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Poloxamer-Stabilized Topical Miconazole Nitrate-Loaded Microemulsion: Formulation Design andCharacterizationKenneth Chibuzor Ofokansi a , Franklin Chimaobi Kenechukwu a & A. B. Isah ba Drug Delivery Research Unit, Department of Pharmaceutics, Faculty of PharmaceuticalSciences , University of Nigeria , Nsukka , Enugu State , Nigeriab Department of Pharmaceutics and Pharmaceutical Microbiology , Ahmadu Bello University ,Zaria , NigeriaAccepted author version posted online: 17 Jan 2013.Published online: 23 Oct 2013.

To cite this article: Kenneth Chibuzor Ofokansi , Franklin Chimaobi Kenechukwu & A. B. Isah (2013) Poloxamer-StabilizedTopical Miconazole Nitrate-Loaded Microemulsion: Formulation Design and Characterization, Journal of Dispersion Science andTechnology, 34:11, 1563-1574, DOI: 10.1080/01932691.2012.752713

To link to this article: http://dx.doi.org/10.1080/01932691.2012.752713

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Poloxamer-Stabilized Topical MiconazoleNitrate-Loaded Microemulsion: Formulation Design andCharacterization

Kenneth Chibuzor Ofokansi,1 Franklin Chimaobi Kenechukwu,1 andA. B. Isah21Drug Delivery Research Unit, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences,University of Nigeria, Nsukka, Enugu State, Nigeria2Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University,Zaria, Nigeria

GRAPHICAL ABSTRACT

Miconazole nitrate-loaded microemulsion based on Tween 80 and propylene glycol, castor oiland water with and without Poloxamer 407 was prepared and characterized with respect to drugcontent, stability, antifungal, and physicochemical properties and ex vivo drug release usinga modified Franz diffusion apparatus. Results obtained revealed homogeneous and stable micro-emulsions with excellent physicochemical properties, permeation coefficients and fluxes, andincreased in vitro antifungal activity (�1.2 times) against clinically isolated Candida albicanswhen compared to a commercial topical miconazole (Fungusol) solution. Poloxamer-stabilizedmicroemulsion could offer a better and more reliable approach of delivering miconazole than boththe unstabilized and Fungusol formulations.

Keywords Globule size, miconazole nitrate, microemulsion, thermodynamic stability

1. INTRODUCTION

A large percentage of new drug candidates in addition tomany existing drugs often show poor solubilizationbehavior which leads to poor oral bioavailability with wideintra- and inter-subject variation and present formulatorswith considerable technical challenges.[1–3] Therefore,producing suitable formulations is very important inimproving the solubility and bioavailability of such drugs.The selection of an appropriate dosage form is criticalbecause a dosage form with poor drug delivery capacitycan make a useful drug worthless. Bioavailability has

important clinical implications as both pharmacologicand toxic effects are proportional to both dose and bio-availability.[4] To overcome the solubility and bioavail-ability problems of such drugs, many approaches havebeen meticulously explored with success in some selectedcases, including use of permeation enhancers, nanoparticlesand solid dispersions, complexation with cyclodextrins, saltformation, particle size reduction (micronization or nano-sizing), solubilization based on cosolvents, surfactants,etc.[5,6] Recently, lipid-based formulations have attracteda great deal of attention in drug discovery and productdevelopment to effectively overcome physical and biologi-cal barriers related to poor aqueous solubility and stability,membrane permeability, drug efflux, and availability.[7–15]

The rapid growth in the use of lipid-based drug deliverysystems is primarily due to the diversity and versatility ofpharmaceutical grade excipients and drug formulations,

Received 21 November 2012; accepted 21 November 2012.Address correspondence to Kenneth Chibuzor Ofokansi, Drug

Delivery Research Unit, Department of Pharmaceutics, Facultyof Pharmaceutical Sciences, University of Nigeria, Nsukka410001, Enugu State, Nigeria. E-mail: kcofokansi@yahoo.com

Journal of Dispersion Science and Technology, 34:1563–1574, 2013

Copyright # Taylor & Francis Group, LLC

ISSN: 0193-2691 print=1532-2351 online

DOI: 10.1080/01932691.2012.752713

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and their compatibility with liquid, semi-solid, and soliddosage forms.[16,17] An array of lipid systems is availableto pharmaceutical formulators. In fact, the most favoredapproach to improve the oral bioavailabilty of lipophilic drugsis to incorporate such drugs into inert lipid vehicles such asoils, surfactant dispersions, microemulsions, self-emulsifyingformulations, self-microemulsifying formulations, andliposomes.[18–21] This could lead to increased solubilizationwith concomitant modification of their pharmacokineticprofiles, leading to increase in therapeutic efficacy.

Microemulsions are liquid dispersions of water and oilthat are made homogenous, transparent (or translucent)and thermodynamically stable by the addition of relativelylarge amounts of a surfactant and a co-surfactant andhaving an average droplet diameter of 1 to 100 mm.[22–25]

The presence of surfactant and cosurfactant in the systemmakes the interfacial tension very low. These carriersappear to represent an intermediate state between thermo-dynamically stable solubilized solutions, that is, micellescontaining solubilized oils and ordinary emulsions whichare relatively unstable.[26] They can be classified as oil-in-water (o=w), water-in-oil (w=o) or bicontinuous systemsdepending on their structure and are characterized byultra-low interfacial tension between oil and water phases.These versatile systems are currently of great technologicaland scientific interest to researchers because of their poten-tial to incorporate a wide range of drug molecules (hydro-philic and hydrophobic) due to the presence of bothlipophilic and hydrophilic domains. In other words, micro-emulsions act as ‘‘supersolvents’’ of drugs.

In addition to oral and intravenous delivery, they areamenable to sustained and targeted delivery through oph-thalmic, dental, pulmonary, vaginal, and topical routes inaddition to their use in cosmetics and biotechnology-basedproducts[27–32] Microemulsions as drug delivery tool showfavorable properties like clarity, thermodynamic stability(long shelf-life), easy formation (zero interfacial tensionand almost spontaneous formation), optical isotropy,ability to be sterilized by filtration, high surface area (highsolubilization capacity), and very small droplet size. Thesmall droplets also provide better adherence to membranesand transport drug molecules in a controlled fashion.Microemulsions are easy to administer to children and topeople who have difficulty swallowing solid oral dosageforms.[33,34] Compared to conventional emulsions ormacroemulsions, these drug carriers have several advan-tages in terms of excellent thermodynamic stability, highdrug solubilization capacity, that is, increased drugloading, ease of preparation due to spontaneous forma-tion, increased bioavailability, protection against enzymatichydrolysis, transparent and elegant appearance, enhancedpenetration through the biological membranes[27,33,34]

and less inter- and intra-individual variability in drugpharmacokinetics.[35]

Microemulsions as a novel drug delivery system can helpovercome the delivery problems of new classes of activemolecules, and may also extend the therapeutic potentialsof established drugs; hence, microemulsion is currently oftremendous interest to pharmaceutical scientists. Theyhave been reported to improve the rate and extent ofabsorption of lipophilic drugs.[18,22,28–30,35–38] Thus, in thisstudy, attempts were made not only to deliver a poorlywater-soluble imidazole antifungal drug, miconazolenitrate using lipid-based carrier (microemulsion) but alsoto stabilize the formulation with Poloxamer 407, a non-ionic triblock copolymer. Because of its low aqueoussolubility, miconazole is commonly applied topically tocure fungal infections as well as infections caused byGram-positive bacilli and cocci, and less commonly usedinternally as tablet for oral and intestinal candidiasis, asoral gel for angular cheilitis, lip disorder as well as for oralor vaginal thrush.[39] Poloxamer 407, a hydrophilic nonio-nic surfactant, is a well-known stabilizer for lipid formula-tions.[40] Topical and oral microemulsions for a poorlywater-soluble antifungal agent, ketoconazole has beendesigned and, subsequently, developed into ketoconazolegel for improved bioavailability of the drug.[41] Since keto-conazole and miconazole possess similar physicochemicalproperties, designing miconazole nitrate-loaded micro-emulsion formulation would improve the bioavailabilityof the latter as well. Regarding bioavailability, a poorlywater-soluble drug in a microemulsion system has betterpenetration of the physiological membrane due to severalfactors such as the enhancing effect of surfactants, theproper balance between hydrophilicity and lipophilicityof the formulation, smaller particle size, the in vivopartition coefficient of the drug between two immisciblephases, the presence of drug in emulsified form, meta-bolism of the oil in the microemulsion, effect of lipidvehicle on gastric emptying time, and drug solubility inthe microemulsion excipients.[27,28–30,33,35–39,42]

Consequently, the purpose of this investigation wasto evaluate miconazole nitrate-loaded microemulsionstabilized with Poloxamer 407 with a view to improvingits bioavailability. The microemulsion in the presentstudy consisted of a vegetable oil, castor oil; a nonionicsurfactant, Tween 80 (HLB 15); a cosurfactant, propy-lene glycol; and water. Pseudoternary phase diagramswere constructed to locate the region of microemulsionformation at different ratios of surfactant to cosurfac-tant. Poloxamer 407 was used to stabilize the formulatedmicroemulsion, which was thereafter characterized interms of globule size, viscosity, pH and conductivity.The ex vivo release of miconazole from the drug-loadedmicroemulsion was carried out using Franz diffusionapparatus in which the donor compartment was sepa-rated from the receptor compartment by freshly excisedhairless rat skin.

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2. MATERIALS AND METHODS

2.1. Materials

The materials used were miconazole nitrate powder(BASF, Ludwighaten, Germany), Fungusol topical powder(Afrab-Chem Limited, Lagos, Nigeria) Tween 80 (Williamand Sons Ltd., UK), propylene glycol (Mark and WilliamsChem., GmbH, Germany), Poloxamer 407 (Synochem,Germany), monobasic potassium phosphate, sodiumhydroxide and hydrochloric acid (BDH, UK) and castoroil ((pharmacopoeial grade). Distilled water was collectedfrom an all-glass still. All other chemicals used were analyti-cal grade and used as received without further purification.

2.2. Construction of Pseudoternary Phase Diagrams

Pseudoternary phase diagrams were constructed toexamine the formation of microemulsions using the watertitration method.[43] Tween 80 and propylene glycol weremixed at volume ratios of A¼ 5:1, B¼ 4:1, C¼ 3:1, andD¼ 2:1 to obtain the surfactant-cosurfactant mixtures,which were then mixed with castor oil at volume ratios of9.5:0.5, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9 to yield10ml each of surfactant (surfactant-cosurfactant)-oilmixture. Then 0.1 g of Poloxamer 407 was added to eachsurfactant-cosurfactant and oil ratio in a beaker, and thesemixtures were titrated dropwise with distilled water andagitated at 50 rpm under magnetic stirring (SR 1UM52188, Remi Equipment, India) at the ambient temperature(25�C) until a fine microemulsion was formed. Theabove procedure was repeated for surfactant-cosurfactantmixture of ratio E¼ 4:1 but without the addition ofPoloxamer 407. The pseudoternary phase diagram wasconstructed by plotting the amounts of water phase, oilphase, and surfactant-cosurfactant phase used in theexperiment. The ternary phase diagrams were constructedusing Sigma Plot 11 Exact Graph and Data Analysis soft-ware (Systat Software Inc., USA). The globule sizes of theemulsions formed were determined by light microscopy tofurther assess their quality.

For convenience, the phase diagrams were constructedby drawing ‘‘water dilution lines’’ representing increasingwater content and decreasing surfactant-cosurfactantlevels.[43] The water was titrated along dilution lines drawnfrom the surfactant-cosurfactant apex (100% surfactant-cosurfactant) to the opposite oil side of the triangle. Theline was arbitrarily denoted as the value of the line inter-section with the oil scale (e.g., 10:90, 40:60). If turbidityappeared followed by a phase separation, the samples wereconsidered to be biphasic. If clear and transparent mixtureswere visualized after stirring, the samples were consideredmonophasic. The samples were marked as points in thephase diagram. The area covered by these points wasconsidered to be the region of microemulsion formation.The combination volume ratios of surfactant-cosurfactant,

oil and water that gave the best microemulsion are shownin Table 1.

2.3. Preformulation Isotropicity Test

The various combinations of the Smix and oil wereformulated at 30–40�C in a test tube under manual shak-ing. The formulations were allowed to stand undisturbedat 28� 2�C and visually assessed for phase separation.The formulations that remained stable were then selectedfor preparing drug-loaded microemulsion.

2.4. Preparation of Miconazole Nitrate-LoadedMicroemulsion Formulation

Appropriate quantities of Tween 80, propylene glycoland castor oil were mixed together in a beaker after which0.1 g of Poloxamer 407 was added to each beaker for thedifferent batches (except batches E1 and E2) followed by100mg each of micronized miconazole nitrate with mildagitation. Then appropriate quantity of distilled waterwas added and the mixture was homogenized at a speedof 3200 rpm for 5 minutes at 25�C using an ultraturaxhomogenizer (Silverson L4R, Adelphi Manufac., UK).The resultant 50ml volume of miconazole nitrate-loadedwater-in-oil microemulsion formulations were packagedin clean transparent bottles. The ternary combinationratios of surfactant-cosurfactant, water and castor oil usedto obtain 50ml volume of the drug-loaded microemulsionare presented in Table 2.

2.5. Absolute Drug Content Analysis

A quantity of the microemulsion (25ml) was emulsifiedby vortexing in 200ml of phosphate buffer (pH 7.4) undermagnetic stirrer hot plate assembly at 50 rpm for 60minutes. One-ml volume was then withdrawn, dilutedand the absorbances measured using a spectrophotometer(SP 6-450 UV-VIS, Pye Unicam, UK) at 210 nm. This

TABLE 1Combination volume ratios of surfactant-cosurfactant,

oil, and water that formed stable microemulsions

Batchcode

Surfactant-cosurfactantvolume ratio

Surfactant-cosurfactant:oilvolume ratio

Water(ml)

Poloxamer(mg)

A 5:1 5:5 8.0 100B1 4:1 3:7 3.0 100B2 4:1 2:8 2.0 100C 3:1 2:8 1.6 100D 2:1 2:8 3.5 100E1 4:1 6:4 7.0 —E2 4:1 5:5 7.2 —

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was repeated at different time intervals and the valuesobtained expressed as percent of the initial drug dose.

2.6. Physicochemical Characterization of theMicroemulsion

2.6.1. Particle Size Analysis and MorphologicalCharacterization

The particle size of the microemulsion was determined72 hours after production by computerized imageanalysis on a polarized light microscope (Lieca, Germany).A known sample from each of the batches was mounted ona slide and observed under a polarized light microscope.With the aid of software in the microscope, the projecteddiameters of the particles corresponding to the particlesizes of the microemulsion were determined and the meancalculated. The particle morphologies were also observedand captured by the microscope.

2.6.2. Viscosity Measurement

The rheological property of the microemulsion wasevaluated using a torsion viscometer (Gallenkamp,Germany) at 25�C using 20ml of each sample. Experimentswere performed in triplicate for each sample.

2.6.3. pH Determiation

The pH of each prepared batch was determined usinga pH meter (Suntex TS-2, Taiwan) at 25�C, 72 hourspost-production.

2.6.4. Electroconductivity Test

The electroconductivity of the samples was measured byan electroconductometer (Suntex, Taiwan) at 25�C using20ml samples of the drug-loaded microemulsion. Triplicatedeterminations were done in each measurement.

2.7. Preparation of the Rat Abdominal Skin

Male Wistar rats were sacrificed under prolongedanesthesia and the abdominal skin of each rat was excised.Abdominal hairs on the skin of the animal were removed

with clipper and full thickness skin was surgically removedand the dermis side was wiped with isopropyl alcohol toremove residual adhering fat. Heat separation technique[44]

was used to separate the epidermis. The technique involvedsoaking the entire abdominal skin in water at 60�C for 1minute followed by careful removal of the epidermis fromdermis with a blunt forceps. The epidermis was washedwith water and wrapped with aluminum foil and storedat �20�C until used. The stored epidermis was allowed tothaw, cut into 4.5� 4.5 cm2 pieces and hydrated by placingin phosphate buffered saline (PBS, pH 7.4) overnightbefore use.

2.8. Ex Vivo Drug Release Studies

A modified Franz diffusion cell was employed for therelease study. In each case, a finite volume (10ml) of themicroemulsion formulations was placed in the donor com-partment of the Franz diffusion cell separated from thereceptor compartment by a freshly excised hairless rat skin(from the preceding section). The effective diffusion areawas 2.27 cm2 and the receiver chamber has a capacity of32ml. The receptor compartment was filled with 20ml offreshly prepared phosphate buffer (pH 7.4) and maintainedat a temperature of 37� 1�C by means of a thermostati-cally controlled water bath, with agitation provided bya magnetic stirrer-hot plate assembly (SR 1 UM 52188,Remi Equipment, India) at 50 rpm. Aliquots (2.0ml) wereremoved and replaced by an equal volume of the receptorphase at hourly intervals for 6 hours and the collectedsamples analyzed spectrophotometrically for drug contentusing a photometer (UNICO UV-2102 PC, USA) ata predetermined wavelength of 210 nm. The drug contentat each time point was calculated by reference to acalibration plot earlier determined for pure sample ofmiconazole nitrate.

2.9. In Vitro Microbiological Test

The in vitro microbiological activities of the microemul-sion containing miconazole were determined using agar

TABLE 2Ternary combination of surfactant-cosurfactant, water, and castor oil to obtain 50ml of drug-loaded microemulsion

Batch codeTween

80=PG (ml)Tween

80=PG (%) Castor oil (ml) Castor oil (%)Distilledwater (ml)

Distilledwater (%)

A 18.40 36.80 7.90 15.80 23.70 47.40B1 11.50 23.00 27.00 54.00 11.50 23.00B2 8.30 16.60 33.40 66.80 8.30 16.60C 8.60 17.20 34.50 69.00 6.90 13.80D 7.40 14.80 29.60 59.20 13.00 25.00E1 17.60 35.20 11.80 23.60 20.60 41.20E2 14.53 29.06 14.53 29.06 20.94 41.88

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diffusion technique. Volumes (0.1ml) of the various micro-emulsion formulations were diluted with distilled water toobtain 10 mg=ml of miconazole. A 10 mg=ml dilution ofFungusol (miconazole nitrate B. P. 2% powder) in DMSOwas also prepared. Sabouraud-dextrose agar plates wereprepared following manufacturer’s specifications andseeded with clinically isolated Candida albicans. Thevarious dilutions of the drug and the formulations werethen inoculated on the plate and the plate incubated for24 hours at 37�C. The various inhibition zone diameters(IZD) generated after incubation were measured planime-trically and the average for each concentration determinedafter replicate determinations.

2.10. Data and Statistical Analysis

All experiment were performed in replicates (at least,n¼ 3) for validity of statistical analysis. Results werepresented as mean� S.D. ANOVA and Student t testswere performed on the data sets generated using SPSS.Differences were considered significant for p-values <0.05.

Figures were processed using Microsoft Excel andSigmaPlot 11 Exact Graph and Data analysis.

3. RESULTS

3.1. Phase Diagram Study

Pseudoternary phase diagrams were constructed by plot-ting amounts of water phase, oil phase, and surfactant-cosurfactant phase used in the experiment and thecorresponding microemulsion regions (area inside thetriangle assigned on the phase diagram) were identified asshown in Figure 1. In order to limit the ambiguity of theexperiment, the best emulsifying regions were chosen.

3.2. Preformulation Isotropicity Test

The result of the isotropicity test shows that formula-tions [i.e., batches A (5:1), B1 (4:1), B2 (4:1), C (3:1), D(2:1), E1 (4:1) and E2 (4:1)] maintained isotropicity within72 hours and were therefore considered stable. This wasalso used to confirm the excipients combination at a fixeddrug dose that gave a stable formulation.

3.3. Absolute Drug Content Analysis

The results show that the amounts of drug entrapped byeach batch of the microemulsion formulations were:97.09� 0.92% for formulation A, 98.12� 0.18% for formu-lation B1, 98.54� 0.37% for formulation B2, 99.60� 0.28%for formulation C and 99.2� 1.83% for formulation D;while the amounts of drug entrapped by the microemulsionformulations without Poloxamer 407 were: 85.27� 0.56%for formulation E1 and 83.96� 2.09% for formulationE2 (Figure 2).

3.4. Physicochemical Characterization ofMicroemulsion

The physicochemical characteristics of the micro-emulsion formulations are shown in Table 3 and the photo-micrographs of the microemulsion formulations arepresented in Figure 3. Both spherical and irregularly-shapedparticles of size range 5.60� 0.63 mm to 14.00� 1.25 mmwere obtained. Figure 4 presents the relationship betweenwater concentrations and conductivity of the differentsamples. The mean conductivity ranged from 92.10�1.00mS=cm to 167.20� 2.63mS=cm, whereas the viscosityof the microemulsion samples ranged from 142.10� 2.06 cPto 283.30� 5.60 cP, indicating that the viscosity of poloxa-mer-stabilized microemulsion systems were low whilethose that contained no Poloxamer 407 had high viscosity.Moreover, the pH of the formulations was in the range of3.20� 0.09 to 5.91� 0.03.

3.5. Ex Vivo Drug Release Studies

Permeation studies carried out on the various batches ofthe microemulsion resulted in the permeation profile shownin Figures 5 and 6 as well as the permeation data presentedin Table 4. As could be seen from the figures, there wascontrolled permeation of miconazole from the micro-emulsion without burst effect in all the formulations. Theamount of miconazole permeated varied with the ratio ofsurfactant-cosurfactant used in preparing the drug-loadedmicroemulsion, with batch C containing 3:1 ratio of Tween80-propylene glycol giving the highest ultimate amountpermeated after 6 hours. The permeability assessment ofthe drug from the microemulsion formulations (Table 4)showed permeation fluxes of 4.139, 4.761, 4.917, 5.161,4.839, 3.572, and 2.378 mg=cm2 �min respectively forbatches A, B1, B2, C, D, E1, and E2. The permeation coeffi-cients of poloxamer-stabilized microemulsion formulationsin phosphate buffer were: 2.070� 10�3, 2.381� 10�3,2.459� 10�3, 2.581� 10�3, and 2.420� 10�3 cm=min forbatches A, B1, B2, C, and D, respectively; while the per-meation coefficients of microemulsion formulations withoutPoloxamer 407 were: 1.786� 10�3 and 1.189� 10�3 forbatches E1 and E2, respectively. The permeation coefficientswere obtained from the steady-state flux values calculatedfrom the linear ascent of the plot of amount permeatedagainst time (the slope), making use of the following equa-tion.[45]

P ¼ J=CO ðcm=minÞ ½1�

where P is the permeation coefficient; CO is the initial drugconcentration in the drug compartment (2000mg=ml): Jrepresents the steady-state flux obtained from equation 2thus:

J ¼ dQ=Adt ðlg=cm2 �minÞ: ½2�

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FIG. 1. Ternary phase diagrams of the Smix (surfactant-cosurfactant mixture). (a) 5:1; (b) 4:1; (c) 3:1; (d) 2:1; (e) 4:1 without poloxamer.

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where Q indicates the quantity of substances crossing theartificial membrane, A is the area of the artificial membraneexposed (2.27 cm2) and t is the time of exposure.

3.6. In Vitro Antifungal Activity

The in vitro activity of the microemulsion formulationswas compared to a solution of miconazole commercialsample (Fungusol powder) in DMSO and the resultrecorded as inhibitory zone diameter (IZD), as presentedin Table 5. IZDs recorded for poloxamer-stabilizedmicroemulsion batches were: A (20.63� 1.25mm), B1

(21.40� 0.97), B2 (21.59� 2.00), C (22.87� 0.91) and D(19.46� 1.00mm); whereas that for microemulsion batcheswithout Poloxamer 407 were: E1 (18.53� 2.06) and E2

(17.24� 1.60).

3.7. Product Stability Assessment

It is always very important to assess the stability ofnovel formulations. Stability could be viewed from thedegradation of the active ingredient or physical propertyof the formulation. In this case, physical stability of theformulations were studied, wherein, the time taken forthe formulation to separate was noted. Antimicrobialassessment was also carried out within this period, whichrevealed no significant difference (p< 0.05) in the potencyof the stored formulation and freshly prepared ones.

4. DISCUSSION

The imidazole antifungal drug, miconazole nitrate wasformulated as a microemulsion to improve its watersolubility and bioavailability. As can be seen from theresult of the physicochemical tests, the microemulsionsformulated with different proportions of the Tween 80,propylene glycol, oil, and water performed differently.Microemulsion containing low amounts of surfactant-cosurfactant was formulated with greater percentage ofcastor oil and smaller quantity of water. The reverse isthe case for microemulsion containing high amounts ofsurfactant-cosurfactant. This indicates that systems withlower content of the surfactant-cosurfactant are mainlywater in oil while those with higher quantities ofsurfactant-cosurfactant formed oil in water microemulsion.Phase diagrams were constructed to determine the com-positions of an aqueous phase, an oil phase, and asurfactant-cosurfactant phase that would yield a microe-mulsion. Microemulsion preparation requires adjustingthe hydrophilic lipophilic balance (HLB) value of theformulation by including a cosurfactant, which makes thepolar solvent less hydrophilic. In this study, propyleneglycol was used as the cosurfactant, Tween 80 as surfactantand castor oil as the oil phase. Poloxamer 407 was alsoincorporated into the microemulsion as a stabilizer andanother cosurfactant to increase the curvature of the oil

TABLE 3Physicochemical properties of miconazole nitrate-loaded microemulsion

Batch code Particle size (mm)a,b Conductivity (mS=cm)a,b Viscosity (cP)a,b pHa,b

A 7.10� 0.90 167.20� 2.63 170.90� 3.50 5.55� 0.12B1 10.80� 1.04 133.50� 1.73 163.20� 3.74 5.23� 0.05B2 11.00� 1.30 114.40� 1.25 154.40� 2.25 4.23� 0.01C 14.00� 1.25 92.10� 1.00 242.80� 4.50 5.91� 0.03D 13.30� 1.16 134.90� 1.98 142.10� 2.06 4.42� 0.10E1 8.80� 1.02 150.30� 2.05 283.30� 5.60 5.23� 0.00E2 5.60� 0.63 155.00� 3.00 259.50� 4.50 3.20� 0.09

aMean� S.D.bn¼ 3, batches A, B1, B2, C, and D contain poloxamer while batches E1 and E2 contain no poloxamer.

FIG. 2. Percentage content of miconazole nitrate in the micro-

emulsion formulations.

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layer.[27] The pseudoternary phase diagram is an effectivemeans of determining the amounts of components ina microemulsion; any combination of the componentscan be plotted as a percent on a pseudoternary phasediagram and the total composition is always 100%.[46]

For simplicity, the microemulsion is assumed to be athree-component system (oil, water, and the mixture ofsurfactant and cosurfactant). Phase behavior investigationsof this system demonstrated a suitable approach to deter-mining the water phase, oil phase, surfactant concen-tration, and cosurfactant concentration with which thetransparent, 1-phase low-viscous microemulsion systemwas formed. The phase study revealed that the maximumproportion of oil was incorporated in microemulsion

systems when the surfactant-to-cosurfactant ratio was 3:1.From a formulation viewpoint, the increased oil contentin microemulsions may provide a greater opportunity forthe solubilization of miconazole nitrate. The optimum for-mulation of microemulsion contained Tween 80 (12.90%),propylene glycol (4.30%), castor oil (69.00%), and water(13.80%). Due to the low water-solubility of miconazolenitrate and the rigidity of oily surface, some amounts ofPoloxamer 407 were added to dissolve the drug and increasethe curvature of the oily layer.[27] The Poloxamer 407 incor-porated into the microemulsion system not only reduced theinterfacial tension between the oil phase and the aqueousphase but also made the lipophilic drug soluble in thesystem. The preformulation istotropicity test was also used

FIG. 3. Photomicrographs of microemulsion batches: (A) A (5:1) (B) B1 (4:1) (C) B2 (4:1) (D) C (3:1) (E) D (2:1) (F) E2 (4:1). (Figure available in

color online.)

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to confirm the excipients combination at a fixed drug dosethat gave a stable formulation. The absolute drug contentanalysis performed on the microemulsion formulations isnecessary to determine the dosing units of the formulations.The values obtained were within acceptable range, whichindicates good control of the formulation processand increase in the level of confidence on the products.

However, there was significant increase in the drug contentof the poloxamer-stabilized microemulsion formulationscompared to the drug content of the microemulsion formula-tionswithout Poloxamer 407, indicating the improved solubi-lizing effect ofmiconazole in themicroemulsion formulations.

The physicochemical characteristics of the formulatedmicroemulsion are shown in Table 3. It is evidentfrom the physicochemical data that the formulated

FIG. 6. Permeation profile of miconazole nitrate from non-

poloxamer-stabilized microemulsion formulations in phosphate buffered

saline. (Figure available in color online.)

FIG. 5. Permeation profile of miconazole nitrate from poloxamer-

stabilized microemulsion formulations in phosphate buffered saline.

(Figure available in color online.)

FIG. 4. Graph of conductivity against water concentration of the

microemulsion formulation. (Figure available in color online.)

TABLE 4Permeation parameters of miconazole nitrate-loaded

microemulsion formulations

Formulationcode

Flux (J)(mg=cm2 �min)a,b

Permeation coefficient(P) (cm=min)a,b

A (5:1) 4.139 2.070� 10�3

B1 (4:1) 4.761 2.381� 10�3

B2 (4:1) 4.917 2.459� 10�3

C (3:1) 5.161 2.581� 10�3

D (2:1) 4.839 2.420� 10�3

E1 (4:1) 3.572 1.786� 10�3

E2 (4:1) 2.378 1.189� 10�3

aMean� S.D.bn¼ 3, batches A, B1, B2, C and D contain poloxamer while

batches E1 and E2 contain no poloxamer.

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microemulsion had particles within the micrometer range.A microemulsion normally contains droplet size diametersranging from 1 to 100 mm; they appear transparent or clearbecause the light-scattering capability of the small particlesis weak.[27] By implication, the formulations are micro-particles, which could improve the dissolution and bio-availability of poorly water-soluble drugs like miconazolenitrate. The micrometric size range of the particles wasretained even after storage for 72 hours, indicating thestability of the formulated microemulsion. It was observedthat spherically shaped microemulsions within the micro-meter range were obtained, although some particles werenot spherical. The reason for the irregular particles isuncertain, but may be related to excessive handling of themicroemulsion. The investigated microemulsion systemcontaining the nonionic surfactant mixture, oil, and watershowed electroconductive behavior in spite of its nonionicnature. The conductivity values of the studied sampleincreased linearly with the amounts of water used. Above14% water, a drastic increase in conductivity was observed;furthermore, a sharp increase in conductivity was found at41% water. The linear increase in conductivity with increas-ing amounts of water is most likely caused by a transitionfrom an oily continuous microemulsion system to a watercontinuous microemulsion system, consistent with an ear-lier report.[47] Poloxamer-stabilized microemulsion hadhigher conductivity due to the viscosity-thinning effect ofthis second cosurfactant (Poloxamer 407), and hencehigher electrolyte shift. It is obvious from the viscosityvalues obtained that all batches of the microemulsion hadhigh and varied viscosities. The viscosity of the formulationwas brought to an optimum by the addition of Poloxamer407 (a viscosity adjusting agent and stabilizer). The formu-lation without poloxamer (i.e., batches E1 and E2) hadhigher viscosities compared to poloxamer-stabilized micro-emulsion formulations. Usually, water=oil microemulsions

have higher viscosities than those of oil=water systems.From the viscosity and electroconductivity studies, it isdiscernible that poloxamer-stabilizedmicroemulsion formu-lation is of the o=w type.[48] The pH values of the formulatedmicroemulsion were in the range of 3.20� 0.09 to5.91� 0.03. Normally, the pH of most topical formulationsis preferably within the range 6.3 to 6.5.[47] There is, there-fore, the need for appropriate buffers to be incorporated inthemicroemulsion especially since these formulations wouldbe developed for topical application.

The ex vivo release studies showed that batch C formu-lation containing surfactant-cosurfactant (Tween 80-propylene glycol) ratio of 3:1 gave the highest permeationflux and coefficients, implying that sustained releasemiconazole dosage form might be developed with this for-mulation. Batches B1 and B2, containing Poloxamer 407(a stabilizing agent) and a surfactant-cosurfactant ratio of4:1 gave a greater permeation fluxes and coefficients thanthe corresponding batches E1 and E2 having the samesurfactant-cosurfactant ratio, but without the stabilizingagent. These values were within the range obtained forsome lipophilic and lipophobic drugs.[45,49] Compared withthe fluxes and coefficients of microemulsions withoutpoloxamer, the poloxamer-stabilized microemulsion for-mulations demonstrated enhanced permeation attestingfurther to the improvement in the in vitro performance ofthe formulations. The in vitro permeation result presentedhere strongly indicates improved permeation of the drugthrough the rat skin membrane which is known to mimicpermeation in vivo. By implication, poloxamer facilitatedthe solubilization as well as the release of drug from themicroemulsion. The presence of surfactant and cosur-factants as well as stabilizer lowers the interfacial tensionbetween water and oil; therefore, the surface area of thedispersed droplets is very large.[27–50] The lower interfacialtension counter-balanced the dispersion entropy; therefore,the microemulsion system became thermodynamicallystable due to the low free energy of the system. The amountsof drug released from themicroemulsionwere low comparedto the drug content of the 2ml used. However, batch Ccontaining a surfactant-cosurfactant ratio of 3:1 gave thebest ex vivo drug release profile and is therefore consideredthe most efficient drug-loaded microemulsion for thedelivery of miconazole nitrate, a lipophilic antifungal drug.A lipophilic drug in a microemulsion system has betterpenetration of the physiologicalmembrane due to the properbalance between hydrophilicity and lipophilicity of theformulation, smaller particle size, the presence of drug inan emulsified form, site of absorption, metabolism of theoil in the microemulsion, effect of lipid vehicle on gastricemptying, drug solubility in the microemulsion excipients,the in vivo partition coefficient of the drug between the twoimmiscible phases and most importantly the enhancingeffect of surfactants which enables the system to achieve

TABLE 5Susceptibility of C. albicans to miconazole in the

microemulsion formulations

Formulation IZD (mm)a,b

A (5:1) 20.63� 1.25B1 (4:1) 21.40� 0.97B2 (4:1) 21.59� 2.00C (3:1) 22.87� 0.91D (2:1) 19.46� 1.00E1 (4:1) 18.53� 2.06E2 (4:1) 17.24� 1.60Fungusol solution 10.92� 0.38

aMean� S.D.bn¼ 3, batches A, B1, B2, C and D contain poloxamer while

batches E1 and E2 contain no poloxamer.

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high drug loading and improved bioavailability, forlipophilic compounds which miconazole nitrate is a goodexample.[27,28–30,33,35–39,42]

The in vitro antifungal activity was determined to ascer-tain the possibility of loss, retention or improvement ofactivity of miconazole as a result of formulation into thisnovel dosage form. The result revealed that there wassignificant (p< 0.05) increase in the in vitro inhibition of C.albicans by all the microemulsion formulations comparedwith the solution made with the commercial miconazole(Fungusol) powder. The poloxamer-stabilized micro-emulsion batch C (3:1) with absolute drug content of99.60� 0.28% showed the greatest activity against Candidaalbicans with IZD of 22.87� 0.91mm as against the leastactivity of 10.91� 0.38mm recorded for the solutionmade with the commercial miconazole (Fungusol) powder.The increase in antifungal activity of the microemulsionformulations may be attributed to the improved permeationof the drug into the fungal cell wall brought about by appro-priate choice and combination of excipients used in the for-mulation of the microemulsion. Moreover, there weresignificant (p< 0.05) increases in the in vitro inhibition ofC. albicans by the poloxamer-stabilized microemulsionformulations compared to microemulsion formulationswithout poloxamer, further indicating the enhancedpermeation effect offered by the poloxamer in the formula-tions. The results of the stability studies show that therewas no significant change in the physical characteristicsand potency of the miconazole nitrate-loaded micro-emulsion over a period of three months, implying that therewas little or no degradation of the drug and=or the excipientsused in the formulations, especially the poloxamer-stabilizedmicroemulsions, within this period of time.

5. CONCLUSIONS

The design and preparation of microemulsions is a newand exciting field of research that seeks to exploit theattractive properties of lipid carriers to improve thedelivery of therapeutic molecules. To date microemulsionshave been shown to be able to protect labile molecules,control drug release, increase drug solubility, increase bio-availability and reduce patient variability. Furthermore, ithas proven possible to formulate preparations suitablefor most routes of administration. The antifungal drug,miconazole nitrate was formulated as a homogenous andthermodynamically stable microemulsion using castor oil,Tween 80, propylene glycol, water, and Poloxamer 407 toimprove its bioavailability. Ex vivo and microbiologicalstudies undertaken with the poloxamer-stabilized micro-emulsion provided positive results, especially with thesystem having a surfactant-cosurfactant ratio of 3:1, thus,encouraging further development and optimization of thisformulation. This study has shown that poloxamer-stabilized

microemulsion could be employed to deliver and improvethe bioavailability of the poorly absorbed drug, miconazolenitrate.

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