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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FRR Aqueous:Drug/Lipid Streams Total Flow Rate (mL/min)

POSTER #: 2357