Formulation and Development of CoQ10- Loaded s

Formulation and Development of CoQ10Loaded s-SNEDDS for Enhancement of Oral Bioavailability Md. Habban Akhter, Ayaz Ahmad, Javed Ali & Govind Mohan

Journal of Pharmaceutical Innovation From R&D to Market ISSN 1872-5120 Volume 9 Number 2 J Pharm Innov (2014) 9:121-131 DOI 10.1007/s12247-014-9179-0

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Author's personal copy J Pharm Innov (2014) 9:121–131 DOI 10.1007/s12247-014-9179-0

RESEARCH ARTICLE

Formulation and Development of CoQ10-Loaded s-SNEDDS for Enhancement of Oral Bioavailability Md. Habban Akhter & Ayaz Ahmad & Javed Ali & Govind Mohan

Published online: 15 April 2014 # Springer Science+Business Media New York 2014

Abstract Purpose Coenzyme (CoQ10) is a poorly soluble drug strategically selected to enrich oral bioavailability by incorporating in solid self-nanoemulsifying drug delivery system (sSNEDDS) comprised of oil, surfactant, and cosurfactant. The conventional self-emulsifying drug delivery system (SEDDS) and liquid SNEDDS (l-SNEDDS) usually have the problem of drug instability and precipitation. Methods The selected oils, surfactant, and cosurfactant with maximum drug solubility were Lauroglycol FCC, Labrasol, and Transcutol P. The ternary phase diagrams were constructed, and selected formulations from ternary phase diagrams were subjected to thermodynamic stability and selfdispersibility test and characterized for emulsion droplet size and droplet size distribution. The optimized formulation was comprised of Lauroglycol FCC 20 % (w/w), Labrasol 10 % (w/w), and Transcutol P 20 % (w/w) as oil, surfactant, and cosurfactant. Results The transmission electron microscopy (TEM) study of optimized l-SNEDDS reported mean globule size of 34 nm was transformed into s-SNEDDS by spray-drying technique using solid carrier. The s-SNEDDS was characterized for differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope M. H. Akhter (*) : A. Ahmad : G. Mohan NIMS Institute of Pharmacy NIMS University, Jaipur 303121, India e-mail: [email protected] A. Ahmad e-mail: [email protected] G. Mohan e-mail: [email protected] J. Ali Department of Pharmaceutics Faculty of Pharmacy Jamia Hamdard, New Delhi, India e-mail: [email protected]

(SEM), and X-ray diffraction (X RD). The in vitro release profile of s-SNEDDS showed drug release (97.5±4.5 %), marketed formulation (57.96±0.54 %), and CoQ10 powder (0.36±0.06 %) in 1 hour. The pharmacokinetic study of optimized s-SNEDDS in male Wistar rats revealed the improved maximum concentration (Cmax) (3.4-fold vs CoQ10 powder; 1.4-fold vs marketed formulation) and area under the curve (AUC) (5-fold vs CoQ10 powder; 2-fold vs marketed formulation). With this result, s-SNEDDS could be of potential to enhance the oral bioavailability of CoQ10. Conclusion Thus, s-SNEDDS in addition to enhancing the dissolution and oral bioavailability often results in low production cost, easy processing, and better patient compliance. Keywords CoQ10 . s-SNEDDS . Ternary phase diagram . Self-dispersibility . Bioavailability

Introduction Oral route remains one of the most popular routes since ancient time due to convenience, but this route often endures the hurdle of lower drug absorption due to poorly aqueous soluble drugs [1]. However, several approaches such as micronization, solubilization, complexation with cyclodextrin, micellar solubilization by surfactants and cosurfactant, microencapsulation, drug dispersion in carriers, solid dispersion, and coprecipitates are being investigated to promote the dissolution rate and absorption of water-insoluble drugs [2]. However, solid self-nanoemulsifying drug delivery system (sSNEDDS) is the better option to improve the solubility and oral bioavailability of lipophilic drugs [3, 4]. CoQ10 are a fat-soluble, vitamin-like, ubiquitous compound that functions as an electron carrier in the mitochondrial respiratory chain, as well as serving as an important endogenous cellular antioxidant. Due to its structure and high

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molecular weight, the aqueous solubility is very low causing slow absorption and low oral bioavailability. The antioxidant property of CoQ10 serves to protect the vital organ heart from circulating low-density lipoprotein. Therapeutically, CoQ10 proved to be useful in cardiovascular disorders, i.e., congestive heart failure, cardiomyopathy, angina pectoris, hypertension, myocardial infarction [5], Parkinson disease [6], diabetes mellitus [7], asthenozoospermia (make infertility or low sperm motility) [8], cancer [9], and periodontal disease [10]. Several work reported for improving bioavailability such as solid dispersion of CoQ10 with tyloxapol [11], formulation with different solubilizing agent, hydrogenated lecithin [12], and complexes with beta cyclodextrin [13]. In most of these formulations, bioavailability is low because of extreme hydrophobic nature of CoQ10. Self-emulsifying drug delivery system (SEDDS) of CoQ10 formulated in peanut oil resulted in twofold increase in oral bioavailability [1]. Balakrishnan and coworkers prepared SEDDS of CoQ10 comprised of 45 % (v/v) Labrasol, 25 % (v/v) Labrafil M 1944 CS, and 10 % (v/v) Capryol 90. The mean oil droplet size of emulsion was 240 nm. The twofold increase in oral bioavailability of formulation compared to powder suspension with drug loading (4 % w/w) was reported [14]. Nazzal and Khan evaluated self-nanoemulsified drug delivery system of ubiquinone by using Polyoxyl 35 castor oil and lemon oil in which drug emulsified within 10 min and drug release range varied from 11 % to 102.3. The percentage of drug loaded was 30 % (w/w) in lemon oil but this oil is volatile in nature [15]. Nepal and coworkers prepared CoQ10-loaded semisolid SNEDDS in a capsule shell which was based on WITEPSOL® H35 as oil phase, Solutol® HS15 as surfactant, and Lauroglycol® FCC as cosurfactant, and 4.4-fold increase in bioavailability was reported. The process of selfemulsification from the capsule shell was delayed due to some part of the formulation embedded in gelled hydroxypropyl methylcellulose (HPMC) pieces [16]. The capsule shell with liquid or semisolid SNEDDS possesses the problem of volatile solvents which has a tendency to evaporate into a shell that lead to drug precipitation [17]. The release profile from such a dosage form may be delayed and altered. The objective of the this study was to enrich the oral bioavailability of poorly soluble drug CoQ10 through optimizing the formulation by constructing phase diagram, particle size estimation, and in vitro and in vivo evaluation.

Materials and Methods CoQ10 was a gift sample from Sami Lab (Bangalore, India). Capryol 90, Transcutol P, Lauroglycol FCC, Labrafac CC, Labrasol, and Plurol olique CC 49 were provided by Colorcon Asia Pacific (Mumbai, India). Aerosil 300 (colloidal silicon

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dioxide) was obtained from Ranbaxy (Pontaside, India). Tween 80 and Avicel were purchased from S.D. Fine Chemical (Mumbai, India). Soyabean oil, almond oil, mustard oil, coconut oil, and rice bran oil were purchased from SigmaAldrich Chemicals (Banglore, India). Male Wistar rats were procured from Central Animal House Facility, NIMS University (Jaipur, India). All other materials and reagents used were of analytical grade. Solubility and Partition Coefficient in Lipid Vehicles The solubility of drug was performed by using shake flask method. An excess amount of CoQ10 was added to cap vial containing 2 ml of the lipid vehicles. After sealing, the mixture was vortexed using a cyclomixer (Remi, India) for 5 min, at a maximum speed to facilitate proper mixing of drug within the lipid vehicle. Mixtures were shaken in a water-bath shaker (Remi, India) maintained at room temperature until equilibrium (72 h) was attained. The resulting mixture was centrifuged at 3,000 rpm for 20 min (Remi, India). The supernatant was separated and extracted in methanol, and quantification of CoQ10 was determined using UV spectrophotometer at 275 nm. Furthermore, the partition coefficient was determined in octanol-water system in a separating funnel. Each phase comprised of equal volume, and 100 mg CoQ10 was transferred to the mixture of solvent. The funnel was shaken vigorously and then clamped in stand for 24 h (shaking in between) to effect partitioning. After 24 h, sample from each solvent was taken and diluted to suitable proportion, and absorbance was measured using UV spectrophotometer at 275 nm. Construction of Ternary Phase Diagram From solubility studies of drug in different lipid vehicle, Lauroglycol FCC was selected as oil phase. Labrasol and Transcutol P were used as surfactant and cosurfactant. Surfactant and cosurfactant (Smix) were mixed in different weight ratios 1:1, 1:2, 1:3, 2:1, 3:1 and 4:1. These Smix were prepared in increasing concentration of surfactant with respect to cosurfactant and vice versa for detailed study of the phase diagrams for identification of self-nanoemulsifying area. For each phase diagram, oil and specific Smix were mixed thoroughly in different weight ratios from 1:9 to 9:1 in different glass vials. The different combinations of oil and Smix 1:9, 2:8 (1:4), 3:7, 4:6 (2:3), 5:5 (1:1), 6:4, 7:3, 8:2 (4:1), and 9:1 were made to delineate the phase boundary. Slow titration with aqueous phase (drop by drop) was made to each weight ratio of oil and Smix, and visual observation was carried out for transparent and easily flowable oil-in-water (o/w) nanoemulsions. The physical state of the formulation was marked on the apices of the triangle of a three-component

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phase diagram representing aqueous phase, oil phase, and Smix ratio. Stress Testing on Selected Formulation from Phase Diagram From each phase diagram constructed, different formulations were selected from self-nanoemulsifying region so that drug could be incorporated into oil phase. The selected dose of CoQ10 was 25 mg for incorporation into oil phase. The oil concentration should be such that it solubilizes the drug (single dose) completely depending on the solubility of the drug in the oil. The thermodynamic stability study was conducted on selected formulations by exposing to high and low temperature 50 and 5 °C separately inside a chamber with maintained temperature for about 48 h. The stable formulations were further centrifuged (Remi, India) at 3,000 rpm for 40 min to observe phase separation, and finally, passed formulations were exposed to freeze thaw cycle in triplicate at each temperature between −21 and +25 °C for not less than 48 h. Those formulations, which passed these thermodynamic stress tests, were further taken for the dispersibility test for assessing the efficiency of self-emulsification. Self-Dispersibility Test The efficiency of self-emulsification of oral nanoemulsion was assessed for each formulation of 0.1 ml was added to 100 ml of water at 37±0.5 °C. A standard stainless steel dissolution paddle rotating at 50 rpm provided gentle agitation. The tendency to emulsify spontaneously and the progress of emulsion droplet spread were visually assessed using the grading criteria. Grade A Rapidly forming (within 1 min) nanoemulsion, having a clear or bluish appearance Grade B Rapidly forming, slightly less clear emulsion, having a bluish white appearance Grade C Fine milky emulsion formed within 2 min. Grade D Dull, grayish white emulsion having slightly oily appearance that is slow to emulsify (>2 min) Grade E Formulation, exhibiting either poor or minimal emulsification with large oil globules present on the surface

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the oil, vortex mixed, and aqueous phase added with gentle agitation, and resulting mixture gave nanoemulsion. Since the prepared formulation was a self-nanoemulsifying system, therefore, water has been excluded from the nanoemulsion. Characterization of l-SNEDDS The l-SNEDDS formulation (0.1 ml) was diluted to 100 ml with distilled water in a volumetric flask. The flask was inverted and shaken gently at room temperature. The emulsion droplet size was measured using a Zetasizer 1000 HS (Malvern Instruments, UK). The light scattered from zigzag movement of the particle in formulation was observed at an angle of 90° at 25 °C. The viscosity of formulation was determined without dilution by Searle type R/SCPS Plus Rheometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA) using spindle # C 50-1 at 25± 0.5 °C. Moreover, the morphology and structure of the lSNEDDS was also studied using transmission electron microscopy (TEM) (Morgagni 268D, Netherland) operating at 200 kV capable of point-to-point resolution. For this study, formulation was diluted up to 100 times with distilled water, a drop of formulation was directly deposited on the holey film grid, and after drying, observation was made. A combination of bright-field imaging at increasing magnification and of diffraction modes was used to reveal the morphology and size of the formulation. In Vitro Drug Release In vitro drug release study of three l-SNEDDSs EN1 (1.5 ml), EN2 (2 ml), and EN3 (3 ml), CoQ10-loaded s-SNEDDS (equivalent to 25 mg CoQ10), marketed formulation, and CoQ10 powder with same dose of CoQ10 (25 mg) were placed in preset assembly of USP dissolution apparatus II containing 900 ml of distilled water at 37±0.5 °C. The speed of the paddle was adjusted to 75 rpm [18]. At predetermined time intervals, 1-ml aliquot of the sample was withdrawn, filtered through membrane filter of size 0.45 μm, and analyzed for CoQ10 content by UV spectrophotometer at 275 nm. The withdrawal amount of sample volume was replaced with a fresh medium, and the entire experiment was repeated in triplicate. Preparation of s-SNEDDS

Preparation of Liquid SNEDDS Those formulations which passed the self-dispersibility test having least Smix concentration were selected at a difference of 5 % w/w of oil from phase diagram. The liquid (l-SNEDDS) formulations were prepared by dissolving 25 mg of CoQ10 in 10, 15, and 20 % of oil, and respective Smix ratio was added to

The formulation amount of AEROSIL 300 (100 mg) was suspended in 100 ml ethanol by magnetic stirring. The lSNEDDS of EN1 formulation (1.5 ml) was introduced with constant stirring at room temperature for 20 min to obtain a good suspension of AEROSIL 300. The suspension was spray-dried in a laboratory spray dryer apparatus (Buchi, Switzerland) under the control condition of inlet temperature

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55 °C, outlet temperature 40 °C, and feeding rate of the suspension 5 ml/min. The final drug content of the sSNEDDS was 10.2 % w/w ratio.

Table 1 Solubility values CoQ10 in various lipid vehicles, mean±SD (n=3) Excipients

Solubility (mg/ml) mean±SD (n=3)

Characterization of s-SNEDDS

Labrasol Capryol 90 Transcutol P Lafrafac CC

117.6±16.60 89±10.50 98±5.50 95±7.40

The s-SNEDDS formulation (equivalent to 25 mg of CoQ10) was diluted to 100 ml with distilled water in a volumetric flask. The flask was inverted and shaken gently at room temperature. The particle size of the nanoemulsion was measured with the same aforementioned instrument. The physical state of CoQ10 in s-SNEDDS was characterized by differential scanning calorimetry (Pyris 4 DSC, Perkin Elmer, USA). The samples of about 5 mg were placed in standard aluminum pans and heated at a scanning rate of 10 °C/min from 30 to 100 °C using dry nitrogen gas as effluent gas. The drugexcipient interaction study was performed using Fourier transform infrared spectroscopy (FT-IR). CoQ10 (20 mg) was triturated with finely powdered and dried potassium bromide (KBr). The mixture was carefully grinded, spread uniformly in a die cavity to give a spectrum of suitable intensity. A background scan of KBr was taken, and subsequently, CoQ10 with various excipients in KBr was carried out in the range of 4,000–40 cm−1. Moreover, shape and surface topography of the s-SNEDDS was investigated by scanning electron microscopy (SEM Hitachi, Japan) fixing the sample on a brass stub using double-sided adhesive tape. The sample was made electro-conductive by coating with gold in vacuum using Hitachi Ion Sputter (E-1030) at 15 mA and particle size investigated using SEM images with an image analysis system (ImageInside ver 2.32). Furthermore, X-ray diffraction (X RD) pattern of s-SNEDDS were carried out with an X’Pert PRO diffractometer (PANalytical X'pert PRO, Netherland) at room temperature using monochromatic CuKa-radiation (k= 1.5406 Å) at 30 mA, 40 kVover a range of 2 theta angles from 0° to 80°. Pharmacokinetic Study The in vivo protocol was approved by the animal ethical committee (approval no. NU/TH/THD/13/99), NIMS University, and their guidelines were followed for studies. The in vivo study of optimized s-SNEDDS, CoQ10 powder, and marketed formulation of CoQ10 were examined in male Wistar rats. The rat weighing 240–300 g obtained from central animal facilities, NIMS University, Jaipur. Animals were free to access water and food under controlled laboratory condition and fasted for 12 h before injecting the different dosage form. They were divided into three groups; each group comprised of three pair of animal. The dose administered was 25 mg/kg from s-SNEDDS, CoQ10 powder, and marketed formulation calculated based on the body weight of the animal that complies with no observed adverse effect level by applying safety

Lauroglycol FCC Tween 80 Plurol olique CC 49 Soybean Almond Rice bran oil Mustard oil Coconut oil

118±13.90 93±2.50 78.9±13.00 76±8.30 90±2.00 102±11.50 59±5.40 93±5.00

factor [19]. Rats were trained to take the liquid formulation voluntarily from a syringe which is effective in accurate dosing [20]. The blood samples were withdrawn for each formulation from the retro-orbital puncture of the rat at predetermined time interval of 0, 0.3, 1, 2, 3, 4, 10, 20, 32, and 50 h in tight screw-capped evacuator tubes coated with disodium EDTA and 200 μl of plasma collected by centrifuging blood samples at 3,500 rpm for 15 min. Plasma samples were stored in the dark at −80 °C until further analysis.

HPLC Analysis Plasma (200 μl) was supplemented with 50 μl of a 1,4benzoquinone solution (2 mg/ml) to oxidize the CoQ10 and vortex mixed. After 10 min, 1 ml of n-propanol was added, vortex mixed, and centrifuged at 10,000 rpm for 2 min so that protein precipitate settled down and the supernatant obtained was transferred in a screwcapped test tube. The vacuum pump evaporator was used to evaporate the supernatant (1 ml). The residue left was reconstituted with 200 μl of n-propanol, and 100 μl of the resulting solution was injected into HPLC for analysis of CoQ10 content. The HPLC system was used (SHIMADZU (AT vp), Japan) consisting of a UV detector (SCL-10A vp), a pump system (LC-10 AT vp), and an injector. The wavelength for the analysis was set at 275 nm, and Colligen®100 column (RP 18 (C18) (5 μm, 250×4.6 mm)) eluted gradiently with a mobile phase, methanol, and n-hexane (80:20 % v/v) at a flow rate of 1 ml/min previously filtered through 0.45-μ membrane filter. The drawn calibration curve was linear over the range of 0.01 to 10 μg/ml and validated within acceptable range (R2 =0.999), and LOD and LOQ were 0.0032 and 0.01 μg/ml, respectively.

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Fig. 1 a–f Ternary phase diagrams of l-SNEDDS for different Smix ratios 1:1 (a), 1:2 (b), 1:3 (c), 2:1 (d), 3:1 (e), and 4:1 (f) representing Smix %, oil %, and water % to the corresponding apices of the triangle. Dark spot indicates o/w self-nanoemulsifying region. Oil, Lauroglycol FCC; Smix, surfactant (Labrasol), cosurfactant (Transcutol P)

Pharmacokinetic Analysis Pharmacokinetic analysis was carried out using the WinNonlin software (version 5.2.1, Pharsight Corp.,

Mountain View, CA, USA) with a noncompartmental model. The maximum concentration (Cmax) of CoQ10 and time to maximum concentration (Tmax) were determined by visual inspection of the concentration-time profile. The area under

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Fig. 2 Emulsion droplet size distribution of l-SNEDDS (a) and s-SNEDDS (b)

the curve (AUC0-12) of plasma concentration-time profile from 0 to 12 h was calculated using the linear/log trapezoidal method. The pharmacokinetic data of different formulations were compared for statistical significance by the one-way ANOVA followed by Tukey-Kramer multiple comparisons test using the GraphPad Instat software (GraphPad Software Inc., CA, USA).

Results and Discussion Screening of Lipid Vehicles The selected excipients were generally regarded as safe (GRAS), approved, and pharmaceutically acceptable for oral administration. The higher solubility of drug in oil is the prerequisite condition for SNEDDS formulation, and chances of drug precipitation are lowered. The oil comprised of a mixture of triglycerides of varying chain length and different degrees of unsaturation [21]. The oil in formulation has a significant role in emulsification process and spreading characteristics and amount of drug loaded in SNEDDS [16, 22]. For determining the solubility of drug in these components, various lipid vehicles of high purity were selected and the result is shown in Table 1. The solubility of CoQ10 in Lauroglycol FCC, Labrasol, and Transcutol P were found 118 ±13.90, 117.6±16.60, and 98±5.50 mg/ml. From these finding, Labrasol was chosen as surfactant because of higher solubilizing capacity, Transcutol P as cosurfactant, and Lauroglycol FCC as oil. The blends of surfactant and cosurfactant (high and low HLB value) are suitable for optimum of l-SNEDDS formulation which shows better drug loading capacity [2, 3]. The effect of Labrasol on the enhanced absorption of insulin in rat is being reported previously [23]. It may be attributed to the fact that it inhibits efflux of CoQ10 from the enterocytes to the lumen of the GI tract and enhances the CoQ10 absorption [24]. Prasad and associates further described Labrasol is a potential absorption enhancer through

enhancing the membrane permeability and maintaining high concentration of drug across the intestinal wall [25]. Labrasol increases absorption of Pg-substrate and enhanced the absorption of rifampicin in rat model [26]. When Transcutol P was combined with Labrasol, a significant amount of oil could be solubilized into the surfactant solutions. Xi and associates investigated the positive effect of Transcutol P as cosurfactant on the droplet size of the stable emulsion. An optimum concentration of cosurfactant is required to form the least droplet size of emulsion [27]. This may be attributed to the fact that addition of cosurfactant along with surfactant causes stabilized interfacial film to expand and further lowers the interfacial tension between oil and water phase [28]. The partition coefficient of CoQ10 in octanol-water system was determined, and log P was found 4.23 indicating a hydrophobic nature of the drug. Octanol is an organic or oily phase corresponding to oil in l-SNEDDS.

Construction of Ternary Phase Diagram In phase diagram of Smix ratio 1:1 (Fig. 1a), when the equal amount of cosurfactant was added with surfactant, only 7 % w/ w of oil could be solubilized with the Smix concentration of 28.1 % w/w. When cosurfactant concentration was further increased to make Smix ratio 1:2 (Fig. 1b), it was observed that the self-nanoemulsifying area increased and oil solubilized up to 27.6 % with Smix concentration of 41.4 % w/w. Moreover, the concentration of cosurfactant increased to make Smix ratio 1:3 in which self-nanoemulsifying area decreased, only 14.8 % w/w of oil remained dissolved at high Smix concentration 59.3 % w/w (Fig. 1c). On reversing the order, i.e., Smix ratio 2:1, surfactant concentration increased twice than cosurfactant as shown in (Fig. 1d), the self-nanoemulsifying area increased slightly as compared to 1:1 and 1:2 Smix ratio, and here, only 18.2 % w/w oil could be solubilized with Smix concentration of 42.4 % w/w. At 3:1 S mix ratio (Fig. 1e), the selfnanoemulsifying area increased and maximum amount of oil that could be solubilized was 20.7 % w/w corresponding to 48.3 % w/w Smix concentration. At 4:1 Smix ratio, self-

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Fig. 3 Negative staining TEM image of l-SNEDDS of EN1 formulation (a) and SEM image of s-SNEDDS (b)

nanoemulsifying area was observed constant as compared to 3:1 Smix ratio followed by the same amount of oil 20.7 % w/w had to be solubilized in 48.3 % w/w Smix concentration (Fig. 1f). Low surfactant concentration with better selfemulsification was considered for studying phase diagram. The surfactant increases interfacial area of oil-water interface and hence alters the dispersion entropy which is required for spontaneity and thermodynamic stability of selfnanoemulsifying system [16, 29]. Selection of Formulation from Phase Diagram Those formulations which formed clear or slightly bluish o/w nanoemulsion in the phase diagram were selected for further studies. In stress testing, formulations were exposed to highspeed centrifugation, changing temperature condition heating or cooling (H/C cycle) and freeze thaw cycle. Those formulations, which survived thermodynamic stability tests, were taken for dispersibility test. In this test, the formulations which were categorized as Grade A and B will form nanoemulsion in the gastrointestinal tract. Keeping the criteria Fig. 4 Overlay DSC curve: CoQ10 (a), CoQ10 with AEROSIL 300 (b), CoQ10 with Avicel (c), and s-SNEDDS (d)

of increasing oil concentration and minimum amount of surfactant used for its solubilization following formulation were selected. The optimized l-SNEDDS have the following composition: (a) Lauroglycol FCC 20 % (w/w), Labrasol 10 % (w/ w), and Transcutol P 20 % (w/w) for formulation EN1 at Smix ratio (1:2) and oil-to-Smix ratio (2:3); (b) Lauroglycol FCC 15 % (w/w), Labrasol 23.34 % (w/w), and Transcutol P 11.67 % (w/w) for formulation EN2 at Smix ratio (2:1) and oil-to-Smix ratio (3:7); and (c) Lauroglycol FCC 10 % (w/w), Labrasol 32 % (w/w), and Transcutol P 8 % (w/w) for formulation EN3 at Smix ratio (4:1) and oil-to-Smix ratio (1:4). The optimized formulations were taken for globule size, viscosity determination, and in vitro release study. Characterization of l-SNEDDS The droplet size of nanoemulsion is considered to be critical for self-emulsification performance and in vivo evaluation of formulation [30, 31]. The emulsion droplet size analysis of the l-SNEDDS formulation showed that size increased with increase in oil concentration in the formulation. The emulsion

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Fig. 5 Fourier transform infrared spectroscopy: CoQ10 (a), CoQ10 with Avicel (b), CoQ10 with AEROSIL 300 (c), and sSNEDDS (d)

droplet size distribution of l-SNEDDS of EN1 formulation and s-SNEDDS formulation are expressed in (Fig. 2). The droplet size of optimized l-SNEDDS was further analyzed by TEM and found that size of emulsion droplet varying considerably with the concentration of oil in the formulation. Three formulations (EN1, EN2, and EN3) containing 20, 15, and 10 % of oil, respectively, were analyzed for droplet size. The mean droplet size of formulation EN1, EN2, and EN3 was appeared as 34, 31.40, and 26.46 nm, respectively. The difference in size was not statistically significant (P>0.05). The polydispersity index (PDI) of EN1, EN2, and EN3 formulations was measured 0.25, 0.39, and 0.42, respectively. The minimum PDI of formulation EN1 confirmed the spherical shape and uniform globule size despite larger globule size. The TEM analysis suggested the droplet size of formulation EN1 was stable and showed similar result with size obtained from Zetasizer as shown in (Fig. 3). Moreover, these formulations were characterized for rheological property. The viscosity of EN1, EN2, and EN3 formulations were determined 12 ± 1.23, 18.5 ± 2.34, and 14.6 ± 2.2 cP indicated that

Fig. 6 X-ray diffraction pattern: CoQ10 powder (a) and s-SNEDDS (b)

optimized EN1 formulation was less viscous than other formulations. The mean droplet size, PDI and viscosity of sSNEDDS on post dilution were determined 35.6 nm, 0.28 and 13±1.03 further substantiated that solid SNEDDS could preserved the characteristics of liquid formulation. Characterization of s-SNEDDS The DSC curve of CoQ10, CoQ10 with AEROSIL 300, CoQ10 with Avicel, and s-SNEDDS are shown in (Fig. 4). The drug has a melting point of 50 °C, and no change was observed with AEROSIL 300 and Avicel to the entire range of temperature (30–100 °C). The absence of conspicuous endothermic peak in s-SNEDDS formulation revealed that drug present in the formulation may be converted to an amorphous form. Furthermore, the characteristic IR peak of CoQ10 with potassium bromide appeared for alkenyl (=CH) stretching at 2,930 cm−1, for alkyl (-CH3) stretching at 2,848 cm−1, for carbonyl (-C=O) stretching at 1,610 cm−1, for methoxy (-OCH3) stretching at 1,382 cm−1, and for ether (-C-O-C-) stretching at 1,238 cm−1. All the peaks in CoQ10 were appeared along with AEROSIL 300 and Avicel indicating no drug-excipient interaction (Fig. 5). The IR spectrum of sSNEDDS formulation has diminished to flat level indicating that compounds remained in a dissolved state in the formulation [32]. The spray-dried s-SNEDDS were also compressed into a tablet; therefore, Avicel was being incorporated with dried SNEDDS as a directly compressible binder. The SEM image of s-SNEDDS was spray-dried. The dried particles were irregular and dispersed because of gellation properties and thixotropic nature of AEROSIL 300 (Fig. 3). AEROSIL

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129 100

Fig. 7 In vitro dissolution profile of three l-SNEDDS formulations (EN1, EN2, and EN3), sSNEDDS, marketed formulation, and CoQ10 powder in distilled water, mean±SD (n=3)

% drug release

80 Formulation EN1 Formulation EN2

60

Formulation EN3 s-SNEDDS 40

Marketed Formulation CoQ10 powder

20

0 0

20

40

60

80

100

120

140

Time (min)

300 improved drug distribution in different particle sizes, and mean diameter of the encapsulated particles depends upon the viscosity of the suspension [33]. The physical state of CoQ10 in s-SNEDDS was further verified from diffraction pattern of X RD. The absence of a distinct peak in s-SNEDDS represented the lack of crystalline structure of CoQ10 in the formulation, and no polymorph of drug was reported on longterm stability (Fig. 6).

significantly higher (P < 0.01) than CoQ10 powder and marketed formulation. The maximum amount of drug released during early time (1.2 h) of dissolution because the selfemulsification time was 10 s or more, and the study was extended for 1 h to observe precipitation of drug from the formulations. For evaluating drug release from such a formulation, surface area of the dispersed oil droplets is considered for in vivo drug absorption and the formed fine oil droplets directly transferred to the intestinal epithelia [34, 35].

In Vitro Drug Release Pharmacokinetic Study Dissolution studies were performed to compare the release of drug from three different l-SNEDDS formulations (EN1 to EN3), s-SNEDDS, marketed formulation, and CoQ10 powder (Fig. 7). Among three l-SNEDDSs, formulation EN1 showed maximum release of drug (98±1.9 %) at the end of 1.20 h converted into s-SNEDDS that gave highest drug release (97.5±4.5 %) at the end of 1.2 h within 2.2 h of study which was not significant statistically (P>0.05). The higher release of drug from EN1 formulation may be attributed to the fact that high oil content (20 % w/w) provided large number of globules, and minimum variation in globule size was due to low PDI, in spite of large globule size of the formulation. CoQ10 powder (0.3±0.06 %) showed negligible release, and marketed formulation showed (57.96±0.54 %) release at the end of 1.2 h. The drug release from s-SNEDDS was

The in vivo study of different formulations s-SNEDDS, lSNEDDS, marketed formulation, and CoQ10 powder was conducted after oral administration in male Wistar rats and pharmacokinetic parameters are shown in Table 2 and Fig. 8. The drug exhibited slow absorption which confirmed the previous reports [14, 16]. The reduction in Tmax implied that rapid absorption of drug from s-SNEDDS due to improved solubilization of drug compared to CoQ10 powder and marketed formulation; however, no statistical significance was established. The s-SNEDDS formulation improved Cmax (3.4-fold), and AUC (5-fold) were significantly higher than CoQ10 powder (P