AU639903B2 - Fibrinolysis - Google Patents

Description

t- i z OPI DATE 13/08/90 AOJP DATE 13/09/90 APPLN. ID 49645 PCT NUMBER PCT/AU90/00013 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 90/07932 A61K 37/02 A l (43) International Publication Date: 26 July 1990 (26.07.90) (21) International Application Number: PCT/AU90/00013 (81) Designated States: AT (European patent), AU, BE (European patent), CA, CH (European patent), DE (Euro- (22) International Filing Date: 19 January 1990 (19.01.90) pean patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (Eu- Priority data: ropean patent), SE (European patent), US.

PJ 2356 20 January 1989 (20.01.89) AU Published (71) Applicant (for all designated States except US): THE UNI- With international search report.

VERSITY OF MELBOURNE [AU/AU]; Gratten Street, Parkville, VIC 3052 (AU).

(72) Inventors; and Inventors/Applicants (for US only) HAMILTON, John, Allan [AU/AU]; 18 Charles Street, Kew, VIC 3101 (AU).

HART, Prudence, Hamilton [AU/AU]; 28 Avenue Street, Millswood, S.A. 5034 (AU).

(74) Agents: CORBETT, Terence, Guy et al.; Davies Collison, I Little Collins Street, Melbourne, VIC 3000 (AU).

(54) Title: FIBRINOLYSIS (57) Abstract A method for degrading fibrin deposits and preventing such deposits associated with pathological conditions is described, which comprises administering to a subject in need of such treatment a therapeutically effective amount of IL-4 activity, optionally in association with one or more pharmaceutically acceptable carriers or excipients. There is also described thrombolytic compositions which comprise IL-4 or a derivative thereof possessing IL-4 activity together with a pharmaceutically acceptable carrier or excipient.

I I:: WO 90/07932 PC17AU90/00013 1

FIBRINOLYSIS

The present invention relates to fibrin degradation or breakdown (which may be referred to as fibrinolysis), and compositions and methods for the treatment of pathological conditions associated with fibrin deposition.

Fibrin plays a crucial role in haemostasis and wound healing, and is laid down in the human and animal body as a result of a complex series of biochemical reactions. Notwithstanding these crucial functions of fibrin, fibrin formation is also a common event in many pathological conditions and inflammatory lesions. For example, fibrin deposition is associated with atherosclerosis, rheumatoid arthritis, glomerulonephritis, systemic lupus erythematosis, myocardial infarcts, pulmonary embolism, deep vein thrombosis, autoimmuine neuropathies, granulomatous disease, parasitic infections and allograft rejection. Metastases, or WO 90/07932 PCT/AU90/00013 so called "secondary" tumours, have been linked with thromboembolism phenomona. The abundant fibrin deposited around some solid tumours in the stroma may serve as a cocoon that hinders lymphocytes, macrophages and other inflammatory cells from reaching tumours.

Fibrin deposition may also be associated with renal disease and hypertrophic scars and keloids.

Fibrous adhesions are also a significant problem in post-operative surgery.

Urokinase and tPA (tissue plasminogen activator) are plasminogen activators (PA's) which have previously been used both experimentally and clinically to effect fibrin lysis, and in particular, the lysis or degradation of fibrin clots. Whilst effective in degrading fibrin, these molecules have attendant disadvantages. Urokinase activates plasminogen to give plasmin independently of the presence of fibrin (unlike tPA), and thus large amounts must be administered to effect fibrinolysis, this being expensive and causing unwanted bleeding.

tPA has a short half life in vivo, and thus large quantities have to be administered over a long period of time, resulting in unwanted bleeding and expense.

It has now surprisingly been found that the lymphokine, interleukin-4 activates both human and animal cells to produce plasminogen activators, which results in fibrin degradation.

Also, IL-4 inhibits the procoagulant activity of human monocytes resulting in decreased fibrin formatioh at the monocyte surface.

IL-4 is a lymphokine, which exhibits both B cell a-d T cell growth factor activities (published SAustralian Patent Application No. 67334/87). This 1__ WO 90/07932 PCT/AU90/00013 3 lymphokine also exhibits suppressive activity, as it supresses human monocyte production of the cytokines IL-1, TNF, and suppression of PGE2' IL-4 has been purified to homogeneity, and the gene encoing this protein has been cloned allowing IL-4 to be produced in large amounts by recombinant DNA technology, as described in published Australian Patent Application No. 67334/87, in the name Schering-Biotech Corporation. IL-4 (human) is commercially available from a number of suppliers.

On the basis of IL-4's activity in suppressing cytokine production, it was most surprising that IL-4 was effective in stimulating plasminogen activator (PA) production, notably, t-PA and urokinase, by appropriate target cells.

In accordance with the present invention, there is provided a method for degrading fibrin deposits and preventing such deposits associated with pathological conditions or which may lead to such conditions, which comprises administering to a subject in need of such treatment a therapeutically effective amount of IL-4 or a derivative thereof possessing IL-4 activity, optionally in association with one or more pharmaceutically acceptable carriers or excipients.

Pathological conditions which may be treated in accordance with the invention are those which are caused wholly or at least in part by fibrin deposition. These include deep vein thrombosis, pulmonary embolism, renal disease, hypertrophic keloid scars, coronary infarction, metastasis, inflammation, disseminated intravascular coagulation, atherosclerosis, rheumatoid arthritis, WO90/07932 PCT/AU90/00013 glomerulonephritis, systematic lupus erythematosis, autoimmune neuropathies, granulomatous disease, parasitic infection, allograft rejection, and other conditions associated with fibrin deposition.

Administering IL-4 to subjects suffering from such conditions may result in increased plasminogen activator, particularly tPA and urokinase (prourokinase) production by target cells, causing activation of plasminogen and subsequent fibrin degradation. Also, fibrin formation in such patients may be lessened.

Notable cell types stimulated to produce t-PA when stimulated with IL-4 are the monocyte/macrophage cell types. Importantly, these cell types are often closely associated with thrombi and other fibrin deposits, and hence, when stimulated to produce plasminogen activators on contact with IL-4, may cause highly efficient localised fibrin degradation.

This avoids the need to administer large amounts of plasminogen activator to a patient, hence reducing side effects of unwanted bleeding, and excess cost due to the administration of large amounts of plasminogen activator.

Endothelial cells may also be stimulated with IL-4 to produce urokinase (prourokinase). This is also of importance as fibrin deposits are generally associated with endothelial cells.

Other target cells within a human or animal subject may also be stimulated with IL-4 to effect fibrin degradation. A number of other target cells besides the monocyte may also loose theiz ability to produce procoagulant activity as a result of IL-4 action.

Preferably, the IL-4 employed in this invention i il WO 90/07932 PCr/AU9/00013 is produced by recombinant DNA technology. Where human subjects are to be treated, it is of course desirable that the IL-4 is of human origin. Where animals are treated, animal IL-4 is preferred.

Any IL-4 derivative or analogue which possesses the B cell, T cell and mast cell stimulatory effects of IL-4, and other characteristic activities of IL-4, as described in published Australian Patent Application No. 67334/87 may be used in this invention. The precise nature of IL-4 derivatives or analogues is not of importance. Rather, the derivatives or analogues must possess the well defined biological activity of IL-4, and, must possess the ability to stimulate target cells such as macrophages/monocytes to produce plasminogen activators. Application No. 67334/87 mentioned above teaches methods for the production of IL-4 derivatives and analogues. As used in this specification the term IL-4 encompasses human IL-4, IL-4 of animal origin such as murine, ovine or bovine IL-4, and analogues or derivatives thereof which possess characteristic IL-4 activity. For convenience these molecules may be referred to hereinafter as "active material".

IL-4 may be administered in a convenient manner such as by the oral, intraveneous, intramuscular, subcutaneous, intraperitoneal, intranasal, intradermal or suppository routes.

IL-4 may also be administered to a human or animal subject by continuous infusion over a predetermined time period, for example, from minutes to 24 hours. Administration may be by way of an intravenous catheter connected to an appropriate pump, or by gravity feed.

'I

WO 90/07932 PCT/AU90/00013 6 The amounts of and dosage regimes of IL-4 which are administered to a subject to effect fibrin degradation will depend on a number of factors such as the mode of administration, the nature of the condition being treated, the body weight of the subject being treated, and the judgement of the prescribing physician or veterinarian. Generally speaking, IL-4 may be administered in an amount between 0.lLg to 2000mg per kilogram of body weight per day. The quantity of active compound in a unit dosage such as a tablet or capsule may vary from about 0.1gg to 100mg.

IL-4 may be coated by, or administered with, a material to prevent its inactivation. For example, the active material may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFP) and trasylol.

Liposymes include water-in-oil-in-water P40 emulsions as well as conventional liposomes.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must INTERNATIONAL SEARCH

REPORT

International Appication No. PC/AU 90/00013 I. CLASSIFICATION OF SU=BECT MATTER (if several classification symbols apply, indicate all) 6 According to International Patent Classification (IPC) or to both National Classification and IPC i- i WO 90/07932 PCT/AU90/00013 7 be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, sterile water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thermerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active material in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the I WO 90/07932 PCT/AU90/00013 8 preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When IL-4 is suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or with an edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active material may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, Oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.

Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or I I PC/AU90/00013 WO 90/07932 9 elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharamceutically pure and substantially non-toxic in the amounts employed. In addition, the active material may be incorporated into sustained-release preparations and formulations.

As used herein, the terms "pharmaceutically acceptable carrier" and "excipient" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like described above. The use of such carriers and excipients is well known in the art, see for example, Remington's Pharmaceutical Science and U.S. Pharmacopeia (1984); Mack Publishing Company, Easton, PA.

The active material may also be administered in association with one or more anti-thrombolytic agents selected from, for example, tPA, prourokinase, urokinase or streptokinase. Potentiation of fibrinolytic activity may take place when IL-4 is administered with such agents.

The active material may also be administered in association with one or more anticoagulant agents, such as heparin, warfarin, aspirin, anisindione, phenindone and bishydroxy coumarin; and/or one or more vasodilators such as nitriles (for example, amylnitrile, nitroglycerin, sodium nitrile, isosorbide dinitrate), papaverine, nicotinic acid and cyclandelate. Anticoagulant and vasodilatory agents may improve access to thrombosis and other fibrin deposits thereby enhancing fibrin degradation.

WO 90/07932 PCT/AU90/00013 The invention is also concerned in another aspect with thrombolytic compositions which comprise IL-4 in association with one or more pharmaceutically acceptable carriers or excipients; and which optionally include one or more anti-thrombolytic agents, and/or one or more anticoagulant agents, and/or one or more vasodilators, as described above.

The action of IL-4 in causing fibrin degradation and preventing fibrin and thrombi formation is surprising and unexpected based on the prior art known to the applicant, and provides new therapies for diseases associated with fibrin formation.

The following Examples further illustrate the invention. It will, of course, be understood that the invention is in no way restricted to the specific embodiments described in these Examples.

EXAMPLE 1 Materials and Methods: Monocvte Isolation and Culture: Monocytea were isolated from peripheral venous blood by countercurrent centrifugal elutriation as described by Hart et al. (1988) J. Immunol. 141: 1516. Cell fractions containing )95% monocytes, identified by morp.ological criteria, by non-specific esterase staining and by their phagocytic properties were pooled and cultured for 18 h as previously detailed (0.8-1,0 x 10 in 1 ml a-Modified Eagle's Medium containing 1% fetal calf serum). To 30 terminate the cultures, the supernatants were centrifuged to remove non-adherent cells; after twice washing with PBS, the adherent and non-adherent cells were pooled and lysed with 0.2% Triton X-100 in PBS.

92 1 223 q:\oper~pas,49645goap2 WO 90/07932 PCT/AU90/00013 11 Assay for PA Activity: Monocyte supernatants or lysates (50 pl) and human plasminogen (0.8 ptg), dissolved in 100 p1 0.1 M Tris-HCl, pH 8.1, were added to 0.28 cm 125 wells previously coated with 125I-fibrin, according to the methods of Hamilton (1981) J. Immunol. 126: 851. After 2-3h, soluble 125 I-fibrin degradation products were measured. PA activity was expressed according to the activity of u-PA standards (Leo Pharmaceutical Products, Denmark). Monocyte-derived plasminogen-independent fibrinolytic activity was always of the plasminogen-dependent activity.

Antibody Analysis: IgGs (immunoglobulins) were isolated from rabbit antisera to human u-PA and to human t-.PA (Lots 100 and 153, respectively provided by Dr. W-D.

Schleuning, CHUV, Lausanne, Switzerland) by standard methods using Protein A Sepharose CL-'S (Pharmacia, Uppsala, Sweden). The mouse myeloma IgG, HOPCY (Dr.

A. Burgess, Ludwig Institute for Cancer Research, Melbourne, Australia), was used as a. irrelevant antibody. Culture supernatants, r'ell lysates or PA standards (u-PA) as above, t-PA as a culture supernatant rom the MM138 melanoma cell line (Dr. R.

Whitehead, Ludwig Institute for Cancer Research, Melbourne), 0.2 IU/ml) were incubated with IgGs (1 ptg/ml final concentration) for 1 h at 37 0 C prior to assay of residual PA activity. In the immunoprecipitation experiments, Protein A Sepharose CL-4B was added, as well as Triton X-100 to a final concentration of 0.25%, After further incubation for min. at room temperature with periodical mixing, i the pellets were washed as previously described in Vassalli, et al. (1984) J. Exp. Med. 152:

I

intravascular coaguadriuan, adntie!u. uV0, Srheumatoid arthritis, glomerulonephritis, systemic lupus erythematosis, autoimmune neuropathies, granulomatous diFsase, parasitic infection, allograft S../2 WO 90/07932 PCT/AU90/00013 12 1643, except that SDS was not included in the wash buffers.

SDS-Casein Zymography: SDS-PAGE zymography was carried out according to the methods of Roche et al. (1983) B.B.A. 745: 82.

The resolving gel contained casein (2 mg/ml, Sigma) and human plasminogen (6 pg/ml) and was pre-electrophoresed at 15 mA for 1 h at room temperature. Samples (20 il), equilibrated in 0.0625 M Tris-HCl, pH 6.8, 1.25% SDS, 10% glycerol, were incubated at 700C for 10 min before loading onto the stacking gel followed by electrophoresis at 12 mA for 2 h using the buffer system of Laemmli, Nature 227: 680 (1970). After electrophoresis, gels were washed for 1 h in 2.5% Triton X-100, then rinsed and incubated in 0.1 M Tris, pH 8.0, for 18 h at 37 0 C. Gels were stained by immersion in 0.25% coomassie blue R-250 in 50% methanol/7% acetic acid for 60 min, then destained for 2 h in methanol/10% acetic acid.

Detection of t-PA.mRNA: Total monocyte RNA was prepared according to the methods of Chirgwin et al. (1979) Biochemistry il: 5292) and fractionated (5 Rg/lane) on a formaldehyde-containing 1% agarose gel prior to transfer to Genescreen Plus nylon membrane (Dupont, Boston, MA). The filter was hybridized overnight at 0 C in a standard hybridization buffer containing >2 6 32 x 10 cpm/ml of P-labelled t-PA.cRNA. The cRNA probe was prepared from a plasmid containing the 2.3 kb Bal II fragment from pPAll 4B (Fisher, R. et al.

(1985) J. Biol. Chem. 26L: 11223), cloned into the vector pGEM-4 blue (Promega, Madison, MA) and linearized with Xbal prior to initiation of II 1 WO 90/07932 PCT/AU90/00013 13 transcription with T7 RNA polymerase (New England Biolabs, Beverly, MA). After hybridization, the filter was washed three times with 2 x SSC prior to treatment with 1 jg/ml RNase A (Boehringer-Mannheim, West Germany) for 20 min at 37 0

C.

Measurement of Procoagulant Activity: Monocyte procoagulant activity is measured by the ability of the cells to shorten the partial thromboplastin (clotting) time of platelet poor plasma. The human monocytes (4 x 106 cells/ml) are activated by lipopolysaccharide or a supernatant from stimulated lymphocytes for 18 h at 37 0 C to generate procoagulant activity on their surface. This I activity of frozen-thawed and sonicated cell samples was tested using citrate-treated platelet poor human plasma in a prothrombin assay. Briefly, the clotting assay was performed by incubating the cell suspension (0.8 x 105 cells/0.1 ml) with 0.1 ml plasma for 1 min. at 37°C. Then CaCI 2 (30mM; 0.lml) was added and the clotting time determined manually. Results are cited as mU of activity/10 cells. A standard curve of the clotting time of human plasma with decreasing dilutions of rabbit brain thrornboplastin was used to calculate mU.

EXAMPLE 2 t-PA Production by IL-4 Stimulated Human Monocytes: Human monocytes prepared according to Example 1 (1 x 10 /mi) were incubated with 0.25U/ml human recombinant IL-4 (Genzyme).

Plasminogen activator (PA) activity (determined 2 by plasminogen-dependent fibrinolytic activity as described in Example 1) was detected in monocyte i .1 WO90/07932 PCT/AU90/00013 14 lysates after exposure to IL-4 for 2 h, and was secreted into the culture supernatants by 3 h, with maximal production of PA occuring 6 h after IL-4 stimulation.

All of the PA activity detected in the monocyte culture supernatants was blocked by anti-tPA IgG, but not by anti-urokinase IgG. By SDS-casein zymography, a technique which determines the apparent molecular weight of PA, a 70kD band migrating in a manner characteristic of the t-PA standard was found.

Accordingly, IL-4 stimulates human monocytes to produce t-PA.

Northern analysis showed the presence of t-PA.mRNA in monocytes treated with IL-4. t-PA mRNA was not detected in unstimulated monocytes.

EXAMPLE 3 Production of PA Activity by Endothelial Cells Stimulated with IL-4: Bovine aortic endothelial cells (105 cells) were incubated in RPMI/10% FCS for 48 h, washed in isotonic saline, and then cultured in a-MEM (1 ml) in the presence or absence of 2.5 units/ml purified human IL-4 (Genzyme). After 24 h, conditioned medium was collected and assayed for PA activity, as a plasminogen-dependent fibrinolytic activity. PA activity was detected in those cells stimulated with IL-4. Results are as follows: WO 90/07932

I

i: I I PCT/AU9/00013 Bovine endothelial cells (Control) Bovine endothelial cells stimulated with 2.5 units/ml PA Activity lu/ml 0.09 0.01 0.32 0.01 IL-4SDS-casein zymography identified urokinase type PA activity and t-PA activity with the increase being in the urokinase type.

EXAMPLE 4 Inhibition of Procoagulant Activity of Human Monocytes by IL-4: Human monocytes (4 x 10 6 cells), prepared according to Example 1, were incubated with 100ng/ml lipopolysaccharide (LPS), with human recombinant IL-4 (Schering Plough), or with LPS together with varying concentrations of IL-4.

Procoagulant activity (PCA) (determined by the shortening of the clotting time of citrated plasma as described in Example 1) was measured in monocyte lysates after exposure to the reagents for 18 h at room temperature.

I

iiii j Australian Patent Application No. 67334/87). This Ii WVO 90/07932 PCr/Au9o/ooo1 3 16

PCA

6 cells Experiment 1 Experiment 2 N.D. N.D.

N.D. N.D.

*Human monocytes (control) Human monocytes treated with units/mi IL-4 Human monocytes treated with: LPS (100ng/mi) LPS (100ng/mi) IL.-4 (2.5U/ml) LPS (100ng/mi) 0.5 LPS (100ng/mi) 0.lU/ml) N.D. not detected.

20.9 ±0.8 4. 6 ±0.1 4.2 0.2 13 .9 0.4 19.4 0. 1.4 0. 1 5. 1 0.2 8. 5 0. 7

Claims (13)

1. A method for degrading fibrin deposits and preventing the formation of such deposits associated with pathological conditions or which may lead to such conditions, which comprises administering to a subject in need of such treatment a therapeutically effective amount of IL-4 or a derivative thereof possessing IL-4 activity, optionally in association with one or more pharmaceut'lcally acceptable carriers or excipients.

2. A method according to claim 1, wherein the pathological conditions treated are selected from deep vein thrombosis, pulmonary embolism, renal disease, hypertrophic keloid sears, coronary infarction, metastasis, inflammation, disseminated intravascular coagulation, atherosclerosis, rheumatoid arthritis, glomerulonephritis, systemic lupus erythematosis, autoimmune neuropathies, granulomatous disease, parasitic infection, allograft rejection, and other conditions associated with fibrin deposition.

3. A method according to claim 1, wherein the IL-4 or a derivative thereof is locally administered at or near the site of fibrin deposition.

4. A method according toclaim 1, where the IL-4 or a derivative thereof is administered by intravenous, intramuscular, intranasal, intradermal, intraperitoneal, suppository or oral route.

A method according to claim 1A where the therapeutically effective amount of IL-4 or I Preferably, the IL-4 employed in this invention J18 18 derivatives thereof is between about O.lpg to about 200 mg per Kg body weight of the subject to be treated.

6. A method according to claim 1, wherein the IL-4 or a derivative thereof is continually infused into a subject for a predetermined period.

7. A method according to claim 1, wherein the IL-4 or a derivative thereof is administered in conjunction with one or more thrombolytic agents or activators of thrombolytic agents selected from tPA, urokinase, streptokinase or prourokinase.

8. A method according to'claim 1, wherein the IL-4 or a derivative thereof is administered in conjunction with one or more anticoagulant agents such as heparin, warfarin, aspirin, anisindione, phenindone and bishydroxy coumarin.

9. A thrombelytie e. iy IL-4 or a derivative thereof possessing IL-4 activity in association .with a pharmaceutically acceptable carrier/or excipient, which composition additionally comprises one or more :thrombolytic agents or activators ther of selected from the group consisting of tPA, urokinase, ourokinase and streptokinase.

10. A thrombolytic compos ion according to claim 9, which further comprises one or more anticoagulant agents.

11. A thrombolytic c position according to claim wherein the anticoagulant agents are selected from the group consisting of hep rin, warfarin, aspirin, anisindone, phenindone and bishyd xy coumarin. i

12. A thromb ytic composition according to any one of claims 9 to 1 which additionally comprises one or more 921223,n.:,'tapr\pAs49645-90.apl8 19 cmoition accordngt n wh i..the vasodilators are select na! htil-fe group consisting of a1rn cotiniC acid and cyclandalate-. DATED this 23rd day of December, 1992 THE UNIVERSITY OF MELBOURNE by its Patent Attorneys DAVIES COLLISON CAVE 921223,q:\oper~pas,4964590.ap.19 INTERNATIONAL SEARCH REPORT International Application No. PCT/AIJ 90/00013 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) 6 I According to International Patent Classification (IPC) or to both National Classification and IPC 4I I nt. Cl. A61K 37/02 II. FIELDS SEAPCHED Minimum Documentation Searched 7 SClassification System Classification Symbols S IPCI A61K* 37/02 I Documentation Searched other than Minimum Documentation Sto the Extent that such Documents are Included in the Fields Searched 8 SAU: IPC as above SIII. DOUMNTS CONSIDERED TO BE RELEVANT 9 Category* Citation of Document, with indication, where appropriate, Relevant to of the relevant passages 12 Claim No

13 SX WO,A2, 89/09621 (RITTER, MARY ALICE and LARCHE, MARK) (9) 19 October 1989 (19.10.89) I I S X WO,A1, 88/04667 (IM NEX CORPORATION) 30 June 1988 (30.06.88) (9) SX WO,A1, 87/07303 (INSTITUT PASTEUR DE LILLE, INSTITUT PASTEUR, (9) INSTITUT NATIONAL DE LA SANTE ET DE LA ECHERCH MEDICALE-INSERM) 3 December 1987 (03.12.87) SX WO,A1, 87/02990 (SCHERNG-BIOTECH CORPORATION) 21 May 1987 (21.05.87) (9) X EP,A2, 0333523 (THE UAB RESEARCH FOUNDATION) 20 September 1989 (9) I(20.09.89) I I (continued) Special categories of cited documents: 10 later document published after the international filing date or priority date document defining the general state of the and not in conflict with the application but art which is not considered to be of cited to understand the principle or theory particular relevance underlying the invention earlier document but published on or document of particular relevance; the S after the international filing date claimed invention cannot be considered novel document which may throw doubts on priority or cannot be considered to involve an S claim(s) or which is cited to establish the inventive step publication date of another citation or document of particular relevance; the S other special reason (as specified) claimed invention cannot be considered to document referring to an oral disclos;re, involve an inventive step when the document use, exhibition or other means is combined with one or more other such document published prior to the documents, such combination being obvious to international filing date but later than a person skilled in the art. the priority date claimed document member of the same patent family IV. CERTIFICATION SDate of the Actual Completion of the Date of Mailing of this International SInternational Search Search Report 16 March 1990 (16.03.90) 26 MAR '190 International Searching Authority Sig ref A thorized Officer I Astra Patent Office Jj HANSON Form PCT/ISA/210 (second sheet) (January 1985) j l=u.i 5 rrom those enumerated above. In the case of sterile powders for the L International App..cation No. PCT/AU 90/00013 I FURTHER INORMATION CONTINUED FROM THE SECOND SHEET X I EP,A1, 0230107 (SCHERING BIOTECH CORPORATION) 29 September 1987 S(29.09.87) (9) (9) I V. OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE 1 This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 1.CX] Claim numbers 1-8, because they relate to subject matter not required to be searched by this Authority, namely: method for treatment of the human or animal body 2.[3 Claim numbers because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: S Claim numbers because they are dependent claims and are not drafted in accordance i with the second and third sentences of PCT Rule 6.4 VI. OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 SThis International Searching Authority found multiple inventions in this international application I as follows: 2.E) I3. I 4.) As all required additional search fees Were timely paid by the applicant, this international search report covers all searchable claims of the international application. As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims of the international application for which fees Were paid, specifically claims: No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claim numbers: As all searchable claims could be searched without effort justifying an additional fee, the International Searching Authority did not invite payment of any additional fee. I Remark on Protest E3 The additional search fees were accompanied by applicant's protest. [3 No protest accompanied the payment of'additional search fees. Form PCT/ISA/210 (supplemental sheet ('January 1985) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL APPLICATION NO. PCT/AU 90/00013 This Annex lists the kno-n publication level patent family members relating to the patent docui-ents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent Document Cited in Search Patent Family Members Report WO 8909621 AU 34301/89 GB 8808015 WO 8804667 AU 10559/88 EP 335900 WO 8707303 AU 74321/87 DK 254/88 EP 246967 FR 2598917 JP 1501597 PT 84913 WO 8702990 AU 67334/87 CN 86108579 DK 3710/87 EP 230107 EP 249613 FI 873141 HU 43630 IL 80678 JP 63501401 NO 872988 PT 83761 ZA 8608748 EP 333523 AU 33433/89 IL 89602 WO 8908449 EP 230107 AU 67334/87 CN 86108579 DK 3710/87 F4P 230107 EP 249613 FI 873141 HU 43630 IL 80678 JP 63501401 NO 872988 PT 83761 WO 8702990 ZA 8608748 END OF ANNEX 5,25DS/183