R e vie w

Author for correspondence: KellSa s.a.s., Str. Campo e Zampe 12, I-13900 Biella, BI, Italy Tel.: +39 015 252 4359 n Fax: +39 015 252 7615 n [email protected]

†

n

Review

Kjell S Sakariassen†, Peteris Alberts, Pierre Fontana, Jessica Mann, Henri Bounameaux & Alexandra Santana Sorensen

Future Cardiology

Effect of pharmaceutical interventions targeting thromboxane receptors and thromboxane synthase in cardiovascular and renal diseases

The present review focuses on the roles of thromboxane A 2 (TxA 2 ) in arterial thrombosis, atherogenesis, vascular stent-related ischemic events and renal proteinuria. Particular emphasis is laid on therapeutic interventions targeting the TxA 2 (TP) receptors and TxA 2 synthase (TS), including dual TP receptor antagonists and TS inhibitors. Their significant inhibitory efficacies on arterial thrombogenesis, atherogenesis, restenosis after stent placement, vasoconstriction and proteinuria indicate novel and improved treatments of cardiovascular and selected renal diseases. New therapeutic interventions of the TxA 2 pathway may also be beneficial for patients with poor biological antiplatelet drug response, for example, to aspirin and/or clopidogrel. These new TP/TS agents offer novel improved treatments to ef ficiently and s imultaneou s ly inter fere with thrombogenesis and atherogenesis, and to enlarge the existing panel of platelet inhibitors for efficient prophylaxis and treatment of arterial thrombosis and renal proteinuria.

The present review describes the prominent roles of the thromboxane A 2 (TxA 2) pathway, TxA 2 (TP) receptors and TxA 2 synthase (TS), in arterial thrombosis, atherogenesis, vascular stent-induced thrombosis and restenosis as well as proteinuria in a wide range of renal disorders, including adult nephrotic syndrome and diabetes Type 1 and Type 2. Particular emphasis is laid on pharmaceutical interventions targeting TP receptors and TS. Platelets play a key role in arterial thrombosis, and antiplatelet drugs are therefore a cornerstone in the treatment of this disease. One strategy to inhibit platelet function is to target the TxA 2 effects [1–5] . TxA 2 is generated from membrane phospholipids through the consecutive action of phospholipase A 2, COX-1 and TS. It mediates its specific effect via the TP receptor, and is one of the most powerful platelet activators known, a potent smooth muscle constrictor and a vascular smooth muscle cell mitogen. Other metabolites of membrane phospholipids include isoprostanes, which are prostanoid derivatives, for example, 8-epi-PGF2 and 9a11b‑PGF2, and hydroxyeicosatetraenoic acids (HETEs), for example, 15(S)-hydroxyeicosatetraenoic acids. Both isoprostanes and HETEs are formed by nonenzymatic peroxidation of membrane 10.2217/FCA.09.33 © 2009 Future Medicine Ltd

phospholipids in platelets, cells of blood vessels and monocytes/macrophages. They are all TP-receptor ligands [6–9] . Binding of TxA 2, isoprostanes or HETEs to TP receptors amplifies platelet aggregation and thereby favors hemostasis. However, the same agonists can also trigger deleterious thrombogenesis owing to platelet activation and aggregation, vasoconstriction and initiation and progression of atherogenesis due to endothelial dysfunction and vascular smooth muscle cell proliferation. The role of TxA 2 production in arterial thrombosis has led to the clinical use of anti-TxA 2 agents, either inhibitors of its biosynthesis and/or TP receptor antagonists. The clinical efficacy of aspirin – the antiplatelet agent most widely used to prevent arterial thrombosis – is based on irreversible acetylation of COX-1 and the inhibition of platelet TxA 2 generation [10] . Although aspirin is considered a reference drug in this setting, no beneficial effects on vasoconstriction, endothelial dysfunction or vascular smooth muscle cell proliferation have been observed at the currently recommended dose range of 75–150 mg/day [11] . TP-receptor antagonists, TS inhibitors and dual TP-receptor antagonists and TS inhibitors have certain pharmacological advantages over aspirin: not only do they block the effect of TxA 2 Future Cardiol. (2009) 5(5), xxx–xxx

Keywords arterial thrombosis n arteriosclerosis n renal proteinuria n thromboxane receptor antagonists n thromboxane synthase inhibitors

part of

ISSN 1479-6678

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Review

Sakariassen, Alberts, Fontana et al.

on platelets, but they also inhibit the deleterious effects of other TP ligands such as isoprostanes and HETEs [6,7] . Moreover, antagonism of TP receptors with or without inhibition of TS has a favorable effect on atherosclerosis progression and arterial plaque stabilization both in animals and in man [12–14] . The TP receptor is one of the prostanoid receptors belonging to the rhodopsin-like G‑protein coupled receptor (GPCR) superfamily. It was previously called the TxA 2 or the prostaglandin H 2 (PGH 2 ) receptor [15] . The TP receptor is expressed in platelets, endothelial cells, smooth muscle, kidneys, bronchi, thymus, spleen and monocytes and macrophages [16–18] . There are two splice variants, TPa and TPb. TPa is the only isoform that has been demonstrated to be expressed in the human platelet cell membrane [17,19,20] , in the human umbilical vein smooth muscle cells [21] , and in human umbilical endothelial cells [19] . The low number of tissue distribution studies might be explained by the fact that few, if any (depending on the definition of selectivity), isoform-selective TP-receptor antagonists have been described [22] . TS (EC 5.3.99.5) is an endoplasmic reticulum membrane protein that is present in several tissues, including platelets, endothelial cells, smooth muscle cells, monocytes/macrophages and kidneys [23,24] . Its function is to catalyze conversion of PGH2 to TxA 2. TP receptors are also present at various anatomical locations in the kidney [25] . Increased production and secretion of TxA 2 by TS and/or activation of the TP receptors are involved in several disorders, including the proteinuric renal diseases of adult nephrotic syndrome and Types 1 and 2 diabetes mellitus, arterial thrombogenesis and atherogenesis. TP-receptor activation may also contribute to the development of hypertension, at least in a variety of gene-modified live mouse models [26,27] . Activation of TP receptors on smooth muscle cells of bronchi airways may trigger bronchoconstriction and asthma. The role of TxA 2, isoprostanes, HETEs, TP receptors and TS has gained considerable interest in cardiovascular and renal diseases during the last 25 years. Several other clinical conditions have been identified for therapeutic interventions of the TxA 2 pathway. These include asthma, rhinitis, pre-eclampsia, septic shock, retinopathy, pulmonary hypertension, lupus nephritis, Raynaud’s disease and inflammatory bowel disease [28,29] . Emerging clinical conditions for antagonists/inhibitors of the TxA 2 pathway are cancers of the neck, breast, lung, 2

Future Cardiol. (2009) 5(5)

pancreas and prostate [29] . This has spurred pharmaceutical interventions of TP receptors and TS, including agents which currently are in clinical development. Role of TxA2 in arterial thrombus formation

The prime physiological roles of TxA 2 are to activate and recruit platelets to secure optimal platelet activation and aggregation to stop bleeding, and thus to support the normal hemostatic process. However, these TxA 2 mediated platelet functions play a central role in thrombus formation, thus in pathological processes considered to be ‘hemostasis at the wrong time and place’ (Figure 1) . Blood platelets synthesize and secrete TxA2. They also possess TP receptors in the membrane [29,30] . Binding of TxA2, the prostaglandin endoperoxides PGG2, PGH2, PGD2 and PGF2, isoprostanes, and HETEs to the platelet TP receptors results in platelet activation and their subsequent aggregation (Figure 1) [6,31–33] . Isoprostanes are in particular formed by clinical conditions of vascular oxidative stress, for example, due to hyperglycaemia in patients suffering from Type 1 and Type 2 diabetes [34] . The platelet TP-receptor density and the corresponding platelet aggregation response are up-regulated by testosterone, and it is suggested that this effect is at least in part responsible for the contribution of the thrombogenicity of androgenic steroids [35] . Moreover, polymorphisms of the TP-receptor gene have been described; some of them are associated with platelet reactivity and some with poor platelet secretion in response to the TxA 2 mimetic U-46619 [36] . However, none of the polymorphisms has been reported to alter the effect of TP antagonists or TS inhibitors. A polymorphism of the TP receptor – Arg60 to Leu – is responsible for a mild bleeding disorder [37] . The platelet TxA 2 activation process induces a phospholipid-rich, negatively charged platelet surface, which promotes coagulation, resulting in increased thrombin generation and fibrin deposition in and around the growing thrombus. Several other agonists, such as ADP, thrombin and fibrillar collagen, act in concert with TxA 2 to further augment the growth and stability of the thrombus. Role of TxA2 in atherogenesis

It is well recognized that TxA 2 biosynthesis and its interaction with vascular TP receptors are intimately linked to the development of vascular future science group

Effect of pharmaceutical interventions targeting thromboxane receptors & thromboxane synthase

Review

PGHS Lox

Ne

COX-1 and 2 PS TS

PGD2S

Potential reasons for aspirin hyporesponsiveness, resistance and/or platelet hyper-reactivity

Vaso- and broncho-constriction

Platelet aggregation

Proliferation of vascular smooth muscle

Mitogenesis Future Cardiol. © Future Science Group (2009)

Figure 1. Biosynthesis of TxA 2 from arachidonic acid and other metabolites, which interact with the TP receptor on platelets, monocytes/macrophages, endothelial cells and vascular smooth muscle cells. Pathological consequences of these interactions are indicated in red. HETE: hydroxyeicosatetraenoic acid; IP: Prostacyclin receptor; Lox: Lipoxygenase; Ne: Nonenzymatic; PGD2S: Prostaglandin D2 synthase; PGHS: Prostaglandin endoperoxides H synthase; PGI2: Prostacyclin; PS: Prostacyclin synthase; TP: Thromboxane receptor; TS: Thromboxane A 2 synthase.

lesions [12,14,38] . TP receptors are present in the membrane of platelets, monocytes/macrophages, endothelial cells and vascular smooth muscle cells [39–42] . TxA 2 is a mitogen for vascular smooth muscle cells and a potent vasoconstrictor [43] . The vasoconstrictor activity increases with age with a corresponding augmentation in TP receptor density. These TxA 2 effects on vascular smooth muscle cells promote atherogenesis and appear to play a role in vascular stent-induced hyperplasia/restenosis [44] . In addition, severe vasoconstriction increases the blood shear rate, which may enhance platelet activation, platelet release reaction and the risk of thrombus formation [45] . In addition, platelets may release PDGF-B, a potent mitogen for smooth muscle cell proliferation [46] . Binding of TxA 2 and the isoprostanes 8-isoPGF(2a), 8-iso-PGE(2) and 8-iso-PGA(2) to TP receptors on endothelium suppresses the signaling of the VEGF, resulting in reduced endothelial cell differentiation and migration, and even a reversal of angiogenesis and induction of apoptosis [29,47,48] . Thus, the process future science group

of vascular re-endothelization is hampered. Furthermore, TxA 2 binding to endothelium stimulates synthesis and expression of the adhesion molecule ICAM-1, which promotes the adhesion of monocytes to the endothelium [12] . This particular cellular interaction is a key step in atherogenesis [46] . These observations all support the unfortunate and prominent deleterious role of TxA 2 and isoprostanes in atherogenesis (Figure 1) . Role of TxA2 in renal proteinuria

The TP receptor in the kidneys [15] is localized to the glomeruli, arterial vessel walls, luminal membranes of thick ascending limbs of the Henle’s loop, the luminal and basolateral membranes of either distal convoluted tubules or connecting tubules, as well as the basolateral membranes of collecting tubules [15,25,49] . Increased TxA 2 production by TS [23,24] and excretion of TxA 2 by the kidneys and/or activation of TP receptors have been implicated in the proteinuric renal diseases such as adult nephrotic syndrome, lupus nephritis and Type 1 and Type 2 diabetes mellitus [23,24,50,51] . www.futuremedicine.com

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According to the WHO, Type 1 and 2 diabetes affect over 170 million people worldwide, and this will rise to 366 million by 2030 [52] . Over the past two decades, there has been a continuous increase in the incidence of end-stage renal disease among patients with Type 2 diabetes. The first clinical sign of renal dysfunction in diabetic patients is microalbuminuria, which in 20–40% of the cases progresses to overt proteinuria. Of patients with proteinuria, 10–50% develop chronic kidney disease, which ultimately requires dialysis or transplantation. Agents that inhibit the renin–angiotensin system, ACE inhibitors and the angiotensin II-receptor blockers are the treatments currently available, but they provide insufficient protection [53] . Isoprostanoids and TxA 2 are upregulated in many renal diseases, for example, diabetic nephropathy and nephrotic syndrome [54,55] . TxA 2 activates TP receptors and causes renal vasoconstriction, decreased renal blood flow, glomerular thrombosis and renal fibrosis, lower single-nephron glomerular filtration rate, contraction of isolated glomeruli and mesangial cells, production of plasminogen activator inhibitor (PAI)-1, and increased glomerular basement membrane permeability to proteins [49,56] . Adult nephrotic syndrome is a life-threatening condition. There are around three new cases per 100,000 adults per year in the Western world [57] . In this condition, damaged capillary walls in the kidney glomeruli result in high levels of proteinuria (>3.5 g/24 h) and low albuminemia (10 mM) [57] . Thromboembolism occurs in 35–40% of nephrotic syndrome patients [58] . Patients with adult nephrotic syndrome have increased release of arachidonic acid, increased production of arachidonic acid metabolites including TxA 2 , shortened platelet survival and hyperaggregable platelets. There is also a reported decrease in intraplatelet serotonin and b-thromboglobulin and a significant negative correlation with plasma total cholesterol, triglycerides and low-density and high-density lipoprotein cholesterol, which may contribute to the increased platelet reactivity [59] . The increased platelet aggregability is promoted by hypoalbuminemia as well, since normally albumin is involved in inhibition of arachidonic acid-induced platelet aggregation [60,61] . It is anticipated that these platelet reactivity-related factors, and in particular the increased platelet TxA 2, may increase the risk of glomerular and microvascular damage of the affected kidney [62] . 4


A number of studies in animal models and in man published during the last 25 years have demonstrated that TP-receptor antagonists and TS inhibitors reduce albuminuria and/or proteinuria, as well as urinary excretion of TxB2, the stable metabolite of TxA 2 [49,56,63–70] . Therefore, antagonizing TP receptors and/or inhibiting TS may have the potential to be beneficial in proteinuric renal diseases. Clinical indications of pharmaceutical TxA2 interventions of arterial thrombus formation

A number of clinical indications for drugs having potent, selective and safe TxA 2 antagonism of thrombus formation have been identified. These indications are discussed with particular regard to the clinical use of aspirin and clopidogrel. A major significant role for TxA 2 in arterial thrombogenesis was already observed in 1950, as aspirin administration was found to reduce the incidence of myocardial infarction [2] . The antiplatelet properties of aspirin were described in 1967 [5] . The more recent Antithrombotic Trialist Collaboration reported that aspirin administration resulted in an average reduction of 25 % of myocardial infarction, ischemic stroke, angina and atrial fibrillation in patients at high risk of developing atherothrombotic vascular events [1] . However, the beneficial effect of aspirin appears more limited in patients with severe arterial stenosis, in other words, at blood flow conditions of extremely high shear [71,72] . It is not known whether this applies to TP receptor antagonists and/or TS inhibitors and clopidogrel. However, both linotroban, a TP-receptor antagonist that reached clinical development, and clopidogrel reduced thrombus formation at blood flow conditions of high shear characteristic of mildly atherosclerotic arteries [4,73] . Both agents affected thrombus formation at a relatively low shear condition, resulting in loosely packed platelet thrombi, whereas aspirin had no effect [4,73,74] . Thus, ADP and TP-receptor antagonists appear to be globally more important than COX-1 inhibition by aspirin in inhibiting thrombus formation at arterial shear conditions [75] . At present there are no TP-receptor antagonists, TS inhibitors or dual TP-receptor antagonists and TS inhibitors registered in USA or in most European countries. The only exception is the dual TP-receptor antagonist and TS inhibitor picotamide, which is registered in Italy for thrombosis and peripheral arterial disease (PAD) (Table 1) [13,38] . In Japan, the dual TP receptor and future science group


PGD2 antagonists ramatroban and seratrodast are both registered for allergic rhinitis and asthma, whereas the TS inhibitor ozagrel is registered for thrombosis and asthma. Other antiplatelet agents registered and in clinical development, including their targets, inhibitory mechanisms, mode of interaction with the targets and clinical indications, are summarized for comparative purposes in Tables 1 & 2. Anticoagulant agents and platelet thrombin-receptor antagonists are not included. An update on oral antiplatelet therapy, including both registered antiplatelet agents and antiplatelet agents in clinical development was recently published [76] . Picotamide demonstrated a 45% reduction in all-cause mortality among patients with PAD and diabetes compared with aspirin in the Drug Evaluation in Atherosclerotic Vascular Disease in Diabetes (DAVID) study [38] . Some limitations to this result came from the fact that neither vascular mortality nor major ischemic cardiovascular events were reduced by picotamide when compared with aspirin. It is likely that the relatively low number of patients of the

Review

study did not confer enough statistical power to detect an effect on these end points, and thus, as pointed out by the authors, confirmatory studies are required to substantiate the observed reduction in mortality, and also to prove an effect on ischemic vascular end points. Terutroban is a potent TP receptor antagonist, currently in clinical development (Table 2) [40] . This compound significantly inhibits TxA 2induced in vitro platelet aggregation and development of vascular lesions in ApoE-deficient mice [12,40] . It is also a more potent inhibitor than aspirin of vascular stent-induced thrombus formation in pigs, although less efficient than clopidogrel [77] . Furthermore, terutroban interacts with TS, but as a relatively weak inhibitor [12,49] . It is anticipated that TS will not be affected by terutroban at clinical effective doses. The understanding of the impact of TP receptors and TS in thrombus formation, atherogenesis and renal proteinuria together with the recognition of the clinical limitations of aspirin and clopidogrel have led to a revival of interest and strategies to block the TxA 2-mediated vascular

Table 1. Registered antiplatelet agents. Compound

Inhibitory mechanism(s) Reversible Pro-drug

Clinical indications

Ref.

Acetylsalicylic acid Worldwide (Aspirin®) Picotamide (Plactidil®) Italy Ramatroban (Baynas®) Japan

Country

COX-1 & COX-2

No

No

[120– 122]

TP and TS TP and PGD2

No Yes

No NP

MI, stroke, angina, vascular stents, atrial fibrillation Arterial thrombosis, PAD Allergic rhinitis, asthma

Seratrodast (Bronica®) Japan Ozagrel (Edaravone®) Japan Triflusal (Disgren®) Spain

TP and PGD2 TS COX-1

No Asthma, allergy Not likely NP Thrombosis, asthma Yes and no AMI, CVA, angina

Clopidogrel (Plavix®)

USA, EU

P2Y12

NP NP Parent – no; metabolite – yes No

Ticlopidine (Ticlid®)

USA

P2Y12

No

Yes

Abciximab (Reopro®)

USA, UK

GPIIb-IIIa, antibody

No

No

Tirofiban (Aggrastat®) USA, UK

GPIIb-IIIa

Yes

No

Eptifibatide (Integrilin®) Cilostazol (Pletal®)

USA, EU

GPIIb-IIIa, heptapeptide

Yes

No

USA

PDE/miscellaneous effects

Yes

NP

Dipyridamole (Persantin®, Aggrenox®)

USA, UK

PDE/miscellaneous effects

Yes

No

Yes

MI, stroke, PAD, acute coronary syndrome, angina, vascular stents, IHD MI, stroke, PAD, acute coronary syndrome, angina, vascular stents, IHD, thrombotic stroke Percutaneous coronary intervention Percutaneous coronary Intervention, MI, coronary syndrome Percutaneous coronary syndrome, angina Intermittent claudication, stroke Postoperative thromboembolic cardiac valve replacement complications, stroke

[13,38] [123, 124] [116] [117] [201]

[57, 118] [118]

[118] [125]

[126] [127] [122]

ADP: Adenosine diphosphate; AMI: Acute myocardial infarction; AMP: Adenosine monophosphate; COX-1: Cyclooxygenase-1; CVA: Cerebral vascular accident; P2Y12: Platelet membrane ADP receptor; GP: Platelet membrane glycoprotein receptor; IHD: Ischemic heart disease; MI: Myocardial infarction; NP: Not published; PAD: Peripheral arterial disease; PAOD: Peripheral arterial occlusive disease; PDE: Phosphodiesterase; TP: TP receptor; TS: Thromboxane A 2 synthase.

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Table 2. Antiplatelet agents in clinical development. Compound Phase Inhibitory Reversible Pro-drug Clinical mechanism(s) indications

Ref.

Arterial thrombosis, vascular stents, atherosclerosis Arterial thrombosis, acute coronary syndrome

[40,128]

Yes

Arterial thrombosis, acute coronary syndrome

[118]

Yes

No

Arterial thrombosis, acute coronary syndrome

[118]

PDE and TS

NP

NP

[129]

II

P2Y12

Yes

No

APD791

I

NP

NP

EV-077– 3201–2TBS

I

5-HT2A antagonist TP and TS

Yes

No

PAOD, intermittent claudication, atherosclerosis Arterial thrombosis, acute coronary syndrome Arterial thrombo-embolic disease NP

Terutroban

III

TP

NP

No

Cangrelor

III

P2Y12

Yes

No

Prasugrel

III

P2Y12

No

Ticagrelor

III

P2Y12

Parogrelil

III

Elinogrel

[118]

[202] [203] [119]

Other approaches to develop platelet inhibitors include inhibition of von Willebrand factor by nanobodies (camel antibodies), ALX-0081 and ALX-0681, and to interfere with the platelet-collagen interaction by a humanized collagen binding protein, PR-15. MI: Myocardial infarction; NP: Not published; P2Y12: Platelet membrane ADP receptor; PAD: Peripheral arterial disease; PAOD: Peripheral arterial occlusive disease; PDE: Phosphodiesterase; TP: TP receptor; TS: Thromboxane A 2 synthase.

and renal actions. Efficient TP-receptor antagonists will inhibit activation of the TP receptors by HETEs and isoprostanes (Figure 1) [33] . This is in contrast to aspirin, which has no effect on the formation of HETEs and isoprostanes and their activation of the platelet TP receptors. It should be emphasized that the synthesis of HETEs and isoprostanes is increased during inflammatory conditions, such as triggered by the continuous inflammation/oxidant stress in atherogenesis and in patients with diabetes [78,204] . Therefore, efficient antagonism of the platelet TP receptor should improve protection against the risk of thrombus formation in individuals with advanced atherosclerosis, since the effect of all known agonists of the TP receptors is most likely neutralized. The antagonism/inhibition of the TxA 2 pathway by picotamide appears more efficient to reduce the progression of vascular lesions than that achieved by the inhibition of COX-1 by aspirin [13] . This may improve the beneficial effect in reduction of potential platelet activation and platelet and fibrin deposition at vascular sites of arterial lesions, since circulating platelets of symptomatic Type 2 diabetics are in a more activated state than in healthy individuals [79] . Antagonism of the platelet TP receptor and TS inhibition may also be of benefit for the acute coronary syndrome. It is also important to note that activated platelets do release the vascular smooth muscle 6


cell mitogen PDGF-B, thus promoting atherogenesis [46] . The potential beneficial effects of the picotamide and terutroban TxA 2 antagonism/ inhibition on vascular endothelium and smooth muscle cells in PAD are discussed below. Another potential clinical indication for TxA 2 antagonism/inhibition is vascular stent-induced thrombus formation. This procedure is haunted by both thrombogenesis and restenosis [44] . A recent study in pigs with a stent inserted into an arteriovenous shunt demonstrated clearly that the TP-receptor antagonist terutroban is a significantly more potent inhibitor than aspirin of stent-induced thrombus formation [77] . However, clopidogrel remained the most potent inhibitor of thrombus formation in this particular study [77] . It should be emphasized that this study was performed at blood flow conditions representative of veins when aspirin is known to have virtually no effect on thrombus formation. Therefore, this information is not really representative of arterial blood flow conditions. Nevertheless, the rapidly increasing number of stent implantations and the unfortunate development of early- and late-stage thrombus formation, as well as restenosis, may represent additional indications for TP-receptor antagonists, and dual TP-receptor antagonists and TS inhibitors owing to their beneficial and simultaneous effects on arterial thrombus formation and stenosis initiation and growth. Such antagonists/inhibitors may in the future science group


future be considered for the currently described prophylaxis/treatment regimens of aspirin and/or aspirin plus clopidogrel [44] . Other clinical indications for TP-receptor antagonists and/or TS inhibitors include patients resistant or hyporesponsive to aspirin and clopidogrel, or to both aspirin and clopidogrel. Unfortunately, the identification and characterization of aspirin hyporesponsiveness and resistance to aspirin and clopidogrel have not yet been clinically well defined. As such, reported frequencies of these phenomena vary from 0 to 57% for aspirin, and from 5 to 44% for clopidogrel [80,81] . The frequency of dual hyporesponders to both aspirin and clopidogrel is reported to be 6% [82] . Potential mechanisms of aspirin hyporesponsiveness/resistance (Box 1) and clopidogrel hyporesponsiveness/resistance (Box 2) are listed below [78,83,84,204] . In addition, it should be mentioned that aspirin ingestion may trigger gastrointestinal discomfort and that both aspirin and clopidogrel, as with all antithrombotic treatments, may trigger hemorrhagic bleeding episodes. In conclusion, targeting the TP receptor, both with or without simultaneous inhibition of TS, is a promising strategy that may result in a significant inhibition of arterial thrombogenesis, atherogenesis and renal proteinuria. This strategy may be particularly beneficial for diabetic patients with PAD, for situations of acute vessel injury with production of isoprostanes (acute coronary syndrome), patients with vascular stents and patients with hyporesponsiveness/ resistance to aspirin and/or clopidogrel. The prominent role played by the TXA 2 pathway in arterial thrombosis supports the notion that TP receptor/TS antiagents represent an attractive

Review

alternative for antithrombotic prophylaxis and treatment of individuals hyporesponsive to aspirin and/or clopidogrel. Clinical indications of pharmaceutical TxA2 interventions in atherogenesis & vascular stent-induced re-stenosis

Targeting the TxA 2 pathway to reduce arterial thrombosis is well established and is recommended in current practice. However, there are less data showing that targeting this pathway is effective to reduce atherogenesis. Continuous administration of aspirin to ApoE-deficient mice was reported to reduce the atherosclerotic lesion at the arterial sinus [85] . However, a recent study with continuous administration of the same dose of aspirin or aspirin and clopidogrel had no effect on the atherosclerotic progression in ApoE-deficient mice [86] . This apparent discrepancy makes it impossible to draw any conclusion with regard to a beneficial effect of aspirin on atherogenesis, at least in this animal model. However, the intervention of TxA 2-mediated atherogenesis with picotamide in patients suffering from Type 2 diabetes mellitus and PAD demonstrated a significantly decreased formation and progression of vascular lesions, despite the low number of patients enrolled [13] . This group of patients may represent a clinical indication for pharmaceutical intervention by potent and selective TxA 2 pathway antagonists/ inhibitors to decrease the formation and progression of atherosclerotic lesions. In this regard it is also interesting to note that exposure of picotamide to vascular endothelium increases the synthesis and secretion of PGI 2 , which is a potent inhibitor of platelet aggregation

Box 1. Mechanisms which may trigger low response to aspirin. Aspirin hyporesponsiveness and/or resistance: reduction or inability of aspirin to inhibit platelet TxA 2 production Concurrent intake of certain non steroidal anti-inflammatory drugs (e.g., ibuprofen and indomethacin) possibly preventing the access of aspirin to the COX-1 binding site. n Increased turnover of platelets. n Polymorphisms of COX-1 gene. n

High on-treatment platelet hyper-reactivity: high platelet reactivity despite adequate inhibition of platelet TxA 2 biosynthesis Amplification pathways that overcome TxA2 pathway blockade, for example, ADP, epinephrine, thrombin and specific G-proteins. n Biosynthesis of TxA by pathways that are not blocked by aspirin, for example, COX-2 in monocytes/ 2 macrophages and vascular endothelial cells. n Biosynthesis of HETEs, (e.g., 11(S)- and 15(S)-hydroxyeicosatetraenoic acids) and isoprostanes (e.g., 8-epi-PGF2 and 9a11ßPGF2), particularly in advanced atherosclerosis, symptomatic Type 2 diabetes and other conditions with high oxidant stress. n Polymorphisms of various platelet-receptor genes. n



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Box 2. Mechanisms that may trigger low response to clopidogrel. Clopidogrel hyporesponsiveness and/or resistance – reduction or inability of clopidogrel to form active P2Y12 receptor antagonist Decreased plasma drug levels due to decreased gastrointestinal absorption. Decreased hepatic metabolism of pro-drug to active drug due to reduced cytochrome P450 activity in genetic variants. n Decreased hepatic metabolism to active metabolite due to inhibition of cytochrome P450 by other drugs. n Polymorphisms in the P2Y12 receptor that reduce its affinity for the active metabolite of clopidogrel. n n

High on-treatment platelet hyper-reactivity – high platelet reactivity despite adequate inhibition of P2Y12 Amplification pathways that overcome ADP pathway blockade, for example, TxA 2, epinephrine, thrombin and specific G-proteins. n Polymorphisms of various platelet-receptor genes. n

and a potent promoter of vasodilatation [87] . Furthermore, it should be emphasized that the beneficial PGI2 synthesis and secretion induced by TS inhibition may also be associated with an increase in the TXA 2 precursors prostaglandin endoperoxides PGG2 and PGH2, which are agonists of the TP receptors (Figure 1) . However, antagonism of the TP receptor will block this activation as was shown in a canine model of coronary thrombosis where TS was inhibited by U63,557a and the TP receptor by L636,499 [88] . Terutroban has been demonstrated to reduce the development of atherosclerotic lesions and regression of advanced atherosclerotic plaques in ApoE-deficient mice and in New Zealand White rabbits, respectively [12,14] . The latter observation suggests that TP-receptor antagonism may not only delay the development of atherosclerotic lesions, but also transform lesions towards a more stable phenotype. These live animal studies do indeed confirm a prominent role of TxA 2 in atherogenesis, and they are in agreement with observations of picotamide in patients with Type 2 diabetes mellitus and PAD [13] . Effects of aspirin and clopidogrel on the vessel wall

Both aspirin and clopidogrel have beneficial effects on the vessel wall. They inhibit platelet activation and the subsequent release of the vascular smooth muscle cell mitogen PDGF-B [43] . In addition, aspirin is reported to protect against the development of diabetic retinopathy in both man and rat [89,90] . This is in contrast to clopidogrel, since studies on potential beneficial effects on streptozotocin-induced diabetic rat retinopathy are not conclusive [89,91] . Another beneficial effect of aspirin is the biosynthesis 8


of the vasodilator nitric oxide (NO) in vascular endothelium [92–94] . On the other hand, aspirin blocks the endothelial biosynthesis of the potent vasodilator and platelet aggregation inhibitor PGI 2 [87] . Clopidogrel has an apparent inhibitory effect on atherosclerosis triggered by transplanted vascular tissue; the hallmark feature of chronic transplant rejection [95] . This is at least the case for mice, since monotherapy with clopidogrel effectively reduces transplant atherosclerosis in a murine aortic allograft model [95] . Finally, and as mentioned above, the potential beneficial effect of aspirin on atherosclerotic progression in ApoE-deficient mice is currently inconclusive, and a study with combined administration of aspirin and clopidogrel had no effect on atherogenesis in the same animals [85,86] . Thus, the direct beneficial effects of the antiplatelet agents aspirin and clopidogrel on the vessel wall appear rather limited when compared with the dual TP-receptor antagonist and TS inhibitor picotamide, and to the TP-receptor antagonist terutroban as considered and discussed above [12–14] . However, it should be emphasized that the beneficial effects of terutroban on the vessel wall have been observed in live animal models, and as such need to be confirmed or not confirmed in man. Increased tendency of thrombogenesis & atherogenesis in patients with Type 2 diabetes mellitus

Patients with Type 2 diabetes mellitus have more reactive and activated platelets in the circulation, increased vascular inflammation, more advanced arteriosclerosis and a higher risk for thrombus formation than their age-matched population [96–98] . future science group


The increase in platelet activation and reactivity is characterized by increased levels of platelet surface adhesion molecules such as GPIIb–IIIa and P-selectin [96–98] . This is paralleled with an increased shear-induced platelet adhesion to the endothelial extracellular matrix [99] . Enhanced platelet TxA 2 biosynthesis and increased levels of isoprostanes derived from arachidonic acid by nonenzymatic lipid peroxidation are also present [100] . This is also the case for patients with nonhyperlipidemic Type 2 diabetes mellitus [101] . In addition, hyperinsulinemia is associated with elevated plasma levels of plasminogen-activated inhibitor-1, impaired fibrinolysis and myocardial reinfarction [102] . Current antithrombotic therapy includes the antiplatelet agents aspirin and clopidogrel, either alone or in combination. The advanced process of arteriosclerosis is apparently promoted by the increased levels of the TxA 2 biosynthesis, the formation of isoprostanes and HETEs and their deleterious effects on vascular endothelium and smooth muscle cells [12,43,47,48] . ACE inhibitors, angiotensin II-receptor blockers, statins and thiazolidinediones, for example, rosiglitazone and pioglitazone, have anti-inflammatory properties that reduce the risk of atherosclerosis independent of their effect on blood pressure, lipids and glucose. It is fair to conclude that potent and selective TxA 2 pathway antagonism/inhibition of the vascular PT receptors and TS could be of significant benefit for the diabetic population. This is a result of their inhibition of platelet activation and, thus, arterial thrombus formation, and their simultaneous inhibition of initiation and growth of arteriosclerotic lesions triggered by TXA 2, particularly by the predominantly formed isoprostanes and HETEs in these patients. Such beneficial effects have already been indicated by two clinical studies with picotamide in patients with Type 2 diabetes mellitus and PAD [13,38] . Clinical indications of pharmaceutical TxA2 intervention of renal proteinuria

Proteinuria has been identified as a renal and cardiovascular risk factor [103] , not only in patients with chronic kidney disease, but also in patients with cardiovascular diseases. This applies to the entire range of proteinuria, from microalbuminuria to overt proteinuria. A recent publication from the Framingham group mentions a threefold-higher risk of cardiovascular disease in patients with albuminuria either at or higher than the gender-specific median [104] . In the African-American Study of Kidney Disease and Hypertension (AASK) study, there was a statistically significant relationship future science group

Review

between increasing levels of proteinuria and faster decline in renal function, as measured by glomerular filtration rate [105] . Patients with diabetic nephropathy from the Reduction of Endpoints in non-insulin–dependent diabetes mellitus with the Angiotensin II Antagonist Losartan (RENAAL) study with proteinuria higher than 1.5 g/g creatinine had a 1.92-fold higher risk for cardiovascular end points and a 2.7-fold higher risk for heart failure when compared with patients with proteinuria less than 1.5 g/g creatinine [106] . Reduction of proteinuria in the RENAAL patients resulted in a statistically significant decrease in cardiovascular end points. In patients with chronic kidney disease, the higher the proteinuria, the more difficult the management of the patient becomes. Common clinical wisdom suggests a cut-off point of approximately 1 g protein/24 h for patients to become more easily manageable. Unfortunately, most patients with proteinuric renal diseases have significantly higher levels of proteinuria, even after treatment. The relationship between TxA 2 and proteinuria has been explored for over 25 years. It has been demonstrated that there is a modification in the metabolism of arachidonic acid in animals with proteinuria [107] . The increased TXB2 synthesis tends to run in parallel with the appearance of proteinuria and reach its peak simultaneously with the highest protein excretion rate. A number of studies, published mainly in the 1990s, have shown the importance of this mode of action. The use of TS inhibitors lowers both proteinuria and the urinary excretion of TXB2 significantly, as discussed above. In patients with Type 2 diabetes, the TP-receptor antagonist and TS inhibitor picotamide reduced exercise-induced albuminuria [108–110] . In summary, there is clinical evidence that TS inhibitors or a dual TP-receptor antagonist and TS inhibitor lower proteinuria. Small proof-ofconcept studies in man have demonstrated that drugs acting on the TP receptor and/or TS do decrease proteinuria – Table 3 lists the available data in the literature, which encompass quite a wide group of proteinuric diseases, from Type 2 diabetes mellitus to nephrotic syndrome, including pre-eclampsia. All of these studies, albeit of short duration, have demonstrated significant decreases in proteinuria. In some of them, such as Barnett’s, a follow-up period devoid of treatment showed that the albumin excretion rate (AER) rose again within a few weeks of stopping the drug, making a stronger case for the link between the use of this type of drug and reduction in proteinuria. www.futuremedicine.com

9

Review


Table 3. Effect of TS inhibitors and a TP antagonist/TS inhibitor in proteinuria. Compound

Mode of action Disease

No. of patients Duration of treatment Proteinuria

Year

Ref.

UK-38,485

TS inhibitor

T1DM

30

16 week

From 32.2 ± 3 µg/min to 9 ± 1 µg/min

1984

[111]

Ozagrel (OKY-046) Ozagrel (OKY-046)

TS inhibitor

Nephrotic syndrome T1DM/DN

11

8 week

1988

[112]

7

8 week

1990

[113]

Picotamide

TP antagonist and T2DMTS inhibitor Exerciseinduced proteinuria

15, 30

10 day, 1 year

From 6.4 ± 4.7 g/day to 2.6 ± 2.0 g/day From 524.9 ± 149.6 mg/gCr to 317.6 ± 90.6 mg/g/Cr 0.05 vs 0.13 (placebo) g/day; to 1/3 day

Rolafagrel (FCE22178)

TS inhibitor

T1DM/DN

24

1 week

From 2.01 ± 0.36 g/day 1993 to 1.6 ± 0.3 g/day

[114]

Ozagrel (OKY-046)

TS inhibitor

Preeclampsia

4

4–12 week

From 8.1 ± 11.5 g/day to 4.9 ± 7.6 g/day

[115]

TS inhibitor

1993, [109,110] 1998

1995

Cr: Creatinine; DN: Diabetic nephropathy; T1DM: Type 1 diabetes; T2DM: Type 2 diabetes; TP: TP receptor; TS: Thromboxane A 2 synthase.

In summary, there is pathophysiological and clinical evidence to support the role of dual TP-receptor antagonists and TS inhibitors in the treatment of proteinuric renal diseases. These drugs could be a useful addition to the therapeutic armamentarium, owing to their antiproteinuric properties, as well as to their potential to decrease cardiovascular morbidity and mortality. Conclusion

Antagonism of TP receptors and inhibition of TS are attractive targets for arterial thrombogenesis, atherogenesis and renal proteinuria

(Tables 1–3) .

Clinical indications include Type 2 diabetes mellitus and PAD, vascular stent implementation, particularly in symptomatic patients with Type 2 diabetes mellitus, acute coronary syndrome and renal proteinuria. The first two groups of patients are at risk of developing thrombogenesis and atherogenesis. Other clinical indications for TP receptor antagonists and/or TS inhibitors are patients with poor response to aspirin, clopidogrel, and to both aspirin and clopidogrel, particularly in clinical conditions where isoprostanes and HETEs are formed.

Executive summary Medical need There is a large unmet medical need for new and improved agents antagonizing/inhibiting the deleterious thromboxane A 2 (TxA 2)mediated effects on arterial thrombogenesis and atherogenesis. n There are virtually no agents registered and only a few agents in clinical development antagonizing the deleterious TxA -mediated 2 effects on endothelium and smooth muscle cells in the process of atherogenesis. n There is a large unmet medical need for new and improved agents for renal proteinuria in adult nephritic syndrome and Type 1 and Type 2 diabetes mellitus.

n

Current status The TxA 2 targets in thrombogenesis are gaining substantial interest, resulting in the development of novel antagonists/inhibitors, since more potent and specific agents than aspirin and picotamide are needed. n The role of TxA in atherogenesis has gradually been recognized, resulting in the development of new antagonists/inhibitors also for 2 this indication – major clinical indications are patients with Type 2 diabetes mellitus and peripheral arterial disease, and patients with implanted vascular stents. n The role of TxA on renal proteinuria in adult nephritic syndrome and diabetes has been established. 2 n

Conclusion TP receptors and TS are attractive targets for arterial thrombogenesis, atherogenesis and renal proteinuria. It is imperative that new efficient and safe TP receptor antagonists and/or TS inhibitors are being developed to battle arterial thrombogenesis, atherogenesis and renal proteinuria.

n n

Future perspective It is anticipated that novel TP receptor antagonists and/or TS inhibitors may reach the clinic as registered agents with dual antithrombotic and antiatherogenic efficacies, as well as with renal antiproteinuric efficacy within a few years.

n

10




Future perspective

It is apparent that the TxA 2 pathway plays a prominent role in thrombogenesis, atherogenesis and renal proteinuria. As such, pharmaceutical intervention targeting TP receptors and TS are gaining interest in cardiovascular and renal diseases. The importance to have selective and safe antagonists and inhibitors of various platelet activation targets are important for the efficient prophylaxis/treatment of patients with poor response to aspirin and/or clopidogrel. This is also the case for a potential simultaneous prophylaxis/ treatment with the same agent of both arterial thrombosis and arteriosclerosis. Therefore, results from on going and future clinical trials with novel TP-receptor antagonists and/or TS inhibitors are eagerly awaited (Table 2) . Bibliography Papers of special note have been highlighted as: n of interest 1.

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Financial & competing interests disclosure

Kjell S Sakariassen and Jessica Mann are members of Evolva Scientific Advisory Board. Pierre Fontana and Henri Bounameaux have received Evolva Grant Research Support. Peteris Alberts and Alexandra Santana Sorensen are Evolva employees. Evolva has a dual TP receptor antagonist and TS inhibitor, EV-077-3201-2TBS, in Phase I studies as indicated in Table 2. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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Kjell S Sakariassen KellSa s.a.s., Str. Campo e Zampe 12, I-13900 Biella, BI, Italy Tel.: +39 015 252 4359 Fax: +39 015 252 7615 [email protected] Peteris Alberts Evolva SA, CH-4123 Allschwil, Switzerland Tel.: +41 61 485 2000 Fax: +41 61 485 2001 [email protected]


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Pierre Fontana Division of Angiology & Haemostasis, Faculty of Medicine, University, Hospitals of Geneva, CH-1211 Geneva, Switzerland Tel.: +41 22 379 59 38 Fax: +41 22 372 92 99 [email protected] Jessica Mann Cardiovascular Development Consulting GmbH, Hirzbodenweg 5, CH-4052, Basel, Switzerland Tel.: +41 78 634 1856 [email protected] Henri Bounameaux Division of Angiology & Haemostasis, Faculty of Medicine, University, Hospitals of Geneva, CH-1211 Geneva, Switzerland Tel.: +41 22 372 92 92 Fax: +41 22 372 92 99 [email protected] Alexandra Santana Sorensen Evolva SA, CH-4123 Allschwil, Switzerland Tel.: +41 61 485 2000 Fax: +41 61 485 2001 [email protected]

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