US 2010.0196348A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0196348 A1 ARMSTRONG et al. (43) Pub. Date: Aug. 5, 2010

(54) COMBINATION TREATMENT WITH T-PA of application No. 1 1/435,230, filed on May 15, 2006, VARLANT AND LOW MOLECULARWEIGHT now abandoned, which is a continuation of application HEPARIN No. 10/371,778, filed on Feb. 21, 2003, now Pat. No. 7,084,118. (76) Inventors: Paul ARMSTRONG, Edmonton (CA); Hal BARRON, San (60) Provisional application No. 60/359,327, filed on Feb. Francisco, CA (US); Silvano BERIOLI, Perugia (IT): 22, 2002. Frederique BIGONZI, 1 Hayles Roses (FR); Erich BLUHMKI, Publication Classification Schwendi (DE); Richard CHIN, (51) Int. Cl. San Francisco, CA (US); A638/48 (2006.01) Christopher GRANGER, Durham, A6IP 9/10 (2006.01) NC (US); Frans VAN DEWERF, A6IP 7/04 (2006.01) Herent (BE); Fong WANG-CLOW, G06O 90/00 (2006.01) Los Altos Hills, CA (US) Correspondence Address: (52) U.S. Cl...... 424/94.64; 705/500 MORRISON & FOERSTER LLP 7SS PAGE MILL RD (57) ABSTRACT PALO ALTO, CA 94304-1018 (US) The invention concerns an improved therapeutic regimen for (21) Appl. No.: 12/701436 the treatment of thrombolytic disorders, such as acute myo cardial infarction (AMI). In particular, the present invention (22) Filed: Feb. 5, 2010 concerns the treatment of thrombolytic disorders, e.g., AMI. with a combination of a tissue plasminogen activator (t-PA) Related U.S. Application Data variant having improved fibrin specificity and extended (63) Continuation of application No. 12/185.588, filed on plasma half-life when compared with wild-type human t-PA Aug. 4, 2008, now abandoned, which is a continuation and a low molecular weight heparin.

Patent Application Publication Aug. 5, 2010 Sheet 2 of 4 US 2010/0196348A1

20

18 r 16 Unfractionated heparin SS 14 a 12 ...EOPE 10 ------Abciximab c S 8 2 6 - 4. 2 log-rank p=0.0001 O O 5 10 15 20 25 30 DAYS TO DEATH, REINFARCTION OR REFRACTORY SCHAEMIA KAPLAN-MEER CURVES FOR PRIMARY EFFICACY ENDPOINT FIG 2

20 18 Unfractionated heparin

16 Abciximab st 14 ------f- 12 ------Enoxaparin - 10 geS 8 i 6 4 2 log-rank p=0.0062 O ------O 5 10 15 20 25 30 DAYS TO DEATH, REINFARCTION, REFRACTORY SCHAEMIA, ICH, OR MAJOR BLEEDING KAPLAN-MEER CURVES FOR PRIMARY EFFICACY PLUS SAFETY ENDPONT FIG 3

Patent Application Publication Aug. 5, 2010 Sheet 4 of 4 US 2010/0196348A1

S'OIH US 2010/0196348 A1 Aug. 5, 2010

COMBINATION TREATMENT WITHT PA major impediment to optimal reperfusion. Accordingly, there VARANT AND LOW MOLECULARWEIGHT is a great need for a thrombolytic regimen that allows early HEPARIN intervention, provides high efficacy, and carries low risk of bleeding side-effects. CROSS-REFERENCE TO RELATED 0008. A particularly successful t-PA variant is tenect APPLICATIONS eplase (TNKaseTM), a t-PA variant with extended half-life and improved fibrin specificity when compared to native human 0001. The present application is a continuation of U.S. t-PA. TNKaseTM (TNK-t-PA: T103N, N117O, KHRR(296 patent application Ser. No. 1 1/435.230, filed on May 15, 299)AAAA t-PA) is a 527 amino acids long glycoprotein 2006, which is a continuation of U.S. patent application Ser. developed by Genentech, Inc., which obtained FDA approval No. 10/371,778, filed Feb. 21, 2003, which claims the benefit on Jun. 2, 2000 for use in the reduction of mortality associated of U.S. Provisional Patent Application Ser. No. 60/359,327, with acute myocardial infarction (AMI). TNKaseTM (tenect filed Feb. 22, 2002, all of which applications are fully incor eplase) is a derivative of wild-type human t-PA, which has a porated herein by reference. threonine (T) replaced by an asparagine at amino acid posi tion 103, adding a glycosylation site at that position, an aspar BACKGROUND OF THE INVENTION agine (N) replaced by glutamine at position 117, removing a 0002 1. Field of the Invention glycosylation site at that position, and four amino acids, 0003. The present invention concerns an improved thera lysine (K), histidine (H), arginine (R), and arginine (R) peutic regimen for the treatment of thrombolytic disorders, replaced by four alanines (A.A.A.A) at amino acid positions Such as acute myocardial infarction (AMI). In particular, the 296–299. A large scale clinical trial (ASSENT-II) has shown present invention concerns the treatment of thrombolytic dis TNKaseTM (tenecteplase) to be the optimal thrombolytic orders, e.g., AMI with a combination of a tissue plasminogen agent, due to its ease of single-bolus administration, equiva activator (t-PA) variant having improved fibrin specificity and lent mortality to t-PA, and lower bleeding risk, as a result of its extended plasma half-life (when compared with wild-type improved fibrin specificity (ASSENT-II Investigators, Lancet human t-PA) and an anti-thrombin agent having anti-Xa and/ 354:716-22 (1999)). While of all thrombolytic agents tested, or anti-IIa activity, Such as a low molecular weight heparin. TNKaseTM (tenecteplase) has been found to have the highest 0004 2. Description of the Related Art fibrin specificity, and its extended half-life allows single 0005 Thrombolytic therapy has been a major advance in bolus dose administration, there is room for further improve the treatment of acute myocardial infarction (AMI). Throm ment by way of improving efficacy without increasing the risk bolytics can re-establish perfusion in occluded arteries, of side-effects, such as intracranial bleeding and stroke. For resulting in Smaller infarct size, improved left ventricular safety of a single bolus administration oftenecteplase see also function, and improved short and long-term Survival. See, Van de Werfet al., Am. Heart J. 137:786-91 (1999). e.g., Braunwald E., Circulation 79:441-1 (1989); Braunwald, N. Engl. J. Med. 329:1650-2 (1993); The GUSTO investiga SUMMARY OF THE INVENTION tors. An international randomized trial comparing four 0009. The present invention concerns an improved thera thrombolytic strategies for acute myocardial infarction. N. peutic regimen for fibrinolytic therapy. In particular, the Engl. J. Med. 329:673-82 (1993). invention concerns the combined administration of a t-PA 0006. However, current regimens of thrombolytic-anti variant Suitable for single-dose administration and an anti thrombic therapy continue to be limited by failure of initial thrombin agent having anti-Xa and/or anti-IIa activity, Such recanalization and reocclusion in about 40-45% of patients as a low molecular weight heparin. (Cannon et al., J. Am. Col. Cardiol. 24:1602-10 (1994); The 0010. Accordingly, in one aspect, the invention concerns a GUSTO Angiographic Investigators, N. Engl. J. Med. 329: method comprising administering to a human patient in need 1615-22 (1993); Cannon and Braunwald, Acta Cardiol. of thrombolytic therapy an effective amount of a combination 49:1-8 (1994)). In addition, intracranial hemorrhage can of a human tissue plasminogen activator (ht-PA) variant Suit occur in over 0.9% of the patients treated with thrombolytics able for single-bolus administration, and a low molecular (ISIS-3 (Third International Study of Infarct Survival) Col weight heparin. laborative Group, Lancet 339:753-70 (1992); The Global Use 0011. In one embodiment, the ht-PA variant is glycosy of Strategies to Open Occluded Coronary Arteries (GUSTO) lated at any of positions 103-105, and devoid of functional IIb Investigators, N. Eng. J. Med. 335:775-82 (1996): carbohydrate structure at position 117 of wild-type ht-PA ASSENT-II Investigators, Lancet 354:716-22 (1999)). amino acid sequence, and exhibits a) extended circulatory 0007 Attempts to increase the efficacy of thrombolytic half-life and substantially retained fibrin-binding, or therapy by increasing the dose of thrombolytic have been improved in vivo fibrinolytic potency, and b) improved fibrin unsuccessful because of unacceptably high incidence of specificity, as compared to wild-type ht-PA. Preferably, the intracranial hemorrhage. Also, an important factor in reduc ht-PA variant has extended circulatory half-life and substan ing mortality associated with AMI is the reduction of time to tially retained fibrin binding as compared to wild-type ht-PA. reperfusion (Rawles, J., BMJ 312:212-15 (1996); Lindereret In another preferred embodiment, the ht-PA variant has al., J. Am. Coll. Cardiol. 22:212-215 (1996); Weaver et al., improved in vivo fibrinolytic potency as compared to wild JAMA 270: 1211-16 (1993)). Guidelines concerning the treat type ht-PA. ment of AMI have suggested a target figure of 90 minutes for 0012. One of the representative t-PA variants useful in the the maximum delay between the patient seeing help and treatment method of the present invention has an N-linked receiving thrombolysis. Despite encouraging results, pre glycosylation at position 103 of the wild-type ht-PA amino hospital thrombolysis has not been widely implemented, and acid sequence. In a preferred t-PA variant, there is asparagine the delay from Symptom onset to arrival to hospital, which as part of an Asn-X-Ser or Asn-X-Thr tripeptidyl sequence, constitutes about two thirds of the overall delay, remains a wherein ASn is asparagine, Ser is serine, Thris threonine, and US 2010/0196348 A1 Aug. 5, 2010

X is any amino acid except proline, at position 103 of the 0022 FIG. 4 shows the relative risks and 95% CIs for wild-type ht-PA amino acid sequence. Another t-PA variant primary efficacy composite endpoint in the total ASSENT-3 has asparagine at position 103, tryptophane at position 104. study population and in prespecified Subgroups. and serine at position 105 of the wild-type ht-PA amino acid 0023 FIG. 5 shows the relative risks and 95% CIs for Sequence. primary efficacy plus safety composite endpoint in the total 0013 ht-PA variants which additionally have an amino ASSENT-3 study population and in prespecified subgroups. acid other than asparagine at position 117 of the wild-type ht-PAamino acid sequence can be advantageously used in the DETAILED DESCRIPTION OF THE PREFERRED treatment methods herein. It is further of advantage to make EMBODIMENT further alterations within the wild-type ht-PA amino acid sequence in order to improve fibrin-specificity, relative to A. Definitions wild-type ht-PA. Such alterations preferably are in the serine 0024. The terms “wild-type human tissue plasminogen protease domain of wild-type ht-PA, in particular within the activator,” “wild-type human t-PA.” and “wild-type ht-PA as amino acid region 296-302 or 274-277 of the wild-type ht-PA used herein, refer to human extrinsic (tissue-type) plasmino amino sequence. ht-PA variants having an alteration (prefer gen activator having fibrinolytic activity that typically has a ably amino acid substitution) in the region 296-299 of the structure with five domains (finger, growth factor, Kringle-1, wild-type h-t-PA amino acid sequence are particularly pre Kringle-2, and protease domains). The nucleotide and amino ferred. In a specific variant, the alteration is the substitution of acid sequences of wild-type (native) human t-PA have been alanine for each of amino acids lysine, histidine, arginine, and reported by Pennica et al., Nature 301:214 (1983) and in U.S. arginine at respective positions 296,297, 298, and 299 of the Pat. No. 4,766,075, issued Aug. 23, 1988. The location of a wild-type ht-PA amino acid sequence. particular amino acid in the polypeptide chain of t-PA is 0014. The treatment method of the present invention can identified by a number. The number refers to the amino acid be carried out with a variety of low molecular weight heparin position in the amino acid sequence of the mature, wild-type preparations, including, for example, enoxaparin, dalteparin, human t-PA polypeptide as disclosed in U.S. Pat. No. 4,766, tinzaparin, certoparin, pamaparin, nadroparin, ardeparin, and 075. In the present application, similarly positioned residues reviparin. A preferred low molecular weight heparin is enox int-PA variants are designated by these numbers even though aparin. the actual residue number is not so numbered due to deletions 00.15 Administration of the low molecular weight heparin or insertions in the molecule. This will occur, for example, and ht-PA variant should take place as soon as possible fol with deletional or insertional variants. The amino acids are lowing the onset of symptoms indicating that thrombolytic identified using the one-letter or three-letter code. Substituted therapy is required. Thus, administration should preferably amino acids are sometimes designated herein by identifying take place within about 8 hours, more preferably within about the wild-type amino acid on the left side of the number denot 6 hours, even more preferably within about 4 hours following ing the position in the polypeptide chain of that amino acid, the onset of symptoms. and identifying the Substituted amino acid on the right side of 0016. The ht-PA variant and the low molecular weight the number. For example, replacement of the amino acid heparin can be administered simultaneously or in either order, threonine (T) by asparagine (N) at amino acid position 103 of following conventional routes of administration. In a specific the wild-type human t-PA molecule yields a t-PA variant embodiment, the low molecular weight heparin is adminis designated T103N t-PA. Similarly, the t-PA variant obtained tered first, followed by administration of the ht-PA variant as by additional Substitution of glutamine (Q) for asparagine (N) a weight-adjusted single bolus. at amino acid position 117 of the wild-type human t-PA 0017. The low molecular weight heparin, e.g., enoxaparin, molecule is designated T103N, N117O t-PA. Deletional vari is typically administered as an intravenous bolus followed by ants are identified by indicating the amino acid residue and Subcutaneous administration, where the Subcutaneous position at either end of the deletion, inclusive, and placing administration may be repeated in periodic intervals in order the Greek letter delta, “A”, to the left of the indicated amino to reduce the likelihood of reocclusion. Thus, enoxaparin may acids. Insertional t-PA variants are designated by the use of be administered as an intravenous bolus of about 30 mg brackets “I” around the inserted amino acids, and the loca immediately followed by a subcutaneous dose of about 1 tion of the insertion is denoted by indicating the position of mg/kg. the amino acid on either side of the insertion. 0018. The condition to be treated can be any condition 0025. The various domains within the wild-type human requiring thrombolytic therapy, such as, for example, myo t-PA (ht-PA) amino acid sequence have been designated, cardial infarction (MI), venous thrombosis, pulmonary starting at the N-terminus of the amino acid sequence of embolism, cerebrovascular accident, and arterial embolism. human tissue plasminogen activator, as: 1) the finger region (F) that has variously been defined as including amino acid 1 upwards of about 44, 2) the growth factor region (G) that has BRIEF DESCRIPTION OF THE DRAWINGS been variously defined as stretching from aboutamino acid 45 upwards of amino acid 91 (based upon its homology with 0019 FIG. 1 shows the design of a randomized clinical EGF), 3) Kringle-1 (K1) that has been defined as stretching trial (ASSENT-3) investigating the efficacy and safety of from aboutamino acid 92 to about 173, 4) Kringle-2 (K2) that tenecteplase in combination with enoxaparin, abciximab, has been defined as stretching from about amino acid 180 to unfractionated heparin. about amino acid 261, and 5) the so-called (serine) protease 0020 FIG. 2 shows the Kaplan-Meier curves for primary domain (P) that generally has been defined as stretching from efficacy endpoint of ASSENT-3. about amino acid 264 to the C-terminal end of the molecule. 0021 FIG. 3 shows the Kaplan-Meier curves for primary These domains are situated contiguously generally of one efficacy plus safety endpoint of ASSENT-3. another, or are separated by short “linker” regions, and US 2010/0196348 A1 Aug. 5, 2010

account for the entire amino acid sequence from about 1 to 0031. The expressions “fibrinolytic activity,” “throm 527 amino acids in its putative mature form. bolytic activity” and "clot lysis activity” are used inter 0026. The term “human tissue plasminogen activator vari changeably and refer to the ability of a t-PA molecule to lyse ant’ or “ht-PA variant' is used to refer to a tissue plasminogen a clot, whether derived from purified fibrin or from plasma, using any in vitro clot lysis assay known in the art, such as the activator, which differs from wild-type ht-PA at least one purified clot lysis assay by Carlson, R. H. et al., Anal. Bio amino acid position, and retains a functional fibrin binding chem. 168, 428-435 (1988) and its modified form described region and serine protease domain. The finger (F), growth by Bennett, W. F. et al., J. Biol. Chem. 2665191-5201 (1991). factor (GF), and (to a lesser extent) Kringle-2 (K2) domains 0032. The expressions “in vivo fibrinolytic potency', in of wild-type ht-PA are known to be involved in fibrin binding. vivo thrombolytic potency” and “in vivo clot lysis potency’ An ht-PA variant having a functional fibrin binding region are used interchangeably and refer to clot lysis per unit dose will retain at least the minimal sequences of these domains oft-PA. “In vivo fibrinolytic potency’ is determined in any that are required for fibrin binding. The serum protease accepted animal model of clot lysis assay, including the ham domain is responsible for the enzymatic activity for wild-type ster pulmonary embolism model (Cohen, D. et al., Thromb. ht-PA. An ht-PA variant having a functional serine protease Haemost. 65:174-180 (1991)), and the rabbit jugular vein domain retains at least the minimal sequences from the serine thrombosis model (Collen, D. et al., J. Clin. Invest. 71:368 protease domain of wild-type ht-PA required for converting 376 (1983)). plasminogen to plasmin in the presence of a plasma clot or in 0033. The expression “substantially retain fibrin binding the presence of fibrin. (affinity). (compared to wild-type human t-PA) and gram 0027. The terms “TNK t-PA,” “T103N, N117O, KHRR matical variants thereofas used herein mean that the fibrin (296–299)AAAA t-PA,” “tenecteplase,” and “TNKaseTM,” binding affinity (apparent K value) of the variant t-PA mol are used interchangeably and designate a human t-PA variant, ecule is within about twofold of the fibrin binding affinity (K, which has a threonine (T) replaced by an asparagine at amino value) for wild-type human t-PA as determined in the same acid position 103, adding a glycosylation site at that position, assay. The expression “substantially improved fibrin bind an asparagine (N) replaced by glutamine at position 117. ing refers to a greater than about four fold increase in the removing a glycosylation site at that position, and four amino fibrin binding affinity (apparent K value) of a t-PA variant, as acids, lysine (K), histidine (H), arginine (R), and arginine (R) compared to that of wild-type t-PA, caused by the inclusion of replaced by four alanines (A.A.A.A) at amino acid positions an additional mutation or set of mutations. The term 296-299 of the wild-type human t-PA amino acid sequence. “improved in vivo fibrinolytic potency” compared to wild TNKaseTM (Genentech, Inc., South San Francisco, Calif.) has type t-PA refers to comparable in vivo clot lysis achieved by been approved by the FDA for use in the reduction of mor the administration of a variant t-PA at about one-third or less tality associated with AMI as a single intravenous bolus. the dose of wild-type t-PA. 0028. The terms “low molecular weight heparin’ and 0034. The terms “clearance rate' and “clearance' refer to “LMW heparin' are used interchangeably, and refer to hep the rate at which the t-PA molecule is removed from the arin fractions typically prepared by fractionation and/or bloodstream. Clearance (rate) is measured with respect to depolymerization of heparin so as to achieve significant native t-PA, Such that decreased clearance (rate) indicates that reduction in average molecular weight as compared with the t-PA variant is cleared more slowly than native t-PA, and whole heparin preparations. Compositions containing, pro increased clearance (rate) indicates that the t-PA variant is cedures for making, and methods for using low molecular cleared more rapidly than native t-PA. weight heparin are described in various patent publications, 0035. The term “circulatory half-life” means the half-life including U.S. Pat. Nos. 4.281,108, 4,687,765, 5,106,734, of a polypeptide of interest or polypeptide variant (e.g., an 4,977,250, 5,576,304, and EP 372969, the contents of which ht-PA variant) circulating in the blood of a given mammal. are hereby expressly incorporated by reference. LMW hep 0036. The expression “higher fibrin specificity' refers to arins for use in the present invention preferably have an the activity of a t-PA variant that exhibits a higher ratio of average molecular weight of about 10 kD or less, more pref fibrin-dependent specific activity to fibrinogen-dependent erably of about 8 kD or less, most preferably less than about specific activity in a S-2251 assay (in either the one-chain or 5 kD. It is further preferred that LMW heparins should be of two-chain form) than wild-typert-PA, and preferably a ratio relatively uniform molecular weight e.g., with at least about of at least about 1.5. 60%, more preferably at least about 80% of polymer units 0037. The expression “higher plasma clot specificity' having a molecular weight within the above defined average refers to the activity of a t-PA variant that exhibits a higher molecular weight limits. ratio of plasma clot-dependent specific activity to plasma 0029. The terms “fibrin binding” and “fibrin binding affin dependent specific activity in a S-2251 assay (in either the ity” refer to the ability of the t-PA molecule to bind fibrin clots one-chain or two-chain form) than wild-typert-PA, and pref in standard fibrin binding assays, such as the method erably a ratio of at least about 1.5. described by Rijken et al., J. Biol. Chem. 257, 2920-2925 0038. The expression “devoid of functional carbohydrate (1982) or its modified versions known in the art. structure at amino acid position 117 of wild-type human 0030. The terms “(t-PA) biological activity”, “biologically t-PA' means complete removal of the carbohydrate at amino active”, “activity” and “active” refer to the ability of the t-PA acid residue 117, as where the glycosylation signal is molecule to convert plasminogen to plasmin in the presence destroyed by site-directed mutagenesis, or substantial of a plasma clot or in the presence of fibrin, as measured by removal, as by treatment with an endoglycosidase which may the S-2288 assay, the plasma clot lysis assay, or other appro leave an intact N-acetylglucosamine residue linked to ASn priate assays. The assay(s) may be conducted in the presence 117, for example. or absence of potential modulators of activity such as fibrin, 0039. The term “thrombolytic disorder is used in the fibrinogen, plasma and/or plasma clots. broadest sense and refers to any condition characterized by US 2010/0196348 A1 Aug. 5, 2010

the formation of a thrombus that obstructs vascular blood flow amount of a compound or combination of compounds, as locally or detaches and embolizes to occlude blood flow applicable, to treat a thrombolytic disorder in a mammal, downstream (thromboembolism). Thrombolytic disorders including humans. The combination of compounds may be, specifically include, without limitation, myocardial infarc but does not have to be, a synergistic combination. "Synergy’ tion (MI), Venous thrombosis, pulmonary embolism, cere as described, for example, by Chou and Talalay, Adv. Enzyme brovascular accident, arterial embolism, etc. Regul. 22:27-55 (1984), occurs when the effect (in the present 0040. The term “myocardial infarction” or “MI is used to case the thrombolytic effect) of the compounds when admin refer to ischemic myocardial necrosis usually resulting from istered in combination is greater than the additive effect of the abrupt reduction in coronary blood flow to a segment of compounds when each is administered alone, as a single myocardium. MI is typically a disease of the left ventricle agent. (LV), but damage may extend to the right ventricle (RV) or 0048. The term “mammal’ refers to any animal classified atria. as a mammal, including humans, domestic and farm animals, 0041. The term “venous thrombosis” is used to include all and Zoo, sports, or pet animals, such as mouse, rat, rabbit, pig, forms of thrombosis, such as thrombosis affecting the Super sheep, goat, cattle and higher primates. ficial veins (superficial thrombophlebitis) and deep vein 0049. The terms “combination.” “combined’ and similar thrombosis (DVT). Since thrombosis is virtually always expressions, when used in reference to the administration of accompanied by phlebitis, the terms “thrombosis” and two or more compounds, mean that the compounds are “thrombophlebitis' are used interchangeably. administered to a Subject concurrently. Concurrent adminis 0042 “Pulmonary embolism' is the sudden lodgment of a tration includes administration at the same time, in the same blood clot in a pulmonary artery with subsequent obstructed formulation or separately, and sequential administration in blood Supply to the lung parenchyma. The most common type any order or at different points in time so as to provide the of pulmonary embolus is a thrombus that usually has desired therapeutic effect. migrated from a leg or pelvic vein. Most of those that cause serious hemodynamic disturbances form in an iliofemoral B. Detailed Description vein, either denovo or by propagation from calf vein thrombi. 0050. According to the World Health Organization, car Thromboemboli originate infrequently in the arm veins or in diovascular diseases account for 12 million deaths in the the right cardiac chambers. world each year. Currently, heart attack is a leading killer of 0043. The term “cerebrovascular accident' is used to refer men and women in developed countries. It is projected that by to stroke and, in general, infarction due to embolism orthrom 2010, heart disease will be the number one cause of death in bosis of intracranial or extracarnial arteries, and associated developing countries. hemorrhage. 0051. In the last decade, thrombolytic therapy has 0044) “Coronary patency’ is evaluated by angiography, emerged as the standard of care for the pharmacological using Thrombolysis. In Myocardial Infarction (TIMI) criteria. management of thrombolytic diseases, such as AMI. How The TIMI flow grades are defined as follows: TIMI flow 0-no ever, despite recent advances, most current thrombolytic regi perfusion; TIMI flow 1-penetration without perfusion; TIMI mens have significant shortcomings due to: (1) low optimal flow 2 partial perfusion with delayed run-off TIMI flow reperfusion rate (TIMI grade 3 flow in about 50% of patients 3-complete perfusion with brisk run-off. treated), (2) time delay to reperfusion averaging 45-120 min 0045. The term “antithrombotic therapy” refers to therapy utes, and (3) reocclusion of the infarct-related artery after aimed at preventing the formation or growth of a blood clot, initial successful reperfusion in 5-10% of the patents treated or partial or complete dissolution of a blood clot already and associated increased mortality; and intracranial bleeding formed. in up to 0.9% of patients treated. See, e.g., Jang et al., J. Am. 0046. The terms “treat' or “treatment” refer to both thera Coll. Cardiol. 33:1879-85 (1999). In order to further improve peutic treatment and prophylactic or preventative measures, treatment outcomes, cardiologists throughout the world are wherein the object is to prevent, slow down (lessen), or investigating various combinations of thrombolytic agents reverse an undesired physiological change or disorder, Such combined with other pharmacological agents, such as anti as the formation of a blood clot and the development of other thrombotics and anti-platelet agents, in an attempt to maxi physiological changes associated with the formation of blood mize artery-opening patency and reduce mortality. clots, e.g., restenosis; reocclusion; hemorrhage; hemody 0.052 The present invention concerns an improved namic disturbances; pain, arrhythmias, sinus node distur therapy regimen for the treatment of thrombolytic disorders, bances, atrioventricular block, etc. associated with MI. For Such as acute myocardial infarction (AMI). In particular, the purposes of this invention, beneficial or desired clinical invention is concerned with a new and improved combination results include, but are not limited to, alleviation of symp therapy using along half-life, fibrin specific t-PA variant (e.g., toms, diminishment of extent of disease, stabilized (i.e., not TNKt-PA) in combination with a low molecular weight hep worsening) state of disease, delay or slowing of disease pro arin. The new treatment regimen combines improved efficacy gression, amelioration or palliation of the disease state, and and safety when compared with other therapeutic approaches remission (whether partial or total), whether detectable or currently used in clinical practice. undetectable. "Treatment can also mean prolonging Survival 0053 B.1 Tissue Plasminogen Activator (t-Pa) Variants as compared to expected Survival if not receiving treatment. 0054 As discussed above, TNKt-PA (TNKaseTM, tenect Those in need of treatment include those already with the eplase: T103N, N117O, KHRR(296–299)AAAA t-PA) is a condition or disorder as well as those prone to have the 527 amino acids long glycoprotein developed by Genentech, condition or disorder or those in which the condition or dis Inc., which obtained FDA approval on Jun. 2, 2000 for use in order is to be prevented. the reduction of mortality associated with acute myocardial 0047. As used herein, the phrase “effective amount’ or infarction (AMI). TNKaseTM (tenecteplase) is a derivative of “therapeutically effective amount” is intended to include an wild-type human t-PA, which has a threonine (T) replaced by US 2010/0196348 A1 Aug. 5, 2010 an asparagine at amino acid position 103, adding a glycosy tage in patients needing emergency intervention after an event lation site at that position, an asparagine (N) replaced by involving thrombus formation, e.g., MI, requires continuous glutamine at position 117, removing a glycosylation site at intravenous administration to maintain therapeutic levels of that position, and four amino acids, lysine (K), histidine (H), t-PA in the blood stream. Variants of wild-type ht-PA that are arginine (R), and arginine (R) replaced by four alanines (A.A. Suitable for single bolus administration are required to have AA) at amino acid positions 296–299. In the 113 patient extended circulatory half-life (decreased clearance) com TIMI 10A trial, TNK t-PA was shown to have a prolonged pared to wild-type ht-PA. In addition, it is desirable that such half-life that allows it to be administered as a single intrave variants have improved fibrin-specificity relative to wild-type nous (IV) bolus (Cannonet al., Circulation 95:351-6 (1997)). ht-PA in order to reduce the likelihood of bleeding incidence Due to this property, TNK t-PA is particularly suitable for associated with bolus administration. ht-PA variants meeting pre-hospital administration. these requirements are disclosed, for example, in EP 643,772, 0055. In the TIMI 10B trial, an angiographic study with the entire disclosure of which is hereby expressly incorpo 886 patients, TNK t-PA was demonstrated to have similar patency and TIMI flow as wild-type human t-PA. Wild-type rated by reference. t-PA was administered as a 15 mg bolus oft-PA, a 0.75 mg/kg 0059 An exemplary ht-PA variant developed for single (up to 50 mg) infusion over 30 minutes, followed by 0.50 bolus administration islanoteplase (nPA), a deletion variant mg/kg (up to 35 mg) infusion over 60 minutes. The 60-minute lacking the finger and epidermal growth factor domains of and 90-minute TIMI flow data are shown in the following wild-type ht-PA (The TIME-II Investigators, Eur: Heart J. Tables 1 and 2. 21:2005-2013 (2000)). This ht-PA variant, developed by Genetics Institute and licensed to Bristol-Myers Squibb and TABLE 1. Suntory, has a longer circulatory half-life than wild-type ht PA but is less fibrin specific. See also Umemura et al., Eur: J. 60 min median 60 min TIMI flow 6-min TIMI flow corrected TIMI Pharmacol. 262:27-31 (1994). The use of lanoteplase is spe Drug? dose grade 2 and 3 grade 3 frame count (n) cifically within the scope herein. Other t-PA variants devel oped for bolus administration and having longer circulatory TNKt-PA 30 mg 78% 48% 37 (N = 176) half-lives than wild-type ht-PA include monteplase (Kawai et TNKt-PA 40 mg 79% S8% 37 al., J. Am. Col. Cardiol. 29:1447-1453 (1997)), in which (n = 84) Cys84 of wild-type ht-PA is substituted by Ser, pamiteplase TNK-t-PA 50 mg 79% 45% 37 (n = 42) (Kawasaki et al., Drug. Dev. Res. 33:33-38 (1994)), having t-PA (n = 65) 78% 48% 40 amino acids 92 to 173 of wild-type ht-PA deleted, an Arg275 substituted by Glu, and a truncated t-PA variant D-K2P (Saito et al., Biotechnol. Prog. 10:472-479 (1994)), missing the TABLE 2 finger and EGF domains. 0060. Further exemplary ht-PA variants suitable for use in 90 min 90 min corrected the combination therapy of the present invention are glyco 90 min TIMI flow TIMI flow median TIMI frame Drug? dose grade 2 and 3 grade 3 count sylated at any of positions 103-105 and lack functional car bohydrate structure at amino acid position 117 of wild-type TNKt-PA 30 mg 76% 55% 38 ht-PA. The removal of the functional carbohydrate structure (n = 297) TNKt-PA 40 mg 79% 64% 30 can be accomplished by any method known in the art, but (n = 131) preferably is achieved by amino acid substitution for at least TNKt-PA 50 mg 88% 66% 34 one residue in the ASn-Ser-Serglycosylation signal at posi (n = 42) tions 117-119 of the wild-type ht-PA amino acid sequence. In t-PA (n = 303) 82% 63% 33 a particularly relevant variant, asparagine at amino acid posi tion 117 is replaced by another amino acid, the preferred 0056. In addition, of all thrombolytic agents tested in large substituent being glutamine (see, also EP 238,304: WO clinical trials, TNK t-PA has the highest fibrin specificity. In 89/04368; and U.S. Pat. No. 5,612,029). In addition, the the TIMI 10A trial, systemic fibrinogen and plasminogen carbohydrate structure at amino acid position 117 of wild levels in patients treated with TNKt-PA fell by only 5-10% at type ht-PA can be substantially removed by the use of an 1 and 3 hours following administration (Tanswell et al., J. Am. endoglycosidase, such as Endoglycolidase H (Endo-H). Coll. Cardiol. 19:1071-5 (1992)). Endo-His only capable of removal of high mannose and 0057 Based on its uniquely advantageous properties, such hybrid oligosaccharides. Accordingly, under appropriate as easy administration as a single rapid bolus, equivalence conditions, Endo-His capable of Substantially removing the compared to wild-type t-PA with respect to mortality and high mannose carbohydrate structure at amino acid residue intracranial hemorrhage, and lower rates of major bleeding 117 of wild-type ht-PA, without functionally affecting the side-effects, TNK t-PA is a new standard for thrombolytic complex structures at amino acid residues 184 and 448. therapy. 0061 Glycosylation in the 103-105 amino acid region is 0058 Although the invention will be illustrated by data preferably accomplished by creating an N-linked glycosyla obtained with TNK t-PA, it is contemplated that other ht-PA tion signal at any of these positions. In particular, such vari variants with similar properties can be used in the treatment ants will have asparagines as part of an Asn-X-Ser, or ASn methods herein. In general, the use of any ht-PA variant X-Thr tripeptidyl sequence (X is any amino acid other than suitable for single bolus administration is specifically within proline) at position 103, 104, or 105 of the wild-type ht-PA the scope of the present invention. The relatively rapid clear amino acid sequence. The added N-linked glycosylation pref ance of wild-type ht-PA from the plasma, while it is an advan erably is at position 103. Such ht-PA variants will have an US 2010/0196348 A1 Aug. 5, 2010 asparagine at position 103, tryptophan at position 104, and lar to those conducted for tenecteplase. Such as, for example, serine at position 105 of the wild-type ht-PA amino acid the ASSENT-II study reported in, Lancet 354:716-22 (1999). Sequence. 0067 B.2 Low Molecular Weight Heparin 0062. As discussed above, it is particularly desirable to use 0068 Antithrombin therapy for patients receiving throm in the combination therapy hereint-PA variants that are more bolytics is believed to be important to both inhibit thrombin fibrin-specific than wild-type ht-PA. Such ht-PA variants will that is already present prior to thrombolysis and that which is act more preferentially at the site of the fibrin clot than wild generated as a consequence of administration of a throm type ht-PA and are, therefore, expected to cause diminished bolytic. Thrombin is one of the main stimuli responsible for side-effects associated with the activation of circulating plas platelet activation in the setting of thrombolysis and plays a minogen, e.g., less severe and less frequent bleeding compli central role in the pathogenesis or coronary rethrombosis. cations. Improved fibrin specificity can be achieved by an Although standard antithrombin therapy with intravenous alteration known in the art. Fibrin specificity can be unfractionated heparin is able to inhibit thrombinactivity, it is improved, for example, by amino acid alterations within the less effective in the inhibition of thrombin regeneration. serine protease domain of wild-type ht-PA. The amino acid Around 50 to 60% of patients has either suboptimal or inad alterations may be, for example, Substitutions at one or more equate anticoagulation with unfractionated heparin during of amino acid positions 296-302, preferably 296-299 of the thrombolysis, and when more intensive unfractionated hep serine protease domain. In a preferred variant, each of the arin regimens were tested, an increased rate of hemorrhages, amino acids lysine, histidine, arginine, arginine at positions including intracerebral hemorrhages, were observed (Ant 296-299 of wild-type ht-PA is replaced by alanine. Inafurther man, E. M., Circulation 90:1624-30 (1994); The Global Use preferred variant, the arginines at positions 298 and 299 are of Strategies to Open Occluded Coronary Arteries (GUSTO) both replaced by glutamic acid. In another preferred variant, II a Investigators, Circulation 90:1631-7 (1994)). the lysine, histidine, and proline at amino acid positions 296. 0069 Low molecular weight heparins (LMWHs) are 297 and 302 of wild-type ht-PA are additionally replaced by obtained from standard unfractionated heparin (UFH), and glutamine, asparagine, and serine, respectively. In a further have been used for the prophylaxis and treatment of venous fibrin-specific variant, phenylalanine, arginine, isoleucine, thromboembolism (see, e.g., Schafer, A.I., Hospital Practice and lysine at amino acid positions 274, 275,276 and 277 of Jan. 15, 1997, pp. 99-106). LMWHs have also be used in the the wild-type ht-PA amino acid sequence are replaced by treatment of unstable angina and non-Q wave myocardial amino acids leucine, histidine, serine, and threonine, respec infarction. Commercially available low molecular weight tively. The latter alteration results in a loss of plasmin cleav heparins include, for example LOVENOX(R) (enoxaparin age site, therefore, the variants are substantially in a single sodium injection, available from Aventis Pharma Inc. chain form. (Bridgewater, N.J.), described in U.S. Pat. No. 5,389,618), 0063. In addition to the alterations resulting in enhanced FRAGMINTM (dalteparin sodium injection, available from circulatory half-life and/or increased fibrin-specificity rela Pharmacia, Inc. (Columbus, Ohio)), INNOHEPR) (tinzaparin tive to wild-type ht-PA, the t-PA variants may contain addi sodium, available from DuPont Pharmaceuticals Company tional amino acid alterations, e.g., Substitutions, insertions (Wilmington, Del.)), ALPHAPARINTM (certoparin, available and/or deletions to further improve their therapeutic proper from Alpha, U.K.), FRAXIPARINETM (nadroparin calcium, ties. available from Sanofi-Synthelabo Canada, Inc.), NORMI 0064. Examples of suitable t-PA variants for use in accor FLOTM (ardeparin, available from Wyeth Laboratories, U.S.), dance with the present invention include T103N, N117Y and CLIVARINETM (reviparin sodium, available from ICN t-PA; S105N, A107S, N117Z t-PA; S105N, A107S, N117Z Pharmaceuticals). t-PA: T103N, N117Z, KHRR(296–299)AAAA t-PA; S105N, 0070 A particularly advantageous low molecular weight A107S, N117Z KHRR(296–299)AAAA t-PA: T103N, heparin preparation is LOVENOXOR (enoxaparin sodium N117Z, R298E, R299E t-PA; S105N, A107S, N117Z, injection), hereinafter referred to as "enoxaparin.” Enox R298E, R299E t-PA: T103N, N117Z, K296Q, H297N, aparin is a low molecular weight heparin produced by depo P301S t-PA; S105N, N117Z, K296Q, H297N, P301S t-PA: lymerization of standard unfractionated heparin (UFH). T103N, N117Z, FRIK(274-277)LHST t-PA; S105N, A107S, Unlike porcine UFH, which has a molecular weight of 12,000 N117Z, FRIK(274-277)LHST t-PA, wherein Z denotes any to 15,000 Daltons, enoxaparin has an average molecular of the 20 naturally occurring amino acids, except asparagine weight of 4,500 Daltons. Compared to UFH, it has more (N). Particularly preferred are the ht-PA variants in which at predictable pharmacokinetics, and a higher ratio of anti-Fac position 117 asparagine (N) is Substituted by glutamine (Q). tor Xa to anti-Factor IIa activity. Enoxaparin is also resistant 0065 Tenecteplase is commercially available from to inactivation by platelet factor 4. In studies examining enox Genentech, Inc., South San Francisco, Calif. The otherht-PA aparin in acute coronary syndrome patients, enoxaparin has variants can be readily prepared by well known techniques of been shown to be safe and more effective than unfractionated recombinant DNA technology and/or chemical synthesis. heparin at reducing coronary events (Cohen et al., N. Engl. J. 0066. The plasma clearance and fibrin specificity of the Med. 337:447-52 (1997); Antman. E. M. and Women's Hosp., t-PA variants can be tested, for example, as described by Boston, Mass., Supplement to Circulation 17:504-2649 Paoni et al., Thrombosis and Haemostasis 70:307-312 (1998)). (1993); Keytet al., Proc. Natl. Acad. Sci. USA 91:3670-3674 (0071 B.3 Combination Therapy (1994); Refino et al., Thrombosis and Haemostasis 70:313 0072 The ht-PA variants and low molecular weight hep 319 (1993); or using a rabbit thrombosed carotid artery model arin used herein can be formulated according to known meth described by Benedict et al., Circulation 92:3032-3040 ods to prepare pharmaceutical compositions, whereby the (1995). Plasma clearance, fibrin binding, and clot lysis activ active ingredient is combined with a pharmaceutically ity can also be tested as described in EP 643,772. The results acceptable carrier. The ht-PA variant formulations include obtained can then be confirmed in human clinical trials, simi sterile aqueous solutions and sterile hydratable powders. Such US 2010/0196348 A1 Aug. 5, 2010

as lyophilized formulations. Typically, an appropriate tenecteplase, the IV and SC doses of the low molecular amount of a pharmaceutically acceptable salt is also used in weight heparin (e.g., enoxaparin) are determined to match the the formulation to render the formulation isotonic. A buffer, targeted thrombolytic disorder, the patient's age, sex, overall Such as arginine base, in combination with phosphoric acid is physical condition, weight, and the like. also typically included at an appropriate concentration to 0077 More specifically, patients are typically given enox maintain a suitable pH, generally from about 5.5 to about 7.5. aparinat about 30 mg if their body weight is less than about 60 In addition or alternatively, a compound Such as glycerol may kg, about 35 mg if it is about 60-69 kg, about 40 mg if it is be included in the formulation to help maintain the shelf-life. about 70-79 kg, about 45 mg if it is about 80-89 kg, and about 0073 Tenecteplase is currently marketed as a sterile, 50 mg if it is about 90 kg or more. white to off-white, lyophilized powder for single IV admin 0078. In a large-scale clinical study (ASSENT-3), enroll istration after reconstitution with Sterile Water for Injection ing 6,095 heart attack patients at more than 500 sites world (SWFI). Each vial of the commercial formulation of tenect wide, details of which are provided in the Examples below, eplase (TNKaseTM) nominally contains 52.5 mg tenecteplase, combination treatment with tenecteplase and enoxaparin 0.55 g L-arginine, 0.17 g phosphoric acid, and 4.3 mg resulted not only in improved clinical efficacy and safety polysorbate 20, which includes a 5% overfill, and each vial benefits, but also yielded an exceptionally low 30-day mor delivers 50 mg of tenecteplase. The reconstituted solution tality rate (5.35%). This is the lowest reported mortality rate contains 5 mg/ml tenecteplase. However, other pharmaceuti reported to date in a large-scale clinical trial of acute myo cal formulations are also specifically within the scope of the cardial infarction. present invention. 007.9 Thrombolytic therapy with a combination of an ht 0074 The t-PA variants of the present invention are typi PA variant and a low molecular weight heparin in accordance cally administered as a single (occasionally repeated) intra with the present invention may be combined with the admin venous (IV) bolus, in combination with a low molecular istration of aspirin as early as possible following the throm weight heparin, which is typically administered intrave botic event, and other therapeutic agents. Such as B-blockers, nously and/or Subcutaneously (SC). Bolus administration has calcium channel blockers, angiotensin-converting enzyme several advantages. The ease of administration facilitates (ACE) inhibitors, intravenous nitrates, B-blockers, angio early intervention and may make more feasible prehospital tensin II inhibitors, statins, ticlopidin/clopidogrel, oral anti treatment with thrombolysis, and the simplicity of dosing coagulants, Abciximab, other gpIIb/IIIa inhibitors, angio may reduce medication errors, thereby reducing associated tensin-receptor blockers, thienopyridines, and thrombolytics, mortality. all conventionally used in cardiac treatment. 0075. In the treatment of thrombolytic disorders, such as 0080 Further details of the invention are provided in the MI, the t-PA variant, such as TNK t-PA (TNKaseTM, tenect following non-limiting example. eplase), hereinafter referred to as “tenecteplase is preferably I0081 All literature and other citations throughout this administered as a single intravenous (IV) bolus, in combina application are hereby expressly incorporated by reference. tion with a low molecule weight heparin, such as LOVENOXOR (enoxaparin sodium injection), hereinafter Example referred to as “enoxaparin.” A typical dose for a t-PA variant Efficacy and Safety of Tenecteplase in Combination Such as tenecteplase is between about 15 mg and about 50 mg. with Enoxaparin in the Treatment of Myocardial depending on the body weight of the patient, although lower Infarction and higher doses are also envisioned. The optimal dose depends on factors like the thrombolytic disorder targeted, 0082 Patients and Methods the patient's sex, age, overall physical condition, the severity I0083 Patients in 575 hospitals in 26 countries were of the disease, and the like. The determination of the optimal recruited. Inclusion criteria were identical to those of the dose is well within the skill of an ordinary physician. In a Assessment of the Safety and Efficacy of a New Throm typical situation, to a patient weighing between about 70 and bolytic Regimen (ASSENT)-2 trial (Lancet 354:716-22 80 kg, a t-PA variant such as tenecteplase is typically admin (1999)), i.e., age 18 years or older, onset of symptoms less istered in a dose of about 40 mg, as a single IV bolus over 5-10 than 6 h before randomization, ST-segment elevation of at seconds, preferably over 5 seconds. For more precise, weight least 0.1 mV in two or more limb leads or at least 0.2 mV in adjusted dosing of the t-PA variant, see also Gibson et al., Am. two or more contiguous pericardial leads, or left bundle J. Cardiol. 84:976-980 (1999). Weight adjusted dose typi branch block. Exclusion criteria on admission were: Systolic cally varies between about 0.2 mg/kg of body weight and blood pressure of more than 180 mm Hg, diastolic blood about 1.25 mg/kg of body weight. pressure of more than 110 mm Hg, or both on repeated mea 0076 Low molecular weight heparin, such as enoxaparin, surements; use of abciximab or other glycoprotein IIb/IIIa is typically administered in conjunction with the t-PA variant, inhibitors within the preceding 7 days; major Surgery, biopsy such as tenecteplase, starting with a fixed IV bolus followed of a parenchymal organ or Substantial trauma within 2 by weight adjusted subcutaneous (SC) injections. For months; any head injury or other trauma occurring after onset example, the low molecular weight heparin Such as enox of current myocardial infarction; any known history of stroke, aparin can be administered as a single IV bolus, followed by transient ischemic attack, or dementia; any known structural SC injections. In a typical dosing regimen, a single IV bolus damage to the central nervous system; current treatment with of about 30 mg low molecular weight heparin (e.g., enox oral anticoagulants; treatment with unfractionated heparin of aparin) is followed by about 1 mg/kg SC injections about more than 5000 U or a therapeutic subcutaneous dose of every 12 hours. In a typical situation, the first SC injection low-molecular-weight heparin within 6 h; known thrombocy immediately follows the IV bolus, and treatment follows until topenia (<100 000 cells/uIL); known renal insufficiency (se the patient's discharge, or until revascularization, or for 7 rum creatinine concentration>221 umol/L for men and >177 days, whichever comes first. Just as with the t-PA variant, e.g., umol/L for women); Sustained cardiopulmonary resuscita US 2010/0196348 A1 Aug. 5, 2010

tion (more than 10 min) in previous 2 weeks; pregnancy, 0093. Data were entered with the use of Oracle Clinical lactation, or parturition in the previous 30 days; active par (version 3.1.1) and electronically transferred to the central ticipation in another investigative drug or device study in the database in Leuven, Belgium. Safety data were reported previous 30 days; previous enrollment in this study; any other monthly to the data and safety monitoring committee. All disorder that would place the patient at increased risk; and stroke cases were reviewed by the same stroke committee that inability to follow the protocol and to comply with the follow reviewed the stroke data in the ASSENT-2 trial. The members up requirements. of this committee were unaware of treatment assignment. 0084. The protocol was approved by each hospital's insti There was no central adjudication for the endpoints of reinf tutional review board, and patients gave informed consent. arction, refractory ischemia, and bleeding complications. 0085 Patients were randomly assigned, via a central com However, definitions were provided to the investigators who, puterized telephone system into one of the following three in addition, had to reconfirm the occurrence of these end groups: points on a special form. I0086 Group I: a bodyweight-adjusted single bolus of 0094 Reinfarction in the first 18h was defined as recurrent tenecteplase with enoxaparin (enoxaparin group). symptoms of ischemia at rest accompanied by new or recur rent ST-segment elevations of 0.1 mV or more in at least two 0087 Group II: low-dose tenecteplase, plus abciximab, contiguous leads, lasting at least 30 min. After 18 h, the plus low-dose unfractionated heparin (abciximab group). definition was: new Q waves in two or more leads, or further 0088 Group III: full-dose tenecteplase with weight-ad increases in concentrations of creatine kinase MB, troponins, justed unfractionated heparin (heparin group). or total creatine kinase above the upper limit of normal and 0089. Each patient was given a unique study number that increased over the previous value. Refractory ischemia was corresponded with the number of a treatment kit. Study treat defined as symptoms of ischemia with ST-segment deviation ments were given on an open-label basis. or T-wave inversion persisting for at least 10 min despite 0090 Tenecteplase was given over 5 seconds according to medical management and not fulfilling the diagnosis of rein bodyweight: patients assigned enoxaparin or unfractionated farction. Non-cerebral bleeding complications were defined heparin were given 30 mgiftheir bodyweight was less than 60 as major (requiring transfusion, intervention because of kg, 35 mg if it was 60-69 kg, 40 mg if it was 70-79 kg, 45 mg hemodynamic compromise, or both) or minor. if it was 80-89 kg, and 50 mg if it was 90 kg or more. In (0095 Statistical Analysis patients assigned combination treatment with abciximab, 0096 Statistical analysis was by intention to treat. No half-dose tenecteplase was given with doses ranging from 15 confirmatory statistical hypothesis was prespecified, but a mg to 25 mg according to the same weight categories as with detailed analysis plan was defined before the database was the full dose. locked. This analysis plan was based on generating risk ratios 0091 Patients assigned weight-adjusted intravenous and CIS for the pairwise comparisons of primary interest. unfractionated heparin received a bolus of 60 U/kg (maxi These comparisons were presented with the two-sided 95% mum of 4000 U) and initial infusion of 12 U/kg per h (maxi CI of the relative risk and with nominal p values. For the mum 1000 U/h) adjusted to maintain an activated partial primary endpoints, Kaplan-Meier curves were constructed thromboplastin time of 50-70 seconds for 48 h with subse and log-rank tests were done. For each endpoint, a two-sided quent heparin administration left to the discretion of the treat 95% CI was also calculated and an overall X test, comparing ing physician. The first blood sample for activated partial the three treatment groups, was done. Comparisons of interest thromboplastin time measurement was drawn after 3 h. were prespecified to first involve the unfractionated heparin Patients assigned enoxaparin co-therapy received an intrave and enoxaparin groups. If these were not different, they were nous bolus of 30 mg immediately followed by the first sub to be pooled and compared with the abciximab group. Oth cutaneous dose of 1 mg/kg. To achieve Sustained anticoagu erwise, each experimental group was to be compared with the lation, this Subcutaneous dose was repeated every 12 h up to unfractionated heparin reference group. hospital discharge or revascularization for a maximum of 7 (0097. On the basis of ASSENT-2, the estimated frequency days. The first two subcutaneous doses could not exceed 100 of the primary efficacy endpoint in the group allocated full mg. Patients assigned abciximab co-therapy received a 0.25 dose tenecteplase and unfractionated heparin was 13.8%. The mg/kg bolus and 0.125 g/kg per min (maximum of 10 frequency of the primary efficacy plus safety endpoint in this ug/min) for 12 h. Because abciximab has an anticoagulant group was 17.7%. On the basis of phase-II studies, it was effect, a lower dose of unfractionated heparin was given: 40 assumed that the two experimental groups would result in a U/kg bolus (maximum of 3000 U) followed by 7 U/kg perh better, or at least similar, outcome when compared with stan (maximum of 800 U/h) to achieve a partial thromboplastin dard treatment. The sample size and power calculations were time between 50 and 70s. Also in this group, the first activated therefore based on non-inferiority of the two experimental partial thromboplastin time was measured after 3 h. Aspirin groups versus the reference group. The study had 80% power (150-325 mg) was given to all patients. Intravenous boluses to exclude, with 95% confidence (one-sided), a 1% higher of unfractionated heparin, enoxaparin, and abciximab were to rate of the primary endpoints compared with the reference be given before bolus tenecteplase. group, provided the point estimate in the experimental treat 0092. The primary endpoints were the composites of ment group was 1.7% lower for the efficacy endpoint and 30-day mortality, in-hospital reinfarction, or in-hospital 2.0% lower for the efficacy plus safety endpoint. refractory ischemia (primary efficacy endpoint) to evaluate 0098 Results efficacy outcomes, and the above plus in-hospital intracranial (0099 6095 patients were enrolled between May, 2000, hemorrhage or in-hospital major bleeding other than intrac and April, 2001, of whom 5989 received study medication ranial bleeding (primary efficacy plus safety endpoint) to (FIG.1). The baseline characteristics were similar in the three evaluate efficacy improvements when safety adverse events groups. Overall, the study populations were similar to those were added to the analysis. of previous trials on thrombolytics. The time from random US 2010/0196348 A1 Aug. 5, 2010 ization to bolus tenecteplase was significantly longer in the abciximab group because of the complexity of the treatments TABLE 3-continued and the need to give the boluses of heparin and abciximab Frequency of composite and single endpoints at hospital discharge before the tenecteplase bolus. Concomitant medications and at 30 days given in hospital included calcium-channel blockers, intrave nous nitrates, B-blockers, ACE inhibitors, angiotensin II Group I Group II Group III inhibitors, statins, aspirin, ticlopidin/clopidogrel, oral antico (n = 2040) (n = 2017) (n = 2038) p agulants, Abciximab, other gpIIb/IIIa inhibitors, and throm refractory (4.6%) (3.2%) (6.5%) bolytics, all conventionally used in cardiac treatment. High ischemia In-hospital ICH 18,2040 19, 2017 19,2038 O.98 proportions of patients received B-blockers, angiotensin-con (0.9%) (0.9%) (0.9%) Verting-enzyme inhibitors, angiotensin-receptor blockers, Major bleeding 62.2040 87.2O16 44,2O3S O.OOOS statins, and thienopyridines. Abciximab and glycoprotein IIb/ (other than ICH) (3.0%) (4.3%) (2/2%) IIIa inhibitors other than study medication were given most frequently in the groups assigned full-dose tenecteplase and ICH = intracranial hemorrhage unfractionated heparin or enoxaparin. Likewise, low-mo 0101 The relative risks in the total population and in the lecular-weight heparins other than study medication were prespecified subgroups are presented in FIGS. 4 and 5. The most frequently given to patients assigned unfractionated rates of the composite endpoints were lower among patients heparin or abciximab. treated with enoxaparin or abciximab than among those 0100. The primary efficacy and efficacy plus safety end treated with unfractionated heparin. Conventional statistical points and their individual components in the three treatment testing for full-dose tenecteplase plus enoxaparin versus full groups are shown in Table 3. The combined efficacy and dose tenecteplase plus unfractionated heparin resulted in p safety outcome in the full-dose tenecteplase plus unfraction values of 0.0002 and 0.0037, respectively, for the primary ated heparin group (Group III) of 17.0% was similar to that efficacy and efficacy plus safety composite endpoints. The estimated (17.7%) before the trial commenced. The Kaplan half-dose tenecteplase plus abciximab versus full-dose Meier curves for these primary endpoints are shown in FIGS. tenecteplase plus unfractionated heparin comparisons for the 2 and 3. Log-rank tests were highly significant. Early after same primary endpoints yielded nominal p values of <0.0001 treatment, the curves for the enoxaparin (Group I) and abcix and 0.0142. After correcting for multiple testing (Bonfer imab (Group II) groups started to separate from that of unfrac roni), conventional significance was reached for the primary tionated heparin (Group III). At 48 h, the end of the unfrac efficacy endpoint in the abciximab group (p=0.0002) but not tionated heparin infusion, differences in the primary for the efficacy plus safety endpoint (p=0.057). For both the endpoints among the three groups were already present. For efficacy and efficacy plus safety endpoints, statistical signifi the primary efficacy endpoint, event rates were 6.1% for cance was reached in the enoxaparin group (p=0.0009 and full-dose tenecteplase plus enoxaparin, 5.2% for half-dose p=0.014.6, respectively). tenecteplase plus abciximab, and 8.8% for full-dose tenect 0102 The lower point estimate of the relative risk of the eplase plus unfractionated heparin (p<0.0001). For the pri composite endpoints was consistent across Subgroups for the mary efficacy plus safety endpoint, the rates were 8.1, 8.2, and combination of full-dose tenecteplase and enoxaparin. For 10.3%, respectively (p=0.022). the combination of half-dose tenecteplase and abciximab, lower point estimates were seen in most Subgroups, except in TABLE 3 patients older than 75 years and those with diabetes (FIGS. 3 and 4). For the efficacy composite endpoint, the test for an Frequency of composite and single endpoints at hospital discharge interaction between treatment and diabetes was significant and at 30 days (p=0.0004). For the efficacy plus safety composite endpoint, Group I Group II Group III the treatment interaction tests were significant for age (p=0. (n = 2040) (n = 2017) (n = 2038) p 0010) and diabetes (p=0.0007). In women, lower point esti 30-day 233,2037 223, 2017 314,2038 O.OOO1 mates of the relative risks of the efficacy composite endpoint mortality, in- (11.4%) (11.1%) (15.4%) were found in both experimental groups, whereas for the hospital efficacy plus safety composite endpoint, the point estimates reinfarction, or in-hospital were on the line of unity. refractory 0103) No significant differences in 30-day mortality were ischemia present (Table 3). In-hospital reinfarction and refractory 30-day 280.2O37 287 2016 347,2036 O.OO81 ischemia occurred less frequently in patients treated with mortality, in- (13.8) (14.2%) (17.0%) hospital enoxaparin or abciximab than in those treated with unfrac reinfarction, in tionated heparin. The rates of in-hospital death or reinfarction hospital were also lower in the enoxaparin and abciximab groups than refractory in the unfractionated heparin group: 138/2040 (6.8%) and ischemia, in hospital ICH, or 148/2017 (7.3%) and 185/2038 (9.1%), respectively (p=0. in-hospital 0.198). No significant reductions in other major cardiac com major bleeds plications were seen, with the exception of a significantly (other than ICH) lower need for urgent percutaneous coronary interventions Death at 30 days 109,2037 133, 2017 122.2O38 O.25 (ischemia-driven percutaneous coronary intervention before (5.4%) (6.6%) (6.0%) In-hospital S4,2040 44,201.7 86,2038 O.OOO9 hospital discharge) in patients on enoxaparin or abciximab reinfarction (2.7%) (2.2%) (4.2%) than in patients on unfractionated heparin. Total in-hospital In-hospital 93.2040 64 2017 132,2O38

0104. Non-cerebral bleeding complications, need for findings in diabetics from the smaller ASSENT-3 study transfusion, and rates of thrombocytopenia were also described in this Example is due to chance or some other recorded. Significantly, more major bleeding complications reason is unknown. Conversely, the data from both trials for (p=0.0002), more transfusions (p=0.001), and a higher rate of this combination in elderly patients are consistent. Taken thrombocytopenia (p=0.0001) were seen in the abciximab together, they suggest that caution should be exercised group (Group II) compared with the unfractionated heparin regarding the use of conjunctive therapy with abciximab in group (Group III). In patients older than 75 years and in elderly patients with an acute myocardial infarction treated diabetics, the rate of major bleeding complications was three with a fibrinolytic agent. Further studies in the important and times higher with abciximab than with unfractionated hep growing population of elderly patients with an acute myocar arin: 1 1/271 (4.1%) versus 31/233 (13.3%), and 8/363 (2.2%) dial infarction are warranted and might involve lower doses of versus 25/355 (7.0%), respectively. More major bleeding these agents and mechanical approaches to reperfusion. The complications and blood transfusions were also seen in the GUSTO-V and current results with half-dose fibrinolytic and enoxaparin group (Group I) compared with unfractionated abciximab Suggest that there might be a role for this combi heparin, although these differences were not significant. nation treatment in younger patients who are likely to There was no excess of thrombocytopenia in this treatment undergo early coronary interventions. This speculation needs group. The total number of readmissions to hospital was to be formally tested in future trials. similar in the three treatment groups: 254/1986 (12.8%) for 0108. The reductions in ischemic complications in the enoxaparin, 221/1946 (11.4%) for abciximab, and 239/1984 full-dose tenecteplase plus enoxaparin group were similar to (12.1%) for unfractionated heparin (p=0.39). A few addi those seen in the abciximab group, but were more consistent. tional strokes occurred after hospital discharge in the three Importantly, no increase in intracranial hemorrhage rate, no groups. Total stroke rates and the rates of death or disabling excess in thrombocytopenia, and only a modest and non stroke at 30 days remained similar: 39/2040 (1.9%) and 122/ significant increase in major bleeding complications was seen 2037 (6.0%) for full-dose tenecteplase and enoxaparin, despite the length of treatment. In view of the present data and 37/2017 (1.8%) and 141/2016 (7.0%) for half-dose tenect the ease of administration, enoxaparin is regarded as an eplase and abciximab, and 34/2038 (1.7%) and 132/2038 attractive alternative anticoagulant treatment when given in (6.5%) for full-dose tenecteplase and unfractionated heparin, combination with tenecteplase. respectively (p=0.83 for total stroke and p=0.43 for death or 0109 The overall 30-day mortality rates in the present disabling stroke). study were low and probably result from selection of patients 0105 Discussion and an improvement in associated medical treatment and 0106 The results of the group treated with full-dose intervention. However, time to treatment remained similar to tenecteplase and weight-adjusted unfractionated heparin that of other large trials of fibrinolytic therapy, emphasizing (Group III) in this trial were very similar to those of ASSENT the opportunity provided by prehospital therapy with simple 2. In ASSENT-2, a higher and not fully weight-adjusted dose regimens. This opportunity is currently being explored in the of unfractionated heparin was given and the first partial ASSENT-3 plus study which will compare the two full-dose thromboplastintime was measured 6 hafter start of treatment. tenecteplase cohorts administered out-of-hospital versus a Nonetheless, total mortality, reinfarction, total stroke, and matched population from the current study. intracranial hemorrhage rates were almost identical in both 0110. Like all clinical studies, the present study has some trials. However, there were fewer major bleeding complica limitations. Ascertainment of selected components of the tions (2.2% vs. 4.7%) and less need for blood transfusion composite endpoints in this open trial was investigator-deter (2.3% vs. 4.3%) in the present trial than in ASSENT-2. These mined and Subject to bias. The primary goal was to examine results indirectly support the use of a more fully weight whether addition of a low-molecular-weight heparin or a adjusted dose of unfractionated heparin, as recommended in platelet glycoprotein IIb/IIIa inhibitor to a fibrinolytic agent the ACC/AHA guidelines, together with earlier monitoring of had promise as a therapeutic approach, and thus statistical the partial thromboplastin time. This unfractionated heparin hypotheses were not defined a priori. Nonetheless, the dosing, however, was not associated with a reduction in the strength and consistency of the results suggest that they are rate of intracranial hemorrhage, by contrast with the findings not due to bias or chance. The observed treatment effects with of a recent post-hoc analysis of the Intravenous tA for the both experimental groups exceeded what was expected in this Treatment of Infarcting Myocardium Early (InTIME)-II data. intermediate-sized trial and raises the question as to whether Guigliano et al., Am. Heart J. 141:742-50 (2001). our exploratory experimental approach will be useful in 0107 Compared with unfractionated heparin, adjunctive future assessments of promising combinations and various therapy with abciximab or enoxaparin reduces ischemic com dosing regimens before large, definitive trials are done. The plications of acute myocardial infarction treated with tenect different duration of heparin therapy in the enoxaparin versus eplase. These reductions were found to be present early after the unfractionated heparin group also deserves comment. We the start of treatment. The results obtained with half-dose chose a 7-day course of enoxaparin to conform with previous tenecteplase plus abciximab are very similar to those with studies in the hope of reducing recurrent ischemic complica half-dose reteplase and abciximab seen in GUSTO-V, and tions and preventing reocclusion; the 48 h infusion of unfrac Support the hypothesis that a more potent antiplatelet agent tionated heparin is a standard antithrombotic strategy used in increases flow in the infarct-related coronary artery. In both previous trials such as ASSENT-2. The longer exposure to trials, these benefits are obtained at the cost of a higher rate of enoxaparin possibly contributed to its increased efficacy and thrombocytopenia, major bleeding complications, and blood to the increased trend for bleeding. transfusions. No benefit, and perhaps even harm, was seen in 0111 Taking into account efficacy and safety, the combi patients older than 75 years and in diabetics. By contrast with nation of full-dose tenecteplase and long-term administration the present study, a 0.6% reduction in 30-day mortality was of enoxaparin emerged as the best treatment, and the most found in diabetic patients enrolled in GUSTO-V. Whether the promising reperfusion therapy regimen, in this trial. This US 2010/0196348 A1 Aug. 5, 2010

easy-to-administer therapy regimen lowered event rates, and 6. The method of claim 4 wherein said ht-PA variant has exhibited an improved safety profile. Because of additional improved in vivo fibrinolytic potency as compared to wild advantages such as the ease of administration and the lack of type ht-PA. need for monitoring of anticoagulation, this combination 7. The method of claim 4 wherein said ht-PA variant is should be regarded as an attractive alternative pharmacologi glycosylated at position 103 of the wild-type ht-PA amino cal reperfusion strategy. acid sequence. 8. The method of claim 7 wherein said glycosylation is SUMMARY N-linked. 0112. In summary, while both Groups I and Group II 9. The method of claim 8 wherein said ht-PA variant has improved the efficacy of the composite endpoint compared to asparagine as part of an Asn-X-Ser or Asn-X-Thr tripeptidyl Group III, only the tenecteplase plus enoxaparin arm (Group sequence, wherein ASn is asparagine, Ser is serine, Thr is I) maintained this benefit when safety adverse events were threonine, and X is any amino acid except proline, at position added to the analysis. Group I demonstrated the best results in 103 of the wild-type ht-PA amino acid sequence. reduction of events associated with the “primary efficacy plus 10. The method of claim 9 wherein said t-PA variant has safety composite endpoint, including 30-day mortality, in asparagine at position 103, tryptophan at position 104, and hospital reinfarction or in-hospital ischemia, and reduction in serine at position 105 of the wild-type ht-PA amino acid in-hospital ICH or major bleeding complications. Sequence. 0113. While Group II (tenecteplase plus unfractionated 11. The method of claim 7 wherein said ht-PA variant has heparin plus abciximab) did demonstrate an improvement in an amino acid other than asparagine at position 117 of the efficacy over Group III, Group II was associated with an wild-type ht-PA amino acid sequence. increase in major bleeding complications including non-ce 12. The method of claim 7 wherein said ht-PA variant has rebral bleeding, need for transfusions and the occurrence of an amino acid other than asparagine at position 117 of the thrombocytopenia, particularly in patients with diabetes or wild-type ht-PA amino acid sequence. age 75 or older, compared to Group I and Group III. Thus, 13. The method of claim 10 wherein said ht-PA variant has Group I showed the best results in the “efficacy plus safety’ an amino acid other than asparagine at position 117 of the analysis, with an event rate of 13.75% compared with Group wild-type ht-PA amino acid sequence. II (14.24%) and Group III (17.04%). 14. The method of claim 11 wherein said variant has 0114 Generally similar across all three groups, the mor improved fibrin specificity as compared to wild-type h-tPA. tality rate, evaluated as part of the combined efficacy plus 15. The method of claim 14 wherein said improved fibrin safety endpoint, was lowest in Group I, at 5.35%, compared to specificity is achieved by an alteration within the amino acid Group II at 6.59% and Group III at 5.99%. The differences region 296-302 or 274-277 of the wild-type ht-PA amino were not statistically significant. Sequence. 0115 Although the present invention is illustrated with 16. The method of claim 15 wherein said alteration is in the reference to certain embodiments, various modifications of region 296-299 of the wild-type h-t-PA amino acid sequence. the invention in addition to those shown and described herein 17. The method of claim 16 wherein said alteration is the will become apparent to those skilled in the art from the Substitution of alanine for each of amino acids lysine, histi foregoing description and general knowledge in the art, and dine, arginine, arginine at positions 296,297, 298, and 299 of fall within the scope of the appended claims. the wild-type h-tPA amino acid sequence. What is claimed is: 18. The method of claim 3 wherein said low molecular 1. A method of selecting a patient for treatment with a weight heparin is selected from the group consisting of enox combination of a human tissue plasminogen activator (ht-PA) aparin, dalteparin, tinzaparin, certoparin, parnaparin, nadro variant Suitable for single bolus administration and low parin, ardeparin, and reviparin. molecular weight heparin, comprising selecting a patient for 19. The method of claim 18 wherein said low molecular said treatment if weight heparin is enoxaparin. (a) the patient has been diagnosed with a thombolytic dis 20. The method of claim 3 wherein said ht-PA variant is order; administered intravenously as a single bolus dose. (b) the patient is 75 years of age or younger, and 21. The method of claim 20 wherein said administration (c) the patient is not diabetic. takes place within about 8 hours following the onset of symp 2. The method of claim 1 wherein said thrombolytic disor toms requiring thrombolytic therapy. der is myocardial infarction (MI). 22. The method of claim 20 wherein the bolus dose is 3. The method of claim 2 further comprising the step of weight adjusted. treating said patient with said combination. 23. The method of claim 22 wherein said bolus is admin 4. The method of claim 3 wherein said ht-PA variant is istered within about 5 seconds. glycosylated at any of positions 103-105, and devoid of func 24. The method of claim 23 wherein said t-PA variant is tional carbohydrate structure at position 117 of wild-type tenecteplase. ht-PA amino acid sequence, and exhibits a) extended circu 25. The method of claim 24 wherein said bolus dose is latory half-life and fibrin-binding affinity within about two about 30 mg for a patient having a bodyweight less than about fold of the wild-type t-PA fibrin binding affinity or improved 60 kg. in vivo fibrinolytic potency, and b) improved fibrin specific 26. The method of claim 24 wherein said bolus dose is ity, as compared to wild-tune ht-PA. about 35 mg for a patient having a bodyweight of about 60 to 5. The method of claim 4 wherein said ht-PA variant has 69 kg. extended circulatory half-life and fibrin-binding affinity 27. The method of claim 24 wherein said bolus dose is within about two-fold of the wild-type t-PA fibrin binding about 40 mg for a patient having a bodyweight of about 70 to affinity as compared to wild-type ht-PA. 79 kg. US 2010/0196348 A1 Aug. 5, 2010

28. The method of claim 24 wherein said bolus dose is 35. The method of claim 34 wherein said enoxaparin is about 45 mg for a patient having a bodyweight of about 80 to administered as an intravenous bolus of about 30 mg imme 89 kg. diately followed by a subcutaneous dose of about 1 mg/kg. 29. The method of claim 24 wherein said bolus dose is 36. The method of claim 35 wherein said subcutaneous dose is repeated about every 12 hours for a maximum of about about 50 mg for a patient having a bodyweight of about 90 kg 7 days. O. O. 37. The method of claim3 wherein said patient is addition 30. The method of claim 20 wherein administration of said ally administered aspirin. low molecular weight heparin takes place before the single 38. The method of claim 3 wherein said patient is moni bolus administration of said ht-PA variant. tored for at least 30 days following said administration. 31. The method of claim 30 wherein said low molecular 39. The method of claim 38 wherein said patient does not weight heparin is administered as an intravenous bolus fol Suffer reinfarction during the period of monitoring. lowed by Subcutaneous administration. 40. The method of claim 38 wherein said patient is not 32. The method of claim 31 wherein said subcutaneous diagnosed with refractory ischemia during the period of administration is repeated. monitoring. 33. The method of claim 32 wherein said subcutaneous 41. The method of claim 40 wherein said patient does not administration is repeated about every 12 hours for a maxi Sufferintracranial hemorrhage or other major bleeding during mum of about 7 days. the period of monitoring. 34. The method of claim 30 wherein said low molecule weight heparin is enoxaparin. c c c c c