Design and stability study of a paediatric oral solution of methotrexate 2mg/ml

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Design and stability study of a paediatric oral solution of methotrexate 2 mg/ml

Sandy Vrignaud

^a,

*, Thomas Briot

^a,b

, Aurélie Launay

^a

, Marie Kempf

^c

, Frédéric Lagarce

^a,b

aPharmacieCentrale,CentreHospitalierUniversitaired’Angers,Angers,Cedex,France

bL’UniversitéNantesAngersLeMans,INSERMU1066,Microetnanomédecinesbiomimétiques,Angers,France

cLaboratoiredeBactériologie,InstitutdeBiologieenSanté–PBH,CentreHospitalierUniversitaired’Angers,Angers,Cedex,France

ARTICLE INFO

Articlehistory:

Received9March2015

Receivedinrevisedform8April2015 Accepted9April2015

Availableonline13April2015

Keywords:

Oralpaediatricsolution Methotrexate HPLC Stabilitystudy

ABSTRACT

Oralpaediatricformsdevelopmentbypharmaceuticalindustryisstillinsufficient.Thepresentstudywas performedtoproposeanadapted andpleasantformulationofliquidoralformulationofMTX.The solutioniscomposedofinjectablemethotrexate,water,OraSweet¹andsodiumbicarbonate.

After120daysstorage,pHremainedstableatabout8inallformulations,insuringnoriskofMTX precipitation. MTX content in solution formulation, determined by high performance liquid chromatographymeasurements,remained inthespecificationsof>90% oftheinitialconcentration whenstoredat4and25C.ForceddegradationofMTXbyheatandacidicconditionsallowedformation anddetectionofdegradationproductsbytheanalyticalmethod.

Microbialstudyofthepreparationshowsthatthesolutionremainsinthespecificationsduringallthe storage,orafteronesampleeachweekduringonemonth,eventuallyindicatingthemicrobialproperties arenotaffectedbypatientuse.

Toconclude,wehereproposeanewMTXliquidformulationstableforatleast120days.

ã2015ElsevierB.V.Allrightsreserved.

1.Introduction

Drugdeliverytochildrenisstilllimitedbythelackoflicensed drugsand non adaptedforms for administration(Fontanet al., 2004).Methotrexate(MTX)iswidelyusedinpaediatrics,mainly for lymphoid acute leukaemia (LAL) treatment or rheumatoid disease(Weissetal.,1998;Ottenetal.,2002).Despiteitsusefor manyyears,noliquidoralformulationsaremarketed.Tabletsare not indicated for children younger than 6 years. Thus, most hospitalpharmacieshavetopreparehardcapsulesbutthereare 3drawbacks:(i)thelackofpossibility toadapt dosesoncethe capsulesareprepared,i.e.,incaseoftoxicity,dosecanbereduced, oronthecontraryagoodtoleranceleadstodosesenhancement, (ii)drugpossiblebadtaste(bitterness..)and(iii)familyexposureto cytotoxicathomewhenthecapsulesareopenedforadministra- tion. Lack of oral liquid forms also leads to use of injectable medicines for oral administration. Such a practice poses the problemofpHincompatibility,ornonadaptedingredientsfororal

use.Forthesereasons,oralsolutionsorsuspensionsareemerging inhospitalpharmacies.Forinstancespironolactone,hydrochloro- thiazide andcaptopril (Fajolleetal., 2005), sildenafil(Provenza etal.,2014),ursodeoxycholicacid(Santovenaetal.,2014),oreven reconstitutablehydrocortisone(Orlu-Guletal.,2013)formulations aredescribed.

Inthepresentstudy,inordertodevelopanewformulation,we focusedonMTXphysicochemicalproperties:MTXispoorlysoluble inwaterandalcohol.MTXprecipitatesatpHlessthan6.6(Allwood etal.,2002).Furthermore,MTXdegradeswhenexposedtolight.In 1979, a formulation of syrup containing sodium bicarbonate, injectionMTX,simplesyrupandchloroformwaterwasproposed, andwasshowntobestableforperiodofupto1month(Stuart etal.,1979).Currently,chloroformwaterisnomoresuitableasitis toxic.Recently,anotherteampreparedMTXsolutioninOraPlus¹ and OraSweet¹ (MegiasVericatetal.,2012).The stabilitywas showntobe25days,whichisinsufficientforanoptimalpatient comfort and for a pre-empted fabrication. Thus, our hospital developed a new oral formulation. The present work studies organoleptic characteristics and chemical stability of MTXoral formulation.AsMTXislikelytobegiventoimmunocompromised children,microbialstudywasalsoconducedtoinsureasatisfying microbialquality.

*Correspondingauthorat:CHUAngers–ServicePharmacie,4ruelarrey,49933 Angers,Cedex9,France.Tel.:+33241353544;fax:+33241354084.

E-mailaddress:[email protected](S.Vrignaud).

http://dx.doi.org/10.1016/j.ijpharm.2015.04.016 0378-5173/ã2015ElsevierB.V.Allrightsreserved.

InternationalJournalofPharmaceutics487(2015)270–273

ContentslistsavailableatScienceDirect

International Journal of Pharmaceutics

j o u r n al h o m ep a g e: w w w . el s e v i e r . c o m / l o c at e / i j p h a r m

2.Materialandmethods

2.1.Reagents

TheoralsolutionwasperformedwithinjectableMTX500mg/

20ml (Mylan, Pittsburg, USA), sodium bicarbonate (COOPER, Melun, France), OraSweet¹ (Paddocklaboratories, Minneapolis, USA) and injectable water (Aguettant, Lyon, France). All ingre- dientswerePharmacopeiagrade.

ForHPLC,acetonitrile(HipersolvChromanorm,VWR,Fontenay sousBoisFrance),potassiumhydroxydeandpotassiumdihydrogen phosphate(ACROSOrganics,Geel,Belgique)wereused.Waterwas obtainedfrom a Prima reverseosmosis system (ElgaLabwater, Antony,France).

Allreagentsandsolventswereanalyticalgrade.

2.2.Storage

MTXsolutionwaspackagedin30mlambertypeIglassbottle, for 120 days, in triplicate. Storage was performed in climatic chamberqualifiedaccordingtoICHat4Cand25Cunder60%

relativehumidity.

2.3.Forceddegradation

MTX solution was incubated with sulphuric acid 0.5M for 60minat70C(n=3).

2.4.Chromatographicconditions

A highpressure liquid chromatography(HPLC) method was developed.ThesystemwascharacterizedbyaPerkinElmerSeries 200 pump, injector and oven. The detector was a diode array detector (Flexar PDA detector, PerkinElmer, Walthman, USA) operatingbetween190and700nm.Chromerasoftware(v4.1.0) (Perkin0Elmer,Walthman,USA)wasusedtoquantifythepeaksof thechromatograms.Themobilephaseconsistedofamixtureof sodiumdihydrogenphosphatebufferwhichpHisadjustedtopH 6.6andacetonitril(90:10v:v).Theflowratewassetto1ml/min.A C18ODSHypersil(250mmx4.6mm,5

m

^m)(Thermo-Scientific, VillebonsurYvette,France)wasusedandmaintainedat20C.The sample injectionvolume was 10

m

^{L and theanalysis time was}

10minutes.MTXdetectionandquantificationwereprocessedat 302nm.

2.5.Methodvalidation

2.5.1.MethodvalidationwasperformedaccordingtoICHQ2 MTXcontent insolutionwas determined bydiluting 500

m

^l

sample with 9.5ml of mobile phase. In order to remove Ora Sweet¹, 5ml of the dilutedmix were centrifugedfor 7minat 4000rpm.100

m

^lofsupernatantwerethendilutedwith900

m

^lof

mobilephase.

Working standard solutions for the calibration curves were preparedusingsameingredientsasinformulation,toreachafinal MTXconcentrationof1, 1.5,2,2.5and3mg/ml.Thentheseworking standardsolutionsunderwentsamedilutionassampleformula- tion,e.g.,inmobilephaseleadingtoconcentrationsbetween5and 15

m

^g/ml.

2.6.Organolepticappreciation

Odor,appearanceandcolorofthepreparationwereassessed.

BecauseoftheintrinsictoxicpropertiesofMTXsolution,tasteand palatabilitywerenotstudied.

2.7.pHstudy

MeasurementswereperformedintriplicateusingapHmeter ConsortC561(Tunhout,Belgium).

2.8.Stabilitystudy

MTXformulationsareconsideredstableifphysicalcharacter- isticshavenotmovedandifdrugconcentrationremainsabove90%

of the initial concentration without absence of characteristic degradationpeaks.

2.9.Microbiologicalstudy

Threeconditions(eachintriplicate)werestudied(Table1):

-Sampledayssameaspatienttakingmedicationathome,e.g., days7,14,21and28.

-Analyseafter120days,correspondingtothephysicochemical stabilityofthesolution.

-Positivecontrol:thesolutioncontainingornotMTXisartificially infectedwithenvironmentbacteriaatday0andanalysedafter 7days.InfectedMTXsolutionswereusedascontrolstoinsure MTXdidnotinhibitmicrobiologicalgrowing.

To performanalysis, eachsample wasdiluted1:10 insterile NaCl 0.9%, and then plated on different media and different conditions:

-Sheep blood agar plate (Thermo Fisher Scientific, United Kingdom),aerobicandanaerobicincubationat37C

-Chromogenicagarplate(BrillianceTMUTIagar–Oxoïd,France), aerobicincubation

-Sabouraud agar plate (Becton Dickinson, Germany), aerobic incubationat29C

-Brainheartinfusion(bioMérieux,France),aerobicincubation According to European Pharmacopoeia monograph of non sterileproducts,liquidoralformulationsmeetmicrobialrequire- mentsifthetotalaerobicmicrobialcountarelessthan10²cfu/ml, thetotalcombinedyeast/mouldcountarelessthan10¹cfu/mland ifthereisnoEscherichiacoli.

3.Resultsanddiscussion

3.1.Methotrexatesolutionformulation

Toinsureabetterhomogeneitysoastosecureadministration, the choice was made to formulate a solution rather than a suspension.ThusinjectableMTXMylan¹wasusedasitisavailable asasolutionanddoesnotcontainanyunsuitableingredientsfor oral use (ingredients composition: injectable water, sodium chloride and sodium hydroxide). On the contrary, pure active MTXisapowderslightlysolubleinwaterthatformssuspension.

Furthermore,we preferredtouseanalready madecommercial injectablesolutionofpharmaceuticalqualityinsteadofapowder tobedissolvedin ordertolimitthechemical contaminationof Table1

MTXsolutioncomposition.

Component Quantity

Methotrexateforinjection500mg/20ml 2.4ml

Sodiumbicarbonate 0.6g

OraSweet¹ 7.5ml

Sterilewater QS30ml

S.Vrignaudetal./InternationalJournalofPharmaceutics487(2015)270–273 271

personalandenvironment.Infact,handlinganinjectablesolution ismuchmoreeasyandsecurethanhandlingapowdertodissolve.

This also limits cross contamination between batches. This procedureisadaptedtohospitalcompoundingpharmacies.

Thefirststepoftheformulationconsistsofdissolvingsodium bicarbonateinwaterinamortar(abeakercanbeusedassociated

withamagneticstirrertoinsuregoodstirring).Next,OraSweet¹is slowlyincorporated.Themixingiskepttoensurethoroughmixing whileaddingMTX(quantitiesinTable1).Concerningtherational of theirchoice,OraSweet¹ isusedbecauseofitspropertiesof flavoring and sweetening oral solutions. pH of this vehicle is approximately 4.2. Keeping in mind that MTX is not stable at pH<6.6,sodiumbicarbonatewasaddedtoreachpH8.

Using this formula, a homogeneous slightly viscous yellow solutionwas obtained. The study of organolepticproperties of solutionsshowedthatthepreparationremainedyellowandthe pleasantcitrus-berrysmellstayedfor120days.BecauseMTXis toxicdrug,wedidnotperformtasteessays, butinpracticethe solution is well accepted by most of treated children (about 20children).

3.2.Stabilitystudy

Whatevertheconservationconditions,noprecipitateoccurred.

After 120 days storage, pH remained stable at about 8 in all formulations. Despite thepresence of a buffer in Ora Sweet¹, sodiumbicarbonateadjustwassufficienttoobtainandmaintain pH8.

Awell-definedandsymmetricpeakwasobtained(Fig.1).

Ontheintervalrange(5–15

m

^{g/ml)thelinearityofthemethod}

was evaluated by constructing the calibration curve at five concentrationslevels.Equationof thecalibrationcurveisarea= 798,730concentration+78,836. The linearity is demonstrated bythe correlationcoefficient obtained, r²=0.999.Moreover the homogeneityofthevariancewastestedwithintheconcentration rangeforallstandardconcentrations.

Therepeatability,assessedonthetestconcentration(10

m

^g/ml)

at18determinations(6replicates/3differentdays),wassystem- aticallyinferiortoacoefficientofvariationof2%.Theaccuracy, assessedatthreeconcentrations,onthreedays(9determinations), wassystematicallysuperiorto90%oftheattendedvalue.MTXis elutedin3.5min,asseeninFig. 1.Themethodisstabilityindicating asdegradationproductsareobtainedanddetectedafteracidicand heatstress(Fig.2).Limitofquantificationdeterminationwasnot necessaryasthisstabilityindicatingmethodhadtheobjectiveto quantifyMTXnear100%concentration.

DrugstabilityinsolutionisindicatedinFig3.MTXcontentin solutionformulationremainedinthespecificationsof10%ofthe initialconcentrationduringallthestudywhen storedat 4and 25Cfor120days.

Fig.1.ChromatogramofMTXformulation2mg/mlatday0.

Fig.2.ChromatogramofMTXformulationexposedtosulphuricacidduring60min at70C.

Fig.3.DrugcontentasapercentofinitialMTXconcentrationinformulationover timeatdifferenttemperatures(%).Square:solutionstoredat4C,triangle:solution storedat25C.

272 S.Vrignaudetal./InternationalJournalofPharmaceutics487(2015)270–273

3.3.Microbialstabilitystudy

BacterialgrowthwasobservedininfectedMTXsolutions.On the other hand, solutions without MTX remained in the specifications.These resultsindicated microbial test performed inthepresentstudytobesterilityindicating.

ConcerningoralsolutionsofMTX,atday0,eachsolutionwasin thePharmacopeiaspecificationsfororalliquidproducts.Whatever theconditions(e.g.,openeach weekfor28daysoronetimeat 120days),solutionsremainedinthespecifications,insuringagood conservationofthesolution.

4.Conclusion

We propose a new formulation of oral MTX. The solution remainsstablefor120daysat4and25C.

Conflictofinterest

Theauthorsindicatehavingnoconflictsofinterestwithregards tothecontentofthisarticle.

Acknowledgment

TheauthorswouldliketothankE.Balduini,I.MainfroidandC.

Truffautfortheirhelp.

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