LU86875A1 - USE OF THE PLASMINOGEN TISSUE ACTIVATOR, ITS ASSOCIATION WITH A SUPEROXIDE-DISMUTASE AND PHARMACEUTICAL FORMULATION CONTAINING THIS ASSOCIATION - Google Patents

Abstract

Tissue plasminogen activator (t-PA), optionally together with superoxide dismutase (SOD), is used to manufacture a medicament for use in the inhibition of damage to jeopardised tissue during blood reperfusion. The medicament is for preventing damage to tissue following ischaemic attack, e.g. in myocardial tissue, when blood supply is restored.

Description

. 87 / B 52 659

"' _ GRAND DUCHY OF LUXEMBOURG

Naked Patent 0 U ....... O / U <k * V Minister of 11 ..... May ...... 1987 of the Economy and the Middle Classes, àr-7-Üiê Intellectual Property Service

____ s Hâÿ LUXEMBOURG

s.

7 · '^ .. Application for Patent of invention ..... ~~ ........ ~ ............. I. Krquitr (11

The company known as: THE WELLCOME FOUNDATION LIMITED, (2) ........ 183-193 Euston Roadf GB-LONDON NW1 2BP (Great Britain), ____________ ........ reErresent.ée .. ..by Mr. „Jacques. ... of..MuY.s.er y ... acting __-_____ ........ in ...... quality ....... of ... jnandataire .... .................................................. ................................. (3) remove (s) this ......... . eleven ...... may ..... 1900 eighty ....... sept______________________________________________________________ (4) to .................... ..1.5 ...... hours, at the Ministry of the Economy and the Middle Classes, in Luxembourg: 1. this request for obtaining a patent for invention concerning: ....... .. "Use of ...... tissue plasminogen activator (5) ......... its ...... association ...... with ...... a superoxide dismutase ...... and pharmaceutical formulation ..... containing ... this ....... as.oc.iation. * i ..______________________________________ 2. description in French .......... language ................................... .............. of the invention in triplicate; 3. ................ 2 ......... ............................. drawing boards, in triplicate; 4.1a receipt of taxes paid to the Registration Office in Luxembourg, 11 May i 1987____________; 5. the delegation of power, dated ......................................... ................................................ the , ___________________________________________; 6. the successor document (authorization); declare) assuming responsibility for this declaration, that the inventor (s) is (are): (6) ........- Henry ...... BERGER Jr., 610. ..... Normandy Street, Cary. North Carolina .................... 2751 1 (United States of America) ___________________________________________________________________________________ ______________________________________ - Crist John FRANGAKIS, 600 Constitution Drive, Durham, ...... ............

_______________ North Carolina 27705 _______ (United States of America) ______________ ______________________________________ claims (s) for the above patent application the priority of one (s) application (s) of (7) ..... patent________________________________________________________________________________________ filed (s) in (8) .in .. States JGtais-jd! .America _____________ on (9) s ........ May 1.2, .1.98.6. and ..... 7 ....... July ....... 1..9..8.6 ....... under ...... on ... ^: o .> ..- B..62, ^ jQ..4 ^ ____________________________ sH »s3ßi $ S; ($ 8t and s May 12. -198 6 and 3 .July .1986 under No — 862,057 in the name of (11) _________ of ....... inventors ...................................... .................................................. .............------------------------------------- ----------------------- elect) domicile for him / her and, if designated, for his / her representative, in Luxembourg________________________________________________________ ........ 35 boulevard Royal ............................................... .................................................. .................................................. ....................... (12) requests (qnt) the grant of a patent for the subject described and represented in the abovementioned appendices, with deferment of this issue to ...____. 6 ....................................... .................................................. .................................................. ...................month. (13) $ antImandatmre: ...... ^ ..................................... ......................................... ......... .................................................. .................... (14) / \ J Vy \ -A_A_XJC / \ Π. Procès-verbal de Dépôt The above application for a patent for invention has been filed with the Ministry of Economy and the Middle Classes, Department of InteJle ^ ue ^ le'a-ijixembourg Property, dated: May 11, 1987 / ; · / U 7 \ Pr. The Minister of Economy and the Middle Classes, at LA hours j ί, · '^ 5j JLp. d.

I5 \ tÆt -11 -Sjl The head of the department ^ O * intellectual property, \ 5> x \ K & is ^ β / / \ y A 68007_d _ / ^ ~ \ Si__ EXPLANATIONS RELATING TO THE FORMULA'PREBETÎÉPÔT ~ ~ * 87 / B 52,659 i!

CLAIM OF PRIORITY

of the patent application / utility model 'feç'AUX: UNITED STATES OF AMERICA From May 12, 1986 and July 7, 1986 (No. 862.046) and From May 12, 1986 and July 7, 1986 (No. 862.057) Description c filed in support of a request for

PATENT

at

Luxembourg

e „. THE WELLCOME FOUNDATION LIMITED

in the name of: LONDON (Great Britain) for: "Use of the tissue activator of plasminogen, its association with a superoxide dismutase and pharmaceutical formulation containing this association." 4

The present invention relates to tissue plasminogen activator, its association with a superoxide dismutase, pharmaceutical formulations containing them and their use in human and veterinary medicine.

There is a dynamic balance between the enzyme system capable of forming blood clots (the coagulator system) and the enzyme system capable of dissolving blood clots (the fibrinolytic system) which maintains an intact vascular bed in the clear state.

To limit blood loss from an injury, blood clots are formed in the injured vessel. After the natural repair of the wound, excess blood clots are dissolved by the action of the fibrinolytic system.

Blood clots may occasionally form in the absence of traumatic injury and may become lodged in large blood vessels thereby doing so partially or even completely; obstacle to the flow of blood. If this occurs in the heart, lung or brain, it can result in a myocardial infarction, pulmonary embolism or stroke. These conditions together constitute the main cause of morbidity and mortality in industrialized nations.

Blood clots consist of a fibrous network which is capable of being dissolved by a proteolytic enzyme, plasmin. This enzyme is derived from an inactive pro-enzyme, plasminogen, a component of blood plasma, under the action of a plasminogen activator. There are two immunologically distinct plasminogen activators in mammals. The intrinsic plasminogen activator, also known as urokinase, is a kidney-produced enzyme which can be isolated from urine. It can also be prepared from a number of tissue cultures that are sources. The extrinsic plasminogen activator, also known as a vascular plasminogen activator and as a tissue plasminogen activator (AP-t), can be isolated from many

AT

2 brous tissue homogenates (in particular of human uterus), of the wall of vascular cells and of certain tissue cultures. In addition to these two types of plasminogen activators, there is also a bacterial product, Streptokinase, prepared from beta-hemolytic streptococci. A major drawback affecting both urokinase and Streptokinase is that they are active throughout the circulation and not just at the site of a blood clot. They can, for example, destroy very-10 blood proteins, such as fibrinogen, prothrombin, factor V and factor VIII, thereby reducing the clotting power of the blood and increasing the risk of bleeding. . In contrast, the biological activity of AP-t depends on the presence of fibrin to which it binds and where it is activated. Maximum activity thus develops only at the location of a blood clot, that is to say in the presence of the fibrin network to be dissolved, and the risk of hemorrhage is largely eliminated.

The interruption of blood flow in a vessel 20 generally leads to the onset of ischemia. In this situation, the tissue is deprived of oxygen and is threatened, a state in which the tissue is altered but still potentially viable. However, if this situation continues for a period of, for example, three hours or more, the tissue will necrosis and, once in this state, cannot be recovered. It is therefore important that re-irrigation, that is, the restoration of blood flow, takes place as soon as possible to save the tissue before it is permanently damaged. Ironically, even if it is done before the tissue becomes necrotic, re-irrigation itself generates a complex set of phenomena, including the presumed formation of the superoxide radical, which exerts a deleterious effect on the hypoxic tissue. Consequently, re-irrigation can only lead to partial recovery of the threatened tissue, the rest being definitively damaged by the occurrence of one or more of these phenomena.

Λ * 3

It has now been discovered that AP-t inhibits the ingestion of threatened tissue during re-irrigation by protecting that tissue from one or more of the above phenomena. The mechanism of action by which AP-t provides this protection has not been explained, but it is not linked to its activity as a thrombolytic agent. This newly discovered property thus gives the possibility of using AP-t / or a pharmaceutical formulation containing it / as an inhibitor of damage to threatened tissue in the circumstances generally exposed here. Accordingly, the present invention provides: (a) a method for inhibiting damage to threatened tissue during re-irrigation in a mammal which comprises administering to the mammal an effective amount of AP-t; (B) the use of AP-t to inhibit damage to threatened tissue during re-irrigation in a mammal; (c) the use of AP-t for the manufacture of a medicament intended for inhibiting the damage of threatened tissue during re-irrigation in a mammal; and (d) a pharmaceutical formulation for use in inhibiting damage to threatened tissue during re-irrigation in a mammal / which comprises AP-t and a pharmaceutically acceptable carrier.

Although the present invention can be implemented to protect any threatened tissue / it is particularly useful for inhibiting damage to threatened myocardial tissue.

The AP-t for use in the present invention can be any biologically active protein corresponding substantially to the AP-t of mammals / and in particular humans / and includes forms with and without glycosylation. It may be one or two chain AP-t / or a mixture of such AP-t / as described in document EP-A-112 122 / and / in the case of fully human AP-t 35 glycosylated / its apparent molecular weight on polyacrylamide gel is approximately 70,000 and its isoelectric point 4 is between 7.5 and 8/0. Preferably / the specific activity of AP-t is approximately 500,000 IU / mg (International Units / mg / the International Unit being a unit of activity defined by the National Institute for 5 Biological Standards and Control / Holly Hill / Hampstead / London / NW3 6RB / United Kingdom / within the framework of the World Health Organization).

The amino acid sequence of AP-t preferably corresponds substantially to that shown in Figure 1 of the accompanying drawings. It is therefore a sequence identical to that of FIG. 1 or comprising one or more deletions / substitutions / insertions / inversions or additions of amino acids / of allelomorphic origin or other / the resulting sequence having at least 80 %, and pre-ference 90%, of homology with the sequence of FIG. 1 and retaining essentially the same biological and immunological properties as this protein. In particular, the sequence is identical to that of FIG. 1 or is the same with the difference that the amino acid of the 245th position from the N-terminal serine is valine instead of methionine , either sequence optionally containing an additional N-terminal polypeptide pre-sequence consisting of Gly-Ala-Arg.

The amino acid sequence shown in Figure 1 contains thirty-five cysteine residues and therefore has the capacity to form seventeen disulfide bridges. On the basis of an analogy made with other proteins whose structure has been determined in more detail, FIG. 2 shows the proposed structure for the sequence (resulting from the formation of disulfide bridges) between the amino -acid of the 90th position and the C-terminal proline. We are less sure of the structure of the N-terminal region, although some proposals have been put forward (Progress in Fibrinolysis, 1983, 6, 269-273: and Proc. Natl. Acad. Sci., 1984, 81, 35 5355 -5359). The most important features of the AP-t structure are the two ring regions (between the 92nd and 173rd amino acids and between the 180th and 261st amino acids), which are responsible for binding the protein to fibrin, and the region of the serine proteases which constitutes the major part of the B chain and which is responsible for the activation of plasminogen. The particularly important amino acids of the serine proteases are those of the catalytic triad His / Asp / Ser. In AP-t, these appear at 322nd, 371st and 463rd positions. The disulfide bridge connecting the cysteine residues at the 264th and 10,395th positions is also important in that it holds the A and B chains together in the double-stranded form of AP-t.

In FIGS. 1 and 2, the amino acid residues are designated by the following conventional one and three letter codes:

Asp D Aspartic acid Cys C Cysteine His H Histidine

Ihr T Thréonine Val V Valine Arg R Arginine

Ser S Serine Ile I Isoleucine Lys K Lysine

Glu E Glutamic acid Leu L Leucine Trp K Tryptophanne 20 Pro P Pro line Tyr Y Tyrosine Gin Q Glutamine

Gly G Glycine Phe F Phenylalanine Met M Methionine

Ala A Alanine Asn N Asparagine

In FIG. 2, the asterisk designates the cleavage site of single-stranded AP-t into double-stranded AP-t in which the A chain contains the two ring regions and the B chain contains the serine protease region.

AP-t can be obtained by any of the techniques described or known in the prior art. For example, it can be obtained from a normal or neoplastic cell line of the type described in Biochemica and

Biophysica Acta, 1979, 580, 140-153: and documents EP-A-41 766 and EP-A-113 319. It is preferred, however, that AP-t be derived from a cultured transformed and transfected cell line, derived using a recombinant DNA technique as described for example in documents EP-A-93 619, EP-A-117 059, EP-A-117 060, EP-A-173 552, EP-A-174 835, EP -A-178 105, WO 86/01538, WO 86/05514 and WO 86/05807.

4 6

Particularly preferred is the use of Chinese hamster ovary (OHC) cells for the production of AP-t / and that these cells are derived as described in Molecular and Cellular Bioloqy, 1985, 5 (7), 1750 -1759. According to this method / the cloned gene is co-transfected with the gene encoding dihydrofolate reductase (dhfr) in dhfr cells of OHC. The transformants expressing dhfr are selected on media lacking nucleosides and are exposed to increasing concentrations of methotrexate.

The dhfr and AP-t genes are thus amplified together leading to a stable cell line capable of expressing high levels of AP-t.

Preferably, the AP-t is purified using any of the techniques described or known in 15. the prior art / for example the techniques described in

Biochemica and Biophysica Acta, 1979, 580, 140-153; J. Biol. Chem. / 1979, 254 (6), 1998-2003; ibid., 1981, 256 (13), 7035-7041 î Eur. J. Biochem., 1983, 132, 681-686; and documents EP-A-41 766, EP-A-113 319 and GB-A-2 122 219.

The AP-t can be used in the manner of the present invention either alone or in combination with another therapeutic agent which also inhibits damage to threatened tissue during re-irrigation. A particularly useful example of such an association is that made with superoxide dismutase (SOD), an enzyme which is known to cause and destroy superoxide radicals which are the agent of one of the phenomena capable of causing tissue damage. In fact, it has also been found that the degree of inhibition offered by the combination of AP-t and SOD is greatly potentiated compared to those provided by AP-t and SOD by themselves. Accordingly, the present invention also provides a combination of AP-t and SOD.

The combination of AP-t and SOD provides a particularly convenient means of achieving both the elimination of blood clots and the inhibition of damage to threatened tissue during subsequent re-irrigation.

7

Thus, from the administration of AP-t and SOD results first of all the elimination of the blood clot under the known thrombolytic action of AP-t, then the inhibition of tissue damage under the combined actions of AP -t and SOD.

The SOD to be used in combination with AP-t can be any biologically active protein corresponding substantially to any one or more of a group of enzymes generally designated by this name. It is preferably of mammalian and in particular bovine or human origin, and is generally combined with a metal cation which is normally used to classify it. Examples of metal cations include those of iron, manganese and copper and, preferably, combinations of copper with other metals such as zinc, cadmium, cobalt or mercury, of which the copper association is preferred. /zinc. The combined manganese and copper / zinc forms of SOD are found naturally in humans. The combined iron and manganese forms of SOD of bacterial origin both have a molecular weight of about 40,000 and are dimers. On the other hand, the manganese combined form of SOD of eukaryotic origin is a tetramer whose molecular weight is approximately 80,000. The copper / zinc combined form of SOD of eukaryotic origin is a dimer which contains a cation copper and a zinc cation per subunit and whose molecular weight is about 32,000. The copper cation is linked by coordination to four histidine residues per subunit and the zinc cation is linked by coordination between a residue histidine and an aspartic acid residue. There is also a copper / zinc combined form of SOD of eukaryotic origin which consists of four subunits and whose molecular weight is approximately 130,000. The molecular weights of the various forms of SOD have been estimated by equilibrium sedimentation, molecular sieving or using polyacrylamide gels. The isoelectric points of the various forms of SOD range from 4 to 6.5, depending on the degree of sulfation and / or deamidation. Preferably, the specific activity of the form combined with copper / zinc of SOD of bovine or human origin is at least 3000 U / mg (the activity unit being as defined in J. Biol. Chem., 1969, 5 244, 6049-6055).

The amino acid sequence of the copper / zinc combined form of SOD of bovine or human origin preferably corresponds substantially to that set out in J. Biol. Chem., 1974, 249 (22), 7326-7338, in the case of bovine origin, 10 and in Biochemistry, 1980, 1 ^, 2310-2316 and FEBS Letters, 1980, 120, 53-55, in the case of human origin. This sequence is therefore identical to one of those exposed in these articles or comprises one or more deletions, substitutions, insertions, inversions or additions of amino acids, of allelomorphic origin or other, the resulting sequence being sufficiently homologous to the published sequence chosen to retain essentially the same biological and immunological properties.

The amino acid sequence of the copper / zinc combined form of bovine SOD contains three cysteine residues per subunit (J ♦ Biol. Chem., 1974, 249 (22), 7326-7338). The intracatenary disulfide bridge is between residues Cys 55 and Cys 144, while the intercatenarian disulfide bridge is between residues Cys 6. The amino acid sequence of the combined copper / zinc form of original SOD Human contains four cysteine residues per subunit (Biochemistry, 1980, 19, 2310-2316, and FEBS Letters, 1980, 120, 53-55). The intracatenary disulfide bridge is between residues Cys 57 and Cys 146, while the intercatenarian di-sulfide bridge is between residues Cys 6.

The Cys 111 residue remains free.

SOD can be obtained by any technique described or known in the prior art. For example, it can be obtained from erythrocytes or from the liver by an extraction method of the type described in GB-A-1 407 807 and GB-A-1 529 890. Alternatively, SOD can be obtained from a transformed or transfected cultured cell line # derived using a recombinant DNA technique as described # for example # in Australian patent application No. 27461/84 and documents WO 85/01503 # EP-A-138 111 # 5 EP-A-164 566, EP-A-173 280 and EP-A-180 964.

The SOD is preferably purified by any suitable technique described or known in the prior art # for example the technique described in document EP-A-112,299.

To use AP-t # or a combination of AP-t and SOD # in the manner of the present invention # it is preferable to use the active ingredient (s) in the form of a pharmaceutical formulation. In the case of the combination # the active ingredients can be in separate formulations which are administered simultaneously or successively. If administered successively # it is best to administer the formulation containing AP-t first and then the formulation containing SOD. In either case, the time for administration of the second of the two formulations should not be such as to lose the benefit of the potentiated tissue damage inhibiting effect which results in vivo association of active ingredients. However # rather than using separate formulations # it is much more convenient to present the two active ingredients together in a single mixed formulation. Accordingly, the present invention also provides a pharmaceutical formulation which includes AP-t and SOD and a pharmaceutically acceptable carrier.

In general # AP-t or the combination of AP-t and SOD will be administered intravascularly, either by infusion or by injection of an embol # and a formulation for parenteral use is therefore necessary. It is preferable to present a lyophilized formulation to the doctor or veterinarian because of the important advantages that such a formulation offers in terms of transport and storage.

The physician or veterinarian can then reconstitute the lyophilized formulation in an appropriate amount of solvent / as needed and when desired.

Pharmaceutical formulations containing AP-t / lyophilized and for parenteral use / are known in the prior art. Examples are provided by documents EP-A-41 766, EP-A-93 619, EP-A-112 122, EP-A-113 319, EP-A-123 304, EP-A-143 081, EP-A-156,169, WO 86/01104, the published Japanese patent No. 57-120523 (application No. 56-6936) and the published Japanese patent No. 58-65218 (application No. 56-163145).

Other preferred examples are provided by GB-A-2 176 702 and GB-A-2 176 703. All of these formulations are also suitable for SOD and for the combination of AP-t and SOD.

Intravascular infusions are normally performed using the parenteral solution contained in an infusion bag or vial or in an electrically operated infusion syringe.

The solution can be delivered to the patient from the sachet or vial by gravity flow or by using an infusion pump. The use of gravity flow infusion systems does not allow the rate of administration of the solution for parenteral use to be controlled sufficiently and the use of an infusion pump is therefore preferred, in particular with solutions whose concentration in active ingredients is relatively high. Even more preferred, however, is the use of an electrically actuated infusion syringe which allows the speed of administration to be controlled even better.

The amount of AP-t, and a combination of AP-t 30 and SOD, which is effective in inhibiting damage to threatened tissue during re-irrigation is obviously a function of a number of factors including, for example , age and weight of the mammal, exact condition requiring treatment and its severity, route of administration, 35 and this amount will ultimately be left to the discretion of the attending physician or veterinarian. However, in 11 in the case of AP-t # an effective dose is generally in the range of 0.2 to 4 mg / kg (i.e. 100,000 to 2,000,000 ül / kg assuming a value of 500,000 IU / mg for the specific activity of AP-t) / preferably from 0/3 to 5 2 mg / kg (that is to say 150,000 to 1,000,000 IU / kg), of the patient's body weight per hour. Thus / for an adult human being weighing 70 kg / an effective amount per hour is preferably between 20 and 140 mg (that is to say between 10,000,000 and 70,000,000 IU), and in particular approximately 10 70 mg (i.e. 35,000,000 IU). In the case where AP-t is associated with SOD / an effective dose of SOD is then generally in the range from 1000 to 50,000 U / kg / preferably from 7000 to 20,000 U / kg / body weight of the patient and per hour. Thus / for an adult human being weighing 70 kg, an effective amount of SOD / per hour # is preferably between 500,000 and 1,500,000 U.

The following nonlimiting examples illustrate the present invention.

Example 1: Preparation of an AP-t Formulation for Parenteral Use

A formulation of AP-t for parenteral use is prepared by proceeding substantially as described in document GB-A-2 176 703; the specific activity of the AP-t used is approximately 300,000 IU / mg.

25 Example 2: Preparation of a SOD Formulation for Use

Parenteral

Bovine SOD # form combined with copper / zinc # supplied in powder form by Sigma Chemical Co. # 30 St Louis # Missouri # U.S.A. # 63178 is dissolved in substantially neutral physiological saline.

Example 3: Preparation of a Formulation of AP-t and SOD for Parenteral Use

The formulations of Examples 1 and 2 are mixed and the resulting mixture is further diluted to achieve the required dosage.

• φ 12

Example 4: Protection of threatened tissue using AP-t and using AP-t and SOD (a) Methodology

Male bigle breed 5 dogs (10-12 kg) are anesthetized with sodium pentobarbital / intubated and ventilated to ambient air using a Harvard respirator. Catheters are implanted for infusion and blood pressure measurements in the left jugular vein and the left carotid artery. A thoracotomy is performed at the level of the fourth intercostal space / the heart is suspended in a pericardial arch and the anterior interventricular artery (AIA) is isolated just below the first main diagonal branch. An electromagnetic flow sensor is placed on 1ΆΙΑ. 11AIA is occluded for 90 minutes by placing a 1/0 silk wire loop downstream of the flow probe. The intravenous treatment is started fifteen minutes before the lifting of the occlusion loop and continues for 45 minutes after the lifting. The thoracotomy is closed and the ani-20 ailments are allowed to recover from the surgical procedures. The animals are again anesthetized 24 hours after the occlusion and the flow rate in the IAA is re-determined. The heart is then removed for post-mortem quantification of the size of the infarction.

25 Four groups of dogs are subjected to evaluation.

Group 1 is made up of controls receiving a saline solution. Group II animals receive 2/5 mg / kg (750,000 ül / kg) of AP-t / Group II animals receive 16,500 U / kg of bovine SOD and Group IV animals receive 2/5 mg / kg ( 750,000 30 ül / kg) of AP-t as well as 16,500 ü / kg of bovine SOD. The formulations used are those described in Examples 1 to 3.

The size of myocardial infarction is quantitatively determined by an extra vivo double perfusion technique. Cannulas are introduced into the IAA immediately downstream of the occlusion site and into the aorta above the coronary orifices. The AIA coronary bed is infused with 1/5% triphenyl tetrazolium hydrochloride (CTT) in 0 / 02M potassium phosphate buffer / pH 7/4.

0/5% Evans blue is perfused in retrograde mode into the aorta. The two regions are perfused with their respective dyes at a constant pressure of 100 mm of mercury for five minutes. The heart is cut into 8 mm slices perpendicular to the point-base axis. The absence of Evans blue identifies the area of the left ventricle which is subject to the risk of infarction due to its anatomical dependence on AIA with regard to blood flow. Within the risk zone / the infarcted myocardial region is distinguished by the lack of coloration of the tissue once it has been subjected to the CTT perfusion / and this due to a loss dehydrogenases.

The cross ventricular sections are carefully drawn on transparent acrylic layers in order to have a permanent recording of the morphology of the infarction and to allow planimetric confirmation of the size of the infarction. The ventricular sections are then stripped of the right ventricular muscle / valves and adipose tissue. The total risk area of the left ventricle and the infarction are separated by careful dissection and weighed. The size of the infarction is expressed as a percentage relative to the anatomical area at risk. A statistical comparison of each group treated with a drug with the control group is carried out by carrying out a one-to-one analysis of variance by the Bonferroni method for multiple comparison (Circulation Research / 1980/47/30 1-9). We take a p value less than 0/05 as the significance criterion.

14 (b) Results

BOARD

NUMBER AREA AT% OF VENTRI-

SUBJECT CULE RISK GROUP

5 DOGS INFARCATED AT RISK * saline0 "5 36.0 + 8.9 37.3 + 7.6 II. AP-t 6 14/3 1 11/7 35.7 ± 5.4 III. SOD 4 13/0 ± 4/6 30/6 ± 2/6 IV. AP-t + SOD 3 2.3 + 1.3 37.2 ± 9.1 10 * The results are expressed as an average ± standard deviation.

According to the analysis of variance, the proportion of left ventricle made ischemic by mechanical occlusion of the IAA did not differ significantly between any of the treated groups and the control group.

(C) Conclusions The use of AP-t significantly inhibits the size of myocardial infarction, which demonstrates its ability to protect threatened tissue during re-irrigation. In addition, by the joint use of AP-t and SOD, we par-20 comes in this regard to produce a synergistic inhibitory effect, the combination providing a higher inhibition rate than that provided by each of AP-t and SOD independently.

Claims (17)

1. Use of a tissue plasminogen activator for the manufacture of a medicament for inhibiting damage to threatened tissue during re-irrigation in a mammal. 2. Use of a tissue plasminogen activator and a superoxide dismutase for the manufacture of a medicament for inhibiting damage to threatened tissue during re-irrigation in a mammal. 3. Use of a tissue plasminogen activator and a superoxide dismutase for the manufacture of a medicament for eliminating a blood clot and for inhibiting damage to threatened tissue during re-irrigation in a mammal. 4. Use according to any one of the preceding claims / the tissue being a myocardial tissue. 5 Leu Thr Trp Glu Tyr Cys Asp Val Pro Ser Cys Ser Thr Cys Gly Leu Arg Gin Tyr Ser Gin Pro Gin Phe Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro Trp Gin Ala Ala Ile Phe Ala Lys His Arg Arg Ser Pro Gly Glu Arg Phe Leu Cys Gly 300 Gly Ile Leu Ile Ser Ser Cys Trp Ile Leu 5er Ala Ala His Cys Phe Gin Glu Arg Phe Pro Pro Hts His Leu Thr Val Ile Leu Gly Arg Thr Tyr Arg Val Val Pro Gly Glu Glu 5. Use according to any one of the preceding claims / the tissue plasminogen activator being in single-stranded form. 6. Use according to any one of claims 1 to 4 / the tissue plasminogen activator being in double-stranded form. 7. Use according to claim 5 or 6, the tissue plasminogen activator having the following amino acid sequence: SerTyr Gin Val Ile Cys Arg Asp Glu Lys Thr Gin Met Ile Tyr Gin Gin His Gin Ser 1 Trp Leu Arg Pro Val Leu Arg Ser Asn Arg Val Glu Tyr Cys Trp Cys Asn Ser Gly b. Arg Ala Gin Cys His Ser Val Pro Val Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn 50 Gly Gly Thr Cys Gin Gin Ala Leu Tyr Phe Ser Asp Phe Val Cys Gin Cys Pro Glu 30 Gly Phe Ala Gly Lys Cys Cys Glu Ile Asp Thr Arg Ala Thr Cys Tyr Glu Asp Gin Gly Ile Ser Tyr Arg Gly Thr Trp Ser Thr Ala Glu Ser Gly Ala Glu Cys Thr Asn Trp 100 Asn Ser Ser Ala Leu Ala Gin Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala Ile Arg Leu Gly Leu Gly Asn His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp 150 Cys Tyr Val Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys k 16 Ser Glu Gly Asn Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg Gly Thr His Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn Ser Met Ile Leu île Gly Lys 200 Val Tyr Thr Ala Gin Asn Pro Ser Ala Gin Âla Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ala Lys Pro Trp Cys His Met Leu Lys Asn Arg Arg 250 8. Use according to claim 2 or 3 / the superoxide dismutase being of the form combined with copper / zinc of bovine or human origin. * 17 9. Combination of tissue plasminogen activator and superoxide dismutase. 10. Combination of tissue plasminogen activator and superoxide dismutase, for use in human or veterinary medicine. 10 Glu Gin Lys Phe Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr 350 Tyr Asp Asn Asp Ile Ala Leu Leu Gin Leu Lys 5er Asp Ser Ser Arg Cys Ala Gin Glu Ser 5er Val Val Arg Thr Val Cys Leu Pro Pro Ala Asp Leu Gin Leu Pro Asp 400 Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Glu Ala Leu Ser Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His Val Arg Leu Tyr Pro 5er Ser Arg Cys Thr Ser 11. Combination of tissue plasminogen activator and superoxide dismutase, intended to be used to inhibit damage to threatened tissue during re-irrigation in a mammal. 12. Combination of tissue plasmin activator and superoxide dismutase, intended to be used to remove a blood clot and to inhibit damage to threatened tissue during re-irrigation in a mammal. 13. Association according to any one of claims 9 to 12, the tissue activator of plasminogen being in single-stranded form. 14. Association according to any one of claims 9 to 12, the tissue activator of plasminogen 20 being in double-stranded form. 15. Association according to claim 13 or 14, the tissue activator of plasminogen having the amino acid sequence set out in claim 7 or having an amino acid sequence identical to the difference that the amino acid of the 245th position from the N-terminal serine is valine instead of methionine, each of these sequences optionally comprising a polypeptide pre-sequence. Additional N-terminal composed of Gly-Ala-Arg. 15 Gin His Leu Leu Asn Arg Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp Thr Arg 450 Ser Gly Gly Pro Gin Ala Asn Leu His Asp Ala Cys Gin Gly Asp Ser Gly Gly Pro Leu Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile Ser Trp Gly Leu 500 Gly Cys Gly Gin Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met Arg Pro 527 20 or having an amino acid sequence identical to the difference that the amino acid of the 245th position from the N-terminal serine is valine instead of methionine / each of these sequences optionally comprising an additional N-terminal polypeptide pre-sequence composed of Gly-Ala-Arg. 16. Association according to any one of claims 9 to 12, the superoxide dismutase being of the form combined with copper / zinc of bovine or human origin. 17. Pharmaceutical formulation comprising a combination according to any one of claims 9 to 16 and a pharmaceutically acceptable carrier.