Mumbai, India). Each formulation was drop wise added to 500 ml of distilled water maintained at 37 ± 2  C. Gentle agitations were provided by standard stainless-steel dissolution paddle rotating at 50 rpm. The emulsification time was assessed visually and graded (Table 1). All the studies were repeated in triplicates with similar observations being made between repeats.

Characterization of NRG loaded SNEDDS

Morphological and structural examination of NRG loaded SNEDDS was carried out using transmission electron microscopy (TEM; Hitachi, Tokyo, Japan) on an H7500 machine operating at 100 kV capable of point-to-point resolution. A droplet (0.5 mL) of the SNEDDS formulation, diluted at 1:100 times with water, was directly positioned on the copper electron microscopy grids supported by formvar films. The excess was drawn off using filter paper. Then the grids were stained with 0.5% aqueous solution of phospho-tungstic acid for 30 s, and the excess was drawn off. The grids were then observed by TEM after drying. Combinations of different bright-field imaging at increasing magnification were used to expose the structure as well as the size of the formed nanoemulsion.

Thermodynamic stability tests Thermodynamic stability studies comprising of centrifugation, heating–cooling cycle and freeze–thaw cycle were performed to evaluate the phase separation and effect of temperature variation on SNEDDS stability. Each of the selected formulation was diluted with water (1:20) (Chaurasiya et al., 2012) and centrifuged (Remi equipments, Mumbai, India) at 3500 rpm for 0.5 h to determine its stability as an isotropic single-phase system. Formulations that showed any sign of phase separation, creaming or cracking were discarded and the remaining formulations were subjected to three heating and three cooling cycles in which samples were incubated at 4  and 45  C for 48 h. The formulations, stable at these temperatures, were further tested in three freeze thaw cycles at temperatures between 20 and 25 ± 2  C in deepfreezer (Vest Frost, Hyderabad, India) for not less than 48 h. Influence of dilution and pH on SNEDDS stability Dilution and pH of the aqueous phase may have substantial consequences on the phase separation and stability of the selfemulsifying systems. In consideration of this, selected formulations were diluted (20 and 1000 times) with various diluents (i.e. deionized water, 0.1 N HCl and phosphate buffer pH 6.8). The diluted formulations were stored for 8 h at 25  C and observed visually for any sign of phase separation or drug precipitation. Droplet size, polydispersity index (PDI) and zeta potential determination The average droplet size and PDI of reconstituted formulations were determined by means of photon correlation spectroscopy (PCS). Measurements were made in triplicate

% Transmittance The NRG-loaded SNEDDS were reconstituted with distilled water and the resulting nanoemulsions were observed visually for any turbidity. Thereafter, its % transmittance was measured at 638.2 nm using UV–vis spectrophotometer against distilled water as the blank. The studies were conducted after 20 times dilution.

Surface morphology and structure

In vitro release profile by dialysis-bag method In vitro release experiments were carried out by dialysis method. Reconstituted liquid (1:20) SNEDDS and NRG suspension (equivalent to 30 mg) were filled in an activated dialysis membrane (MWCO 12 000 g/mole; Sigma, St. Louis, MO, USA) and immersed in 900 ml of phosphate buffer (pH 6.8) at 37 ± 0.5  C using USP-II dissolution apparatus (Veego, Mumbai, India). A weight of 1 g each was affixed to dialysis membrane sack to hold it within the release medium (Ghai & Sinha, 2012). Throughout the experiment, 5 ml of aliquots were withdrawn at different time intervals from the release medium and substituted with an equal volume of fresh buffer maintained at similar temperature. The samples were filtered through a 0.45 mm membrane filter and amount of NRG released was determined by high-pressure liquid chromatography (HPLC) method at 289 nm. In vivo pharmacokinetics studies In vivo pharmacokinetic studies were performed in 6–8 weekold male Albino Wistar rats weighing 200 ± 20 g. The rats

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Table 1. Observation for dispersibility test. S. No Grade

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1 2 3 4 5

A B C D E

Dispersibility and appearance

Time of self-emulsification (min)

Rapidly forming nanoemulsion, having a clear or bluish appearance Rapidly forming, slightly less clear nanoemulsion, having a bluish white appearance Fine milky emulsion Dull grayish white emulsion having slightly oily appearance that is slow to emulsify Formulation exhibiting either poor or minimal emulsification with large oil globules present on the surface

51 52 52 43 43

were housed with six rats per cage and acclimatized for minimum of 3 d on a standard 12 h light/dark cycle at a room temperature of 25 ± 1  C and relative humidity of 50 ± 10%. Rats were deprived of food overnight (12 h), but allowed free access to water before the experiment was carried out. The protocols for the animal studies were approved by the Institutional Animal Ethics Committee, Hamdard University, New Delhi, India (Project No: 850 dated 29-Mar-2012). The animals were randomly divided into two groups, each having six Albino Wistar rats. Group-1 was administered SN 1, and Group-2 was administered drug by suspending in 1 ml of carboxy methyl cellulose solution (0.5% w/v) (Zhao et al., 2013) and given orally using oral feeding canula. The rats were anesthetized using ether, and blood samples were withdrawn from the tail vein at time intervals of 0.25, 0.5, 0.75, 1, 2, 4, 8 and 24 h. The samples were collected in heparinized tubes, and plasma samples were separated immediately by centrifugation at 4,000 rpm for 20 min and stored at 20  C until drug analysis by HPLC. About 100 ml aliquot plasma sample was transferred to a clean glass tube. About 33.33 ml of 2% formic acid was added and vortexed for 2 min. Then 600 ml ethylacetate was added to the tube and vortexed again for 2 min. The sample was centrifuged at 4000  g for 10 min at 4  C and the upper organic layer was transferred into a 5 ml glass tube. The organic layer was dried with nitrogen at 37  C 15 psi pressure. The dried residue was dissolved in 100 ml of the mobile phase. A 20 ml aliquot of the sample was injected into HPLC system and analyzed using methanol water (7:3) as mobile phase. Pharmacokinetic data analysis and statistical evaluation The pharmacokinetic profiles, including area under drug-concentration time-curve (AUC), Cmax, Tmax, and halflife (t1/2) of each rat were analyzed by non-compartmental analysis. The maximum plasma concentration (Cmax) and time of its occurrence (tmax) were directly computed from the plasma concentration versus time plot. All the data are expressed as mean ± standard deviation. To demonstrate statistical difference among the pharmacokinetic parameters, level of significance (p50.05) among three means was calculated using analysis of variance test.

Results and discussion SNEDDS are isotropic mixtures of natural or synthetic oils, solid or liquid surfactants or alternatively, one or more hydrophilic solvents and co-solvents/surfactants that have a unique ability of forming fine oil-in-water (o/w) emulsions on mild agitation followed by dilution in aqueous media, such as

Figure 1. Solubility of NRG in different oils.

GI fluids (Singh et al., 2009). Self-nanoemulsifying properties of SNEDDS strongly depend upon the selected lipids, surfactants and their relative amounts. The utilization of lipid and surfactant(s) mixtures gives the possibility to optimize the SNEDDS for a particular drug. Selection of oil The key factor for the screening of components of SNEDDS formulation is the solubility of the poorly water soluble flavonoid in oil. The superior solubility of drug in the oil phase was important to keep the drug in the solubilized form and it also avoids precipitation of drug on dilution in the gut lumen. Higher solubility of drug in oil will ensure lesser amount of oil in formulation and consequently lesser amount of surfactants and co-surfactants is required for emulsification of drug loaded oil droplets. It was observed that the solubility of NRG was higher in the semi-synthetically derived oils in comparison to the natural oils (Figure 1). This may be attributed to the polarity of the poorly soluble drugs that favor their solubilization in small/medium molar volume oils, such as medium-chain triglycerides or mono- or diglycerides (Lawrence & Rees, 2000). Because of superior solubility novel semi synthetic medium chain derivatives with surfactant properties are progressively and effectively replacing the regular natural and medium chain triglyceride oils (Constantinides, 1995). Among the different oils, Triacetin was found to solubilize the maximum amount of NRG (145 ± 0.51 mg/ml). The solubility in Triacetin was

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consequently improves the thermodynamic stability of the nanoemulsion formulation. Construction of pseudo-ternary phase diagrams

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Figure 2. Oil solubilized (weight %) by different surfactants.

significantly higher than other oils (p50.001), hence it was preferred for further study to prepare SNEDDS. Selection of surfactant and co-surfactant It is possible that the surfactant with good solubilizing properties for drugs may not have equally good affinity for the oil. Thus, the selection of the surfactant and co-surfactant was governed by the emulsification efficiency for oil rather than the ability to solubilize NRG. Different surfactants having HLB in the range of 8–16 were screened on the basis of both, their ability to solubilize the drug and to emulsify selected oil phase (Figure 2). Nonionic surfactants are generally considered safer than the ionic surfactants and are usually accepted for oral ingestion (Nazzal et al., 2002). They are also reported to provide better stability to emulsion over a wider range of pH and ionic strength. Among the screened surfactants, Tween 80 and Cremophor EL showed maximum oil solubilization capacity, i.e., 1.47 and 1.20 w% (p50.001) and were selected for further studies. The selected non-ionic surfactants (Tween 80 and Cremophor EL) were reported to possess bioenhancing activity. Zhang et al. (2003) reported an increased AUC and Cmax for orally administered digoxin in rats when co-administered with Tween 80. Increasing the concentration of Tween 80 increased the extent of absorption. AUC was increased by 30 and 61% after dosing in 1 and 10% Tween 80 solution respectively. Cremophor was reported to have a role in improving bioavailability of atorvastatin formulated as self-emulsifying formulations (Shen & Zhong, 2006). The co-surfactants were selected based on their ability to maximize the nanoemusion area with the selected surfactant. Therefore, the ternary phase diagrams were constructed using a mixture of surfactant and various co-surfactants at 1:1 Smix ratio. Transcutol HP showed maximum nanoemulsion area due to its better compatibility with selected surfactants compared with other co-surfactants tested. The presence of co-surfactant increases the nanoemulsion area (Bali et al., 2010) and hence a higher emulsification efficiency of the system is achieved. Therefore, Transcutol HP was selected as the co-surfactant for SNEDDS formulations development. Surfactant and co-surfactant get preferentially adsorbed at the interface, reducing the interfacial energy as well as providing a mechanical barrier to coalescence. The decrease in free energy required for the nanoemulsion formation

The relationship between the phase behavior and the composition of the SNEDDS mixtures was studied utilizing pseudo-ternary phase diagrams. Pseudo-ternary phase diagrams were constructed in the absence of NRG to identify the self-emulsifying regions and to optimize the percentage of oil, surfactant, and co-surfactant for the SNEDDS formulations. The pseudo-ternary phase diagrams of the systems containing Triacetin as oil, either Tween 80 or Cremophor EL as surfactant and Transcutol HP as co-surfactant are shown in Figure 3. It was observed that self-emulsifying region first increases as surfactant to co-surfactant ratio is increased and then decreases. Increase in nanoemulsion area might be due to the increased adsorption of surfactant molecules at the oil–water interface leading to a decrease in the interfacial tension, which facilitates the formation of smaller droplets. The other reason has been attributed to a greater amount of surfactant molecules diffusing from the oil phase to the aqueous phase, thereby leading to the formation of finer oil droplets at the boundary (Anton & Vandamme, 2009). At higher surfactant concentration decreased in NE area was observed due to the formation of a highly viscous liquid crystalline phase, which makes spontaneous breakup of the oil–water interface more difficult (Wang et al., 2009). Maximum self-emulsifying region existed at Smix ratio of 2:1 whether Tween 80 or Cremophor EL is used as surfactant. There was not much difference in self-emulsifying region between Smix ratio of 1:1 and 1:2 of surfactant:co-surfactant. Different combinations in which amount of oil varies from 20–35% and Smix ratio varying from 1:2 to 4:1 were selected from the phase diagrams. The formulations were diluted with water at ratio of 1:20 and 15 formulations which were showing either grade A or B were selected for drug loading (Table 2). Characterization of NRG-loaded SNEDDS Thermodynamic stability tests SNEDDS system undergoes in situ solubilization to form thermodynamically stable nanoemulsion system, with no sign of phase separation, creaming or cracking. However, if the formulation is metastable or sometimes upon storage, the drug may precipitate out from the emulsion formed upon dilution. Thermodynamic stability study was done to identify and avoid metastable formulations. The observation for thermodynamic stability studies are given in Table 2. Formulations, which did not pass the thermodynamic tests, were dropped out and the remaining formulations were subjected to dilution study. When Tween 80 was used as surfactant, five formulations i.e. T4, T8, T13, T14 and T19 passed while in case of Cremophor EL as surfactant only two formulations i.e., TC14 and TC19 passed the thermodynamic stability test. Influence of dilution and pH on SNEDDS stability SNEDDS are pre-concentrates that on mild agitation followed by dilution form fine o/w emulsions. Thus, when the

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Figure 3. Ternary phase diagrams indicating o/w nanoemulsion region at different Smix ratios (A) Triacetin, Smix (Tween80 and Transcutol HP) and water (B) Triacetin, Smix (Cremophor EL and Transcutol HP) and water.

formulation undergoes infinite dilution in the GI fluids, it is very probable that it might phase separate resulting in the precipitation of the drug owing to its poor aqueous solubility. As the formulation passes through the gastro intestinal tract

there is wide variations in pH of GI tract from acidic environment in stomach to alkaline pH in intestine. The variation in pH may cause the precipitation of drugs that show pH dependent solubility. To avoid such a situation, dilution

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Table 2. Thermodynamic stability tests of drug-loaded formulations.

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Components ratio (v/v)

Evaluation

Formulation code

Oil

Surfactant

Co-surfactant

Dilution with water

Cent

F/T

H/C

Inference

T2 T3 T4 T7 T8 T9 T12 T13 T14 T18 T19 TC4 TC9 TC14 TC19

20 20 20 25 25 25 25 30 30 35 35 20 25 30 35

64 60 53 60 56 50 56 52.5 47 49 42.5 53 50 47 42.5

16 20 27 15 19 25 14 17.5 23 16 22.5 27 25 23 22.5

A A A A A A A B A B B A B B B

ˇ ˇ ˇ x ˇ x ˇ ˇ ˇ x ˇ ˇ x ˇ ˇ

x x ˇ ˇ ˇ ˇ ˇ ˇ ˇ ˇ ˇ x x ˇ ˇ

ˇ x ˇ x ˇ x x ˇ ˇ x ˇ x ˇ ˇ ˇ

Failed Failed Passed Failed Passed Failed Failed Passed Passed Failed Passed Failed Failed Passed Passed

Cent: centrifugation; F/T: Freeze-thaw cycle; H/C: heating–cooling cycle. Table 3. In vitro characterization of optimized SNEDDS formulations. Formulation T8 T13 T14 T19 TC14 TC19

Emulsification time (s)

Mean Droplet size (nm)

Poly dispersity index

Zeta potential (mV)

% Transmittance

46.76 ± 3.61 72.42 ± 2.42 57.87 ± 2.56 64.69 ± 4.01 90.67 ± 2.56 96.34 ± 2.13

36.2 ± 3.63 34.5 ± 2.91 38.2 ± 4.17 42.1 ± 2.47 136.23 ± 5.36 153.21 ± 6.23

0.252 ± 0.003 0.433 ± 0.001 0.381 ± 0.002 0.365 ± 0.004 0.515 ± 0.003 0.486 ± 0.002

15.37 ± 3.4 19.67 ± 2.3 23.33 ± 1.9 18.21 ± 3.7 13.26 ± 2.7 12.36 ± 2.6

92.31 ± 0.21 90.57 ± 0.43 96.21 ± 0.17 95.31 ± 0.48 75.31 ± 0.34 78.13 ± 0.57

study in double distilled water, 0.1 N HCl and phosphate buffer pH 6.8 were carried out. No phase separation or drug precipitation was observed after dilution except in T4 which turns hazy after dilution. Formation of nanoemulsions occurs at a specific concentration of oil, water and surfactant. The lesser amount of oil in T4 may be reason for such observation. The quantity of oil in T4 may not be sufficient to solubilize the required drug load and showed precipitation on dilution. Droplet size and zeta potential It has been reported that droplet size distribution is one of the most important characteristics affecting the in vivo fate of emulsions. The globule size of the emulsion also determines the rate and extent of drug release (Gupta et al., 2011). The mean droplet size of all the SNEDDS formulation was found to be in nanometric range (5200 nm). The Formulations containing Tween 80 as surfactant showed globule size well below 50 nm (34.5 ± 2.91) with PDI value of 0.372 ± 0.002 indicating narrow size distribution (Table 3) while the droplet size was more than 100 nm (153.21 ± 6.23) when Cremophor EL was used as surfactant. Even though the HLB values of the used surfactants were close, the difference observed in the particle size may be attributed to difference in their emulsifying ability owing to the difference in their structure and chain length (Date & Nagarsenker, 2007). The nano droplet size is considered ideal to result in lower emulsification time, enhanced absorption through lymphatics and subsequent augmentation in the therapeutic efficacy of drugs (Bandyopadhyay et al, 2012).

Emulsion droplet polarity is also a very important factor in characterizing emulsification efficiency. Since zeta potential signifies degree of repulsion between neighboring, like charged particles in dispersion, it can be related to the stability of colloidal dispersions. The increase in electrostatic repulsive forces between the globules avoids the coalescence of nanoemulsion. On the contrary, reduction in electrostatic repulsive forces can cause phase separation. For molecules and particles that are small enough a high zeta potential will confer stability, i.e. the solution or dispersion will oppose aggregation (Bali, 2011). Negative values of zeta potential of the optimized formulations showed that the formulations were negatively charged (Table 3). % Transmittance The percentage transmittance is an important parameter to determine the isotropic nature of the system. No significant (p40.05) difference was observed in the percentage transmittance value amongst the formulations of similar group of surfactants. However, there was a significant difference (p50.001) in % transmittance value between the two groups of surfactants (Tween 80 and Cremophor EL). Formulations containing Tween 80 as surfactant showed more than 90% value and formulation T14 was found to have the highest percentage transmittance. Percentage Transmittance for formulations having Cremophor EL was found to be less than 80%. A value closer to 100% indicates an isotropic formulation and gives an indication of globule size in nanometer range.

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Time for self-nanoemulsification A perfect SNEDDS formulation should posses the ability to disperse completely and quickly when subjected to dilution under mild agitation. The rate of emulsification is related to rate and extent of water penetration into the different phases (liquid crystalline or gel phase) formed on the surface of the droplet. Self emulsification takes place when the entropy change favoring dispersion is greater than the energy required to increase the surface area between the oil and aqueous phases of the dispersion. For self-emulsifying system, when the oil phase is introduced into the aqueous phase with gentle agitation, the aqueous phase will penetrate through the interface into oil phase until the interface of the two phases is disrupted. Consequently, oil droplets are formed resulting in emulsification (Rang & Miller, 1999). The emulsification time of formulations containing Tween 80 was found to be less than 1 min while for Cremophor EL it lies between 1–2 min. This could be explained by higher solubilization capacity of Tween 80 compared to Cremophor EL (Figure 2). Surface morphology and structure Transmission electron microscopy is the most important technique for the study of microstructures as it can directly generates images at high resolution and can also capture any

Figure 4. Transmission electron microscopic (TEM) image of T14 Formulation.

concomitant structures as well as microstructure transitions. Morphology and structure of the reconstituted SNEDDS formulations was observed using TEM. It reveals discrete, spherical oil globules, with diameters ranging from 40 to 55 nm and appears darker with bright surroundings. Figure 4 shows droplet sizes of some of the dispersed oil globules of nanoemulsion T14 measured using TEM. The droplet sizes were in proximity with the results obtained using PCS. In vitro dissolution profile The release of drug from the selected formulations is shown in Figure 5. The release of drug from the nanoemulsion formulations was found to be highly significant (p50.001) in contrast to the drug suspension. All the SNEDDS formulations showed better results as compared to drug suspension. More than 60% of the drug was released from all of the SNEDDS formulations in the initial 10 min of the dissolution study in comparison to the drug suspension which showed incomplete release. Formulation T14 showed complete drug release in 45 min amongst the group containing Tween 80 as surfactant (Figure 5). This could be attributed to the small globule size in case of nanoemulsion formulations which provided large surface area for the release of drug and thus permitting faster rate of drug release. Release of drug from T19 was lower than that from T14. High concentration of oil and bigger droplet size could be the reason for such a release pattern. There was incomplete drug release from the formulations containing Cremophor EL as surfactant and the rate of drug release was slow at all points compared with Tween 80 containing formulations. This could be attributed to the higher globule size of the formulations, which slow down the release of the drug from nanoemulsion formulation and lesser solubilization capacity of Cremophor EL for oily phase than Tween 80. Still the release was much higher than that obtained from the drug suspension that showed less than 15% release after 2 h. Thus, presentation of NRG at the molecular level in the form of the nanoemulsion formulation led to an increased solubilization and enhanced drug release. Formulation T14 was selected for in vivo studies as it was having highest drug release (100.52%) and optimum globule size (38.2 ± 4.17 nm).

Figure 5. In vitro drug release in phosphate buffer (pH 6.8).

Self-nanoemulsifying drug delivery system of Naringenin

DOI: 10.3109/10717544.2013.878003

9

Table 4. Pharmacokinetics parameters of optimized formulation and NRG. Formulation

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Drug SNEDDS T14

Cmax (ng/ml)

Tmax (h)

AUC0–24 (ngh/ml)

AUC0–1 (ngh/ml)

T1/2 (h)

5745.79 ± 78 28345.48 ± 134

0.50 0.25

33109.82 ± 1920 93237.94 ± 3311

37347.29 ± 1619 105911.1 ± 2832

7.98 ± 0.24 7.59 ± 0.23

poly-dispersity, higher solubility as well as higher bioavailability. Rapid and complete drug release was achieved from the optimized SNEDDS formulation which was significantly higher than NRG suspension. Furthermore, area under the drug concentration time-curve (AUC) of NRG from SNEDDS revealed a significant increase in NRG absorption compared to NRG suspension. Thus, the developed system could be used an effective approach for enhancing the solubility and bioavailability of poorly water-soluble grapefruit flavonoid NRG. Figure 6. Drug plasma concentration time profile after oral administration of SNEDDS and NRG suspension.

Pharmacokinetics and bioavailability The pharmacokinetic parameters were studied to determine the effect of SNEDDS formulation on the oral bioavailability of NRG. Plasma concentration profile of NRG for SNEDDS showed a great improvement of drug absorption than the drug suspension. NRG was poorly absorbed upon oral administration (Figure 6) and Cmax was reached at 0.5 h after oral administration. After reaching the maximum concentration at 0.5 h, a swift decline and then a slight increment, especially after 2 h, was observed in the plasma concentration. This may possibly be due to the extensive glucuronidation of NRG in the liver that is rapidly eliminated via bile, which is repeatedly delivered back to the lumen of the intestinal tract via enterohepatic recirculation (Ma et al., 2006) Total plasma concentration of NRG SNEDDS were significantly higher than those of NRG suspension at all the points (p50.05). The pharmacokinetic parameters clearly indicated enhanced bioavailability of NRG SNEDDS as compared to pure drug suspension (Table 4). The peak plasma concentration (Cmax) of NRG in T14 and NRG suspension was 28345.48 ± 134 & 5745.79 ± 78 ng/ml, respectively. The Tmax was relatively faster in the SNEDDS compared to suspension. Due to poor aqueous solubility of NRG, it was readily exposed in the GI tract from drug suspension. However, in the case of SNEDDS the drug was in solubilized form, thereby resulting in a low Tmax value. The AUC of NRG from SNEDDS was 2.82 times higher compared to pure drug suspension (AUC0–24 and AUC0–1 were found to be 93237.94 ± 3311, 33109.82 ± 1920 ngh/ml and 105911.1 ± 2832, 37347.29 ± 1619 ngh/ml respectively). Thus, the developed SNEDDS show a potential for enhancing oral bioavailability of NRG.

Conclusion In the present study, NRG SNEDDS comprising of Triacetin (oily phase), Tween 80 (surfactant) and Transcutol HP (co-surfactant) were prepared for enhancing the dissolution and bioavailability of NRG. SNEDDS were optimized based on increased dissolution rate, optimum globule size and

Declaration of interest The authors declare no conflict of interest. The authors are grateful to the Life Sciences Research Board, Government of India for Providing financial assistance to AW KHAN.

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