Thrombolytic approaches to stroke: Thrombosis arises from the activation of the coagulation cascade and the adhesion and aggregation of platelets. The coagulation cascade is made up of a series of enzymatic reactions, which cause the build up of insoluble fibrin. In healthy individuals, the fibrinolysis pathway is activated almost immediately after the initiation of the clotting process. Central to this pathway is plasminogen. Plasminogen is a single-chain glycoprotein with 791 amino acid residues circulating in the bloodstream. Conversion to the active form of plasminogen, plasmin, involves cleavage at the arg-val bond between residues 561 and 562, resulting in the formation of a 2-chain plasmin molecule held together by 2 disulfide linkages. The heavier chain contains 485 residues and the lighter chain 233. The main function of plasmin is the digestion of fibrin in blood clots. Plasmin is a proteolytic enzyme with a similar specificity to that of trypsin. Like trypsin, plasmin belongs to the family of serine proteinases, in which the active site is situated in the light chain. Plasminogen activation also plays critical roles in cell migration related to tumor growth and metastasis. Consequently the plasminogen-plasmin system is under tight control:

• Controlled activation of plasminogen: The conversion of plasminogen to plasmin normally only occurs when the thrombolytic pathway is switched on. This switch mechanism involves tissue plasminogen activator (t-PA) and urokinase, both of which cleave arg-val bond between residues 561 and 562.

• Plasmin Neutralization: A number of circulating proteins neutralize plasmin. The most well known of which, a-2-antiplasmin inhibitor (also termed primary plasmin inhibitor), is the most potent and rapidly acting of the plasmin inhibitors and is thought to be important in the regulation of fibrinolysis in vivo. The importance of this protein is manifested by Miyasato disease, a genetically inherited disease characterized by reduced levels of a-2-antiplasmin and mild spontaneous bleeding (Koie et al, 1978). a-2-antiplasmin and plasmin rapidly form a completely inactive 1:1 stoichometric complex through reaction with the B chain (light chain) of the enzyme, which contains the active center.

• Negative feedback controls: The production of plasmin reduces the expression of t-PA and increases the expression of plasminogen activator inhibitor (Shi et al, 1992). The latter inhibits t-PA and hence both effects represent negative feedback control mechanisms.

The ability of t-PA to activate plasmin has been exploited through the therapeutic development of various forms of t-PA (eg Alteplase, Reteplase & Tenecteplase), each of which convert plasminogen to plasmin (step 2 in the above figure), which in turn degrades fibrin (step 3), the main component of the clot. The thrombolytic pathway can also be activated by streptokinase, a 414-residue protein secreted by hemolytic strains of Streptococci, however the mechanism through which this occurs is different to that of t-PA. Streptokinase has no proteolytic activity and instead it forms a complex with plasminogen (or plasmin) causing a conformational change which exposes the active site - thus evoking autocatalytic conversion of plasminogen to plasmin. Hence, the thrombolytic activity of streptokinase is considered to be indirect. The first generation streptokinase was introduced to the market in the 1960's. More recently, second generation drugs now offer significant improvements (e.g. front-loaded administration). Third generation drugs have offered still greater opportunities through the production of modified recombinant proteins that can be delivered as single bolus agents (e.g. Tenecteplase, Reteplase). Of marketed or experimental thrombolytic agents, Alteplase remains the only one that has been demonstrated to be beneficial when administered intravenously for the treatment of acute ischemic stroke (NINDS study 1995). Based on available data, the stroke council (a component of the American College of Cardiology) concluded in 1996 that recombinant t-PA, given within 3 hours of symptom onset, can improve neurologic outcome of selected acute stroke patients. However since symptomatic and fatal hemorrhage is more common in patients receiving t-PA than in those receiving placebo, it was recommended that thrombolytics be used only in the event that emergency back-up resources were available to handle such complications. This along with concerns of bleeding within the physician community underlie US figures showing that t-PA is administered to less than 3% of acute ischemic stroke patients (Katzan et al, 2000; Morgenstern et al, 2002).

Therapeutic options in the treatment of neurodegeneration secondary to ischemia: Once thrombotic occlusion occurs, the ischemic cascade is activated (depicted in the figure to the left after Lyden & Wahlgren, 2000). These authors offer an excellent overview of the therapeutic approach to the various steps in this cascade. During ischemia there is excessive activity of excitatory amino acids, especially glutamate. Within minutes of onset of ischemia, there is a marked increase in glutamate concentration in the affected portion of the brain. There are 2 main reasons for this surge of glutamate. First, the failure of mitochondrial adenosine triphosphate (ATP) synthesis causes neurons to depolarize and fire action potentials, leading to the release of glutamate. The second process may be the reversal of the glutamate uptake transporter, caused by the increase in extracellular potassium. Under physiologic conditions, glutamate does not cause neuronal damage. When ATP generation fails and the membrane potential falls, glutamate becomes neurotoxic. Although the final step from excitotoxicity to neural cell death is reflected in the increase in calcium levels, molecules that target calcium channels or transporters have to date proved to be without use in stroke. This is due to the wide variety of such proteins and the inability to target relevant proteins with sufficient specificity. Thus, the modulation of glutamate is more likely to represent a potential therapeutic strategy. Glutamate and it's receptors are attractive targets for neuroprotective drugs because glutamate plays a central role in the excitotoxic cascade. Glutamate release is regulated by sodium and hence sodium channel blockers, such as Sipatrigine, have been assessed in formal clinical trials. However, little therapeutic activity has been demonstrated. In contrast, dose-limiting neuropsychiatric effects were reported (Muir et al, 2000). The NMDA receptor is associated with a cation channel, which will only open in response to glutamate if glycine and polyamines are already bound to these obligatory modulatory sites. Gavestinel is selective for the glycine-binding site, and eliprodil for the polyamine site. Clinical data from stroke trials have yet to be published for Eliprodil. Gavestinel on the other hand failed to deliver any beneficial effects in patients with acute ischemic stroke (GAIN International, 2000). Another proposed strategy for blocking NMDA receptor function is the infusion of magnesium. The NMDA receptor is normally blocked by magnesium ions and will only respond to glutamate when this magnesium-induced block is removed on depolarization. A large clinical trial to investigate possible neuroprotection by magnesium is underway. Perhaps the most promising approach to date has been to target the GABA receptor. This receptor is the major inhibitory neurotransmitter in the brain opening the chloride channel subunit of the GABA-A receptor. The resulting increase in chloride conductance leads to hyperpolarization of neurons, including those that release glutamate. One advanced GABA-A agonist, Clomethiazole was investigated in a large, randomized, double-blind study (CLASS, 2002). Once again, clinical studies failed to demonstrate a therapeutic effect in stroke patients. In summary, a number of initially promising approaches have been taken focusing on neuronal excitotoxicity and its potential relevance in ischemic stroke, however little success has been noted in the clinic to date with any compound.

A further area of study has, paradoxically, focused on the ability of key components of the thrombolytic pathway to mediate neural degeneration in response to excitotoxic molecules/cerebral ischemia. In a ground-breaking study, Tsirka et al (1995) demonstrated that mice deficient in t-PA are resistant to neuronal degeneration produced by exitotoxic molecules. This phenomenon was suggested to involve the activation of plasminogen and consistent with this proposal was the finding that infusion of plasminogen activator inhibitor-1 was able to protect against exitotoxic degeneration. Likewise both gene deletion, and plasmin neutralization with (Tsirka et al, 1997).

Further studies examined whether these results can be extrapolated to more specific models of stroke. Wang et al (1997) recently demonstrated that neuronal damage after focal cerebral ischemia was reduced in mice with t-PA deficiency and exacerbated by t-PA infusion. These findings were further validated during key studies reported by University of Leuven researchers (Nagai et al, 1999). This group has developed a complete panel of knock-out mice which contain genetic deletions in each of the components of the coagulation cascade in order to investigate the role that key thrombolytic molecules play in the response to cerebral artery occlusion. These studies demonstrated that inactivation of the t-PA gene reduced the size of infarcts, while knocking-out the plasminogen activating inhibitor (which indirectly increases t-PA) increased infarct size. Likewise, reactivation of these genes by gene transfer resulted in the formation of larger and smaller infarcts respectively. These data all support the hypothesis that the plasmin(ogen) system contributes to ischemic cell death and is entirely consistent with the conclusions drawn by Tsirka et al (1997). Paradoxically however, and at discordance with earlier conclusions, plasmin(ogen) itself appeared to protect mice from the effects of focal cerebral ischemia. This was demonstrated directly - the inactivation of plasminogen resulted in larger infarct - and indirectly - knocking-out a-2-antiplasmin reduced infarct size, a phenomenon reversed by re-administering recombinant a-2-antiplasmin. The authors explain this paradox by suggesting that key regulators of the thrombolytic pathway (eg t-PA) have neurodegenerative activities which are independent of plasminogen activation. It has subsequently been shown that rt-PA may be a direct activator of certain ion channels which increase excitotoxic mediator release into ischemic tissue (Nicole et al, 2001). In addition, t-PA was shown to stimulate microglia through a plasmin(ogen)-independent mechanism (Tsirka et al, 1997). Furthermore the activation of microglia is also likely to be detrimental since these cells participate directly in neuronal degeneration (Lang & Bishop, 1993) through the secretion of glutamate (Streit et al., 1992; Patrizio and Levi, 1994), reactive oxygen intermediates (Piani et al., 1992), or cytokines (Prehn and Krieglstein, 1994). Finally, in contrast to t-PA deletion, knocking out the u-PA gene failed to reduce infarct size, suggesting that there was a selective involvement of rt-PA and antiplasmin.

Thus, in addition to presenting a risk of hemorrhage, t-PA may also worsen excitotoxic damage following stroke. Consequently, although t-PA can re-establish cerebral perfusion, it may also compound the neurodegenerative effects of ischemia in the absence of arterial reperfusion. This may explain in part the short therapeutic window for t-PA, and furthermore it suggests that if patency is not rapidly established, t-PA may actually worsen the clinical outcome. Instead of administration of t-PA, patients may therefore benefit more through the use of other strategies that increase plasmin and/or reduce a-2-antiplasmin, thereby reducing rather than increasing exitotoxic neurodegeneration. Unfortunately the use of plasmin itself is unlikely to fulfill such requirements since it is too large and complex a molecule, making production and administration complicated. Two alternate options have been investigated, the blockade of a-2-antiplasmin or the administration of mini/microplasmin.

Market competition: The results of a search of pharmaceutical databases performed to identify the level of activity focused on stroke is shown in the figure to the left. Not too surprisingly, the pharmaceutical attention paid to this indication is considerable, with a total of 296 products listed. This equates to about 3% of all molecules in development across the pharmaceutical industry. The majority of molecules are in preclinical development, as is the case for all drug classes, however it is of note that an above average proportion of molecules are in this phase (51% compared to an industry average of 42%). Conversely, a below average proportion of molecules is on the market (10% versus 28%). This suggests that pharmaceutical activity within the stroke market is currently biased towards early development. This mirrors the limited pharmaceutical arsenal currently available to clinicians, and the continuing high human and economic cost associated with stroke. A second observation is that the pharmaceutical approach to stroke is extremely broad. Although neuroprotective agents represent an important class of stroke therapies in development, they represent only a small proportion of total stroke drugs. Those in advanced development are given in the table to the left. Thrombolytics have attracted significant interest (approximately 37 molecules are in development or on the market) however most have been developed for myocardial ischemia. The number of such drugs in development for stroke is limited to only a handful of molecules including A74187, BB-10153, Monteplase and Pamiteplase. Likewise, very few thrombolytic agents have been developed for PAOD even through the FDA have suggested that thrombolytics should be assessed for this indication irrespective of their primary target indication.

Comparison of microplasmin with other stroke treatments: Currently recommended pharmacological therapies for stroke focus on the use of t-PA; however, the time frame for effective use is short and consequently many patients are unsuitable for thrombolytic treatment. The additional problem of bleeding associated with this class of drugs means that as few as 3% of patients with ischemic stroke are treated with rt-PA. Perhaps even more concerning is the possibility that rt-PA may potentially worsen the neuronal damage associated with the activation of the ischemic cascade. An alternate approach is the use of neuroprotective agents; however, such molecules have been relatively ineffective in the clinic to date. Microplasmin appears to represent the advantages of both approaches but lacks their limitations. Microplasmin has thrombolytic activity and is thus expected to be able to recanalize blocked vessels. In contrast to rt-PA, it does not cause neuronal damage. In fact the opposite occurs, since it actually limits infarct size following thrombus free occlusion of the middle cerebral artery. Consequently, microplasmin offers a wide window of therapeutic opportunity limiting both phases of cerebral ischemia (ie ischemia and the response to ischemia). In this respect it is unique. In addition to stroke, microplasmin offers an alternative to rt-PA for the treatment of PAOD with a potentially reduced risk of hemorrhagic side effects.Finally, it represents an effective adjunct for vitreo-retinal surgery - an indication for which little competition exists.

Summary & strategic analysis: Stroke is the second most common cause of cardiovascular-related mortality after myocardial infarction. About 700,000 Americans suffer a new or recurrent stroke each year due to atherosclerosis in the carotid/vertebral arteries. Between 15-30% of victims are permanently disabled and 20% require institutional care. As a result stroke is the most common cause of long-term serious disability in the US and represents an economic burden similar in scale to myocardial infarction. Clinical guidelines have largely restricted pharmaceutical options to measures that limit the development of ischemia (ie thrombolytics/anticoagulants) however the window of therapeutic opportunity is vary narrow and the risk of side effects high. Hence thrombolytics are given to as few as 3% of patients. The current market is currently around $1.4 billion, however improving options to reach the remaining 70% of patients could increase the market to more than $3.8 billion by 2007. Current development is diverse incorporating multiple targets. The field of neuroprotection has emerged as one area receiving much attention although clinical success has been limited to date. ThromboGenics’ scientists have been developing microplasmin as a novel treatment of stroke. This molecule is a thrombolytic, and should therefore offer opportunities for the immediate treatment of stroke. However in direct contrast to rt-PA, microplasmin reduces neuronal damage in a model of thrombus free occlusion of the middle cerebral artery. Thus microplasmin represents the first stroke candidate that not only offers a window of opportunity that is consistent with the time frame of clinical needs, but also targets both the development of and the response to ischemia. Given these promising preclinical results, microplasmin is entering Phase I investigation in man in H2/2002 and will be advanced as a treatment strategy for acute ischemic stroke.

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