WO1992010206A1

WO1992010206A1 - Use of plasminogen activator inhibitor type 2 in the treatment of cancer

Info

Publication number: WO1992010206A1
Application number: PCT/GB1991/002177
Authority: WO
Inventors: Mark Noel Forsyth Shinkfield
Original assignee: Delta Biotechnology Limited
Priority date: 1990-12-08
Filing date: 1991-12-09
Publication date: 1992-06-25
Also published as: GB9026737D0

Abstract

PAI-2 (placental-type plasminogen activator inhibitor; plasminogen activator inhibitor type 2; minactivin) may be used at low doses to combat cancers, especially peritoneal cancers. It may be administered via an implanted depot or delivery device for controlled release.

Description

Use of pl asmi nogen activator inhi bitor type 2 in the treatment of cancer

Background of the Invention

Production of Plasminogen Activators (PA) may be an important component of the invasive and metastatic behaviour of malignant tumours, due to the ability of these enzymes to initiate broad-spectrum proteolytic activity. Cell-surface receptors for PAs and plasmin enable localisation of this activity to the close environment of the tumour cell.

The interaction between Uro inase Plasminogen Activator

(uPA) and its receptor may also result in increased growth of tumour cells; an intact PA active site appears to be required as well as the receptor binding part of the molecule.

Prior Art

Inhibition of the PA activity of uPA therefore has the theoretical potential to modify the invasive and metastatic behaviour of malignant tumours. Ossowski and Reich have demonstrated that antibodies to urokinase-plasminogen activator (uPA) reduced metastasis of a human tumour cell line in the chicken chorioallantoic membrane-embryo model¹. Hearing et al showed that colonisation of mouse lungs by intravenously injected mouse melanoma cells was inhibited by uPA antibodies². More recently, Axelrod et al have reported the successful expression of uPA in a transformed cell line which was associated with an increase in lung colonisation when the cells were injected intravenously in nude mice³.

Summary of the Invention

We have investigated the behaviour of human tumour cell lines in cell cultures and after transplantation into nude mice, with attempts to modify this with Recombinant Plasminogen Activator Inhibitor 2 (rPAI2) . Surprisingly, we have found that low doses of rPAI2 are more effective than high doses.

One aspect of the invention provides a method of combatting a cancer in a mammal comprising the administration of plasminogen activator inhibitor type 2 at a cancer combatting dose.

Detailed Description of Preferred τ_mbn<_iments

In some instances the cancer is readily accessible to an anticancer agent via normal routes of administration. For example, when the cancer is at least partly located in the peritoneal cavity of the mammal, an agent can be injected into the peritoneal cavity and thus readily gain access to the tumour tissue. In such instances the dosage administered is preferably 100-100000 units/kg body weight/day, more preferably 1000-50000 units/kg body weight/day, 1000-10000 units/kg body weight/day or about 5000 units/kg body weight/day. Surprisingly, we have found that high doses of PAI-2 are ineffective in a mouse peritoneal tumour model system.

In the instances of cancers which cannot be directly accessed via normal routes of administration (which includes most solid tumours other than intraperitoneal tumours and, possibly, melanomas) it may be necessary to increase the dosage level, but the principle of using low doses of PAI-2 remains. For instance, administration intravenously may be used to treat cancers of the lung despite the PAI-2 being effectively diluted throughout the entire circulation. This dilution would necessarily lead to a higher dosage (for example 2-20 times the dose) being used compared to the dose used when the compound is administered by the IP route.

A "unit" of PAI-2 is the amount of PAI-2 which will inhibit the amidolytic action of an international unit of uPA. A suitable assay is disclosed in Steven, J. , Cottingham, I.R. , Berry, S.J., Chinery, S.A., Goodey, A.R. , Courtney, K. and Ballance, D.J. (1991) Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cereviεiae . Eur. J . Biochem . 196, 431-438. The cancer is, for example, present in the peritoneal cavity of the mammal. The PAI-2 is preferably administered, for example by injection, directly into the peritoneal cavity.

The methods of the invention may be especially effective during surgery to remove tumours, especially those in the peritoneal cavity. Thus, PAI-2 may be administered before, during or within 1.0-24.0 hours after surgery in order to reduce the growth of metastasis and/or to prevent metastasis of the tumour that is being removed.

The term "PAI-Z" is used herein to embrace the natural product, (for example, as obtained from placentae or U937 cells, as disclosed in WO 86/01212, which is incorporated herein by reference) , the nature-identical product produced by recombinant DNA methods (for example prepared by the methods of EP-A-238 275 and PCT/GB90/01229 which are incorporated herein by reference) and all analogues and fragments of PAI-2 which (a) have 80% homology (preferably

>

85%, 90%, 95% or 99%) with PAI-2 for the. respective corresponding amino acid sequences of the two molecules, the sequences being at least 350 (preferably at least 400) amino acids long, (b) have an arginine residue in the reactive centre at the position corresponding to the amino acid immediately amino terminal to the peptide bond cleaved by the enzyme in its natural substrate and (c) have a second order rate constant for urokinase-type plasminogen activator inhibition of at least 10⁵ M-'s-¹ and generally up to at least 2 x lO'M-'s^-1, for example about 2 x 10⁶ M'ε"¹, as measured in the method of Thorsen et al (1988) Eur . J . Biochem . 175, 33-39 (see Table 1 especially). The PAI-2 may be unglycosylated or glycosylated. "Analogues" of PAI-2 include fusions thereof to other polypeptides such as uPA or a fragment of uPA (eg the 1-47 or 20-30 fragments) , for example to target the PAI-2 to the uPA receptors on the tumour cells.

The PAI-2 is generally administered in a sterile, non- pyrogenic medium suitable for the mode of administration, for example intra-peritoneal, intravenous, intramuscular or subcutaneous. A slow release depot may be implanted in the patient, for example in the peritoneum, using a biocompatible polymer such as an ethylene-vinyl acetate co- polymer, a polyanhydride or a polyamino acid. Such medicaments may be prepared by the methods of US Patents Nos 5 030 457, 4 947 904, 4 981 696, 4 888 176, 4 900 556, 4 898 732, 4 994 213, 4 888 413, US 4 975 280, 4 975 284, or 4 933 186, all of which are incorporated herein by reference. Silicone rubber and hydroxylated acrylate compounds such as the commercially available "Hydron" (Trademark) material are generally suitable. Continuous or pulsed delivery devices may be used, for example those disclosed in US Patent No 4 931 050, and transdermal or percutaneous administration may be achieved using the various delivery devices marketed by the Alza Corp. or those disclosed in for example, US Patents Nos 5 028 435 and 4 851 545. All of these patents are incorporated herein by reference.

5 The delivery of the PAI-2 is continued for as long as is deemed desirable by the physician. Other drugs to combat the cancer may be administered as well, for example other inhibitors of uPA, such as anti-uPA antibodies.

10 Experimental Model

The A431 cell line was originally derived from a human squamous cell carcinoma of the vulva, as described in Glard, D.J. , Aaronson, S.A. , Todaro, G.J., Arnstein, P.,

15 Kersey, J.H., Dosik, H. and Parks, W.P. (1973) In vitro cultivation of human tumours: establishment of cell lines derived from a series of solid tumours. J. Natl . Cancer Inεt . 51, 1417-1423. The cell line is available from the ATCC as #CCL1555. It is maintained in monolayer culture in

2.0 plastic tissue culture flasks with HEPES-buffered Dulbecco's modification of Eagle medium, supplemented with 10% foetal bovine serum and antibiotics. Cell suspensions for experiments were obtained by detaching monolayers with ImM EDTA and'then with 0.05% Trypsin and EDTA, followed by 5 suspension in growth medium. The suspensions were treated as detailed in the individual experiments before intraperitoneal injection in nude mice. Quantitation was by counting in a haemocytometer chamber. Athymic nude mice (BALB/c nu/nu) were obtained from a continuous breeding colony in the United Medical & Dental School, London, housed. in a sterile isolator. The mice were supplied at 3-4 weeks of age arid transferred to a separate isolator for the duration of the experiments. Random allocation to treatment groups was performed.

Cells were injected into the peritoneal cavity of the mice as indicated below. Any further treatment was then administered by the same route of injection; this offers the potential for direct contact between the treatment agent and the tumour cells, as well as absorption into the systemic circulation. The duration of experiments was determined by the rate of tumour growth as judged by regular examination of the mice; large tumour masses in the abdomen, cachexia or other signs of ill-health were indications for terminating the experiment by evaluation of all mice.

Evaluation was performed by killing the mice by cervical dislocation and dissecting the abdomen. All tumour deposits visible to the naked eye were removed systematically and weighed to give an index of tumour mass. These dissections were performed "blind". The mass of tumour produced was compared between control and treatment groups, and any differences analysed by parametric and non- parametric statistical methods. Comparative Example l: High dose of rPAI2 in the mouse

A series of experiments was performed to investigate the effects of rPAI2 on the growth of i^'ntraperitoneal A431 xenografts in nude mice. The intraperitoneal route allows direct contact between grafted cells and administered rPAI2, making knowledge of the pharmacokinetics of rPAI2 in the mouse unnecessary for the purposes of these experiments. The A431 cell line shows consistent growth when injected by this route.

Cells were grown in monolayer culture, harvested by trypsinisation and suspended at the required concentration in balanced salt solution. Cell counts were performed on a haemocytometer with assessment of viability by Trypan blue exclusion. Control or rPAI2 incubation was performed prior to injection into the peritoneal cavity of nude mice, previously allocated at random to control or rPAI2 groups. Further treatment was administered by intraperitoneal injection. After 3 weeks the mice were killed by cervical dislocation and dissected. All deposits of tumour in the peritoneal cavity were removed and weighed to the nearest mg. Specimens of tumour and lung tissue were snap-frozen for later analysis of PA content. All dissections were performed blind. Experiment i

A431 cells were incubated in control solution or with rPAI2 10 units/ml for 1 hour at 37°C.

10⁶ cells were injected in a volume of 0.2 ml in 7 mice for control and rPAI2 groups.

The following day each mouse received an intraperitoneal injection of phosphate-buffered saline (PBS) (control) or PBS containing 50 units/ml rPAI2, 0.5 ml volume. The mice which received cells incubated in PBS received PBS injections and those which received cells incubated in PAI-2 received PAI-2 injections.

Experiment ii

A 31 cells were incubated in control solution or with rPAI2 500 units/ml for 1 hour at 37°C.

5 x 10⁵ cells were injected in a volume of 0.5 ml in 13 mice for control and rPAI2 groups.

Experiment iii

A431 cells were mixed with control solution or with rPAI2 10000 units/ml. 5 x 10⁵ cells were injected in a volume of 0.5 ml in 11 mice for control and rPAI2 groups.

Daily intraperitoneal injections of PBS (control) or PBS containing rPAI2 10000 units/ml were given for 20 days, 0.5 ml volume.

No statistically significant difference was seen in the weights of tumour tissue recovered between control and rPAI2 groups in any of these experiments. Statistical analysis was by the Mann-Whitney U test, with a significance level of p = 0.05.

These three experiments were designed to produce progressively greater exposure to rPAI2 in terms of dose and frequency. The lack of any effect on tumour growth might suggest that inhibition of uPA activity does - not influence tumour cell proliferation, and might imply that uPA secreted by the tumour cell does not act as a growth factor in this animal model.

Comparative Example 2; Effects of rPAI2 on the growth of tumour cells in culture

A431 cells were harvested by trypsinisation and cultured in 24-well plates which had previously been coated with poly- d-lysine. Each well was seeded with 2 x 10⁴ cells in protein-free medium (Coon's modification of Ham F12) . Following overnight incubation the cells were washed with PBS and then incubated with 0.5 ml of the same medium (control) or with the addition of uPA 60 i.u./ml, or a titration equivalent inhibitory conce^'ntration of rPAI2. Cells were washed and the medium replaced at daily intervals for 7 days. Eight replicate wells for each treatment were fixed in methanol at intervals and stained with Giemsa. Cell numbers were estimated by counting the cells in 4 high-power microscope fields at the centre of each well.

A statistically significant increase in cell numbers was seen with uPA and rPAI2 treatment at 3 days compared with controls (Mann-Whitney U test, p < 0.05). The stimulatory effect of uPA was still significant at 5 days, but increased scattering of the other results led to loss of any statistically significant difference. At higher cell densities it became apparent that the cell growth was less uniform, with loss of cells from the central part of the wells in some cases. This may have been due to overgrowth of the monolayer with consequent loss of viability and detachment.

Example 1

A431 cells were harvested and the suspension centrifuged. The cell pellet was resuspended in Hank's balanced salt solution (BSS) to a concentration of 2 x 10⁶ ml'¹. This was injected in 38 nude mice aged 12-18 weeks randomly allocated to 4 groups with age stratification, 0.5 ml per mouse (10⁶ cells) .

The following day treatment was commenced with daily intraperitoneal injections of 0.5 ml of the following solutions: PBS (control) , rPAI2 200 units ml^*1, Urokinase (uPA) 200 units ml"¹, Tranexamic acid 3 mg ml^'1.

After 17 days all mice were killed and the tumour masses measured.

Results: Tumour masses in grams

rPAI2 UPA

10 9

0.291 1.946

0.438 1.954

0.432 1.282

0.137 0.427

10

.541 0.169-0.707 1.117-2.791 0.712-1.876

These results demonstrate that a daily limited dose of rPAI-2 is effective in controll

15 tumour growth.

Example 2; Synthesis of polypropylene σlycol fumarate (PPF) from PFP trimer or BPB (MPM) tri er and FA

A slow release formulation is made by the method of US Patent No 4 888 413 (incorporated herein by reference) .

Bis-propylene glycol fumarate (PFP trimer) and propylene glycol dibutenoic ester, BPB (MPM) trimer, are prepared by the reaction of fumaric acid and propylene oxide (PFP) and the reaction of propylene glycol and aleic anhydride (MPM) , respectively. These trimers are used to synthesise PPF polymer by melt polymerisation or step polymerisation.

Preparation of PFP trimer. In a 1000 ml three neck round bottom flask equipped with an overhead mechanical stirrer (vacuum sealed) and condenser with drying tube, 1 mole fumaric acid (116 g) is dissolved in 150 ml -methyl 2- pentanone. The flask is placed in a heating mantle. At reflux, three ml pyridine is added as a catalyst. Propylene oxide is slowly added dropwise from a dripping funnel. Excess propylene oxide is used (150 ml) . At six hours another 150 ml portion of propylene oxide is added dropwise. The reaction continues for twenty hours, until the reaction mixture is a clear yellow liquid, totally devoid of solid fumaric acid. Immediately thereafter, 300 ml 10% Na₂HP0₄ solution is added. The mixture is placed in a 1000 ml separating funnel and vigorously shaken. The mixture is then set aside until there is a distinct separation of the phases. The lower water phase is discarded while the upper phase, which contains the product, is washed twice with 500 ml 5% KCl solution. The phases are again allowed to separate. ^* The lower phase is discarded while the upper phase is filtered over magnesium sulphate. The product is then placed in a tared 1000 ml round bottom flask and rotary evaporated until all solvents have been removed. The resulting product is then lyophilised to remove trace quantities of solvents.

Preparation of MPM Trimer. One mole maleic anhydride (98 g) is dissolved in 100 ml toluene in a 500 ml three neck round bottom flask equipped with a condenser. The flask is submersed in a 100^βC oilbath. 0.5 mole propylene glycol (39 g) is slowly dripped into this sealed system. The reaction is stirred for the duration of the reaction, about 24 hours. After the appropriate time, the reaction vessel is removed from the oilbath. The reaction mixture is transferred to a 1000 ml separating funnel and allowed to settle until two distinct phases are observed. The lower phase contains the product dissolved in ether. The upper toluene phase is discarded. Hexane is added to the ether reaction mixture and the phases again allowed to settle. The lower phase, containing the product, is then transferred to a tared round bottom flask and rotary evaporated until all excess solvent is removed. The product is then lysophilised to remove trace quantities of solvents. Reaction between PFP trimer and Fumaric Acid (FA) (1:0.65).

In a large test tube equipped with a small magnetic stirbar, 3.25 g fumaric acid is added to 10 g PFP trimer. The tube is placed in a 100°C oilbath, and stirred vigorously. Every 20 minutes, water produced in the reaction is removed by wiping the inside of the tube with a Kimwipe (Regd. T.M.). Samples are taken through a long Pasteur Pipette. After 2 hours, the tubes are removed from the oilbath.

The molecular weight of the polymer increases gradually between two and four hours. The weight average molecular weight (Mw) does not change, while the number average molecular weight (Mn) changes slightly, indicating that the reaction is not dramatically sensitive to the reaction time. This simplifies its preparation. The polymer formed after two hours is still liquid and can be used as a paste.

It also demonstrates high mechanical strength, 15.5 MPa.

The PPF reacted at a molar ratio of PFP trimer:fumaric acid of 1:0.65 for two hours at 180°C shows excellent reproducibility in all parameters. Mechanical strength ranges from 12.5 to 15 MPa.

PAI-2 is incorporated while the PPF is still a paste, at a level giving the desired dosage level when implanted in a patient. Exam le 3

Slow release polymers based on those disclosed in US Patent No 4 997 904 (incorporated herein by reference) are used.

Polymer synthesis.

Purification of diacids. Iso-phthalic acid (ISO) and terephthalic acid (TA) are extracted with an acetone-water (1:1) mixture for 24 hours at room temperature, and Soxhlet-extracted with ethanol for 12 hours. 1,3-bis- carboxyphenoxypropane (CPP) is synthesised and purified as described by A.J.Domb and R. Langer, J. Poly. Sci . 25, 3373 (1987) . Fumaric acid (FA) is recrystallised from water. Sebacic acid (SA) , 1,10-dodecanedioic acid (DD) , and adipic acid (AA) are recrystallised from ethanol. All acid monomers are dried in a dry desiccator under vacuum for 4S hours before use.

Preparation of prepoly ers. Iso-phthalic acid prepolymer is prepared as follows: 50 g of the acid powder is added to 500 ml refluxing acetic anhydride with constant stirring. After 10 in under reflux, most of the powder is dissolved and the reaction mixture is removed from the heat bath and filtered. The solution is evaporated to drynesε using an evaporator equipped with a vacuum pump and a water bath at 65°C. The remaining liquid is mixed with 20 ml of dry toluene and left to crystallise at room temperature for 24 hours. The white precipitate is filtered and extracted with 200 ml diethyl ether for 12 hours, to yield 40 g of prepolymer.

Terephthalic acid prepolymers prepa^'red under similar conditions have a high melting point product (>300°C) which is not useful for polymerisation at 180°C.

Isophthalic-terephthalic acids (80:20 and 75:25 molar ratio) , terephthalic acid-fumaric acid (45:55 molar ratio) , terephthalic acid-CPP (45:55 molar ratio), and terephthalic acid-εebacic acid (30:70 molar ratio) mixed prepolymers are prepared by reacting 50 g of the mixture of acids in 500 ml refluxing acetic anhydride for 10 min and isolated as above. The mixed prepolymers are purified by recrystallisation from toluene solution.

CPP, fumaric acid, and aliphatic acid prepolymers are prepared as described by A.J. Domb and R. Langer, J. Poly. Sci . 25, 3373 (1987) .

Polymerisation. Polymers are synthesised by condensation polymerisation of the prepolymers at 180°C for 90 min under high vacuum (greater than 100 mm Hg) as described by A.J. Domb and R. Langer, J. Poly. Sci . 25, 3373 (1987). At the end of the polymerisations a viscous melt is obtained. Copolymers with terephthalic acid are synthesised from the corresponding mixed prepolymers. Polymers can also be synthesised by solution polymerisation as described by A.J. Domb, E. Ron, and R. Langer, Macromoleculeε 21, 1925 (1988) . When catalyst is used, 2 weight % of finely powdered cadmium acetate dihydrate is mixed with the prepolymers and the prepolymers polymerised.

The prepolymers of CPP, fumaric acid, isophthalic acid, and aliphatic acids melt at low temperatures. However, the prepolymer of terephthalic acid melts at a temperature above 200°C and is not useful for polymerisation at 180°C. To overcome this problem, mixed prepolymers of terephthalic acid with CPP, isophthalic acid, fumaric acid, and sebacic acid are prepared which melt at temperatures below 120°C. The mixed prepolymers (dicarboxylic acid diacetyl anhydride) are purified successfully by recrystallisation from toluene. Weight average molecular weights of up to 40,000 may be obtained. Incorporation of an aliphatic diacid yields polymers with higher molecular weight. Polymerisation in the presence of a coordinative catalyst, such as cadmium acetate, increases the polymerisation rate with a slight increase in molecular weight.

Release and Degradation. PAI-2 is dispersed in 5 ml of a dichloromethane solution containing one gram of either Poly(CPP-ISO)20:80[polymer A] or poly(CPP-ISO- SA)14:58:28[polymer B] , and cast into a 5 cm diameter Teflon (Regd. T.M.) coated petri dish. After solvent evaporation at room temperature for 10 hours, a film 0.5 mm thick may be obtained. Pieces of this film may be placed in the peritoneum of a patient for slow release of PAI-2. The level of PAI-2 in the polymer and the size of the implant may be chosen to provide the desired low dose of PAI-2 to the patient.

Egg phosphatidylcholine (egg PC) may be obtained from Lipid KG (Federal Republic of Germany) , and egg phosphatidylglycerol (egg PG) may be obtained from Avanti Lipids (Birmingham, Ala.). Reverse osmosis membranes, #SK2P473E5, may be obtained from Millipore Corp (Bedford, Mass.) . A Prolab reverse osmosis (RO) filtration apparatus may be*obtained from Millipore, Model #MSDPROLAB.

Egg PC (38 g) and egg PG (2 g) are dissolved in 473 ml 100% ethanol in a 1 litre flask. After addition of 100 ml distilled water containing PAI-2, with stirring, the lipid/ethanol/water mixture is brought to 1 litre with ethanol. The resulting mixture is approximately 50 μmole/ml lipid in 90% ethanol.

The mixture is placed in the processing tank of a Prolab filtration apparatus for reverse osmosis (RO) filtration. The RO filter is flushed with a.90% ethanol solution prior to use. The system is run at 80% crossflow with 500 psi back pressure. NaCl (9 g) is dissolved in 1 litre of distilled water, and this solution is used as the first wash volume, for replacement of ethanol and water lost by RO filtration. Subsequent washes are with 4 litres of distilled water. Samples from the process vessel are collected every minute. Volume replacement with the five aqueous-media volumes is complete after about 110 minutes, and the filtration rate varied from about 30 to 60 ml/min during the five-volume replacement. The final concentration of ethanol in the filtered mixture is less than 1%.

250 nm liposomes may be obtained, containing a suitable level of PAI-2.

Example 5

Optically active R- and S-enantiomers of lactic acid and of the lactides are commercially available and can be homopolymerised or copolymerised by known methods such as bulk (co)polymerisation usually in a dry, inert atmosphere with an ionic catalyst such as stannous octanoate. The resultant enantiomeric poly(lactides) , after purification eg by precipitation from solution in a suitable solvent such as methylene chloride or chloroform by addition of a non-solvent such as diethyl ether, have crystalline melting points of 173° to 177°C. Lactide copolymers will generally have lower crystalline melting points, depending on lactide content but may be.amorphous. The monomers R-lactide and S-lactide are recrystallised from toluene and dried in vacuo before polymerisation.

Approximately 372 g of R- or S-lactide-is charged to 1500 ml resin kettle fitted with a mechanical stirrer, a serum stopper, and a gas inlet through which a dry nitrogen atmosphere was maintained. The resin kettle is placed in an oil bath maintained at 200°C and the lactide is rapidly stirred until completely melted (approx 5 minutes) . Stannous octanoate (0.160 g) and l-dodecanol (0.085 g) are then added via syringe and the contents of the kettle are maintained at 200°C for 40 minutes with constant stirring during the first 30 minutes. After 30 minutes the contents become too viscous to stir. The kettle is removed from the oil bath and allowed to cool to room temperature following which the reaction mixture is removed from the kettle and dissolved in approx 2000 ml of ethylene chloride. The resulting solution is filtered, then slowly added to a Waring blender operating at high speed and containing a volume of PAI-2-containing methanol equal to three times the volume of the methylene chloride solution. The resulting- precipitated poly-S-lactide is isolated via filtration and dried overnight at ambient temperature. References

1. Ossowski L. & Reich E.

Antibodies to plasminogen activator inhibit human metastasis.

Cell 1983; 35, 611-619

2. Hearing V.J., Law L.W. , Corti A., Appella E., Blasi F. Modulation of metastatic potential by cell surface urokinase of urine melanoma cells. Cancer Research 1988; 48, 1270-1278

3. Axelrod J.H. , Reich R. , Miskin R. Expression of human recombinant plasminogen activators enhances invastion and experimental metastasis of H-ras-transformed NIH 3T3 cells. Mol . Cell . Biol . 1989; 9, 2133-2141

Claims

1. A method of combatting a cancer in a mammal comprising the administration of plasminogen activator type 2 at a cancer-combatting dose.

2. A method of combatting a cancer in a mammal comprising the administration of plasminogen activator type 2 at a dosage level of between 100 units/kg body weight/day and 100000 units/kg body weight/day.

3. A method according to Claim 2 wherein the dosage is between 1000 units/kg body weight/day and 50000 units/kg body weight/day.

4. A method according to Claim 3 wherein the dosage is between 1000 units/kg body weight/day and 10000 units/kg body weight/day.

5. A method according to Claim 4 wherein the dosage is about 5000 units/kg body weight/day.

6. A method according to any one of the preceding claims wherein the cancer is at least partly located in the peritoneal cavity of the mammal.

7. A method according to Claim 6 wherein the PAI-2 is administered directly into the peritoneal cavity.

8. A method of removing a tumour from a mammal comprising removing the tumour- whilst there is a tumour-growth-preventing or tumour-metastasis- preventing amount of PAI-2 present in the body.

9. A controlled release depot or delivery device for implantation in the body of a mammal and delivery thereto of PAI-2 at a cancer-combatting dosage rate.

10. The use of PAI-2 in the manufacture of a medicament for the combatting of a cancer at a dose of 100 units/kg body weight/day to 100000 units/kg body weight/day.

11. Any novel feature or combination of features disclosed herein.