This dossier has been prepared for ThromboGenics by LeadDiscovery

Project number 0246

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Abstract One of the current priorities for cardiovascular drug discovery teams is to identify anti-thromotic candidates which carry a reduced risk of causing hemorrhage. The University of Leuvens in collaboration with ThromboGenics Ltd has developed a genetically modified mouse lacking the Gas6 gene product. Gas6-/- mice suffered reduced venous and arterial thrombosis and were protected against experimental thrombo-embolism. These mice did not, however, suffer spontaneous bleeding and had normal bleeding times after tail clipping. Data were confirmed through the use of anti-Gas6 monoclonal antibodies. The therapeutic effects of Gas6 blockade were shown to be due to the ability of Gas6 to amplify platelet aggregation and secretion in response to known agonists. Gas6 therefore appears to represent a novel cardiovascular target with an excellent preclinical proof of concept. An appropriate screening architecture has been validated and ThromboGenics is now seeking industrial partners to expedite the development of therapeutic candidates.

Background: A fully comprehensive overview of US cardiovascular statistics is available from the American Heart Association (click here). According to the definitions of this organization, cardiovascular disease includes 1. coronary heart disease (ischemia), 2. hypertensive disease, 3. rheumatic fever/rheumatic heart disease and; 4. cerebrovascular ischemia stroke. This classification does not include pulmonary embolism, which results from deep vein thrombosis (secondary to air travel and long-term hospitalization). A staggering 60,300,000 (or 1 in 5) Americans suffer some form of cardiovascular disease and this can be broken down into: High blood pressure (50,000,000); Coronary heart disease (12,400,000 including Myocardial Infarction and/or Angina); Stroke (4,500,000); Congenital cardiovascular defects (1,000,000) and; Congestive heart failure (4,700,000)

In order to appreciate the development and usage of pharmaceutical intervention it is first necessary to understand the process of hemostasis which is excellently reviewed in Merck's online manual of diagnosis & therapy (click here). Following injury or rupture of atherosclerotic plaques, platelet glycoprotein Ib receptors bind to von Willebrand factor on the exposed subendothelium of the damaged vascular wall causing adhesion. In addition platelet aggregation occurs as a result of platelet surface glycoprotein IIb/IIIa receptors binding fibrinogen and also due to increased sheer stress as platelets move through narrowed vessels. This accumulation contributes to the hemastatic plug. The subsequent stimulation of platelets by mediators such as ADP, PAF and thromboxane intermediates results in the release of factors that recruit additional platelets and also cause vasoconstriction. Finally a number of platelet derived factors such as platelet factor V stimulate production of thrombin and hence fibrin through the coagulation cascade. The production of polymeric fibrin bulks out the clot. A diagrammatic version of the cascade can be found at a number of web sites including the Stroke Center and is made up of two impinging pathways. The intrinsic pathway is initiated by factor XIa which converts IX to IXa and then Xa. The extrinsic pathway is activated by vascular tissue factors released from the endothelial wall upon injury. Tissue factors in the presence of factor VIIa also converts IX to IXa and then Xa, as well as converting factor X to Xa. At this point the two pathways converge by converting prothrombin to thrombin which in turn causes the generation of clots from fibrin. Countering this process is the fibrinolysis pathway which is initiated at the same time as clot formation by a number of mediators such as tissue plasminogen activator (tPA) and urokinase. These proteins convert plasminogen to plasmin which in turn degrades fibrin, the main component of the clot.

Treatments options are well reviewed on line (see for example Jones & Robinson, 2000).

Antithrombotic drugs: The earliest example of antithrombotic drugs is aspirin, which prevents thromboxane A2 formation and consequent platelet aggregation and mediator release. Thrombin-induced platelet aggregation remains unaffected however and furthermore, aspirin does not inhibit shear-induced platelet aggregation. Despite sub-maximal inhibition of platelet function, aspirin can reduce the incidence of myocardial infarction in unstable angina patients by 50% (Lewis et al, 1983) and it is recommended for all patients with suspect unstable angina. Likewise aspirin reduces the incidence of stroke in patients with a prior history of transient ischemic attack or stroke by 18% (Forbes, 1998) The newest class of antithrombotics inhibit platelet glycoprotein IIb/IIIa receptors (GRIs) which as mentioned above are responsible for platelet aggregation. Like aspirin and heparin, GRIs do not have thrombolytic activity and in addition they do not prevent platelet adhesion. Studies have shown that GRIs such lamifiban and tirobifan can reduce adverse ischemic events when administered to unstable angina patients in combination with aspirin (Paragon investigators, 1998; Prism Investigators, 1998). Similar studies have not been performed in patients at risk of stroke.

Anticoagulant drugs: The most common representatives from this class are the heparins which facilitate the action of circulating antithrombin III (AT III in figure above), an enzyme that inhibits thrombin and several other activated factors essential for the clotting cascade. Unlike aspirin, heparin directly blocks thrombin action. Although this action prevents new clot formation, it does not dissolve existing thrombus. Fractionated heparin (such as enoxaparin and dalteparin) is more attractive than unfractionated heparin, and has been reported to reduce myocardial infarction when given to patients with unstable angina as an adjunct to aspirin (FRISC study group, 1993) or alone (Antman et al, 1999). Heparins have not been shown to offer improved benefit to patients at risk of stroke.

Thrombolytic drugs: Fibrin-selective thrombolytic agents such as recombinant tissue-type plasminogen activator (rt-PA) were shown to be more effective than non-fibrin-selective agents such as Streptokinase (SK) for the early coronary artery recanalization in patients with evolving myocardial infarction. Treatment with non-fibrin-selective agents induces exhaustive plasminogen activation which results in fibrinogen breakdown, increasing the bleeding tendency, and which deprives the thrombus of plasminogen ("plasminogen-steal"), reducing the thrombolytic effect. The Global Utilization of Streptokinase and t-PA for Occluded coronary arteries (GUSTO) trial, which randomized approximately 10,000 patients revealed that 30-days mortality was significantly (p=0.001) lower with fibrin-selective rt-PA and intravenous heparin (6.3%) than with non-fibrin specific SK plus either intravenous or subcutaneous heparin (7.3%), thus saving an additional 10 lives per 1000 treated (The GUSTO investigators, 1993).

Even current fibrin-selective thrombolytic agents still suffer from a number of limitations. The average time to recanalization is at least 45 minutes, resistance to complete recanalization (TIMI flow) within 90 minutes is nearly 50%, and significant bleeding remains a problem. Several strategies including the construction of t-PA mutants with domain deletion or amino acid substitution have been undertaken to increase the potency or the specific activity of plasminogen activators. On the basis of our present understanding of molecular mechanisms of fibrinolysis, domain deletion and substitution mutants of t-PA (eg Reteplase or TNK-t-PA) will not constitute superior thrombolytic agents, although they have the advantage of bolus application. (ASSENT-2 investigators, 1999). Each of these agents remains very expensive (US$ 1,200-2,000 per dose) and despite their demonstrated efficacy and life-saving properties many countries have not adopted them as default therapy for acute myocardial infarction. In Europe SK (US$ 30 per dose) remains the drug of choice.

Anti-Ischemic Therapy: The earliest example of anti-ischemic drugs is nitroglycerin, first used for the relief of angina in 1879. Nitroglycerin relaxes vascular smooth muscle in veins, arteries, and arterioles. It causes systemic venous pooling, resulting in decreased preload and decreased myocardial oxygen demand. It may also vasodilate coronary arteries, thereby improving myocardial oxygen supply. Although there is no evidence that nitrates improve prognosis in unstable angina, a meta-analysis of trials of nitroglycerin in acute myocardial infarction demonstrates a 35% reduction in mortality (Yusuf et al, 1988). Beta-Blockers reduce myocardial oxygen demand by lowering heart rate and myocardial contractility. Beta-blockers have been shown to reduce mortality and reinfarction in post-infarction patients. They have been shown to reduce the risk of acute myocardial infarction in patient with unstable angina started acutely on beta-blockers by 13% (Yusuf et al, 1988b). Calcium channel blockers prevent vasospasm in angina and decrease myocardial oxygen demand by decreasing afterload, contractility, and heart rate. They may also cause AV node blockade and peripheral vasodilatation leading to hypotension. There are few data on the efficacy of calcium channel blockers in unstable angina and their use is limited to symptom control. Calcium channel blockers, preferably diltiazem, should be used only after nitrates and beta-blockers have failed to relieve symptoms.

Gas6 inhibitors as improved antithrombotic treatments: In 1988 a new family of genes was identified that appeared to play an important role in controlling the function of cells during cell arrest following the withdrawal of growth factors. The reintroduction of growth factors caused the repression of these genes termed growth-arrest specific genes (Schneider et al, 1988). Gas6 is a member of this family that was first characterized 5 years later (Manfioletti et al, 1993). The Gas6 protein is related to vitamin K-dependent protein S a well known anticoagulant whose mutation carries a severe risk of developing thrombosis (Borgel et al, 1997).

In 1995, both protein S and Gas6 were shown to bind Tyro 3 (alternatively called Sky, rse, brt, or tif) receptors expressed by osteoclasts, the testes, lung carcinoma malignant plasma cells and CNS cells (Nakamura et al, 1998; Schulz et al, 1995; Wimmel et al, 1999; De Vos et al, 2001; Lai et al, 1994). Gas6 also binds Axl (alternatively, Ark or UFO) receptors (Stitt et al, 1995) which are expressed by myeloid and erythro-megakaryocytic leukemias, endothelial cells and thyroid carcinoma but not in lymphoid malignancies (Challier et al, 1996; Melaragno et al, 1998, Ito et al, 1999). In 1996 Gas6 was reported to bind a third receptor, Mer (Nagata et al, 1996) which is expressed in the testes (Chan et al, 2000)

Despite the homology of Gas6 and vitamin K-dependent protein S it was considered unlikely that Gas6 should play a role in coagulation since it differs from protein S in that a peptide loop crucial for the anticoagulant activity of the latter is missing (Manfioletti et al, 1993). Thus unlike mice which lack Tyro3, Axl and Mer receptors, those lacking Gas6 do not suffer spontaneous bleeding or thrombosis (Angelillo-Scherrer et al, 2001) The exact role of Gas6:Axl binding has thus remained elusive. Receptor binding does not appear to play a mitogenic role in hematopoietic tissue (Avanzi et al, 1997). Gas6 has however been shown to evoke a variety of effects in endothelial cells including the:

 

• protection of growth arrested endothelial cells from apoptosis and the stimulation of their proliferation (Nakano et al, 1996 & 1997)

• inhibition of inflammatory cell adhesion (Avanzi et al, 1998)

• chemoattraction of endothelial cells (Fridell et al, 1998)

Each of these findings, along with the inability of Gas6 deficiency to promote bleeding implicates Gas6 as a therapeutic target for vascular injury and indeed it was subsequently shown that balloon injury induced Axl and Gas6 expression with a similar time course which paralleled that of neointima formation (Melaragno et al, 1998). Gas6 was further tied in with vascular protection by Ishimoto & Nakano (2000) who reported that platelets are able to release Gas6 upon stimulation with thrombin, ADP or collagen. Platelets were not only able to release Gas6 but they also expressed Tyro3, Axl and Mer receptors suggesting the presence of a positive feedback mechanism (Angelillo-Scherrer et al, 2001). This study also reported a number of additional data that together offers the greatest support to date for targeting Gas6 in cadiovascular disease.

• The effect of Gas6 deficiency on thrombosis was determined in three separate animal models: ligation of the abdominal vena cava; photochemical denudation of the carotid artery and; platelet-dependent thrombo embolism. In the first two models, the size of thrombi was reduced by 60-85% and in the third model mortality was reduced from 80% to under 20% in Gas6 deficient mice. Reduced thrombotic size was not due to increased thrombolytic activity.
  

• Compared to platelets from wild type mice those from Gas6-deficient mice displayed greatly reduced sensitivity to ADP, collagen or the thromboxane A2 agonist, U46619 with respect to irreversible aggregation. This defect appeared to be related to decreased fibrinogen release and consequent aggregation.
  

• Likewise, platelets from Gas6 deficient mice displayed considerably diminished granular release in response to stimuli including ADP, thrombin, U46619 and collagen.
  

• Gas6 deficient mice do not suffer exaggerated bleeding compared to wild type littermates, losing 166 and 172ml of blood respectively in tail clip experiments. Thus, Gas6 appears to be redundant for baseline hemostasis, but constitutes an important `amplification' system in pathological conditions.

  Gas6 neutralizing antibodies produced much the same response as displayed by Gas6 deficient mice further supporting the targeting of Gas6 as a means of reducing thrombosis without increasing the risk of potentially fatal hemorrhage.
  

• Human platelets expressed both Gas6 and all three known Gas6 receptors. Furthermore Gas6 antibodies prevented ADP-induced platelet aggregation

Patent position: A patent application (WO 00/76309) to protect the use of Gas6 deficient mice and also the development of Gas6 inhibitors for the treatment of endothelial dysfunction has been made but not granted. This should not preclude the development of specific proprietary inhibitors of Gas6.

Market size: According to ThromboGenics, in 1996, cardiovascular drugs had a global market of US$ 35 billion. Among the top 20 selling drugs, 6 were for cardiovascular indications, but none were antithrombotics. In 1999, annual sales of drugs for the treatment of thrombosis were US$ 6.5 billion. Antithrombotic sales can be broken down as follows:

In addition thrombolytics such as t-PA (US$ 400 million globally alone, with an estimated market size approaching US$1 billion) and anticoagulants (US$ 2.3 billion) represent a significant market. For ischemic stroke, approximately 600 million US$ are spent yearly, mostly on secondary prevention, while there is no generally effective treatment of acute ischemic stroke. The market for antithrombotic agents will be increased by the introduction of new drugs with improved efficacy/safety ratio, new administration routes (e.g. bolus injection allowing out of hospital application) and better cost-effectiveness.

Market competition: Gas6 can be considered an antithrombotic agent and consequently an analysis of representatives from this class was performed. In addition a review of anticoagulants was also conducted. At present 134 antithrombotics are listed as being in development. The graph to the right shows that the development of this class is in decline. If drugs in development are analysed across all classes the majority of molecules are in preclinical development (Pre) reflecting scientific advances and the identification of new targets. The proportion of molecules in clinical development (Clin) is generally low as molecules either progress to the clinical or they are replaced by improved candidates emerging from preclinical phase. This is not the case for antithrombotics whose success has been declining sharply over the past 4 years. In other words 50% less compounds are in preclinical development than 4 years ago, while very few new molecules have progressed through clinical trial. A breakdown of more advanced molecules is given in table 2 where it can be seen that the emergence of GP IIb/IIIa antagonists as a viable target and the continued development of thrombin inhibitors represents an isolated area of success. New molecules are therefore required to maintain the development of the antithrombotic field, especially those with improved safety profiles. On the other hand the development of platelet aggregation antagonists and eicosanoid related target has peeked. Fewer (51) anticoagulants are listed as in development or on the market however distribution paterns are similar to general pharmaceutical trends. Development of this field is however going through a resurgence. Although an above average proportion of products are on the market few molecules are in the clinic. On the other hand significant preclinical activity is observed. This trend largely reflects the development of new molecules related to the blood factors.

Comparison of Gas6 inhibitors with other targets:

Comparison of Gas6 inhibitors with other targets: Despite the usefulness of antithrombotic drugs in reducing the incidence of myocardial infarction and stroke in at risk patients, this benefit must be weighed up against the risk of hemorrhage. For example the oral GP IIb/IIIa blocker, lefradafiban at doses which tended to reduce cardiac events also caused bleeding in 7% of patients and further dose escalation produced an unacceptable risk (Akkerhuis et al, 2000). A second GP IIb/IIIa blocker, sibradafiban also caused serious bleeding in 5-6% of patients compared to 4% treated with aspirin (Symphony investigators, 2000). Fractionated heparins such as enoxaparin, like the other classes of antithrombotics caused major bleeding in about 6.5% of patients although minor bleeding was considerably higher with an incidence approaching 14% (Cohen et al, 1998). Like the antithrombotics, the thrombolytics also carry a risk of hemorrhage (about 4% for streptokinase) Consequently the primary aim in the development of new antithrombotic agents is to separate the inhibition of thrombus formation from the risk of hemorrhage. This is the clear advantage of targeting Gas6 which does not appear to carry a risk of bleeding.

Strategic analysis and suggested further studies: From this dossier it is clear that targeting Gas6 receptors is an excellent approach to thrombosis. This conclusion is based on data obtained from Gas6 deficient mice and evidence is therefore direct and unrelated to non-specific effects that can be problematic in pharmacological proof of concept studies. Furthermore these data is supported by antibody based studies. These mice were resistant to thrombosis in a range of different animal models and furthermore platelets, which play a key role in thrombosis appear to be directly involved in this phenomenon. It can therefore be predicted with confidence that Gas6 receptor antagonists will be antithrombotic. Perhaps of greatest importance is the observation that Gas6 deficiency was not related to hemorrhage. This is distinctly different to animals deficient in the three known Gas6 receptors and also antithrombotic molecules in clinical use. Gas6 receptor antagonists are therefore likely to carry significant market advantage. A number of points should be made clear however. Firstly Gas6 receptor antagonists have not yet been developed and furthermore it is not known which of the Gas6 receptors should be targeted. Each of the three known receptors are expressed by human platelets, however it is clear that targeting all three receptors would likely cause problems. Thus further studies should be performed to identify the preferred target receptor(s). Secondly it is not clear whether Gas6 blockade diminishes platelet responsivness similarly in health and disease. It may therefore be of use to compare the behaviour of platelets from healthy volunteers and angina patients in the presence and absence of neutralizing anti-Gas6 antibodies. If screening of Gas6 receptor antagonists is initiated, the following architecture is suggested.

Figure 1: Proposed screening architecture for the development of antithrombotic Gas6 receptor antagonists