Rajivgandhiuniversityofhealthsciences,Karnataka

FORMULATIONANDEVALUATIONOFNANO PARTICLESCONTAINING RAMIPRIL

M. PharmDissertationProtocolSubmittedto

RajivGandhiUniversityofHealthSciences,Karnataka

Bangalore– 560041

Ms. M.SHIVALEELAB.Pharm

UndertheGuidanceof

Mr.VenkateshD.PM.Pharm,(Ph.D)

Asst.Professor

Department of Pharmaceutics

AcharyaB.M.ReddyCollegeofPharmacy,Soldevanahalli,Chikkabanavara(Post)HesaraghattaMainRoad,Bangalore–560090.

2011-2013

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FORREGISTRATIONOFSUBJECTSFORDISSERTATION

1. / NameoftheCandidate andAddress / Ms.M.SHIVALEELA
#5-198
Nalgonda(Dist)
Matampally(mandal) Pedaveedu(Post), Andhrapradesh.
Andhrapradesh
2. / NameoftheInstitution / ACHARYAB.M. REDDYCOLLEGEOF PHARMACY,
Soldevanahalli,HesaraghattaMainRoad, Chikkabanavara Post.
Bangalore-560090
3. / CourseofStudyandSubject / M.Pharm
(Pharmaceutics)
4. / DateofAdmission / 14-12-2011
5. TITLE OF THEPROJECT:-
FORMULATION ANDEVALUATIONOFNANOPARTICLES CONTAININGRAMIPRIL

6. BRIEF RESUME OF INTENDED WORK:

6.1 NEED FOR THE STUDY:

Nanoparticlesaresolidcolloidalparticlesrangingfrom(1to1000nm)insize;they consistofmacromolecularmaterialsinwhichtheactiveingredients(drugorbiologicallyactive material)is dissolved,entrapped, orencapsulated,or adsorbed1.

Advantages ofnanoparticles:

Nanoparticledrugcarriers havehigherstabilities.

Nanoparticles havehighercarriercapacity.

Feasibilityofincorporationofbothhydrophilicandhydrophobicsubstances.

Feasibilityofvariable routesofadministration.

Nanoparticles are biodegradable, non-toxic and capable of being stored for longer periods.

Nanoparticles canalsobeusedforcontrolleddeliveryofdrugs.

Nanoparticles reducedosingfrequencyandhavehigherbioavailability.

Noveldrugdeliverysystemsgiveanopportunityforformulationscientiststoovercome the manychallengesassociated with Antihypertensivedrug therapy, thereby improving the managementofpatients withhypertension.

CurrentlyavailableAntihypertensivedrugscanbeclassifiedintothesecategories:ACE- inhibitors,angio-tensinantagonist,calciumchannelblocker,diuretics,centralsympathomimetics alpha-adrenergicblocker, vasodilator,betaadrenergicblocker.Mostofthedrugsbearsome significantdrawbackssuchasrelatively shorthalf-life,lowbioavailability, Poorpermeability

andundesirableeffects.2

Ramiprilisanangiotensin converting enzyme inhibitordrugusedmainlytotreathighbloodpressure(hypertension). Ramipril is a potent, competitive inhibitor of ACE,the enzyme responsible for the conversion of angiotensin I(AT-I)to angiotensin II (AT-II). AT-II regulates blood pressure and is a key component of the renin-angiotensin-aldosterone system(RASS). Ramipril may be used in the treatment of hypertension,congestive heart failure,nephropathy,and to reduce the rate of death. Ramipriladministrationresultsinadecreaseintotalperipheral resistance(afterload)andcardiacvenousreturn (preload)Allofthephysiologicaleffectsof angiotensinII,includingstimulationofreleaseofaldosterone,areantagonizedinthepresenceof Ramipril. Reduction in blood pressure occurs independently of the status of the renin- angiotensinsystem.

Ramipril is highly lipophilic poorly water soluble drug with absolute bioavailability of 28-35%. The half life of Ramipril is 2 to 4 h. Ramipril is well absorbed following oral administration and undergoes significant first-pass metabolism3.

Therefore we plan to formulate nanoparticles containing Ramipril to increase bioavailability and to reduce dosing frequency.

6.2REVIEWOF LITERATURE:

JawaharNand other coworkerspreparedPLGAnanoparticlesofcarvedilolthatwillimprovethe bioavailabilityofcarvedilolandsustainedreleasetoreducetheinitialhypotensivepeak andtoprolongtheantihypertensiveeffectofthedrug.Nanoparticleswereexaminedfor physicochemicalcharacteristicssuchasparticlessize,shape,evaluationparameterssuch as drug entrapment efficiency, drug loading capacity, drug and polymer interaction studiesin-vitroreleasekineticsstudieswerestudied.In-vitrocumulative releasefromthenanoparticles wasfoundtobe72%at24h1.

SaikatDetal.,formulatedaspirinloadedalbuminnanoparticlesbycoacervationmethod.

PhotoncorrelationspectroscopyandtransmissionElectronMicroscopyofthesamples were done to characterize the nanoparticles. itisconcludedthatalbumin nanoparticlesbycoacervationmethodiswell suitedandthepreparativevariablesoftheprocedureis finetuneddependingonthe clinicalapplicationsuchasintra-articulartherapyinarthritisorintraocularreleaseagentfordiabetic retinopathy2.

BenedictoADet al.,formulatedcubicnanoparticlesofglycerylmonooleate (GMO)/poloxamer407) as potential oral drug deliverysystems toenhancethe bioavailabilityofthewater-insolublemodeldrugsimvastatin. Thesimvastatin-loaded cubicnanoparticleswerepreparedthroughfragmentationoftheGMO/poloxamer407bulk cubic-phasegelusing highpressurehomogenization.Theinternalstructureofthecubic nanoparticles was identified by cryo-transmission electron microscopy. Pharmacokineticprofilesbeagledogs showedsustainedplasmalevelsofsimvastatincubicnanoparticlescalculatedonthebasis of area under the curve was 241% compared to simvastatin crystal powder. The enhancement of simvastatin bioavailability was possibly attributable to facilitated absorptionbylipidsintheformulationratherthanimprovedrelease.3
KharRKand other coworkerspreparedcyclosporine(cya)loadedEudragitRL100nanoparticlesusingsolventevaporationtechniquewith2%PVAasstabilizer.Fourbatchesofnanoparticles withvarying drugconcentrationCYN-1,CYN-2,CYN-3andCYN-4wereprepared.Theinvivoresultof formulationCYN-2revealedthatthedrugloadednanoparticles showedpreferentialdrugtargetingtoliverfollowedbyspleen,lungs andkidney.Stability studiesshowedthatmaximumdrugcontentandclosestinvitroreleasetoinitialdatawas foundinthesample (formulationCYN-2)storedat40C.4

DoraCPetal.,formulatednanoparticles(NPs)Glibenclamide(GB)usingEudragitL100.TheNPswerepreparedbysolventdisplacementmethod.The NPs were evaluated for particle size, size distribution, zeta potential, drug loading and encapsulation efficiency. Encapsulation efficiencyanddrugloadingcapacitywerefoundtobeincreasedasdrug concentration increaseswithrespecttopolymer.DissolutionstudyrevealedincreasedreleaseofGBfrom NPs.TEMstudyrevealedsphericalmorphologyof thedevelopedNPs. DevelopedNPsrevealedadecreasedtminand enhancedbioavailability.ThedevelopedNPscouldreducedosefrequency,decreasesideeffectsandimprovepatientcompliance5.

Tamizhrasi Sand other coresearchers preparedandevaluatedpolymethacrylicacidnanoparticles containing lamivudineindifferentdrugtopolymerratiobynanoprecipitationmethod.No appreciable differencewasobservedintheextentofdegradationofproductduring60daysinwhich, nanoparticleswerestoredatvarioustemperatures. Thein-vitro releasebehaviorfromallthedrug loaded batcheswas found to bezero order and providedsustainedreleaseoveraperiodof24h.Thedevelopedformulationovercomeand alleviates thedrawbacksandlimitationsoflamivudinesustainedreleaseformulationsand couldpossibilitybeadvantageousinterms ofincreasedbioavailabilityoflamivudine6.

Gaikwad A et al., formulated furosemide nanoparticlesto reducedosing frequency.

Polymericnanoparticleshavereceivedmoreattentionforpreparingsustaineddosage formsbecause of theirinertness,solubilityinrelativelynon-toxicsolvent.Frusemide loaded EudragitRS100nanoparticles werepreparedbynanoprecipitationmethodfororal delivery. Formulation were prepared in different ratios of drug and carrier, and characterizedforparticlesize,shape,percentageyield,drugentrapment,stabilitystudies, zetapotential, FT-IRstudy, in-vitro drugreleaseandrelease kinetics.Thein-vitrodrugreleasestudyrevealedthat sustainedreleaseofsomeformulationlastupto24 h7.

LiuDetal.,intheirworkreportedthatthepreparationofsolidlipidnanoparticles (SLNs)suffersfromthedrawbackofPoorincorporationofwater-solubledrugs.Theaim ofthisstudywasto assessvarious formulation andprocessparametersto enhancetheincorporationofawatersoluble drug (diclofenacsodium,DS)intoSLNs prepared by the emulsion/solvent evaporation method. Results showed that the entrapmentefficiency(EE)ofDSwasincreasedtoapproximately100%byloweringthe pHofdispersedphase.Stabilizersandtheir combinationwithPEG400inthedispersedphasealsoresultedinhigherEEanddrug loading(DL).EEincreasedand DLdecreased asthephospholipids/DSratio became greater,whiletheamountofDShadanoppositeeffect.Ethanolturnedouttobetheideal solvent makingDS-SLNs.According to the investigations,drugsolubilityindispersionmediumplayedthemostimportantroleinimproving EE8.

VandanaSinghand other co-researcherspreparedRosiglitazoneloadedgelatinnanoparticlesbytwostep desolvationmethod.Thenanoparticleswerecharacterizedforvariousparameters.ThereleasepatternfollowedtheKorsmeyer Peppas equation. Gelatin nanoparticles also exhibited excellent redispersibility and minimalincreaseinparticlesize.Theresultsindicatedthattwostepdesolvationmethod iswellsuitedtopreparegelatinnanoparticlesandtheprocessvariablesoftheprocedure canbefinetuneddependingontheclinicalapplications9.

HyvonenSetal.,intheirworkreportedthatamodifiednanoprecipitation(interfacial

polymer deposition following solvent displacement) method was used to produce nanoparticles from low molecular weight poly (l-lactic acid). Model drugs, eithersalbutamolsulphateorbeclomethasonedipropionate,wereencapsulatedintheparticles. Theinfluenceofthepreparationmethodonthephysicochemicalstatesofthepolymer randthedrugsaswellasonthedrug–polymer interactionswerestudiedbyelectron microscopy, X-ray diffractometry, thermal analysis and infrared spectroscopy. Although changesinthe crystallinityofthepolymerandthemodeldrugswereseen,noclear interactionsbetweenthepolymerandthedrugweredetected10.

DeviKVetal.,intheirworkreportedthatAcyclovirnanoparticleswerepreparedby solvent deposition method and optimized using 3² full factorial design. From the preliminarytrials,theconstraintsforindependentvariablesX1(amountofPLGA)and X2(amountofPluronicF68)have beenfixed.Thederivedpolynomialequationsfor Particlesizeand%drugentrapment wereverified bycheckpoint formulation. The prepared formulationswerefurtherevaluated fordrugcontent,in vitro drugrelease pattern, and short-term stability and drug-excipient interactions. Thereleasewas foundto followfirstorderreleasekineticswith fickiandiffusionmechanismforall batches.Thesepreliminaryresultsindicatethatacyclovirloaded PLGAnanoparticlescouldbeeffectiveinsustainingdrugreleaseforaprolongedperiod11.

Sivabalan M et al., formulated and evaluated 5-Fluorouracil loaded Chitosan and Eudragit Nanoparticles for Cancer therapy. The goal of the investigation was to formulateandevaluatechitosanandEudragitnanoparticlesof5-Fluorouracilforcancertherapy.Nanoparticlesof5-Fluorouracilwerepreparedusingchitosan,EudragitS100, liquid paraffin and Tween -20 using Emulsion droplet coalescence method. The nanoparticles prepared were evaluated for morphology, loading efficiency, in vitro releaseandinvitroanticanceractivities. In-vitro anticancer study revealed that the formulated nanoparticles were found to have good cidalactivityoncancer cells insustainedmanner.12

Park J and other co-researcher reported that the efficacy of doxorubicin (DOX) can be maximized and dose-limiting cardiotoxicity minimized by controlled release from PEGylated nanoparticles. To test this hypothesis, a unique surface modification technique was used to create PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating DOX. An avidin-biotin coupling system was used to control poly(ethylene glycol) conjugation to the surface of PLGA nanoparticles, of diameter ~130 nm, loaded with DOX to 5% (wt/wt). Administration of 18 mg/kg of free DOX induced a sevenfold increase in CPK levels and significant decreases in left ventricular fractional shortening over control animals, whereas nanoparticles-encapsulated DOX produced none of these pathological changes.13

6.3OBJECTIVE OF THE STUDY:-

Themainobjectiveofthepresentworkistoformulateanddevelopthesustainedrelease nanoparticles ofantihypertensivedrugRamipril byusingdifferentpolymers.

Theplanofworkis:

1. IdentificationandconfirmationforpurityofdrugbyUV/HPLC

2. Preformulationstudies.

CharacterizationofdrugandpolymersbyFTIR/DSCstudies

3. Formulation of nanoparticles of Ramipril

4. Evaluationparameters

Particle size analysis and shape morphology
Entrapment efficiency
Content uniformity
Zeta potential

5. To carry out in-vitro dissolution studies of nanoparticles formulations.

6. To carryout Stabilitystudies as perICHguidelines.

7.0MATERIALSANDMETHODS:

7.1SOURCE OF DATA:-

Reviewofliteraturefrom:

a. Journals–suchas

JournalofPharmaceutical Research

InternationalJournalofPharmaTechResearch

EuropeanJournalofPharmaceutics andBiopharmaceutics

JournalofNanoparticleResearch

InternationalJournalofPharmaceuticalSciencesandResearch

InternationalJournalofPharmaceutics

b.

c. World WideWeb .

d. Standardbooks fromourlibraryAcharyaandBM Reddycollegeofpharmacy e.J-Gate@Helinet

7.2 MATERIALS ANDMETHODS:

MATERIALS:

Drug: Ramipril

Polymer:PEG, Chitosan,EudragitRL andEudragitRS100etc

Solvents:methanol,ethanol,n-hexane,chloroform,n-butanol,acetone,wateretc.

Allotherchemicals andreagentwillbeofanalyticalgrade.

METHOD OF COLLECTION OF DATA:

METHODS

1. Preformulation studies

Authentification of drug:

Solubility studies
Melting point determination
Identification and purity of drug by UV/HPLC.
Drug and polymer interaction studies by FTIR.

2.Preparationofnanoparticles willbeconductedbyfollowingmethods.

 Homogenizationmethod/Precipitationmethod6.

 Ionicgelationmethod

 Modifiedsolventinjectionmethod

 Theresultant productisfreezedriedusing lyophilizerand finalproductthus obtainedwillbesubjectedtoevaluationparameter7.

3.Evaluationparameters

 Percentageyieldandcontentassay8.

 Entrapment efficiency9.

 Content uniformity10.

 Particle size and shape morphology by Scanning Electron Microscopy.

 Zeta potential by using zeta sizer11.

4.To carry out in-vitro dissolution study of Nanoparticle formulations12.

5.Stabilitystudies as perICHguidelines.

7.3DOESTHESTUDYREQUIRESANYINVESTIGATIONORINVESTIGATIONS TOBECONDUCTEDONPATIENT OROTHERHUMANSORANIMALS?

“NO”

7.4HASETHICALCLEARANCEBEENOBTAINEDFROMYOURINSTITUTIONIN CASE OF 7.3?

“NOT APPLICABLE”

8.0 REFERENCES:

1. Jawahar N, Nagasamy VD, Sureshkumar R, Senthil V, Ganesh GNK, Vinodh P.

DevelopmentandcharacterizationofPLGA-nanoparticlescontainingcarvedilol.JPharm

SciRes 2009;1(3):123-8.

2. SaikatD,RintiB,JayeshB.AspirinLoadedAlbuminNanoparticlesbyCoacervation: ImplicationsinDrugDelivery.Trends BiomaterArtifOrgans2005;18(2):203-11.

3. BenedictoAD,O’BrienDF.Bicontinouscubicmorphologiesinblockcopolymersand amphiphile water systems: mathematical description through the minimal surfaces. Macromol1997;30(11):3395-402.

4. Khar RK,andDiwanM.TargeteddeliveryofdrugsEuroJ PharmBiopharm2006;

72:254-8.

5.DoraCP,SinghSK,kumarS,AshokKD,AakashD.Developmentandcharacterizationof nanoparticles of glibenclamide by solvent displacement method. Acta poloniae Pharmaceutica-DrugRes 2010;67(3):283-90.

6. TamizhrasiS,ShuklaA,ShivkumarT,RathiV,RathiJC. Formulationandevaluationof lamivudine loaded polymethacrylicacidnanoparticles.IntJPharmaTechRes2009July- Sept;1(3):411-5.

7. Gaikwad A, Tamizhrasi S, Sorti A, Gavali P, Gajanan MH. Formulation and In vitro characterizationofpolymethacrylicacidnanoparticlecontainingfrusemide.IntJPharmTech Res 2010;2(1):300-04.

8. LiuD,JiangS,ShenH,QinS,LiuJ,ZhangQ, Li R, Xu Qw.Diclofenacsodium-loadedsolidlipid nanoparticlespreparedbyemulsion/solventevaporationmethod. JNanoparticleRes2011; 13(6):2375-86.

9. Singh Vand Chaudhary AK. Development and characterization ofrosiglitazone loadedgelatinnanoparticlesusingtwostepdesolvationmethod.IntJ PharmSciReviewRes

2010;5(1):100-03.

10.HyvonenS,PeltonenL,KarjalainenM,HirvonenJ.Effectofnanoprecipitationonthe physicochemicalpropertiesflowmolecularweightpoly(l-lacticacid)nanoparticlesloaded withsalbutamolsulphateandbeclomethasonedipropionate.IntJ Pharm2005;(1-2)295:269-81.

11.Devi KV,BhosaleUV.Formulationandoptimizationofpolymericnanodrugdelivery systemofacyclovirusing3²Fullfactorialdesign.IntJ PharmTechRes 2009;1(3):644-53.

12.SivabalanM, Shering A, Phaneendhar Reddy, Vasudevaiah, Anup Jose, G Nigila. “Formulationandevaluationof5-fluorouracilloadedchitosanand eudragitnanoparticlesforcancertherapy”.Int.J.ComprehensivePharmacy 2011;2(1):1-4.

13. Park J, Fong PM, Lu J, Russel KS, Booth CJ, Saltzman WM PEGylated PLGA nanoparticles for the improved delivery of doxorubicin. Int J Nanomedicine.2009Dec;5(4):410-18.

14. ICH guidelines [online][cited on 20/06/2012]availablefrom

ICH guidelines online sited on

9 / Signatureofthecandidate:
10 / Remarks oftheGuide: / This topic selected for dissertation is satisfactory.
Adequate equipments and chemicals are available
to carry out the project work.
11 / NameandDesignationof:
11.1 / InstitutionalGuide: / Mr.VenkateshD.PM.Pharm(Ph.D)
Asst. Professor.
Dept. ofPharmaceutics
11.2 / Signature:
11.3 / Co-Guide:
11.4 / Signature:
11.5 / HeadoftheDepartment: / Dr.SHIVANANDKALYANAPPAM.pharm,Ph.D
ProfessorandH.O.D
Dept.ofPharmaceutics
r
11.6 / Signature
12 / 12.1 / Remarks ofthe Principal
12.2 / Signature /

Dr. Divakar GoliM.Pharm, Ph.D

Principal

Acharya & B.M. Reddy College of Pharmacy,
Bangalore-90.