2013

http://informahealthcare.com/ddiISSN: 0363-9045 (print), 1520-5762 (electronic)

Drug Dev Ind Pharm, Early Online: 1–10! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.787536

RESEARCH ARTICLE

Pharmaceutical optimization of lipid-based dosage forms for theimprovement of taste-masking, chemical stability and solubilizingcapacity of phenobarbital

Ezequiel Monteagudo1, Mariana Langenheim1, Claudia Salerno1, Fabian Buontempo1,2, Carlos Bregni1,and Adriana Carlucci1

1Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina and2Hospital de Pediatrıa S.A.M.I.C. ‘‘Prof. Dr. Juan P. Garrahan’’, Buenos Aires, Argentina

Abstract

Microemulsions (MEs) and self-emulsifying drug delivery systems (SEEDS) containing pheno-barbital (Phe) were developed to improve its chemical stability, solubilizing capacity andtaste-masking in oral liquid dosage forms. Cremophor� RH40 and Labrasol� were used assurfactants for the screening of ME regions, Capmul� MCM L, Captex� 355, Imwitor� 408,Myglyol� 840 and Isopropyl myristate were the oil phases assayed; Transcutol� P, Polyethylene-glycol 400, glycerol, Propylene-glycol and ethanol the cosurfactants. Phe stability assay wascarried out (20:4:20:56% and 20:4:35:41% (w/w); surfactant:oily phase:cosurfactant:water) forboth surfactants; only one containing ethanol showed significant dismissing in its drug content.Solubility capacity for these selected formulations were also evaluated, an amount between17 and 58 mg/mL of Phe could be loaded. At last, an optimized ME formulation withCremophor� RH40 20%, Capmul� MCM L 4%, PEG 400 35% and sucralose 2% (w/w) was chosenin order to optimize taste-masking using an electronic tongue. Strawberry along with bananaand tutti-frutti flavors plus mint flavor proved to be the best ones. Labrasol-based pre-concentrates were tested for (micro)emulsifying properties; all of them resulted to behave asSEDDS. In summary, a rationale experimental design conducted to an optimized ME for Phe oralpediatric administration which was able to load 5-fold times the currently used dose (4 mg/mL),with no sign of physical or chemical instability and with improved taste; SEDDS for capsulefilling were also obtained. The biopharmaceutical advantages described for these dosage formsencourage furthering in vivo evaluation.

Keywords

Biopharmaceutical advantages, chemicaldegradation, electronic tongue,microemulsion, SEDDS

History

Received 5 December 2012Revised 4 March 2013Accepted 5 March 2013Published online 22 April 2013

Introduction

Phenobarbital (Phe) is still the most commonly prescribedantiepileptic drug in the world. As a result of its efficacy andlow cost, it is recommended by the World Health Organization(WHO) as first line drug for partial and tonic-clonic seizures indeveloping countries in all age groups. It seems to act in arelatively non-selective manner, both limiting the spread ofepileptic activity and elevating the seizure threshold. Phe bindsstrongly to the gGABA receptor, and its major action is at thisreceptor, post-synaptically, where it increases the duration ofchannel opening without affecting the frequency of opening1,2;it also has sedative and hypnotic properties3.

Phe (5-ethyl 5-phenyl barbituric acid, molecular weight232.32) is a substituted barbituric acid with a crystalline structure.It is a free acid, which has poor water solubility (1 mg/mL) but it

is freely soluble in ethanol (100–125 mg/mL)3; the drug has a pKacited as 7.3, 7.41 and 7.64.

Phe absorption is affected by the type of presentationadministered (e.g. free acid or salt, crystal size), gastric bloodflow, gastric emptying time, gastric acidity and also by thepresence of food and neutralizing agents. In newborns and younginfants, bioavailability is reduced. Pharmacies usually preparecapsules with doses corresponding to the age and weight ofchildren. To reduce individualized preparations, oral liquids havebeen developed. Elixirs containing different concentrations ofdrug such as 3 and 4 mg/mL or a solution with Propylene glycoland water (90%/10%) are in common use in order to increase itssolubility1,3,4.

However, some problems appeared in pediatrics adminis-tration; in children the usual starting dose is 3 mg/kg per day, withmaintenance doses in the range 3–6 mg/kg per day. Twice-dailydosing may be necessary in younger children because of theshorter half-life. The drug is widely used in neonatal seizures,where rapid seizure control is needed, and IV administrationis used. In pediatric hospital environment, a liquid oral dosageform containing 20 mg/mL is currently used in Neonatologydivision.

Address for correspondence: Dra. Adriana M. Carlucci, Department ofPharmaceutical Technology, Faculty of Pharmacy and Biochemistry,University of Buenos Aires, Junın 956, 1113ADD Buenos Aires,Argentina. Tel/fax: 54 11 49648271. E-mail: adrianac@ffyb.uba.ar,amcarlucci@gmail.com

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Phe is stable to air but subject to hydrolysis, which is the mostimportant degradation route. The hydrolysis rate is accelerated insolutions with a pH above 9. The presence of a co-solvent in theformulations resulted critical for the chemical stability of thedrug4,5. The biggest limitation of co-solvency is the toxicity ofmost water-miscible solvents that have a high potential forincreasing drug solubility3; considering the doses currently usedin Neonatology division, the toxicity appears as the main problemto resolve. Additionally to the low water solubility of the drug, ithas a narrow therapeutic index6. Therefore, the formulation of thispoorly water-soluble drug represents a challenging task forformulation experts7.

Microemulsions (MEs) are defined as colloidal dispersions,thermodynamically stable systems that are isotropic and have lowviscosity. Their structure consists in micro domains of lipids orwater stabilized by an interfacial film of surfactant and co-surfactant molecules. They present various advantages such as theability to solubilize hydrophobic drugs, spontaneity of formation,long-term physical stability and ease of manufacturing. They canbe classified as oil in water (o/w) or water in oil (w/o) and thedroplet size is lower than150 nm8.

In recent years, there has also been an increasing interest forthe study of S(M)EDDS (Self-(micro)emulsifying drug deliverysystem) which are isotropic mixtures of oil, surfactant, co-surfactant and drug with ability to form oil in water (micro)emul-sion upon mild agitation following dilution with aqueous phase9.Among the lipid-based systems, S(M)EDDS is a promisingtechnology to improve the rate and extent of the absorption ofpoorly water-soluble drugs7. The efficiency of oral absorption ofthese active compounds from such type of formulation depends onmany formulation-related parameters, such as surfactant concen-tration, oil/surfactant ratio, polarity of the emulsion, droplet sizeand charge, all of which in essence determine the self-emulsification ability10. Additionally, the taste of a formulationhas an important role in the development of oral pharmaceuticals,since taste has major influence on the therapy compliance. Taste-masking is defined as a perceived reduction of an undesirabletaste that would otherwise exist11. Nowadays, taste assessmentsare frequently used as a quality-control parameter to evaluatetaste-masked formulations. These taste assessments are performedwith taste-sensing analytical devices and complemented with tastesensory panels. The need for palatable drugs is particularly moreimportant when formulating oral dosage forms for childrenand elderly patients. Taste assessment of pharmaceuticalproducts typically requires a trained taste panel. For thecase of pediatric patients, special legal issues are involved.These tests require the same health safeguards as a clinicaltrial; because of that, Electronic Tongue (e-tongue) technol-ogy raised as a tool that can address these difficulties.These analytical systems provide a fast, objective and simpleassessment that has proven correlation to human taste panelevaluation12.

The objective of the present work was to design o/w MEscontaining the extensively reported hydrophilic co-solvents,which were also able to load up to 20 mg/mL of Phe.The physical and chemical stability of selected compositionswere studied according with WHO recommendations during6 months. An optimized formulation was developed, so asto obtain a taste-masked formulation containing the activecompound based on a screening of different taste maskingapproaches by guidance of an e-tongue. Additionally, some pre-concentrate formulations designed in basis of optimizedME compositions were evaluated for their physicochemicalproperties in order to infer the feasibility of being incorporatedas S(M)EEDS in a soft capsule for administration in adultpatients.

Material and methods

Reagents and excipients

Polyoxyl 40 hydrogenated castor oil (Cremophor� RH40),caprylocaproyl polyoxyl-8-glycerides (Labrasol�) andTranscutol� P (diethylene glycol monoethyl ether) were pur-chased from Gattefosse, France. Isopropyl myristate, propyleneglycol, polyethylene glycol 400 (PEG 400) and glycerol werebought at BASF, Florham Park, NJ; ethanol was from J.T.Baker,Center Valley, PA, Capmul� MCM L (glycerol monocaprylocap-rate) and Captex� 355 (caprylic/capric triglyceride) werepurchased from Abitec, Columbus, OH. Imwitor�408 (propyleneglycol caprylate) and Miglyol�840 (propylene glycol dicaprylate/dicaprate) were bought at Sasol, Witten, Germany. Phe wasobtained from Saporiti S.A., Buenos Aires, Argentina. Distilledwater was obtained from a Milli-Q equipment. Sucralose waspurchased at Merisan Argentina and all other flavors were fromGivaudan�, Argentina.

Methods

Preliminary solubility screening

To determine the equilibrium solubility of Phe in selectedexcipients, drug was added until an excess could be observed.The samples were left to equilibrate for 96 h using a RotatingBottle apparatus (Varian, Palo Alto, CA) at 5 rpm. Five oil phases,Capmul� MCM L, Captex�355, Imwitor�408, Myglyol�840 andIsopropyl Myristate; two surfactants, Labrasol� and Cremophor�

RH40; and five co-surfactants, Transcutol� P, Polyethylene glycol400 (PEG 400), glycerol, ethanol and Propylene-glycol were usedin this preliminary solubility test. The test was carried out at 37 �Cusing a thermal bath.

Screening of ME regions

Different ratios of Labrasol� or Cremophor�RH40, with each oneof the co-surfactants and the oil phase which have shown the bestdrug solubilizing capacity were mixed using magnetic stirrer.Then, distilled water was added and the samples were left toequilibrate using a thermal bath at 37 �C. The adopted criterionfor considering MEs was the observation of clear, single phase,isotropic and low viscous systems.

Selection of ME formulations

In order to evaluate the influence of each one of the selectedexcipients on Phe chemical stability, the same relative compos-itions of surfactant, oil phase, co-surfactant and water werechosen for both tensioactives. It was also considered that thesecompositions would contain the least possible amount of surfac-tant, the same relative proportion of oil phase (4% w/w); and twodifferent concentrations (20% and 35% w/w) for each one of theco-surfactants. The last point to be considered for formulationselection was that they had to be able to solubilize 20 mg/mL ofdrug. Recommendations for liquid products containing Phe, suchas packaging in tight containers, storage at controlled roomtemperature and protection from light were carried out.

Physicochemical properties for selected ME formulations

Density was measured using a Mettler Toledo 30 px; pH offormulations was determined with a Mettler Toledo Seven EasypHmeter. Conductivity was measured using Accumet researchar20 at 25 �C and for rheological measurements a Brookfield DV-III Ultra at 25 �C was used. Polarization microscopy wasperformed using an Olympus BH microscope, so as to evaluateformulation isotropy. Droplet size measurements were evaluated

2 E. Monteagudo et al. Drug Dev Ind Pharm, Early Online: 1–10

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with a Nanozetasizer ZS, Malvern Instruments, Worcestershire,UK. Samples were not diluted to perform the measurements andassays were performed at 25 �C.

The morphology of ME was studied using TransmissionElectron Microscopy (TEM). To perform TEM observations, theconcentrated MEs were first diluted in water (1:40), then a sampledrop was placed onto a grid covered with Fomvar film and theexcess was drawn off with a filter paper. Samples weresubsequently stained with uranyl acetate solution for 30 s.Samples were finally dried in a closed container with silica geland analyzed. The droplet diameter was estimated using acalibrated scale.

To determine the equilibrium solubility of Phe in selectedMEs, drug in excess was added. The procedure carried out was thesame as described above for evaluating solubilizing capacities ofeach excipient.

Chemical and physical stability evaluation

Chemical stability and solubility studies were performed using aShimadzu Class VP HPLC. Samples containing 20 mg/mL of theactive compound were stored at different conditions for 6 months(25 �C/60% R.H., 40 �C/75% R.H. and 50 �C/75% R.H.), duringthis period of time drug content was re-analyzed monthly.Physicochemical properties were re-determined after 6 monthsto evaluate physical stability of formulations. All experimentswith Phe-loaded formulations were carried out using amber glassmaterial as the drug is photosensitive6.

Electronic tongue evaluation

Drug bitterness prediction. E-tongue provides relative meas-urements of non-volatile or low volatile molecules and dissolvedorganic compounds that are responsible for taste and flavorsensations. It is an analytical instrument comprising an array ofnonspecific, low-selective, chemical sensors with high stabilityand cross-sensitivity to different species in solution12.

The bitterness evaluation of Phe 0.25 mg/mL in distilled waterwas performed with a-Astree II e-tongue (Alpha MOS, Toulouse,France) equipped with an Alpha MOS sensor set # 3 (TasteMasking Application – KIT #3 for pharmaceutical application(ZZ, AB, BA, BB, CA, DA and JE) from Alpha MOS Inc., knownas bitterness prediction module (BPM), a 48-position auto-sampler and a stirrer. The sensor set consisted of seven liquidsensors (BD, EB, JA, JG, KA, OA and OB) (KIT#2 forpharmaceutical application) for bitterness prediction measure-ments, based on chemically modified field effect transistors(ChemFET) and the potentiometric measurements were per-formed using an Ag/AgCl reference electrode (Metrohm AG). Thesystem was equipped with a data acquisition and analysis softwarepackage (AlphaSoft V 7.0 software). The bitterness modelconsists of a linear regression performed with aqueous solutionsof well-known bitter compounds (caffeine, quinine, acetamino-phen and prednisolone) at different concentrations; this allowsbuilding a bitterness scale ranging from 1 to 20 bitterness units(BU) that correlates with a trained human panel response, provenby AMOS13.

After the equipment conditioning and calibration steps, themodel was validated with two loperamide standard solutions andlater six replicates of each sample were measured with anacquisition time of 2 min. For bitterness assessment, the averageof the last 20 s of the last three replicates were considered andanalyzed with AlphaSoft V 7.0 software.

Taste-masking evaluation. The taste masking evaluation wasperformed with the a-Astree II e-tongue previously described,

with a pharmaceutical sensor set (sensors ZZ, AB, BA, BB, CA,DA and JE). These sensors are also based on ChemFET withdifferent co-polymer coatings allowing sensors cross-selectivity.In this way, each sensor can respond to substances responsible forthe five basic taste sensations: sweet, sour, bitter, salty and umamiwith different intensity.

The potentiometric measurements of each sensor versus Ag/AgCl reference electrode are collected and the raw data isexpressed as voltage versus time. The average data of the last 20 sof a total measurement time of 120 s per sample was used and thelast three of six total replicates were used in the data analysis. Thesensors were rinsed in two beakers of distilled water followingeach analysis.

After data acquisition samples were analyzed with AlphaSoftV 7.0 software using multivariate evaluation techniques, asPrincipal Component Analysis. This is a tool to summarize andreduce data by transforming a number of variables, in this casesensors responses, to a smaller number of variables, so calledprincipal components in order to describe the measured samples,in a new space with less dimensions14.

Sensors functionality test. A diagnostic analysis was performedto check sensor functionality. Five diagnostic solutions (sodiumchloride 0.01 M, hydrochloric acid 0.01 M, monosodium glutam-ate 0.01 M, sucralose 2% w/w and quinine hydrochloride1� 10�4M) were employed to verify sensor discriminationbetween salty, sour, umami, sweet and bitter taste. Each sampleacquisition time was 120 s followed by water cleanings, eachsolution was tested six times. The average sensor values between100 and 120 s from the last three replicates of each standardsolution were evaluated by Principal Component Analysis. Thefirst principal component (PC1) represents 92.37% of datavariability, while the second principal component (PC2) repre-sents 5.97%. As a result, the two dimension Principal ComponentAnalysis map, PC1 versus PC2, represents the 98.34% of the totalinformation. The Principal Component Analysis discriminationindex obtained for these samples was 100, which confirms asatisfactory performance of the sensors with a good discrimin-ation between diagnostic solutions.

Evaluation of alternative compositions for taste-masking. Theoptimized ME formulation was chosen after chemical andphysical stability evaluation and a taste-masking evaluation wasperformed for this formula with different flavoring options,containing sucralose 2% w/w as sweetener. The flavoring optionsselected considering pediatric preference were: tutti-frutti, straw-berry, banana and also the addition of a reduced amount of mintflavor were evaluated for mouth-feel and aftertaste improvement.

Each flavored formula (Table 1) with their correspondingplacebo was evaluated with the e-tongue taste masking application.

Table 1. Composition of flavored formulation candidates based on theoptimized ME.

ComponentsFormula 1(% w/w)

Formula 2(% w/w)

Formula 3(% w/w)

Formula 4(% w/w)

Phe 2 2 2 2Cremophor RH40 20 20 20 20Capmul MCM 4 4 4 4PEG 400 35 35 35 35Sucralose 2 2 2 2Strawberry flavor 1 1 – –Tutti-frutti flavor – – 1 1Banana flavor 1 1 – –Mint flavor 0.15 – 0.15 –Purified water qs 100 mL 100 mL 100 mL 100 mL

DOI: 10.3109/03639045.2013.787536 Lipid-based dosage forms for phenobarbital 3

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After data collection, sample measurements were evaluated usingPrincipal component analysis multivariate analysis and theEuclidean distances (D) (Equation (1)) between sample groupswere studied. The aim was to obtain a reduced Euclidean distancebetween the flavored formulation and the corresponding placebo,since reduced distances between formulation and placebo pairsindicates better taste masking performance12.

D ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðp1 � q1Þ2 þ ðp2 � q2Þ2 þ � � � þ ðpn � qnÞ2

qð1Þ

where P¼ (p1, p2, . . . , pn) and Q¼ (q1, q2, . . . , qn)

Optimization of S(M)EDDS formulation

On the basis of the data obtained during physical and chemicalstability, the tensioactive which had shown the better behaviorwas selected. Then, pre-concentrates corresponding to every MEcontaining this surfactant were prepared and loaded with 20 mg/mL of the active compound. For that purpose, amount exactlyweighted of the surfactant, co-surfactant and oil phase selectedwere mixed under magnetic stirring during 10 min at 37 �C usinga thermal bath (Varian).

The parameters evaluated were rate of (micro)emulsificationand droplet size after incubation in simulated gastric fluid. Forin vitro evaluation, we considered 200 mg, contained in 10 mL offormula containing 20 mg/mL of Phe, as the corresponding dosefor a 12-year-old child whose weight is 40 kg (recommended dailydose in pediatrics is 5 mg/kg/d, distributed once or twice daily).For S(M)EDDS evaluation, it is always considered evaluating adose in 250 mL of fluid, so 10 mL of each one of the selectedSMEDDS was delivered to 250 mL of simulated gastric fluid indissolution test equipment with USP paddle method.

Thermodynamic Stability Studies were also carried out. Theobjective of thermodynamic stability was to evaluate phaseseparation and effect of temperature variation on S(M)EDDSformulation. All the S(M)EDDS prepared were diluted withdeionized water (1:20) and centrifuged (Eppendorf Centrifuge5810) at 15 000 rpm for 15 min, and formulations were observedvisually for phase separation. Formulations that did not show anysign of phase separation after centrifugation were subjected tofreeze thaw cycle. In the freeze thaw study, SMEDDS containingdrug were diluted with deionized water (1:20) and two freezethaw cycle between (�20 �C and þ25 �C) with storage at eachtemperature for not less than 4 h were done15.

Statistical analysis

Statistical calculations were performed with the GraphPad InStat(San Diego, California, USA) statistical package for Windows.Data shown in tables and figures of in vitro properties evaluationrepresent mean of three determinations� standard deviation (SD).Statistical significance of the differences between the groups wascalculated by the Tukey–Kramer multiple comparison test and aprobability value of p smaller than 0.05 indicated a statisticallysignificant difference.

Results

Preliminary solubility screening

Labrasol� exhibited better Phe solubilizing capacity thanCremophor�RH40. Oil phases showed the following increasingorder for that parameter: Captex� 355, Miglyol� 840, Isopropylmyristate, Imwitor� and Capmul� MCML. In consequence, thelater was selected for the forthcoming evaluation. PropyleneGlycol, PEG 400, ethanol and Transcutol� P showed similarvalues of solubilization which can be related with the log P of theactive compound reported as 1.53 at pH 7.416. Glycerol was the

co-surfactant which shown the lowest capacity of drug solubil-ization and was discarded for the following evaluations.

In the present work, the four alternative co-surfactants wereused so as to evaluate Phe chemical stability in liquid oral MEs.Even though ethanol is not an excipient allowed for pediatricdosage forms, it was included in the co-surfactants evaluationbecause it is reported as the one able to give the most protectiveeffect against Phe hydrolysis. Besides, ethanol is a component inthe commercially available elixir. Propylene-glycol, on the otherhand, is the most used hydro-miscible solvent in oral liquidpreparations containing Phe. PEG 400 and Transcutol� P, twocurrently used co-solvents in this type of dosage form, have alsoshown a significant drug solubilizing capacity, so they werefurther evaluated.

Screening of ME region

Both for Labrasol� and Cremophor�RH40, ethanol andTranscutol�P were the co-surfactants that showed the largerareas of MEs. Cremophor�RH40-based MEs were obtained at arelative proportion of surfactant from 25% to 5%, and Labrasol�

ones were obtained between a range of 25% and 15%. Anexception was observed for Transcutol� P, which was able to formMEs with 10% of the surfactant. As a result, Cremophor�RH40was the excipient that showed significantly higher areas of MEscompared with the systems based on Labrasol�.

It was also observed for both tensioactives, that ethanol andTranscutol�P were the co-surfactants which have shown thegreater ME area, followed by Propylene-Glycol; PEG 400 was notable to form MEs with Labrasol� and Cremophor�RH40 showedthe smallest ME region in Cremophor�RH40-MEs.

Finally, not only in Cremophor�RH40 systems but also inLabrasol� ones, the highest amount of Capmul� MCML that waspossible to incorporate was 16% w/w; these results convert thesesystems in promising drug delivery ones because of theirpotentially high solubilizing capacity for poorly water-solubledrugs.

Selection of ME formulations

Based on the ME areas observed in the screening mentionedabove, and according with the criteria based on propertiescomparison of similar compositions, 14 compositions wereselected. The excipient ratios (surfactant:oil phase:cosurfactant:-water) were 20:4:20:56 and 20:4:35:41 (Table 2).

Table 2. Compositions of selected MEs after screening with excipients(% w/w).

FormulaCR RH

40 Labrasol�Capmul�

MCM L Ethanol PGPEG400 Transcutol� P

1 20 4 202 20 4 203 20 4 204 20 4 355 20 4 356 20 4 357 20 4 208 20 4 209 20 4 20

10 20 4 3511 20 4 3512 20 4 3513 20 4 2014 20 4 35

CR RH40: Cremophor RH40; PG: Propylene glycol; PEG 400:Polyethylene glycol 400.

4 E. Monteagudo et al. Drug Dev Ind Pharm, Early Online: 1–10

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In this way, the relative proportion of surfactant selected was20% for all the compositions, the percentage of Capmul� MCM-L, was fixed in 4%, and the two relative proportions chosen foreach one of the co-surfactant were 20% and 35%.

Physicochemical properties for selected ME formulations

Table 3 summarizes the results of physicochemical characteriza-tion of the selected MEs. Compositions containingCremophor�RH40 were the ones which showed the highestviscosity. It was also observed that formulations containingPropylene-Glycol and Transcutol�P showed a slightly increasedviscosity when containing 30% of the co-surfactant. Compositionswith ethanol showed a decrease in viscosity value at the higher co-surfactant content. It was remarkable the pronounced increase ofviscosity shown by ME N� 14 (35% PEG 400) comparing withME N� 13 (20% PEG 400). For both surfactants, the highercontent of any of the selected co-surfactant, the lower conduct-ivity values observed. MEs droplet sizes were between 20.66 and145.7 nm, compositions based on Cremophor�RH40 were theones which exhibited the more broad range of mean droplet sizes.The values for this parameter showed by Labrasol-based ME werebetween 27.62 and 57.26 nm.

Labrasol-based MEs with the higher concentration of co-surfactant were able to solubilize nearly two-fold more activecompound (32–58 mg/mL versus 20–26 mg/mL, respectively).There was no significant difference between the solubilizingcapacity exhibited by ethanol and Transcutol�P systems.Cremophor�RH40 systems showed a solubilizing capacitybetween 17 and 24 mg/mL for MEs containing 20% of the

co-surfactant and 33–54 mg/mL for the ones elaborated with 35%.Better solubilizing behavior for ethanol and Transcutol�P wasalso observed, with no significant differences between them.Propylene-glycol was the cosurfactant with less drug solubilizingcapacity for both surfactants and the two different concentrationstested. PEG 400 showed the same poor capacity of solubilizationin Cremophor�RH40 systems (Table 4); it was no possible toobtain MEs containing PEG 400 and Labrasol�. It seems, then,that the solubilizing behavior mainly depends on the nature andthe concentration of co-surfactant. It is also remarkable that 10 ofthe 14 selected compositions were able to solubilize more than20 mg/mL of Phe, the amount proposed in the objectives of thepresent work.

Images obtained by TEM evaluation for ME N� 1 are shown inFigure 1. Mean droplet size obtained using a calibrated scale wasaround 140 nm for this ME which indicates that there were nosignificant differences between Dynamic Light Scattering andTEM results.

Chemical and physical stability evaluation

Table 5 summarizes the results obtained during Phe chemicalstability testing carried out for all the selected compositionsduring six months at different storage conditions. The compos-ition corresponding to Cremophor�RH 40:Capmul� MCM-L:ethanol 20:4:20 was the only one which exhibited significantdecrease in Phe content after 6 months at 50 �C (87%), but it isnoteworthy that no significant change in total content wasobserved either at room temperature or at 40 �C during thesame time. The rest of Cremophor-based compositions evaluatedexhibited no changes in the conditions tested.

Figure 1. TEM microphotograph of ME N� 1 (dilution 1:40).

Table 3. Physicochemical parameters measured in the selectedformulations.

FormulaViscosity(mPa.s)

Conductivity(uS/cm)

Mean droplet size(nm) PDI

1 5.77� 0.07 116.0� 0.1 31.06 0.3982 9.96� 0.12 151.0� 0.2 73.99 0.1343 8.28� 0.09 102.0� 1.2 57.26 0.3984 4.95� 0.03 64.1� 1.6 27.62 0.2905 12.80� 0.07 69.4� 1.3 50.47 0.3846 9.86� 0.05 66.3� 1.1 46.45 0.2527 22.10� 0.10 163.0� 1.4 127.90 0.0738 23.30� 0.06 160.0� 1.3 33.42 0.4599 26.10� 0.11 164.0� 1.5 141.10 0.121

10 15.40� 0.09 100.0� 1.6 20.66 0.31011 41.80� 0.09 81.0� 0.7 79.80 0.52012 32.30� 0.10 92.6� 1.1 99.30 0.51013 27.50� 0.07 154.0� 2.0 45.11 0.34814 89.20� 0.14 74.7� 1.9 145.70 0.373

Mean� SD (n¼ 3); PDI: poly-dispersity index.

Table 4. Phe solubility in the selected MEcompositions.

Formula Phe solubility (mg/mL)

1 24.39� 0.422 20.03� 0.533 25.98� 0.624 58.74� 0.555 32.01� 0.726 58.09� 0.347 24.14� 0.378 18.27� 0.479 20.90� 0.81

10 49.64� 1.2111 33.31� 0.3912 54.49� 0.5613 17.13� 0.2414 32.82� 0.72

Mean� SD (n¼ 3).

DOI: 10.3109/03639045.2013.787536 Lipid-based dosage forms for phenobarbital 5

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In relation to Labrasol-based compositions, the ones contain-ing the lowest proportion of cosurfactants (20% w/w) showed aslight but not significant decrease in Phe content at the highesttemperature; no difference of importance was observed in theones containing 35% w/w of each one of the cosurfactants.Compositions with Labrasol� showed better solubilizing behaviorduring 6 months.

Concerning physical stability, compositions containing 20% ofPropylene-glycol showed drug precipitation, not only forCremophor�RH40 but also for Labrasol� systems.Cremophor�RH40-ME containing 20% of Transcutol�P or PEG400 also showed drug precipitation during the period ofevaluation. Propylene-glycol was the co-surfactant that showedthe worst physical stability results, this phenomenon is probablyrelated to its poor drug solubilizing capacity.

Taste-masking optimization

Drug bitterness prediction

In the present work, the bitterness of an aqueous Phe solution wasevaluated to establish if the drug bitterness could impact onmedicine acceptability; then one ME formulation (N� 14) waschosen, based on physicochemical properties and stability evalu-ation, to optimize taste masking.

As described above, a BPM analysis was performed to assessdrug bitterness. A partial least squares regression was performedwith the bitterness calibration solutions and an acceptable linearregression was obtained for the model (R2:0.81140.800), then thelast three replicates of the test solution were projected. Thebitterness of Phe 0.25 mg/ml in distilled water was 13.3� 1.6 BUand ranked between acceptable and limited acceptable bitterness(Figure 2). This result indicates that it is advisable to considertaste masking during formulation.

Different flavoring options were selected considering fre-quently accepted flavors by pediatric population (tutti-fruttisimilar to chewing gum flavor, strawberry and banana); also areduced addition of mint flavor was considered, since this couldpartially avoid a slight burning sensation produced by someexcipients present in the formula improving mouth-feel andalso leave a slightly fresh aftertaste after taking themedication. In addition, sucralose was selected since it is anon-cariogenic sweetener, more than 500 times sweeter thansucrose, and widely used in food industry and pharmaceuticalapplications.

The success of taste masking (masking efficiency) was laterscreened by the sensor array measurements using the e-tongue tastemasking application and each flavored candidate formulation wascompared to the corresponding placebo formulation.

Electronic tongue – taste-masking analysis

A complete sample set: formulations, placebos (test samples) anddiagnostic samples were tested. After data collection, the valuesmeasured by each sensor were evaluated with principal compo-nent analysis and visualized in a two dimensional map includingtest samples and diagnostic samples, PC1 versus PC2 (Figure 3).Later, to improve sample groups visualization, another taste mapwas performed excluding the diagnostic samples (Figure 4).Finally, the Euclidean distance evaluation between groups wasperformed with the complete sample set. The Euclidean distancebetween flavored formula and the corresponding placebo arelisted in Table 6. The flavored formulations n� 1, n� 3 and n� 4evidenced reduced distance with their respective placebos, inother words, higher taste masking capacity, while the flavoredformula n� 2 and the current formulation evidenced reduced taste-masking capacity.

Optimization of S(M)EDDS formulation

From the physical and chemical stability results it can beconcluded that the most interesting compositions for furtherS(M)EDDS optimization were Formulations N� 1, 3, 4, 5 and 6.Table 7 summarizes the final compositions that were evaluatedwhich are in relation to new components ratios, considering theabsence of water in these formulations.

After the release of 10 mL of each one of the pre-concentratesin 250 mL of simulated gastric fluid, a rapid and uniformdistribution was observed for every composition, but dispersionswith emulsions characteristics were obtained for all of them. So,the pre-concentrates belong to SEDDS composition. It isremarkable that no precipitation was observed after 20 min ofagitation at room temperature and at 15 rpm. Another interestingdata to point out is that no instability sign was observed after thethermodynamic stability test.

Discussion

Co-solvency technique is currently and extensively used toincrease Phe solubility. It had been reported mixtures of glycerin,propylene-glycol and water without addition of alcohol which hadno crystal growth; additionally, it was demonstrated that somecompositions containing 4 mg/mL of drug were able to maintainacceptable stability characteristics for 2 years3.

Our results showed that Phe can be dissolved easily in anumber of MEs and also in the selected SEDDS at a concentrationof 20 mg/mL. The selection of the ME oil phase is very importantbecause drug solubility in the formulation depends mainly on thisphase and this property results fundamental in the search for highsolubilizing capacity systems17,18. The droplet size of the MEs

Table 5. Chemical stability.

1 month 6 months

Formulation T¼ 0 RT 40 �C 50 �C RT 40 �C 50 �C

1 100� 0.66 99.8� 0.18 99.7� 0.16 100.5� 0.78 99.8� 0.22 99.5� 0.83 98.2� 1.783 100� 0.28 100.4� 0.49 100.0� 1.04 99.8� 0.88 100.5� 0.66 101.1� 0.08 97.2� 4.174 100� 0.80 101.7� 0.48 101.2� 0.20 101.1� 0.75 101.0� 0.73 100.1� 0.58 99.7� 3.155 100� 0.53 102.0� 0.19 100.8� 0.28 100.2� 0.21 99.4� 0.04 100.6� 0.57 101.0� 1.866 100� 0.82 101.1� 1.37 100.7� 0.44 101.9� 0.79 98.9� 1.53 100.4� 0.08 102.5� 0.277 100� 0.51 104.6� 0.05 101.7� 1.07 104.9� 0.05 102.8� 0.12 102.0� 0.17 87.0� 0.55

10 100� 0.12 102.0� 0.16 99.3� 0.63 103.3� 0.08 100.8� 0.33 101.1� 0.30 103.5� 0.1811 100� 0.19 102,2� 1.18 102,7� 0.04 104,5� 0.31 100,9� 0.40 100,5� 0.33 103,1� 0.0212 100� 0.16 102.5� 0.85 101.1� 0.37 103.2� 0.30 101.0� 0.27 101.2� 0.44 104.1� 0.0514 100� 0.55 100.5� 1.55 99.8� 0.85 99.1� 0.46 101.0� 1.91 101.1� 1.41 100.2� 1.02

Phe content (mean %� SD; n¼ 3); RT: Room Temperature.

6 E. Monteagudo et al. Drug Dev Ind Pharm, Early Online: 1–10

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Figure 3. Test and diagnostic samples in principal component analysis map (PC1 versus PC2).

Figure 2. BPM – Phe 0.25 mg/mL.

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makes them also very promising in relation to biopharmaceuticaladvantages8,19–24. Besides, the two selected dosage forms for thepresent work have also shown advantages from the chemicalstability point of view. Dietz et al. reported the stability of severaloral liquid forms with Phe 4 mg/mL including emulsions andaqueous solutions with and without Propylene-glycol. HPLCanalysis of these oral liquids found that the emulsion containingPropylene-Glycol was stable throughout 56 weeks, exhibiting noloss of Phe content; this result was also verified for the

commercial elixir. However, the emulsion without Propylene-glycol resulted in a decline of Phe content; Phe loss was 34% in4 weeks for the emulsion4. Considering these antecedents, theMEs developed constitute a dosage form that can significantlyavoid hydrolytic degradation. It has also been shown that Phestability in solutions is promoted by using a mixed solventcomposed of water and generic solvents such as alcohol,Propylene-glycol, glycerin or PEG3,4. Gupta reported the follow-ing decreasing order for maintaining Phe chemical stability inhydro-miscible solutions: ethanol, propylene-glycol and glycerol5.On the contrary, the results shown in the present workdemonstrated that a composition containing ethanol was theonly one which has shown chemical instability; this situationdemonstrates the particular characteristics that MEs are able togive to the carried drugs. It is also remarkable that ethanol mustnot be used for children formulations, and its inclusion in thepresent work was only for comparison reasons. Moreover, it wasrecently proposed the necessity to develop these dosage forms notonly with lower surfactant content, the component related withhigher toxicity, but also with no addition of cosolvents25. As aconsequence of the increasing interest that this type of dosageforms have presented in the last years, a number of approaches forthe selection of the most appropriate lipid system(s) and the roleof various excipients for improved delivery of dosage form wererecently reported10.

The development of S(M)EDDS is another area of pharma-ceutical research which shows increasing interest for the oraladministration: olmesartan medoxomil26 and bicyclol27 weresuccessfully evaluated in vivo. A S(M)EDDS containing tacroli-mus was optimized choosing excipients which were able to inhibitthe efllux of glycoprotein P for the drug28. It is well-known thatonly very specific pharmaceutical excipient combinations wouldlead to efficient self-emulsifying systems. There is an increasingneed for guidelines in excipient selection to obtain effective

Figure 4. Test samples in principal component analysis map (PC1 versus PC2).

Table 6. Euclidean distances between flavored formula and thecorresponding placebo.

FormulaEuclidean distance

(BU)Euclidean distance

(S.D.)

Flavored Formula 1 66.3 4.6Flavored Formula 2 188.0 5.9Flavored Formula 3 65.4 2.6Flavored Formula 4 55.0 4.1Current Formula 100.0 1.0

Table 7. Compositions corresponding to S(M)EDDS evaluated byphysicochemical in vitro parameters (expressed as % w/w).

Formula Labrasol Capmul� MCM L Ethanol PG TCL� P

1 45.5 9 45.53 45.5 9 45.54 34 7 595 34 7 596 34 7 59

PG: Propylene-glycol; TCL P�: Transcutol P�.

8 E. Monteagudo et al. Drug Dev Ind Pharm, Early Online: 1–10

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delivery system with improved bioavailability, we did this selectionin basis to the stability behavior of related-compositions MEs10.

Epilepsy is a chronic condition requiring long-term treatmentwith drugs that have intrinsic limitations18. If we consider thedoses currently used in Neonatology division, toxicity appears asthe main problem to resolve. Because of this, Jelveghari reporteda liquid oral dosage form of Phe without alcohol for neonates3.MEs and S(M)EDDS are dosage forms that can be useful toimprove Phe biopharmaceutical characteristics, specially for therequirement of high dose administration in pediatric patients withan appropriate chemical stability. There are no antecedents inliterature about MEs or S(M)EDDS containing Phe, despite theancient use of the active compound and the extensive research inthese pharmaceutical dosage forms.

Finally, some other clinical advantage must also be consideredfor these dosage forms. Taste-masking is an area of knowledge ofincreasing interest, especially for pediatric patients and for long-term administration, as is the case of Phe29. Taste qualityperception and recognition is based on building or recognition ofactivated sensory nerve patterns by the brain. This step isachieved by the e-tongue statistical software which interprets thesensor data into taste patterns12. It could also assist or evenreplace the sensory evaluation in the development of fast-dissolving films with bitter taste30. As mentioned above, palatabledrugs are particularly important when formulating oral dosageforms for children and considering the sensory differencesbetween adults and children, it is evident that children as targetpopulation are regarded as the most suitable panel for tasteassessment of pediatric formulations. Palatability studies are notdescribed in any regulatory guidance but must be considered asclinical studies performed by qualified personnel with EthicalCommittee approval and informed consent from parents orguardians and assent from the child as appropriate. There maybe ethical difficulties in designing suitable safe studies in whichchildren can easily participate31.

Recently, e-tongue analysis has been utilized as an in vitromethod to address these difficulties during formulation develop-ment for taste-masking evaluation; although further studies arerequired to provide additional correlation data between humantaste assessment and e-tongue prediction, formulation develop-ment could be rationalized and simplified which makes e-tonguespromising tools to reduce human taste assessment tests32.

Sensory testing techniques consist of formalized, structuredand a codified methodologies which are widely used for newproduct development or formula modification. There are twomain types of sensory methods: analytical or affective tests.Analytical tests include the evaluation of specific productattributes in terms of discrimination or difference and description.Affective tests on the other hand, involve panelist’s preference andattempt to qualify the degree of liking or disliking of a product.Affective testing requires a larger amount of volunteers thananalytical testing and the larger panel size arises due to the highvariability of individual preferences and thus a need to compen-sate with increased number of people to ensure statistical powerand test sensitivity33,34. Regarding our work, it is remarkable thatformulation N� 14, which was selected one for this taste-maskingevaluation, because of its drug solubilizing capacity, stabilitybehavior and the nature of its components, is the one which hasshown the highest viscosity (89.20 mPa.s) and also the biggestmean droplet size (145 nm).

The preliminary values obtained for SEDDS characterizationand the ME selected compositions encourage further in vivoevaluation; bioavailability of these dosage forms must be knownbefore proposing a new protocol for oral administration. It wouldlikely be necessary an adjustment of dose, because not onlySEDDS but also MEs have been related to improvement of

biopharmaceutical characteristics. Another important conse-quence to take in consideration after in vivo studies is the actualtoxicity that a new protocol of administration may represent forpatients of all ages, but especially for newborn.

Conclusions

Cost is one issue that clearly determines antiepileptic drugselection, and it is reasonable to recommend one of the traditionaland cheaper one as first-line therapy. A number of MEcompositions with approved and widely used excipients for theoral route were designed through pharmaceutical development todecrease the hydrolytic degradation Phe undergoes in oral liquidformulations. In addition, it was possible to load the currentlydose used in pediatrics and especially in neonatology (20 mg/mL)that is five-fold the value of the commercialized elixir. No sign ofphysical instability was observed for the loaded compositionsafter 6 months in different storage conditions that includedevaluation at 50 �C. After the ME screening, SEDDS compos-itions were evaluated through their characteristic parameters.Further evaluation is necessary so as to optimize a SEDDScomposition before being incorporated into soft capsules for Pheadministration to adult patients.

It is remarkable that both type of dosage forms proposed in thepresent work, MEs and SEDDS, present interesting characteristicsas drug delivery systems that have already included independentabsorption in fasting conditions. Among lipid-based systems, bothdosage forms are promising technology to improve the rate andextent of poorly water-soluble drugs absorption; nanometric sizeof internal phase constitutes another interesting characteristic.

At the same time, they would offer acceptable physicalstability as they showed to avoid the typical hydrolytic degrad-ation even at relative high contents of water in the formulations,although these results were obtained carrying out a rapidexperimental design.

The multichannel taste sensor or e-Tongue has demonstrated tobe a useful tool to assess drug bitterness intensity and also toevaluate the taste-masking efficiency for several ME formulationcandidates during pharmaceutical product optimization.

In conclusion, two different dosage forms containing Phe fororal administrations were prepared that provide excellent drugsolubilizing capacity and stability characteristics; in vivo studywould be followed so as to evaluate their bioavailability.

Declaration of interest

The authors report no declarations of interest.Support was provided by the National Agency of Scientific and

Technological Promotion (ANPCyT) and the University of Buenos Aires.

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