Enhancing mechanism of intestinal absorption of highly lipophilic compounds using microemulsion-Quantitative analysis of the partitioning to the mesenteric lymph in intestinal cells

Kazunori Iwanaga,Yoichi Higashiyama,Makoto Miyazaki, Masawo Kakemi

Division of Pharmaceutics,Osaka University of Pharmaceutical Sciences,4-20-1 Nasahara,Takatsuki, Osaka 569-1094,Japan

Original Research Paper

Enhancing mechanism of intestinal absorption of highly lipophilic compounds using microemulsion-Quantitative analysis of the partitioning to the mesenteric lymph in intestinal cells

Kazunori Iwanaga＊,Yoichi Higashiyama,Makoto Miyazaki, Masawo Kakemi

Division of Pharmaceutics,Osaka University of Pharmaceutical Sciences,4-20-1 Nasahara,Takatsuki, Osaka 569-1094,Japan

ARTICLEINFO

Article history:

Received 10 September 2014

Received in revised form

11 December 2014

Accepted 14 December 2014

Available online 30 December 2014

Microemulsion

Intestinal absorption

Mesenteric lymph

Partition

Quantitative analysis

The purpose of this study was to quantify the effect of the fatty acid alkyl-chain length of a polyethylene glycol(PEG)glyceryl ester,which was used as a microemulsion oil component,on the partitioning of highly lipophilic compounds to the mesenteric lymph after oral administration.Oil blue N,a highly lipophilic anthraquinone derivative,was orally administered to lymph duct-cannulated and untreated rats in two kinds of different microemulsions.Gelucire®50/13 and Gelucire®44/14 were used as the oil component with long chain and medium chain fatty acid portions,respectively,of PEG glyceryl esters in microemulsions.The cumulative amount of oil blue N in lymph f l uid was almost the same between the two microemulsions,although the transferred amount of oil component (triglyceride)in the lymph after administration of the Gelucire®50/13 microemulsion was signif i cantly higher than that of the Gelucire®44/14 microemulsion.On the other hand,the solubility of oil blue N in Gelucire®44/14 was much higher than that in Gelucire®50/13.No signif i cant differences were observed between microemulsions in the bioavailability of oil blue N.From these data,the partitioning of oil blue N to the lymph was calculated using a mathematical model,showing that the partitioning ratios of oil blue N to the lymph f l uid were almost the same for both microemulsions.The solubility of oil blue N to the oil component of the microemulsions and the transfer of triglycerides to the lymph after administration of the microemulsions counteract each other,leading to similar partitioning ratios of oil blue N to the lymph.

©2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

1.Introduction

Recently,changes in the strategies for new drug development have led to advances and a higher number of lipophilic,or poorly water-soluble,compounds being tested as drug candidates[1].In the case of extremely poorly water-soluble compound which cannot be expected to show enough bioavailability,it has to be developed as parenteral formulations such as an injection,or the def i nition of maximum tolerated dose and toxicity testing are also impracticable due to low solubility because higher dose is needed for these study;therefore,strategies for developing new oral dosage forms are necessary to increase the intestinal absorption.One of the ways to resolve this issue,the utilization of lipid dispersion systems,is promising because of their potential to dissolve highly lipophilic drugs[2-4].

Triglycerides can be absorbed as a component of a formulation that permeates the intestinal epithelial cell membrane after hydrolysis to free fatty acids and monoglycerides by pancreatic lipase in the intestinal tract,as well as following ingestion as dietary lipids.Subsequently,triglycerides are reconstructed in the endoplasmic reticulum of the intestinal epithelium and released into the mesenteric lymph duct as chylomicrons,comprising apolipoprotein and cholesterol in the Golgi apparatus.Triglycerides transferred to the mesenteric lymph f i nally reach the systemic circulation via the thoracic lymph;therefore,the intestinal absorption of the lipophilic drugs administered in lipid dispersion systems, such as emulsions,can be affected by the absorption of triglycerides concomitantly administered as a component of formulations[5,6].By means of a model-based quantitative analysis,we previously clarif i ed that the amount of a compound absorbed via the lymph ducts is related to both the partitioning of the compound loaded in the emulsion to the lymph route after oral administration,which is determined by the solubility of the compound in the triglyceride in the emulsion,and the amount of triglyceride transferred into the lymph duct[7].We also showed that the longer the fatty acid alkyl-chain length of the triglyceride making up the emulsion, the more triglyceride transferred to the lymph f l uid[7].

In recent years,microemulsions have been much anticipatedas a novel dosageformulation for highly lipophilicdrugs [8,9].A microemulsion is def i ned as a dispersion system composed of oil/surfactant/co-surfactant/water,which is transparent and thermodynamically stable[10];therefore,it can dissolve highly lipophilic drugs in its core oil phase.For example,Neoral®,a self-microemulsifying drug delivery system(SMEDDS)including cyclosporin A,and has been used clinically[11].It has been reported that orally administered Neoral®forms a microemulsion by contacting and mixing with the intestinal f l uid,thereby improving the intestinal absorption of cyclosporin A and decreasing the variation in the amount of intestinally absorbed drug[12].

Gelucire®s are the most frequently used excipients as they are powerful solubilizing agents and bioavailability enhancers by oral and topical routes.Both Gelucire®44/14(lauroyl macrogolglycerides)andGelucire®50/13(stearoylmacrogolglycerides)have unique compositions of amphiphilic lipids producedbyinductionofpolyethyleneglycol(PEG)to glyceride groups;therefore,they are used for oral dosage formulationsof highlylipophilic drugseither by themselvesor in combination with other co-surfactants.However,the effects of the fatty acid alkyl-chain length of a PEG glyceryl ester in a microemulsion on the drugabsorptionmechanismand/or drug absorption enhancement mechanism are not fully understood in situations where a microemulsion is administered as a lipid-based formulation.

In this study,we prepared microemulsions using Gelucire®44/14 or Gelucire®50/13 as an oil phase,a mixture of Cremophor ELR/ethanol=3/1 as a surfactant/co-surfactant,and water,and used oil blue N as a model highly lipophilic compound(Fig.1).The effect of the fatty acid alkyl-chain length of a PEG glyceryl ester in a microemulsion on both the partitioning of highly lipophilic compounds to the mesenteric lymph and intestinal absorption enhancement were examined in rats.

2.Materials and methods

2.1.Materials

Oil blue N and Cremophor®EL were purchased from Sigma Chemical Co.,Ltd.(St.Louis,MO,USA).Gelucire®44/14 (lauroyl macrogolglycerides)and Gelucire®50/13(stearoyl macrogolglyceride)were kind gifts from Gattefosse(Gennevilliers Cedex,France).The ethanol was from Nacalai Tesque Co.,Ltd.(Kyoto,Japan).The purity of oil blue N and ethanol were more than 98%and 99.5%,respectively,and that of other chemicals including Cremophor®EL,Gelucire®44/14,and Gelucire®50/13 were more than 95%.

2.2.Construction of a pseudo-ternary phase diagram of microemulsions

The pseudo-ternary phase diagrams of oil,surfactant/cosurfactant,and water were constructed at room temperature usingthe watertitration method[13,14].Brief l y,mixtureswith various compositions of oil and surfactant/co-surfactant were prepared at 60°C.The adequate volume of water was added while stirring at 60°C.The mixture was sonicated at 40 W for 2 min using an Ultrasonic Disrupter UD-201(Tomy Co.,Tokyo, Japan)and put into the well of a titration plate.The plate was left for 24 h at room temperature.The turbidity of each sample was determined by measuring the absorbance at 600 nm. The samples that showed no turbidity were identif i ed as microemulsions.

2.3.Preparation of the microemulsion formulationsOil blue N(clogP=4.73,Sci.Finder;CAS,Columbus,OH)was used as a model poorly water-soluble compound.The chemical structure of oil blue N is shown in Fig.1.The weight ratio of each composition of microemulsion used in this study was fi xed to oil/(surfactant/co-surfactant)/water=1/1/8 according to the results of the pseudo-ternary phase diagram.Either Gelucire®44/14 or Gelucire®50/13 was used as the oil and Cremophor®EL and ethanol were used as a surfactant and cosurfactant,respectively.The weight ratio of surfactant/cosurfactant was fi xed to 3/1 for both microemulsion formulations because the highest solubility of oil blue N was observed at this ratio(data not shown).The preparation of the microemulsions was performed according to the method of Ito et al. [15].Brie fl y,the adequate weights of oil,surfactant,and co-surfactant were mixed at 60°C.Oil blue N was added to the mixture and sonicated at 40 W for 2 min.Water was then added to the mixture while stirring at 60°C.The oil blue N concentration in each microemulsion was adjusted to 1.2 mg/ml.

2.4.Measurement of the droplet size of the microemulsions

The droplet size of the microemulsions was measured at 37°C using the dynamic light scattering technique with a Zeta sizer nano-S(Malvern instruments,Ltd.,Worcestershire,UK).

2.5.In vivo oral administration to rats with noncannulated lymph ducts

All animal experiments were approved by the Animal Experimentation Committee of the Osaka University of Pharmaceutical Sciences.Male Wistar rats weighing 300-350 g were obtained from Japan SLC,Inc.(Shizuoka,Japan).Rats were fed and given drinking water ad libitum,and housed under a 12-h light-dark cycle for at least 2 weeks before the experiment. Rats were divided into two groups with 4 rats in each group for all the experiments.The day before the experiment,the rats were lightly anesthetized with ether,and were implanted surgically with a combination of phicon(Fuji Systems Ltd, Tokyo,Japan)and PE50(Clay Adams,Parsippany,NJ,USA)in a catheter,which was inserted into the right jugular vein for blood sampling.The catheter was externalized through the back of the neck and secured.After recovery,rats were fasted but drinking water was supplied ad libitum for 16 h before the experiment.After a suff i cient recovery period,0.5 ml of each microemulsion formulation was orally administered using a stomach catheter and 400 μl of blood was periodically withdrawn from the jugular vein at a predetermined time interval for 8 h.Blood was centrifuged at 7400 g for 5 min to obtain a plasma sample.Plasma samples were kept at-20°C until use in the assay.For calculating the bioavailability,the concentration of oil blue N in the plasma after intravenous injection of 120-μg oil blue N was used as per data we previously reported[7].To keep the rats with cannulated lymph ducts under the same conditions,the rat abdomens were incised and sutured as a sham operation.

2.6.In vivo oral administration to rats with cannulated lymph ducts

To measure the transfer of oil blue N into the lymph f l uid,the rat abdomen was incised and a catheter(0.8 mm,Natsume Seisakusho Co.,Ltd.,Tokyo,Japan)was inserted into the thoracic lymph duct under sodium pentobarbital anesthesia (i.p.50 mg/kg of body weight).After the catheter was externalized,the abdominal incision was sutured.The rats were restrained in a Ballman cage and were fasted for 16 h before the experiment,but drinking water was supplied ad libitum. Each microemulsion formulation(0.5 ml)was orally administeredandlymphf l uidwascollectedintoa clearglasstubefor 8 h,with the glass tube exchanged for a new one every hour. The endpoint of blood and lymph f l uid sampling was set at 8 h because we found,in a preliminary experiment,that both intestinal absorption and the lymphatic transfer of oil blue N ended within 8 h of oral administration.Lymph f l uid and plasma samples were kept at-20°C until use in the assay.

2.7.Determination of oil blue N concentration in the plasma

An 800-μl aliquot of phosphate-buffered saline(pH 7.4)and 10 μl of internal standardsolution(Solventblue 59 dissolvedin dimethyl sulfoxide[20 μg/ml])were added to the plasma sample(200 μl).Subsequently,2 ml of chloroform was added, and the mixture was vigorously shaken for 10 min.After centrifugation at 1200 g for 10 min,the organic phase was transferred into another tube and evaporated in vacuo.Methanol(150 μl)was added for reconstitution and a 50-μl sample was injected into a high-performance liquid chromatography (HPLC)system(JASCO Co.,Tokyo,Japan).The area under the compound concentration-time curve(AUC)was calculated using the linear trapezoidal method,and the AUC extending beyondthe last data samplingpointwas estimatedby dividing the concentration in the plasma at 8 h after administration by the terminal slope.

2.8.Determination of oil blue N and triglyceride concentration in the lymph f l uid

Methanol(500 μl)was added to 20 μl of the lymph fl uid sample, and the mixture was vigorously shaken for 1 min.The mixture was centrifuged at 5300 g for 4 min,and 50 μl of the supernatant was injected into the HPLC system.

The amount of triglyceride transferred to the mesenteric lymph after oral administration of each microemulsion to rats wasdeterminedbymeasuringthetriglycerideconcentrationin the lymph fl uid using a commercial kit(Triglyceride E Test WAKO,Wako Pure Chemicals Co.,Ltd.,Osaka,Japan).As a controlexperiment,thetriglycerideconcentrationinthelymph fl uid after oral administration of 0.5 ml of water was determinedinasimilarmannertothatusedforthemicroemulsions.

2.9.Solubility of oil blue N in the Gelucire®s

The solubility of oil blue N in Gelucire®44/14 and Gelucire®50/ 13wasmeasuredusingtheconventionalf l ask-shaking method.Gelucire®44/14 and Gelucire®50/13 are consideredto form the core region of the chylomicron after reconstruction in the intestinal epithelium.To determine the solubility, 10%Gelucire®s in water was used because they are semi-solid at 37°C.An excess amount of oil blue N was added to 3 ml of each solution and stirred for 12 h at 37°C.The samples were centrifuged at 7400 g for 5 min.The supernatant was f i ltered and diluted with acetonitrile and the oil blue N concentration was measured using a spectrophotometric method.The absorbance of oil blue N in acetonitrile was measured at spectrum of 644 nm.

2.10.Evaluation of the effects of the microemulsions on the intracellular disposition of oil blue N

An orally administered highly lipophilic drug is taken up by epithelial cells;thereafter,a fraction of the drug enters the chylomicron and is released into the mesenteric lymph duct. Consequently,drugs absorbed via the lymphatic route avoid the hepatic f i rst-pass effect.We previously reported that quantitative analysis of the partitioning of highly lipophilic compounds to the mesenteric lymph in intestinal cells possible using the following equations[7].The total absorbed amount of a highly lipophilic drug after oral administration can be quantitatively described by the sum of the drug amount absorbedvia the portal route andthat absorbedby the lymphatic route(Fig.2);therefore,the following equation (eq.(1))is obtained:

where,Fais the fraction of the dose taken up by the intestinal cells,and Fgand Fhare the fractions of the dose that are not metabolized in the intestine and liver,respectively.fportaland flymphare the ratio of the amount transferredto the portal vein and mesenteric lymph duct,respectively,to the amount that is not metabolized in the intestinal epithelial cells.The relationship between fportaland flymphcan be expressedas follows:

In the experiment in which rats with cannulated lymph ducts were used,the total amount of drug absorbed via the lymphatic route(Alymph)can be obtained.The ratio of the amount absorbed via the lymphatic route to the total amount absorbed after oral administration(ROL)can be described using eqs.(1)and(2)as follows:

A value of 0.824±0.021 is used as the Fhfrom our previous report[7].By substituting Alymphand F obtained from the experiments in eq.(3),the flymphwas calculated to consider the effect of each microemulsion on the intracellular disposition of oil blue N.

2.11.HPLC conditions

The concentrations of oil blue N in the samples were determined by reversed-phase HPLC on a Mightysil RP-18 GP column(250×4.0 mm,5 μm).The HPLC system consisted of a PU-980 pump,UV-970 UV-VIS spectrophotometric detector (JASCO Co.,Tokyo,Japan),and C-R6A integrator(Shimadzu Co.,Kyoto,Japan).The mobile phase was acetonitrile(100%) and was run at a f l ow rate of 1.5 ml/min.The UV-VIS detector was set at 644 nm.The method exhibited a linear range of 10-1000 ng/ml in blank rat plasma and lymph f l uid with lower detection limit of 5 ng/ml.The intra-and inter-assay coeff i cients of variation did not exceed±12%from the nominal concentration.The accuracy of oil blue N was within±9%of the theoretical value.

2.12.Statistical analysis

All values are expressed as the mean±standard deviation of the mean(SD).The statistical analyses were performed using the Mann-Whitney U test.The level of signif i cance was set at p＜0.05.

3.Results and discussion

3.1.Characterization of Gelucire®44/14 and Gelucire®50/13 microemulsion

Microemulsion is the most promising formulation to enhance theoralbioavailabilityofhighlylipophilicdrugs[16,17]because itcandissolvethesecompounds inits coreoilphase;however, it is composed of oil/surfactant/co-surfactant/water and is a more complex system than emulsion.we prepared microemulsions using Gelucire®44/14 or Gelucire®50/13 as an oilphase,a mixture of Cremophor ELR/ethanol=3/1 as a surfactant/co-surfactant,and water.

Table 1-Effects of fatty acid alkyl-chain length of the oil component in microemulsions on the partitioning of oil blue N to the mesenteric lymph.

The pseudo-ternary phase diagrams of oil,surfactant/cosurfactant,and water used for the formation of the microemulsions are shown in Fig.3.Microemulsions were formed using both Gelucire®s as an oil composition.The region of microemulsion formationforGelucire®44/14was greaterthan that for Gelucire®50/13.According to this result,the composition of the microemulsions was determined as Gelucire®44/ 14 or Gelucire®50/13/(Cremophor EL/ethanol)/water=1/1/8 and the Cremophor®EL/ethanol ratio was f i xed to 3/1 for further experiments.

The mean volume diameter and size distribution of each microemulsion was measured using the dynamic light scattering method.The mean volume diameters of the Gelucire®44/14 and Gelucire®50/13microemulsions were 12.3±2.62 nm and 10.6±1.62 nm,respectively.No signif i cant differences in mean diameter and size distribution were observed between microemulsions.

3.2.Solubility of oil blue N to the oil component ofmicroemulsions

The solubility of oil blue N to 10%Gelucire®s in water is listed in Table 1.The solubility of oil blue N to 10%Gelucire®44/14 is approximately 3-fold higher than that to 10%Gelucire®50/13. This result shows that oil blue N has a greater aff i nity for theGelucire®44/14,which is an oil component with medium fatty acid alkyl-chain length.

3.3.Effect of the alkyl-chain length of the fatty acid portion of a PEG glyceryl ester in microemulsion on the transfer of oil blue N to the lymph f l uid

The enhanced bioavailability of lipophilic compounds in microemulsions may also be caused by absorption via the lymphatic route.It is very important for the development of formulations to clarify the difference in the mechanism of enhancing intestinal absorption for highly lipophilic drugs between emulsion and microemulsion preparations.

The time course of the cumulative amount of oil blue N recovered in the lymph f l uid after oral administration to rats with cannulated lymph ducts is shown in Fig.4.When administered in each microemulsion,oil blue N was detected in the lymph f l uid as early as 1 h after administration,and the recovered amounts gradually increased.The cumulative amount of oil blue N stabilized within 8 h of administration. The total amounts of oil blue N recovered in the lymph f l uid afteradministration inthe Gelucire®44/14andGelucire®50/13 microemulsions were 35.7±0.7%and 29.2±4.3%,respectively.No signif i cant difference in total recovered amount was observed between the two microemulsions.This result shows thatmicroemulsionas wellas emulsionpreparations promote the absorption of oil blue N via the lymphatic route since no absorption was observed after administration as a suspension form[7].We previously showed that the cumulative amount of oil blue N in the lymph f l uid after administration in emulsion was dependent on the chain length of the fatty acid portion of a triglyceride in an emulsion,and that a long-chain triglyceride emulsion showed a value of 23.6±3.1%[7].This suggests that a microemulsion preparation provides an enhancement to the transfer of oil blue N to the lymph than does an emulsion preparation.Generally,it is considered that the highly lipophilic compounds show the high aff i nity to the oil component with a longer fatty acid alkyl-chain length.In fact,the order of the alkyl-chain length of fatty acid portion of oil was good agreement with that of the cumulative amount of oil blue N in the lymphatic f l uid after administration in emulsions[7].On the other hand,no tendency was shown for themicroemulsions;therefore,themechanismofthe lymphatic transfer of highly lipophilic compound is probably different between emulsions and microemulsions.

3.4.Comparison of the amount of triglyceride in microemulsion transferred to the lymph f l uid after oral administration

Highly lipophilic compounds,such as oil blue N used in this study,possibly transfer to the mesenteric lymph with the f l ow oftheoilcomponent(alkyl-macrogolglycerides)ofthe microemulsion.We measured the amount of triglyceride in the lymph f l uid after oral administration of the microemulsions as an index of the transfer of lipid formulation to the lymph.As shown in Fig.5,the cumulative amount of triglyceride in the lymph f l uid 8 h after administration of Gelucire®44/14 and Gelucire®50/13 microemulsions reached 42.03±2.92 mg and 62.21±6.48 mg,respectively.In the control group,the cumulative amount of triglyceride in the lymph was 21.55±2.39 mg.Compared to the control group,both microemulsionssignif i cantlyincreasedtheamountof triglyceride transferred to the lymph.The concentration of triglyceride in the lymph f l uid after administration of the Gelucire®50/13 microemulsion was signif i cantly higher than that after the Gelucire®44/14 emulsion.This suggests that microemulsions composed of oil with a long fatty acid alkylchain provide greater enhancement of the transfer of triglyceride to the lymph than those with a medium alkyl-chain triglyceride portion.This result is in agreement with our previous reports using emulsions[7].Khoo et al.[18]also reported that the triglyceride amount transferred into the lymph f l uid after oral administration of a microemulsion composed of long-alkyl chain triglycerides as an oil component to dogs was approximately 4-fold higher than that of a microemulsion with a medium-alkyl chain triglyceride.The drugs absorbed via this route can avoid the hepatic f i rst-pass effect;therefore, not only emulsions but also microemulsions are expected tobe formulations that enable improved oral bioavailability of highly lipophilic compounds.

3.5.Enhancing effect of intestinal absorption of oil blue N by microemulsionDifferences in the amount of a drug absorbed via the lymphatic route may alter its bioavailability after oral administration becausedrugsabsorbedviathisroutecanavoidthehepatic fi rstpass effect.To con fi rm the effects of microemulsions on the bioavailability of oil blue N,we measured the oil blue N concentration in plasma after oral administration in rats without cannulated lymph ducts.The concentration of oil blue N in the plasma after oral administration are shown in Fig.6.When administeredinbothmicroemulsions,oilblueNwasdetectedin the plasma samples.The concentration of oil blue N in plasma was ranked Gelucire®44/14 microemulsion≥Gelucire®50/13 microemulsion for each sampling point.The calculated bioavailabilities of oil blue N are listed in Table 1.No signi fi cant differences in bioavailability were observed between the Gelucire®44/14(0.496±0.046)and Gelucire®50/13(0.385±0.036) microemulsions;however,these values were signi fi cantly higher than that in the long-chain triglyceride emulsion that showedthe highestbioavailability(0.284±0.028)ofoilblueNin our previous study[7].

Taken together,the af fi nity(i.e.,solubility)of oil blue N to Gelucire®44/14 is higher than that to 50/13 Gelucire®.On the other hand,the triglyceride amount transferred to the lymph after administration of the 50/13 Gelucire®emulsion was higher than that of the Gelucire®44/14 emulsion,with the result that the amount of oil blue N transferred to the lymph fl uid was comparable between both microemulsions because of these factors counteracting.

3.6.Quantitative evaluation of the partitioning of oil blue N to the lymphatic route after oral administration in microemulsion

An orally administered drug is taken up by the intestinal epithelial cells,and thereafter,partitioned into the lymph; therefore,the drug amount taken up in the epithelial cells greatly affects the amount of drug transferred to the lymph. We performed quantitative analysis of the drug disposition in the intestinal cells using the results of in vivo oral administration experiments.

By substitution of Alymph,the bioavailability and Fhvalues in eq.(3),the ROL,and flymphwere calculated and these are listed in Table 1.The flymphvalues after administration in the Gelucire®44/14 and Gelucire®50/13 microemulsions were 0.679±0.046 and 0.724±0.022,respectively.The ROL values for Gelucire®44/14 and Gelucire®50/13 microemulsion were 0.720 and 0.761,respectively.No signif i cant differences were observed between microemulsions for either parameter.A reasonable explanation for the comparable flymphvalues between the 50/13 and 44/14 Gelucire®microemulsions is that the aff i nity of oil blue N to the oil component of the microemulsions and the transfer of triglyceride to the lymph after their administration counteract each other.To increase intestinal absorption via the lymph route,it is effective to use a formulation including an oil component that increases both drug solubility and the f l ow of triglyceride to the lymph.

4.Conclusion

We clarif i ed that the partitioning of highly lipophilic compoundsintheintestinalcellstothemesentericlymphafteroral administration in microemulsion is independent on the chain length of the fatty acid portion of an oil component of microemulsions using the mathematical model.The enhancing mechanism of intestinal absorption of highly lipophilic compounds by micromelusions is probably different from that by emulsions.

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*Corresponding author.Division of Pharmaceutics,Osaka University of Pharmaceutical Sciences,4-20-1 Nasahara,Takatsuki,Osaka 569-1094,Japan.Tel./fax:+81 72 690 1049.

E-mail address:iwanaga@gly.oups.ac.jp(K.Iwanaga).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2014.12.005

1818-0876/©2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).