CA1297009C

CA1297009C - Use of t-pa for the inhibition of damage to jeopardized tissue during reperfusion in a mammal

Info

Publication number: CA1297009C
Application number: CA000536803A
Authority: CA
Inventors: Henry Berger, Jr.; Crist John Frangakis
Original assignee: Wellcome Foundation Ltd
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 1986-05-12
Filing date: 1987-05-11
Publication date: 1992-03-10
Anticipated expiration: 2009-03-10
Also published as: US4976959A

Abstract

ABSTRACT

Use of t-PA, optionally in combination with SOD, in the inhibition of damage to jeopardized tissue during blood reperfusion.

Description

The present invention relates to tissue plasminogen activator, to its combination with supero~ide dismutase, to pharmaceutical formulations thereof, and to the use thereof in human and veterinary medicine.

There exists a dynamic equllibrium between the enzyme system capable of forming blood clots, the coagulation system, and the en~yme system capable of dissolving blood clots, the fibrinolytic system, whlch maintains an intact patent vnscular bed. To limit loss of blood from in~ury, blood clots are formed in the in~ured vessel. After natural repair of the in~ury, thesuperfluous blood clots are dissolved through operation of the fibrinolytic system. Occasionally, blood clots form without traumatic lnJury and may lodge in ma~or blood vessels resulting in a partial or even total obstruction to blood flow. When this occurs in the heart, lung or brain, the result may be a myocardlal infarction, pulmonary embolism or stroke. These conditions combined are the leading cause of morbidity and mortality ln the industrialised nations.

Blood clots consist of a fibrous network that is capable of dissolution by the proteolytic enzyme plasmin. The enzyme is derived from the inactive proenzyme, plasminogen, a component of blood plasma, by the action of a plasminogen activator. There are two immunologically distinct mammalian plasminogen activators. Intrlnsic plasminogen activator, also known as urokinase~ is an enzyme produced by the kidney and can be isolated from urlne. It can also be prepared from a number of tissue culture sources. Extrinsic plasminogen actlvator, also known as vascular plasminogen activator and as tlssue plasminogen sctivator (t-PA), can be isolated from many tisstle homogenates (notably human uterus), the vascular cell wall and from some cell cultures. In addition to these two kinds of plasmlnogen activator, there is also a bacterial product, streptokinase, prepared from beta-haemolytic streptococci. A ma~or drawback with both urokinase and streptokinase is that they are activethroughout the circulation and not ~ust at the site of a blood clot. They can, for example, destroy other blood proteins, such as fibrlnogen, prothrombin, factor V and factor VIII so reducing blood clotting ability and increasing the risk of haemorrhage. In contrast, the biological activity of t-~A ls dependent .
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on the presence of fibrin to whlch it binds and where it is activated. Maximum activity is thus developed only at the site of a blood clot, l.e. in the presence of the fibrin network to be dissolved, and this greatly avoids the ris~ of haemorrhage.

The lnterruption of blood flow in a vessel generally leads to the onset of an ischaemic event. In this condition the tissue is deprived of oxygen and becomes jeopardized, a state in which the tissue is injured but stilLpotentially viable. If however the condit:Lon is maintained for a period of, say, three of more hours, the tissue becomes necrotic and, once in this state, cannot be recovered. It is therefore important that reperfusion, i.e. the restoration of blood flow, takes place as soon as possible to salvage the tissue beore it becomes permanently damaged. Ironically, reperfusion itself, even if carried out before the tissue becomes necrotlc, results in a complex group of phenomena, including the putative formation of the superoxlde radical, that have a deleterlous effect on hypoxic tissue. Consequently, reperfusion can lead only to the partial recovery of jeopardized tissue, the remainder being permanently damaged by the occurrence of one or more of these phenomena.

It has now been found that t-PA inhibits the damage to jeopardizad tissue during reperfusion by protecting it against one or more of the aforsmentioned phenomena. The mechanism of action of t-PA in affording such protection has not been elucidated but it is independent of its action as a thrombolytic agent. This ne~ly discovered property thus enables t-PA, or a pharmaceutical formulation thereof, to be used as an inhibitor of dama~e to jeopardized tlssue in the circumstances outlined herein. Accordingly, the present inventionprovides:-(a) A method for inhibitlng damage to jeopardlzed tissue during reperfusionin a mammal, which comprises administering to the mammal an effective amount of t-PA;

(b) Use of t-PA in inhibiting the damage to jeopardized tissue during reperfusion in a mammal;

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(c) Use of t-PA for the manufacture of a medicament Eor the inhibition of damage to ~eopardi~ed ti.ssue during reperfusion in a mammal; or (d) A pharmaceu~ical formula~ion for use in inhibiting damage to jeopardized tissue during reperfusion in a mammal, which comprises t-PA and a pharmaceutically acceptable carrier.

Although the present invention may be used to protect any jeopardized tissue, it is particularly useful in inhibiting damage to jeopardized myocardial tissue.

The t-PA of use with the present invention may be any bioactive protein substantially corresponding to mammalian, and especially human, t-PA andincludes forms with and without glycosylation. It may be one- or two-chain t-PA, or a mixture thereof, as described in EP-A-112 122, and, in the case of fully glycosylated human t-PA, has an apparent molecular weight on polyacrylamide gel of about 70,000 and an isoelectric point of between 7.5 and 8Ø Preferably the t-PA has a specific activity of about 500,000 IU/mg (International Units/mg, the International Unit being a unit of activity as defined by WH0, National Institute for Biological Standards and Control, Holly Hill, Hampstead, London, NW3 6RB, U.K.).

The amino acid sequence of t-PA preferably substantially corresponds to that set forth in Figure 1. The sequence is thus identical to that in Figure 1 or contains one or more amino acid deletions, substitutions, insertions, inversions or additions of allelic origin or otherwise, the resulting sequence having at least 80%, and preferably 90~, homology with the sequenca in Figure 1 and retaining essentially the same biological and immunological properties of the protein. In particular, the sequence is identical to that in Figure 1 or has the same sequence but with the amino arid in the 245th position from the ~erine N-terminus being valine instead of methionlne, either sequence optionally having an additional polypeptide N-terminal presequence of , Gly-Ala-Arg.

The amino acid sequence set forth in Flgure 1 has thirty-~five cysteine residuesand thus the potential for forming seventeen disulphide bridges. Based on :-: .' :
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' . , , - 4 - B487/48~3 analogy with other proteins whose structure has been determined in more detail, the postulated structure for the sequence (arising from disulphide bondformation) between the amino acid in the 90th position and the proline C-terminus is set forth in Figure 2. The structure of the N-terminal region is less certain although some proposals have been put forward (Pro~ress in Fibrinolysis, 1983, 6, 269-273; and _oc. Natl. Acad. Sc ., 1984, 81, 5355-5359). The most important features of the structure of t-PA are the two kringle regions (between the 92nd and the 173rd amino acids and between the 180th and 261st amino aclds), which are responsible for the binding of the protein to fibrin, and the serine protease region, which comprises the major part of the B-chain and which ls responsible for the activation of plasminogen.
The amino acids of special significance in serine proteases are the catalytic triad, His/Asp/Ser. In t-PA these occur at the 322nd, the 371st and the 463rd positions. The disulphide bridge between the 264th and 395th cysteine amino acid residues is also important in that it holds together the A- and the B-chains in the two-chain form of t-PA.

In Figures 1 and 2, the conventional one and three letter codes have been employed for the amino acid residues as follows:

Asp D Aspratic acid Cys C Cystein Arg R Arginine Thr T Threonine Val V Valine Lys K Lysine Ser S Serine Ile I Isoleucine Trp W Tryptophan Glu E Glutamic acid Leu L Leucine Gln Q Glutamine Pro P Proline Tyr Y Tyrosine Net M Methionine Gly G Glycine Phe F Phenylalanine Asn N Asparagine Ala A Alanine His H Histidine The t-PA may be obtained by any of th~ procedures described or known in the art. For example, it may be obtained from a normal or neoplastic cell line of the kind described in Biochimica et Biophysica Acta, 1979, 580, 140-153;RP-A-41 766 or EP-A-113 319. It is preferred, however, that t-PA is obtained from a cultured transformed or transfected cell line derived using recombinant DNA technology as described in, for example, EP-A-93 619; EP-A-117 059;EP-A-117 060; EP-A-173 552; EP-A-174 835; EP-A-178 105 W0 86/01538; ~0 . .
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86/05514; or WO 86/05807. It is particularly preferred that Chinese hamster ovary (CHO) cal].s are used for the production of t-PA and are derived in the manner as described in Molecular and Cellular Biologv, 1985, 5(7~, 1750-1759.
In this way, the cloned gene is cotransfected with the gene encoding dihydrofolate reductase (dhfr) into dhfr CHO cells. Transformants expressing dhfr are selected on media lacking nucleosides and are exposed to increasing concentrations of methotrexate. The dhfr and t-PA genes are thus coamplified leading to a stable cell line capable of expressing high levels of t-PA.

The t-PA is, preferably, purified using lmy of the procedures described or known in the art, such as the procedures described in Biochimlca et Biophyslca Acta, 1979, 580, 140-153; J. Biol. Chem.,1979, 254~6~, 1998-2003; ibid, 1981, 256(13), 7035-7041; Eur. J. Biochem., 1983, _2, 681-686; EP A-41 766; EP-A-113 319; or GB-A-2 122 219.

t-PA may be used in the manner of the present invention either alone or in combination with another therapeutic agent which also inhibits damage to jeopardized tissue during reperfusion. A particularly useful example of such a combination is with supero~ide dismutase (SOD), an enzyme that is known to scavenge and destroy superoxide radicals, one of the phenomena capable of causing tissue damage. Indeed, it has also been found that the combination of t-PA and SOD provides a significantly potentiated level of inhibition compared with that provided by t-PA or SOD per se. Accordingly the present invention also provides a combination of t-PA and SOD.

The combination of t-PA and SOD affords a particularly convenient means both for the removal of blood clots and for the inhibition of damage to ~eopardized tissue during subsequent reperfusion. Thus, the administration of t-PA and SOD
will result first in the removal of the blood clot through the kno~nthrombolytic action of t-PA and then in the inhibition of tissue damage through the combined action of t-PA and SOD.

The SOD of use in combination with t-PA may be any bioactive proteinsubstantially corresponding to any one or more of a group of en~ymes known generally by this name. It is preferably of mammalian, and especially of bovine or human, origin and is generally associated ~ith a me~al cation by MJStOLM/B487/488 .: , ' ' :' . ' ~ ~3~

which it is normally classified. Examples of a metal cation include lron, manganese, copper and preferably combinations of copper with other metals, such as zinc, cadmium, cobalt or mercury, of which a copper/zinc combination is preferred. Both the manganese and the copper/zinc forms of SOD occur naturally in humans. The iron and manganese forms of SOD of bacterial origin both have a molecular weight of about 40,000 and are dimers. The manganese form of SOD of eukaryotic origin on the other hand has a molecular weight of about 80,000 and is a tetramer. The copper/zinc form of SOD of eu~aryotic origin has amolecular weight of about 32,000 and ls a climer with one copper cation and one zinc cation per subunlt. The copper cation is ligated to four his-tidine resldues per subunit and the zinc cation is ligated between histidine and aspartlc acid. There is also a copper/zinc form of SOD of eukaryotic orlgin whlch has a molecular weight of about 130,000 and which consists of four subunits. The molecular weights of the various forms of SOD ~ere estimated using sedimentatlon equillbrium, molecular sleving or using polyacrylamide gels. The isoelectric polnts of the various forms of SOD range from 4 to 6.5 depending on the degree of sulphation and/or deamidation. PreEerably, the specific activity of the copper/zlnc form of SOD of bo~ine or human origin is at least 3000 U/mg (the unit of activity being as defined in J.Biol. Chem., 1969, 244, 6049-6055).

The amino acid sequence of the copper/zinc form of SOD of b~vine or human origin preferably substantially corresponds to that set forth in J. Biol~
Chem., 1974, 249(22), 7326 to 7338, in the case of that of bovine origin, and Biochemistr~, 1980, 19, 2310 to 2316 and FEBS Le~ters, 1980, 120, 53 to 55, in the case of that of human origin. The sequenca is thus identical to that set forth in these articles or contains one or more amino acid deletions,substitutions, insertions, inversions or additions of allelic origin or otherwise, th~ resulting sequence havlng suficient homology with the published sequence so as to retain essentially t~e same biological and immunological properties.

The amino acid sequence of the copper/zlnc form of SOD of bovine origin contains three cysteine residues per subunit (J. Biol. Chem., 1974, 249(22)l 7326-7338). The intrachain disulphide bridge occurs between the Cys 55 and Cys 144 residues while the interchain disulphide bridge occurs between the Cys 6 ~ .

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, ~2~3~ 3 residues. The amino acid sequence of the copper/zinc iorm of SOD of human origin contains four cysteine residues per subunit (BiochemistrY, 1980, lg, 2310 to 2316 and F S Letters, 1980, 120, 53 to 55). The intrachain disulphide bridge occurs between the Cys 5~ and Cys 146 residues while the interchain disulphide bridge occurs also between the Cys 6 residues. The Cys 111 residue remains free.

The SOD may be obtained by any suitable procedure described or known in the art. For example, it may be obtained from erythrocytes or from liver by an extraction procedure of the kind described in GB-A-l ll07 807 and GB-A-l 529 890. Alternatively, SOD may be obtained from a cultured transformed or transfected cell line, derived using recombinant DNA technology as described in, for example, Australian patent application 27461/84, WO 85/01503, EP-A-138 lll, EP-A-164 566, EP-A-173 280 and EP-A-180 964.

The SOD is preferably purified using any suitable procedure described or known in the art, such as the procedure described in EP-A-112 299.

In using t-PA, or a combination of t-PA and SOD, in the manner of the present invention, it is preferred to employ the active ingredient(s) in the form of a pharmaceutical formulation. In the case of the combination, the activeingredients may be employed in separate formulations which may be administered simultaneously or sequentially. If they are administered sequentially, it is preferred to administer thc t-PA formulation first and then the SOD
formulation. In any event, the delay in administering the second of the two formulations should not be such or to lose the benefit of a potentiated effect of the combination of the active ingredients in vivo in inhi.biting tissue damage. However, rather than use separate formulations, it is much moreconvenient to present both active ingredients together in a single combined formulation. Accordingly, the present invention also provides a pharmaceutical formulation, which comprises t-PA and SOD and a pharmaceutically acceptable carrier.

Generally, t-PA, or the combination of t-PA and SOD, will be administered by the intravascular route, whether by infusion or by bolus in~ection, and thus a parenteral formulation is required. It is preferred to present a lyophilised , . ' :. ' .
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a s487/488 formulation to the physician or veterlnarian because of the significanttransportatLon and storagc advantages that it affords. The physician or veterinarian may then reconstitute the lyophilised formulation in an appropriate amount of solvent as and when required.

Parenteral and lyophilised pharmaceutical formulations contalning t-PA are known in the art. Examples of such art include EP-A-41 766; EP-A-93 619;
EP-A-112 122; . EP-A-113 319; EP-A-123 304; EP-~-143 081; EP-A-156 1.69; WO
86/01104; Japanese patent publi.cation 57--120523 (application 56-6936) and Japanese patent publication 58-65218 (application 56-163145). Addi.tionalpreferred examples include GB-A-2 176 702 and GB-A-2 176 703. All such formulations are also suitable for SOD and for the combination of t-PA and SOD.

Intravascular infusions are normally carrled out with the parenteral solution contained within an infusion bag or bottle or within an electrically operated infusion syringe. The solution may be delivered from the infusion bag or bottle to the pati.ent by gravity feed or by the use of an infusion pump. The use of gravity feed infuslon systems does not afford sufficient control over the rate of administration of the parenteral solution and, thercfore, the use of an infusion pump is preferred especially with solutions containing relatively high concentrations of active ingredients. More preferred, however, is the use of an electrically operated infusion syringe which offers even greater control over the rate of administration.

An effective amount of t-PA, and of a combination of t-PA and SOD, to inhibi-t damage to jeopardized tissue during reperfusion will of course depend upon a number of factors including, for example, the age and weight of the mammal, the precise condition requiring treatment and lts severity, the route of administration, and will. ultimately be at the discretion of the at~endant physician or veterinarian. An effective dose, however, in the case of t-PA is generally in the range from 0.2 to 4 mg/kg ~i.e. 100,000 to 2,000,000 IU/kg assuming a speciflc activlty for t-PA of 500,000 IU/mg), preferably from 0.3 to 2 mg/kg ~i.e. 150,000 to 1,000,000 IU/kg), bodywelght of patient per hour.
Thus for a 70kg adult human being, an effective amount per hour will preferably be from 20 to 1~0 mg ~i.e. 10,000,000 to 70,000,000 IU), especially about 70 mg (i.e. 35,000,000). If SOD is used in combination w~th t-PA, then an effective MJS/OL~/B487/488 - "'' ' .
' 9 ~ 30g dose of SOD is generally in the range from 1000 to 50,000 U/kg, preferably from 7000 to 20,000 U/kg, bodyweight of patient per hour. Thus for a 70 kg adult human being, an effective amount of SOD per hour will preferably be from 500,000 to 1,500,000 U.

The following examples are provided in illustration of ~he present invention and should not be construed in any way as constitu-ting a limitation thereof.

Example 1: PreParation of Parenteral Formulation of t-PA

A parenteral formulation of t-PA was prepared substantially as described in GB-A-2 176 703. The t-PA had a specific activity of about 300,000 IU/mg.

Example 2: Preparation of Parenteral Formulation of SOD

Bovine SOD of the copper/zinc form was obtained from Sigma Chemical Co., St Louis, Missouri, U.S.A., 63178, as a powder and dissolved in substantially neutral physiological saline solution.

Example 3: Prepara~ion of Parenteral Formulation of ~-PA and SOD

The formulations of Examples 1 and 2 were mixed and further diluted in physiological saline solution to achieve the required dosage.

E~ample 4: Protection of Jeopardized Tissue bY t-PA and by t-PA and SOD

(a) Methodolo~

Male beagle dogs (10-12kg) were anaesthetized with pentobarbital sodium,intubated, and ventilated with room air via a Harvard respirator. Catheters for infusion and arterial blood pressure measurements were implanted in the left ~ugular vein and left carotid arter~. A thoracotomy was performed at the fourth intercostal space, the heart suspended in a pericardial cradle and the left anterior descending artery (LAD) isolated just below the first major diagonal branch. An electromagnetic flow probe was placed on the LAD. A 90 minute occlusion of the LAD ~as produced by placing a snare of 1/0 silk sutre ~': ., ~: . ~
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, 9g dlstal to the flow probe. Treatment was lnltlated intravenously fifteen minut~s prior to release of this snare occlusion and continued for 45 minutes after release. The thoracotomy was closed, and the animals were allowed to recover from the surgical procedures. The animals were reanaesthetized 24 hours after the occlusion, and the flow ln t:he LAD reassessed. Then the heart was removed for post mortem quantification oE infarct size.

Four groups of dogs were evaluated. Group 1 conslsted of saline controls. Group II were administered 2.5 mg/~g (750,000 IU/kg) t-PA, ~roup III were administered 16,500 U/kg bovine SOD, and Group IV were administered both 2.5 mg/kg (750,000 IU/kg) t-PA and 16,500 U/kg bovlne SOD. The formulations used were as described in Examples 1 to 3.

Nyocardial infarct slze was quantifled by an ex vivo dual reperfusion technique. Cannulas were inserted into the LAD immediately distal to the site of occlusion and into the aorta above the coronary ostia. The LAD coronary bed that was perfused with 1.5% triphenyl tetrazolium hydrochloride (TTC) in 0.02 M
potassium phosphate buffer, pH 7.4. The aorta was perfused in a retrograde manner with 0.5% Evans blue dye. Both regions were perfused ~ith their respective stalns at a constant pressure of lOO mm mercury for five minu~es.
The heart was cut into 8 mm slices perpendicular to the apex-base axis. The area of the left ventrlcle at risk of infarction due to anatomlcal dependsnce on the LAD for blood flow is identified by the lack of Evans blue in this region. The region of infaxcted myocardium within the area at risk was demarcated by the lack of stalning of the tissue when perfused with TTC due to a loss of dehydrogenase enzymes.

The transverse ventricular sections were traced carefully on to clear acrylic overlays to provide a permanent record of infarct morphology and to allow planimetric confirmation of infarct size. Ventricular sectlons then were trimmed of right ventricular muscle, valvular, and fatty tissue. The total left ventrlcle area at rlsk and infarct was separated by careful dissection and weighed. Infarct size was expressed as a percentage of the anatomic area at risk. Stat~stical comparison of the drug treaement group to the control group was made using a one way analysis of variance (anova) using Bonferroni's method MJS/OL~/B487/488 .
.

, for m~ltlple comparlson (Circulation Research, 1980, 47, 1-9). A p value of less than 0.05 was taken as the crlterlon of slgnlflcance.

(b) Results TABLE

______ __ ___ __ ______ _ _ _ _ _ _ _ _ _ __ _ _ GROUP NUMBER AREA AT RISK ~ YENT~ICLE*
OF DOGS INFARCTED AT RISK
_________ __ _____ __ ____ _ ____ ___ . __ _ I. Sallne 5 36.0 + 8.9 37.3 ~ 7.6 II. t-PA 6 14.3 + 11.7 35.7 + 5.4 III. SOD 4 13.0 + 4.6 30.6 + 2.6 IV. t-PA+ 3 2.3 + 1.3 37.2 + 9.1 SOD

______________ _ __ _______ __ _ ___ __ _ __ _ ____ _ _ _ * Data are expressed as means + standard errors.

The proportion of the left ventricle made ischaemic by mechanical occlusion of the LAD was not signlficantly different bet~een any of the treat~ent groups and th control group by ANOVA.

(c) Concluslons The use of t-PA signiflcantly lnhibited the myocardial infarct size ~husdemonstrating its ability to protect ~aopardized tissue during reperfusion. In additlon, the combined use of t-PA and SOD achleved a synergistic inhibitory effect ln this regard, wlth the combination provldlng a level of lnhibition greater than that provlded by each of t-PA or SOD on lts own.

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Claims

1. Use of t-PA for the manufacture of a medicament for the inhibition of damage to jeopar-dized tissue during reperfusion in a mammal.

2. Use of t-PA and SOD for the manufacture of a medicament for the inhibition of damage to jeopar-dized tissue during reperfusion in a mammal.

3. Use of t-PA and SOD for the manufacture of a medicament for the removal of a blood clot and for the inhibition of damage to jeopardized tissue during reperfusion in a mammal.

4. Use according to claim 1, 2 or 3, wherein the tissue is myocardial tissue.

5. Use according to claim 1, 2 or 3, wherein the t-PA is in the one-chain form.

6. Use according to claim 4, wherein the t-PA
is in the one-chain form.

7. Use according to claim 1, 2 or 3, wherein the t-PA is in the two-chain form.

8. Use according to claim 4, wherein the t-PA
is in the two-chain form.

9. Use according to claim 5, wherein the t-PA
has the amino acid sequence set forth in Figure 1 or has the same amino acid sequence but with the amino acid in the 245th position from the serine N-terminus being valine instead of methionine, either sequence optionally having an additional polypeptide N-terminal presequence of Gly-Ala-Arg.

10. Use according to claim 6, wherein the t-PA
has the amino acid sequence set forth in Figure 1 or has the same amino acid sequence but with the amino acid in the 245th position from the serine N-terminus being valine instead of methionine, either sequence optionally having an additional polypeptide N-terminal presequence of Gly-Ala-Arg.

11. Use according to claim 2 or 3, wherein the SOD is the copper/zinc form of bovine or human origin.

12. A combination of t-PA and SOD.

13. A combination of t-PA and SOD for use in human and veterinary medicine.

14. A combination of t-PA and SOD for use in the inhibition of damage to jeopardized tissue during reperfusion in a mammal.

15. A combination of t-PA and SOD for use in the removal of a blood clot and in the inhibition of damage to jeopardized tissue during reperfusion in a mammal.

16. A combination according to claim 12, 13, 14 or 15, wherein the t-PA is in the one-chain form.

17. A combination according to claim 12, 13, 14 or 15, wherein the t-PA is in the two-chain form.

18. A combination according to claim 13, wherein the t-PA has the amino acid sequence set forth in Figure 1 or has the same amino acid sequence but with the amino acid in the 245th position from the serine N-terminus being valine instead of methio-nine, either sequence optionally having an additional polypeptide N-terminal presequence of Gly-Ala-Arg.

19. A combination according to claim 17, wherein the t-PA has the amino acid sequence set forth in Figure 1 or has the same amino acid sequence but with the amino acid in the 245th position from the serine N-terminus being valine instead of methio-nine, either sequence optionally having an additional polypeptide N-terminal presequence of Gly-Ala-Arg.

20. A combination according to claim 12, 13, 14 or 15, wherein the SOD is the copper/zinc form of bovine or human origin.

21. A pharmaceutical formulation which com-prises a combination according to claim 12, 13, 14, 15, 18 or 19, and a pharmaceutically acceptable carrier.

22. A pharmaceutical formulation which com-prises a combination according to claim 16, and a pharmaceutically acceptable carrier.

23. A pharmaceutical formulation which com-prises a combination according to claim 17, and a pharmaceutically acceptable carrier.

24. A pharmaceutical formulation which com-prises a combination according to claim 20, and a pharmaceutically acceptable carrier.

25. Use of t-PA for the inhibition of damage to jeopardized tissue during reperfusion in a mammal.

26. A blood clot removing pharmaceutical formulation comprising a combination of t-PA and SOD
in an acceptable and effective amount for removal of blood clots, in association with a pharmaceutically acceptable carrier.

27. A formulation according to claim 26, wherein the t-PA is in the one-chain form.

28. A formulation according to claim 26, wherein the t-PA is in the two-chain form.

29. A formulation according to claim 26, wherein the t-PA has the amino acid sequence set forth in Figure 1 or has the same amino acid sequence but with the amino acid in the 245th position from the serine N-terminus being valine instead of methio-nine, either sequence optionally having an additional polypeptide N-terminal presequence of Gly-Ala-Arg.

30. A formulation according to claim 26, 27, 28 or 29, wherein the SOD is the copper/zinc form of bovine or human origin.

31. An inhibitor of damage to jeopardized tissue during reperfusion in a mammal pharmaceutical formulation comprising a combination of t-PA and SOD
in an acceptable and effective amount for inhibition of damage to jeopardized tissue during reperfusion in a mammal, in association with a pharmaceutically acceptable carrier.

32. A formulation according to claim 31, wherein the t-PA is in the one-chain form.

33. A formulation according to claim 31, wherein the t-PA is in the two-chain form.

34. A formulation according to claim 31, wherein the t-PA has the amino acid sequence set forth in Figure 1 or has the same amino acid sequence but with the amino acid in the 245th position from the serine N-terminus being valine instead of methio-nine, either sequence optionally having an additional polypeptide N-terminal presequence of Gly-Ala-Arg.

35. A formulation according to claim 31, 32, 33 or 34, wherein the SOD is the copper/zinc form of bovine or human origin.