SY161, a cost-effective treatment of acute myocardial infarction

Project number THR002

This dossier has been prepared for ThromboGenics by LeadDiscovery

This dossier is internet interactive - click on underlined terms for more details. References are linked free of charge to the PubMed service of the NCBI and will open up in a separate window named "Entrez". LeadDiscovery's PaperSet service allows readers to link through to entire bibliographies of relevant publications. Internet addresses often change so if you have any problems with links please contact us.

Important notes concerning this document: This document is best viewed using the medium sized font setting (Explorer users) or the "use document specified fonts" setting (Netscape Navigator users). This document strictly reflects the opinion of LeadDiscovery's editorial panel. While all reasonable efforts are made to ensure the accuracy of information provided LeadDiscovery takes no responsibility for incorrect or misleading information. LeadDiscovery is designed for educational and drug development purposes only and is not intended or designed to offer medical advice or advice of any sort, and must not be used for such purpose. The information provided through LeadDiscovery should not be used for diagnosing or treating a health problem or a disease and no reliance should be placed on any information contained in this abstract or elsewhere on LeadDiscovery's website. It is not intended to be a substitute for professional care. If you have or suspect you may have a health problem, you should consult your physician or other health care provider

Aim: LeadDiscovery is a company of industrial scientists dedicated to identifying areas of research with pharmaceutical/biotech potential. Using our two key services, TherapeuticAdvances and DiscoveryDossiers, we use our experience to help academic scientists or biotech companies highlight this potential. Equally we provide an impartial and non-commission based service to industry identifying field leaders and by suggesting how their research areas can be adapted to product development.

Important note for correct viewing of this document: This document is best viewed using the medium sized font setting (Explorer users) or the "use document specified fonts" setting (Netscape Navigator users).

Abstract Although streptokinase (SK) remains the most widely prescribed thrombolytic agent for acute myocardial infarction (AMI) in many parts of the world due to it's low cost, it is generally accepted that it demonstrates lower efficacy in terms of arterial patency and an absolute difference of 1% in mortality compared to more expensive agents such as t-PA. In addition, SK has a high incidence of allergic reactions, lacks thrombus specificity, and can only be administered once.

Staphylokinase, a molecule completely unrelated to SK, offers an alternative with considerably reduced antigenicity, greater fibrin-specificity, and an efficacy at least as good as t-PA in terms of arterial patency. The mechanism of action of staphylokinase is well-characterized, and to date it remains the most fibrin-selective plasminogen activator known. ThromboGenics’ scientists have exploited these differences to produce a proprietary variant of staphylokinase, SY161-P5. This variant exhibits a superior profile over wild-type staphylokinase, with reduced antigenicity, an extended half-life, and the ability to be administered as a single bolus. This property will allow physicians to rapidly administer this 3rd generation thrombolytic – even in an ambulance setting - which represents a distinct advantage over SK. In a recently completed Phase II clinical trial, SY161-P5 was as effective as Alteplase in terms of arterial patency at 60 minutes. SY 161-P5 has undergone extensive GMP development and toxicology, and can be produced at a low cost. In other words, SY161-P5 offers the therapeutic advantages of 3rd generation thrombolytics at a price which has so far only been able to be placed on less effective 1st generation products. SY161-P5 is currently under Phase II B evaluation and pharmaceutical partners are currently being sought to take this most exciting candidate through further rounds of investigation towards the clinic.

Human and economic costs of myocardial infarction: Cardiovascular diseases have been the most common cause of death in the US each year with the exception of 1918. In 1998, this group of diseases claimed almost 1 million deaths which translates to 40% of all deaths and 1 death every 30 seconds. Strikingly more people die from cardiovascular disease than the next six leading causes of death combined, and if cardiovascular disease were to be eliminated, the average life expectancy would rise by 7 years. As shown in the inset (above) coronary heart disease is by far the leading cause of deaths from cardiovascular disease. Of the 12 million or so Americans with coronary heart disease, some 1 million suffer new or recurrent coronary attacks each year. Forty percent of these patients die, 220,000 before even reaching hospital, making coronary heart disease the single leading cause of death in America (one of every 5 deaths). Correspondingly, the socio-economic cost of coronary heart disease is a profound economic burden and is broken down in table 1.

As mentioned above the effects of myocardial ischemia range in severity from angina pectoris to unstable angina and possibly acute myocardial infarction; in many cases this can result in sudden cardiac death. Mirroring this progression is a gradual shift in clinical approach. Treatments of angina aim to slow the formation of thrombi or to break up existing thrombi; to reduce ischemic damage by vasodilating coronary blood vessels or; by reducing the oxygen demands of cardiac tissue. Such treatments frequently involve the use of antithrombotic approaches. One novel target (still in early preclinical development) promising excellent efficacy and predicted to be without the risk of bleeding, a side effect of most current therapies, is described in an accompanying dossier (click here to access Inhibitors of Gas6 as improved anti-thrombosis candidates). On the other hand the initial aims in treating myocardial infarction are to minimize ischemic damage. Hence emphasis is focused on breaking up existing thrombi (thrombolysis) and slowing formation of further thrombi as an immediate and long-term objective. The two processes of thrombosis and thrombolysis are in a state of obligatory balance in order to prevent either excessive bleeding or clotting. The coagulation cascade is made up of a series of enzymatic reactions, which promote the build up of insoluble fibrin. Almost immediately after the initiation of the clotting process, the fibrinolysis pathway is activated. 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 the 2-chain plasmin molecule held together by 2 disulfide linkages. The main function of plasmin is the digestion of fibrin in blood clots. Plasmin is a proteolytic enzyme with a specificity similar to that of trypsin. Like trypsin, plasmin belongs to the family of serine proteases, 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, cerebral hemorrhage and some other pathologies related to cellular migration and adhesion. Consequently plasminogen activation is under tight physiologic control and normally only occurs when the thrombolytic pathway is switched on. This process 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 distinctly different from 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, subsequently promoting autocatalytic conversion of plasminogen to plasmin. Thus streptokinase is considered to be an indirect activator of plasminogen.

Excellent overviews of the various thrombolytics can be accessed on-line at both Merck's manual of diagnosis & therapy and also the US Pharmacist. The requirements of effective thrombolytics are clear. Fifty percent of deaths from acute myocardial infarction occur within 3 to 4 h of onset of the clinical syndrome, and thrombolytic treatments designed to break up thrombi should be quickly delivered. As a rule, effort should be made to achieve a "door to needle" time of <30 minutes. Rapid administration alone can reduce hospital mortality between 30 and 50%. In addition to rapid administration, thrombolytics should recanalize affected vessels rapidly and completely. Furthermore their half-life should be long enough to avoid the risk of clot reformation or the requirement of multiple administrations. Balanced against the benefits of thrombolytics, treatment carries a risk of significant side effects. Plasminogen occurs in two phases, fibrin-bound (ie in thrombi) and in the free plasma phase. Activation of circulating plasminogen may result in degradation of unbound fibrinogen as well as inactivation of clotting factors V and VIII, thus producing a systemic lytic state. This can increase the risk of hemorrhage. However, specific activation of fibrin-bound plasminogen through effective recanalization of the infarct-related artery has been shown to save an additional 10 lives/1000 treated. In addition to the risk of hemorrhage, some of the thrombolytics evoke an allergic reaction which is not only problematic in it's own right but the generation of neutralizing antibodies can diminish the efficacy of repeat administrations. A further issue that should be addressed is cost. Given the high incidence of myocardial infarction, price reduction could have an impressive global impact on the cost of treatment.

Four principal thrombolytics are currently available including Streptokinase (SK), Alteplase, Reteplase and Tenecteplase. Urokinase was previously available from Abbot (see warning letter). The first generation thrombolytic, streptokinase, was introduced to the market in the 1960's. More recently, second generation drugs have been developed that offer significant improvements and can be readily produced through the use of recombinant DNA technology. Third generation drugs have offered still greater opportunities through the production of modified recombinant proteins that can be administered as single bolus agents.

 

• Streptokinase (click here for full prescribing information) is a thrombolytic agent that is derived from Group C beta hemolytic streptococci and acts indirectly by first forming an "activator complex" with plasminogen. This results in a conformational change such that the complex becomes an active enzyme, which then activates other plasminogen molecules.

• Alteplase (rt-PA, Activase) (click here for full prescribing information) is a tissue plasminogen activator produced by recombinant DNA technology. It is synthesized using the complementary DNA for natural human tissue-type plasminogen activator obtained from a human melanoma cell line. The manufacturing process involves the secretion of the enzyme Alteplase into the culture medium by Chinese Hamster Ovary cells into which the cDNA for Alteplase has been genetically inserted. Alteplase produces limited conversion of plasminogen in the absence of fibrin. When introduced into the systemic circulation at pharmacologic concentration, Alteplase binds to fibrin in a thrombus and converts the entrapped plasminogen to plasmin. This initiates local fibrinolysis with limited systemic proteolysis.

The therapeutic advantage of fibrin-selective thrombolytic agents such as Alteplase over non-fibrin-selective agents such as Streptokinase has been assessed in two major megatrials. The multicenter, randomized GISSI-2 trial showed that the two therapies were similarly efficacious and also had similar major cardiac side-effect incidence (angina and reinfarction). Incidence of bleeding was however higher in the patients receiving Streptokinase and heparin (Gissi-2 investigators). In a subsequent GUSTO trial (The GUSTO investigators, 1993) the use of "accelerated" Alteplase was evaluated in 41,021 patients. This protocol involved delivering up to 100 mg of drug within 90 minutes. Under these conditions, 24-hour, 30-day and one year mortality was significantly lower with Alteplase and intravenous heparin than with the non-fibrin specific agent Streptokinase plus either intravenous or subcutaneous heparin. The difference in 30-day and one year mortality was 1% thus saving an additional 10 lives per 1000 treated.

• Reteplase (Retavase) (click here for a monograph) is a single-chain recombinant variant of rt-PA produced by Escherichia coli cells. Reteplase differs from the Alteplase molecule by the deletion of specific domains. Reteplase contains 355 of the 527 amino acids of native t-PA including the kringle 2 and the protease domains of human t-PA. This variant has a longer half life, decreased fibrin affinity and increased thrombolytic potency compared to Alteplase. The mechanism of action of Reteplase is similar to Alteplase. Reteplase is less fibrin specific than Alteplase. This reduces the binding of the surface of fibrin of a thrombus, which may allow improved clot penetration and was postulated to explain the increased potency and enhanced thrombolysis of Reteplase.
• Tenecteplase (TNKase) (click here for a monograph) is a recombinant form of Alteplase. Tenecteplase is structurally and pharmacologically related to rt-PA, but it exhibits higher fibrin selectivity, greater resistance to plasminogen-activator inhibitors (e.g., PAI-1), and has a longer plasma half-life than Alteplase – thus Tenecteplase can be administered as a single, weight-adjusted bolus. This property greatly simplifies the care of acutely ill patients.

The efficacy of Tenecteplase was compared to that of "accelerated" Alteplase in the ASSENT-2 trial (The ASSENT investigators) and it was concluded that the two strategies were equivalent for 30-day mortality. The ease of administration of Tenecteplase may facilitate more rapid treatment in and out of hospital. In a subsequent study (Angeja et al, 2001) the two strategies were also found to be associated with a similar incidence of hemorrhage.

The characteristics of various available thrombolytics are described in the table below (data is compiled from the US Pharmacist, Merck's online manual of diagnosis and "therapy & ACC/AHA Guidelines for the Management of Patients With Acute Myocardial Infarction" (available on-line at the American College of Cardiology).

*Costs taken from the American Heart Association; **Costs taken from AHFSfirst

In summary, all thrombolytics suffer from the following limitations : time to recanalization remains unacceptably long, resistance to complete recanalization is still too common, and significant bleeding (primarily intracranial hemorrhage) remains a devastating side affect of all thrombolytics. The principal advantage of third generation thrombolytics is that they can be administered as a single bolus. (ASSENT-2 investigators, 1999). Each of these agents remains very expensive. Hence, many countries have not adopted them as default therapy for acute myocardial infarction. In Europe streptokinase remains the drug most frequently prescribed. It is clear therefore that third generation thrombolytics require further improvement, and in particular, emphasis should be placed on reducing the cost of treatment while at the same time maintaining or improving the reduced mortality rate seen with second generation versus first generation treatment.

SY161-P5 (Polyethylene Glycol-derivatised Staphylokinase Variant), an cost-effective Single Bolus Treatment of Acute Myocardial Infarction with Improved Thrombus Specificity:

One of the significant limitations related to streptokinase use has been the lack of effective coronary recanalization compared to other agents. It also lacks significant fibrin specificity. Activation of circulating plasminogen may result in degradation of unbound fibrinogen as well as inactivation of clotting factors V and VIII, thus producing a lytic state. Furthermore high doses are required since only a fraction of the administered dose becomes bound to fibrin-associated plasminogen. Towards the beginning of the 1990's researchers at the University of Leuven started to investigate the mechanism by which a second bacteria-derived plasminogen activator, staphylokinase, evoked thrombolysis. Staphylokinase, like streptokinase, forms a stoichiometric complex with and activates plasminogen however unlike the situation where streptokinase binds plasminogen, this does not expose the active site. Furthermore, it was subsequently shown that in solution, staphylokinase bound plasmin with an affinity at least 150-times that of plasminogen (Sakharov et al, 1996). In a clot environment plasminogen is partially degraded which results in conformational changes whereby binding with staphylokinase becomes stronger. Staphylokinase is therefore highly specific forming a complex with forms of plasmin(ogen) generally only found around thrombi. Furthermore it was found that alpha 2-antiplasmin, a major component of plasma, inhibits the enzymatic activity of the plasmin(ogen)-staphylokinase complex (Lijnen et al, 1991). Such an interaction does not occur with the plasminogen-streptokinase complex. Fibrin competes with alpha 2-antiplasmin for binding to the plasmin-staphylokinase complex (Lijnen et al, 1992) such that in the clot environment the inhibition of active site exposure is reduced. Perhaps even more important is the related finding that alpha 2-antiplasmin binding causes dissociation of staphylokinase from the complex allowing it to recycle. Thus, not only is the proteolytic activity of staphylokinase greater in a clot environment, but it also actively accumulates around the clot. Both streptokinase and staphylokinase induce a dose-dependent lysis of a human plasma clot however staphylokinase is considerably more potent (17nM Vs 68nM). In vivo, this translated to a 3-fold advantage in a rabbit model of thrombosis (Lijnen et al, 1991). Furthermore, as a result of the control mechanisms described above, outside a clot environment, streptokinase evoked a large degradation of fibrinogen (EC50=4.4nM). In dramatic contrast, the EC50 for staphylokinase was 790nM.

Reduced pro-hemorrhagic activity: The excellent thrombus specificity of staphylokinase suggests that the risk of hemorrhage should be low. This was investigated directly using rabbit bleeding time assays. In contrast to Alteplase, which significantly increased bleeding time, staphylokinase had no effect (Vanderschueren & Collen).

Reduced antigenicity and drug resistance: In addition to low thrombus specificity, the use of streptokinase is hampered by it's antigenicity. Using the baboon as a model of thrombosis recombinant staphylokinase and streptokinase were found to have similar thrombolytic potency however staphylokinase was less immunogenic and less allergenic and furthermore it does not induce resistance to lysis upon repeated administration (Collen et al, 1993). Antigenicity was reduced still further through the development of mutant forms of staphylokinase (Collen et al, 1996). Using this site directed mutagenic approach it was possible to reduce antigenicity while at the same time maintaining efficacy. Moreover, by reducing antigenicity, the effects of antibody-related neutralization could be overcome more quickly meaning that drug resistance became more short-lived (Vanderschueren et al, 1996). Perhaps the most exciting molecule developed to date is SY161-P5, a molecule with reduced antigenicity developed through site directed mutagenesis with an increased half-life resulting from PEGylating the derived molecule (Collen et al, 2000). As a result of PEGylation, SY161-P5 displayed a half-life of 13 minutes.

Drug safety studies: SY161-P5 was tested in a battery of drug safety studies. Intravenous single bolus injections of up to 100-fold therapeutic equivalent, as well as repeated injections during 7 to 28 days revealed no significant pathological findings in mice, rats or hamsters. However, New Zealand white rabbits developed clinically silent, multifocal myocarditis following single or repeat doses of SY 161-P5. These findings were dose-independent and reversible. A similar species-specific cardiotoxic effect has previously been described for other proteolytic proteins, including the approved drug streptokinase. The large experience with these drugs, as well as the clinical data accumulated both with PEGylated and non-PEGylated rSak variants to date, do not indicate cardiotoxic hazards associated with the use of these drugs in humans.

Reduced cost: A general problem with thrombolytic therapies is cost. This does not appear to be a problem for staphylokinase which can be produced as gram quantities of highly purified protein from E. coli transfected with the staphylokinase gene (Schlott et al, 1994). Consequently, it is anticipated that SY161-P5 can be supplied at competitive prices relative to existing therapies.

Improved clinical activity: In view of promising experimental data, a pilot clinical trial was initiated and data published in 1993 (Collen & Van de Werf). In this study, 10 mg of iv recombinant staphylokinase recanalized the arteries in four of five patients with acute myocardial infarction within 40 minutes in. Plasma fibrinogen and alpha 2-antiplasmin levels were unaffected, and allergic reactions were not observed. Post-infusion disappearance of recombinant staphylokinase antigen followed a biphasic mode with a t1/2 alpha of 3 minutes corresponding to a plasma clearance of 270 mL/min. Neutralizing antibodies against recombinant staphylokinase antigen could not be measured for up to 6 days after infusion, however antibodies did appear after this time point. Data from this pilot trial prompted a larger multicenter randomized open trial designed to assess the thrombolytic efficacy, safety, and fibrin specificity of recombinant staphylokinase relative to accelerated Alteplase (Vanderschueren et al, 1995). The main end points were coronary artery patency and plasma fibrinogen levels at 90 minutes. Thrombolysis expressed as TIM 3 perfusion at 90 minutes was achieved in 58% of Alteplase treated patients. This is similar to previously reported values (see table above). Thrombolysis was achieved in 50% of patients treated with 10mg recombinant staphylokinase and 74% of patients treated with 20mg recombinant staphylokinase patients. Furthermore, plasma fibrinogen levels were unchanged following treatment with recombinant staphylokinase compared to a 32% reduction in patients treated with considerably Alteplase. This suggested that compared to Alteplase, recombinant staphylokinase could offer improved efficacy with reduced side effects related to the activation of plasma fibrinogen and the subsequent induction of a thrombolytic and pro-hemorrhagic state.

The use of SY161-P5 in patients yielded even more exciting data. At considerably lower doses (5 mg vs 20mg for unmodified staphylokinase) SY161-P5 as a single bolus restored TIMI-3 flow within 60 minutes in 14 of 18 (78%) acute myocardial infarct patients (this level of success was achieved in 90 minutes with unmodified staphylokinase). By way of comparison, Alteplase restores blood flow in only 40-50% of cases. SY161-P5 failed to promote fibrinogen degradation and immunogenicity was significantly reduced (Collen et al, 2000).

Current development status: GMP manufacture of SY161-P5 for clinical evaluation as a single bolus has been completed. Enrollment in CAPTORS II, a multinational clinical trial in eight countries, has been completed and analysis of data from these 500 patients (divided into SY161-P5 and Alteplase treatment groups) is underway. Preliminary data suggests that SY161-P5 in doses ranging from 20 to 50 micrograms per kg body weight is similar in in terms of efficacy and safety to accelerated t-PA.

Patent position:The use of staphylokinase and new derivatives of staphylokinase is protected by a solid patent portfolio consisting of individual patents assigned to Desire Collen and Leuven Research & Development, which has sub-licensed the technology to ThromboGenics. Patents include EP 0525252 B1 and US 5336495 for staphylokinase and EP 0721982 A1, EP 0721013 A1, EP 0793723 A2, US 5695754, US 5951980, WO 9621016 A3, WO 9940198 A3 and AU705119 B2 for new staphylokinase derivatives. Equivalents to WO 9940198 are pending in individual states (New Zealand; Australia; USA; Canada; Korea; Romania; China; Poland; Hungary and Turkey). Likewise the following patents are also pending EP 98200323.8; EP 98200365.9; US 08/499,092; US 08/860,788 and US 09/020,018

Market size: First generation thrombolytics such as t-PA are estimated to generate revenues of the order of US$ 400 million globally, with an estimated market size approaching US$1 billion. In March 2002, Credit Suisse estimated the 2001 to 2002 sales of the second generation Tenecteplase to total $210 million, reiterating Morgan Stanley Dean Witter's peak year revenue estimates for the drug of $200 million. These figures correlate closely with the Piper Jaffray forecast of 2002 Tenecteplase revenues of $240 million (see current drug discovery).

Pamiteplase and Monteplase are both derivatives of tissue-type plasminogen activator that have recently been launched in Japan. Amediplase is a 2nd-generation t-PA, under development by Menarini for use as a single iv bolus thrombolytic in the treatment of myocardial infarction. It is a recombinant hybrid plasminogen activator, consisting of kringle 2 from t-PA fused to the protease domain of scuPA. Defibrotide has been launched as a prophylaxis of deep vein thrombosis and for venous disease and is now in phase II development for myocardial infarction.

Comparison of SY161-P5 with other products: As described above and summarized in the table below SY161-P5 represents a cost-effective alternative to 2nd and 3rd generation thrombolytics and even the 1st generation, streptokinase. Like the 3rd generation drugs, single bolus administration is possible, allergic reactions are rare and recanalization is excellent.

Summary & strategic analysis: Streptokinase remains the most widely prescribed thrombolytic of choice in many parts of the world due to it's low cost, even though major trials have demonstrated lower arterial patience, high incidence of allergy and lack of fibrin specificity. Third generation agents such as Alteplase, Reteplase and Tenecteplase offer ease of administration and a mortality advantage of approximately 1% when compared to SK, but are all very expensive agents. PEGylated staphylokinase offers an attractive alternative to all of these agents, with excellent arterial patency at 60 minutes (comparable to any known t-PA derivative), virtually nonexistent allergic reactions, considerably reduced antigenicity and is the most fibrin-selective thrombolytic known. ThromboGenics’ scientists have exploited these differences to produce a proprietary variant of staphylokinase, SY161-P5. This variant exhibits an excellent profile over wild-type staphylokinase for acute AMI. Consequently, SY161-P5 can be given as a single bolus which is one of the few advantages that 3rd generation thrombolytics have over the main alternative to streptokinase. SY161-P5 appears to be at least as effective as Alteplase in terms of arterial patency, yet it can be produced at a low cost. SY161-P5 has currently completed enrolment in a phase II trial and pharmaceutical partners are currently being sought to take this most exciting candidate through further rounds of investigation towards the clinic.