Research Article Intestinal Permeability Studies and...

Research ArticleIn Vitro Intestinal Permeability Studies and PharmacokineticEvaluation of Famotidine Microemulsion for Oral Delivery

Sajal Kumar Jha1 Roopa Karki2 Venkatesh Dinnekere Puttegowda2 and D Harinarayana3

1Department of Pharmaceutics Bengal College of Pharmaceutical Sciences amp Research BRB Sarani BidhannagarDurgapur 713212 India2Department of Industrial Pharmacy Acharya amp BM Reddy College of Pharmacy Soldevanahalli Bangalore 560090 India3Nishka Scientific amp Reference Laboratories Hyderabad India

Correspondence should be addressed to Sajal kumar Jha sajalkumarjhagmailcom

Received 12 July 2014 Revised 18 November 2014 Accepted 18 November 2014 Published 7 December 2014

Academic Editor Faiyaz Shakeel

Copyright copy 2014 Sajal Kumar Jha et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The absolute bioavailability of famotidine after oral administration is about 40ndash45 and absorbance only in the initial part ofsmall intestine may be due to low intestinal permeability Hence an olive oil based microemulsion formulation with Tween-80 assurfactant and PEG-400 as cosurfactant was developed by using water titration method with the aim of enhancing the intestinalpermeability as well as oral bioavailability In vitro drug permeation in intestine after 8 h for all formulations varied from 3042 to7839 and most of the formulations showed enhanced permeation compared to pure drug (4892) Famotidine microemulsionexhibited the higher absorption and 119862max achieved from the optimized famotidine formulation (45620 ngsdothml) was higherthan the standard (12680 ngsdothmL) It was found that AUC

0ndash24 h obtained from the optimized famotidine test formulation(30235 ngsdothmL) was significantly higher than the standard famotidine (16633 ngsdothmL) F-1 demonstrated a longer (6 h) 119879maxcompared with standard drug (2 h) and sustained the release of drug over 24 h The bioavailability of F-1 formulation was about18-fold higher than that of standard drug This enhanced bioavailability of famotidine loaded in microemulsion system might bedue to increased intestinal permeability

1 Introduction

Peptic ulcer comprises heterogeneous disorders which man-ifest as a break in the lining of the gastrointestinal mucosabathed by acid and pepsin It is the most predominant ofthe gastrointestinal diseases with a worldwide prevalenceof about 40 in the developed countries and 80 inthe developing countries In recent years large advance inchemical and pharmacological studies has contributed tothe knowledge about new therapeutically active compoundsand control drug delivery systems for peptic ulcer Outof the available category of drugs for the treatment ofulcer H

2antagonistrsquos class of drugs like famotidine and

ranitidine is considered to be the safest drugs available[1] Hence these drugs have promising future if controlledrelease formulations are made Famotidine is N1015840-(aminosulfonyl)-3-[[[2-[(diamine methylene) amino]-4-thiazolyl]

methyl] thio] propanimidamide a model BCS class-III drugIt is a potent H

2receptor antagonist used to treat peptic

ulcer and hence effectively heals gastric and duodenal ulcersand is also effective in Zollinger-Ellison Syndrome The BCSclassification provided new quantitative data of importancefor modern drug development especially within the areaof drug permeability It gives clear and easy applied rulesfor determining the rate limiting factors of GI absorptionprocess For a BCS class-III drug we need to increase itspermeability to improve its oral bioavailability because herein class III drugs they have high solubility but low perme-ability The absolute bioavailability of famotidine after oraladministration is about 40ndash45 [2] and absorbance only inthe initial part of small intestine may be due to low intestinalpermeability Microemulsions as colloidal carriers are oneof the promising systems that have nowadays attractedthe main interest in intestinal permeability enhancement

Hindawi Publishing CorporationInternational Scholarly Research NoticesVolume 2014 Article ID 452051 7 pageshttpdxdoiorg1011552014452051

2 International Scholarly Research Notices

They are optically isotropic transparent and thermodynami-cally stable homogeneous solutions of oil andwater stabilizedby addition of a surfactant and usually a cosurfactant [34] Hence an olive oil based microemulsion formulationwith Tween-80 as surfactant and PEG-400 as cosurfactantwas developed by using water titration method with anaim of enhancing the intestinal permeability as well as oralbioavailability [5] The surfactant and cosurfactant (Tween-80 and PEG-400) may have contributed to an increase inthe permeability of the intestinal membrane or improved theaffinity between lipid particles and the intestinal membrane

2 Materials and Methods

21 Materials Famotidine USP was obtained from MicroLabs (Bangalore India) as free gift sample PEG-400 waspurchased from BD Pharmaceuticals Ltd (Kolkata India)Tween-80 was purchased from Merck Specialties Pvt Ltd(Mumbai India) HPLC-grade methanol ammonium ace-tate and ethyl acetate were purchased from Sigma-AldrichIndia All other chemicals used in this study were obtainedcommercially and were of analytical (AR) grade

22 Preparation and Characterization of Formulations Oliveoil basedmicroemulsion formulationswere developed (phasetitration method) with Tween-80 as surfactant and PEG-400 as cosurfactant keeping constant weight ratio of surfac-tantcosurfactant (2 1) Drug loaded microemulsion system(F-1) was prepared by dispersing famotidine (40mg) intothe mixture of surfactant cosurfactant and oil followed byprecise addition of water drop by drop to the oily phases withmagnetic stirring at ambient temperature After the resultingsystems were equilibrated with gentle magnetic stirring theywere ultrasonicated Droplet size distribution of optimizedmicroemulsion was determined by using a Delsa Nano-C(Beckman Coulter Instruments) based on light scatteringphenomenon which analyzes the fluctuations in light scat-tering Percentage transmittance of samples was measured at650 and 400 nm with distilled water taken as blank and threereplicates were performed for each sample The pH values ofthe microemulsion were measured by a pH meter (DigitalSystronics Mumbai India) at ambient temperature withglass electrode The viscosity measurement of the preparedmicroemulsion was performed using Brookfieldrsquos viscometer(Brookfield LVDV-II + proviscometer) at single mode usingspindle number CPE41 at 32 plusmn 05∘C All aspects of testingwere controlled using Rheocalc software

23 In Vitro Intestinal Permeation Studies To check theintraduodenal permeability the duodenal part of the intes-tine was isolated and taken for the in vitro permeation studyThen this tissue was thoroughly washed with phosphate-buffered saline (pH 68) solution to remove the mucous andlumen contentsThemicroemulsion sample of approximately1mL was injected into the lumen of the duodenum usingsyringe and the two sides of the intestine were tightlyclosed The receiver compartment was filled with 45mL ofphosphate-buffered saline (pH 68) with continuous aeration

and a constant temperature of 37∘C Mixing was performedby means of a magnetic stirrer at 50 rpm 1mL samples werewithdrawn periodically from the receiver compartment attime intervals of 30 minutes 1 h 2 h 3 h 4 h 5 h 6 h and7 h up to 8 h and diluted to 10mL with phosphate-bufferedsaline (pH 68) solution and replaced with an equal volumeof fresh transport medium The absorbance was measuredusing a UV-VIS spectrophotometer at wavelength of 265 nmkeeping the respective blanks (phosphate-buffered saline) [6]

24 Kinetics of Intestinal Permeation Studies In order topredict and correlate the in vitro intestinal permeation behav-ior from these famotidine-loaded microemulsions throughexcised goat intestine it is necessary to fit into a suitablemathematical model The in vitro famotidine permeationdata from microemulsions containing famotidine throughexcised goat intestine were evaluated kinetically by variousmathematical models like zero-order first-order Higuchiand Korsmeyer-Peppas model

25 In Vivo Pharmacokinetic Studies All animal procedureswere performed in accordance with protocols reviewed andapproved by the Committee for the Purpose of Controland Supervision on Experimental Animals (CPCSEA) Thepharmacokinetic study of the microemulsion containingfamotidine was conducted in New Zealand rabbits weighing25ndash30 Kg The rabbits have been chosen as the model forstudy because there have been many bioavailability studiesdone using this animal model The rabbits were housedindividually with free access to food and water A 12 hlight12 h dark cycle was held to keep a normal circadianrhythm in the animals

Six rabbits were divided into three groups and fastedfor 24 hours Control batch was fed with normal salinetest batch was fed with 6mgkg famotidine (pure drug)and the test batch was given the formulation equivalent to6mgkg of drug Water was given ad libitum during fastingand throughout the experiment The blood samples (approx-imately 300ndash400 120583L) were collected from the marginal earvein of the rabbits using heparinized needle (20ndash24 size) atpredetermined time intervals specifically at 05 2 6 8 1012 and 24 hours after oral administration The heparinizedblood samples were immediately collected in centrifugationtubes (5mL) and centrifuged at 20000 rpm at 0∘C for 15minutes Supernatant layer of plasma was separated intoanother centrifugation tube and stored atminus20∘Cuntil analysis[7]

26 LC-MSMS Instrument The 1200 Series HPLC System(Agilent Technologies Waldbronn Germany) was usedMass spectrometric detection was performed on an API 3200triple-quadrupole instrument (Applied BiosystemsMDSSCIEX Toronto Canada) Data processingwas performed onAnalyst 142 software package (SCIEX) [8]

27 Chromatographic Method Conditions Agilent ZorbaxSB-CN (50mm times 21mm ID 5micron) was selected asthe analytical column The mobile phase was composed of

International Scholarly Research Notices 3

Table 1 Chromatographic condition for pharmacokinetic study

Chromatographic conditionsLC-MSMS API 3200 MDS SCIEXIonization source ESI

Column Agilent Zorbax SB-CN (50mm times21mm ID 5micron)

Mobile phase Methanol 20mM ammoniumacetate (55 45 vv)

Flow rate Isocratic flow rate of 06mLminDetection MSMSExtraction solvent 3mL of ethyl acetateInjection volume 10 120583LRetention time offamotidine 137min

Linearity range 5 to 5000 ngmLRabbit plasma volumeused 50 120583L

methanol 20mM ammonium acetate (55 45 vv) The flowrate of the mobile phase was set at 06mLmin and theinjection volume was 10 120583L The column temperature was setat 20∘C [8]The retention time of famotidine was found to beapproximately 137min as shown in Table 1

28 Sample PreparationExtraction Procedure An aliquot50120583L plasmawas used for analysis All samples and standardswere made slightly acidic by addition of 10120583L of 01Maqueous ammonium acetate (pH 6) and were extracted into3mL of ethyl acetate The extraction tubes were shaken athigh speed for 5min followed by centrifugation at 6000 rpmfor 5min The organic phase was transferred to clean glasstubes and evaporated to dryness in a 45∘C water bath undera nitrogen stream The samples were reconstituted within200120583L ofmobile phase and vortexed for 30 sec After transferinto glass inserts of autosampler vials an aliquot of 10 120583L ofeach sample was injected onto the LC-MSMS system [9]

29 Pharmacokinetic Data Analysis After oral adminis-tration of the microemulsion and standard drug plasmasamples were analyzed by LC-MSMS for their famotidinecontent A curve of cumulative drug absorbedVs time curvedfrom 0 to 24 hours was plotted to calculate the area undercurve (AUC) Other pharmacokinetic parameters that ispeak plasma level (119862max) and time to reach peak plasma level(119879max) were obtained after analysis of the individual plasmaconcentration-time curves The calculations were made bycomputer using Win Nonlin TM Professional version 31software (Pharsight Corporation California USA)

3 Results and Discussions

31 Physicochemical Evaluation of Developed FormulationsMicroemulsion formulation containing olive oil was pre-pared using Tween-80 and PEG 400 fixed Smix ratios of 2 1

Table 2 Composition of optimized famotidine microemulsion

Ingredients (by wt) Famotidine microemulsion(F-1)

Famotidine (mgmL) 40Olive oil 714Smix (Tween-80 PEG 400) 6429Water 2857

0102030405060708090

0 2 4 6 8 10Cum

ulat

ive d

rug

perm

eate

d (

)

Time (h)

F-1F-2F-3

Figure 1 Intestinal permeability studies of formulations F-1 F-2and F-3

(Table 2) Formulation was evaluated for the various physico-chemical parameters (Table 3)The narrow globule size rangeof 1701 plusmn 1 nm and polydispersity index 0415 plusmn 0029 for F-1 indicated that the microemulsion approached a monodis-persed stable system and could deliver the drug effectivelyowing to larger surface areaThe presence of zeta potential tothe tune of minus658 plusmn 032mV on the globules of F-1 conferredphysical stability to the system The microemulsions wereexpected to have good physical stability (phase separation) aszeta potential is less than minus30 to minus40mV [10ndash12] A percent-age transmittance of 958 for F-1 indicated clear dispersionThe pH of the optimized famotidine microemulsion wasfound to be 712 plusmn 146 approximating the normal blood pH(74) It was observed that the viscosity of the microemulsionformulation generally was very low (1385plusmn 096 cp)This wasexpected because one of the characteristics ofmicroemulsionformulations is of lower viscosity [13ndash15]

32 In Vitro Intestinal Permeation Studies In vitro drug per-meation was examined through goat intestine over a periodof 8 h in 01 (N) HCl solution at 37 plusmn 05∘C for the optimizedmicroemulsions and the corresponding pure drug Therewas an increase in intestinal permeability for optimizedformulation compared to pure drug due to presence ofsurfactants and cosurfactants combination generally used aspermeation enhancers

In vitro drug permeation in intestine after 8 h for all for-mulations varied from 3042 to 7839 (Table 4 and Figures1 2 and 3) and most of the formulations showed enhancedpermeation compared to pure drug (4892) (Table 5 and


Table 3 Evaluation parameters of optimized microemulsion (119899 = 3)

Formulation pH Globule size(nm) plusmn SEM PDI plusmn SEM Zeta potential

(mV) plusmn SEM Viscosity (cp) Percentagetransmittance

F-1 712 plusmn 146 1701 plusmn 1 0415 plusmn 0029 minus658 plusmn 032 1385 plusmn 096 958

Table 4 In vitro intestinal permeation studies for famotidine microemulsions

F code cumulative drug permeated0 h 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h

F-1 0 1829 plusmn 216 2764 plusmn 371 3358 plusmn 206 4367 plusmn 391 5274 plusmn 337 5968 plusmn 280 6734 plusmn 260 7839 plusmn 252









Data are presented as mean plusmn SD

Table 5 In vitro intestinal permeation studies for pure famotidine


Pure drug(famotidine) 0 1451 plusmn 231 2292 plusmn 219 2845 plusmn 226 3389 plusmn 168 3753 plusmn 216 4115 plusmn 210 4553 plusmn 287 4892 plusmn 236

010203040506070

0 2 4 6 8 10

Cum

ulat

ive d

rug

perm

eate

d (

)

Time (h)

F-4F-5F-6


Figure 4) Formulation F-1 (7839) shows highest perme-ation and F-9 (3042) shows lowest permeation in intestineThe maximum permeation could be due to having the lowestdroplet size and lowest viscosity of all the formulations Thusthe drug diffused at a faster rate from the microemulsionsystem

0

105

1520

30

40

25

35

45

0 2 4 6 8 10

Cum

ulat

ive d

rug

perm

eate

d (

)

Time (h)

F-7F-8F-9


33 Kinetics of Intestinal Permeation Studies In order topredict and correlate the in vitro intestinal permeation behav-ior from these famotidine-loaded microemulsions throughexcised goat intestine it is necessary to fit into a suitablemathematicalmodelThe in vitro famotidine permeation datafrommicroemulsions containing famotidine through excised


Table 6 Famotidine microemulsions intestinal permeation kinetics data

F code Zero-order1198772

First-order1198772

Higuchi kinetics1198772

Korsmeyer-Peppas1198772

Diffusion exponent(119899)

F-1 0986 0984 0774 0977 1283F-2 0979 0983 0777 0990 1329F-3 0996 0998 0926 0998 0849F-4 0877 0947 0993 0989 0610F-5 0992 0995 0929 0986 0784F-6 0993 0994 0928 0991 0812F-7 0994 0998 0925 0988 0752

Pure drug

0102030405060708090

0 2 4 6 8 10

Cum

ulat

ive d

rug

perm

eate

d (

)

Time (h)

F-1

Figure 4 Intestinal permeability studies of pure drug and optimizedformulation

goat intestine were evaluated kinetically by various math-ematical models like zero-order first-order Higuchi andKorsmeyer-Peppas model Based on intestinal permeationbehavior of famotidine-loadedmicroemulsions formulationsF-1 to F-7 have been selected for kinetic evaluation Theresults of the curve fitting into these above-mentioned math-ematical models indicate the in vitro intestinal permeationbehavior of famotidine-loaded microemulsions (F-1 to F-7)shown in Table 6 When respective correlation coefficientswere compared F-1 followed the zero-order model (1198772 =0986) whereas F-2 F-3 and F-4 formulations followedKorsmeyer-Peppas model (1198772 = 0990 0998 and 0989) andformulation F-5 F-6 and F-7 followed the first-order release(1198772 = 0995 0994 and 0998) over a period of 8 hours Thedetermined values of diffusion exponent (119899) ranged between0610 and 1329 indicating that the intestinal drug permeationfrom these famotidine-loaded microemulsions followed theSupercase II transport

34 In Vivo Pharmacokinetic Studies The in vivo study wasperformed to quantify famotidine after oral administrationof formulation containing drug The plasma concentrationtime profiles of the drug in male New Zealand albinorabbits following oral administration of the microemulsionformulation (F-1) and standard drugwere compared Figure 5

050

100150200250300350400450500

0 05 2 6 8 10 12 24

Mea

n pl

asm

a con

cent

ratio

n (n

gm

L)

Time (h)

Famotidine standardFamotidine test

Figure 5 Comparison of pharmacokinetic profiles of standard drugand oral microemulsion of famotidine (F-1)

shows mean plasma concentration-time curve of famotidineafter a single oral administration of standard drug andtest formulation The oral pharmacokinetic parameters arepresented in Tables 7 and 8 Famotidine microemulsions (F-1) demonstrated a longer 119879max (6 h) compared with standarddrugs (2 h) and sustained the release of drugs over 24 hbecause the drug needs to be released out from the oilphase thereby resulting in a delayed 119879max [16] After oraladministration F-1 exhibited the higher absorption and119862maxachieved from the optimized famotidine test formulation(45620 ngsdothmL) represents greater improvement than thestandard drug (12680 ngsdothmL) The drug content of thetest formulation was significantly higher at all time periodsafter administration than that of the standard formulationIt was found that AUC

0ndash24 h obtained from the optimizedfamotidine test formulation (30235 ngsdothmL) was signifi-cantly higher than the standard famotidine (16633 ngsdothmL)Area under the curve (AUC) for microemulsion showedalmost a 18-fold increment from AUC generated afteradministering standard famotidine indicating a significantenhancement of famotidine bioavailability when given orallyas microemulsions [17] The obtained result confirms thesuperior bioavailability of test formulation than the standard


Table 7 Results of pharmacokinetics study on standard famotidineand test formulation

Time (h) Concentration (ngmL)Famotidine standard Famotidine test (F-1)

000 0000 0000050 2340 plusmn 512 3860 plusmn 616200 8620 plusmn 708 45620 plusmn 568600 12680 plusmn 617 31200 plusmn 654800 10620 plusmn 532 15600 plusmn 5731000 8920 plusmn 840 7820 plusmn 5491200 6520 plusmn 626 3610 plusmn 6102400 2920 plusmn 561 1220 plusmn 534Data are presented as mean plusmn SD

Table 8 Pharmacokinetic data of standard famotidine and testformulation

Pharmacokinetic parameters Famotidinestandard

Famotidine test(F-1)

AUC(0ndash24) (ngsdothmL) 16633 30235

119862max (ngmL) 12680 45620119879max (h) 2000 6000Data are presented as mean plusmn SD of four animals

drug The significant differences of the factors leading drugabsorption in vivo between the microemulsion preparationsand standard drugs were probably attributed to the following

Famotidine belongs to BCS class-III drug and the oralabsorption as well as the bioavailability of both drugs ismainly limited due to low intestinal permeability The sur-factant and cosurfactant (Tween-80 and PEG 400) may havecontributed to an increase in the permeability of the intestinalmembrane or improved the affinity between lipid particlesand the intestinal membrane Further due to small particlesize microemulsions may adhere to the gut membrane orenter the inter-villar spaces thus extending gastrointestinalresidence time in the gastrointestinal tract [18] Moreovermicrosized preparation ensures greater surface area and alsothe presence of Tween-80 as a surfactant in the microemul-sion formulation might modulate the intestinal membranepermeability through apically polarized efflux system leadingto enhanced oral bioavailability [19]

4 Conclusion

The results from these studies demonstrated that microemul-sion is a viable approach for developing a liquid dosageform of famotidine with enhanced intestinal permeabilityas well as bioavailability Enhancing permeability correlateswith improved pharmacokinetic profile The pharmacoki-netic studies reveal that the oral administration of famotidinemicroemulsion sustained the release of drugs over 24 h As aconsequence of this decrease in the dose and frequency ofadministration for drugs is possible to achieve the desiredtherapeutic activity This study proved the utilization ofmicroemulsion as a carrier for oral delivery of famotidine

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to Micro Labs (Bangalore India)for their kind gift samples of the drug famotidine They arealso grateful to Nishka Scientific amp Reference LaboratoriesHyderabad India for their kind help in pharmacokineticstudies

References

[1] S Ramchandran G Poovi and M D Dhanaraju ldquoEvaluationof gastric and duodenal antiulcer activity of famotidine formu-lation in experimental animalsrdquo Journal of Pharmacological andToxicological vol 6 no 2 pp 189ndash195 2011

[2] R K Goyal Elements of Pharmacology BS Shah PrakashanNew Delhi India 2008

[3] A A Badawi S A Nour W S Sakran and S M S El-MancyldquoPreparation and evaluation ofmicroemulsion systems contain-ing salicylic acidrdquo AAPS PharmSciTech vol 10 no 4 pp 1081ndash1084 2009

[4] M F Nazar AM Khan and S S Shah ldquoMicroemulsion systemwith improved loading of piroxicam a study ofmicrostructurerdquoAAPS PharmSciTech vol 10 no 4 pp 1286ndash1294 2009

[5] S K Jha R Karki D P Venkatesh B Sajeev and A Geethalak-shmi ldquoCharacterization of olive oil based microemulsion drugdelivery system for oral delivery of antiulcer agentrdquo AmericanJournal of Pharm Tech Research vol 4 no 2 pp 190ndash201 2014

[6] P KGhosh R JMajithiyaM LUmrethia andR S RMurthyldquoDesign and development of microemulsion drug deliverysystem of acyclovir for improvement of oral bioavailabilityrdquoAAPS PharmSciTech vol 7 no 3 pp E172ndashE177 2006

[7] S G Patel S J Rajput A Groshev and V B Sutariya ldquoPrepa-ration and characterization of microemulsion of cilostazol forenhancement of oral bioavailabilityrdquoCurrent DrugDelivery vol11 no 3 pp 531ndash540 2014

[8] Q Cai H Sun Y Peng et al ldquoA potent and orally active antag-onist of multiple inhibitor of apoptosis proteins (IAPs) (SM-406AT-406) in clinical development for cancer treatmentrdquoJournal of Medicinal Chemistry vol 54 no 8 pp 2714ndash27262011

[9] R P Shaik S B Puttagunta C B Kothapalli B Z S Awenand B R Challa ldquoA validated LCndashMSMS method for thedetermination of tolterodine and its metabolite in rat plasmaand application to pharmacokinetic studyrdquo Journal of Pharma-ceutical Analysis vol 3 no 6 pp 489ndash499 2013

[10] R G Thorne and W H Frey ldquoDelivery of neurotrophic factorsto the central nervous system pharmacokinetic considerationsrdquoClinical Pharmacokinetics vol 40 no 12 pp 907ndash946 2001

[11] J Born T Lange W Kern G P McGregor U Bickel and HL Fehm ldquoSniffing neuropeptides a transnasal approach to thehuman brainrdquo Nature Neuroscience vol 5 no 6 pp 514ndash5162002

[12] L Illum ldquoNasal drug delivery new developments and strate-giesrdquo Drug Discovery Today vol 7 no 23 pp 1184ndash1189 2002

[13] M R Patel R B Patel J R Parikh A B Solanki and BG PatelldquoEffect of formulation components on the in vitro permeation


of microemulsion drug delivery system of fluconazolerdquo AAPSPharmSciTech vol 10 no 3 pp 917ndash923 2009

[14] M R Patel R B Patel and J R Parikh ldquoInvestigation ofefficiency of isopropyl myristate-based oil in water microemul-sions for topical delivery of fluconazolerdquo International Journal ofBiomedical and Pharmaceutical Sciences vol 3 pp 60ndash68 2009

[15] M R Patel R B Patel J R Parikh A B Solanki and BG PatelldquoInvestigating effect of microemulsion components in vitropermeation of ketoconazolerdquo Pharmaceutical Development andTechnology vol 16 no 3 pp 250ndash258 2011

[16] Y-M Yin F-D Cui C-F Mu et al ldquoDocetaxel microemulsionfor enhanced oral bioavailability preparation and in vitro andin vivo evaluationrdquo Journal of Controlled Release vol 140 no 2pp 86ndash94 2009

[17] G Sharma K Wilson C F van der Walle N Sattar J R Pet-rie and M N V Kumar ldquoMicroemulsions for oral delivery ofinsulin design development and evaluation in streptozotocininduced diabetic ratsrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 76 no 2 pp 159ndash169 2010

[18] L Hu H Wu F Niu C Yan X Yang and Y Jia ldquoDesign offenofibrate microemulsion for improved bioavailabilityrdquo Inter-national Journal of Pharmaceutics vol 420 no 2 pp 251ndash2552011

[19] H Araya M Tomita and M Hayashi ldquoThe novel formulationdesign of OW microemulsion for improving the gastrointesti-nal absorption of poorly water soluble compoundsrdquo Interna-tional Journal of Pharmaceutics vol 305 no 1-2 pp 61ndash74 2005

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MEDIATORSINFLAMMATION

of


They are optically isotropic transparent and thermodynami-cally stable homogeneous solutions of oil andwater stabilizedby addition of a surfactant and usually a cosurfactant [34] Hence an olive oil based microemulsion formulationwith Tween-80 as surfactant and PEG-400 as cosurfactantwas developed by using water titration method with anaim of enhancing the intestinal permeability as well as oralbioavailability [5] The surfactant and cosurfactant (Tween-80 and PEG-400) may have contributed to an increase inthe permeability of the intestinal membrane or improved theaffinity between lipid particles and the intestinal membrane

2 Materials and Methods

21 Materials Famotidine USP was obtained from MicroLabs (Bangalore India) as free gift sample PEG-400 waspurchased from BD Pharmaceuticals Ltd (Kolkata India)Tween-80 was purchased from Merck Specialties Pvt Ltd(Mumbai India) HPLC-grade methanol ammonium ace-tate and ethyl acetate were purchased from Sigma-AldrichIndia All other chemicals used in this study were obtainedcommercially and were of analytical (AR) grade

22 Preparation and Characterization of Formulations Oliveoil basedmicroemulsion formulationswere developed (phasetitration method) with Tween-80 as surfactant and PEG-400 as cosurfactant keeping constant weight ratio of surfac-tantcosurfactant (2 1) Drug loaded microemulsion system(F-1) was prepared by dispersing famotidine (40mg) intothe mixture of surfactant cosurfactant and oil followed byprecise addition of water drop by drop to the oily phases withmagnetic stirring at ambient temperature After the resultingsystems were equilibrated with gentle magnetic stirring theywere ultrasonicated Droplet size distribution of optimizedmicroemulsion was determined by using a Delsa Nano-C(Beckman Coulter Instruments) based on light scatteringphenomenon which analyzes the fluctuations in light scat-tering Percentage transmittance of samples was measured at650 and 400 nm with distilled water taken as blank and threereplicates were performed for each sample The pH values ofthe microemulsion were measured by a pH meter (DigitalSystronics Mumbai India) at ambient temperature withglass electrode The viscosity measurement of the preparedmicroemulsion was performed using Brookfieldrsquos viscometer(Brookfield LVDV-II + proviscometer) at single mode usingspindle number CPE41 at 32 plusmn 05∘C All aspects of testingwere controlled using Rheocalc software

23 In Vitro Intestinal Permeation Studies To check theintraduodenal permeability the duodenal part of the intes-tine was isolated and taken for the in vitro permeation studyThen this tissue was thoroughly washed with phosphate-buffered saline (pH 68) solution to remove the mucous andlumen contentsThemicroemulsion sample of approximately1mL was injected into the lumen of the duodenum usingsyringe and the two sides of the intestine were tightlyclosed The receiver compartment was filled with 45mL ofphosphate-buffered saline (pH 68) with continuous aeration

and a constant temperature of 37∘C Mixing was performedby means of a magnetic stirrer at 50 rpm 1mL samples werewithdrawn periodically from the receiver compartment attime intervals of 30 minutes 1 h 2 h 3 h 4 h 5 h 6 h and7 h up to 8 h and diluted to 10mL with phosphate-bufferedsaline (pH 68) solution and replaced with an equal volumeof fresh transport medium The absorbance was measuredusing a UV-VIS spectrophotometer at wavelength of 265 nmkeeping the respective blanks (phosphate-buffered saline) [6]

24 Kinetics of Intestinal Permeation Studies In order topredict and correlate the in vitro intestinal permeation behav-ior from these famotidine-loaded microemulsions throughexcised goat intestine it is necessary to fit into a suitablemathematical model The in vitro famotidine permeationdata from microemulsions containing famotidine throughexcised goat intestine were evaluated kinetically by variousmathematical models like zero-order first-order Higuchiand Korsmeyer-Peppas model

25 In Vivo Pharmacokinetic Studies All animal procedureswere performed in accordance with protocols reviewed andapproved by the Committee for the Purpose of Controland Supervision on Experimental Animals (CPCSEA) Thepharmacokinetic study of the microemulsion containingfamotidine was conducted in New Zealand rabbits weighing25ndash30 Kg The rabbits have been chosen as the model forstudy because there have been many bioavailability studiesdone using this animal model The rabbits were housedindividually with free access to food and water A 12 hlight12 h dark cycle was held to keep a normal circadianrhythm in the animals

Six rabbits were divided into three groups and fastedfor 24 hours Control batch was fed with normal salinetest batch was fed with 6mgkg famotidine (pure drug)and the test batch was given the formulation equivalent to6mgkg of drug Water was given ad libitum during fastingand throughout the experiment The blood samples (approx-imately 300ndash400 120583L) were collected from the marginal earvein of the rabbits using heparinized needle (20ndash24 size) atpredetermined time intervals specifically at 05 2 6 8 1012 and 24 hours after oral administration The heparinizedblood samples were immediately collected in centrifugationtubes (5mL) and centrifuged at 20000 rpm at 0∘C for 15minutes Supernatant layer of plasma was separated intoanother centrifugation tube and stored atminus20∘Cuntil analysis[7]

26 LC-MSMS Instrument The 1200 Series HPLC System(Agilent Technologies Waldbronn Germany) was usedMass spectrometric detection was performed on an API 3200triple-quadrupole instrument (Applied BiosystemsMDSSCIEX Toronto Canada) Data processingwas performed onAnalyst 142 software package (SCIEX) [8]

27 Chromatographic Method Conditions Agilent ZorbaxSB-CN (50mm times 21mm ID 5micron) was selected asthe analytical column The mobile phase was composed of
















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4 Conclusion




Acknowledgments


References


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















0102030405060708090

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F-1 0986 0984 0774 0977 1283F-2 0979 0983 0777 0990 1329F-3 0996 0998 0926 0998 0849F-4 0877 0947 0993 0989 0610F-5 0992 0995 0929 0986 0784F-6 0993 0994 0928 0991 0812F-7 0994 0998 0925 0988 0752

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n pl

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cent

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n (n

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4 Conclusion




Acknowledgments


References


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





















010203040506070

0 2 4 6 8 10

Cum

ulat

ive d

rug

perm

eate

d (

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Time (h)

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0

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1520

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40

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F-1 0986 0984 0774 0977 1283F-2 0979 0983 0777 0990 1329F-3 0996 0998 0926 0998 0849F-4 0877 0947 0993 0989 0610F-5 0992 0995 0929 0986 0784F-6 0993 0994 0928 0991 0812F-7 0994 0998 0925 0988 0752

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100150200250300350400450500

0 05 2 6 8 10 12 24

Mea

n pl

asm

a con

cent

ratio

n (n

gm

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Time (h)
















4 Conclusion




Acknowledgments


References


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




First-order1198772




F-1 0986 0984 0774 0977 1283F-2 0979 0983 0777 0990 1329F-3 0996 0998 0926 0998 0849F-4 0877 0947 0993 0989 0610F-5 0992 0995 0929 0986 0784F-6 0993 0994 0928 0991 0812F-7 0994 0998 0925 0988 0752

Pure drug

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ive d

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Time (h)

F-1




050

100150200250300350400450500

0 05 2 6 8 10 12 24

Mea

n pl

asm

a con

cent

ratio

n (n

gm

L)

Time (h)
















4 Conclusion




Acknowledgments


References


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












4 Conclusion




Acknowledgments


References


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Intestinal Permeability Studies and...

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