enhanced intestinal absorption of etoposide by self-microemulsifying drug delivery systems: roles of...

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European Journal of Pharmaceutical Sciences xxx (2013) xxx–xxx

PHASCI 2843 No. of Pages 11, Model 5G

29 August 2013

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier .com/ locate/e jps

Enhanced intestinal absorption of etoposide by self-microemulsifyingdrug delivery systems: Roles of P-glycoprotein and cytochromeP450 3A inhibition

0928-0987/$ - see front matter � 2013 Published by Elsevier B.V.http://dx.doi.org/10.1016/j.ejps.2013.08.016

⇑ Corresponding author. Tel.: +86 27 83657550; fax: +86 27 83692892.E-mail address: [email protected] (L. Si).

Please cite this article in press as: Zhao, G., et al. Enhanced intestinal absorption of etoposide by self-microemulsifying drug delivery systems: Rolglycoprotein and cytochrome P450 3A inhibition. Eur. J. Pharm. Sci. (2013), http://dx.doi.org/10.1016/j.ejps.2013.08.016

Gang Zhao, Jiangeng Huang, Kewen Xue, Luqin Si ⇑, Gao LiSchool of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China

a r t i c l e i n f o

272829303132333435363738

Article history:Received 10 April 2013Received in revised form 16 July 2013Accepted 10 August 2013Available online xxxx

Keywords:EtoposideSelf-microemulsifying drug delivery systemAbsorptionP-glycoproteinCytochrome P450 3A

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a b s t r a c t

Etoposide is recognized as a dual P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) substrate drugwith poor water-solubility. To improve its solubility and bioavailability, three novel self-microemulsify-ing drug delivery systems (SMEDDS) contained the known P-gp and CYP3A inhibitory surfactants,Cremophor RH40, Cremophor EL, or Polysorbate 80, were prepared. This work aims to evaluate theenhanced intestinal absorption of etoposide SMEDDS as well as to explore the roles of P-gp and CYP3Ainhibition in the absorption process. Etoposide SMEDDS were orally administered to rats for in vivo bio-availability investigation. In situ single-pass intestinal perfusion with mesenteric vein cannulation wasemployed to study the drug permeability and intestinal metabolism. In vitro Caco-2 cell models wereapplied to study the effects of P-gp and CYP3A inhibition by SMEDDS on the cellular accumulation of eto-poside. It was found that the bioavailability and in situ intestinal absorption were significantly enhancedby SMEDDS with the order of Polysorbate 80-based SMEDDS > Cremophor EL-based SMEDDS > Cremo-phor RH40-based SMEDDS. In addition, there was a dramatically high linear correlation between theAUC0–t values and the apparent permeability coefficient values based on the appearance of the drug inmesenteric vein blood. Cellular uptake studies demonstrated that P-gp inhibition by SMEDDS playedan important role in etoposide uptake. Moreover, etoposide metabolism was demonstrated to be dramat-ically inhibited by the three kinds of SMEDDS. These finding may assist in the improvement of the intes-tinal absorption of P-gp and/or CYP3A substrate drugs.

� 2013 Published by Elsevier B.V.

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1. Introduction

Intestinal P-glycoprotein (P-gp) efflux and first-pass metabo-lism by cytochrome P450 3A (CYP3A) play critical roles in limitingthe absorption and bioavailability of orally administered drugs.P-gp decreases intracellular drug accumulation by actively extrud-ing them from the enterocytes. Metabolism of the drug moleculeby intestinal CYP3A is associated with the fraction of the absorbeddose that crosses the gut in the unmetabolized form. Despite thelower CYP3A expression levels in intestine in comparison to thatin liver, intestinal CYP3A metabolism has also been found to playa significant role in the disposition of some substrate drugs (Linet al., 1999), such as midazolam (Paine et al., 1996), cyclosporineA (Wu et al., 1995) and baicalein (Zhang et al., 2005). Owing tothe spatial relationship of CYP3A enzymes and P-gp transporter,repeated exposure of substrate drugs to metabolism by CYP3Amay be possible and regulated by passive absorption and activeP-gp efflux. Furthermore, the role of intestinal CYP3A metabolism

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may be more important in subjects with active P-gp efflux (Wacheret al., 1998). Consequently, the barriers of intestinal P-gp efflux andCYP3A metabolism will be more disadvantageous for intestinalabsorption of the dual substrate drugs.

Etoposide, a semisynthetic epipodophyllotoxin derivative, iscommonly used in the treatment of small cell lung cancer, lympho-mas, and leukemia (Henwood and Brogden, 1990). It has beendemonstrated to be a substrate of both P-gp and CYP3A (Kishiet al., 2004; Najar et al., 2011). Etoposide is clinically used via bothoral and intravenous routes. However, intravenous routes are lim-ited due to drug precipitation from the parenteral solution whendiluted with intravenous fluids. Hence, oral formulations havebeen developed to ensure administration safety. Recently, severalreports (Najar et al., 2011; Toffoli et al., 2004) showed that the oralbioavailability of etoposide in humans is low (about 50%) withhuge inter- and intra-patient variability. Its low bioavailabilitymay result from P-gp efflux and CYP3A metabolism (Lee et al.,2011; Zhang et al., 2011), while the variation of bioavailabilitymay be arisen from individual difference of P-gp and CYP3Aexpression (Najar et al., 2011). A variety of researchers have dem-onstrated that the bioavailability of etoposide can be significantly

es of P-

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2 G. Zhao et al. / European Journal of Pharmaceutical Sciences xxx (2013) xxx–xxx


29 August 2013

improved by inhibiting P-gp and CYP3A (Lee et al., 2011; Najaret al., 2011). In addition, due to the poor water-solubility and acidinstability of etoposide (Shah et al., 1989), its oral formulation re-quires some improvement. Based on these considerations, self-microemulsifying drug delivery systems (SMEDDS) were appliedto increase the solubility, stability and intestinal absorption of eto-poside in this study.

Recently, a number of pharmaceutical excipients, which arecommonly used in pharmaceutical formulations, have been identi-fied as potential inhibitors of P-gp and/or CYP3A. For example,Cremophor RH40 (Cr RH40), Cremophor EL (Cr EL), and Polysorbate80 (PS 80) have been verified to possess inhibitory effects on bothP-gp and CYP3A in vitro and in vivo (Bravo González et al., 2004;Ren et al., 2008; Yamagata et al., 2007; Zhang et al., 2003). As sur-factants are used for improving the solubility of poorly water-soluble drugs, these excipients are considered to be incorporatedin S(M)EDDS to increase the intestinal absorption of the dual P-gp and CYP3A substrate, etoposide. Very few studies about the P-gp and/or CYP3A inhibitory effect of S(M)EDDS are performed asyet. Therefore, the aim of this study is to evaluate the enhancedabsorption of etoposide SMEDDS as well as to explore the rolesof P-gp and CYP3A inhibition in the absorption process.

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2. Materials and methods

2.1. Materials

Etoposide was obtained from Borui Fine Chemical Co., Ltd. (Wu-han, China). Etoposide catechol metabolite (30-O-desmethyl etopo-side) was purchased from International Laboratory Ltd. (San Bruno,CA, USA). Cr RH40, Cr EL, and PS 80 were obtained from Cognis UKLtd. (Southampton, Hampshire, UK). PEG 400 was from Dow Chem-ical Ltd. (Midland, MI, USA). Ethyl oleate was obtained from Qing-shengda Chemical Industry Co., Ltd. (Beijing, China). Medium chaintriglyceride was obtained from Sins-swed Pharmaceutical Co., Ltd.(Beijing, China). Verapamil hydrochloride was obtained from Cen-tralpharm Inc. (Tianjin, China). Ketoconazole was from JiadePharmaceutical Co. (Beijing, China). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide andrhodamine 123 were purchased from Sigma–Aldrich (St. Louis,MO, USA). HPLC grade methanol was purchased from Fisher Scien-tific (Fair Lawn, NJ, USA). All other chemicals were of commercialanalytical reagent grade and were used without additionalpurification.

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2.2. Preparation and characterization of etoposide SMEDDS

A series of etoposide SMEDDS were prepared as follows: prede-termined quantity of etoposide was initially dissolved in co-sol-vent PEG 400; oily phase and surfactant were subsequentlyadded with gentle stirring and incubation at 37 �C until homoge-neous mixtures were formed. The mixtures, etoposide SMEDDS,were stored at ordinary temperature until they were used. As neg-ative controls, three optimized formulations of blank SMEDDSwere prepared in each formulation with varying the ratio of oil,surfactant, and co-solvent (Table 1).

Microemulsions were formed following 1:100 dilution of etopo-side SMEDDS (1%, w/w) with blank Krebs–Ringer buffer. Thedroplet sizes and zeta potential of the resulting microemulsionswere measured utilizing a Zeta Potential/Particle Size analyzer(Brookhaven Co., Holtsville, NY, USA). The measurement conditionswere as follows: angle, 90�; temperature, 25 �C; wavelength,658.0 nm; reflection index, 1.330. Microprecipitation of the drugfollowing 1:250 aqueous dilution of the SMEDDS formulationwas checked by comparing etoposide concentrations in the result-

Please cite this article in press as: Zhao, G., et al. Enhanced intestinal absorptioglycoprotein and cytochrome P450 3A inhibition. Eur. J. Pharm. Sci. (2013), ht

ing microemulsion prior to and after centrifugation at 3000g for10 min. The supernatant was collected for HPLC analysis.

The in vitro dispersion profiles of etoposide SMEDDS were mon-itored in Krebs–Ringer buffer (pH 7.4) using a Chinese Pharmaco-poeia XC paddle method. In brief, a mass of etoposide SMEDDS(0.25 g) was adhered onto a glass slide and introduced into the dis-persion medium (200 ml) at 37 �C with stirring at 50 rpm. Analiquot (1 ml) of the dispersion medium was withdrawn andreplaced with an equal volume of fresh dispersion medium at def-inite intervals. After filtering through a membrane filter (0.22 lm,Millipore, MA, USA), the dispersion medium was mixed with anequal volume of methanol and centrifuged at 12,000g for 10 min,the supernatant was injected into HPLC system for analysis.

The chemical stability study was carried out by keeping the eto-poside solution or etoposide SMEDDS in Krebs–Ringer buffer (pH7.4) or artificial gastric juice (pH 1.5) in a shaker bath at 37 �C.The initial concentration of etoposide was 50 lg/ml. An aliquot(0.4 ml) of etoposide sample was withdrawn and mixed with anequal volume of methanol at definite intervals. The mixture wascentrifuged at 12,000g for 10 min. The supernatant was then iso-lated and analyzed by HPLC.

2.3. Animal experiments

2.3.1. AnimalsAnimal experiments were performed on male Sprague–Dawley

rats weighing 280–320 g. The rats were obtained from the CentralAnimal Laboratory of Huazhong University of Science and Technol-ogy, and maintained in a temperature-controlled environmentwith a 12-h light/dark cycle, with free access to food and tap water.In vivo and in situ studies were approved by the ExperimentalAnimal Ethical Committee of Tongji Medical College, HuazhongUniversity of Science and Technology, and all animal experimentsadhered to the National Institutes of Health Guide for Care andUse of Laboratory Animals.

2.3.2. In vivo bioavailability studiesAnimals were randomly allocated into five treatment groups

with six animals in each group. Before the experiment, rats werefasted overnight for 12 h with free access to water. For oral admin-istration groups, rats received different etoposide SMEDDS (1%, w/w) at a dose of 12 mg/kg by gavage. Following administration, 3 mlof water was given to rats for spontaneous formulation of micro-emulsion in gastrointestinal tract. Etoposide suspension (1.5 mg/ml, suspended in 0.5% sodium carboxymethyl cellulose solution)was administered at a dose of 12 mg/kg as a control. Blood samples(0.2 ml each time) were obtained from tail vein and collected inheparinized Eppendorf tubes at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12and 24 h after administration. For intravenous injection, etoposidewas initially dissolved in PEG 400 and then diluted with isotonicsaline solution (2 mg/ml) for intravenous injection. Rats received4 mg/kg etoposide intravenous injection via the tail vein. Bloodsamples were obtained from retro-orbital plexus and collected inheparinized Eppendorf tubes at 0.08, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12and 24 h after injection. Plasma was isolated by centrifugation at6000g for 10 min and stored at �20 �C until analysis.

2.3.3. In situ single-pass intestinal perfusion studiesAnimals were randomly allocated into six treatment groups

with five animals in each group. Single-pass intestinal perfusionwith mesenteric vein cannulation was performed as reported byCummins et al. (2003). Briefly, jugular vein was cannulated forinfusion of donor blood. One segment of ileum was then isolatedfor drug perfusion. Meanwhile, mesenteric vein originated fromthe isolated intestinal segment was cannulated for continuous col-lection of blood. Different dilutions of etoposide SMEDDS with

n of etoposide by self-microemulsifying drug delivery systems: Roles of P-tp://dx.doi.org/10.1016/j.ejps.2013.08.016


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Table 1Formulations and characterization of SMEDDS.

Items Formulations (%, w/w)

Cr RH40-based SMEDDS Cr EL-based SMEDDS PS 80-based SMEDDS

Ethyl oleate – 25 25Medium-chain triglyceride 14 – –Cremophor RH40 (Cr RH40) 43 – –Cremophor EL (Cr EL) – 50 –Polysorbate 80 (PS 80) – – 50PEG 400 43 25 25Droplet size (nm) 24.8 ± 0.7 21.3 ± 0.9 20.7 ± 1.3Zeta potential (mV) �4.0 ± 1.0 �11.2 ± 1.2 �11.9 ± 0.6Solubilized dose after 5 min dispersion (%) 91.1 ± 1.7 82.5 ± 1.3 92.2 ± 2.5Solubilized dose after 30 min dispersion (%) 99.2 ± 1.7 98.8 ± 2.0 98.0 ± 2.5

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Krebs–Ringer buffer (1:100) was perfused through the isolatedileum segment. Etoposide solution was performed as a control.Verapamil hydrochloride (100 lg/ml) or ketoconazole (15 lg/ml)was added in etoposide solution to serve as a positive P-gp orCYP3A inhibitor, respectively. The final concentration of etoposidein all perfusate was 40 lg/ml. During perfusion, lumenal perfusatesamples and mesenteric vein blood samples were harvested forcontinuous 30 min. The blood samples were centrifuged at 6000gfor 10 min, and the plasma was isolated and stored at �20 �C untilanalysis.

2.4. Cell experiments

2.4.1. Cell culture and cytotoxicity evaluationDulbelcco’s Modified Eagle’s Medium containing Nutrient Mix-

ture F-12 (DMEM/F-12, 1:1) was purchased from HyClone Labora-tories, Inc. (Logan, UT, USA). Fetal bovine serum (FBS) was obtainedfrom Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou,China). Hank’s balanced salts solution (HBSS) supplemented with1 mg/ml glucose was adjusted to a pH of 6.8 with HEPES and usedin all cell experiments. Caco-2 cells, obtained from the Institute ofBiochemistry and Cell Biology (Shanghai, China), were used atpassages between 34 and 60. Cells were cultured in DMEM/F-12supplemented with 10% (v/v) FBS, 100 U/ml penicillin and0.1 mg/ml streptomycin at 37 �C under an atmosphere of 5% CO2

and 95% relative humidity.Toxicities of etoposide SMEDDS on Caco-2 cells were evaluated

by MTT assay (Mosmann, 1983). The MTT assay determined cellu-lar metabolic activity by the reduction of MTT. Briefly, cells wereseeded in 96-well plates (Corning, NY, USA) at a density of5 � 104 cells per well and cultured over 24 h to allow attachment.Etoposide SMEDDS with 1:2000, 1:1000, 1:500, 1:250, 1:100, or1:50 dilution was prepared following dilution of etoposideSMEDDS with blank HBSS. The final concentrations of etoposidein all dilutions were 50 lg/ml. After incubating with etoposideSMEDDS for 4 h or 48 h, cells were rinsed with PBS three times.20 ll of MTT solution (5 mg/ml) was then added into each well.After incubation for 4 h at 37 �C, the supernatant was removedand the formazan product was dissolved in 200 ll dimethyl sulfox-ide. The absorbance of each well was measured at 570 nm using amicroplate reader (SpectraMaxM5, Molecular Devices Co., USA).The net absorbance was taken as index of cell viability. The absor-bance taken from the wells with cells untreated with etoposideSMEDDS was served as a control and designated as 100%. Cellviability was expressed as a percentage of control.

2.4.2. Cellular uptake and metabolism studiesCaco-2 cells were applied to explore the roles of P-gp and

CYP3A inhibition in the etoposide accumulation. Cells were seededin 24-well plates at a density of 5 � 104 cells/cm2 and cultured for16 d. For measurement of drug metabolism, Caco-2 cells were cul-


tured in DMEM/F-12, which contained 1a,25-dihydroxy vitamin-D3 (Sigma-Aldrich, 250 nM), at day 11 for six consecutive days toinduce CYP3A expression (Fan et al., 2009). CYP3A protein levelin the cells treated with 1a,25-dihydroxy vitamin-D3 or blankHBSS was determined by Western blot analysis. Cells were lysedwith the RIPA Lysis Buffer (Beyotime, Hangzhou, China). Cellprotein content was determined by a BCA protein assay, and90 lg of the protein was mixed with 2� gel loading buffer (Beyo-time), separated on a 9% SDS-polyacrylamide gel at 120 V for 2 h,and then transferred to nitrocellulose membranes (Millipore).The nitrocellulose membranes were incubated with 5% skim milkin Tris-buffer saline with 0.1% Tween 20 at room temperature.After washing twice for 10 min with Tris-buffer saline, the mem-branes were incubated with the appropriate dilutions of mousemonoclonal antibody against CYP3A4 (Millipore) or GAPDH (Beyo-time) overnight at 4 �C. After washing, the membranes were incu-bated with the appropriate secondary antibody (goat anti-mouseIgG, Beyotime) for 2 h at 37 �C. Immunoreactive proteins werevisualized using Ultra ECL kit (Liankebio, Hangzhou, China) andanalyzed with the Quantity One software (BioRad, CA, USA). Pro-tein expression was presented as the ratio of CYP3A band intensityto GAPDH band intensity in the same blot.

Testosterone is a well-known probe substrate of CYP3A. Sobefore the cellular metabolism study, the rate of CYP3A-mediatedtestosterone 6b-hydroxylation was assessed to determine theactivity of CYP3A in Caco-2 cells with or without modification.After rinsing with blank HBSS, the cells were incubated with tes-tosterone (70 lg/ml) for 2 h at 37 �C. At the end of the incubation,cells were scraped and sonicated for 30 s. The metabolite,6b-hydroxytestosterone, in cell lysate was extracted with dic-hlormethane (1:5, v/v) and assayed by a validated HPLC methodusing an Agilent 1100 HPLC system (Agilent, MA, USA). The separa-tion was performed on a BDS Hypersil C18 (250 mm � 4.6 mm,5 lm) column. The mobile phase was methanol:10 mM disodicphosphate (60:40, v/v) at a flow rate of 1.0 ml/min. The eluatewas detected at 254 nm. The activity of CYP3A was calculated astotal mass of 6b-hydroxytestosterone normalized to the corre-sponding cell protein mass and incubation time.

Cells were preincubated with blank HBSS. After 0.5 h, the HBSSwas discarded, and 1:1000, 1:500, or 1:250 dilution of etoposideSMEDDS was added to incubate with cells for 0.5 h. Cells wereincubated with etoposide solution as control. Verapamil hydro-chloride (100 lg/ml) or ketoconazole (15 lg/ml) was added in eto-poside solution to serve as a positive P-gp or CYP3A inhibitor,respectively. To evaluate the factor of P-gp efflux, Caco-2 cells withor without verapamil hydrochloride treatment were employed tocarry out the incubation of etoposide SMEDDS. The final concentra-tions of etoposide in all wells were adjusted to be 50 lg/ml. At theend of the incubation, cells were rinsed with ice-cold blank HBSSthree times and then scraped and sonicated for 30 s. Etoposideconcentration in cell lysate was analyzed using HPLC system, while



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etoposide metabolite was analyzed by LC–MS/MS. The proteincontent in the lysate was determined by a BCA protein assay usingbovine serum albumin as a protein standard via absorbance deter-mination at 570 nm. Uptake rates were expressed as accumulativemass of etoposide normalized to the corresponding cell proteinmass and incubation time. The metabolism rate of etoposide wascalculated as total mass of metabolite normalized to the corre-sponding cell protein mass and incubation time.

2.4.3. Cell membrane fluidity measurementsFluorescence polarization techniques were applied to deter-

mine the effects of SMEDDS on Caco-2 cell membrane fluidityusing a common fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene(DPH, Sigma–Aldrich, USA). Caco-2 cells were seeded in 75 cm2

flasks (Corning, NY, USA) at a density of 5 � 104 cells/cm2 and cul-tured for consecutive 16 d. Cells were preincubated with blankHBSS for 0.5 h, followed by digestion using trypsin-EDTA solution.After rinsing with blank HBSS, cells were suspended in HBSS at aconcentration of 2 � 105 cells/ml. Then DPH stock solution(2 mM, prepared in tetrahydrofuran) was added in the cell suspen-sion to get a final concentration of 2 lM and equilibrated for 1 h at37 �C in darkness. At the end of equilibration, cells were centri-fuged at 600g for 5 min and rinsed with blank HBSS twice toremove uncombined DPH. Cells were then resuspended in HBSSat a concentration of 2 � 105 cells/ml. Three formulations ofSMEDDS were then diluted by 1000-, 500-, or 250-fold with theDPH-labeled cell suspension and incubated for 1.5 h at 37 �C. Cellsincubated with 0.2 mM cholesterol or 30 mM benzyl alcohol wereapplied as positive controls for the cell membrane fluidity decreaseor increase measurements, respectively. Cells incubated with blankHBSS were used as negative controls. The fluorescence intensitywas measured at 365 nm for excitation and 426 nm for emissionwith both slit widths of 10 nm using a LS 55 Fluorescence Spec-trometer (PerkinElmer, Waltham, MA, USA).

2.4.4. Subcellular distribution by confocal microscopyTo observe the uptake extent and distribution of P-gp substrate-

loading SMEDDS in Caco-2 cells, a well known P-gp substrate, rhoda-mine 123, was selected in confocal microscopy studies. Localizationof rhodamine 123 in Caco-2 cells was carried out on a LMS 510 Metaconfocal laser scanning microscope (Carl Zeiss, Jena, Germany). Cellswere cultured on cover slips and incubated with the dilutions of rho-damine 123 SMEDDS (0.1%, w/w) at 37 �C for 0.5 h. Rhodamine 123solution was added with or without verapamil hydrochloride(100 lg/ml) to evaluate the contribution of P-gp inhibition in the up-take of rhodamine 123. The final concentration of rhodamine 123 ineach petri dish was 2 lg/ml. After rinsing with blank HBSS at the endof the incubation, cells were stained with Hoechst 33342 (Sigma–Al-drich) and imaged by confocal microscopy.

2.5. Sample preparation

For etoposide extraction, plasma samples and cell lysate sam-ples were extracted with dichlormethane (1:5, v/v). After vortexand centrifugation at 12,000g for 10 min, the organic layers wereisolated and dried using a nitrogen stream. The residues wereredissolved with mobile phase and centrifugated at 12,000g for10 min, 5 ll of the supernatant was injected into the LC–MS/MSsystem for analysis. For the extraction of etoposide metabolite,ascorbic acid (Sigma–Aldrich, 0.5 mg/ml) was added to avoidoxidation of etoposide metabolite during the sample preparation.

2.6. Sample analysis

Quantitation of etoposide and its metabolite was performed onan ultra performance liquid chromatography system equipped


with an Agilent 6460 triple-quadrupole mass spectrometer (LC–MS/MS system, MA, USA). Separations were performed by isocraticelution on an Agilent SB-C18 (75 mm � 2.1 mm, 2.7 lm) column.The mobile phase was methanol:0.1% formic acid (55:45, v/v). Sep-arations were performed at 40 �C with a flow rate of 0.3 ml/min.Ionization was achieved using electrospray in the positive modewith a spray voltage of 4000 V. Nitrogen was used as nebulizergas and nebulizer pressure was set at 35 psi. Desolvation gas(nitrogen) was heated to 325 �C and delivered at a flow rate of10 l/min. The multiple reaction monitoring (MRM) mode was usedto monitor the transition of etoposide molecule m/z 589.5 [M + H]+

to 229.0 and etoposide metabolite molecule m/z 575.1 [M + H]+ to229.0. The LC–MS/MS assay validation results are listed as follows:the LLOQ was 20 ng/ml for etoposide and 2 ng/ml for 30-O-desm-ethyl etoposide. The assay was linear over the concentrationranges of 0.02–8 lg/ml for etoposide and 0.002–0.8 lg/ml for30-O-desmethyl etoposide. The correlation coefficients for bothanalytes in plasma and cell lysate were higher than 0.99. The intra-and inter-day accuracy (RE%) for both etoposide and its metabolitein plasma and cell lysate were within ±8%, the intra- and inter-dayprecision (RSD%) were less than 6% (Details see Supplementary ta-ble). The recovery of etoposide and 30-O-desmethyl etoposide atlow, medium and high concentration levels in plasma and celllysate were all above 77% for etoposide and above 90% for 30-O-desmethyl etoposide. The matrix effect for both etoposide and30-O-desmethyl etoposide was found to be 92–105%.

2.7. Data analysis

For pharmacokinetic studies, the peak plasma concentration(Cmax) and the time to reach the peak concentration (Tmax) were di-rectly noted from the individual plasma concentration versus timecurves. The area under the plasma concentration–time curve from0 to t (AUC0–t) was calculated following linear trapezoidal method.These pharmacokinetic parameters were calculated using DAS ver2.1 (Medical College of Wannan, China). The absolute bioavailabil-ity of etoposide was calculated as a ratio of the AUC0–t after oraland intravenous administration corrected for differences in actualadministered dose: F = (AUC0–t(oral)/doseoral)/(AUC0–t(intravenous)/doseintravenous).

The permeability coefficient of etoposide across rat intestinewas calculated based on the disappearance of the drug in perfusate(Plumen) as well as the appearance of the drug in mesenteric veinblood (apparent permeability coefficient, Pblood) using the follow-ing equations (Cummins et al., 2003):

Plumen ¼ �QA

lnCoutðcorrÞ

Cinð1Þ

where Q is the flow rate of drug through the intestine (0.24 ml/min),A is the area of the perfused intestine segment (cm2). Cin is the drugconcentration in the inlet of the perfusate entering the intestinalsegment. Cout(corr) is the corrected drug concentration calculatedby following equations:

CoutðcorrÞ ¼Cout � Q out

Q inð2Þ

where Cout and Qout are the drug concentration and the measuredflow (ml/min) in the efflux perfusate, respectively. Qin is themeasured flow (ml/min) in the inlet of the perfusate entering theintestinal segment.

Pblood ¼dX=dt

A < C >ð3Þ

where dX/dt is the rate of drug appearance in mesenteric vein blood(lg/s), A is the area of the intestine segment (cm2), and <C> is thelogarithmic mean concentration of drug in the intestinal lumen.



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To evaluate the metabolism extent of etoposide by intestinalCYP3A, an extraction ratio (ER, described in Eq. (4)) was calculatedas the total amount of metabolite formed during perfusion dividedby the sum of the parent drug in the mesenteric vein blood andmetabolite in both blood and perfusates (Cummins et al., 2003).The amount of the drug absorbed and metabolized in this part isnormalized to the surface area of the intestinal segment from indi-vidual experiments.

ER ¼ metabolitemetaboliteþ parent drug

ð4Þ

For the cell membrane fluidity measurements, fluorescencepolarization (P) was calculated as follows:

P ¼ IVV � IVH

IVV þ GIVHð5Þ

where IVV and IVH represented the measured fluorescence intensi-ties with emission polarizer vertically and horizontally oriented,respectively when the excitation polarizer was vertically oriented;G = IHV/IHH, IHV and IHH represented the measured fluorescenceintensities with emission polarizer vertically and horizontallyoriented, respectively when the excitation polarizer was horizon-tally oriented.

All results were expressed as mean ± SD. Student’s t test wasemployed to evaluate statistically significant difference betweentwo groups. Values of p < 0.05 and p < 0.01 were considered statis-tically significant and highly significant for all tests, respectively.

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3. Results and discussion

3.1. Characterization of etoposide SMEDDS

Following by 1:100 dilution of etoposide SMEDDS with blankKrebs–Ringer buffer, the resulting microemulsions were negativelycharged. The pH values were around 5.6. The zeta potential anddroplet sizes of the microemulsions were listed in Table 1. As themost important property of SMEDDS, the particle sizes of the threeformulations were all around 22 nm after microemulsification.From the results, little differences between the particle sizes ofthe three kinds of SMEDDS could be found. The aqueous dilutionsof 1:250–1:1000 were also characterized. And the results indicatedthat there was no significant impact of dilution fold on the dropletsizes of microemulsions (data not shown). Through centrifugation,no microprecipitation of etoposide following dilution of etoposideSMEDDS was found, suggesting that SMEDDS exhibited a satisfac-tory solubilization capacity for etoposide. Furthermore, after 1:250aqueous dilution of etoposide SMEDDS (1%, w/w), the concentra-tion of etoposide in the resulting microemulsion was 0.04 mg/ml,which was less than one third of the saturated concentration ofetoposide in Krebs–Ringer buffer at 37 �C (about 0.15 mg/ml).Thus, it was unlikely that supersaturation took place under suchlow concentration of etoposide. All the formulations of etoposideSMEDDS were observed to rapidly disperse to form microemul-sions. The percentage of dispersed dose was >80% within 5 minand >95% within 30 min (Table 1). Considering the relatively high-er saturated concentration of etoposide in Krebs–Ringer buffer,sink conditions were fulfilled by a wide margin in this releasestudy.

Chemical stability of etoposide is a prerequisite for the nextin situ and in vitro experiments. The stability of etoposide underthe in vitro culture conditions has been systematically studied (Ma-der et al., 1991). It has been found that etoposide degradation isdependent on the ionic strength of buffer solutions. An increaseddegradation could be found over time in 67 mM phosphate orHEPES buffer at 45 �C and pH = 8 (Mader et al., 1991). Therefore,


it was necessary to examine the stability of etoposide SMEDDSunder our experiment conditions. Furthermore, as etoposide wasorally administered, acid instability of etoposide in artificial gastricjuice also needed to be examined (Shah et al., 1989). Our resultsshowed that nearly no etoposide degradation from etoposidesolution or etoposide SMEDDS was found after 4 h incubation.Therefore, the degradation of etoposide could be neglected underthe conditions of the next in situ and in vitro experiments.However, the degradation extent of etoposide solution was about14% after incubation for 12 h, while that of etoposide SMEDDSwas less than 5%. In artificial gastric juice (containing hydrochloricacid and pepsin, pH1.5), the degradation extent of etoposide wasabout 39% for etoposide solution and 57–72% for etoposideSMEDDS after incubation for 12 h. The result indicated that etopo-side degradation could be prevented by SMEDDS. It is possible thatwhen SMEDDS were diluted with aqueous solution, the spontane-ously formed o/w microemulsion could encapsulate etoposide inthe core and protect the drug from interaction with ionic solution.This could explain the improved stability of etoposide SMEDDS.

3.2. Animal experiments

3.2.1. In vivo bioavailability studiesAs an orally administered formulation, SMEDDS can be sponta-

neously diluted by gastrointestinal fluid under the digestive motil-ity of the stomach and intestine to form oil-in-water (o/w)microemulsion with small droplet sizes and large interfacial areafor drug absorption (Patel et al., 2009). The resulting microemul-sions were therefore beneficial for etoposide absorption. Thein vivo bioavailability studies were carried out to investigate theincrease extent of the bioavailability of etoposide by SMEDDS. Plas-ma etoposide concentration versus time profile in rats followingintravenous injection of etoposide solution is presented inFig. 1a. The pivotal pharmacokinetic parameters were AUC0–24h of3.02 ± 0.94 h lg/ml and apparent volumes of distribution of14.82 ± 8.44 l/kg. Plasma etoposide concentration versus time pro-files after oral administration of etoposide SMEDDS to rats areshown in Fig. 1b. The main pharmacokinetic parameters are listedin Table 2. AUC0–24h and Cmax for the three kinds of etoposideSMEDDS were significantly increased in comparison with that foretoposide suspension. No statistical difference between Tmax forall formulations was found. But it could be observed from Table 2,Tmax for Cr RH40- and PS 80-based SMEDDS were relatively shorterthan that for Cr EL-based SMEDDS, suggesting that Cr RH40- or PS80-based SMEDDS showed a fairly rapid onset. In addition, the oralbioavailability of etoposide from Cr RH40-, Cr EL-, or PS 80-basedSMEDDS was 1.4-, 1.7-, or 2.5-fold that of etoposide suspension,respectively. These results indicated the significant enhancementof etoposide bioavailability by SMEDDS with an order of PS80-based SMEDDS > Cr EL-based SMEDDS > Cr RH40-basedSMEDDS. Owing to the little volume of obtained plasma samples,extremely low concentration level of etoposide metabolite couldbe detected. Thus in this part, plasma concentration versus timeprofile of etoposide metabolite was not presented.

3.2.2. In situ intestinal absorption and metabolism studiesTo evaluate the effect of SMEDDS on intestinal absorption of

etoposide, in situ single-pass intestinal perfusion with mesentericvein cannulation was carried out. It has been demonstrated thatthe absorptive and secretory permeability of etoposide in ileumsegment both higher than that in other segments (Kunta et al.,2000). Thus the ileum segment was utilized for the single-passperfusion studies. In this in situ study, the apparent permeabilitybased on the appearance of the drug in the mesenteric vein bloodis related to the drug entering into the portal blood. Therefore,intestinal P-gp and CYP3A are both involved in this apparent



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Fig. 1. Plasma concentration versus time profiles of etoposide obtained followingintravenous injection of 4 mg/kg etoposide injection (-�-) or oral administration of12 mg/kg etoposide suspension (-4-), Cr RH40-based (-j-), Cr EL-based (-s-), or PS80-based etoposide SMEDDS (-d-) to rats. Values were expressed as mean ± SD(n = 6).

Fig. 2. Perfusate concentration versus time profiles of etoposide from outlet of theintestine for etoposide alone (-s-), etoposide with verapamil hydrochloride (-d-).Cr RH40-based (-N-), Cr EL-based (-}-), or PS 80-based etoposide SMEDDS (-j-).Etoposide concentration from inlet of the intestine was 40 lg/ml. Results wereexpressed as mean ± SD (n = 5).



29 August 2013

permeability, whereas the permeability based on the disappear-ance of the drug in perfusate is only related to the fraction ab-sorbed from the intestinal lumen. To expore the effects of P-gpand CYP3A inhibition by SMEDDS on etoposide entering into theportal blood, the apparent permeability coefficient Pblood was thusapplied.

For intestinal perfusion with mesenteric vein cannulation, sam-ples were obtained from outlet of the intestine at 5 min intervalsup to 30 min as well as from the mesenteric vein for continuous30 min. The profiles of etoposide concentration from outlet of theintestine for etoposide SMEDDS are shown in Fig. 2. The apparentpermeability results of etoposide SMEDDS are shown in Fig. 3.The Pblood of etoposide was calculated to be (3.67 ± 0.40) � 10�6

cm/s for etoposide solution and increased to (9.07 ± 1.41) � 10�6

cm/s in the presence of verapamil hydrochloride, suggesting thatP-gp was effectively limiting the absorption of etoposide acrossthe intestine, and verapamil hydrochloride could evidently inhibitthis process. From Fig. 3, the three kinds of SMEDDS were all ob-served to enhance the intestinal absorption of etoposide by varyingdegrees. Especially, PS 80-based SMEDDS resulted in a 2.7-fold in-crease of permeability.

Table 2Pharmacokinetic parameters obtained following oral administration of etoposide suspensi

Etoposide suspension Etoposide SMED

Cr RH40-based

AUC0–24h (h lg/ml) 2.20 ± 0.60 3.05 ± 0.39�

Cmax (lg/ml) 0.39 ± 0.07 0.50 ± 0.07�

Tmax (h) 1.00 ± 0.52 0.88 ± 0.14Bioavailability (%) 25.44 ± 6.94 35.30 ± 4.09�

All values were expressed as mean ± SD, n = 6.�p < 0.05, ��p < 0.01, statistically different from the corresponding parameter of etoposid


The increased apparent permeability of etoposide may beattributed to the combined effects of solubilization improvement,membrane fluidity increase, P-gp and CYP3A inhibition bySMEDDS. However, the detailed mechanisms of intestinal absorp-tion of SMEDDS were still unknown. It can be expected that chargeshielding was not involved because of the surfactants were non-ionic. From previous reported mechanisms (Lu et al., 2012), theenhanced absorption of SMEDDS may attribute to the solubiliza-tion improvement and membrane fluidity increase by surfactantcomponents (Rege et al., 2002). In addition, as etoposide is a dualP-gp and CYP3A substrate, its oral bioavailability could be en-hanced through inhibiting P-gp efflux and CYP3A metabolism (Na-jar et al., 2011). Besides, it should be noted that the solubility ofetoposide in long-chain triglyceride lipid was low (<50 mg/g).And the log P value of etoposide was 0.6, which was far less than5 (Hansch et al., 1995). These properties indicated that nearly nolymphatic transport process was involved in the intestinal absorp-tion of etoposide SMEDDS (Charman et al., 1986).

The major pathway of etoposide metabolism was O-demethyla-tion by CYP3A. The metabolite, 30-O-desmethyl etoposide, was anetoposide catechol, which may undergo sequential oxidation (vanMaanen et al., 1988; Zhuo et al., 2004). So during the sample prep-aration, 0.5 mg/ml ascorbic acid was added to avoid oxidation ofetoposide metabolite (Pang et al., 2001). The extraction ratio wascalculated (using Eq. (4)) to represent the metabolism extent ofetoposide in this study, and the results are shown in Fig. 4. Theextraction ratio of etoposide solution was calculated to be0.125 ± 0.040, indicating that little amount of etoposide wasmetabolized. The reason for this relative low metabolism levelmight be related to the intestine segment chosen. As ileum isknown to express a lower level of CYP3A enzymes compared withthe upper intestine segment in rats (Li et al., 2002). However, themetabolism extent of etoposide in this segment was observed tobe significantly reduced by CYP3A inhibitors. The extraction ratioof etoposide in the presence of 15 lg/ml ketoconazole was reduced

on or etoposide SMEDDS to rats (12 mg/kg).

DS

SMEDDS Cr EL-based SMEDDS PS 80-based SMEDDS

3.65 ± 0.92�� 5.46 ± 1.30��

0.52 ± 0.10� 1.37 ± 0.64��

1.17 ± 0.68 0.83 ± 0.1342.17 ± 9.74�� 63.21 ± 13.75��

e suspension group.



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Fig. 3. The permeability coefficient of etoposide SMEDDS based on the disappear-ance of the drug in perfusate (Plumen) as well as the appearance of the drug inmesenteric vein blood (apparent permeability coefficient, Pblood). The permeabilitycoefficients were calculated using the corresponding equations in the manuscript.The permeability of etoposide alone was served as control. Verapamil hydrochloride(100 lg/ml) was added in etoposide perfusate as a positive P-gp inhibitor. �p < 0.05,��p < 0.01, statistically different from that of control. Results were expressed asmean ± SD (n = 5).



29 August 2013

to 0.011 ± 0.007. In comparison to this strong CYP3A inhibitor, thethree kinds of SMEDDS reduced the extraction ratio of etoposidefrom 4.6- to 8.6-fold (Fig. 4). Furthermore, the high variability ofintestinal CYP3A enzymes expression may result in highly variablemetabolism results. Owing to the inhibition of CYP3A enzymes, itcould be observed that the variability (standard deviation value)of etoposide biotransformation was evidently reduced by SMEDDS.

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Fig. 4. Extraction ratios of etoposide SMEDDS. The extraction ratio was calculatedas the total amount of metabolite formed during perfusion divided by the sum ofthe parent drug in the mesenteric vein blood and metabolite in both blood andperfusates. The amount of etoposide absorbed and metabolized was normalized tothe surface area of the intestinal segment. The extraction ratio for etoposide alonewas served as control. Ketoconazole (15 lg/ml) was added in etoposide perfusate toserve as a positive CYP3A inhibitor. ��p < 0.01, statistically different from that ofcontrol. Results were expressed as mean ± SD (n = 5).


It has been demonstrated that P-gp protein expression showsan overall increase from proximal to distal parts in rat and humansmall intestine (Dahan et al., 2009). However, CYP3A proteinexpression shows an opposite trend (Li et al., 2002; Paine et al.,1997). So P-gp content in ileum is the most abundant in smallintestine. This may result in the most significant change of P-gpmodulation by inhibitors, as the positive inhibitor verapamilhydrochloride is expected to cause the most dramatic increase ofetoposide permeability (Fig. 3). Oppositely, the low expressionlevel of CYP3A in ileum resulted in a weak metabolite level. How-ever, the effect of SMEDDS on CYP3A may be directly reflectedfrom metabolite production. And the sensitive LC–MS/MS methodwas adequate to satisfy the quantitation of etoposide metabolite.The results of cumulative amount of etoposide metabolite in per-fusate and mesenteric vein blood were listed in Table 3. Theseresults indicated that etoposide metabolism by ileum CYP3A wassignificantly inhibited by three kinds of SMEDDS.

The linear correlation between the AUC0–24h values and thePblood values was then evaluated, and a dramatically high linearcorrelation (r = 0.9973) was found (Fig. 5). However, a relativelylower linear correlation (r = 0.9646) was obtained between theAUC0–24h values and the Plumen values. Therefore, it seems betterto employ Pblood value to predict the in vivo trends. This result sug-gested that the single-pass intestinal perfusion approach withmesenteric vein cannulation could be applied to predict thein vivo pharmacokinetic results.

3.3. Cell experiments

3.3.1. Cytotoxicity studiesNon-toxic concentration is a basic principle to study the effects

of SMEDDS on drug absorption using cell models. It is widelyknown that a number of surfactant-containing carriers have poten-tial toxicity on cancer cells (Gursoy et al., 2003). So before thecellular uptake experiments, the toxicities of etoposide SMEDDSon Caco-2 cells need to be evaluated. In this study, the viabilitiesof Caco-2 cells incubated with different dilutions of etoposideSMEDDS were determined. The MTT assay results are shown inFig. 6. From the results, a dose-dependent cytotoxicity of the threeformulations could be observed in the range from 1:2000 to 1:50dilutions during the incubation for 48 h, suggesting that SMEDDSwith dilution ratio of >1:50 were capable of inducing toxicity onCaco-2 cells. However, after incubation for 4 h, the viabilities ofCaco-2 cells incubated with etoposide SMEDDS (with dilution ratio>1:50) were higher than 80%. The cell viability of >80% was consid-ered to be non-toxic to the cells (Wahlang et al., 2011). The similarresults were also observed by Sha et al. (2005), who found that thenegatively charged microemulsions formed by diluting SMEDDS(containing Cr EL and Labrasol) with blank HBSS (1:50–1:2000)had no toxic effect on Caco-2 cells after 2 h incubation. In addition,Yin et al. (2009) found that cell viability was generally not affectedby Cr EL at concentration lower than 2% (w/v, about 1:50 dilution).Therefore, during the next cell experiments, etoposide SMEDDS didnot induce any toxic effect on cells and the results could be re-garded as reliable.

In addition, the viabilities of Caco-2 cells incubated with theetoposide solution for 4 h were also evaluated. It was noteworthythat as an anticancer agent, etoposide with concentration of<50 lg/ml was found to possess nearly no toxicity on colon cancercells, Caco-2 cells (cell viability >80%). It had been reported thatetoposide with concentration of >25 lM (about 14.7 lg/ml) couldcause the viabilities of HCT 116 cells (derived from human colorec-tal cancer) to decrease to 80% or less after incubation for 24 h (Parket al., 2005). In our study, the low cytotoxicity of etoposide mayattribute to the short time of incubation and the lower sensitivityof Caco-2 cells.



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Table 3Etoposide appearance in mesenteric vein blood and etoposide metabolite formationin perfusate and mesenteric vein blood after perfusion for continuous 30 min.

Perfusion solution Cumulative amountof etoposide inblood (ng/cm2)

Cumulative amountof metabolite inperfusate andblood (ng/cm2)

Etoposide alone 10.57 ± 1.16 1.54 ± 0.60Etoposide with verapamil 26.15 ± 3.51 0.93 ± 0.22Etoposide with ketoconazole 19.36 ± 2.81 0.32 ± 0.18Cr RH40-based SMEDDS 18.03 ± 2.30 0.52 ± 0.23Cr EL-based SMEDDS 14.84 ± 2.53 0.34 ± 0.17PS 80-based SMEDDS 28.71 ± 3.65 0.42 ± 0.15

All values were expressed as mean ± SD, n = 5.

Fig. 5. Linear correlation between the AUC0–24h values and the Pblood values as wellas between the AUC0–24h values and the Plumen values. Pblood and Plumen values werecalculated based on the appearance of etoposide in mesenteric vein blood and thedisappearance of etoposide in perfusate, respectively.

Fig. 6. Toxicity results of various dilutions of Cr RH40-based (open column), Cr EL-based (gray column), or PS 80-based etoposide SMEDDS (dark column) on Caco-2cells after incubation for 48 h or 4 h. The final concentrations of etoposide in alldilutions of SMEDDS were 50 lg/ml. Results were represented as cell viability% ± SD(n = 5).



29 August 2013

3.3.2. Cellular uptake and metabolism studiesThe uptake rate results of etoposide SMEDDS on Caco-2 cells

were shown in Fig. 7. The results showed that etoposide SMEDDSincubation resulted in higher levels of accumulation of etoposidein Caco-2 cells than etoposide solution incubation, suggesting thatthe three kinds of SMEDDS were all capable of enhancing theuptake rate of etoposide. In addition, dose-dependent increasesof uptake rates for three formulations of etoposide SMEDDS couldbe observed from Fig. 7. The fold increase of uptake rates over con-trol were the highest for PS 80-based etoposide SMEDDS and thelowest for Cr RH40-based etoposide SMEDDS under the same dilu-tion folds. The well-known P-gp inhibitor, verapamil, made a 74%increase on the uptake rate of etoposide.

The uptake rates of etoposide SMEDDS on Caco-2 cells with thetreatment of verapamil hydrochloride were also studied and theresults were shown in Fig. 7. The uptake rates of etoposideSMEDDS on Caco-2 cells without treatment were all observed tobe lower with varying extent than that on verapamil treated cells,in spite of no statistic difference for 1:1000 and 1:500 dilutions ofCr RH40- and Cr EL-based SMEDDS and 1:1000 dilution of PS80-based SMEDDS. These enhancements on cells with verapamiltreatment reflected the effects of P-gp inhibition by SMEDDS. From


Fig. 7, PS 80-based SMEDDS showed a strongest inhibitory effect onP-gp. In addition, from the results of 1:250 dilution of SMEDDS, itcould be observed that the uptake rates of etoposide were in-creased with an order of PS 80-based SMEDDS > Cr EL-basedSMEDDS > Cr RH40-based SMEDDS. According to previous studies(Lu et al., 2012; Rege et al., 2002), the solubilization improvementand membrane fluidity increase by surfactant components maycontribute to the increased uptake rate. The solubility of etoposidein Cr RH40, Cr EL, and PS 80 were tested to be 0.63, 0.66, and1.01 mg/ml, respectively. In addition, there was no statistic differ-ence between the cell membrane fluidity treated with eachSMEDDS at the same dilution (Fig. 10). To sum up, PS 80-basedSMEDDS possessed the strongest P-gp inhibitory effect and stron-gest effects of solubilization in comparison with the other twoSMEDDS. Thus the uptake rates of etoposide were dramatically in-creased by PS 80-based SMEDDS.

The role of CYP3A metabolism in etoposide uptake was investi-gated on Caco-2 cells, which was known to under-express CYP3Aenzymes. Treatment of Caco-2 cells with 1a,25-dihydroxy vita-min-D3 (250 nM) may result in an increase of CYP3A mRNA andprotein expression (Fan et al., 2009). This modified cell modelwas thus employed to study the metabolism of etoposide. CYP3A4protein expression in Caco-2 cells with 1a,25-dihydroxy vitamin-D3 treatment was foud to be induced by about 3.7-fold in compar-ison to that with blank HBSS treatment (Fig. 8). The activity ofCYP3A in the modified Caco-2 cells was compared with that inthe cells without modification. The formation rate of 6b-hydroxy-testosterone in the modified cells was obviously increased to be75.76 ± 8.49 ng/mg protein/h, whereas nearly no metabolite couldbe detected in normal Caco-2 cells. These results demonstratedthat the protein level and activity of CYP3A in the modified Caco-2 cells were significantly increased.

From our previous studies (Ren et al., 2008, 2009), some com-mon nonionic surfactants including Cr RH40, Cr EL, and PS 80had been demonstrated to be strong CYP3A inhibitors in vitroand in vivo. In the present study, these surfactants were incorpo-



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Fig. 7. Uptake rates of etoposide SMEDDS on Caco-2 cells in the absence (graycolumn) or presence (dark column) of verapamil. Cells were incubated with (a) CrRH40-, (b) Cr EL- or (c) PS 80-based etoposide SMEDDS for 0.5 h. SMEDDS withdilutions of 1:1000, 1:500, or 1:250 were studied. Etoposide solution with orwithout verapamil treatment was incubated as a controls. The final concentrationsof etoposide in all solutions were adjusted to be 50 lg/ml. Results were representedas fold increase of uptake rate over the corresponding control (n = 3). ��p < 0.01,statistically different from the control without verapamil treatment; #p < 0.05,##p < 0.01, statistically different from the control with verapamil treatment; �

p < 0.05, ��p < 0.01, statistically different between the results on Caco-2 cells with orwithout verapamil treatment.

Fig. 8. Effect of 1a,25-dihydroxy vitamin-D3 (Vit D3) on CYP3A4 protein expres-sion. Caco-2 cells were treated with or without 250 nM 1a,25-dihydroxy vitamin-D3 for six consecutive days. At the end of the incubation, cells were harvested, andthe protein level was determined by Western blot analysis. Results were expressedas mean ± SD (n = 3). ��p < 0.01, statistically different from the group withouttreatment.

Fig. 9. Metabolism rates of different dilutions of etoposide SMEDDS based on theappearance of etoposide metabolite in Caco-2 cells. The metabolism rate ofetoposide solution was served as control. Ketoconazole (15 lg/ml) was added inetoposide solution as a positive CYP3A inhibitor. Results were expressed asmean ± SD (n = 3). The differences of all groups of values were highly significantin comparison with the control (p < 0.01).



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rated in SMEDDS to investigate the inhibitory effects of theSMEDDS on CYP3A enzymes using CYP3A-expressing Caco-2 cellmodes. The results were shown in Fig. 9. The metabolism rate ofetoposide solution was calculated to be (0.54 ± 0.08) lg/mgprotein/h, and was markedly reduced to (0.16 ± 0.01) lg/mg pro-tein/h by the positive control, ketoconazole (p < 0.01). For the threekinds of etoposide SMEDDS, dose-dependent decrease of metabo-lism rate could be observed. Especially, 1:250 dilutions of all for-mulations presented stronger inhibitory effects on CYP3A thanketoconazole. Furthermore, Cr RH40-based SMEDDS resulted inthe largest decrease of the metabolism rate of etoposide.

The absolute amount of etoposide metabolite for all groups wasvery small in comparison to the parent drug (<0.3%). Thus theeffect of CYP3A on the cellular uptake of etoposide was little. Thismay result from the low CYP3A expression on Caco-2 cells.However, due to the relatively large amount of CYP3A expressionin the intestine, especially in the duodenum, first-pass metabolismof etoposide in the small intestine could be obviously reduced byinhibition of intestinal CYP3A activity (Lee et al., 2011; Yanget al., 2013). Therefore, when the etoposide SMEDDS were orallyadministered, the inhibitory effects of SMEDDS on intestinal CYP3A


may play an important role in the intestinal absorption ofetoposide.

3.3.3. Cell membrane fluidity measurementsThe fluorescence anisotropy results for Cr EL-, Cr RH40-, or PS

80-based SMEDDS or positive controls were shown in Fig. 10.The results of positive control experiments showed the appropriateincrease in fluorescence anisotropy for cholesterol and thedecrease in fluorescence anisotropy for benzyl alcohol, indicatingthe decrease or increase in the membrane fluidity by cholesterolor benzyl alcohol, respectively. As shown in Fig. 10, three formula-tions of SMEDDS all caused dose-dependent reductions in the fluo-rescence anisotropy of DPH (p < 0.01), suggesting that the Caco-2cell membrane fluidity could be significantly increased by theseSMEDDS. In addition, there was no statistic difference betweenthe cell membrane fluidity treated with each SMEDDS at the samedilution.



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Fig. 10. Fluorescence anisotropy of Caco-2 cells measured with DPH as a probe.Cells treated with 1:1000, 1:500, or 1:250 dilution of Cr RH40-, Cr EL-, or PS 80-based SMEDDS for 1.5 h at 37 �C were investigated. Cells incubated with blank HBSSwere served as negative controls. And cells incubated with cholesterol or benzylalcohol were applied as positive controls for the cell membrane fluidity decrease orincrease measurements, respectively. Results were expressed as mean ± SD (n = 3).The differences of all groups of values were highly significant in comparison withthe negative control (p < 0.01).



29 August 2013

At present, the underlying mechanisms of surfactant-biologicalmembrane interaction are not fully understood. It had been knownthat surfactants may exert their P-gp inhibitory effects by non-specific mechanisms involving alteration of membrane fluidity(Hugger et al., 2002; Li et al., 2011). Surfactant-P-gp interactionwas a complex result of direct P-gp inhibition via blocking P-gp/substrate binding and indirect inhibition by altering membranefluidity. And surfactants could change the comformation of mem-brane bound P-gp transporter through altering membrane fluidity(Dudeja et al., 1995). In addition, this comformation change was

Fig. 11. Confocal micrographs of Caco-2 cells after incubation with (a) rhodamine 12330 min. The concentration of rhodamine 123 in all incubation systems was 2 lg/ml. Bfluorescence represented rhodamine 123. Scale bars: 50 lm. (For interpretation of the refarticle.)


associated with the reduction of P-gp ATPase activity throughmembrane fluidization. And the membrane fluidization usuallyresulted in the decrease of P-gp activity. Therefore, membrane flu-idity was an important index to evaluate the effect of inhibitorycomposition on P-gp. Our results showed that all formulations ofSMEDDS could dose-dependently increase the cell membranefluidity. These results agreed well with the finding of Huggeret al. (40), who reported that Cr EL and PS 80 possessed inhibitoryeffect on P-gp activity of Caco-2 cells by increasing membrane flu-idity. As contained oxyethylene groups, Cr EL and PS 80 could alterthe lipid phase of the membrane or change the fluidity of the polarhead group regions of cell membranes. Therefore, such mecha-nisms may be involved in the P-gp malfunction by Cr RH40-, CrEL-, or PS 80-based SMEDDS.

3.3.4. Subcellular distributionTo directly illuminate the accumulation and distribution of P-gp

substrate in Caco-2 cells, confocal microscopy was employed.Allowing for the short excitation wavelength of etoposide, a classi-cal P-gp substrate, rhodamine 123 with long excitation wave-length, was selected for the next confocal microscopy studies.From all confocal micrographs of Caco-2 cells, rhodamine 123was found to be around cell nucleus (Fig. 11). All micrographs dis-played no accumulation of rhodamine 123 on tight junctions. FromFig. 11, the cells incubated with rhodamine 123 SMEDDS showedobviously higher density of red fluorescence signals than that incu-bated with rhodamine 123 solution, suggesting that the cellularaccumulation of rhodamine 123 was significantly enhance by thethree kinds of SMEDDS.

In conclusion, the oral bioavailability of etoposide in rats couldbe significantly increased by Cr RH40-, Cr EL-, and PS 80-basedSMEDDS, especially PS 80-based SMEDDS was the best choice to

solution or (b) Cr RH40-, (c) Cr EL-, or (d) PS 80-based rhodamine 123 SMEDDS forlue fluorescence represented nucleus that was labeled with Hoechst 33342; Red

erences to color in this figure legend, the reader is referred to the web version of this



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increase the bioavailability and cellular uptake of etoposide. Cellu-lar uptake studies demonstrated that P-gp inhibition by SMEDDSplayed an important role in etoposide uptake. Moreover, metabo-lism studies in situ and in vitro demonstrated that etoposidemetabolism was dramatically inhibited by the three kinds ofSMEDDS. Therefore, Cr RH40, Cr EL, and PS 80 still possess signifi-cant inhibitory effects on P-gp and CYP3A when used in SMEDDS.And it was an effective strategy to incorporate P-gp and/or CYP3Ainhibitory excipients into SMEDDS for improving the intestinalabsorption of the substrate drugs.

4. Uncited references

Charman and Stella (1986), Dahan and Amidon (2009), Park andKim (2005) and Patel and Sawant (2009).

Acknowledgement

This research work was supported by the National Natural Sci-ence Foundation of China (Grant number: 30873171).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ejps.2013.08.016.

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