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The discovery of prostacyclin (1976). An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation Nature, 1976 1.

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the discovery of prostacyclin 1976
The discovery of prostacyclin (1976)

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

Nature, 19761

“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

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

Nobel Laureate

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

pharmacological effects of prostacyclin
Pharmacological effects of prostacyclin

HOOC

O

OH

OH

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
Prostacyclin stimulates cAMP production in platelets: Anti-thrombotic effects

Platelet-rich plasma

25

Washed platelets

20

15

Multiples of basal cAMP

10

5

Platelet disaggregation

0

3

10

30

100

300

1000

3000

PGI2 (nM)

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

Data are mean ±standard error

cAMP, 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

prostacyclin inhibits adhesion of platelets exposed to blood vessel wall
Prostacyclin inhibits adhesion of platelets exposed to blood vessel wall

Control1

+ PGI2

Platelets

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

GPIb, platelet glycoprotein 1b; GPIIb/IIIa, platelet integrin IIbβ3; PGI2, prostacyclin

Images 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

bp lowering effects of prostacyclin in systemic and pulmonary circulation
BP-lowering effects of prostacyclin in systemic and pulmonary circulation

30

25

20

15

10

5

0

Systemic

Anaesthetised rat1

PulmonaryRabbit perfused lung2

Intra-arterial

90

Intravenous

80

70

60

Control

U46619

U46619 + prostacyclin

(0.5 μg/kg)

Mean fall in diastolic BP (mmHg)

50

PAP (mmHg)

40

30

20

10

n=6

0

0

40

80

120

160

200

240

280

320

0.5

0.125

2

8

0.25

4

1

Prostacyclin dose (µg/kg)

Time (min)

Vasodilatory effects mediated through potassium channels

Data are mean ±standard error. U46619 is a thromboxane-A2 mimetic

BP, 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

prostacyclins have many diverse cellular functions
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
Pathophysiology of PAH:Pathways of disease

Endothelin pathway

Prostacyclin pathway

Vessel

lumen

Endothelialcells

Nitric oxide pathway

Prostaglandin I2

Arachidonic acid

Pre-proendothelin

Proendothelin

L-arginine

L-citrulline

Endothelin

receptor A

Prostacyclin

(prostaglandin I2)

Endothelin-1

Nitric oxide

+

+

Prostacyclin derivatives

cAMP

Endothelin-receptor antagonists

Exogenous nitric oxide

cGMP

Endothelin

receptor B

Vasodilatation and antiproliferation

Phosphodiesterase type 5

Vasoconstriction and proliferation

Vasodilatation and antiproliferation

Smooth muscle cells

Phosphodiesterase type 5 inhibitor

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
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

alterations between prostacyclin and thromboxane homeostasis in pah
Alterations between prostacyclin and thromboxane homeostasis in PAH

Prostacyclin

Thromboxane

800

10,000

8000

600

p<0.05*

6000

2,3-Dinor-6-keto-PGF1a(pg/mg of creatine)

11-Dehydro-thromboxane B2(pg/mg of creatine)

400

4000

200

2000

p<0.05†

n=9

n=20

n=5

n=2

n=14

n=20

n=8

n=6

0

0

Control

IPAH

APAH

PH-CVD

Control

IPAH

APAH

PH-CVD

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

Data are mean ±standard error. Statistical significance assessed using two-tailed Mann-Whitney test; *versus normal control; †versus other 3 groups

APAH, 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

prostacyclin synthase expression is reduced in pah
Prostacyclin synthase expression is reduced in PAH

Frequency of PGI2 synthase expression

100

Normal (n=7)

IPAH (n=12)

p=0.03

80

p=0.015

60

PGI2 synthase (% positive vessels)

40

20

0

Large

Medium

Small

Pulmonary arteries

Data are mean ±standard error. Statistical significance assessed using unpaired two-tailed t-test

IPAH, idiopathic PAH; PAH, pulmonary arterial hypertension; PGI2, prostacyclin

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

loss of ip receptor function may depress analogue efficacy in pah
Loss of IP receptor function may depress analogue efficacy in PAH

Whole lung1

250

*p<0.05 vs control2

0.6

200

0.4

p<0.01

Control

IPAH

150

0.3

SPH

cAMP (pmol/mg protein)

IP/GAPDH ratio

100

0.2

*

*

50

0.1

n=3

n=3

n=3

0

0.0

-7.0

-6.5

-6.0

-5.5

-5.0

-4.5

-4.0

Donor

IPAH

SMC

Log [Beraprost] (M)

n=4

Data are mean ±standard error. Statistical significance assessed using Student’s t-test

cAMP, 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

role of ppar in the prostacyclin signalling pathway
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 independent1

cAMP, 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

key functions of ppars in the lung
Key functions of PPARs in the lung
  •  Adhesion molecules
  • Endothelium
  • Leukocytes
  •  Proinflammatory mediators
  • Macrophage
  • T-lymphocyte
  • Dendritic cells

PPAR

PPAR, 

PPAR

PPAR

PPAR, , /

PPARs andPGI2 analogues

  •  Growth factors
  • Vascular cells
  • Fibroblasts
  •  ECM remodelling
  • Smooth muscle
  • Fibroblasts

Inhibition of cellular

proliferation, migration and apoptosis

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

ppar expression is diminished in lung tissue of patients with ph
PPAR expression is diminishedin lung tissue of patients with PH

PPAR in lung endothelium

PPAR protein expression

Normal

IPAH

2nd PH

COPD

PPAR

Normal

Control

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

Diseased

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

decreased expression of k v channels disrupts pulmonary vascular tone
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

mice fed iloprost and ppar oe transgenic mice develop fewer lung tumours
Mice fed iloprost and PPAR OE transgenic mice develop fewer lung tumours

Control chow

Iloprost chow

Iloprost chow delayed

14

12

10

*p<0.05 vs wild-type control

**p<0.001 vs wild-type control

8

*

*

Tumour multiplicity

6

**

4

**

**

2

0

Wild type

PPAR OE

Data are mean ±standard error. Statistical significance assessed using Student’s unpaired t-test

OE, over-expressing; PPAR, peroxisome proliferator-activated receptor

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

importance of prostacyclin in pah pathophysiology
Importance of prostacyclin in PAH pathophysiology

Anti-proliferation

Vasodilatation

Anti-thrombosis

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

slide18

Prostacyclin pathways

Nitric oxide-cGMP pathway

Endothelin pathway

PPAR mediated activity

cAMP mediated activity

cGMP mediated activity

Anti-proliferation

Vasodilatation

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; PGH2, 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

mechanism of action
Mechanism of action

+

  • 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

Prostacyclin pathway

Arachidonic acid Prostaglandin I2

Prostacyclin (PGI2)

Prostacyclin derivatives

cAMP

Vasodilation and anti-proliferation

Pulmonary artery in patient with PAH3

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

instability of epoprostenol
Instability of epoprostenol

100

Epoprostenol

Tyrode’s solution pH 7.7

80

Washed human platelets(2x108 ml-1)

  • Unstable at physiological temperatures and pH2
  • Light sensitive2
  • Hydrolysed to 6-oxo-PGF1α2
  • Elimination half-life of approximately 3 minutes2

60

% Control

40

20

10

20

30

Incubation time (min)

Decay of prostacyclin at 37°C in vitro1

Data are mean ±standard error

PGF, 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
Modifications to the prostacyclin side chain for increased stability

Treprostinil

O

COOH

Stability

HOOC

Beraprost

CH3

O

OH

OH

OH

Iloprost

CH3

Prostacyclin

CH3

OH

prostacyclin analogues chemical structures and plasma half lives
Prostacyclin analogues:Chemical structures and plasma half-lives

COOH

COOH

O

CH3

OH

OH

OH

OH

PGI2 (t½= 2 min)1

Iloprost (t½= ~30 min)2

COOH

COO*

O

O

Na+

CH3

HO

OH

OH

OH

Treprostinil (t½= ~240 min)3

Beraprost (t½= ~30 min)2

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

k i nm values of various pgi 2 analogues for human prostanoid receptors 1 2
Ki (nM) values of various PGI2 analogues for human prostanoid receptors1,2

Blank means low affinity with Ki >2000 nMRed= 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

treprostinil activates ppar and inhibits proliferation
Treprostinil activates PPARand inhibits proliferation

Stimulation of PPAR

Inhibition of proliferation

4

─ PPAR

*p<0.001 vs control

100

*

+ PPAR

*

3

75

*

p<0.05

Fold increase in relativeluciferase activity

2

% Cell proliferation

50

1

25

0

0

+PPAR antagonist

+TRE

+PPARantagonist+TRE

Serum

10-7

10-6

10-5

Serum

Treprostinil [M]

  • ~2.5-fold ↑ in PPAR in treprostinil-stimulated cells
  • PPAR inhibition partially reverses anti-proliferative effects of treprostinil

Data are mean ±standard error. Statistical significance assessed using One-way ANOVA

PPAR, peroxisome proliferator-activated receptor; TRE, treprostinil

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

treprostinil camp generation through ep 2 but not ep 4 receptors
Treprostinil cAMP generation through EP2but not EP4 receptors

Rat alveolar macrophages

p<0.001

16

14

12

10

cAMP(pmol/million cells)

8

6

4

2

N.D.

0

Treprostinil

+

EP4 antagonist

(AE3-208)

Treprostinil

Treprostinil

+

EP2 antagonist

(AH-6809)

Vehicle

Statistical significance assessed using ANOVA followed by Bonferroni correction

cAMP, cyclic adenosine monophosphate; ND, no data; TRE, treprostinil

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

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

cAMP generation

Smooth muscle cell growth

400

100

Treprostinil

Beraprost

80

300

Iloprost

Cicaprost

60

200

% Cell growth

cAMP (pmol/mg protein)

40

100

20

*

0

0

n=5–12

-12

-11

-10

-9

-8

-7

-6

-5

-4

-12

-11

-10

-9

-8

-7

-6

-5

Prostacyclin analogue (log M)

Prostacyclin analogue (log M)

*p<0.02 vs treprostinil

†p<0.01 vs iloprost

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

cAMP, cyclic adenosine monophosphate

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

differential effects on camp production
Differential effects on cAMP production

400

Treprostinil

Beraprost

Iloprost

300

Cicaprost

200

cAMP (pmol/mg protein)

100

0

-12

-11

-10

-9

-8

-7

-6

-5

Prostacyclin analogue (log M)

Data are mean ±standard error of 6–12 determinations

cAMP, 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
Differential effects on smooth muscle cell proliferation

Smooth muscle cell growth

Treprostinil

100

Beraprost

Iloprost

80

Cicaprost

60

% Cell growth

40

20

*p<0.02 vs treprostinil

†p<0.01 vs iloprost

0

n=5–12

-12

-11

-10

-9

-8

-7

-6

-5

-4

Prostacyclin analogue (log M)

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

prostacyclin inhibits proinflammatory cytokines and chemokines
Prostacyclin inhibits proinflammatory cytokines and chemokines

Indomethacin 40 nM

150

*

*

*

*

*

*

Iloprost 40 nM

120

Cicaprost 10 nM

100

Treprostinil 40 nM

% of vehicle

75

*p<0.05 vs vehicle-treated cells

50

25

0

TNF-α

IL-12 p70

IL-1α

IL-6

MIP-1α

MCP-1

  • PGI2 analogues neutralise proinflammatory proteins and promote anti-inflammatory proteins through  NFB
  • IP receptor dependent involving in part cAMP

Data are mean ±standard deviation of four experiments. Statistical significance assessed using unpaired Student t test

cAMP, cyclic adenosine monophosphate; IL, interleukin; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NFB, nuclear factor kappa B; PGI2, prostacyclin; TNF, tumour necrosis factor

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

differential effects on il 10 production
Differential effects on IL-10 production

p<0.05 compared to vehicle treated

600

500

400

% of vehicle

300

200

100

0

1 nM

4 nM

4 nM

40 nM

10 nM

40 nM

0.4 nM

0.1 nM

0.4 nM

400 nM

400 nM

100 nM

400 nM

Cicaprost

Indo

Iloprost

Treprostinil

  • Prostacyclin analogues promote anti-inflammatory protein expression

Data are mean ±standard deviation

IL, interleukin; Indo, indomethacin

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

summary
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