Treprostinil Pharmacokinetics in Rats Are Extended Using Inhaled Prodrug Formulations V Malinin1, Z Li1, RW Chapman1, F Leifer1, D Omiatek1, J Ong1, D Salvail2, C-E Laurent2, WR Perkins1

1Insmed, Inc., Bridgewater, NJ, USA

2IPS Therapeutique Inc., Sherbrooke, QC, Canada

INTRODUCTION• The current prostanoid inhalation therapies for pulmonary arterial hypertension (PAH) require

frequent dosing (treprostinil, Tyvaso®, 4 times daily1; iloprost, Ventavis®, 6 to 9 times daily2) due to fast elimination of the active drug.

• A sustained-release formulation of treprostinil (TRE) may potentially reduce frequency of dosing to once daily, reduce peak concentration in blood, and thus lessen some of the adverse effects of treatment.

• To reduce dosing frequency and provide a more consistent dose level, we initially developed an inhalable lipid nanoparticle (LNP) formulation of treprostinil acid that displayed a slowed-release profile of TRE (see poster by Omiatek et al, ERS Poster #2357), and in an attempt to further extend the therapeutic effect, we synthesised prodrug versions of TRE (Figure 1).

• In a rat model of acute hypoxia-induced PAH (see poster by Leifer et al, ERS Poster #2356), inhaled LNP formulations of treprostinil prodrugs (TPD-LNPs) were found to have a longer duration of activity (>2 hours) compared with TRE. TPDs with alkyl chains of lengths C12 and C16 yielded the longest extensions in activity.

AIMS• To evaluate the plasma and lung pharmacokinetics in rats that received a single inhaled dose

of these TPDs

METHODS• Nebulised TRE solution and TPD formulations were administered (at 15 nmol/kg, or 6 mg/kg TRE

equivalent) to anaesthetised-ventilated rats (6-hour studies) or to conscious rats by nose-only inhalation (24-hour studies).

• Blood and lung samples were collected at specified time points. – TRE and TPD concentrations in blood plasma and lung tissue were measured by

HPLC/MS/MS analysis.

CONCLUSIONS• Inhaled TPDs are present in the lungs for an extended duration and are associated with a slow,

sustained release of TRE into the blood. • This duration of activity is increased with TPD formulated in nanoparticles. • The pharmacokinetics profile of TPD is quite different than inhaled TRE and strongly suggests that

long alkyl chain prodrug formulations of TRE will likely have prolonged pulmonary vasodilator activity in humans.

REFERENCES 1. Tyvaso (treprostinil) [package insert]. Research Triangle Park, NC: United Therapeutics Corp; 2014.2. Ventavis (iloprost) [package insert]. South San Francisco, CA: Actelion Pharmaceuticals US, Inc; 2013.

Please see other posters in this series:• Leifer F, et al, ERS Poster #2356 • Omiatek D, et al, ERS Poster #2357 • Chen K-J, et al, ERS Poster #2358

ACKNOWLEDGEMENTSThe authors would like to acknowledge Connexion Healthcare (Newtown, PA) for providing editorial, layout, and design support. Insmed, Inc. (Bridgewater, NJ) provided funding to Connexion Healthcare for these services.

Poster presented at the European Respiratory Society (ERS) International Congress, 6-10 September, 2014, Munich, Germany.

Release Kinetics of TRE From Inhaled C16TR-LNP Over 24 Hours Is Independent of Dose

Figure 7. Blood plasma TRE (A) and lung total TRE + TPD (B) kinetics in rats after dosing in nose-only system with nebulised C16TR-LNP at different estimated pulmonary doses (see key)a6 mL suspension was nebulised for a period of 30 to 60 minutes. Time corresponds to the time after beginning of nebulisation.LNP, lipid nanoparticle; TPD, treprostinil prodrug; TRE, treprostinil.

TRE

C12TR-LNP

C14TR-LNP

C16TR-LNP

0

100

200

300

400

500

TRE

and

TPD

inLu

ng(T

RE

Eq-n

g/g)

TRETPD

NotDetected

Inhaled TPD Demonstrates Long Residence Time in Lung

AUC, area under the curve; Cmax, maximum concentration; Tmax, time to maximum plasma concentration. TPD-LNP, treprostinil prodrug–lipid nanoparticle; TRE, treprostinil.Figure 5. TRE and TPD in the lung of

ventilated rats after dosing with nebulised TRE solution or TPD-LNP suspension.a

TRE detected in the lung was speculated to be generated due to TPD hydrolysis during sample preparationaLungs were collected at 6 h after dosing. TPD concentration is presented as TRE equivalent on a mole base.TPD-LNP, treprostinil prodrug–lipid nanoparticle; TPD, treprostinil prodrug; TRE, treprostinil.

Inhaled TPD provides sustained release of TRE through 24 hours

Figure 6. Blood plasma TRE (A) and lung total TRE + TPD (B) in rats after dosing with nose-only inhalation with nebulised TPD-LNP formulationsa

a6 mL suspension was nebulised for a period of 30 to 60 minutes. Time corresponds to the time after beginning of nebulisation.TPD-LNP, treprostinil prodrug–lipid nanoparticle; TPD, treprostinil prodrug; TRE, treprostinil.

Table 1. Plasma Pharmacokinetics of TRE in Ventilated Rats After Dosing With Nebu-lised TRE Solution or TPD-LNP Suspension at an Estimated Pulmonary Dose of 6 μg/kg

Solution Nanoparticles Micelles

TRE C12TR C14TR C16TR C12TR C14TR C16TRAUC 0-6 h (ng*h/mL) 2.13 4.47 3.89 1.84 9.72 9.11 5.35

Cmax (ng/mL) 3.37 0.56 0.34 0.22 2.40 1.20 0.84

Tmax (h) 0.05 0.5 0.5 1.0 0.05 0.5 1.0

Figure 2. Ventilation system for dosing of rats by nebulisation and collection of blood in 6-hour pharmacokinetics study

0.01

0.1

1

10

0 5 10 15 20 25

TRE

in B

lood

Pla

sma

(ng/

mL)

Time (h)

C16TRC14TRC12TR

10

100

1000

10000

0 5 10 15 20 25

CxTR

+TRE

in L

ung

(Eq-

ng/g

)

Time (h)

C16TRC14TRC12TR

A B

A B

Figure 1. (A) Chemical structure of tresprostinil prodrug (TPD) and (B) schematic representation of TPD– lipid nanoparticles (TPD–LNPs)

TPD TPD-LNPsA BR = C12, C14, or C16

aActual lung dose is calculated based on the measured total lung concentration at 1 hour. AUC is calculated by trapezoidal method.AUC, area under the curve; Cmax, maximum concentration; IPD, isocentre point dose; Tmax, time to maximum plasma concentration, TPD-LNP, treprostinil prodrug–lipid nanoparticle; TRE, treprostinil.

Table 2. Pharmacokinetics of TRE in Rats After Dosing in Nose-Only System With Nebulised TPD-LNP Formulations at an Estimated Pulmonary Dose of 6 μg/kga

Compound C12TR C14TR C16TR

Lung dose IPD (µg/kg) 6.2 10.4 17.9

Lung apparent ellimination rate (h-1) 0.42 0.15 0.10

Plasma Cmax IPD (ng/mL) 4.03 4.93 3.46

Plasma apparent elimination rate (h-1) 0.30 0.18 0.14

AUC 1-24 (ng*h/mL) 11.9 24.0 17.9

0 2 4 60.01

0.1

1

10

Time (h)

Blo

od P

lasm

aTR

E(n

g/m

L) TREC12TR-LNPC14TR-LNPC16TR-LNP

0 2 4 60.01

0.1

1

10

Time (h)

TREC12TR-MC14TR-MC16TR-M

Blo

od P

lasm

aTR

E(n

g/m

L)

Inhaled TPD Formulated in LNPs Extends Plasma Pharmacokinetics and

Reduces Peak Levels of TRE

Inhaled TPD Micelles Extend Plasma Pharmacokinetics of TRE

Figure 4. Blood plasma TRE in ventilated rats after dosing with (A) nebulised TRE solution and TPD-LNP or (B) TPD-M formulations LNPs, lipid nanoparticles; M, micellar; TPD, treprostinil prodrug; TPD-M, treprostinil prodrug–micellar, TPD-LNP, treprostinil prodrug–lipid nanoparticle; TRE, treprostinil.

A B

Conscious Nose-Only Inhalation• Male Sprague Dawley rats were placed in restraining tubes and exposed to nebulised drugs via the

Jaeger-NYU Nose-Only Directed-Flow Inhalation Exposure System (CH Technologies, Westwood, NJ) (Figure 3).

• Test articles (6 mL at specific concentration) were nebulised using the Aeroneb nebuliser to deliver a predetermined estimated pulmonary dose.

• Blood and lung tissue samples were taken at selected times after nebulisation of the drugs over a 24-hour period.

Figure 3. Jaeger-NYU Nose-Only Directed-Flow Inhalation Exposure System (CH Technologies, Westwood, NJ) used for 24-hour pharmacokinetics study. Reproduced from www.onares.org with permission.

RESULTS• Ventilated rats treated with nebulised TRE solution had the highest blood plasma concentration (Cmax)

(3.5 ng/mL), which occurred immediately after dosing (Figure 4A). Measurable levels of TRE were not seen beyond 4 hours in the blood plasma and by 6 hours in the lungs.

• In contrast, ventilated rats treated with nebulised TPD-LNP had lower blood plasma TRE Cmax values, ranging from 0.2 ng/mL to 0.6 ng/mL (Table 1). At 6 hours, TPD remained in the lung at levels that ranged from 100 ng/g to 400 ng/g tissue of TRE equivalent (Figure 5).

• When dosing with micellar TPD, blood plasma levels of TRE were higher than with TPD-LNP, indicating that nanoparticles play an additional role in slow-release effect (Figure 4B).

• In the 24-hour studies in rats dosed with TPD-LNP (nose-only inhalation), TRE Cmax was higher than in ventilated animals and showed close to first-order exponential decline. Blood plasma concentrations of TRE in rats that received dosing with C14- and C16-TPDs were maintained at greater than 0.1 ng/mL for up to 24 hours, ie, levels corresponding to activity in acute hypoxia studies.

• Lung levels of total TRE + TPD were approximately 103 higher than plasma TRE and also exhibited exponential decline.

Anaesthetised, Ventilated Rats• Male Sprague Dawley rats were anaesthetised and

prepared with endotracheal tube for ventilation (Figure 2). The right femoral vein was cannulated to facilitate blood collections. Terminal lung samples were taken for analysis only 6 hours after dosing.

• Aeroneb® nebuliser and a controller (Aerogen, Gal-way, Ireland) were used to produce aerosol of a mass median aerodynamic diameter (MMAD) between 2.5 µm and 4 µm and at a rate of 0.1 mL/min.

• A SAR-830/AP Small Animal Ventilator (CWE Inc., Ardmore, PA) set up at ventilator tidal volume (VT) of 8 mL/kg, rate of 90 breaths/min, was used to deliver nebulised test articles of volume 250 µL.

0.01

0.1

1

10

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0 5 10 15 20 25

TRE

in B

lood

Pla

sma

(ng/

mL)

Time (h)

C16TR 18 µg/kgC16TR 6 µg/kgC16TR 1.8 µg/kgC16TR 0.6 µg/kg

100

1000

10000

100000

0 5 10 15 20 25

TRE+

CxTR

in L

ung

(Eq- ng

/g)

Time (h)

C16TR 18 µg/kgC16TR 6 µg/kgC16TR 1.8 µg/kgC16TR 0.6 µg/kg

PHOSPHOLIPID

HYDROPHOBICFILLER

PEGYLATEDLIPID

TREPROSTINILPRODRUG

POSTER #: 2367