Formulation and characterization of a 0.1% rapamycin cream for the treatment of Tuberous Sclerosis Complex-related angio fi bromas
Guillaume Bouguéon
a, Frédéric Lagarce
a,b, Ludovic Martin
c, Hélène Pailhoriès
d, Guillaume Bastiat
b, Sandy Vrignaud
a,*
aPharmacieCentrale,CentreHospitalierUniversitaired’Angers,AngersCedex,France
bL'UniversitéNantesAngersLeMans,INSERMU1066,Microetnanomédecinesbiomimétiques,Angers,France
cServicedeDermatologie,CentreHospitalierUniversitaired’Angers,AngersCedex,France
dLaboratoiredeBactériologie,InstitutdeBiologieenSanté—PBH,CentreHospitalierUniversitaired’Angers,AngersCedex,France
ARTICLE INFO
Articlehistory:
Received21January2016
Receivedinrevisedform25May2016 Accepted29May2016
Availableonline1June2016
Keywords:
Rapamycin Sirolimus TuberousSclerosis m-Torinhibitor Topicformulation Stabilitystudy
ABSTRACT
Medicinesforthetreatmentofrarediseasesfrequentlydonotattracttheinterestofthepharmaceutical industry,and hospitalpharmacists arethusoftenrequestedby physiciansto preparepersonalized medicines.TuberousSclerosisComplex(TSC)isararediseasethatcausesdisfiguringlesionsnamedfacial angiofibromas.Varioustopicalformulationsofrapamycin(=sirolimus)havebeenprovedeffectivein treatingthesechangesinsmallcaseseries.Thepresentstudyprovidesforthefirsttimecharacterization ofa0.1%rapamycincreamformulationpresentinggoodrapamycinsolubilisation.Thefirststepofthe formulationissolubilisationofrapamycininTranscutol1,andthesecondstepistheincorporationofthe mixtureinanoil-in-watercream.
AHPLCstability-indicatingmethodwasdeveloped.Rapamycinconcentrationinthecreamwastested byHPLCandconfirmedthatitremainedabove95%oftheinitialconcentrationforatleast85days, without characteristic degradationpeaks. The preparation metEuropean Pharmacopoeia microbial specificationsthroughoutstorageinaluminumtubes,includingwhenpatientusewassimulated.Odour, appearanceandcolourofthepreparationwereassessedandnochangewasevidencedduringstorage.
Therheologicalpropertiesofthecreamalsoremainedstablethroughoutstorage.
Toconclude,wereportpreparationofanovelcreamformulationpresentingsatisfactoryrapamycin solubilisation forthetreatmentofTSCcutaneousmanifestations, withstabilitydata.Thecream is currentlybeingusedbyourpatients.Efficacyandtolerancewillbereportedlater.
ã2016ElsevierB.V.Allrightsreserved.
1.Introduction
TuberousSclerosisComplex(TSC),alsoknownasBourneville’s disease,isa raregenetic autosomaldominantdisorderwithan estimatedfrequencyofbetween1/6000and1/10,000livebirths (JacksandWitman,2015).Angiofibromasdevelopovertimeand are very disfiguring, affecting the patient both physically and psychologically.
After observation of the effects of systemic rapamycin on angiofibromas(Bissleretal.,2008;Hofbaueretal.,2008),topical sirolimuswastestedonmousemodels(Rauktysetal.,2008)and rapidlyformulatedforhumans(Haemeletal.,2010).Fromthenon,
severaltopicalformshavebeendevelopedusingmTORinhibitors, namelyrapamycinandeverolimus(Madke,2013;Balestri etal., 2015).
Forthelastfiveyears,morethantenformulationshavebeen reported in different pharmaceutical forms (ointment, creams, solutions, etc.) at different concentrations (0.003–1%), from crushedtabletstooralsolution,whicharenotoptimalfortheir tolerance and efficacy. However, all authors described patient improvement, with minimal side effects (except with the solutions), inconsistent percutaneous absorption and systemic diffusion,butrecordedrecurrenceshortlyafterstoppingtreatment (Madke,2013;Balestrietal.,2015;Bouguéonetal.,2015).
We therefore decided to develop a topical treatment and focusedourresearchonthreeaspects.First,wewantedtooffera formulationcontainingrapamycininitssolubilizedform,inorder toobtainimmediatebioavailabilityoftheactivemoleculeandthus allowing dose optimization and avoiding the risk of bleeding
* Correspondence to: CHUAngers—ServicePharmacie, 4 rue Larrey, 49933 AngersCedex9,France.
E-mailaddresses:savrignaud@chu-angers.fr,sandy-vrignaud@hotmail.fr (S.Vrignaud).
http://dx.doi.org/10.1016/j.ijpharm.2016.05.064 0378-5173/ã2016ElsevierB.V.Allrightsreserved.
ContentslistsavailableatScienceDirect
International Journal of Pharmaceutics
j o u r n a lh o m e p a g e : w w w . e l s e vi e r . c o m / l o c a t e/ i j p h a r m
attributedtocrushedtablets(Tuetal.,2014).Secondly,wewanted to offer the patient an appealing topical treatment of good appearanceinordertoimprovepatientobservanceand compli- ance with treatment.Indeed, some patients had reported that ointmentwasdifficulttoapplyorrough(Park etal.,2014)and sometimes parents decided to reduce the frequency to avoid sendingchildrentoschoolwithanoilyskin.Thirdly,wewantedto characterizeourformulationandassessitsstabilityovertimeto ensureitsefficacyinusebypatients.
Acreamformulationwasthereforedevelopedandastability- indicatingmethodwasthen designedand astability studywas performed.
2.Materialsandmethods 2.1.Materials
Rapamycinpowder,polysorbate80,andsorbitantrioleatewere providedbyInresa,(Bartenheim,France).Transcutol1P,oliveoil, castoroil,liquidparaffinandsweetalmondoilwereprovidedby COOPER(Melun,France).Excipialhydrocrème1wasprovidedby Galderma (Laboratoires Spirig SAS, Toulouse, France). All exci- pientswereofPharmacopoeiagrade.
Methanol (HipersolvChromanorm,VWR,FontenaysousBois France),was used for HPLC. Waterwas obtainedfroma Prima reverse osmosis system (Elga Labwater, Antony, France). All reagentsandsolventswereofanalyticalgrade.
2.2.Rapamycinsolubilisationinvariousoilsandsurfactants Rapamycinpowder(10mg)wasincubatedwithQS500mgof oils,solventsorsurfactants(Table1).Mixingwasperformedunder stirringfor60minatroomtemperature.
2.3.Creamstorage
Thecreamwaspackagedin30mlaluminum tubes(COOPER, Melun,France)andstoredinaclimaticchamberconformingtoICH (International Consensus on Harmonization, 2015) at 252C under605%relativehumidity.
2.4.Samplepreparation:rapamycinextractionandrecovery Analiquotofcream(2g)wasintroducedintoanErlenmeyer flaskwith10mlof methanoland maintainedunder stirringfor 10min.Allexperimentswereperformedintriplicate.Theresulting solutionwascentrifugedat900gfor10minandthesupernatant wasremovedand analyzedforrapamycin contentcompared to rapamycininmethanol.
2.5.Chromatographicconditions
A highpressure liquid chromatography (HPLC) method was developed, based on Ricciutelli’s publication (Ricciutelli et al.,
2006).ThesystemwascharacterizedbyaPerkinElmerSeries200 pump,injectorandoven.Thedetectorwasadiodearraydetector (Flexar PDA detector, Perkin Elmer, Walthman, USA) operating between190–700nm.Chromerasoftware(v4.1.0) (PerkinElmer, Walthman,USA)wasusedtoquantifythepeaksofthechromato- grams.Themobilephaseconsistedofamixtureofmethanoland water (80:20v:v). The flow rate was set at 1ml/min. A C18 Supelcosilcolumn(150mm4.6mm,5
m
m)(Supelco1Analytical, Sigma-Aldrich1, Bellefonte, USA) was used and maintained at 50C.Thesampleinjectionvolumewas0.01mlandtheanalysis time was 10min.Rapamycin detection and quantification were processedat278nm.2.6.Methodvalidation
ThemethodwasvalidatedaccordingtoICHQ2R1(International ConsensusonHarmonization,2015).
Astandardcurvewasestablishedwithfivedifferentrapamycin concentrations in cream: 0.06%, 0.08%, 0.1%, 0.12% and 0.14%.
Extraction followed by rapamycin determination was then performed.Thelinearityof themethodwas evaluatedonthree differentstandardcurves.
Therepeatabilityofthemethodwasevaluatedbypreparingsix creamsamplesconcentratedat0.1%rapamycinonthreedifferent days.Eachsampleunderwentrapamycinextractionanddetermi- nation.
The accuracy of the method was established using three concentrationlevels (0.08,0.1 and 0.12%) in triplicateonthree differentdays.Eachsampleunderwentrapamycinextractionand determination.
2.7.Forceddegradation
Sensitivitytoheat:a2gsampleofcreamwasheatedat90Cfor 60min (n=3). Then the sample was subjected to the same extraction method as for rapamycin determination in cream.
Transcutol1PandExcipialhydrocrème1werealsotestedalonefor heat degradation. No degradation products were observed on chromatograms.
2.8.Physico-chemicalstability
Rapamycindeterminationinthecreamwasassessedatday0,3, 7,14,21,28,63and85(n=3foreachday)andmeanconcentration wasexpressedas95%confidenceintervalofthemean.Themean andconfidenceintervalwereconsideredacceptableifgreaterthan 95% of the initial concentration, without the existence of characteristicdegradationpeaks.
2.9.Organolepticappreciation
Odour,appearanceandcolourofthepreparationwereassessed atdays0,3,7,14,21,28,63and85(n=3foreachday).Odourwas measuredas described in theEuropean Pharmacopoeia, i.e. by spreading1.5gofcreamona6cmwatchglassandsmellingthe creamafter15min.
2.10.Rheologicalmeasurements
ExperimentswereperformedaccordingtoEuropeanPharma- copoeiaMonograph2.2.10.Thenon-steadyflowpropertyofthe creams was studied using a Kinexus1 rheometer (Malvern Instruments S.A., United Kingdom), with cone plate geometry (diameter 50mm, angle: 2) and with controlled shear rates rangingfrom 0.5to50s 1 atroomtemperature. Two cyclesof increasinganddecreasingshearratewereperformed.
Table1
Rapamycinsolubilityindifferentsolvents,oilsandsurfactants.
oil/surfactant/solventtested Rapamycinsolubility
Virginoliveoil <2mg/ml
Castoroil <2mg/ml
Sweetalmondoil <2mg/ml
Liquidparaffin <2mg/ml
Sorbitantrioleate(Span185) <2mg/ml Polysorbate80(Tween180) <2mg/ml
DiethyleneglycolmonoethyletherP(Transcutol1) Fullysoluble(20.2mg/ml)
The evolution of viscosity profiles was studied in triplicate directlyaftertheformulation process and after28 and 84days incubationatroomtemperature,andthencomparedtothecream used as the main ingredient: Excipial Hydrocrème1. Viscosity resultswereexpressedasmeanandstandarddeviation.
2.11.Microbiologicalstudy
Allstudiesformicrobialcontaminationswereperformedbya certifiedlaboratoryfollowing theEuropean Pharmacopoeia 8.0:
Monograph5.1.3:“efficacyofantimicrobialstorageforcutaneous preparations”andMonograph2.6.1:“sterility”.
Threeconditionswerestudied(eachintriplicate):
–Sampledaysthesameasthoseforapatientusingamedication athome,i.e.openedandremovedeachdaythentestedatdays7, 14,21and28.
–Analysis after 28 and 85days (85days corresponding to the physicochemicalstabilityofthecream).
–Positive control: Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 9027) were growth on Blood agar plate for 24h at 37C before being suspended for inoculation(105 UFC/gofcream). Creameithercontainingor notcontainingrapamycinwasartificiallyinfectedwithStaphy- lococcusaureusorPseudomonasaeruginosaatday0andanalyzed after 7days. Infected rapamycin formulations were used as controls to ensurerapamycin didnot inhibit microbiological growth.Thecriterionforevaluationofantimicrobialactivityis logarithmic decrease of viable microorganism’s number as comparedtoinoculumvalue.
To perform analysis, 1g of cream was diluted in 5ml of injectablewater.Then0.4mlwasplatedonasheep’sbloodagar plate (n=5) (Thermo Fisher Scientific, United Kingdom), and incubatedat37C.
AccordingtotheEuropeanPharmacopoeiamonographfornon- sterileproducts,theformulationmeetsmicrobialrequirementsif thetotalaerobicmicrobialcountsarelessthan102cfu/mlorcfu/g, thetotalcombinedyeast/mouldcountsarelessthan102cfu/mlor cfu/gandthereisnoStaphylococcusaureusandnoPseudomonas aeruginosa.
3.Resultsanddiscussion
The absence of commercially available medicines for the treatmentofmainrarediseaseislikelytoleadtothedevelopment ofapreparationinahospitalpharmacy.Designingasuitablecream forpatientsincludesthechoiceofanappealingformulation,study ofthedrug’sphysicochemicalstabilityinthecream,andensuring satisfactorymicrobialquality.
3.1.Solubilityofrapamycininvariousoils,solventsandsurfactants Facialangiofibromasappearduringinfancy,between2and5 yearsofage,andgrowprogressivelyuptoteenageyears.Inviewof children’s and adolescents’ acne-prone skin, an oil-in-water emulsion basis appeared to be the best compromise for TSC patienttreatment.However,rapamycinispoorlysolubleinwater.
The first step in making a compound was therefore the solubilization of rapamycin in an appropriate solvent prior to topicformulation.Solubilizationofthedrugintheformulationis importantbothtoallowitsdiffusionthroughtheskinandalsofor ease of application of the cream. Different ingredients were selectedtoprepareasafeformulationfortopicalapplication(listed inTable1).
Differentreportsindicatethatrapamyciniseasilyincorporated in liquid paraffin prior to topical formulation (Madke, 2013;
Balestrietal.,2015).Wethereforefirsttestedrapamycinsolubility inliquidparaffin,andobservedlessthan2mg/mlsolubility.
Wethenassesseddifferentsafeingredientsusablefortopical application. Table 1 shows rapamycin solubilityin three ingre- dients. The best candidate was Transcutol1 that solubilizes rapamycinextemporaneously,whereasthedrugwasonlyslightly soluble in other ingredients. Transcutol1, i.e. diethylene glycol monoethylether,isalreadyusedinmarketedmedicines.Further- more, Transcutol1 has been shown to be a drug penetration enhancerintheskin(Muraetal.,2000).Thispropertyappeared very interesting to exploit, as it could improve treatment of angiofibromas.Therestofthestudywasthereforeperformedwith Transcutol1.
Because Rapamycinissolubilized in asolventwhich is then incorporated in an emulsion (the cream excipient), its level of solubilityshouldbehigherthan1%(i.e10mg/ml)inordertoobtain afinalconcentrationof0.1%activedrugwhileaddinglessthan10%
of solvent in the cream, which is important to maintain the stability of a cream which is an emulsion. The solubility of rapamycininTranscutol1alloweduseofonly5%ofthisexcipient inthefinalformulationasexplainedbelow.
3.2.Formulationofrapamycincream
The cream was prepared by first solubilizing rapamycin in Transcutol1.ThismixturewasthenprogressivelyaddedtoExcipial Hydrocrème1 under manual stirring for several minutes. The formulationispresentedinTable2.Therapamycindosageinthe creamwas0.1%(w/w),whichisinthelowrangeofthepreviously publishedformulationsbutheretheactivedrugwas solubilized and a much higher levelof activity was thereforeexpected.As explained above, the high level of solubility of rapamycin in Transcutol1 would allow a dosage of 0.4% if needed without affectingtheappearanceofthecreamorphysicalstability.
Becauseofrapamycintoxicity,preparationwasperformedina low pressurizedglove-boxto protect thepharmacy technician.
Excipial Hydrocrème1 was selected fromvarious commercially availableformulationsasitisanoil-in-wateremulsioncomposed ofsafeingredients,isnon-comedogenicandnotunpleasanttouse.
Thefinalcreamhasaslightoilodour,ahomogeneousappearance and is whitein colour.Thesecharacteristicsremained constant throughoutthestudy.
3.3.Stabilitystudy
Once the cream had been prepared, a stability study was performed. The physico-chemical stability study of rapamycin requiresdrugextractionandHPLCmethodvalidationtodetermine rapamycinconcentration. Drugrecoveryafterrapamycin extrac- tionbymethanolfromthecreamwas64.2%1.2%.Thismethod didnotdetectthetotalityofrapamycininthecream.Nevertheless, suchextractionwasrepeatableandalloweddetectionofdegrada- tionproducts,asshowninFig.1b.
Table2
Compositionofrapamycincream.
Component Quantity
Rapamycin 0.03g
Transcutol1 1.5g
ExcipialHydrocrème1 QS30g
Awell-definedandsymmetricpeakofrapamycinwasobtained using the HPLC method (Fig. 1a). The correlation coefficient, (r2=0.994)indicatedthelinearityintheintervalrangeof[0.06– 0.14%]. Forced degradation was obtained by heating (Fig. 1b) method,and permittedvalidation of the method as indicating stability, as it allowed detection of degradation products at retention times between 1 and 2min. Heat was chosen as a non-specificdegradationprocessasmanydegradationpathways followArrhenius’lawandarethusgovernedbytemperature.
Repeatability, assessed at 18 determinations of the test concentration(0.1%)(6replicates/3differentdays),wassystemat- icallylowerthanacoefficientofvariationof7%.Accuracy,assessed at three concentrations (0.08–0.1 and 0.12%), on three days (9 determinations), was systematically higher than 95% of the expectedvalue.Rapamycinwaselutedin6min,asshowninFig. 1a.
Theevolutionoftherapamycinconcentrationintheprepara- tionis indicated inTable 3.The meanpercentage and the95%
confidenceintervalaroundthemeanweresystematicallyhigher than95%of theinitialconcentration,indicating thatrapamycin wasstable inthecream. Furthermore,nodegradationproducts weredetectedonchromatogramsthroughoutthestudy.
3.4.Rheologicalpropertiesofthecream
Theviscosityofinitialandformulatedcreamsdecreasedwhen theshearrateincreasedandviceversa,showingtherheofluidifiant
properties of the formulations (Fig. 2). In addition, the low standard deviations for the 3 independent formulated creams provedtherepeatabilityof theformulationprocess.Finally,the flowprofilesovertimeforthe3independentformulatedcreams remainedunchanged,showingthetimestabilityoftheformulated creams(Fig.3).Wilcoxon(forFig.2data)andKruskal-Wallis(for Fig.3data)statisticaltestsdidnotshowanysignificantdifference regardingtheformulationnature,thetimeandshearrateramp.
Suchpropertiesaresatisfactoryforpatientuse.
3.5.Microbialstabilitystudy
The rapamycincreamremainedwithin European Pharmaco- poeiaspecificationsforcontaminationthroughoutstudyafter28 and 85days when stored at 30C (Table 4). No Staphylococcus aureusor Pseudomonasaeruginosaweredetected inany case.A positive control was performed with artificially Pseudomonas aeruginosaorStaphylococcusaureus-ontaminatedcream.Microbial growth was observed, demonstrating that rapamycin did not inhibit growth. After 28days incubation, no increase of viable microorganismswasobservedascomparedtoday2.
Wealsotestedconservationmimickingpatientuse,i.e.thetube was opened everydayfor one month.Microbialcontamination assessed at days 7-14-21 and 28 was also satisfactory (below European Pharmacopoeia limits, Table 5). This result was attributed to (i) triclosan, disodium EDTA and chlorhexidine Fig.1.Examplesofrapamycinchromatogram:(a)referencecream0.1%,(b)forceddegradationbyheat.
Table3
Sirolimusconcentrationinthecreamexpressedas95%confidenceinterval(CI)(n=3)relativetoconcentrationonday0.
Time(days) 3 7 14 21 28 63 85
CI95(%comparedtoday0) 101.5–102.5 102.8–103.2 103.9–104.1 106.7–107.3 105.5–106.5 99.7–100.3 100.4–101.6
Fig.2.RheologicalcomparisonbetweenExcipialHydrocrème1andcreamcontainingrapamycin,atday0.
Legend:blueline:ExcipialHydrocrème1shearrateincrease,redline:ExcipialHydrocrème1shearratedecrease,greenline:rapamycincreamshearrateincrease,purpleline:
rapamycincreamshearratedecrease.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
Fig.3.Rheologicalpropertiesofcreamcontainingrapamycinovertime.
Legend:Day0:Darkpurpleline:shearrateincrease.Lightpurpleline:shearratedecrease.Day28:Darkorangeline:shearrateincrease.Lightorangeline:shearratedecrease.
Day85:Darkblueline:shearrateincrease.Lightblueline:shearratedecrease.
Table4
Microbialcontaminationofcreamduringstorage.CFU/g=Colonyformingunits/
gramofcream.Expressedasaverageof3replicatesforeachday.
Day 0 28 85
NumberofCFU/g 16.711.8 11.4 00
Pseudomonasaeruginosa none none none
Staphylococcusaureus none none none
Table5
Microbialcontaminationofcreaminpatientusecondition.UFC/g=Colonyforming units/gramofcream.Expressedasaverageof3replicatesforeachday.
Day 7 14 21 28
NumberofCFU/g 4.23.1 11.4 00 11.4
Pseudomonasaeruginosa none none none none
Staphylococcusaureus none none none none
dihydrochloride, which are preservatives present in Excipial Hydrocrème1and(ii)appropriatepackaginginaluminumtubes, limitingcontactwithcontamination.
4.Conclusion
We present forthefirst time aformulation withsolubilized rapamycin.Todate,publicationshaveonlyreportedformulations madefromcrushedtabletsororalsolutionsofrapamycin,which havepresentedmajordrawbacks:(i)whenusingtabletsthedrugis partiallyorevennotsolubilizedinthetopicalformulation,anditis thusless(orevennot)bioavailabletoexertitseffect.Furthermore, skindamage has beenreported due to inadequatecrushing of tablets.(ii) When using oral formulations, oral ingredients are presentinthetopicalcompositionand arelikelytocauseside- effects.
Incorporation ofrapamycinandhomogeneousdistributionin the topical formulation are more effective if the rapamycin is previouslysolubilizedina solvent.Moreover,skindiffusionand drug bioavailability will be enhanced. Our best results were obtained with Transcutol1 (highly purified diethylene glycol monoethyl ether) commonly used as an ingredient in topical formulations.Transcutol1is alsoanexcellentpermeationagent thatenhancesdrugdiffusionthroughtheskin(Muraetal.,2000).
Absolutegaleniccontrolofourpreparationwillpermitthebest drugdistribution,willincreasebioavailabilityandefficiency,will reducethenecessaryconcentrationofrapamycinanditscost.
Wereporthereforthefirsttimeastabilitystudyofatopical formulationcontainingrapamycin.Thephysico-chemicalstability ofthecreamwas85days.Nodegradationproductsweredetected during this time. Microbiological stability studies showed that patientscan usethe creamwithout risk of infection. Viscosity measurementsarerecommendedintheEuropeanpharmacopoeia.
The experiments revealed that incorporating rapamycin and Transcutol1inthecreamdidnotchangeitsrheologicalproperties.
Furthermore,theorganolepticcharacteristics(odour,appearance andcolour)wereunaffected.
Suchapreparationwarrantsclinicalevaluation,withtheaims of optimizing dosage and proposinga long term maintenance schemetoavoidrecurrenceofcutaneousmanifestationsofTSC.
The0.1%rapamycincreamhasbeenusedforseveralmonthsbyTSC patientsinourhospital.Theefficacyandtolerancewillbereported later.
Conflictofinterest
The authorsconfirmthey have noconflictsof interest with regardtothecontentofthisarticle.
Acknowledgments
TheauthorswouldliketothankChristineTruffautandThomas Briotfortheirhelp.
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