Professor Sir John Vane (1927–2004) F.R.S.

Nobel Laureate

“John Vane has discovered prostacyclin and has carried out detailed analyses of its

biological effects and function. In addition, Vane has made the fundamental discovery that antiinflammatory compounds such as

aspirin act by blocking the formation of prostaglandins and thromboxanes”

The Nobel Prize in Physiology or Medicine 19822

The discovery of prostacyclin (1976)

1. Moncada et al. Nature. 1976;263:663-665; 2. http://nobelprize.org/nobel_prizes/medicine/laureates/1982/press.html. Accessed May 2010

An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation

Nature, 19761

Anti-inflammato

ry

Anti-thrombotic

Vasodilatation

Anti-proliferative

Pharmacological effects of prostacyclin

O

OH OH

HOOC

Prostacyclin

Gomberg-Maitland et al. Eur Respir J. 2008;31:891-901; Zardi et al. Int Immunopharmacol. 2005;5:437-459; Ghofrani et al. J Am Coll Cardiol. 2004;43:68S-72S; Humbert et al. J Am Coll Cardiol. 2004;43:13S-24S

Prostacyclin stimulates cAMP production

in platelets: Anti-thrombotic effects

0

5

10

15

20

25

3 10 30 100 300 1000 3000PGI2 (nM)

Mul

tipl

es o

f bas

al c

AMP

Platelet-rich plasmaWashed platelets

Platelet disaggregation

Data are mean ±standard errorcAMP, cyclic adenosine monophosphate; IP, prostacyclin receptor; PGI2, prostacyclin

Adapted from Moncada et al. In: Westwick et al, eds. Mechanisms of Stimulus-Response Coupling in Platelets. 1985;159:337–358

• Elevation of platelet cAMP following 1 min incubation at 37°C with prostacyclin in human platelet-rich plasma or washed platelets

Prostacyclin inhibits adhesion of platelets exposed to blood vessel wall

Control1

+ PGI2

Platelets

GPIb, platelet glycoprotein 1b; GPIIb/IIIa, platelet integrin IIbβ3; PGI2, prostacyclinImages from Brendan Whittle, Dept of Prostaglandin Research, Wellcome, Beckenham. With permission.

1. Dept of Prostaglandin Research, Wellcome, Beckenham; 2. Zardi et al. Int Immunopharmacol. 2005;5:437–459

• Prostacyclin analogues play an important role as regulators of endothelial function including maintaining vascular homeostasis of the microcirculation2

BP-lowering effects of prostacyclin in systemic and pulmonary circulation

PAP

(mm

Hg)

30

20

15

10

5

0

25

Time (min)

0 40 80 120 160 200 240 280 320

ControlU46619U46619 + prostacyclin (0.5 μg/kg)

PulmonaryRabbit perfused lung2

SystemicAnaesthetised rat1

Mea

n fa

ll in

dia

stol

ic B

P (m

mHg

)

Prostacyclin dose (µg/kg)

9080706050

0

0.250.5 2 8

Vasodilatory effects mediated through potassium channels

Intra-arterialIntravenous

Data are mean ±standard error. U46619 is a thromboxane-A2 mimeticBP, blood pressure; PAP, pulmonary arterial pressure

1. Adapted from Armstrong et al. Br J Pharmacol. 1978;62:125-130; 2. Adapted from Schermuly et al. Respir Res. 2007;8:4

410.125

40302010

n=6

Prostacyclin effect Cellular response Mechanism

Classical functions

Vessel tone ↑ Vasodilation ↑ cAMP, ↑ K+, ↓ Ca2+

Anti-proliferative↓ SMC proliferation↓ Fibroblast growth↑ Apoptosis

↑ cAMP, ↑ K+, ↓ ET-1↑ PPAR (?)

Anti-thrombotic↓ Platelet aggregation↓ Platelet adherence to vessel wall

↓ Thromboxane A2↓ PDGF, ↓ AMs

Novel functions

Anti-inflammatory ↓ Pro-inflammatory cytokines↑ Anti-inflammatory cytokines

↓ IL-1, IL-6↑ IL-10

Anti-mitogenic ↓ ECM remodelling↓ Fibrosis

↓ MMP 2 & 9 ↓ TGF-, ↓ CTGF

Anti-tumourigenic ↓ Tumour formation and metastasis ↑ PPAR

Prostacyclins have many diverse cellular functions

AM, adhesion molecule; Ca2+, calcium; cAMP, cyclic adenosine monophosphate; CTGF, connective tissue growth factor; ECM, extracellular matrix; ET-1, endothelin 1; IL, interleukin; K+, potassium; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; PPAR, peroxisome proliferator-activated receptor; SMC, smooth muscle cell; TGF, transforming growth factor

Gomberg-Maitland et al. Eur Respir J. 2008;31:891-901; Zardi et al. Int Immunopharmacol. 2005;5:437-459; Ghofrani et al. J Am Coll Cardiol. 2004;43:68S-72S; Humbert et al. J Am Coll Cardiol. 2004;43:13S-24S

Pathophysiology of PAH:Pathways of disease

Endothelin pathway

Nitric oxide pathway

Prostacyclin pathwayVessel

lumenEndothelialcells

L-arginine L-citrulline

Pre-proendothelin

Proendothelin

Arachidonic acid

Prostaglandin I2

Prostacyclin(prostaglandin

I2)Nitric oxideEndothelin-1

Endothelinreceptor A

Endothelinreceptor B Phosphodiesteras

e type 5

cGMPcAMP

Smooth muscle cells

Vasoconstriction and

proliferationVasodilatation

and antiproliferation

Vasodilatation and

antiproliferation

Phosphodiesterase type 5 inhibitor

Endothelin-

receptor antagonis

ts

+

+–

–

–

–Exogenou

s nitric oxide

Prostacyclin

derivatives

cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; PAH, pulmonary arterial hypertension

Humbert et al. N Engl J Med. 2004;351:1425-1436

Therapy targets for PAH:Prostacyclin pathway

cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; PAH, pulmonary arterial hypertension

Humbert et al. N Engl J Med. 2004;351:1425-1436

• Pulmonary hypertension is associated with a decrease in prostacyclin levels and an increase in thromboxane levels

Alterations between prostacyclin and thromboxane homeostasis in PAH

10,000

8000

6000

4000

2000

0Control IPAH APAH PH-CVD11

-Deh

ydro

-thr

ombo

xane

B2 (

pg/m

g of

cr

eatin

e)

p<0.05†

n=14 n=20 n=8 n=6

Thromboxane800

600

400

200

2,3-

Din

or-6

-ket

o-PG

F 1a (

pg/m

g of

cr

eatin

e)

0Control IPAH APAH PH-CVD

p<0.05*

n=9 n=20 n=5 n=2

Prostacyclin

Data are mean ±standard error. Statistical significance assessed using two-tailed Mann-Whitney test; *versus normal control; †versus other 3 groupsAPAH, associated pulmonary arterial hypertension; IPAH, idiopathic pulmonary arterial hypertension; PAH, pulmonary arterial hypertension; PGF, prostaglandin F; PH-CVD, pulmonary hypertension associated with collagen vascular disease

Adapted from Christman et al. N Engl J Med. 1992;327:70-75

Frequency of PGI2 synthase expression

Normal (n=7)IPAH (n=12)

100

80

60

40

20

0

PGI 2

synt

hase

(% p

ositi

ve

vess

els)

Large Medium SmallPulmonary arteries

p=0.03

p=0.015

Prostacyclin synthase expression is reduced in PAH

Data are mean ±standard error. Statistical significance assessed using unpaired two-tailed t-testIPAH, idiopathic PAH; PAH, pulmonary arterial hypertension; PGI2, prostacyclin

Adapted from Tuder et al. Am J Respir Crit Care Med. 1999;159:1925-1932

*p<0.05 vs control2

Loss of IP receptor function may depress analogue efficacy in PAH

Data are mean ±standard error. Statistical significance assessed using Student’s t-testcAMP, cyclic adenosine monophosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, prostacyclin receptor; IPAH, idiopathic PAH; PAH, pulmonary arterial hypertension; SMC, smooth muscle cell; SPH, secondary pulmonary hypertension

Adapted from 1. Lai et al. Am J Respir Crit Care Med. 2008;178:188-96; 2. Murray F. Am J Physiol Lung Cell Mol Physiol. 2007;292:L294-L303

Whole lung1

0.6

0.4

0.3

0.2

0.1

0.0Donor IPAH SMC

IP/G

APD

H r

atio p<0.01

n=3 n=3 n=3

n=4

-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0Log [Beraprost] (M)

cAM

P (p

mol

/mg

prot

ein)

0

50

100

150

200

250

Control

SPH

* *

IPAH

Role of PPAR in the prostacyclin signalling pathway

• Peroxisome proliferator-activated receptors (PPARs)– Family of nuclear transcription factors

PPAR, PPAR, PPAR/1,2

– Regulate genes involved in cellular proliferation, apoptosis, migration and inflammation1-3

• PPAR expression is frequently decreased in PAH3

– Loss of PPAR gives rise to apoptotic-resistant cells3

• Prostacyclin activates PPAR– IP-receptor dependent1

– cAMP independent1cAMP, cyclic adenosine monophosphate; IP, prostacyclin receptor; PAH, pulmonary arterial hypertension; PPAR, peroxisome proliferator-activated receptor

1. Falcetti et al. Biochem Biophys Res Commun. 2007;360:821-827; 2. Belvisi et al. Chest. 2008;134:152-157; 3. Ameshima et al. Circ Res. 2003;92:1162-1169

Adhesion molecules

• Endothelium• Leukocytes

PPAR

PPARPPARs andPGI2 analogues

Inhibition of cellularproliferation, migration and

apoptosis

PPAR

Proinflammatory mediators

• Macrophage• T-lymphocyte• Dendritic cells

PPAR,

PPAR,

, /

Key functions of PPARs in the lung

ECM, extracellular matrix; PGI2, prostacyclin; PPAR, peroxisome proliferator-activated receptor

Belvisi et al. Chest. 2008;134:152-157; Becker et al. Fundam Clin Pharmacol. 2006;20:429-447

Growth factors• Vascular cells• Fibroblasts

ECM remodelling• Smooth muscle• Fibroblasts

Control

Normal IPAH 2nd PH COPD

PPAR protein expression

PPAR in lung endothelium

Normal

Diseased

PPAR expression is diminishedin lung tissue of patients with PH

• 38 plexiform lesions from 9 patients with severe PH expressed little to no PPAR

2nd PH, secondary pulmonary hypertension; COPD, chronic obstructive pulmonary disease; IPAH, idiopathic pulmonary arterial hypertension; PH, pulmonary hypertension; PPAR, peroxisome proliferator-activated receptor

Ameshima et al. Circ Res. 2003;92:1162-1169

PPAR

PAH cell type Agent Channel Cellular response

HPASM1,2 – ↓ Kv1.5Cell depolarisation

↓ Kv current↓ Apoptosis

RPASM3

Rat lungHuman lung2

Anorexic agent ↓ Kv1.5

Cell depolarisationVasoconstriction

↑ Incidence of PAH

Rat lung4 Hypoxia ↓ Kv1.5 ↓ Kv2.1

Pulmonary remodelling

↑ PVR

Decreased expression of Kv channels disrupts pulmonary vascular tone

HPASM, human pulmonary arterial smooth muscle; Kv, voltage-gated potassium channel; PAH, pulmonary arterial hypertension; PVR, pulmonary vascular resistance; RPASM, rat pulmonary arterial smooth muscle

1. Yuan et al. Circulation. 1998;98:1400-1406; 2. Moudgil et al. Microcirculation. 2006;13:615-632; 3. Weir et al. Circulation. 1996;94:2216-2220; 4. Michelakis et al. Circulation. 2002;105:244-250

Control chowIloprost chowIloprost chow delayed

*

14

12

10

8

Tum

our

mul

tipl

icit

y

4

2

0

Wild type PPAR OE

6*

****

**

Mice fed iloprost and PPAR OE transgenic mice develop fewer lung

tumours

Data are mean ±standard error. Statistical significance assessed using Student’s unpaired t-testOE, over-expressing; PPAR, peroxisome proliferator-activated receptor

Adapted from Nemenoff et al. Cancer Prev Res (Phila Pa). 2008;1:349-356

*p<0.05 vs wild-type control**p<0.001 vs wild-type control

Importance of prostacyclin in PAH pathophysiology

AC, adenylyl cyclase; AMP, adenosine monophosphate; cAMP, cyclic adenosine monophosphate; IP; prostaglandin receptor; P, arachidonic acid; PAH, pulmonary arterial hypertension; PDE, phosphodiesterase; PGH2, prostaglandin H2; PGI2, prostacyclin; PPAR, peroxisome proliferator-activated receptor

1. Humbert et al. N Engl J Med. 2004;351:1425-1436; 2. Ghofrani et al. J Am Coll Cardiol. 2004;43:68S-72S; 3. Mitchell et al. Exp Physiol. 2007;93:141-147

VasodilatationAnti-proliferation

Anti-thrombosis

AC, adenylyl cyclase; AMP, adenosine monophosphate; Ca2+, calcium; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; ECE, endothelin converting enzyme; ET-1, endothelin-1; ETA/B, endothelin receptor; ETRA, endothelin receptor antagonists; GC, guanylyl cyclases; GMP, guanosine monophosphate; IP; prostaglandin receptor; IP3, inositol trisphophate; L-Arg, L-Arginine; NO, nitric oxide; P, arachidonic acid; PDE, phosphodiesterase; PDE-5I, phosphodiesterase type 5 inhibitor; PGH 2, prostaglandin H2; PGI2, prostacyclin; PPAR, peroxisome proliferator-activated receptor; Pro-Endo, pro-endothelin1. Humbert et al. N Engl J Med. 2004;351:1425-1436; 2. Ghofrani et al. J Am Coll Cardiol. 2004;43:68S-72S; 3. Mitchell et al. Exp Physiol. 2007;93:141-147

Prostacyclin pathways

Nitric oxide-cGMP pathway

Endothelin pathwayPPAR mediated

activitycAMP mediated

activitycGMP mediated

activity

Anti-proliferation

Vasodilatation Anti-thrombosis

• Direct vasodilation of the pulmonary and systemic arterial vascular beds1

• Vasodilatory effects reduce right and left ventricular afterload and increase cardiac output and stroke volume (as demonstrated in animal studies)1

• Inhibits platelet aggregation1

• Inhibits proliferation of human pulmonary artery smooth muscle cells in vitro2

Mechanism of action

Prostacyclin pathway

Pulmonary artery in patient

with PAH3

Arachidonic acid Prostaglandin I2

Prostacyclin (PGI2)

cAMP

Prostacyclin derivatives

+

Vasodilation and anti-proliferation

Smooth muscle cells

cAMP = cyclic adenosine monophosphate

1. Remodulin® (treprostinil sodium) Summary of Product Characteristics, United Therapeutics Europe Ltd. April 2010; 2. Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201; 3. Humbert et al. N Engl J Med. 2004;351:1425-1436

Decay of prostacyclin at 37°C in vitro1

• Unstable at physiological temperatures and pH2

• Light sensitive2

• Hydrolysed to 6-oxo-PGF1α2

• Elimination half-life of approximately 3 minutes2

Epoprostenol

10

20

40

60

80

100

% C

ontr

ol

20 30Incubation time (min)

Tyrode’s solution pH 7.7Washed human platelets(2x108 ml-1)

Instability of epoprostenol

Data are mean ±standard errorPGF, prostaglandin F

1. Adapted from Whittle BJR; 1983. Actions of prostacyclin and thromboxanes: Products of the arachidonic acid cascade. In: Hormones and cell regulation, Volume 7. Eds Dumont, J.E., Nunez, J., Denton, R.M. Elsevier Biomedical Press; Amsterdam, pp 3-23; 2. Flolan® (epoprostenol sodium) Summary of Product Characteristics, GlaxoSmithKline. March 2006

Modifications to the prostacyclin

side chain for increased stability

O

OH OH

HOOC

Prostacyclin

OH

CH3IloprostCH3

Stab

ilit

y

COOHO

Treprostinil

Beraprost

OH

CH3

OH

COOH

OH

O

PGI2 (t½= 2 min)1

OH

COOH

OH

O

Treprostinil (t½= ~240 min)3Beraprost (t½= ~30 min)2

COO*

Na+O

CH3

HO OH

COOH

OHOH

Iloprost (t½= ~30 min)2

CH3

PGI2, prostacyclin; t1/2, half-life

1. Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201; 2. Gomberg-Maitland et al. Eur Respir J. 2008;31:891-901; 3. Remodulin® (treprostinil) US prescribing information; United Therapeutics Corp. January 2010

Prostacyclin analogues:Chemical structures and plasma half-

lives

Ki (nM) values of various PGI2 analogues

for human prostanoid receptors1,2

Ligand DP IP FP EP1 EP2 EP3 EP4

Cicaprost1 >1340 17 >1340 >1340 >1340 255 44

Iloprost1 1035 11 619 11 1870 56 284

Beraprost2 16 110

Carbacyclin1 132 275 427 23 942 14 352

PGE21 307 119 9 5 0.3 0.8Blank means low affinity with Ki >2000 nM

Red= Ki in mouse

Ki, inhibition constant

1. Adapted from Abramovitz et al. Biochim Biophys Acta. 2000;1483:285-293; 2. Adapted from Kiriyama et al. Br J Pharmacol. 1997;122:217-224

p<0.05

0

25

50

75

100

Serum +PPAR

antagonist

+TRE

+PPAR

antagonist+

TRE

% C

ell p

rolif

erat

ion

Stimulation of PPAR

Inhibition of proliferation

*p<0.001 vs control

0

1

2

3

4

Serum 10-7 10-6 10-5

Treprostinil [M]

Fold

incr

ease

in r

elat

ive

luci

fera

se a

ctiv

ity + PPAR

*

* *─ PPAR

Treprostinil activates PPAR and inhibits proliferation

Data are mean ±standard error. Statistical significance assessed using One-way ANOVAPPAR, peroxisome proliferator-activated receptor; TRE, treprostinil

Adapted from Falcetti et al. Biochem Biophys Res Commun. 2007;360:821-827

• ~2.5-fold ↑ in PPAR in treprostinil-stimulated cells

• PPAR inhibition partially reverses anti-proliferative effects of treprostinil

Treprostinil cAMP generation through EP2

but not EP4 receptorsRat alveolar macrophages

p<0.001

Statistical significance assessed using ANOVA followed by Bonferroni correctioncAMP, cyclic adenosine monophosphate; ND, no data; TRE, treprostinil

Adapted from Aronoff et al. J Immunol. 2007;178:1628-1634

Vehicle Treprostinil Treprostinil+

EP2 antagonist(AH-6809)

Treprostinil+

EP4 antagonist(AE3-208)

N.D.0

2

4

6

8

10

12

14

16

cAM

P(p

mol

/milli

on c

ells)

Differential effects on cAMP production and cell proliferation in smooth muscle cells

cAMP generation

Data are mean ±standard error (of 6–12 determinations for first graph). Statistical significance assessed using one- or two-way ANOVAcAMP, cyclic adenosine monophosphate

Adapted from Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201

*p<0.02 vs treprostinil†p<0.01 vs iloprost

*†

n=5–12

TreprostinilBeraprostIloprostCicaprost

cAM

P (p

mol

/mg

prot

ein)

400

300

200

100

0

-12 -11 -10 -9 -8 -7 -6 -5Prostacyclin analogue (log M)

Smooth muscle cell growth

% C

ell g

row

th

100

80

60

20

0

-12 -11 -10 -9 -8 -7 -6 -4

40

-5Prostacyclin analogue (log M)

Differential effects on cAMP production

Treprostinil

IloprostBeraprost

Cicaprost

cAM

P (p

mol

/mg

prot

ein)

400

300

200

100

0

-12 -11 -10 -9 -8 -7 -6 -5Prostacyclin analogue (log M)

Data are mean ±standard error of 6–12 determinationscAMP, cyclic adenosine monophosphate

Adapted from Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201

Differential effects on smooth muscle cell proliferation

Data are mean ±standard error. Statistical significance assessed using one- or two-way ANOVA

Adapted from Clapp et al. Am J Respir Cell Mol Biol. 2002;26:194-201

Smooth muscle cell growth%

Cel

l gro

wth

100

80

60

20

0

-12 -11 -10 -9 -8 -7 -6 -4

40

-5Prostacyclin analogue (log M)

Treprostinil

IloprostBeraprost

Cicaprost

*p<0.02 vs treprostinil†p<0.01 vs iloprost

n=5–12

Prostacyclin inhibits proinflammatory cytokines and chemokines

*p<0.05 vs vehicle-treated cells

0

25

50

75

100

120

150

% o

f veh

icle

IL-12 p70TNF-α IL-1α IL-6 MIP-1α MCP-1

* * * * * * Indomethacin 40 nM

Iloprost 40 nM

Cicaprost 10 nM

Treprostinil 40 nM

Data are mean ±standard deviation of four experiments. Statistical significance assessed using unpaired Student t testcAMP, cyclic adenosine monophosphate; IL, interleukin; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NFB, nuclear factor kappa B; PGI2, prostacyclin; TNF, tumour necrosis factor

Adapted from Zhou et al. J Immunol. 2007;178:702-710

• PGI2 analogues neutralise proinflammatory proteins and promote anti-inflammatory proteins through NFB

• IP receptor dependent involving in part cAMP

Differential effects on IL-10 production

Indo Iloprost Cicaprost Treprostinil

400

nM

0.4

nM

4 nM

400

nM40

nM

0.1

nM

1 nM

100

nM

10 n

M

0.4

nM4

nM

400

nM

40 n

M

% o

f veh

icle

0100200300400500600

p<0.05 compared to vehicle treated

Data are mean ±standard deviationIL, interleukin; Indo, indomethacin

Adapted from Zhou et al. J Immunol. 2007;178:702-710

• Prostacyclin analogues promote anti-inflammatory protein expression

Summary

• Prostacyclins are a heterogeneous class of agents with different half-lives and receptor specificities • PPAR represents an important intracellular target for prostacyclins• Non-classical effects suggest a broader clinical application• Prostacyclin pathway remains a key target to modify PAH disease