Poster: PA2485

NP-UK-00074

INTRODUCTION• Treprostinil (TRE) is a pulmonary vasodilator for the treatment of pulmonary arterial hypertension (PAH) with

delivery by the subcutaneous, intravenous, oral, and inhaled routes of administration. It is not certain to what extent the pulmonary vasodilation produced by inhaled TRE is due to systemic vs local activity. A component of local activity has been previously suggested.1

• INS1009 is hexadecyl-treprostinil (C16TR) that is formulated in a lipid nanoparticle for inhaled delivery as a long-acting pulmonary vasodilator (Figure 1). C16TR is a prodrug of TRE.

• In anesthetized rats, pulmonary vasodilation was more prolonged with INS1009 compared with TRE with activity up to 3 hours, the maximum duration for this model.2

AIMS• To assess whether a single inhaled dose of INS1009 in rats has pulmonary vasodilator activity up to 24 hours.• To investigate whether local activity within the lung contributes to the prolonged pulmonary vasodilation

with inhaled INS1009.

C16TR Chemical Formula: C39H66O5

Molecular weight: 614.95

TRE Chemical Formula: C23H34O5

Molecular weight: 390.52

Figure 1. INS1009 (lipid nanoparticle C16TR), and chemical formula and molecular weight for C16TR and treprostinil (TRE).

METHODSAerosol Delivery• Male Wistar Han rats were exposed to INS1009 at pulmonary doses of 0.57, 2.84, and 28.4 µg/kg (0.36, 1.8, and 18 µg/kg

TRE equivalent) or phosphate buffered saline (PBS) using an Aeroneb® Pro Nebulizer (Aerogen, Galway, Ireland) delivered through a 12-port nose-only exposure system (CH Technologies, Westwood, NJ, USA) (Figure 2).

• At times of immediate post dose (IPD), 6, 12, and 24 hours after dosing, pulmonary and systemic vascular hemodynamics were measured in response to a challenge with the thromboxane mimetic U46619 (see Vascular Hemodynamics). At the end of the study, lungs were excised and TRE and C16TR lung levels as well as TRE plasma levels were assayed by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS-MS).

• The mass median aerodynamic diameter (MMAD) of the aerosol droplet size was measured at the port of chamber using a Marple Impactor at 2 L/min sampling fl ow rate.

Inhaled INS1009 Demonstrates Localized Pulmonary VasodilationZhili Li1, Richard W. Chapman1, Vladimir S. Malinin1, Michel R. Corboz1, Adam J. Plaunt1, Charles E. Laurent2, Han Yin2, Dany Salvail2, Walter R. Perkins1

1Insmed Incorporated, Bridgewater, NJ, USA; 2IPS Therapeutique Inc., Sherbrooke, Canada.

Poster presented at the European Respiratory Society (ERS) International Congress 2016, 3-7 September, London, UK.

Exhaust (Upper swivel)

Trumpet Connector cone

Outer plenum

Inner plenumFresh Aerosol

Exhaled BreathTest article inlet (Bottom swivel)

Figure 2. Nose-only exposure system.

Intravenous Infusion• For intravenous (IV) studies, TRE was infused at a rate between 2.5 and 1250 ng/kg/min to anesthetized rats

in order to achieve target plasma TRE concentrations between 0.1 and 50 ng/mL. TRE was infused for the duration of the study, which involved infusion at 30 minutes before and during the assessment of pulmonary vascular hemodynamics in response to U46619 challenge (see Vascular Hemodynamics). At the end of the study the TRE plasma levels were assayed by HPLC/MS-MS.

Vascular Hemodynamics• Pulmonary arterial pressure (PAP), systemic arterial blood pressure, and heart rate were measured in anesthetized,

ventilated rats before (baseline) and after intra-arterial injection of the thromboxane mimetic U46619.3 Each rat was given 3 doses of U46619 (2.5, 5, and 10 µg/kg); each U46619 dose was infused over a 20-second period, with each dose separated by a 30-minute interval. Pulmonary vascular responses to U46619 were reported as the % increase in PAP from baseline.

RESULTS• Aerosol Droplet Size Distribution of INS1009

– The average MMAD of the aerosol delivered to the nose port was 2.36 ± 0.16 µm. Data are captured in Table 1, Figure 3, and Table 2.

• Vascular Hemodynamics With U46619 – U46619 produced a dose-dependent (2.5, 5, and 10 µg/kg) increase in PAP (Figure 4A). – INS1009 inhibited the U46619-induced increase in PAP (Figure 4B).

• Aerosol INS1009 – INS1009 (0.57 and 2.84 µg/kg) inhibited the U46619-induced increase in PAP up to 12 hours with a nonsignifi cant

trend at 24 hours (Figure 5). – At the highest dose (28.4 µg/kg), INS1009 inhibited the U46619-induced increase in PAP up to 24 hours (Figure 5). – INS1009 produced a dose-dependent increase in plasma TRE and C16TR in the lungs (Figures 6 and 7).

• Intravenous TRE – Higher TRE plasma concentrations (≈ 90x) were required to inhibit the U46619-induced increase in PAP with IV

TRE compared with inhaled INS1009 (Figure 8).

Table 1. Percent of C16TR Deposited on Each Stage of Marple Impactor4

Stage Run 1 Run 2 Run 3 Average SD

1 0.1 0.2 0.2 0.2 0.0

2 0.1 0.3 0.2 0.2 0.1

3 0.6 1.7 1.3 1.2 0.6

4 3.1 13.7 7.3 8.1 5.3

5 25.5 21.9 22.1 23.2 2.0

6 43.3 43.9 48.8 45.3 3.0

7 19.6 9.6 11.5 13.6 5.3

8 4.6 5.8 5.6 5.3 0.6

Filter 3.1 2.9 2.9 2.9 0.1

SD, standard deviation.

1 2 3 4 5 6 7 8 Filter0

10

20

30

40

50

C16

TR%

Impactor Stage

C16TR%

Figure 3. Aerosol droplet size distribution of C16TR after nebulization of INS1009 measured at a port of aerosol chamber.4

C16TR, hexadecyl-treprostinil.

Table 2. Summary of Aerosol Droplet Distributions: Mass Median Aerodynamic Diameter (MMAD), Geometric Standard Deviation (GSD), and Fine Particle Fraction (FPF)4

Run 1 Run 2 Run 3 Average SDMMAD (µm) 2.19 2.52 2.38 2.36 0.16

GSD 2.00 2.06 2.05 2.03 0.03

FPF (<5 µm), % 88.4 82.9 85.0 85.4 2.7

806040200Time (min)

Pul

Pre

ss(m

mH

g)

0

20

40

60

80

100

120

PAP

(m

mH

g)

U46619 2.5 g/kg

U46619 5.0 g/kg

U46619 10.0 g/kg

Time (min)

A. Inhaled PBS

U46619 2.5 g/kg

U46619 5.0 g/kg

U46619 10.0 g/kg

806040200Time (min)

Pul P

ress

(mmH

g)

0

20

40

60

80

100

120

B. Inhaled INS1009

PAP

(m

mH

g)

Time (min)

Figure 4. Vascular hemodynamics with U46619 challenge.PAP, pulmonary arterial pressure; PBS, phosphate buffered saline.

*

*

*

*

***

*

*

*

PBS 0.57 2.84 28.40

10

20

30

40

50

60

PA

P (%

Incr

ease

with

U46

619

Cha

lleng

e)

PBS IPD 6 h 12 h 24 h

INS1009 (μg/kg)

Figure 5. Effect of inhaled INS1009 over 24 hours on PAP increase with U46619 (5 µg/kg).4,a

IPD, immediately post dose; PAP, pulmonary arterial pressure; PBS, phosphate buffered saline.aThe number of rats in each group over time as follows: PBS: n = 12; IPD, 6 h, 12 h, and 24 h: 0.57 µg/kg: n = 5, n = 5, n = 4, n = 4; 2.84 µg/kg: n = 6, n = 6, n = 5, n = 4; 28.4 µg/kg: n = 4, n = 4, n = 4, n = 4.

IPD 6 h 12 h 24 h

0.1

1

10

Pla

sma

TRE

(ng/

mL)

Time post Inhalation

Plasma 0.57 µg/kg 2.84 µg/kg 28.4 µg/kg

IPD 6 h 12 h 24 h

10

100

1000

10000

C16

TR E

quiv

alen

t in

the

Lung

(ug/

g)

Time post Inhalation

0.57 µg/kg 2.84 µg/kg 28.4 µg/kg

Lung

Figure 6. Treprostinil (TRE) in plasma post inhalation of INS1009.4

IPD, immediately post dose.

Figure 7. C16TR equivalent (C16TR + treprostinil) in the lung post inhalation of INS1009.4

C16TR, hexadecyl-treprostinil.

0.01 0.1 1 10 10000

10

20

30

40

50

60

70

80

90

100

PA

P (%

Incr

ease

with

U46

619

Cha

lleng

e)

Plasma TRE (ng/mL)

IV Infused Treprostinil: EC50 = 7.02 ng/mL

0.01 0.1 1 10 10000

20

40

60

80

100

PA

P (%

Incr

ease

with

U46

619

Cha

lleng

e)

Plasma TRE (ng/mL)

Inhaled INS1009: EC50 = 0.09 ng/mL

Figure 8. Comparison between inhaled INS1009 (left) and intravenous infused treprostinil (TRE) (right) on the PAP increase (percent) with U46619 (5 µg/kg) as a function of plasma TRE concentrations.4,a

aThe curves were obtained by fi tting the data using logistic5 regression.

CONCLUSIONS• Inhaled INS1009 protects from U46619-induced pulmonary vasoconstriction up to 24 hours after a single inhaled dose.• The approximately 90x difference in EC50 for plasma TRE concentration between inhaled INS1009 and infused TRE

for protection from U46619-induced pulmonary vasoconstriction suggests that inhaled INS1009 has signifi cant local activity within the lung.

• Mechanisms for local activity in the lung may involve direct effects of TRE on pulmonary vascular smooth muscle or indirect effects on endogenous pulmonary vasodilators/constrictors [nitric oxide, acetylcholine, phosphodiesterase type 5 (PDE5)].

REFERENCES1. Sandifer BL, Brigham KL, Lawrence EC, et al. Potent effects of aerosol compared with intravenous treprostinil on the pulmonary

circulation. J Appl Physiol. 2005;99(6):2363-2368.2. Li Z, Chapman RW, Malinin V, et al. Effi cacy of Nebulized INS1009 Lipid Nanoparticle Formulation in Acute Hypoxia-Induced

Rat Pulmonary Hypertension. Poster presented at: Respiratory Drug Delivery; 5-8 May, 2015; Nice, France. 3. John GW, Valentin JP: Analysis of the pulmonary hypertensive effects of the isoprostane derivative, 8-iso-PGF2α, in the rat.

Br J Pharmacol. 1997;122(5):899-905.4. Data on fi le. Insmed Incorporated, Bridgewater, NJ.

ACKNOWLEDGMENTSThe authors acknowledge Connexion Healthcare (Newtown, PA) for providing layout and design support. Insmed Incorporated (Bridgewater, NJ) provided funding to Connexion Healthcare for these services. The research was funded by Insmed Incorporated.

DISCLOSURES Zhili Li, Richard W. Chapman, Vladimir S. Malinin, Michel R. Corboz, Adam J. Plaunt, and Walter R. Perkins are employees of Insmed Incorporated.

Charles E. Laurent, Han Yin, and Dany Salvail are employees of IPS Therapeutique Inc.

266 ERS Poster (Li)_FINAL.indd 1 8/30/16 10:47 AM