Suppression of inhibitors

Abstract

The present invention relates to methods for inhibiting malignant tumour growth, invasion and/or metastasis in a patient, the method comprising suppressing the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme (IPNME) in malignant tumour tissue or potential malignant tumour tissue. The suppression may be brought about by administering compounds interacting with the IPNME, but also administration of compounds interacting with transcription of genes encoding the IPNME is a possibility. The invention also relates to methods of selecting and identifying compounds in the therapeutical methods, as well of the use of such compounds in the treatment of malignancies.

1 . A method of inhibiting malignant tumour growth, invasion and/or metastasis in a patient who has been established to have a high risk of developing a malignant tumour or who has developed a malignant tumour, the method comprising suppressing the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue.  
     
     
         2 . A method according to  claim 1 , wherein the suppression of the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue allows the protease or the non-proteolytic matrix-degrading enzyme directly or indirectly to degrade the malignant tumour tissue or the potential malignant tumour tissue.  
     
     
         3 . A method according to  claim 1  or  2 , wherein the suppression of the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue results in interference with the process of tumour angiogenesis.  
     
     
         4 . A method according to any of claims  1 - 3 , wherein the suppression of the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue results in interference with the migrating capacity of malignant tumour cells or of other cells in the tumour stroma.  
     
     
         5 . A method according to any of claims  1 - 4 , wherein the patient is a patient who has been established to have a carcinoma in situ.  
     
     
         6 . A method according to any of claims  1 - 5 , which is performed as a neoadjuvant treatment, and/or as preoperative treatment, and/or as adjuvant treatment.  
     
     
         7 . A method according to any of claims  1 - 6 , wherein the patient is a patient who has been established to have a high risk of developing a malignant tumour by having a high-risk-indicating score of a tumour marker such as a serum/plasma tumour marker or by having a gene or gene product which indicates that the patient is at high risk of developing a malignant tumour.  
     
     
         8 . A method according to any of claims  1 - 7 , wherein the patient is a patient who has an increased concentration of the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme in the malignant tumour tissue and/or in plasma and/or serum.  
     
     
         9 . A method according to  claim 8 , wherein the increased concentration of the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme has been established to be a prognostic factor indicating a poor prognosis for the patient having the type of malignant tumour in question.  
     
     
         10 . A method according to  claim 8 , wherein the increased concentration of the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme has been established to be a predictive factor indicating efficiency of the treatment of the type of malignant tumour in question by suppressing the said inhibitor.  
     
     
         11 . A method according to any of the preceding claims, wherein the malignant tumour is selected from the group consisting of mammary carcinomas, urological carcinomas e.g. prostate carcinoma and bladder carcinoma, gynaecological carcinomas e.g. ovarian carcinoma and cervical carcinoma, non-small cell lung tumours, gastrointestinal cancers e.g. colon adenocarcinoma and gastric cancers, brain tumours, sarcomas, haematological malignancy e.g. lymphoma and skin cancers e.g. melanoma and squamous cell skin cancer.  
     
     
         12 . A method according to any of claims  2 - 11 , wherein the malignant tumour tissue degraded, including micrometastases, is tissue comprised of the malignant tumour cells themselves, the extracellular matrix and/or the stromal cells of the malignant tumour, e.g. endothelial cells, fibroblasts, macrophages, leucocytes; and substantially no other tissue than malignant tumour tissue is degraded to an extent or degree which give unacceptable toxic side effects.  
     
     
         13 . A method according to any of the preceding claims, wherein the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme is an inhibitor of a serine protease such as uPA or plasmin.  
     
     
         14 . A method according to any of claims  1 - 12 , wherein the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme is an inhibitor of a metalloprotease such as the inhibitors TIMP-1 or TIMP-2.  
     
     
         15 . A method according to any of claims  1 - 12 , wherein the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme is an inhibitor of a cysteine protease (thiol protease), an aspartic protease such as cathepsin D or another matrix-degrading enzyme, such as heparanase.  
     
     
         16 . A method according to  claim 13 , wherein the serine protease inhibitor is selected from the group consisting of plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), and protease nexin 1.  
     
     
         17 . A method according to  claim 16 , wherein the serine protease inhibitor is PAI-1.  
     
     
         18 . A method according to any of the preceding claims, wherein the suppression of the inhibitory activity of the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme is obtained by administering to the patient a compound which suppresses the inhibitory activity of the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme.  
     
     
         19 . A method according to  claim 18 , wherein the activity of PAI-1 in the tumour tissue is inhibited by administering to the patient a compound which suppresses the plasminogen activator-inhibitory activity of PAI-1.  
     
     
         20 . A method according to  claim 19 , wherein the compound is a compound which suppresses the plasminogen activator-inhibitory activity of PAI-1 by inhibiting the binding of PAI-1 to vitronectin.  
     
     
         21 . A method according to  claim 19 , wherein the compound is a compound which inhibits the effect of PAI-1 on uPA, but does not inhibit the binding of uPA to uPAR.  
     
     
         22 . A method according to  claim 21 , wherein the compound is a compound which is capable of binding to PAI-1 and not capable of converting plasminogen into plasmin.  
     
     
         23 . A method according to  claim 22 , wherein the compound is a derivative or variant of uPA or pro-uPA.  
     
     
         24 . A method according to  claim 22 , wherein the compound is an antibody which is capable of binding to PAI-1, such as the PAI-1 antibody clone 2 disclosed in WO 87/00549, an active fragment thereof and/or an immunological equivalent thereof.  
     
     
         25 . A method according to any of claims  22 - 24 , wherein the compound which is capable of binding to PAI-1 has a cytotoxic drug coupled thereto.  
     
     
         26 . A method according to any of claims  16 - 25 , wherein malignant cells of the malignant tumour produce uPA, the PAI-1 being produced by other cells in the malignant tumour.  
     
     
         27 . A method according to any of claims  16 - 25 , wherein malignant cells of the malignant tumour produce PAI-1, the uPA being supplied by other cells in the malignant tumour.  
     
     
         28 . A method according to any of claims  16 - 25 , wherein malignant cells of the malignant tumour produce neither uPA nor PAI-1, the uPA and the PAI-1 being supplied by other cells in the malignant tumour.  
     
     
         29 . A method according to any of claims  16 - 25 , wherein malignant cells of the malignant tumour produce both uPA and PAI-1.  
     
     
         30 . A method according to any of claims  1 - 17 , wherein the activity of the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme is suppressed by administering, locally or systemically, to the patient, stromal cells which have been transformed with a genetic construct expressing a gene product suppressing the inhibitor of the protease or of-the non-proteolytic matrix-degrading enzyme.  
     
     
         31 . A method according to  claim 30 , wherein the stromal cells are capable of finding and infiltrating a malignant tumour of a mammal genotype, in particular human genotype, in an immunodeficient non-human recipient vertebrate into which malignant tumour cells of the said mammal genotype have been introduced, the stromal cells containing 
 1) a gene which, when it is expressed in the immunodeficient recipient vertebrate by stromal cells which are capable of finding and colonizing, in the recipient vertebrate, colonies of malignant tumour cells of the said mammal genotype, is capable of controlling the progression of the malignant tumour cells of the said mammal, and    2) a promoter securing expression of the gene product when the stromal cells have been transferred to the said mammal.    
     
     
         32 . A method according to  claim 30  or  31 , wherein the stromal cells are tumour-infiltrating stromal cells which are capable of finding malignant tumour cells in the patient.  
     
     
         33 . A method according to any of claims  30 - 32 , wherein the stromal cells are stromal cells of a tissue type which is compatible with the tissue type of the patient.  
     
     
         34 . A method according to  claim 33 , wherein the stromal cells are stromal cells from the patient.  
     
     
         35 . A method for selecting a compound for inhibiting malignant tumour growth, invasion and/or metastasis in a patient who has been established to have a high risk of developing a malignant tumour or who has developed a malignant tumour, the compound being capable of suppressing the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue, the method comprising one or more of the following steps: 
 1) a screening assay in which the possible suppression of the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme is determined by adding the compound to a system comprising immobilized inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme and solubilized protease or non-proteolytic matrix-degrading enzyme, the protease or the non-proteolytic matrix-degrading enzyme bound to the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme being detected by the enzyme being labelled or by means of a labelled antibody directed to the protease or the non-proteolytic matrix-degrading enzyme, or adding the compound to a system comprising immobilized protease or non-proteolytic matrix-degrading enzyme and solubilized inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme, inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme bound to the protease or the non-proteolytic matrix-degrading enzyme being detected by the inhibitor being labelled or by means of a labelled antibody directed to the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme,    2) an assay in which the possible suppression of the inhibitory activity of the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme in the tumour tissue is determined by adding the compound to a system comprising radiolabelled inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme and tumour cells expressing the protease or the non-proteolytic matrix-degrading enzyme and detecting any inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme binding to the protease or the non-proteolytic matrix-degrading enzyme by gamma counting of the cells,    3) a screening assay, in which the potential suppression of the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme by the compound is determined by using whole cells to which the protease or the non-proteolytic matrix-degrading enzyme is bound on the surface, e.g. by a protease receptor, such as uPA to the uPA receptor,    4) administering a compound which has been established to suppress the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in the tumour tissue to a nude mouse or a nude rat which is inoculated with human malignant tumour cells which are capable of invasion and/or metastasis in the mouse or rat in the presence of the protease or the non-proteolytic matrix-degrading enzyme and the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme, and selecting, as a suitable compound, a compound inhibiting the growth and/or invasion and/or metastasis of the human malignant tumour cells in the mouse or the rat.    
     
     
         36 . A method according to  claim 35 , wherein the inhibitor of the protease is PAI-1.  
     
     
         37 . A method according to  claim 36 , wherein the human malignant tumour cells are cells which produce uPA, the PAI-1 being supplied by other human cells with which the mouse is inoculated.  
     
     
         38 . A method according to  claim 36 , wherein the human malignant tumour cells are cells which produce PAI-1, the uPA being supplied by other human cells with which the mouse is inoculated.  
     
     
         39 . A method according to  claim 36 , wherein the human malignant tumour cells are cells which produce neither uPA nor PAI-1, the uPA and the PAI-1 being supplied by other cells.  
     
     
         40 . A method according to  claim 39 , wherein the human malignant tumour cells are cells which produce both uPA and PAI-1.  
     
     
         41 . A compound which is a suppressor of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue for use as a medicament.  
     
     
         42 . A compound according to  claim 41  for use for inhibiting malignant tumour growth, invasion and/or metastasis in a patient who has been established to have a high risk of developing a malignant tumour or who has developed a malignant tumour.  
     
     
         43 . A compound according to  claim 41  or  42 , which allows the protease or the non-proteolytic matrix-degrading enzyme directly or indirectly to degrade the malignant tumour tissue or the potential malignant tumour tissue by the suppression of the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue.  
     
     
         44 . A compound according to any of claims  41 - 43 , which 
 1) suppresses the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme as determined by adding the compound to a system comprising immobilized inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme and solubilized protease or non-proteolytic matrix-degrading enzyme, the protease or the non-proteolytic matrix-degrading enzyme bound to the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme being detected by the enzyme being labelled or by means of a labelled antibody directed to the protease or the non-proteolytic matrix-degrading enzyme, or by adding the compound to a system comprising immobilized protease or non-proteolytic matrix-degrading enzyme and solubilized inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme, inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme bound to the protease or the non-proteolytic matrix-degrading enzyme being detected by the inhibitor being labelled or by means of a labelled antibody directed to the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme,    2) suppresses the inhibitory activity of the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme in the tumour tissue as determined by adding the compound to a system comprising radiolabelled inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme and tumour cells expressing the protease or the non-proteolytic matrix-degrading enzyme and detecting any inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme binding to the protease or the non-proteolytic matrix-degrading enzyme by gamma counting of the cells,    3) suppresses the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme as determined by using whole cells to which the protease or the non-proteolytic matrix-degrading enzyme is bound on the surface, e.g. by a protease receptor, such as uPA to the uPA receptor, and/or    4) inhibits the growth and/or invasion and/or metastasis of human malignant tumour cells in a nude mouse or a nude rat as determined by administering to the nude mouse or the nude rat, which has been inoculated with human malignant tumour cells which are known to invade and/or metastasize in the presence of the protease or the non-proteolytic matrix-degrading enzyme and the inhibitor of the protease or of the non-proteolytic matrix-degrading enzyme and which are capable of invasion and/or metastasis in the mouse, the compound which has been established to suppress the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in the tumour tissue.    
     
     
         45 . A compound according to any of claims  41 - 44  which is the antibody PAI-1 clone 2 disclosed in WO 87/00549, an active fragment thereof, and/or an immunological equivalent thereof.  
     
     
         46 . The use of a compound, which 
 suppresses the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue,    for preparing a composition for inhibiting malignant tumour growth, invasion and/or metastasis in a patient who has been established to have a high risk of developing a malignant tumour or who has developed a malignant tumour.    
     
     
         47 . The use according to  claim 46 , wherein the compound allows the protease or the non-proteolytic matrix-degrading enzyme directly or indirectly to degrade the malignant tumour tissue or the potential malignant tumour tissue by suppressing the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue.  
     
     
         48 . The use according to  claim 46  or  47 , wherein the compound 
 1) inhibits the uPA-binding activity of PAI-1 as determined by adding the compound to a system comprising immobilized PAI-1 and solubilized uPA, uPA bound to PAI-1 being detected by being labelled or by means of a labelled anti-uPA antibody, or adding the compound to a system comprising immobilized uPA and solubilized PAI-1, PAI-1 bound to uPA being detected by being labelled or by means of a labelled anti-PAI-1 antibody, and/or  
 2) inhibits the binding of PAI-1 to uPA as determined by adding the compound to a system comprising radiolabelled PAI-1 or a derivative thereof and cells having uPA bound to the cell surface to uPA receptors and detecting any PAI-1 or derivative binding to uPA by gamma counting of the cells, and/or  
 3) suppresses the inhibitory activity of PAI-1 on uPA as determined by adding the compound to a system comprising PAI-1 and cells having uPA bound on the cell surface to uPA receptors and detecting any effect of PAI-1 either directly by a chromogenic uPA substrate or indirectly by adding plasminogen and measuring plasmin generation using a plasmin-specific substrate such as the fluorogenic substrate H-D-Val-Leu-Lys-7-amido-0.4-methylcoumarin,  
 for preparing a composition for treating a patient who has been established to have a high risk of developing a malignant tumour or who has developed a malignant tumour by inhibiting the plasminogen activator-inhibitory activity of PAI-1 in malignant tumour tissue or potential malignant tumour tissue.  
 
     
     
         49 . The use according to  claim 48 , wherein the compound additionally 
 4) inhibits the activity of PAI-1 in tumour tissue as determined by adding the compound to the malignant cells or other cells in the tumour tissue expressing PAI-1 and subsequently adding uPA, followed by measurement of uPA activity either directly by a chromogenic uPA substrate or indirectly by adding plasminogen and measuring plasmin generation.    
     
     
         50 . The use according to  claim 48  or  47 , wherein the compound additionally 
 5) is capable of inhibiting the growth and/or invasion and/or metastasis of human malignant tumour cells when administered to a nude mouse or a nude rat which is inoculated with human malignant tumour cells which are known to invade and/or metastasize in the presence of uPA and PAI-1 and which are capable of invasion and/or metastasis in the mouse.  
 
     
     
         51 . A method for predicting the therapeutical efficacy of a method according to any of claims  1 - 34 , the method comprising 
 1) determining the level of PAI-1 in malignant or potentially malignant tissue or another sample, such as plasma, serum, or urine, from a subject which potentially is to be subjected to a method according to any of claims  1 - 34 , and    2) establishing that the said therapeutical efficacy is high if the PAI-1 level is beyond or equal to a pre-determined threshold value, and that the said therapeutical efficacy is low if the PAI-1 level is below the pre-determined threshold value.    
     
     
         52 . A method according to  claim 51 , wherein the determination of the PAI-1 level is performed by using an immunoassay, such as an ELISA or RIA, or by using an activity assay.  
     
     
         53 . A method according to any of claims  18 - 34 , wherein the compound is obtainable by performing the following steps: 
 a) identifying atomic group(s) of PAI-1 which is/are essential in the binding between PAI-1 and a substance which is a compound according to any of claims  41 - 45  or a compound selected by the method according to any of claims  36 - 40 , e.g. by providing mutated or truncated variants of PAI-1 and by exclusion identifying the essential atomic group(s) taking part in the binding,    b) providing a fragment of PAI-1 exhibiting a substantial binding to the substance, by using the information obtained in step a),    c) determining the binding free energy of the substance bound to the PAI-1 fragment provided in step b), e.g. by microcalometry, and selecting a threshold value which is at least 50% of the determined binding free energy or indicates substantial interaction between PAI-1 and a substance of a defined molecular weight,    d) co-crystallizing the PAI-1 fragment with the substance,    e) performing an X-ray crystallographic analysis of the co-crystallized product obtained in step d),    f) identifying the atomic groups of the PAI-1 fragment and of the substance taking part in the interaction between PAI-1 and the substance,    g) providing a 3-dimensional representation (X) of the atomic groups identified in step f),    h) modifying the 3-dimensional representation (X) by exchanging, adding or removing at least one atomic group in the 3-dimensional representation provided in step g),    i) predicting the binding free energy of the PAI-1 fragment bound to the modified version of X,    j) selecting as a candidate substance a modified version of the substance (X) resulting in a predicted binding free energy in step i) which is higher than the threshold value selected in step c),    k) synthesizing the candidate substance,    l) determining the binding free energy of PAI-1 bound to the candidate substance, e.g. by microcalometry,    m) establishing that the candidate substance results in a binding free energy determined in step l) which is higher than the threshold value selected in step c),    n) optionally subjecting the candidate substance to the method according to any of claims  35 - 40  in order to confirm that the candidate substance is capable of suppressing the inhibitory activity of an inhibitor of a protease or of a non-proteolytic matrix-degrading enzyme in malignant tumour tissue or potential malignant tumour tissue, and    o) electing as the compound the candidate substance.