Journal of Drug Delivery Science and Technology · ground [16]. The optical clarity of each point in the phase diagram was measured to conﬁrm the clarity of the observed nano-emulsion

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Journal of Drug Delivery Science and Technology

journal homepage: www.elsevier.com/locate/jddst

Bioavailability enhanced clopidogrel -loaded solid SNEDDS: Developmentand in-vitro/in-vivo characterization

Eman Abd-Elhakeem, Mahmoud HM. Teaima, Ghada A. Abdelbary∗, Galal M. El MahroukDepartment of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt

A R T I C L E I N F O

Keywords:ClopidogrelS-SNEDDSBioenhancerPharmacokinetic studyLC/MS/MS

A B S T R A C T

The purpose of this study is to develop solid self-nanoemulsifying drug delivery systems (S-SNEDDS) for oralbioavailability enhancement of Clopidogrel (CLP). CLP is an anti-platelet drug used to prevent heart strokes. Itsuffers from low bioavailability due to extensive hepatic metabolism. Two pseudo-ternary phase diagrams wereconstructed using different oils, bioenhancing surfactants and co-surfactants. SNEDDS were evaluated for theirglobule size, polydispersity index and in-vitro drug release. The optimum SNEDDS were adsorbed ontoAeroperl®300 to produce S-SNEDDS. Results showed that SNEDDS-8 (SII8) consisting of 10% Capryol™90, 10%Cremophore®EL and 80% Transcutol®HP possessed the smallest globule size and high % drug release.Furthermore, the optimum S-SNEDDS formula was characterized using differential scanning calorimetry andFourier Transform Infra-Red which indicated that CLP was molecularly dispersed within the solid nano-systemwithout any signs of interactions. CLP bioavailability in male albino rabbits after oral administration of theoptimum CLP-loaded S-SNEDDS formulation compared to the commercially CLP tablets (Plavix®) was in-vestigated. Results revealed that the optimum prepared system exhibited nine folds increment in CLP bioa-vailability compared to the conventional Plavix® tablets. In conclusion, CLP-loaded S-SNEDDS formulationscontaining bioenhancing surfactants proved to be a promising approach to improve the oral bioavailability ofCLP.

1. Introduction

Clopidogrel (CLP) is an oral antiplatelet drug belonging to thieno-pyridine class, it is an important therapeutic agent for patients who areat risk for cardiovascular events like myocardial infarction, peripheralvascular disease, and stroke [1]. Since CLP is a prodrug, it needs to beactivated first in order to exert its pharmacological effect. The activemetabolite binds to the P2Y12 adenosine diphosphate receptor of theplatelets and results in reduced ADP-mediated activation of the glyco-protein IIb/IIIa complex [2]. Conversion to the active metabolite iscatalyzed by hepatic enzymes and different cytochromes P450s (CYPs)[3]. Pharmacokinetic studies indicated that approximately 15% of CLP'sactive metabolite is available and the remaining 85% is hydrolyzed toan inactive carboxylic acid derivative compound by esterase para-oxonase-1 (PON1) [4], hence it is difficult to determine its plasmaconcentration. Clopidogrel bisulfate (CLP B) salt is widely used inpharmaceutical industry due to its good water solubility being in thesalt and its pellucid form [5]. Unfortunately, this bisulfate salt form hasbeen reported to have poor stability and significantly degrades underaccelerated moisture and heat conditions [6]. Clopidogrel base exists in

an oily form which is difficult to purify and formulate. Furthermore,CLP has been reported to have poor oral bioavailability due to extensivehepatic metabolism with mean absolute bioavailability ̴ 50%.

Nanoemulsions including self-emulsifying (SEDDS) and self nano-emulsifying drug delivery systems (SNEDDS) are among the approachesthat can be used to enhance the bioavailability of such orally ad-ministered drug candidates [7]. SNEDDS are combination of natural orsynthetic lipids, surfactants, co-surfactants, and drugs that rapidly forman oil/water nanoemulsion upon stirring in an aqueous medium [8].Upon oral administration SNEDDS mix with GIT fluids during peri-staltic movement and spontaneously emulsify to form o/w emulsion,this results in a generation of nano-metric droplets with average size20–200 nm [9].

Formulation of liquid CLP-loaded SNEDDS could be considered asan attempt to enhance its oral systemic availability through improvingpermeability across intestinal membrane, eliminating or reducing foodeffect in addition to the reduction in droplet size and high drug dis-solution [10]. The lymphatic delivery of the drug can be achievedthrough the intestine avoiding the first-pass hepatic metabolism [11].Moreover SNEDDS formulations that contain certain types of

https://doi.org/10.1016/j.jddst.2018.12.027Received 12 October 2018; Received in revised form 12 December 2018; Accepted 24 December 2018

∗ Corresponding author. 8 avenue Bertie Albrecht, 75008, Paris, France.E-mail addresses: [email protected], [email protected] (G.A. Abdelbary).

Journal of Drug Delivery Science and Technology 49 (2019) 603–614

Available online 28 December 20181773-2247/ Published by Elsevier B.V.

T

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surfactants termed bioenhancers such as Cremophores, Tween®80, andLabrasol® were reported to further improve the bioavailability of ab-sorbed compounds by facilitating transcellular and paracellular ab-sorption through acting as p-glycoprotein and/or CYP450 enzymes in-hibitors thus decreasing intestinal efflux and drug biotransformation[12]. Solid SNEDDS (S-SNEDDS) combine the advantages of liquidSNEDDS which are solubility and bioavailability enhancement in ad-dition to enhancing the stability, patient compliance and accuratedosing [13]. S-SNEDDS can be prepared by various techniques in-cluding spray-drying, freeze-drying, or by direct adsorption of nanoe-mulsion preconcentrates onto microporous solid carriers [14]. There isa lack of data about the use of SNEDDS for enhancement of the oralbioavailability of CLP. Based on the aforementioned, this study aimedto formulate and optimize CLP-loaded SNEDDS and S-SNEDDS con-taining surfactants reported to be bioenhancers. All prepared formulaewere in-vitro evaluated and pharmacokinetic parameters for the op-timum selected solid system was determined and compared to that ofthe market product following the oral administration to male albinorabbits.

2. Experimental section

2.1. Materials

Clopidogrel bisulfate (CLP B) {Hydrogen sulfate salt of Methyl-(+)-(S) - α-(o-chlorophenyl)-6, 7-dihydrothieno [3, 2-c] pyridine-5(4H)-acetate} was received as a kind gift from Hikma Pharma, Egypt.Methanol was purchased from Sigma-Aldrich Co., St. Louis, USA.Capryol™90 {Propylene Glycol Monocaprylate (TypeII}, Oleic acid,Labrafil®M 1944 CS {Oleoyl Polyoxyl-6 Glycerides NF}, Labrafac®PG{Propylene Glycol Dicaprylate/Dicaprate}, Labrasol® {CaprylocaproylPolyoxylglycerides}, Transcutol™ HP{Diethyleneglycol MonoethylEther} and Lauroglycol™90{Propylene Glycol Monolaurate (Type II)}were received as a kind gift from Gattefosse Co, St-Priest, France.Polyethylene Glycol (PEG) 400 was purchased from Fluka, Switzerland.Cremophore®RH40 {Polyoxyl 40 Hydrogenated Castor Oil USP/NF}and Cremophore®EL {Polyoxyl 35 Hydrogenated Castor Oil USP/NF},were purchased from BASF, Germany. Tween®80{Polyoxyethylene (20)sorbitan monooleate}, Tween®20{Polyoxyethylene (20) sorbitanmonolaurate} and hydrochloric acid were purchased from El-NasrPharmaceuticals (Cairo, Egypt). Aeroperl®300 Pharma {silicon dioxide}was kindly gifted by Evonik (Essen, Germany). Plavix® tablets con-taining 75mg CLP (SANOFI, Cairo, Egypt, Batch No.:7A683).Transparent gelatin capsule shells (size 0) were supplied by the Centerof Applied Research and Advanced Studies, Faculty of Pharmacy, CairoUniversity (CARAS).

All other reagents and chemicals used were of analytical reagentgrade and were used without further purification.

2.2. Solubility study

Solubility study of CLP B salt in different vehicles (listed in Table 1)was executed by adding an excess known amount (approximately100mg) of CLP B in a 2ml microtube (Axy-gen MCT-200). Aftersealing, the mixture of the drug and 1ml of the vehicle was vortexedand kept for 48 h at 25 °C in thermostatically controlled shaking waterbath (Model 1083, GLFCorp., Germany) to endorse drug solubilization.The samples were then centrifuged at 10,000×g for 5min and filteredthrough a Millipore membrane filter (0.45 μm) to separate excess un-dissolved CLP. The supernatant was diluted with methanol for spec-trophotometric quantitative determination of dissolved CLP (UV-1601 PC, Shimadzu, Japan) via a validated method at 278 nm with thesame dilution ratio of the pure vehicle under study in methanol used asa blank. Each experiment was repeated three times and S.D. was cal-culated.

2.3. Construction of pseudo-ternary phase diagrams

Based on the solubility study results, Capryol™90 was chosen as oilyphase Tween®80 and Cremophore®EL as surfactants, PEG 400 andTranscutol®HP as co-surfactants to construct two phase diagrams.Systems that could emulsify spontaneously upon dilution and formclear or translucent o/w nano-emulsion were identified from ternaryphase diagrams constructed using infinite dilution method where 36possible ratios of SNEDDS were prepared with varying concentrationsof oil, surfactant and co-surfactant till reaching final total concentrationof 100% w/w [15]. Each system (one gm) was poured in glass beakercontaining 200ml distilled water at 37 °C and gently agitated. Thespontaneous emulsification was visually examined against dark back-ground [16]. The optical clarity of each point in the phase diagram wasmeasured to confirm the clarity of the observed nano-emulsion. Systemswith an average size of 200 nm or lower and produced clear (with %transmittance more than or equal 95%) or translucent (with % trans-mittance more than or equal 90%) dispersions were considered in thenanoemulsion region of the diagram (referred to as formulations ofgrade A and B, respectively) [17,18], and selected for further evalua-tion and preparation of CLP-loaded SNEDDS. The two-phase diagrams(Fig. 1) were drawn using CHEMIX ternary plot software (CHEMIXSchool Ver. 3.60, Pub. Arne Standnes).

2.4. Formulation of CLP-loaded SNEDDS

Self nano-emulsifying liquid systems were prepared by dissolvingconstant dose of CLP B (37.5 mg) in an accurately weighed mixture ofoil, surfactant, and co-surfactant in a stoppered glass vial and mixedwith a vortex mixer to ensure uniform drug distribution. Clear (grade A)and translucent (grade B) drug loaded formulae were further char-acterized and evaluated.

2.5. Characterization and evaluation of CLP-loaded SNEDDS

2.5.1. Spectroscopic characterization of optical clarityThe percentage transmittance as a determinant of optical clarity for

each diluted nano-emulsion loaded preconcentrate was measured at638 nm using distilled water as blank [19].

2.5.2. Robustness to dilutionThe prepared CLP-loaded SNEDDS were diluted 100 times with

0.1 N HCl then observed for phase separation or drug precipitationthrough 24 h storage period [20]. The formulae which showed no phaseseparation or precipitation through the specified time were considered“robust to dilution” [16]. This was good indication for the stability ofCLP-loaded SNEDDS at infinite dilution [21].

2.5.3. Determination of self-emulsification timeOne gm of each of selected systems was diluted with 200ml distilled

Table 1Saturated solubility of CLP in different SNEDDS components.

Type of vehicle Solubility (mg/ml)± S.D., n= 3

Capryol™ 90 77.24 ± 0.35Labrafac® PG 44.28 ± 0.22Oleic acid 34.35 ± 0.21Lauroglycol™90 12.86 ± 0.19Cremophore® EL 75.00 ± 0.22Tween®80 58.69 ± 2.04Labrafill®1944 M 14.80 ± .186Labrasol 11.50 ± 1.54Cremophore® RH40 10.53 ± 2.59Tween®20 1.20 ± 0.52Transcutol™HP 85.00 ± 0.24PEG 400 75.00 ± 0.36

E. Abd-Elhakeem et al. Journal of Drug Delivery Science and Technology 49 (2019) 603–614

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water and gently agitated at 125 rpm and 37ᵒC. The systems wereevaluated by monitoring the time required for the disappearance of thepreconcentrate and formation of clear dispersion of nanosized o/wglobules [22].

2.5.4. Determination of globule size (GS), polydispersity index (PDI)Globule size is an important indicator of the physical stability of a

nanoemulsion; a small globule size indicates non-flocculation in thesystem. It is a critical factor in self-emulsification performance [23] andalso affects the rate and extent of drug release. PDI indicates uniformityof size distribution within each formula. Clear (grade A) and translu-cent (grade B) drug loaded formulae were selected for particle sizeanalysis where they were diluted using 200ml distilled water andanalyzed using Malvern Zetasizer (Malvern Instruments,UK) wherelight scattering was monitored at a temperature of 25ᵒC and an angle172ᵒ [24]. The globule size was expressed as average globule size ofdroplets in the system (Z-average) and polydispersity index which in-dicates the width of size distribution [17]. One-way ANOVA was em-ployed to test significance of Z-averages between different formulationsin each system.

2.5.5. Viscosity determinationBrookfield viscometer (Japan) DV-E at 100 rpm and 25 ± 0.5ᵒC

was used to measure the viscosity of each of the selected systems beforeand after dilution to determine the type of formed nanoemulsion [25].

2.5.6. In-vitro drug release studyThis study was performed according to U.S. Pharmacopeia for the

dissolution of CLP with minor modifications. Selected liquid systemswere filled in hard gelatin capsules size (0). CLP release from the pre-pared capsules was evaluated in HCl buffer (pH=2) maintained at37 ± 0.5ᵒC, USP Type I dissolution apparatus (Erweka DT600 sixspindle dissolution tester) with baskets rotating at 50 rpm. At 5, 10, 15,20, 30, 45 and 60min five mls from the dissolution medium werewithdrawn and an equivalent volume of buffer was added to keep thetotal volume of release medium constant throughout the experiment.

The amount of CLP was calculated spectrophotometrically at 278 nmusing HCl buffer as a blank. One-way ANOVA was done to test sig-nificance in cumulative amount of drug released.

2.5.7. Transmission electron microscopy (TEM)The morphology of the selected CLP-loaded SNEDDS was observed

by TEM (Joel, JEM-100 CX electron microscope, Joel, Tokyo, Japan). Asample drop of selected liquid drug loaded SNEDDS was applied to acollodion-coated 300 mesh copper grid and left for 5min to allow someof the dispersed SNEDDS to adhere to collodion. The remaining liquidwas removed by adsorbing the drop with the corner of a piece of filterpaper. A drop of 1% phosphotungstic acid aqueous solution was appliedand left for 1min then the remaining was removed and the sample wasair-dried and examined [26].

2.6. Preparation and characterization of CLP-loaded S- SNEDDS

Based on physicochemical characterization and evaluation of thedrug loaded SNEDDS formulae, one optimized liquid formula was se-lected for the preparation of S-SNEDDS by adsorption technique usingAeroperl®300 Pharma as solid carrier. In brief, the drug-loaded pre-concentrate was adsorbed on Aeroperl®300 Pharma by direct triturationin a glass mortar using a preconcentrate adsorbent ratio of 1:0.75 [27].Solid state characterization for S-SNEDDS was done; this included dif-ferential scanning calorimetry (DSC), FTIR, and scanning electron mi-croscopy (SEM) in addition to powder micromeritic properties evalua-tion.

2.6.1. Differential scanning calorimetry (DSC)Samples of pure CLP B, Aeroperl®300, physical mixture of both at

ratio (1:1) and the selected solid formula were run on DSC (Differentialscanning calorimeter DC-60; Schimadzu, Kyoto, Japan). Samples wereprocessed at a temperature ramp increase of 10ᵒC/min from 0 to 400ᵒC[28].

Fig. 1. Pseudo-ternary phase diagrams.


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2.6.2. Scanning electron microscopy (SEM)Surface morphology for selected solid formula was examined using

Jeol JSM-5200 scanning electron microscope (Tokyo, Japan). Thesample was fixed on brass stub using double-sided adhesive tape andthen made electrically conductive by coating in a vacuum with a thinlayer of gold (1̴50Å) for 30 s. The pictures were taken at an excitationvoltage of 25 Kv. SEM was done for CLP B, Aeroperl®300, and selectedformula [8].

2.6.3. Fourier transform infrared spectroscopy (FTIR)The FTIR spectrum was recorded using FTIR spectrophotometer

(Genesis II, Mattson, USA). The FTIR spectrum of the selected systemwas compared to that of plain drug and adsorbent. Potassium bromidepellets were prepared by gentle mixing 1mg sample with 200mg KBr.Each sample was placed in light path of sample cell and the spectrumwas recorded [29].

2.6.4. Micromeritic properties of S-SNEDDS2.6.4.1. 2.6.4.1. Angle of repose. Funnel method was used to determinethe angle of repose of S-SNEDDS. Briefly funnel height was firstadjusted in such way that the tip of the funnel is separated from thepowder by a distance of 5 cm, the powder was allowed to flow freelythrough the funnel. The diameter of the resulted powder cone wasmeasured and the angle of repose was calculated using the followingequation [30].

tan Ɵ=h/r

where,

h= the powder cone height,r= the cone radius.

2.6.4.2. Carr's index. The test was done using tapping method. Theselected solid formula was weighed and then placed in 100ml cylinder.The powder was carefully leveled without compacting and the apparentvolume of Vₒ was recorded. The cylinder containing the sample wastapped until the volume was unchanged and then tapped volume Vf wascalculated. Bulk density and tapped density were calculated using thefollowing formula:

ρb=M / Vₒ

ρtap=M /Vf

Where (ρb) is bulk density, M is a weight of the sample, Vₒ is the ap-parent volume of powder, (ρtap) is tapped density, M is a weight of thesample and Vf is tapped volume of powder.

Carr's index was then calculated using the value for bulk density andtapped density as follow:

Carr's index (%) = (ρtap- ρb) *100/ ρtapWhere ρtap is tapped density and ρb is bulk density [30].

2.6.5. In -vitro drug dissolution of CLP-loaded solid SNEDDSThe release of CLP from capsule containing the selected S-SNEDDS

was done at 37° and a rotation rate of 50 rpm using the USP DissolutionTester, apparatus I (DIS-6000; Copley, UK) with 900ml HCl buffer(pH=2) as a dissolution medium. Five mls were withdrawn at 5, 10,15, 20, 30, 45 and 60min and replaced with an equal volume of a freshmedium. The withdrawn samples were filtered through 0.22 μmMillipore filter and analyzed for their clopidogrel content spectro-photometrically at 278 nm using HCl buffer as a blank. Triplicate stu-dies were used. One sample t-test was done for comparison between theoptimum solid formula and market product (Plavix®).

2.7. In-vivo pharmacokinetic study

The study design was an open-label, randomized parallel design,two groups received two treatments. 2.5 ± 0.2 kg healthy male albinorabbits were used for this study. The rabbits were housed for 12 h dark/light cycle in a room with controlled temperature (25ᵒC) and humidity(55% ± 5%). All the procedures used in the present study were con-ducted according to the guidelines approved by the Institutional AnimalCare and Ethical Committee of Cairo University (Approval No.871). Therabbits were randomly divided into two groups and deprived of foodbut had free access to water 24 h before the day of the experiment.

They were treated orally as follows:Group I: Market conventional tablet (Reference), equivalent to

75mg drug.Group II: Optimized S-SNEDDS formula filled in hard gelatin cap-

sule size 0 (Test) [8], equivalent to 37.5 mg drug.Blood samples (1 mL) were withdrawn after oral administration via

the marginal ear vein at time points (0, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10,12 and 24 h) and placed in heparinized microcentrifuge tubes. Theblood samples were immediately centrifuged at 5000 rpm for 10min toseparate the plasma. The plasma samples were collected and stored at−20ᵒC until drug analysis. Frozen plasma samples were thawed atroom temperature then 1.0ml of plasma mixed with 100μlTorsemide asinternal standard (50 ng/ml) and 3ml acetonitrile to precipitate theplasma protein, vortexed and centrifuged at 5000 rpm for 5min theninjected to be analyzed into LC-MS/MS model LC-20AT.Chromatographic separations were performed on a reversed-phaseAguilant column (50mm, 5 μm diameter). The mobile phase consistedof 80% acetonitrile, 20% water with 0.1% formic acid. The detectionwas done using triple quadrupole detector MS/MS.

The analysis was operated at the MRM (multiple reaction mon-itoring) mode. The plasma concentrations vs. time profiles were ana-lyzed using pharmacokinetic software KineticaTM2000 software (ver3,InnaPhase Corporation, USA) [31]. Data from the plasma concentra-tion-time curve within 24 h after drug intake were employed to esti-mate the following pharmacokinetic parameters for individual rabbit ineach group, peak plasma concentration (Cmax), time to reach peakplasma concentration (Tmax), area under the plasma concentration vs.time curve from zero to the last sampling time (AUC0–24h) and half-life(t1/2) for both S-SNEDDS and Plavix®.

3. Results and discussion

3.1. Solubility studies

SNEDDS are monophasic liquid state systems composed of an oil, asurfactant, a co-surfactant and the drug under the investigation [32],hence, proper selection of SNEDDS components to ensure the highestdrug loading and solubilization is a key determinant factor. The satu-rated solubility studies of CLP B in different SNEDDS components wereillustrated in Table (1). Amongst the different oils screened, Ca-pryol™90 showed the highest drug solubilization, therefore it waschosen as an oily phase. Capryol ™90 is a saturated medium chain tri-glyceride(eight carbon) with an amphiphilic nature which could impartsurfactant properties and efficient self-emulsification properties [30],which could help in the formation of self-emulsifying systems con-taining the drug, similar finding was reported by Balakrishnan et al.[19]. Moreover, the presence of similar aliphatic chains in both struc-tures of CLP and Capryol™90 resulted in a stacking effect in addition tothe dipole-dipole interactions between the carbonyl groups found inboth structures.

Among the tested surfactants, two surfactants were selected namelyTween®80 and Cremophore®EL, based on their highest drug equilibriumsolubility (Table 1).

PEG 400 and Transcutol®HP were selected as co-surfactants owingto their highest solubilization power for the drug (Table 1). PEG 400


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showed good solubilising power which could be attributed to the inter-molecular interaction such as hydrogen bond between the proton (H+)in CLP B and the ether bonds found in PEG 400 structure. Transcu-tol®HP had good drug solubilising power which ensure high drugloading beside its absorption and permeability enhancement [33].

3.2. Construction of pseudoternary phase diagram

Figure (1) demonstrates the phase diagrams for both systems. Basedupon preliminary studies, System I comprised Capryol™90/Tween®80/PEG400, while system II comprised Capryol™90/Cremophore®EL/Transcutol®HP. Visual observation showed clear and translucent sys-tems (grades A and B formulae) that maintained their physical ap-pearance 24 h post-dilution confirming nano-emulsion stability. Thepseudo-phase diagram showed that an oil content of 10% resulted inclear systems, while higher oil percentage resulted in a biphasic system.Only clear plain systems (grade A) with % transmittance ≥95% andtranslucent plain systems (grade B) with % transmittance ≥90% wereloaded with CLP B and subjected to further evaluation and character-ization.

From the pseudo-phase diagrams, it can be observed that system IIhad larger clear area than system I, this may be attributed to higherbranching and higher unsaturation of the alkyl chain of Cremophore®ELcompared to that of Tween®80 which increased the flexibility of oil/water interface thus leading to further reduction in the nano-emulsionsize and greater clarity [34].

3.3. Characterization of the selected clopidogrel loaded SNEDDS

3.3.1. Spectroscopic characterization of optical clarityThe different plain SNEDDS were visually assessed based on the

observed opacity and percentage transmittance measured upon dilutionwith 200ml water. Only clear and translucent mixtures [grades A, andB] were selected for further evaluation and loaded with CLP while othermixtures were rejected. Systems selected post drug loading are illu-strated on Fig. 1 using a red dot. It is clear from the results, Table (2A,B) that grades A and B formulae exhibited high %transmittance above90% indicating good emulsification ability with globule size in nano-range. None of the prepared systems loaded with CLP showed any drugprecipitation within 24 h storage period after diluting 200 times withdistilled water which confirm the stability of the prepared systems.

As the value of percent transmittance being close to 100%, thissignifies the clarity and transparency of the prepared systems and en-sures that the prepared systems have a size in the nano-range which in

turn confirms highest drug release and absorption due to the smallsurface area provided with nanoglobules [30].

3.3.2. Robustness to dilutionRobustness to dilution was assessed for two selected SNEDDS sys-

tems and the results are reported in Table (2). No signs of precipitationor phase separation were observed upon diluting the tested systems(except SI8) with 200-fold with 0.1 N HCl. These results confirm thestability and suitability of the formed nano-emulsion for oral use [21].In addition, this confirms that both surfactant and co-surfactant have norole in drug solubilization, only the oil used is responsible for drugsolubilization as the dilution of the nanoemulsion by gastrointestinaltract fluids leads to lowering solvent capacity of the surfactant or co-surfactant. Thus, to formulate stable SNEDDS, it is an important to re-cognize what affects drug loading capacity while maintaining theability of the system to undergo monophasic liquid state upon dilutionwith either water or buffer and minimizing the drug precipitation orcrystallization in diluted system.

3.3.3. Self-emulsification timeEmulsification time determines the emulsification efficiency of the

prepared nano-emulsion [19]. The emulsification process is consideredthe rate-limiting step to absorption which is similar to the disintegra-tion of tablets. Assessment of emulsification time of the selected CLPloaded SNEDDS does not only indicate the time needed for emulsifi-cation process under gentle mixing, but also ensures the dispersion ofCLP within the dispersed o/w emulsion without further precipitation.The formation of liquid crystals and gel phases are the first steps ofemulsification process which considerably influences globule formationand accessibility of interface for drug partition [35]. The self-emulsi-fication time results of CLP loaded SNEDDS are presented in Table (2).From Table (2), it is evident that all the tested formulae had shortemulsification time. This short emulsification time may be attributed tothe low oil content used which did not exceed 10% [21]. As previouslyreported, increasing the oil concentration in the SNEDDS resulted in anincrease in the system viscosity that require greater shear force fordispersion and thus slow down the emulsification process [36]. Asshown in the results, increasing the concentration of surfactant in-creases the time of emulsification which could be attributed to theviscosity of surfactants used that delay the mixing of the componentstogether. As reported by Baslious et al. [12],Tween®80 and Cremo-phore®EL had high emulsification efficiency which in turn resulted inshort emulsification time. This clarifies that both oil and surfactanthave an important role in emulsification process.

Table 2Composition and characterization of selected liquid CLP-loaded SNEDDS.

Formula No. Sample composition Visual observation(Grading)

PercentageTransmittance

Self-emulsification time± S.D. (sec)

Robustness todilution

Globule size(nm)± S.D.

PDI± S.D.Oila SAb Co-SAc

A; system I

SI8 10 10 80 B 90.87 15.00 ± 0.79 No 186.50 ± 0.71 0.58 ± 0.02SI15 10 20 70 B 94.36 25.00 ± 0.22 Yes 127.50 ± 3.54 0.400 ± 0.00SI21 10 30 60 B 92.33 26.00 ± 0.15 Yes 156.55 ± 2.12 0.57 ± 0.04SI33 10 60 30 A 97.85 54.00 ± 0.18 Yes 102.00 ± 0.42 0.53 ± 0.23SI34 20 70 10 B 92.76 65.00 ± 0.47 Yes 102.25 ± 1.06 0.39 ± 0.01SI36 10 80 10 A 99.59 85.00 ± 0.38 Yes 105.00 ± 0.00 0.59 ± 0.00a (Capryol™90),b (Tween®80),c (PEG400)

B; system II

SII8 10 10 80 A 99.87 10.98 ± 0.29 Yes 20.45 ± 0.28 0.29 ± 0.02SII14 20 20 60 A 95.55 13.92 ± 0.23 Yes 35.43 ± 1.13 0.12 ± 0.01SII15 10 20 70 A 97.99 15.97 ± 0.15 Yes 40.68 ± 8.17 0.29 ± 0.14SII21 10 30 60 B 92.76 16.87 ± 0.44 Yes 22.15 ± 4.05 0.25 ± 0.11SII26 10 40 50 B 94.23 17.32 ± 0.11 Yes 14.00 ± 0.15 0.10 ± 0.01SII30 10 50 40 A 98.48 18.25 ± 0.43 Yes 13.86 ± 0.13 0.10 ± 0.02

a (Capryol™90),b (Cremophore®EL),c (Transcutol®HP).


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3.3.4. Globule size (GS) and polydispersity index (PDI)Globule size measurement (Table 2) revealed that all clear systems

had a Z-average value below 200 nm thus fulfilling the criteria ofSNEDDS [18]. Globule size for system I ranged from 102.00 ± 0.00 to186.50 ± 0.70 nm while that for system II from 13.86 ± 0.13 to40.68 ± 8.17 nm. System II globule size was smaller than that ofsystem I, although it was previously reported that surfactants with closeranges of HLB would not affect globule size [37], (HLB of Cremo-phore®EL ranges between 12 and 14 and for Tween®80 is 15). Howeverthis difference in size could be attributed to the steric hindrance effectof Cremophore®EL, as it possess a highly branched chemical structurecompared to Tween®80, which improves the flexibility of the oil/waterinterface and promotes the formation of reduced size globules [38]. Agood relation could be seen between the globule size and the clearappearance of the diluted preconcentrate as reported by Taha et al.[39].

Polydispersity index (PDI) signifies a good uniformity in globulesize distribution within the system. PDI values for system I ranged from0.39 ± 0.01 to 0.59 ± 0.00 while those for system II ranged from0.10 ± 0.01 to 0.29 ± 0.14, indicating good uniformity [40].

ANOVA test was used to evaluate the significance of different for-mulation variables on the globule size of the prepared SNEDDS systems.ANOVA results for system I showed that SI33, SI34 and SI36 had sig-nificantly the lowest Z-average (p < 0.05) followed by SI15 and SI21.This might be attributed to the higher emulsifier concentration whichproduced nano-emulsion with smaller globule size due to the greaterreduction in the interfacial energy [41]. ANOVA results for system IIshowed that all tested systems did not show significant difference fromeach other (p > 0.05).

3.3.5. Viscosity determinationViscosity evaluation is an important parameter, first to decide the

type of the prepared nanoemulsion (o/w) or (w/o), second to char-acterize the physical stability of the system. SNEDDS with high viscositydisperse slowly while those with a lower viscosity disperse im-mediately. This is in accordance with Eid et al. [36] findings who re-ported that SNEDDS with high viscosity form gel upon contact withwater resulting in slow dispersion and emulsification, while systemswith low viscosity did not form a gel and disperse immediately [42].Results of the viscosity measurements are illustrated in Table (3), theviscosity of system I before dilution ranged from 22.00 ± 0.84 to29.00 ± 1.69 cps, while those for system II from 18.00 ± 2.03 to26.00 ± 0.78 cps. However, the viscosity values of the diluted nano-systems decreased and ranged from 9.00 ± 0.56 to 16.00 ± 0.69 cpsfor system I and from 7.00 ± 0.10 to 14.00 ± 0.37 cps for system II.As a result of the above findings, it could be concluded that all preparedSNEDDS formulae were of O/W nano-emulsion type [30].

3.3.6. In -vitro release study of the selected loaded liquid SNEDDSThe in-vitro dissolution studies of CLP from the selected SNEDDS

filled into capsules (size 0) were performed in order to ensure the quickrelease into the dissolution medium and give an idea about the self-nanoemulsification efficiency of the developed system [43].

The release profiles of CLP from two selected SNEDDS systems areshown in figures (3A and B). Results shows that capsules containingSI15 and SII8 exhibited 96.505% and 97.97% respectively in-vitro re-lease in HCl buffer (pH=2) within 1 h. ANOVA analysis of the per-centage drug released after 20min showed that the mentioned for-mulations (SI15 system I and SII8 system II) exhibited significantlyhigher Q20% than other formulae. This might be due to rapid self-emulsification of both formulae (had shortest emulsification time) andthe instantaneous dispersion of preconcentrate in the medium aftercomplete dissolution of the capsule shell [19]. The hydrophilicity andhigh HLB value of the used surfactants (HLB values of Tween®80 andCremophore®EL are 15 and 13.5, respectively) [44], lead to in-stantaneous diffusion of the formulation into the aqueous media, andimmediate formation of O/W nano-emulsifying system.

Moreover both formulae contain low surfactant concentration 20%Tween®80 in SI15 and 10% Cremophore®EL in SII8, these low con-centrations helped in the increase in the cumulative drug release due tothe fact that, systems with high surfactant concentration were con-sidered as the most viscous systems resulting in gel formation uponcontact with water, consequently slow dispersion and release, whilethose with low concentration did not form gel and disperse immediately[42]. ANOVA results showed insignificant difference between the per-centage drug released from both SI15 and SII8 (P= 0.123). SII8 hadslightly higher Q20% (97.97 ± 0.368) than SI15 (96.505 ± 0.714),this may be due to the high Transcutol®HP (80%) concentration alongwith the low surfactant concentration resulting in the creation of voidspaces among surfactant molecules which in turn facilitates the diffu-sion of CLP molecules into the aqueous medium [45].

ANOVA analysis for Z-average revealed that SII8 had significantlower average size than SI15 from system I (P= 0.001). Based on theabove results SII8 from system II was chosen as a candidate to be for-mulated into S-SNEDDS and for in-vivo evaluation.

3.3.7. Transmission electron microscopy (TEM)The TEM image of SII8 is shown in figure (2A-2); it reveals regular

spherical dark globules in a clear surrounding. The results of globulesize measurement of the preconcentrate showed close size to thoseobtained by the zeta sizer (Figs. (2A)-1). TEM image of the globules inSII8 showed a thick layer surrounding each globule indicating thepresence of surfactant/co-surfactant monolayer around nano-emulsionglobule which forms a barrier to coalescence resulting in a stable na-noemulsion [26]. No signs of drug precipitation were observed.

3.4. Preparation of CLP-loaded solid SNEDDS formulation

The selected liquid SNEDDS SII8 (containing 10% Capryol™90,10%Cremophore®EL and 80% Transcutol®HP) was converted into solidSNEDDS through adsorption onto the surface of the solid carrierAeroperl®300 pharma [46]. The amount of carrier required to produce acompatible powder with good flowability was determined based onseveral trials. The selected loading ratio was 1:0.75(w/w) liquid pre-concentrate to carrier.

3.5. Characterization of CLP-loaded solid SNEDDS

3.5.1. DSCThe DSC thermograms of CLP B, Aeroperl®300, the selected solid

system (S-SNEDDS 8) and its corresponding physical mixture are gra-phically illustrated in figure (2B). The DSC thermogram of clopidogrelshowed a sharp endothermic peak at 175ᵒC corresponding to the drugmelting point [47]. The DSC thermogram of Aeroperl®300 showed a

Table 3Viscosity measurements of selected liquid CLP -loaded SNEDDS before and afterdilution.

Formula No Viscosity beforedilution(cps)± S.D.

Viscosity afterdilution(cps)± S.D.

System I SI15 23.00 ± 0.57 11.00 ± 0.98SI21 22.00 ± 0.84 9.00 ± 0.56SI33 29.00 ± 1.69 15.00 ± 0.48SI34 25.00 ± 0.68 16.00 ± 0.69SI36 26.00 ± 0.78 15.00 ± 0.17

System II SII8 18.00 ± 2.03 7.00 ± 0.10SII14 24.00 ± 0.85 9.00 ± 0.63SII15 25.00 ± 0.38 11.00 ± 0.42SII21 25.00 ± 0.75 14.00 ± 0.37SII26 21.00 ± 0.47 10.00 ± 0.39SII30 26.00 ± 0.78 12.00 ± 0.37


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broad endothermic peak at 75ᵒC [48]. The DSC thermogram of CLP-Aeroperel®300 physical mixture at ratio 1:1 showed the super-imposition of the individual components. Both CLP and Aeroperl®300peaks are present, which indicates absence of any drug-carrier inter-action. The characteristic endothermic peak of CLP disappeared in thethermogram of the selected optimum formula confirming that the drugmolecularly diffused within the formula. Similar results were pre-viously reported in the literature [32,49].

3.5.2. Scanning electron microscopy (SEM)Images of CLP B, Aeroperl®300 and the selected solid system(S-

SNEDDS 8) are shown in figure (2C). CLP B plain drug showed round-shaped crystals with spongy very rough surface, while Aeroperl®300appeared as void granules with spherical and smooth surface [50]. Thisstructure increases its surface area and enhances its adsorption capacityresulting in a powerful adsorbent. The rough surface of CLP B com-pletely disappeared in the scanning electron micrograph of S-SNEDDS8, confirming that the drug had been completely dispersed within the

solid nano-emulsion with no precipitation or crystallization of the drugoccurring within the formula. Investigation of this formula at a highermagnification power showed that the smooth surface of the carriergranules was overspread with the nano-emulsifying system which in-dicates the success and the efficiency of the method used for the pre-paration of S-SNEDDS [8].

3.5.3. Fourier transform infrared (FTIR) spectroscopyThe infrared spectrum of CLP B shown in Figure (2D), it exhibited

characteristic peak at 1750 cm¯1 due to c= o stretching vibration (estergroup) and bands associated with CeO stretching appeared atnearby1156 cm¯1. The spectrum of Aeroperl®300 showed the samepattern of bands as documented in literature [51]. All the character-istics peaks of CLP were retained in the FTIR spectra of optimum solidformula, confirming that there was no chemical incompatibility be-tween the drug and the used carrier [32].

Fig. 2A. (A-1) intensity size distribution curve of SII8 loaded nanoemulsion after dilution, (A-2) transmission electron micrograph of SII8 loaded nanoemulsion afterdilution.

Fig. 2B. Differential scanning calorimetric thermograms of CLPB, Aeroperel, their physical mixture and solid SII8.


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Fig. 2C. Scanning electron micrographs of CLP, Aeroperl and solid SII8.

Fig. 2D. Fourier transform IR spectrums of CLPB, Aeroperel, their physical mixture and solid SII8.


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3.5.4. Micromeritics of S-SNEDDS3.5.4.1. Angle of repose. The calculated value for angle of repose for theselected solid system was 21.97 ± 3.25° which confirms the goodflowability of the powder [30].

3.5.4.2. Carr's index. The prepared solid preconcentrate of SII8 hadCarr's index from 20.37 ± 4.23 to 18.04 ± 3.83% which indicatesgood flowability [52].

3.5.5. In-vitro dissolution of CLP loaded S-SNEDDSThe in-vitro dissolution profile of CLP from the selected solid

SNEDDS system SII8 filled into hard gelatin capsule size 0 is illustratedin figure (3C) in comparison with that of the market product (Plavix®

tablets). Statistical analysis was done to test significance difference inpercent drug released. One sample t-test at confidence interval> 95%did not identify any significant difference between optimum solid for-mula and market product. The previous findings confirm the success ofthe prepared SNEDDS.

The high drug release from S-SNEDDS may be due to rapid forma-tion of the self nano-emulsifying preconcentrate upon desorption ofliquid system of SII8 from Aeroperel®300 surface after exposure to therelease medium. Aeroperl®300 as a solid carrier has high specific sur-face area (≈300m2/g) [53], this surface area contributed to the highdrug release. Moreover, its porous structure allow dispersion of CLP inthe release medium after wetting of the solid system [54].

3.6. In-vivo evaluation

CLP is an inactive prodrug, its active metabolites (2-oxo-clopidogreland the thiol derivative) are responsible for the in-vivo effects [55].However, these metabolites are undetectable in plasma as they arehighly labile. In addition, this drug is metabolized to its major inactiveclopidogrel carboxylic acid derivative by esterases in animals [56].Hence, the pharmacokinetic parameters of clopidogrel could be de-termined indirectly through the quantitative determination of this in-active metabolite [57]. LC/MS/MS method was used for clopidogrel'smetabolite detection and was validated using international guidelinesand proven to be accurate, sensitive and selective. CLP's metabolite andits internal standard torsemide had retention times of 0.567min and0.557min, respectively. A plasma calibration curve was constructed byplotting peak area ratios (drug to IS) against CLP's metabolite con-centrations. The standard calibration curve was linear with a correla-tion coefficient (r2) 0.997.

The CLP average plasma-concentration time profiles followingsingle oral dose administration of CLP loaded S-SNEDDS filled in a hardgelatin capsule size 0 and the market product (Plavix® tablet) are shownin Figure (4). CLP S-SNEDDS showed higher Cmax and AUC and lowertmax values compared to Plavix®. Non-compartmental analysis was im-plemented for determination of the mean pharmacokinetic parametersof CLP after oral administration of S-SNEDDS and the market product.The average Cmax value following the oral administration of CLP S-SNEDDS was 24.81 ± 3.07 μg/mL reached after 0.5 h, whereas theaverage Cmax for Plavix® tablets was 1.68 ± 0.39 μg/mL and it wasattained at a tmax of 1 h. ANOVA statistical analysis showed statistically

Fig. 3. In-vitro CLP release from the prepared liquid formulae, (A) System I, (B) System II. (C) In-vitro release of CLP from solid SII8 capsule in comparison toPlavix®tablet.


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significant differences (p < 0.05) between both treatments for Cmaxand the median tmax. Regarding the extent of drug absorption, sig-nificantly higher AUC (0-∞) values were obtained from the CLP S-SNEDDS, AUC (0-∞) was 48.79 ± 14.84 μg h/mL compared to10.75 ± 1.88 μg h/mL in the case of Plavix®. The area under plasmaconcentration time curve produced from CLP S-SNEDDS was 9.07 timesgreater than that of plavix® tablet. Moreover, there was a non-sig-nificant difference between the two treatments for the terminal elim-ination rate constant (λz) and the elimination half-life (t1/2).

From the above-mentioned findings, it could be concluded that theenhanced bioavailability of CLP S-SNEDDS with a 9.07 times more thanthat obtained after administration of Plavix® with higher Cmax andshortened tmax could be due to drug lymphatic uptake to the systemiccirculation of being nano-metric formulation [58]. Furthermore, en-hancement of CLP bioavailability could be achieved through the in-hibitory effect of CYP3A4-mediated hepatic first-pass metabolism bypresenting a higher proportion of the drug to pass to the systemic cir-culation, this effect attributed to the increase in the extent of CLP ab-sorption.

Capryol™90is a medium chain fatty acids which enhance para-cellular transport [11,59] through its effect on the tight junctions of theepithelial cells [30]. Cremophore®EL was reported to possess bio-en-hancing activities which involve inhibitory effects on P-gp and CYP450enzymes decreasing intestinal efflux and drug biotransformation and/orfacilitating transcellular and paracellular absorption [12]. Such find-ings could be supported by a study made on atorvastatin [60], whereCremophore was reported to enhance its oral bioavailability and uti-lized in formulating a new SNEDDS market product namely; Neoral®

[61]. Higher lymphatic uptake is attributed to average nanosize, SII8was of size 20.45 ± 0.28 nm and PDI value of 0.29 ± 0.02 thus fa-voring lymphatic uptake. As reported by Oussoren et al., a particle sizeof 10–100 nm is the most favorable for lymphatic uptake [62]. Thisdirect relationship between particle size and enhancement of bioa-vailability could be confirmed in the study made on myricetin wherebetter bioavailability was noticed in SNEDDS with a small particle size[63]. Also, in some studies to enhance bioavailability smaller droplet

size nano-emulsions were appraised [64]. On the contrary, bioavail-ability has not been directly related to nano-emulsion droplet size insome other research [65]. In view of the mentioned results, it could beconcluded that S-SNEDDS based CLP dosage forms could be formulatedwith lower dose due to the enhanced CLP bioavailability.

4. Conclusion

In this study S-SNEDDS was formulated for the bioavailability en-hancement of drugs suffering from first pass effect like CLP. All systemswere in the nano-metric size range with short emulsification time andacceptable in-vitro release profiles. Moreover, the in-vivo study showedprominent 9.07 folds increase in the bioavailability after the oral ad-ministration of the optimum CLP S-SNEDDS formula compared to themarket product Plavix®. Hence, the developed S-SNEDDS proved to be apromising carrier for CLP for the effective management of cardiovas-cular events.

Declarations of interest

None.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jddst.2018.12.027.

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Bioavailability enhanced clopidogrel -loaded solid SNEDDS: Development and in-vitro/in-vivo characterizationIntroductionExperimental sectionMaterialsSolubility studyConstruction of pseudo-ternary phase diagramsFormulation of CLP-loaded SNEDDSCharacterization and evaluation of CLP-loaded SNEDDSSpectroscopic characterization of optical clarityRobustness to dilutionDetermination of self-emulsification timeDetermination of globule size (GS), polydispersity index (PDI)Viscosity determinationIn-vitro drug release studyTransmission electron microscopy (TEM)

Preparation and characterization of CLP-loaded S- SNEDDSDifferential scanning calorimetry (DSC)Scanning electron microscopy (SEM)Fourier transform infrared spectroscopy (FTIR)Micromeritic properties of S-SNEDDS2.6.4.1. Angle of reposeCarr's indexIn -vitro drug dissolution of CLP-loaded solid SNEDDS

In-vivo pharmacokinetic study

Results and discussionSolubility studiesConstruction of pseudoternary phase diagramCharacterization of the selected clopidogrel loaded SNEDDSSpectroscopic characterization of optical clarityRobustness to dilutionSelf-emulsification timeGlobule size (GS) and polydispersity index (PDI)Viscosity determinationIn -vitro release study of the selected loaded liquid SNEDDSTransmission electron microscopy (TEM)

Preparation of CLP-loaded solid SNEDDS formulationCharacterization of CLP-loaded solid SNEDDSDSCScanning electron microscopy (SEM)Fourier transform infrared (FTIR) spectroscopyMicromeritics of S-SNEDDSAngle of reposeCarr's indexIn-vitro dissolution of CLP loaded S-SNEDDS

In-vivo evaluation

ConclusionDeclarations of interestSupplementary dataReferences

Documents

Journal of Drug Delivery Science and Technology · ground [16]. The optical clarity of each point in the phase diagram was measured to conﬁrm the clarity of the observed nano-emulsion