Incorporation Into Lipid Nanoparticles Extends the ...
Transcript of Incorporation Into Lipid Nanoparticles Extends the ...
Incorporation Into Lipid Nanoparticles Extends the Duration of Activity of Treprostinil in an Acute Hypoxia Rat Model of Pulmonary Arterial Hypertension
D Omiatek1, F Leifer1, V Malinin1, J Ong1, T Henn1, Z Li1, RW Chapman1, D Salvail2, CE Laurent2, WR Perkins1 1Insmed, Inc., Bridgewater, NJ, USA
2IPS Therapeutique Inc., Sherbrooke, QC, Canada
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LNPs effectively retained TRE to provide sustained release
In vitro bioavailability of TRE was slowed for TRE in LNPs
Figure 4. TRE-LNP drug release screened by dialysis
LNPs, lipid nanoparticles; TRE, treprostinil.
Figure 5. TRE-LNP efficacy screened by cAMP assay on CHO-K1 cells
Concentration of cAMP data was 1 µM TRE for each sample. cAMP, cyclic adenosine monophosphate; CHO-K1, Chinese hamster ovary; LNP, lipid nanoparticle; TRE, treprostinil; TRE-LNP, treprostinil lipid nanoparticle.
In Vivo TRE-LNP Characterisation Results
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Inhaled TRE-LNPs extend vasodilatory effect as compared with TRE
Inclusion into LNP extends blood plasma levels of inhaled TRE
Figure 6. TRE-LNP efficacy screened in a rat model of pulmonary arterial hypertensionmPAP, mean pulmonary arterial pressure; PBS, phosphate-buffered saline, TRE, treprostinil; TRE-LNP, treprostinil lipid nanoparticle.
Figure 7. TRE-LNP pharmacokinetic profile in a rat model of pulmonary arterial hypertension
TRE, treprostinil; TRE-LNP, treprostinil lipid nanoparticle.
Figure 3. Manufacturing conditions of flow rate ratio (FRR) (A) and total flow rate (B) affect treprostinil–lipid nanoparticle (TRE-LNP) particle size
TREPROSTINIL
CATIONIC LIPID
PHOSPHOLIPID
HYDROPHOBICFILLER
PEGYLATEDLIPID
Figure 1. (A) Chemical structure of treprostinil (TRE) and (B) schematic representation of treprostinil lipid nanoparticles (TRE-LNPs)
INTRODUCTION• Treprostinil(TRE)isaprostacyclinanalogueusedtotreatpulmonaryarterialhypertension(PAH).• Inthisstudy,wedesignedaTREdeliverysystembasedonthedevelopmentofalipidnanoparticle(LNP)suspensionforencapsulationandsustainedreleaseofthedrug.
• Uniform,nanoscaletreprostinil–lipidnanoparticles(TRE-LNPs)(Figure 1)werecomposedof(i)TRE,(ii)acationiclipidtoamalgamatewiththelipophilicandcomplementarynegativelychargedterminusoftheTREmolecule,(iii)apolymericcoatingtostabiliseparticlesandenhancetheirbioavailability,(iv)ahydrophobic“filler”moleculetostabilisethecoreoftheparticle,and(v)anominalconcentrationofphospholipidtoaffixthepegylatedlipidtotheLNPcomposite.
AIMS• TodesignaninhalableTREformulationforthetreatmentofPAHthatwouldhaveanimprovedpharmacokinetic(PK)profilerelativetothecurrentinhaledTREtherapyforPAH,Tyvaso®,tofacilitateaonce-dailydosingschedule
–Toachievethis,wedevelopedabioavailableTREvehiclecarrierwithanoptimiseddrugpayload andreleaseprofiletosupportasustainedvasodilatoryresponserelativetofreeTRE.
Nanoparticle Formation at Miscible Fluid Interface
Lipid/ Organic Solvent Inlet
NanoparticleOutlet
1 mm
25 nm
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Figure 2. Manufacture of treprostinil–lipid nanoparticles (TRE-LNPs) by solvent flash precipitation
METHODSTRE-LNP Production• Solventflashprecipitationviamicroscaleflowfocusingwasusedfortheone-step,continuousflowsynthesisofuniformnanoscaleTRE-LNPs(Figure 2).
• Inthisprocess,acenterstreamofalcohol-solvatedTREandlipidisimpingedwithaqueousstreamspositionedperpendiculartotheTREstream.
–Astheaqueousstreamsmeetwithandlaterallyfocusthemiscible-solvatedlipidstream,theorganicandaqueousphasesinterdiffuse,producingasolventcompositioninwhichthelipidsareincreasinglylesssoluble.
–Thiscausesthelipidstoself-associateintointermediateassembliesthateventuallycloseonthemselvesintosphericalnanoscaleLNPs.
In Vitro TRE-LNP Characterisation Methods • Releasekineticsscreenedbydialysis –50kDamolecularweightcutoffforcelluloseacetatemembrane –InitialconcentrationofTREwas100μM –1mLofsamplewasdialysedagainst1Lof1xphosphate-bufferedsaline(PBS)for24hours.
• Efficacyscreenedinamammaliancellassay(foracompletedescriptionofthesemethods,seeposterbyChenK-J,etal,ERSPoster#2358)
–Chinesehamsterovary(CHO-K1)cells(ATCC®CCL-61)weretransientlyco-transfectedwiththepGloSensorTM-22FcAMPplasmid(PromegaCorporation,Madison,WI)andtheprostanoidreceptorEP2-plasmid.
–TransfectedcellswerethentreatedwithTREanddifferentformulationsofTRE-LNPs. –Cyclicadenosinemonophosphate(cAMP)levelsweremeasuredevery5minutesforthedura-
tionofthestudies,andtheincreaseinactivationrelativetothecontrolsampleswascalculated.
LNP Size Controlled Via Process Variables• Bycontrollingtheflowrateratio(FRR)oftheaqueous:alcohol-solvateddrugstreamsandthetotal flowrateofthesystem,theTRE-LNPparticlesizecanbefine-tuned(Figure 3). –Particlesizeisknowntoplayaroleinformulationbioavailability.
In Vivo TRE-LNP Characterisation Methods• RatmodelofPAH –MaleSpragueDawleyratswereanaesthetised,artificiallyventilatedandpreparedformeasure-
mentofmeanpulmonaryarterialpressure(mPAP),meansystemicbloodpressure(mSAP),heartrate(HR),andarterialoxygensaturation(SaO2).
–Physiologicparametersweremeasuredduringnormoxia(fractionofinspiredoxygen[FiO2]=0.21,SaO2≈90%)andfor2to3hoursduringhypoxia(FiO2=0.10,SaO2≈50%).
–BloodsamplesweretakenovertimeandlungtissueharvestedattheendofthestudytomeasureTREconcentration(HPLC/MS/MSanalysis).
–CompoundsweredeliveredviaAeroneb®(Aerogen,Galway,Ireland)nebuliserinterposedinaventilatorcircuit(seeposterbyMalininetal,ERSPoster#2367)atanestimatedpulmonarydoseaof2µg/kg.
aMeasuredfromTREconcentrationinlungimmediatelypostdose.
RESULTSIn Vitro TRE-LNP Characterisation Results
CONCLUSIONS• Inthepresentstudy,wesoughttodevelopaninhaledTRE-LNPformulationforthetreatmentofPAHtoimprovethedurationofthetherapeuticbenefitandthetolerabilityofTRE.OptimisationofTRE-LNPformulationwasbasedonparticlesizeandTREreleasekinetics.ActivitywasassessedinvitroinCHO-K1cellsusingapGloSensorassayandinvivoinanacutehypoxiaratmodelofPAH.
• AgradualincreaseincAMPactivationofCHO-K1cellssuggestedaslowed-releaseprofileoftheTREnanoparticleformulationrelativetothefreedrug.
• Inthehypoxicratmodel,thepulmonaryvasodilatoryactivityofinhaledTRE-containingLNPswasextendedbeyondthatofinhaledTREinsolution,whichisconsistentwithanextendedPKprofileofthedrugobservedinexcisedbloodplasma.
• PackagingTREintoananoparticleformulationincreaseddurationofthevasodilatoryeffectrelativetothefreedrug,butisunlikelytofacilitateaonce-dailydosingschedulebasedonthepharmacokineticprofileobserved.
• TofurtherimprovenanoparticleretentionofTRE,wedevelopedaderivatisedTREprodrugmadebycovalentattachmentofalkylchains(seeposterbyLeiferetal,ERSPoster#2356).WebelievethatthisapproachwillresultinasustainedvasodilatoryresponsewellbeyondthatobservedwithTRE-LNPsandfreedrug.
Please see other posters in this series:• Leifer F, et al, ERS Poster #2356 • Chen K-J, et al, ERS Poster #2358 • Malinin V, et al, ERS Poster #2367
ACKNOWLEDGEMENTSTheauthorswouldliketoacknowledgeConnexionHealthcare(Newtown,PA)forprovidingeditorial,layout,anddesignsupport.Insmed,Inc.(Bridgewater,NJ)providedfundingtoConnexionHealthcarefortheseservices.
Poster presented at the European Respiratory Society (ERS) International Congress, 6-10 September, 2014, Munich, Germany.
Table 1. TRE-LNP Formulation Characteristics
Formulation
Mole Percent of TRE-LNP
Particle Size (nm)TRE Cationic Lipid
Hydrophobic Filler Phospholipid
Pegylated Lipid
TRE-LNP-1 15 30 35 0 10 47
TRE-LNP-2 5 15 60 10 10 59
TRE-LNP-3 2 10 68 10 10 45
TRE TRE-LNPsA B
TRE-LNP, treprostinil lipid nanoparticle.
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POSTER #: 2357