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 strate- gically selected to enrich oral bioavailability by incorporating in solid self-nanoemulsifying drug delivery system (s- SNEDDS) 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 construct- ed, and selected formulations from ternary phase diagrams were subjected to thermodynamic stability and self- dispersibility 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 (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 im- proved maximum concentration (C max ) (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 poten- tial to enhance the oral bioavailability of CoQ10. Conclusion Thus, s-SNEDDS in addition to enhancing the dissolution and oral bioavailability often results in low pro- duction 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 cyclodex- trin, micellar solubilization by surfactants and cosurfactant, microencapsulation, drug dispersion in carriers, solid disper- sion, and coprecipitates are being investigated to promote the dissolution rate and absorption of water-insoluble drugs [2]. However, solid self-nanoemulsifying drug delivery system (s- SNEDDS) is the better option to improve the solubility and oral bioavailability of lipophilic drugs [3, 4]. CoQ10 are a fat-soluble, vitamin-like, ubiquitous com- pound that functions as an electron carrier in the mitochondrial respiratory chain, as well as serving as an important endoge- nous cellular antioxidant. Due to its structure and high 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]J Pharm Innov (2014) 9:121–131 DOI 10.1007/s12247-014-9179-0
Transcript
RESEARCH ARTICLE
Formulation and Development of CoQ10-Loaded s-SNEDDSfor 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
AbstractPurpose Coenzyme (CoQ10) is a poorly soluble drug strate-gically selected to enrich oral bioavailability by incorporatingin solid self-nanoemulsifying drug delivery system (s-SNEDDS) comprised of oil, surfactant, and cosurfactant.The conventional self-emulsifying drug delivery system(SEDDS) and liquid SNEDDS (l-SNEDDS) usually havethe problem of drug instability and precipitation.Methods The selected oils, surfactant, and cosurfactant withmaximum drug solubility were Lauroglycol FCC, Labrasol,and Transcutol P. The ternary phase diagrams were construct-ed, and selected formulations from ternary phase diagramswere subjected to thermodynamic stability and self-dispersibility test and characterized for emulsion droplet sizeand droplet size distribution. The optimized formulation wascomprised of Lauroglycol FCC 20 % (w/w), Labrasol 10 %(w/w), and Transcutol P 20 % (w/w) as oil, surfactant, andcosurfactant.Results The transmission electron microscopy (TEM) studyof optimized l-SNEDDS reported mean globule size of 34 nmwas transformed into s-SNEDDS by spray-drying techniqueusing solid carrier. The s-SNEDDS was characterized fordifferential scanning calorimetry (DSC), Fourier transforminfrared spectroscopy (FT-IR), scanning electron microscope
(SEM), and X-ray diffraction (X RD). The in vitro releaseprofile 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 ofoptimized s-SNEDDS in male Wistar rats revealed the im-proved maximum concentration (Cmax) (3.4-fold vs CoQ10powder; 1.4-fold vs marketed formulation) and area under thecurve (AUC) (5-fold vs CoQ10 powder; 2-fold vs marketedformulation). With this result, s-SNEDDS could be of poten-tial to enhance the oral bioavailability of CoQ10.Conclusion Thus, s-SNEDDS in addition to enhancing thedissolution and oral bioavailability often results in low pro-duction cost, easy processing, and better patient compliance.
Oral route remains one of the most popular routes sinceancient time due to convenience, but this route often enduresthe hurdle of lower drug absorption due to poorly aqueoussoluble drugs [1]. However, several approaches such asmicronization, solubilization, complexation with cyclodex-trin, micellar solubilization by surfactants and cosurfactant,microencapsulation, drug dispersion in carriers, solid disper-sion, and coprecipitates are being investigated to promote thedissolution rate and absorption of water-insoluble drugs [2].However, solid self-nanoemulsifying drug delivery system (s-SNEDDS) is the better option to improve the solubility andoral bioavailability of lipophilic drugs [3, 4].
CoQ10 are a fat-soluble, vitamin-like, ubiquitous com-pound that functions as an electron carrier in the mitochondrialrespiratory chain, as well as serving as an important endoge-nous cellular antioxidant. Due to its structure and high
M. H. Akhter (*) :A. Ahmad :G. MohanNIMS Institute of Pharmacy NIMS University, Jaipur 303121, Indiae-mail: [email protected]
molecular weight, the aqueous solubility is very low causingslow absorption and low oral bioavailability. The antioxidantproperty of CoQ10 serves to protect the vital organ heart fromcirculating low-density lipoprotein. Therapeutically, CoQ10proved to be useful in cardiovascular disorders, i.e., conges-tive heart failure, cardiomyopathy, angina pectoris, hyperten-sion, myocardial infarction [5], Parkinson disease [6], diabetesmellitus [7], asthenozoospermia (make infertility or low spermmotility) [8], cancer [9], and periodontal disease [10]. Severalwork reported for improving bioavailability such as soliddispersion of CoQ10 with tyloxapol [11], formulation withdifferent solubilizing agent, hydrogenated lecithin [12], andcomplexes with beta cyclodextrin [13]. In most of these for-mulations, bioavailability is low because of extreme hydro-phobic nature of CoQ10.
Self-emulsifying drug delivery system (SEDDS) of CoQ10formulated in peanut oil resulted in twofold increase in oralbioavailability [1]. Balakrishnan and coworkers preparedSEDDS of CoQ10 comprised of 45 % (v/v) Labrasol, 25 %(v/v) Labrafil M 1944 CS, and 10 % (v/v) Capryol 90. Themean oil droplet size of emulsion was 240 nm. The twofoldincrease in oral bioavailability of formulation compared topowder suspension with drug loading (4 % w/w) was reported[14]. Nazzal and Khan evaluated self-nanoemulsified drugdelivery system of ubiquinone by using Polyoxyl 35 castoroil and lemon oil in which drug emulsified within 10 min anddrug release range varied from 11 % to 102.3. The percentageof drug loaded was 30 % (w/w) in lemon oil but this oil isvolatile in nature [15].
Nepal and coworkers prepared CoQ10-loaded semisolidSNEDDS in a capsule shell which was based onWITEPSOL®
H35 as oil phase, Solutol® HS15 as surfactant, andLauroglycol® FCC as cosurfactant, and 4.4-fold increase inbioavailability was reported. The process of self-emulsification from the capsule shell was delayed due to somepart of the formulation embedded in gelled hydroxypropylmethylcellulose (HPMC) pieces [16]. The capsule shell withliquid or semisolid SNEDDS possesses the problem of vola-tile solvents which has a tendency to evaporate into a shell thatlead to drug precipitation [17]. The release profile from such adosage form may be delayed and altered.
The objective of the this study was to enrich the oralbioavailability of poorly soluble drug CoQ10 through opti-mizing the formulation by constructing phase diagram, parti-cle 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 ColorconAsia Pacific (Mumbai, India). Aerosil 300 (colloidal silicon
dioxide) was obtained from Ranbaxy (Pontaside, India).Tween 80 and Avicel were purchased from S.D. Fine Chem-ical (Mumbai, India). Soyabean oil, almond oil, mustard oil,coconut oil, and rice bran oil were purchased from Sigma-Aldrich Chemicals (Banglore, India). Male Wistar rats wereprocured from Central Animal House Facility, NIMS Univer-sity (Jaipur, India). All other materials and reagents used wereof analytical grade.
Solubility and Partition Coefficient in Lipid Vehicles
The solubility of drug was performed by using shake flaskmethod. An excess amount of CoQ10 was added to cap vialcontaining 2ml of the lipid vehicles. After sealing, the mixturewas vortexed using a cyclomixer (Remi, India) for 5 min, at amaximum speed to facilitate proper mixing of drug within thelipid vehicle. Mixtures were shaken in a water-bath shaker(Remi, India) maintained at room temperature until equilibri-um (72 h) was attained. The resulting mixture was centrifugedat 3,000 rpm for 20 min (Remi, India). The supernatant wasseparated and extracted in methanol, and quantification ofCoQ10 was determined using UV spectrophotometer at275 nm.
Furthermore, the partition coefficient was determined inoctanol-water system in a separating funnel. Each phase com-prised of equal volume, and 100 mg CoQ10 was transferred tothe mixture of solvent. The funnel was shaken vigorously andthen clamped in stand for 24 h (shaking in between) to effectpartitioning. After 24 h, sample from each solvent was takenand diluted to suitable proportion, and absorbance was mea-sured 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 andTranscutol P were used as surfactant and cosurfactant. Surfac-tant and cosurfactant (Smix) were mixed in different weightratios 1:1, 1:2, 1:3, 2:1, 3:1 and 4:1. These Smix were preparedin increasing concentration of surfactant with respect to co-surfactant and vice versa for detailed study of the phasediagrams for identification of self-nanoemulsifying area. Foreach phase diagram, oil and specific Smix were mixed thor-oughly in different weight ratios from 1:9 to 9:1 in differentglass 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 weremade to delineate the phase boundary. Slow titration withaqueous phase (drop by drop) was made to each weight ratioof oil and Smix, and visual observation was carried out fortransparent and easily flowable oil-in-water (o/w)nanoemulsions. The physical state of the formulation wasmarked 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 formulationswere selected from self-nanoemulsifying region so that drugcould be incorporated into oil phase. The selected dose ofCoQ10 was 25 mg for incorporation into oil phase. The oilconcentration should be such that it solubilizes the drug (sin-gle dose) completely depending on the solubility of the drug inthe oil.
The thermodynamic stability study was conducted on se-lected formulations by exposing to high and low temperature50 and 5 °C separately inside a chamber with maintainedtemperature for about 48 h. The stable formulations werefurther centrifuged (Remi, India) at 3,000 rpm for 40 min toobserve phase separation, and finally, passed formulationswere exposed to freeze thaw cycle in triplicate at each tem-perature between −21 and +25 °C for not less than 48 h. Thoseformulations, which passed these thermodynamic stress tests,were further taken for the dispersibility test for assessing theefficiency of self-emulsification.
Self-Dispersibility Test
The efficiency of self-emulsification of oral nanoemulsionwas assessed for each formulation of 0.1 ml was added to100 ml of water at 37±0.5 °C. A standard stainless steeldissolution paddle rotating at 50 rpm provided gentle agita-tion. The tendency to emulsify spontaneously and the progressof emulsion droplet spread were visually assessed using thegrading criteria.
Grade A Rapidly forming (within 1 min) nanoemulsion,having a clear or bluish appearance
Grade B Rapidly forming, slightly less clear emulsion, hav-ing 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 onthe surface
Preparation of Liquid SNEDDS
Those formulations which passed the self-dispersibility testhaving least Smix concentration were selected at a difference of5 % w/w of oil from phase diagram. The liquid (l-SNEDDS)formulations were prepared by dissolving 25 mg of CoQ10 in10, 15, and 20 % of oil, and respective Smix ratio was added to
the oil, vortex mixed, and aqueous phase added with gentleagitation, and resulting mixture gave nanoemulsion. Since theprepared 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 mlwith distilled water in a volumetric flask. The flask wasinverted and shaken gently at room temperature. The emulsiondroplet size was measured using a Zetasizer 1000 HS(Malvern Instruments, UK). The light scattered from zigzagmovement of the particle in formulation was observed at anangle of 90° at 25 °C. The viscosity of formulation wasdetermined without dilution by Searle type R/SCPS PlusRheometer (Brookfield Engineering Laboratories, Inc.,Middleboro, MA, USA) using spindle # C 50-1 at 25±0.5 °C. Moreover, the morphology and structure of the l-SNEDDS was also studied using transmission electron mi-croscopy (TEM) (Morgagni 268D, Netherland) operating at200 kV capable of point-to-point resolution. For this study,formulation was diluted up to 100 times with distilled water, adrop of formulation was directly deposited on the holey filmgrid, and after drying, observation was made. A combinationof bright-field imaging at increasing magnification and ofdiffraction modes was used to reveal the morphology and sizeof 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, andCoQ10 powder with same dose of CoQ10 (25 mg) wereplaced in preset assembly of USP dissolution apparatus IIcontaining 900 ml of distilled water at 37±0.5 °C. The speedof the paddle was adjusted to 75 rpm [18]. At predeterminedtime intervals, 1-ml aliquot of the sample was withdrawn,filtered through membrane filter of size 0.45 μm, and ana-lyzed for CoQ10 content byUV spectrophotometer at 275 nm.The withdrawal amount of sample volume was replaced witha fresh medium, and the entire experiment was repeated intriplicate.
Preparation of s-SNEDDS
The formulation amount of AEROSIL 300 (100 mg) wassuspended in 100 ml ethanol by magnetic stirring. The l-SNEDDS of EN1 formulation (1.5 ml) was introduced withconstant stirring at room temperature for 20 min to obtain agood suspension of AEROSIL 300. The suspension wasspray-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 thesuspension 5 ml/min. The final drug content of the s-SNEDDS was 10.2 % w/w ratio.
Characterization of s-SNEDDS
The s-SNEDDS formulation (equivalent to 25 mg of CoQ10)was diluted to 100 ml with distilled water in a volumetricflask. The flask was inverted and shaken gently at roomtemperature. The particle size of the nanoemulsion was mea-sured with the same aforementioned instrument. The physicalstate of CoQ10 in s-SNEDDS was characterized by differen-tial scanning calorimetry (Pyris 4 DSC, Perkin Elmer, USA).The samples of about 5 mg were placed in standard aluminumpans and heated at a scanning rate of 10 °C/min from 30 to100 °C using dry nitrogen gas as effluent gas. The drug-excipient interaction study was performed using Fourier trans-form infrared spectroscopy (FT-IR). CoQ10 (20 mg) wastriturated with finely powdered and dried potassium bromide(KBr). Themixture was carefully grinded, spread uniformly ina die cavity to give a spectrum of suitable intensity. A back-ground scan of KBr was taken, and subsequently, CoQ10 withvarious excipients in KBr was carried out in the range of4,000–40 cm−1. Moreover, shape and surface topography ofthe s-SNEDDS was investigated by scanning electron micros-copy (SEM Hitachi, Japan) fixing the sample on a brass stubusing double-sided adhesive tape. The sample was madeelectro-conductive by coating with gold in vacuum usingHitachi Ion Sputter (E-1030) at 15 mA and particle sizeinvestigated using SEM imageswith an image analysis system(ImageInside ver 2.32). Furthermore, X-ray diffraction (XRD) pattern of s-SNEDDS were carried out with an X’PertPRO diffractometer (PANalytical X'pert PRO, Netherland) atroom temperature using monochromatic CuKa-radiation (k=1.5406 Å) at 30 mA, 40 kVover a range of 2 theta angles from0° to 80°.
Pharmacokinetic Study
The in vivo protocol was approved by the animal ethicalcommittee (approval no. NU/TH/THD/13/99), NIMS Univer-sity, and their guidelines were followed for studies. Thein vivo study of optimized s-SNEDDS, CoQ10 powder, andmarketed formulation of CoQ10 were examined in maleWistar rats. The rat weighing 240–300 g obtained from centralanimal facilities, NIMS University, Jaipur. Animals were freeto access water and food under controlled laboratory conditionand fasted for 12 h before injecting the different dosage form.They were divided into three groups; each group comprised ofthree pair of animal. The dose administered was 25 mg/kgfrom s-SNEDDS, CoQ10 powder, and marketed formulationcalculated based on the body weight of the animal that com-plies with no observed adverse effect level by applying safety
factor [19]. Rats were trained to take the liquid formulationvoluntarily from a syringe which is effective in accuratedosing [20]. The blood samples were withdrawn for eachformulation from the retro-orbital puncture of the rat atpredetermined time interval of 0, 0.3, 1, 2, 3, 4, 10, 20, 32,and 50 h in tight screw-capped evacuator tubes coated withdisodium EDTA and 200 μl of plasma collected by centrifug-ing blood samples at 3,500 rpm for 15 min. Plasma sampleswere stored in the dark at −80 °C until further analysis.
HPLC Analysis
Plasma (200 μl) was supplemented with 50 μl of a 1,4-benzoquinone solution (2 mg/ml) to oxidize the CoQ10and vortex mixed. After 10 min, 1 ml of n-propanolwas added, vortex mixed, and centrifuged at 10,000 rpmfor 2 min so that protein precipitate settled down andthe supernatant obtained was transferred in a screw-capped test tube. The vacuum pump evaporator wasused to evaporate the supernatant (1 ml). The residueleft was reconstituted with 200 μl of n-propanol, and100 μl of the resulting solution was injected into HPLCfor analysis of CoQ10 content. The HPLC system wasused (SHIMADZU (AT vp), Japan) consisting of a UVdetector (SCL-10A vp), a pump system (LC-10 AT vp),and an injector. The wavelength for the analysis was setat 275 nm, and Colligen®100 column (RP 18 (C18)(5 μm, 250×4.6 mm)) eluted gradiently with a mobilephase, methanol, and n-hexane (80:20 % v/v) at a flowrate of 1 ml/min previously filtered through 0.45-μmembrane filter. The drawn calibration curve was linearover the range of 0.01 to 10 μg/ml and validated withinacceptable range (R2=0.999), and LOD and LOQ were0.0032 and 0.01 μg/ml, respectively.
Table 1 Solubility values CoQ10 in various lipid vehicles, mean±SD(n=3)
Excipients Solubility (mg/ml) mean±SD (n=3)
Labrasol 117.6±16.60
Capryol 90 89±10.50
Transcutol P 98±5.50
Lafrafac CC 95±7.40
Lauroglycol FCC 118±13.90
Tween 80 93±2.50
Plurol olique CC 49 78.9±13.00
Soybean 76±8.30
Almond 90±2.00
Rice bran oil 102±11.50
Mustard oil 59±5.40
Coconut oil 93±5.00
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Pharmacokinetic Analysis
Pharmacokinetic analysis was carried out using theWinNonlin software (version 5.2.1, Pharsight Corp.,
Mountain View, CA, USA) with a noncompartmental model.The maximum concentration (Cmax) of CoQ10 and time tomaximum concentration (Tmax) were determined by visualinspection of the concentration-time profile. The area under
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)
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the curve (AUC0-12) of plasma concentration-time profilefrom 0 to 12 h was calculated using the linear/log trapezoidalmethod. The pharmacokinetic data of different formulationswere compared for statistical significance by the one-wayANOVA followed by Tukey-Kramer multiple comparisonstest using the GraphPad Instat software (GraphPad SoftwareInc., CA, USA).
Results and Discussion
Screening of Lipid Vehicles
The selected excipients were generally regarded as safe(GRAS), approved, and pharmaceutically acceptable for oraladministration. The higher solubility of drug in oil is theprerequisite condition for SNEDDS formulation, and chancesof drug precipitation are lowered. The oil comprised of amixture of triglycerides of varying chain length and differentdegrees of unsaturation [21]. The oil in formulation has asignificant role in emulsification process and spreading char-acteristics 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 theresult is shown in Table 1. The solubility of CoQ10 inLauroglycol FCC, Labrasol, and Transcutol P were found118 ±13.90, 117.6±16.60, and 98±5.50 mg/ml. From thesefinding, Labrasol was chosen as surfactant because of highersolubilizing capacity, Transcutol P as cosurfactant, andLauroglycol FCC as oil. The blends of surfactant and cosur-factant (high and low HLB value) are suitable for optimum ofl-SNEDDS formulation which shows better drug loadingcapacity [2, 3].
The effect of Labrasol on the enhanced absorption ofinsulin in rat is being reported previously [23]. It may beattributed to the fact that it inhibits efflux of CoQ10 from theenterocytes to the lumen of the GI tract and enhances theCoQ10 absorption [24]. Prasad and associates further de-scribed Labrasol is a potential absorption enhancer through
enhancing the membrane permeability and maintaining highconcentration of drug across the intestinal wall [25]. Labrasolincreases absorption of Pg-substrate and enhanced the absorp-tion of rifampicin in rat model [26]. When Transcutol P wascombined with Labrasol, a significant amount of oil could besolubilized into the surfactant solutions. Xi and associatesinvestigated the positive effect of Transcutol P as cosurfactanton the droplet size of the stable emulsion. An optimum con-centration of cosurfactant is required to form the least dropletsize of emulsion [27]. This may be attributed to the fact thataddition of cosurfactant along with surfactant causes stabi-lized interfacial film to expand and further lowers the interfa-cial tension between oil and water phase [28]. The partitioncoefficient of CoQ10 in octanol-water system was deter-mined, and log P was found 4.23 indicating a hydrophobicnature of the drug. Octanol is an organic or oily phase corre-sponding to oil in l-SNEDDS.
Construction of Ternary Phase Diagram
In phase diagram of Smix ratio 1:1 (Fig. 1a), when the equalamount of cosurfactant was added with surfactant, only 7 % w/w of oil could be solubilized with the Smix concentration of28.1 % w/w. When cosurfactant concentration was furtherincreased to make Smix ratio 1:2 (Fig. 1b), it was observed thatthe self-nanoemulsifying area increased and oil solubilized upto 27.6 % with Smix concentration of 41.4 % w/w. Moreover,the concentration of cosurfactant increased to make Smix ratio1:3 in which self-nanoemulsifying area decreased, only 14.8 %w/w of oil remained dissolved at high Smix concentration59.3 % w/w (Fig. 1c). On reversing the order, i.e., Smix ratio2:1, surfactant concentration increased twice than cosurfactantas shown in (Fig. 1d), the self-nanoemulsifying area increasedslightly as compared to 1:1 and 1:2 Smix ratio, and here, only18.2 % w/w oil could be solubilized with Smix concentration of42.4 % w/w. At 3:1 Smix ratio (Fig. 1e), the self-nanoemulsifying area increased and maximum amount of oilthat could be solubilized was 20.7 % w/w corresponding to48.3 % w/w Smix concentration. At 4:1 Smix ratio, self-
Fig. 2 Emulsion droplet size distribution of l-SNEDDS (a) and s-SNEDDS (b)
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nanoemulsifying area was observed constant as compared to3:1 Smix ratio followed by the same amount of oil 20.7 % w/whad to be solubilized in 48.3 % w/w Smix concentration(Fig. 1f). Low surfactant concentration with better self-emulsification was considered for studying phase diagram.The surfactant increases interfacial area of oil-water interfaceand hence alters the dispersion entropy which is required forspontaneity and thermodynamic stability of self-nanoemulsifying system [16, 29].
Selection of Formulation from Phase Diagram
Those formulations which formed clear or slightly bluish o/wnanoemulsion in the phase diagram were selected for furtherstudies. In stress testing, formulations were exposed to high-speed centrifugation, changing temperature condition heatingor cooling (H/C cycle) and freeze thaw cycle. Those formu-lations, which survived thermodynamic stability tests, weretaken for dispersibility test. In this test, the formulationswhich were categorized as Grade A and B will formnanoemulsion in the gastrointestinal tract. Keeping the criteria
of increasing oil concentration and minimum amount of sur-factant used for its solubilization following formulation wereselected. The optimized l-SNEDDS have the following com-position: (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 FCC15 % (w/w), Labrasol 23.34 % (w/w), and Transcutol P11.67 % (w/w) for formulation EN2 at Smix ratio (2:1) andoil-to-Smix ratio (3:7); and (c) Lauroglycol FCC 10 % (w/w),Labrasol 32 % (w/w), and Transcutol P 8 % (w/w) forformulation 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 criticalfor self-emulsification performance and in vivo evaluation offormulation [30, 31]. The emulsion droplet size analysis of thel-SNEDDS formulation showed that size increased with in-crease in oil concentration in the formulation. The emulsion
Fig. 3 Negative staining TEM image of l-SNEDDS of EN1 formulation (a) and SEM image of s-SNEDDS (b)
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droplet size distribution of l-SNEDDS of EN1 formulationand s-SNEDDS formulation are expressed in (Fig. 2). Thedroplet size of optimized l-SNEDDS was further analyzed byTEM and found that size of emulsion droplet varying consid-erably with the concentration of oil in the formulation. Threeformulations (EN1, EN2, and EN3) containing 20, 15, and10 % of oil, respectively, were analyzed for droplet size. Themean droplet size of formulation EN1, EN2, and EN3 wasappeared as 34, 31.40, and 26.46 nm, respectively. The dif-ference in size was not statistically significant (P>0.05). Thepolydispersity index (PDI) of EN1, EN2, and EN3 formula-tions was measured 0.25, 0.39, and 0.42, respectively. Theminimum PDI of formulation EN1 confirmed the sphericalshape and uniform globule size despite larger globule size.The TEM analysis suggested the droplet size of formulationEN1 was stable and showed similar result with size obtainedfrom Zetasizer as shown in (Fig. 3). Moreover, these formu-lations were characterized for rheological property. The vis-cosity of EN1, EN2, and EN3 formulations were determined12±1.23, 18.5±2.34, and 14.6±2.2 cP indicated that
optimized EN1 formulation was less viscous than other for-mulations. The mean droplet size, PDI and viscosity of s-SNEDDS on post dilution were determined 35.6 nm, 0.28and 13±1.03 further substantiated that solid SNEDDS couldpreserved 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 wasobserved with AEROSIL 300 and Avicel to the entire range oftemperature (30–100 °C). The absence of conspicuous endo-thermic peak in s-SNEDDS formulation revealed that drugpresent in the formulation may be converted to an amorphousform. Furthermore, the characteristic IR peak of CoQ10 withpotassium bromide appeared for alkenyl (=CH) stretching at2,930 cm−1, for alkyl (-CH3) stretching at 2,848 cm−1, forcarbonyl (-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 ap-peared along with AEROSIL 300 and Avicel indicating nodrug-excipient interaction (Fig. 5). The IR spectrum of s-SNEDDS formulation has diminished to flat level indicatingthat compounds remained in a dissolved state in the formula-tion [32]. The spray-dried s-SNEDDS were also compressedinto a tablet; therefore, Avicel was being incorporated withdried SNEDDS as a directly compressible binder. The SEMimage of s-SNEDDS was spray-dried. The dried particleswere irregular and dispersed because of gellation propertiesand thixotropic nature of AEROSIL 300 (Fig. 3). AEROSILFig. 6 X-ray diffraction pattern: CoQ10 powder (a) and s-SNEDDS (b)
300 improved drug distribution in different particle sizes, andmean diameter of the encapsulated particles depends upon theviscosity of the suspension [33]. The physical state of CoQ10in s-SNEDDS was further verified from diffraction pattern ofX RD. The absence of a distinct peak in s-SNEDDS repre-sented the lack of crystalline structure of CoQ10 in the for-mulation, and no polymorph of drug was reported on long-term stability (Fig. 6).
In Vitro Drug Release
Dissolution studies were performed to compare the release ofdrug from three different l-SNEDDS formulations (EN1 toEN3), s-SNEDDS, marketed formulation, and CoQ10 powder(Fig. 7). Among three l-SNEDDSs, formulation EN1 showedmaximum release of drug (98±1.9 %) at the end of 1.20 hconverted 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 whichwas not significant statistically (P>0.05). The higher releaseof drug from EN1 formulation may be attributed to the factthat high oil content (20 % w/w) provided large number ofglobules, and minimum variation in globule size was due tolow PDI, in spite of large globule size of the formulation.CoQ10 powder (0.3±0.06 %) showed negligible release, andmarketed formulation showed (57.96±0.54 %) release at theend of 1.2 h. The drug release from s-SNEDDS was
significantly higher (P<0.01) than CoQ10 powder andmarketed formulation. Themaximum amount of drug releasedduring early time (1.2 h) of dissolution because the self-emulsification time was 10 s or more, and the study wasextended for 1 h to observe precipitation of drug from theformulations. For evaluating drug release from such a formu-lation, surface area of the dispersed oil droplets is consideredfor in vivo drug absorption and the formed fine oil dropletsdirectly transferred to the intestinal epithelia [34, 35].
Pharmacokinetic Study
The in vivo study of different formulations s-SNEDDS, l-SNEDDS, marketed formulation, and CoQ10 powder wasconducted after oral administration in male Wistar rats andpharmacokinetic parameters are shown in Table 2 and Fig. 8.The drug exhibited slow absorption which confirmed theprevious reports [14, 16]. The reduction in Tmax implied thatrapid absorption of drug from s-SNEDDS due to improvedsolubilization of drug compared to CoQ10 powder andmarketed formulation; however, no statistical significancewas established. The s-SNEDDS formulation improved Cmax
(3.4-fold), and AUC (5-fold) were significantly higher thanCoQ10 powder (P<0.001). Compared to marketed formula-tion, s-SNEDDS formulation gave significantly higher Cmax
(1.4-fold) (P<0.01) and AUC (∼2-fold) (P<0.001).
0
20
40
60
80
100
0 20 40 60 80 100 120 140
Time (min)
% d
rug
rel
ease
Formulation EN1
Formulation EN2
Formulation EN3
s-SNEDDS
Marketed Formulation
CoQ10 powder
Fig. 7 In vitro dissolution profileof three l-SNEDDS formulations(EN1, EN2, and EN3), s-SNEDDS, marketed formulation,and CoQ10 powder in distilledwater, mean±SD (n=3)
J Pharm Innov (2014) 9:121–131 129
Table 2 Pharmacokinetic parameters of s-SNEDDS, CoQ10 powder, and marketed formulation after an oral administration in rats, mean±SD (n=3)
**P<0.01, compared with marketed formulation; *** P<0.001, compared with CoQ10 powder
The fine droplets of o/w nanoemulsion formed on mildagitation of s-SNEDDS in GI fluid rationalize the advantageof laying out the drug in a molecular state with a largeinterfacial surface area for absorption and better rate of drugpartitioning into the aqueous GI fluids [36, 37]. The previouswork on SEDDS [14] and SNEDDS [16] of CoQ10 can becomparable to this study pertaining to improvement in AUC(5-fold of s-SNEDDS vs 2.4-fold of SEDDS) (5-fold s-SNEDDS vs 4.4-fold SNEDDS) and reduction in Tmax
(4 h s-SNEDDS vs 6 h SNEDDS). Comparative plasmaprofile showed that Cmax of s-SNEDDS was 2±0.30 μg/mlvs 480±347 ng/ml, and AUC of s-SNEDDS was 38.55±3.42 μgh/ml vs 5,070±2,770 ngh/ml of SNEDDS. The drugrelease from SNEDDS of HPMC capsule triggered with initiallag in self-emulsification (∼10 min) to burst probably imbib-ing of GI fluid into the capsule shell followed by gellation,shell rupture, and release. The parts of SNEDDS formulationcling to the gelled HPMC pieces which may delay the drugrelease and impair release profile [38]. Furthermore, the ad-vantage of converting into solid form strategically is to main-tain the stability, robustness, encompassing the problem ofleakage, and interaction of liquid component with capsuleshell [17, 39]. With this result, it was concluded that s-SNEDDS is an effective formulation approach to improvethe oral bioavailability of CoQ10.
Stability Study
In this study, percentage degradation of CoQ10 was assessedin s-SNEDDS and l-SNEDDS. The formulations were placedin stability chamber at 5±3 °C, 30±2 °C/65±5 % RH, and 40±2 °C/75±5%RH and analyzed for drug content in 0, 30, and90 days, respectively, as per ICHguideline is shown inTable 3.The stability result showed that maximum degradation of drugfrom s-SNEDDS and l-SENDDS were 0.66 and 3.13 % at 40±2 °C at end of 90 days.
Conclusion
In this study, CoQ10-loaded s-SNEDDS was prepared byspray-drying technique in which optimized l-SNEDDS wastransformed into s-SNEDDS using AEROSIL 300 as a poroussolid carrier. The s-SNEDDS preserved all the characteristicsof l-SNEDDS in the formulation. The DSC FT-IR studyrevealed that CoQ10 was present in a molecularly dissolvedstate in the formulation. The X RD of s-SNEDDS reportedthat no polymorph of CoQ10 was formed in s-SNEDDS.Comparative in vitro study of the optimized s-SNEDDSshowed faster release than CoQ10 powder and marketedformulation. The in vivo study in rats gave significantly higherAUC and Cmax than CoQ10 powder and marketed formula-tion. Therefore, this technology could be explored in enhanc-ing the dissolution and oral bioavailability of poorly aqueoussoluble drug, CoQ10 by developing in solid form.
Acknowledgments Authors are grateful to Jamia Hamdard (HamdardUniversity), All India Medical Science (AIIMS), Advanced Instrumenta-tion Research Facility (AIRF), Jawaharlal Nehru University (JNU), NewDelhi, and GLA University for providing facilities of Zeta sizer, TEM,SEM, X RD, and FT-IR. The authors are also thankful to Sami Lab(Bangalore, India) for providing a gift sample of CoQ10.
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