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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, 1976 1.

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The discovery of prostacyclin (1976)

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  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  21. 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

  22. 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

  23. 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

  24. 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

  25. 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

  26. 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

  27. 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

  28. 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

  29. 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

  30. 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

  31. 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

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