BACKGROUND Plasminogen activation and matrix metalloproteinase activity may be a central point for the regulation of many cellular activities such as wound healing, angiogenesis, metastatic cancer spread, blood clotting (thrombosis) and progression of vascular diseases including atherosclerosis, neointimal hyperplasia and aneurismal arterial dilation. One activity associated with plasminogen activation and matrix metalloproteinase (MMP) activity is extracellular matrix (ECM) degradation. Excessive ECM degradation is observed in tumour cells with metastatic potential and contributes to the breakdown of physiological barriers to tumour cell migration and thus facilitates the metastatic process.

In some patients, diagnosis of a primary tumour may lead to excision and eradication if detected early enough. The patient, with minimum preventative treatment, such as chemotherapy, may survive and show signs of definite regression of the cancer or total absence and recovery from the cancer.

Most treatments for cancer focus on reducing proliferation of cancer cells by direct cytotoxicity. Whilst this is an important means of reducing the primary tumour size, it does not recognise that most of the mortality associated with

cancer is due to metastatic or secondary disease spread to other vital parts of the body including lungs, brain, bone and liver.

Therefore, if a cancer is discovered late, the tumour may have progressed to a malignant metastatic phenotype characterised by excessive and uncontrolled extracellular matrix degradation. It is this phenotype, which often results in the spread of tumour cells to other parts of the body and formation of secondary tumours. These"secondaries"are often the ultimate cause of death to the patient.

ECM degradation is associated with a cell surface, urokinase (u-PA)- mediated, plasminogen activation which is a process integral to ECM degradation. The primary inhibitor of u-PA activity in the extracellular matrix is plasminogen activator inhibitor type-2 (PAI-2), a serine protease inhibitor. u-PA, bound to its cell surface receptor u-PAR, is central to this process while the serine protease inhibitor PAI-2 is the primary inhibitory regulator of u-PA activity. In addition to its central role in cell surface plasminogen activation u-PA bound to its cell surface receptor u-PAR is responsible for mitogenic and cell adhesion events also involved in the metastatic phenotype.

Tumour cell invasion and the metastatic process have been associated with elevated levels of cell-surface u-PA mediated plasminogen activation whilst in vitro, in vivo and clinical studies suggest that inhibition of cell-surface u-PA by PAI-2 is associated with reduced tumour cell invasion, metastasis and improved clinical outcome.

Tumour cell invasion and metastasis has also been associated with elevated levels of components of the Matrix Metalloproteinase (MMP) enzyme system. In breast cancer particularly elevated levels of MMP-2 and MMP-9 are prognostic of a poor outcome in this condition. Akin to the plasminogen activating system, inhibition of excessive MMP activity or induction of the natural inhibitors of MMPs, tissue inhibitors of matrix metalloproteinases (TIMPS) in the setting of malignant disease may be of benefit in inhibiting metastatic progression.

As mentioned inhibition of the malignant metastatic phenotype via induction of PAI-2/TIMP's expression and/or inhibition of u-PA/MMP expression and/or reduced activity of u-PA/MMP may represent a novel means via which the metastatic phenotype can be arrested. Agents capable of inducing PAI- 2/TIMPS and/or inhibiting u-PA/MMP expression and/or activity may restrict u- PA/MMP-mediated tumour cell proteolysis and facilitate in the development of therapeutic strategies to combat malignant disease.

It would be desirable to identify a compound capable of modulating expression of components of the plasminogen activating and MMP systems including PAI- 2/TIMPs and u-PA/MMP particularly, to prevent or reduce associated conditions such as metastatic spread. In the case of cancer, present methods of eradicating the primary tumour do not alleviate metastatic spread. Moreover, all primary tumours may not be identified for removal. A system that prevents spread should reduce the incidence of secondary tumours and possibly increase life expectancy.

The identification of agents capable of modulating the plasminogen activating and MMP systems such as inducing PAI-2/TIMPs and inhibiting u-PA/MMP expression and/or activity may therefore have a therapeutic role in the management of associated conditions such as the malignant metastatic phenotype.

Accordingly, it is an object of the present invention to overcome at least some of the problems of the prior art and to provide a new approach to the treatment of metastatic cancer spread, leukaemia, lymphom, myeloma and associated haemoncological conditions.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in

the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION In one aspect of the present invention, there is provided a method of modulating a plasminogen-activation/MMP associated condition involving a plasminogen activating/MMP system, said method comprising administering an effective amount of oxamflatin or an oxamflatin derivative to a patient in need thereof.

In another aspect of the present invention there is provided a method of preventing and treating a plasminogen-activation/MMP associated condition involving a plasminogen activating/MMP system, said method comprising administering an effective amount of oxamflatin or an oxamflatin derivative to a patient in need thereof.

This can be applied to any preferred forms of plasminogen activation/MMP associated conditions such as, but not limited to extracellular matrix degradation, metastatic cancer spread, leukaemia, lymphom, myeloma and associated haemoncological conditions.

In a further preferred aspect of the present invention, there is provided a method of modulating extracellular matrix degradation, said method comprising administering to a patient in need, an effective amount of oxamflatin or an oxamflatin derivative in conjunction with a cytotoxic agent.

In yet another aspect of the present invention, there is provided a composition for use in the modulation of a plasminogen-activation/MMP associated condition, said composition comprising oxamflatin or an oxamflatin derivative and a carrier or diluent.

The invention also includes oxamflatin derivatives that behave in a similar manner to oxamflatin, that may be more potent for inhibiting metastatic spread and cell mobility. One example of an oxamflatin derivative is MCT-1, a compound having the following structure:

IN THE FIGURES Figure 1 shows that Oxamflatin treatment induces PAI-2 mRNA expression in U- 937 cells and potentiates PMA mediated induction of PAI-2 mRNA.

Total RNA was extracted from U-937 cells and subjected to Northern blotting.

The filter was hybridised with a labelled cDNA probe complementary to PAI-2 mRNA. The same filter was reprobed for ß-Actin mRNA.

(Lane 1) Untreated cells.

(Lane 2) Cells treated for 16 hours with 1 M oxamflatin.

(Lane 3) Cells treated for 16 hours with 25 nM PMA.

(Lane 4) Cells treated for 16 hours with a combination of 25nM PMA and 1 M oxamflatin Figure 2 shows that Oxamflatin treatment potentiates PMA-mediated induction of endogenous PAI-2 mRNA expression in HT-1080 cells.

Total RNA was extracted from HT-1080 cells and subjected to Northern blotting.

The filter was hybridised with a labelled cDNA probe complementary to PAI-2 mRNA. The same filter was reprobed for B-Actin mRNA (Lane 1) Untreated cells (Lane 2) Cells treated for 16 hours with 1 zM oxamflatin (Lane 3) Cells treated for 16 hours with 5 M oxamflatin (Lane 4) Cells treated for 16 hours 25 nM PMA (Lane 5) Cells treated for 16 hours with a combination of 25 nM PMA and 1 M oxamflatin

(Lane 6) Cells treated for 16 hours with a combination of 25nM PMA and 5 M oxamflatin Figure 3 shows that Oxamflatin treatment inhibits u-PA mRNA expression in U- 937 and HT-1080 cells.

Panel A: RNA extracted from U-937 cells and used in Figure 1, was also utilised in this experiment The filter was hybridised with a labelled cDNA probe complementary to u-PA mRNA. The same membrane was reprobed for ß-Actin (Lane 1) Untreated cells.

(Lane 2) Cells treated for 16 hours with 1 zM oxamflatin (Lane 3) Cells treated for 16 hours with 5 p. M oxamftatin (Lane 4) Cells treated for 16 hours with 25 nM PMA (Lane 5) Cells treated for 16 hours with a combination of 25 nM PMA with 1 jj. M oxamflatin.

Panel B: The same filter as shown in Figure 2 (Derived from HT-1080 cell total mRNA) was stripped and reprobed with a u-PA cDNA fragment. Subsequently the same membrane was reprobed with ß-Actin.

(Lane 1) Untreated (Lane 2) Cells treated for 16 hours with 1 M oxamflatin (Lane 3) Cells treated for 16 hours with 5 M oxamflatin (Lane 4) Cells treated for 16 hours with 25 nM PMA (Lane 5) Cells treated for 16 hours with a combination of 25nM PMA with 1 M oxamflatin (Lane 6) Cells treated for 16 hours with a combination of 25nM PMA with 5 M oxamflatin.

Figure 4 shows that Oxamflatin transactivates the PAI-2 gene promoter in transfected HT-1080 cells.

HT-1080 cells were stably transfected, in duplicate, with the wild type-219 PAI- 2 promoter-CAT constructs. As indicated, transfected cells were stimulated for

16 hours with 1 or 5M oxamflatin, 25 nM PMA or a combination of 5 tM oxamflatin and 25 nM PMA. CAT activities were quantitated by phosphorimaging of the TLC plates and results expressed in arbitrary units.

Error bars represent standard error of the mean of samples with four data points.

Figure 5 shows that the effects of oxamflatin treatment on the binding and identity of nuclear proteins binding to the CRE and AP-1a sites in the PAI-2 gene promoter.

Nuclear proteins extracted from HT-1080 cells untreated (Con) or treated for 1 hr with medium (serum 10%) containing 1pM oxamflatin (Ox) or 25nM PMA (PMA) were subjected to supershift analysis using monoclonal antibodies directed against either c-Jun or Jun D and labelled oligonucleotides harbouring either the PAI-2 AP-1a or CRE binding sites. The position of the supershifted c- Jun complex is indicated by the lower right broken arrow. The position of the supeshifted Jun D complex is indicated by the upper right solid arrow.

Lanes 1-9 use the PAI-2 AP-1 a oligonucleotide as a probe.

Lane 1. Control + no antibody Lane 2. Control + c-jun antibody Lane 3. Control + Jun D antibody Lane 4. Ox + no antibody Lane 5. Ox +c-jun antibody Lane 6. Ox +Jun D antibody Lane 7. PMA +no antibody Lane 8. PMA + c-jun antibody Lane 9. PMA + Jun D antibody Lanes 10-18 use the PAI-2 CRE oligonucleotide as a probe.

Lane 10. Control + no antibody Lane. 11. Control + c-jun antibody Lane 12. Control + Jun D antibody Lane 13. Ox + no antibody

Lane 14. Ox +c-jun antibody Lane 15. Ox +Jun D antibody Lane 16. PMA +no antibody Lane 17. PMA + c-jun antibody Lane 18. PMA + Jun D antibody Figure 6 shows that Oxamflatin treatment of HT-1080 cells is associated with reduced u-PA-mediated proteolytic activity in zymography studies.

Panel A Conditioned medium from HT-1080 cells untreated (0), or treated with 1,2. 5,5. 0 or 7. 5, uM Oxamflatin for 24 hrs was subjected to zymographic analysis. A u-PA standard at 10 units/ml (u-PA std) was used to confirm the position of u-PA- mediated proteolytic activity Molecular weight markers are shown to the right of the panel. The position of u-PA-mediated proteolytic activity is indicated to the left of the panel. The incubation period used for u-PA-mediated proteolysis was 6 hours.

Panel B Conditioned medium from HT-1080 cells untreated (0), or treated with 1,2. 5,5. 0 or 7. 5M Oxamflatin for 24 hrs was subjected to zymographic analysis. A u-PA standard at 10 units/ml (u-PA std) was used to confirm the position of u-PA- mediated proteolytic activity. Molecular weight markers are shown to the right of the panel. The position of u-PA-mediated proteolytic activity is indicated to the left of the panel. The incubation period used for u-PA-mediated proteolysis was 24 hours.

Figure 7 Oxamflatin treatment increases PAI-2 protein expression in U-937 cells.

Cytosolic extracts were obtained from U-937 cells untreated, or treated with increasing concentrations of Oxamflatin or 25 nM PMA for 24 hours. Western blot was performed using an anti-PAI-2 antibody.

Panel A Lane 1 24 hours untreated cells Lane 2 cells treated for 24 hours with 1 M Oxamflatin Lane 3 cells treated for 24 hours with 2.5 M Oxamflatin Lane 4 cells treated for 24 hours with 5.0 M Oxamflatin Lane 5 cells treated for 24 hours with 7.5 zM Oxamflatin Lane 6 cells treated for 24 hours with 25nM PMA Panel B Coomassie stain of SDS-PAGE gel of above western blot indicating balanced protein loading.

Lanes as for Panel A Figure 8 shows the effect of oxamflatin and MCT-1 on u-PA-mediated proteolytic activity in conditioned medium from HT 1080 fibrosarcoma cells.

Figure 9 shows the effect of oxamflatin on u-PA-mediated proteolytic activity on u-PA-mediated proteolytic activity in conditioned medium from MB-MDA-231 Metastatic Breast Cancer Cells.

Figure 10 shows the effect of oxamflatin and MCT-1 on u-PA-mediated proteolytic activity in conditioned medium from PC-3 Metastatic Prostate Cancer Cells.

Figure 11 shows the effect of oxamflatin and MCT-1 on MMP-mediated proteolytic activity in conditioned medium from MB-MDA-231 metastatic breast cancer cells.

Figure 12 shows the effect of Oxamflatin and MCT-1 on MMP-2 and MMP-9 mRNA levels in MB-MDA-231 metastatic breast cancer cells.

Figure 13 shows the effect of oxamflatin and MCT-1 on primary human placenta fibroblast cells.

Figure 14 shows growth, viability, and invasion of cells in the presence of oxamflatin (0. 1 M) over 40 hours. Panel B shows growth, viability, and invasion of cells in the presence of MCT-1 (0. 1 M) over 40 hours. The response for growth is shown by the left-most bar, viability by the middle bar and invasion on the right-most bar.

Figure 15 shows invasion of 4T1. 13con2 cells (murine mammary breast cancer cells) through matrigel with and without oxamflatin and MCT-1.

Figure 16 shows the inhibitory activity of oxamflatin and MCT-1 on histone deacetylase.

Figure 17 shows u-PA mRNA expression levels as measured by northern analysis in HT-1080, MDA-MB-231 and PC-3 cells after treatment with 5M of Ox or MCT-1 for 16 hrs. Each bar of the histogram represents a minimum of 3 experiments and the data are presented as the mean value +/-SD * p<0. 05, ** p<0.01 compared to level in non-treated cells.

Figure 18 shows transcript levels of PAI-1, PAI-2, u-PA and u-PAR mRNA after treatment of PC-3 (A) and HT-1080 (B) cells with 5uM Ox or MCT-1 for 24 hrs.

Transcript levels were quantitated relative to GAPDH mRNA using real time RT-PCR. Data are presented as the mean and standard deviation of a minimum of 3 experiments.

Figure 19 shows levels of mRNA for MMP-2 and MMP-9 after treatment of HT- 1080 and MDA-MB-231 cells with 5M Ox or MCT-1 for 16 hrs. RNA levels were quantitated from northern blots and expressed as a percentage of untreated cells. The results are shown as the mean +/-SD of a minimum of 3 experiments * p<0.05, ** p<0.01 compared to level in non-treated cells.

Figure 20 shows effect of Ox and MCT-1 on u-PA and MMP-mediated proteolytic activity in HT-1080, MDA-MB-231 and PC-3 cells. A. Fibrin zymography of conditioned medium from HT-1080 cells treated with Ox or MCT-1 at various concentrations for 16 hr. Molecular weight markers identify u-

PA at approximately 54 kDa. B. Averaged data from five separate fibrin zymography experiments and from four separate gelatin zymography experiments in HT-1080 cells.

C. Averaged data from three separate fibrin zymography experiments and from three separate gelatin zymography experiments in MDA-MB-231 cells.

D. Results of four separate fibrin zymography experiments and from four separate gelatin zymography experiments in PC-3 cells.

The results are shown as the mean +/-SD of experiments * p<0.05, ** p<0.01 compared to level in non-treated cells.

Figure 21 shows invasion through Matrigel and viability in HT-1080, PC-3 and MDA-MB-231 cells untreated or treated with either Ox or MCT-1. (A). Extent of invasion and viability (Trypan blue staining at 40 hrs when the invasion assay was completed) in HT-1080 cells treated with 5 uM of either Ox or MCT-1. (B and C). Growth (SRB assay), viability (Trypan blue staining) and invasion through matrigel of PC-3, HT-1080 and MDA-MB-231 cells treated with 0.1 uM Ox (B) or 0.1 uM MCT-1 (C). The results are shown as the mean +/-SD of experiments * p<0.05, ** p<0.01 compared to level in non-treated cells.

Figure 22 shows the in vivo anti-invasive/metastatic potential of Ox and MCT-1 using an orthotopic model of breast cancer.

DESCRIPTION OF THE INVENTION In one aspect of the present invention, there is provided a method of modulating a plasminogen-activation/MMP associated condition involving a plasminogen activating/MMP enzyme system, said method comprising administering an effective amount of oxamflatin or an oxamflatin derivative to a patient in need thereof.

Throughout the description and claims of this specification, the word"comprise" and variations of the word, such as"comprising"and"comprises", is not intended to exclude other additives, components, integers or steps.

A"plasminogen activation/MMP associated condition"as used herein may be any condition which requires activation of plasminogen/MMP by plasminogen activation factors such as plasminogen activators including u-PA and t-PA and by MMP enzymes. Such activation involves a plasminogen activating/MMP enzyme system. Plasminogen/MMP activation may be important in conditions which involve fibrin including clotting diseases, clot formation, extracellular matrix degradation, wound healing, angiogenesis and cell mobility such as those related to fighting infections and vascular disease and therefore, such conditions are included in the scope of the present invention as plasminogen activation/MMP associated conditions.

Preferred plasminogen activation/MMP associated conditions may be selected from the group including metastatic cancer spread, leukaemia, lymphom, myeloma and associated haemoncological conditions, and extracellular matrix degradation,.

In another aspect of the present invention there is provided a method of preventing and treating a plasminogen-activation/MMP associated condition involving a plasminogen activating/MMP system, said method comprising administering an effective amount of oxamflatin or an oxamflatin derivative to a patient.

The plasminogen activating/MMP system may include inhibitors and activators of plasminogen/MMP activation. One such inhibitor is plasminogen activator inhibitor Type 2 (PAI-2), a serine protease inhibitor. Hence, without being restricted by theory, modulation of the plasminogen activation/MMP associated condition may be via modulation of PAI-2. In this manner, plasminogen activation may be inhibited to prevent or treat a plasminogen activation/MMP associated condition when production of PAI-2/TIMPs is increased.

Another component of the plasminogen activating system is urokinase (u-PA).

This enzyme is central to the process of extracellular matrix (ECM) degradation.

By inhibiting production of u-PA mRNA and thereby u-PA expression or by inhibiting u-PA activity, the plasminogen activating/MMP system may be

modulated by inhibition of activation. Again, by targeting u-PA, plasminogen activation may be inhibited to prevent or treat a plasminogen activation/MMP associated condition.

Components of the matrix metalloproteinase (MMP) enzyme system are also central to the process of ECM degradation and thus may also be useful to target in the treatment of metastatic cancer. Preferably, collagenases, elastases, MMP-2 and MMP-9 are targeted.

Applicants have found that the hydroxamic acid derivative oxamflatin, previously noted to revert the malignant phenotype in K-ras transformed NIH-3T3 cells, and a synthetic derivative MCT-1, described in this application, are capable of differentially upregulating PAI-2 and suppressing u-PA and MMP 2 and MMP-9 mRNA expression. Oxamflatin and MCT-1 treatment was also found by the applicants to result in a significant reduction in u-PA and MMP-mediated proteolytic activity. This finding that compounds (Oxamflatin and MCT-1) inhibited u-PA and MMP gene expression and activity and induced PAI-2 expression and protein was very significant since this combination of effects have not been previously described and thus oxamflatin and its derivatives may be potent inhibitors of ECM degradation preferably in the case of metastatic malignant disease.

Therefore in a preferred embodiment, a plasminogen activating/MMP system may include any one or a combination of PAI-2/TIMPs or u-PA/MMP and modulation of the system may include modulation of any one or combination of PAI-2/TIMPs or u-PA/MMP by modulating the gene expression and/or activity.

Preferably modulation of the plasminogen activating/MMP system involves upregulating PAI-2/TIMPs gene expression and down regulating u-PA/MMP gene expression or activity.

The term"modulating a plasminogen-activation/MMP associated condition"may include inhibiting the condition or enhancing or stimulating the condition. For instance, inhibiting a plasminogen activation/MMP associated condition may include inhibiting the processes involved in plasminogen/MMP activation.

Urokinase (u-PA) is integral to the process of plasminogen/MMP activation.

Therefore, inhibiting u-PA activity or genes encoding u-PA may modulate in an inhibitory manner, a plasminogen activation condition. Plasminogen activation inhibitor type 2 (PAI-2) is an inhibitor of u-PA activity. Therefore, inhibition of processes involved in plasminogen activation may also involve stimulation of the PAI-2 expression. An enhanced production of PAI-2 may manifest as enhanced gene or protein expression of PAI-2.

Oxamflatin is a previously known compound. However as described above, its full potential has not been elucidated. Moreover, the active potential of the compound and its derivatives has not been fully known.

This class of compounds known as the hydroxamic acid derivatives have previously been demonstrated to modulate gene expression of extracellular matrix proteins (Sonoda, H et al (1996) Oncogene 13,143-149). These compounds may show activities such as cell growth inhibitory activity, vascularization inhibitory activity and are useful for prophylaxis and therapy of various inflammatory diseases, primary tumours, arteriosclerosis, peptic ulcer, diabetic retinopathy and additional vascular diseases.

Oxamflatin ( (2E)-5- [3- (phenylsulfonylamino) phenyl] pent-2-ene-4-ynohydroxamic acid) has the following formula: This compound has been found in US 5,534, 654 to possess the following inhibitory activities against the growth of vascular endothelial cells and the expression of lymphocyte adhesive factors, detransforming activity of cells transformed by ras gene, inhibition of cell growth and have effect on

inflammation and on tumours. It has previously been shown to reduce primary tumour growth but has not been indicated to have any benefit in the inhibition of metastatic disease spread. Cell growth inhibition as demonstrated by this compound is quite different to cellular spread that requires an element of mobility not present in cell growth. Hence, applicants have identified an unsuspected property which allows this component to be used in modulating metastatic spread or any cell mobility associated condition.

US patent 5,534, 654 describes oxamflatin as one of the aromatic sulfonamide- type hydroxamic acid derivatives. Structural requirements for activity are not evident from this patent. There is no consistency for predicting active compounds that will have effect on any one of the activities described above.

Compound 1-18 (oxamflatin) is considered the most active, having more than ten times the activity of its nearest rival utilising ras transformation inhibition assays.

In another aspect of the present invention, there is provided an oxamflatin derivative having a hydroxamic acid and/or a sulphonamide held spatially apart by an unsaturated carbon skeleton. Preferably, where the carbon skeleton contains a benzene ring the groups are orientated meta to each other.

If the hydroxamic acid and the sulphonamide are fairly closely aligned the activity is decreased. Accordingly, alignment of these groups provides a means to generate any number of derivatives having varying activities.

In a further preferred aspect the sulphonamide nitrogen at position 3 is left unsubstituted in an oxamflatin derivative.

Derivatives of oxamflatin or intermediates in the synthesis of the derivatives, that may be useful in the present invention may have the following structure:

Accordingly, oxamflatin derivatives that may be useful in modulating the plasminogen activation/MMP associated conditions according to the present invention, may be synthesized by modifying hydroxamic acid and/or sulphonamide groups, or have the alkenyl chain constrained in a ring structure or have the sulphonamide nitrogen left unsubstituted.

Suitable oxamflatin derivatives of the present invention may include any one of the following :

Other modifications of oxamflatin derivatives may include those where the central benzene ring is replaced with an indole. Examples may include :

Additional modifications may include the hydroxamic moiety being reversed such as in the following structure:

Synthetic routes for these oxamflatin derivatives are available to the skilled addressee and each one may be prepared by standard chemical transformations.

In a further preferred aspect of the present invention there is provided a method of modulating extracellular matrix degradation, said method comprising administering to a patient in need, an effective amount of oxamflatin or an oxamflatin derivative.

Extracellular matrix degradation, a preferred form of plasminogen activation/MMP associated condition, may occur by the action of cell surface, u- PA-mediated plasminogen and MMP activation. This process has been recognised as a process integral to ECM degradation. u-PA, bound to its cell surface receptor, u-PAR, is regulated by the serine protease inhibitor PAI-2 which is the primary inhibitory regulator of u-PA activity. Hence, by targeting either u-PA, MMP's or PAI-2 or TIMPs, extracellular matrix degradation may be regulated or modulated by oxamflatin or a derivative of oxamflatin as described above.

ECM degradation may also occur in a number of conditions. These may include metastatic cancer spread, leukaemia, lymphom, myeloma and associated haemoncological conditions, would healing, angiogenesis. Hence, it is preferred that the use of oxamflatin or derivatives will be useful for modulating any of these conditions.

In another preferred aspect of the present invention there is provided a method of reducing malignant metastatic spread, said method comprising administering to a patient in need, an effective amount of oxamflatin or an oxamflatin derivative.

Treatment with Oxamflatin or MCT-1 inhibited in vitro invasion of HT-1080, MDA-MB-231 and PC-3 cells. This occurred without a marked effect on cell

viability or proliferation (Figure 21). MCT-1 was a more potent inhibitor of invasion than Oxamflatin in two of the three cell lines tested (PC-3 and MDA- MB-231 cells). Previous reports demonstrate the widespread effect of acetylation as a post-translational modification to many cellular proteins with associated functional sequelae (Polevoda B, Sherman F. , Genome<BR> Biol. 3: 0006.1-0006. 6 (2002) ). Applicants have demonstrated herein that oxamflatin and MCT-1 inhibit histone deactylase activity (see Figure 16).

It is also possible that both these agents are modulating expression of other genes integral to cellular movement. Previous studies have demonstrated modulation of genes, whose protein products are involved in cellular migration, by histone deacetylase inhibitors (Crazzolara R, Johrer K, Johnstone RW, Greil R, Ofler RK, Meister B, Bernhard D. , Br J Haematol. 119 : 965-9 (2002) ). These genes including the CXCR4 chemokine receptor and Intercellular adhesion molecule 1 (ICAM-1) (Crazzolara R, Johrer K, Johnstone RW, Greil R, Ofier RK, Meister B, Bernhard D. , Br J Haematol. 119 : 965-9 (2002) ) (Park JH and Faller<BR> DV. , Virology 303: 345-63 2002) may also be regulated by Oxamflatin and/or MCT-1 and explain the inhibition of invasion at concentrations that do not inhibit u-PA or MMP gene expression i. e at 0.1 zM (Figure 21). To examine more broadly the effects of our agents on the modulation of gene expression Applicants have commenced a cDNA microarray analysis using a 10.8 K human array and both MCT-1 and Oxamflatin.

These studies identify Oxamflatin and its synthetic derivative MCT-1 as able to inhibit expression of components of the PA and MMP enzyme systems whilst simultaneously up regulating expression of the inhibitor PAI-2. These observations correlate with reduced metastatic cancer cell invasion with minimal toxicity and suggest that these compounds may have a therapeutic benefit in malignant disease. In addition, as inhibitory activity by Oxamflatin and MCT-1 has been demonstrated in two proteolytic enzyme systems deemed critical to the pathogenesis of metastasis these agents may be of benefit in circumventing the current problems confronting clinical application of single enzyme system inhibitors.

Excessive and uncontrolled tumour associated u-PA/MMP mediated extracellular matrix degradation is associated with the malignant metastatic phenotype which progresses from the primary tumour to give rise to secondary disease spread to other vital parts of the body. Applicants have demonstrated herein that oxamflatin and MCT-1 have anti-invasive and anti-metastatic activities using an orthotopic model of breast cancer. This system is a model for the dissemination of a breast tumour cell to remote sites in the body.

In a further preferred aspect of the present invention, there is provided a method of preventing and treating metastatic spread said method comprising administering to a patient in need, an effective amount of oxamflatin or an oxamflatin derivative.

Metastatic spread may be prevented or treated or modulated in any tumour including metastatic cells. Preferably the tumour is a solid tumour including breast, bowel, lung, prostate and bone tumours. The metastatic cells may be selected from a group including fibro sarcoma cells, prostate cancer cells, lymphom cells or breast cancer cells. Where the cells are fibro sarcoma cells, it is preferred the cell is a HT-1080 fibro sarcoma cell. The metastatic spread may also derive from a haematological malignancy. Preferably the malignancy is from a histolytic lymphom cell. More preferably the cell is a U937 histiocytic lymphom cell. Where the metastatic cell is a breast cancer cell it is preferably the MB-MDA-231 metastatic breast cancer cell. If the metastatic cell is a prostate cancer cell, it is preferred to be the PC-3 metastatic prostate cell.

As described above, u-PA activity is also regulated by PAI-2. Accordingly, oxamflatin or oxamflatin derivatives as described above may be used to inhibit u-PA activity or gene expression as well as induce PAI-2 expression. Induction of inhibition of these regulators of plasminogen/MMP activation not only modulate plasminogen/MMP activation associated conditions, but more specifically tumour cell proteolysis which facilitates the development of therapeutic strategies to combat malignant diseases.

It has been observed that the primary tumour displays ECM degradation which appears responsible for the secondary disease spread to other vital parts of the body including lungs, brain, bone and liver. By inhibiting or reducing ECM degradation, either by inhibiting or reducing uPA/MMP gene expression or protein expression and activity, and/or inhibiting or reducing PAI-2 expression, ECM degradation may be prevented or reduced.

Whilst this description shows an inhibition of u-PA/MMP expression and activity or increasing PAI-2 expression in respect of reducing or inhibiting metastatic spread, this invention is not limited to this application. This invention also includes within its scope the prevention or treatment of leukaemia, lymphom, myeloma and associated haemoncological conditions. Leukaemia, lymphom and myeloma cells express u-PA/MMP on their cell surface and treatment with oxamflatin may affect the expression of this enzyme in the progression of the disease. Clearly, u-PA has the capacity to convert plasminogen to plasmin and this condition is integral to a number of conditions relating to degradation of fibrin. Hence the present invention includes within its scope, the use of oxamflatin and derivatives on any conditions associated with plasminogen activation and wherein the plasminogen/MMP activation is regulated by u-PA or PAI-2.

With respect to the method of reducing malignant metastatic spread, where malignant metastatic spread may have already begun, the use of oxamflatin or derivatives thereof, may prevent further metastatic spread and treat the spread that has already occurred. In many cases, identification of a primary tumour may be too late, in which case, some ECM degradation may have already occurred. Applicants have found that oxamflatin and its derivatives described above, will be useful to prevent further metastatic spread and to treat the spread which has already occurred.

In yet another aspect of the invention there is provided a method of preventing and treating vascular disease involving a plasminogen activating/MMP system, said method comprising administering an effective amount of oxamflatin or an oxamfiatin derivative to a patient.

Methods of administering the oxamflatin may vary depending on parameters such as the type of tumour and location of the tumour. Administration may be by any suitable route such as intravenous, intranasal, intraperitoneal, intramuscular, intradermal, infusion, suppository, implant and oral including slow release capsules and tablets. Oxamflatin or its derivatives may be administered alone or in combination with a carrier which facilitates its delivery to the site that requires treatment. Oxamflatin or its derivatives may be conjugated to a carrier molecule which is capable of targeting the tumour, preferably a primary tumour to bring the oxamflatin or a derivative thereof to the site of treatment. Such carrier molecules may include antibodies or biological compounds having cellular receptors such as cytokines targeted to their respective receptors.

The effective amount of oxamflatin or an oxamflatin derivative as described above will depend on the patient in need, the condition to be treated and the mode of administration. In the case of a tumour and prevention of metastatic spread, amounts ranging from 2 to 50 mg/kg body weight may be used, however dosages as low as 1 mg/kg may be efficacious.

The methods of the present invention further contemplates the administration of oxamflatin and oxamflatin derivatives as a prophylactic agent prior to, for example, surgery, chemotherapy, or irradiation of a primary tumour. The doses used may be similar to the effective amounts described above and may be given up to 72 hours prior to surgery, chemotherapy or irradiation.

Alternatively, the oxamflatin or the derivative as described above, may be administered after surgery, chemotherapy or irradiation in a similar manner to that described above for prophylaxis.

However, the invention generally contemplates the use of oxamflatin or its derivatives as a prophylactic treatment particularly for situations where the discovery of a small tumour which may be too small to excise may require a treatment to prevent further metastatic spread. The invention also

contemplates the use of oxamflatin in an already established disease to prevent further progression of the disease.

In a further preferred aspect of the present invention, there is provided a method of modulating extracellular matrix degradation, said method comprising administering to a patient in need, an effective amount of oxamflatin or an oxamflatin derivative in conjunction with a cytotoxic agent.

In a further preferred aspect of the present invention, there is provided a method of reducing malignant metastatic spread said method comprising administering to a patient in need, an effective amount of oxamflatin or an oxamflatin derivative in conjunction with a cytotoxic agent in an effective amount to inhibit the proliferation of a tumour.

Suitable cytotoxic agents include standard cytotoxic agents utilized in metastatic conditions such as adriamycin, cisplatin, 5FIuorouracil, etoposide, cyclophosphamide, and capecitabine.

In another aspect of the present invention, there is provided a use of oxamflatin and oxamflatin derivatives in the preparation of a medicament for the modulation of a plasminogen-activation associated condition. In a preferred aspect, the plasminogen/MMP activation associated condition is ECM degradation. More preferably, the plasminogen/MMP activation associated condition is malignant metastatic spread and use of the oxamflatin or derivative may inhibit u-PA/MMP expression or u-PA/MMP activity and/or increase PAI- 2/TIMP expression thereby reducing metastatic spread.

In yet another aspect of the present invention, there is provided a composition for use in the modulation of a plasminogen/MMP-activation associated condition, said composition comprising oxamflatin or an oxamflatin derivative and a carrier or diluent.

Preferably, the plasminogen/MMP-activation associated condition is ECM degradation. More preferably, it is malignant metastatic spread.

The carrier or diluent may be a pharmaceutical acceptable carrier. As used herein"pharmaceutically acceptable carriers and/or diluents"may include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying or enhancing agents and the like. Supplementary active ingredients may also be incorporated into the compositions.

Carriers and diluents may also be suitable for delivering the oxamflatin directly to a site which requires treatment.

The term"treatment"is used herein in its broadest sense to include prophylaxis (ie. prevention) treatment as well as treatment designed to ameliorate the effects of any plasminogen/MMP-activation associated condition, preferably ECM degradation. More preferably, it is metastatic spread. The treatment by use of oxamflatin or derivative is preferably aimed at reducing or inhibiting metastatic spread.

In yet another aspect of the present invention, there is provided a method of preparing a composition for use in the modulation of a plasminogen/MMP- activation associated condition, said composition comprising oxamflatin or an oxamflatin derivative and a carrier or diluent said method comprising mixing a suitable amount of oxamflatin or an oxamflatin derivative with a suitable carrier or diluent to provide an effective amount of oxamflatin or oxamflatin derivative to modulate the plasminogen/MMP-activation associated condition.

Preferably the plasminogen/MMP-activation associated condition is ECM degradation. More preferably, the plasminogen/MMP-activation associated condition is metastatic spread.

In another aspect the present invention provides a compound having a formula selected from the group consisting of

In a further aspect of the present invention there is provided a composition comprising a compound disclosed above.

The present invention will now be more fully described with reference the following examples. It should be understood however that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES Example 1-Oxamflatin increases PAI-2 mRNA in U-937 cells and potentiates PMA-mediated induction of PAI-2 mRNA expression in U-937 and HT-1080 cells.

To determine whether oxamflatin treatment was associated with a concomitant increase in endogenous PAI-2 mRNA expression, Northern blot experiments were performed using RNA extracted from U-937 and HT-1080 cells treated with oxamflatin for 16 hrs alone or in combination with PMA.

Human HT-1080 fibrosarcoma cells (American Type Culture Collection, Rockville, MD) were cultured to confluence at 37°C in Nunclon cell culture

dishes according to standard techniques in 10 ml Dulbecco's modified Eagles Medium (DMEM) supplemented with 2 mM glutamin and 10% heat-inactivated foetal calf serum (HI-FCS, Gibco BRL, Australia). Human U-937 histiocytic lymphom cells (American Tissue Culture Collection, Rockville, MD) were grown in suspension in Nunclon cell culture flasks at 37°C according to standard techniques in 30 mi of RPMI-1640 medium (Gibco BRL, Australia) supplemented with 2 mM glutamin and 10 % HI-FCS.

Oxamflatin was kindly donated by Dr Hikaru Sonoda of Shionogi Research Laboratories Shionogi and Co Ltd, Sagisu 5-12-4, Fukushima-ku, Osaka 533, Japan.

The isolation of total RNA from HT-1080 cells was performed by the method of Chomczynski and Sacchi (1987) Anal. Biochem, 162,156-159. 10 wog of RNA was loaded to each lane and electrophoresed through a 1 % agarose gel containing 20% formaldehyde before being transferred to Hybond-N+ membrane (Amersham, Australia). Filters were hybridised overnight of 42°C in a standard 50% formamide hybridisation buffer (Medcalf, R. L. et al (1986) EMBO J. 5,2217- 2222) containing 32P-labeled cDNA inserts. The cDNAs used for this procedure included the 1.9 kb Eco R1 fragment of the plasmid pJ7 containing the near-full length PAI-2 cDNA (Schleuning, W-D et al (1987) Mol. Cell. Biol. 53,4564-4567), the 2.5 kb Eco-R1 fragment of u-PA (Cajot, J-F et al (1991) Proc. Acad. Natl. Sci.

87,6939-6943) and-actin (Medcalf, R. L. et al (1990) J. Biol. Chem. 24 14618 - 14626). After hybridisation, the membranes were washed by standard techniques and exposed to Kodak BioMax film (Eastman Kodak, Rochester, NY) at-80°C with an intensifying screen.

Treatment of U-937 cells with oxamflatin produced a significant increase in PAI- 2 mRNA, equivalent to that observed with PMA (Figure 1 lanes 2 and 3). The effect of oxamflatin treatment was additive with respect to PMA-mediated induction of PAI-2 mRNA (Figure 1, lane 4). HT-1080 cells treated with oxamflatin alone did not to increase PAI-2 mRNA levels, however oxamflatin treatment profoundly augmented PMA-mediated induction of PAI-2 mRNA

(Figure 2 lanes 5 and 6). Internal control for RNA loading was carried out by hybridising the filter with a labelled ß-actin c-DNA probe.

Example 2-Oxamflatin treatment suppresses u-PA mRNA expression and PMA-mediated induction of u-PA mRNA.

The primary role of extracellular PAI-2 is thought to be the inhibition of u-PA- mediated extracellular matrix degradation therefore in the effect of oxamflatin treatment on u-PA gene expression was investigated. Subsequently, using the same RNA extracted from U-937 cells and used to produce the Northern Blot illustrated in Figure 1. A Northern Blot using a labelled u-PA cDNA fragment as a probe was performed (see Figure 3 Panel A). Results demonstrate that oxamflatin treatment resulted in inhibition of constitutive u-PA mRNA levels (Figure 3 Panel A lanes 1-3). A small but reproducible increase in u-PA mRNA at the 16 hr time point in response to PMA stimulation was noted (Figure 3 Panel A lane 4). Figure 3 Panel A lane 5 also suggests a degree of inhibition of PMA-mediated induction of u-PA mRNA by oxamflatin treatment. In addition the Northern Blot filter used to produce Figure 2 was reprobed with a u-PA cDNA fragment to assess the effect of oxamflatin on u-PA expression in HT- 1080 cells (Figure 3 Panel B). Akin to results illustrated in Figure 3 Panel A, inhibition of constitutive u-PA mRNA expression was also noted in these cells.

P-Actin represents the internal control in both these experiments.

Example 3-Oxamflatin transactivates the PAI-2 gene promoter in transfected HT-1080 cells.

The effects of oxamflatin treatment on transactivation of the PAI-2 gene promoter were determined. HT-1080 cells were stably transfected with a-219 bp PAI-2 gene promoter construct fused to the CAT reporter gene.

A PAI-2 promoter construct harbouring the first 219 bp of the PAI-2 gene promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene was used in this study. This construct has been previously described in Cousin, E et al (1991) Nucal. Acids Res. 19, 3881-3886.

The-219 PAI-2 promoter harbours essential regulatory elements including the Cyclic AMP Response Element (CRE) and Activation Protein-1 (AP-1) binding sites.

HT-1080 cells were stably transfected with the-219-PAI-2 promoter-CAT construct using the calcium phosphate precipitation method as previously described in Sambrook, J et al, Molecular cloning a laboratory manual 2"d Edn, Cold Spring Harbour Laboratory, Cold Spring Harbour, New York, 1989.

Plasmid pNTneo, expressing resistance to neomycin, (kindly provided by Dr Phil Bird, Monash University, Box Hill Hospital Department of Medicine) was co- transfected with the PAI-2 gene promoter construct. Transfected cells were treated with 600 pg/ml G418 and pooled colonies harvested after approximately 3 weeks.

Transfected cells were subsequently incubated for 16 hours in either serum-free medium or serum-free medium supplemented with 1 or 5M oxamflatin alone or in combination with 25 nM PMA. Cells were harvested and cytoplasmic extracts prepared by three freeze-thaw cycles. Protein concentration of the cytoplasmic extracts was assessed using the BioRad dye reagent system (BioRad, Australia). Samples were either used immediately or stored at-80°C.

Transfected cells were treated with oxamflatin (1 uM or 5M), 25 nM PMA, or a combination of both agents.

Cytoplasmic extracts of transfected cells (usually between 10-20 g in 40 lli of 250 mM Tris-HCI, pH 7.4) were incubated with 5 tl of 4.4 mM acetyl coenzyme A (Boehringer Mannheim) and 1 ul 14C-chloramphenicol (Du-Pont) for 4 hours at 37°C. The samples were processed by standard techniques and subjected to analysis by thin layer chromatography. The percentage conversion of 14C- chloramphenicol to its acetylated products was quantified by phosphorimaging using a Fujix BAS 1000 phosphorimager.

As shown in Figure 4, stimulation of transfected cells with 1 or 5 M oxamflatin alone resulted in an approximate 2-fold induction in CAT activity as determined

by phosphorimaging analysis. PMA-treatment, used as a positive control, resulted in a 2-2. 5-fold increase in CAT activity. A 12-fold increase in CAT activity was observed in cells treated with a combination of 5 zM oxamflatin and PMA indicating a likely synergistic effect of this drug combination. Taken together, these data indicate that oxamflatin is capable of transactivating the PAI-2 gene promoter. In addition the data indicate that oxamflatin may stabilise PAI-2 mRNA levels accounting for the observed differences in the extent of oxamflatin-mediated induction of PAI-2 promoter activity and PAI-2 mRNA levels observed by Northern Blot.

Example 4-The effects of oxamflatin treatment on the binding of nuclear proteins to the CRE and AP-1a binding sites in the PAI-2 gene promoter.

Identification of oxamflatin as capable in inducing both PAI-2 promoter activity and mRNA levels, together with previous reports of oxamflatin-mediated induction of Jun-D expression resulted in an investigation that this agent may be capable of modulating binding of nuclear proteins to cis-acting elements within the PAI-2 gene promoter.

The preparation of nuclear proteins from HT-1080 or U-937 cells was performed as previously described in Osborn, L et al (1989) Proc. Natl Acad. Sci, 86,2336- 2340.

Double stranded oligonucleotides containing either an AP-1 binding consensus sequence or the PAI-2 CRE-site within the PAI-2 gene promoter were synthesised. Oligonucleotides were gel purified by electrophoresis through a 15% polyacrylamide gel containing 7M urea and labelled with T4 polynucleotide kinase (Sambrook J. et al (1989) Molecular cloning : a laboratory manual. 2nd edn.

Cold Spring Harbour Laboratory. Cold Spring Harbour, New York). Annealing of complementary single stranded oligomers was performed as previously described (Dear, A. E. et al (1996) Eur. J. Biochem, 241 93-100). The sequences (upper strand only shown) of oligonucleotides used for this study were: 5'-GATTCAATGACTCACGGCTGTG-3', (AP-1a oligomer, complimentary to the region between-110 and-90 in PAI-2 gene promoter) and 5'- TTCAGAGTGACCTCATCCTCC-3' (CRE oligomer, complimentary to the region

between-176 and-196 in the PAI-2 gene promoter (Cousin, E. et al. (1991) Nucl. Acids. Res. 19 3881-3886). Underlined regions within the AP-1a and CRE oligonucleotides indicate the consensus AP-1 and CRE core elements, respectively.

The sequence of the unrelated double stranded oligomer used for the competition experiments was: 5'-CTGGGGCTGACAGATTTTAGCT-3' (upper strand only shown).

4R1 of HT-1080 nuclear protein extract containing 4g of protein in Osborn buffer D (Medcalf, R. L. et al (1990) J. Biol. Chem 24,14618-14626) were incubated at 4°C for 15 minutes with 1p1 (500 ng to 1, ug) of poly d (I-C) (Boehringer Mannheim, Australia) and zip of SMK buffer (12 mM spermidine, 1.2 mM MgCI2 and 200 mM KCL (Medcalf, R. L. et al (1990) J. Biol. Chem 24, 14618-14626). 4111 of y32P-labeled probe (100 cps diluted in buffer D (above) was added and the mixtures incubated on ice for a further 15 minutes before being applied to a 5% polyacrylamide gel prepared in 0.25 x Tris/boric acid/EDTA (TBE) buffer (Medcalf, R. L. et al (1988) J. Cell Biol. 106 971-978) and subjected to electrophoresis. The gels were dried and autoradiographed at - 70°C overnight with an intensifying screen. For competition experiments, nuclear extracts were incubated with a 10-to 100-fold excess of unlabeled double stranded oligonucleotides 15 minutes after addition of poly d (I-C) competitor.

HT-1080 cells were treated with oxamflatin (1pM) for 1 hr and supershift analysis performed (Figure 5). Oxamflatin treatment was not associated with enhancement of nuclear protein binding to either the PAI-2 CRE or AP-1 a sites, nor was modulation or induction of c-Jun or Jun-D binding observed. Induction of nuclear protein binding was observed with PMA treatment, used as a positive control in this experiment. Hence oxamflatin-mediated transcriptional regulation of PAI-2 gene expression is not associated with altered binding of nuclear proteins to two integral cis-acting sites within the PAI-2 gene promoter.

Example 5-Oxamflatin treatment of HT-1080 cells is associated with reduced u-PA-mediated proteolytic activity in zymography studies.

In order to assess the sequelae of oxamflatin-mediated inhibition of u-PA mRNA zymographic studies were undertaken using supernatant's from oxamflatin treated HT-1080 cells.

Zymographic analysis was performed using the supernatants from HT-1080 cells treated with oxamflatin, PMA or a combination of both agents, together with untreated control cells. 5x103 HT-1080 cells were seeded into 60 mm Nunc petri dishes. 2 ml of DMEM medium supplemented with 10% foetal calf serum was added. Zymographic analysis was undertaken using a previously published protocol (Granelli-Piperno, A. and Reich, E. (1978) J. Exp. Med 148,223-234).

20 jui of supernatant samples were run through an SDS-PAGE gel and zymographic analysis undertaken.

Inhibition of u-PA-mediated proteolytic activity as evidenced by reduced lysis of a fibrin based matrix is noted when using supernatant's from HT-1080 cells treated with oxamflatin in a dose dependent manner over a 24 hour time course (Figure 6 Panels A and B). Higher molecular weight species are noted in both Panel A and B and are thought to represent tissue type plasminogen activation (t-PA)/PAI-1 complexes. The molecular weight of these complexes (approximately 110 kD) are in accordance with those previously published (Medcalf, R. L. et al, (1988) J. Cell Biol, 106,971-978). t-PA/PAI-1 complexes are unaffected by Oxamflatin treatment in comparison with u-PA mediated proteolytic activity this result acting as an internal control for the effect of Oxamflatin. Addition of oxamflatin directly to u-PA had no effect on u-PA- mediated proteolysis (data not shown).

Example 6-Oxamflatin treatment increases PAI-2 protein expression in U-937 cells To determine if Oxamflatin-mediated induction of PAI-2 mRNA in U-937 cells was associated with increased PAI-2 protein expression Western Blot analysis was undertaken utilising cytosolic extracts from untreated U-937 cells, or cells treated with increasing concentrations of oxamflatin (1pu, 2. 5 [tM, 5liM, 7. 5plu).

Figure 7 demonstrates a dose dependent increase in PAI-2 protein expression upon treatment with Oxamflatin, over untreated cells. Induction of PAI-2 antigen after treatment with 25 nM PMA was used as a positive control. Lower molecular weight bands present in each lane represent PAI-2 cleavage products. Coomassie staining of the SDS-PAGE gel indicates that equal amount of protein are loaded into each lane.

Example 7-Effect of oxamflatin on u-PA-mediated proteolytic activity from MDA-MB231 Metastatic Breast Cancer Cells MDA-MB231 metastatic breast cancer cells were cultured under normal conditions suggested by the supplier. Conditioned medium was removed and u-PA mediated proteolytic activity was measured in the presence of oxamflatin.

A control (0, uM), 1pM and 5M oxamflatin were tested and proteolytic activity measured. Figure 9 shows reduced u-PA-mediated proteolytic activity in conditioned medium when treated with oxamflatin.

Example 8-Effect of oxamflatin on u-PA-mediated proteolytic activity from PC-3 Metastatic Prostate Cancer Cells PC-3 metastatic prostate cancer cells were cultured under normal conditions suggested by the supplier. Conditioned medium was removed and u-PA mediated proteolytic activity was measured in the presence of oxamflatin. A control (0pM), 1pM and 5pM oxamflatin were tested and proteolytic activity measured. Figure 10 shows reduced u-PA-mediated proteolytic activity in conditioned medium when treated with oxamflatin.

Example 9-Synthesis of MCT-1: (2E)-5- [3- (Methylsulfonylamino) phenyl] pent-2-en-4yonhydroxamic Acid.

Compound 2

To a solution of 25.20 g (0.153 mol) of 3-aminoethylbenzoate in 600 ml of dioxane are added 800 mi of 5% NaHCO3 solution and 15 g of NaHCO3 and the mixture is stirred vigorously. An 80 ml solution of 48.7 ml (0.382 mol) of methanesulfonyl chloride in dioxane is added gradually at room temperature and the resultant mixture is stirred for about 7 hr. The reaction mixture is partitioned between ethyl acetate and 2N HCI. The organic layer is washed with water and saturated saline, dried over. MgS04 and concentrated to obtain a crude compound 2. Recrystallization from toluene/hexane give 29.24 g (0.0958 mi ; yield, 63%) of compound 2.

Compound 2 Compound 3 To a 300 ml solution of 28.85 g (0.0945 mol) of compound 2 in THF is carefully added 5.32 g (0.140 mmol) of lithium aluminium hydride and the mixture is stirred for about 1 hr at room temperature. To the reaction mixture was added ethyl acetate and water under ice-cooling to decompose the excess of reducing agents. It was then partitioned between ethyl acetate and 2N HCI. the organic layer is washed with water and a saline, dried over MgS04, filtered and concentrated under reduced pressure to obtain a crude product.

Recrystallization from methylene chloride/ether gives 22.34 g (0.0848 mol ; yield, 90%) of compound 3.

Compound 3 Compound 4 To a 1500 ml solution of 22.06 g (83.8 mmol) of compound 3 in methylene chloride are added 44 g of molecular sieve 4A (powder) and 32.5 g (151 mmol) of pyridinium chlorochromate and the mixture is stirred fro 70 min at room temperature. The reaction mixture is purified by column chromatography on silica gel eluting with methylene chloride to yield 20.46 g (78.3 mmol ; yield, 94% of compound 4 Compound 4 Compound 5 To a 300 ml solution of 29.79 g (95.4 mmol) of compound 4 in benzene are added 80 ml of triethylamine, 0.671 g (0.956 mmol) of palladium bistriphenylenephosphine dichloride, 0.091 g (0.478 mmol) of copper iodide and 16.7 ml (287 mmol) or propargyl alcohol and the mixture is heated to reflux overnight. The reaction mixture is concentrated under reduced pressure. The residue is combined with ether and filtered to remove insoluble materials.

Purification by chromatography on silica gel gives 5.19 g (18.1 mmol ; yield, 19%) of the objective compound 5 as an oil. Although it contains a little solvent, it is used in the next reaction as it is.

Compound 5 Compound 6 To a 150 mi solution of the crude material from the previous reaction is methylene chloride are added 5g of molecular sieve 4A (powder) and 6.4g (30 mmol) of pyridinium chlorochromate and the mixture is stirred for 70 min at room temperature. The reaction mixture is purified by column chromatography on silica gel eluting with methylene chloride to yield 20.46 g (78.3 mmol ; yield, 94% of the required compound 6.

Compound 6 Compound 7 To a 50 mi solution of 1.75 mi (10.8 mmol) of trimethyl phosphonoacetate in THF is added at once 0.420 g (10.5 mmol) of 60% (in oil) sodium hydride at 0° C. in a atmosphere of nitrogen. After stirring for 1 hr at room temperature, a 12 ml solution of 1.03 g (3.62 mmol) of compound 6 in THF is added to the mixture gradually. After stirring for 60 min at room temperature, the reaction mixture is partitioned between ethyl acetate and 2N HCI. The organic layer is washed with water and a saturated saline, dried, filtered and concentrated. The residue, when purified by chromatography on silica gel and recrystallised from methylene chloride/ether/hexane, gives 0.872 g (2.55 mol ; yield, 71%) of the desired compound 7.

Compound 7 Compound 8 To a 5.0 mi suspension of 0.500 g (1.35 mmol) of compound 7 in methanol is added 2.7 ml (2.7 mmol) of 1 N potassium hydroxide solution and the mixture is stirred overnight at room temperature. When the starting materials still remain in the mixture, the stirring is continued for another 4.5 hr at 40° C. after the addition of 2.0 ml of DMSO and 1.35 ml (1.35 mmol) of potassium hydroxide.

The reaction solution is partitioned between ethyl acetate and water and the organic layer is washed with water. The aqueous layers are combined and partitioned between 2N HCI and ethyl acetate. The organic layer is washed with water (x3) and a saturated saline (x1), dried, filtered and concentrated.

Recrystallization from ethyl acetate/methanol provides 0.333g (0.938 mmol ; yield, 69%) of the desired compound 8.

Compound 8 Compound 9 To an 8 ml suspension of 645 mg (1.96 mmol) of compound 8 in methylene chloride is added 0.60 mi (6.88 mmol) of oxalyl chloride and one drop of DMF.

The mixture is stirred for 30 min at room temperature and then for 1 hr at 40° C. and concentrated under reduced pressure to obtain acid chloride. In another vessel, a suspension of 695 mg (10.0 mmol) of hydroxylamine in 12 mi of THF

is prepared, which is combined with 8.0 ml of saturated NaHCO3 solution and stirred for 5 min at room temperature. To the solution is added the previously prepared 8.0 ml of solution of acid chloride in THF and stirred vigorously for 30 min at room temperature. The reaction mixture is partitioned between ethyl acetate and 2N HCI. The organic layer is washed with water and a saturated saline, concentrated under reduced pressure and allowed to crystallize to obtain 400 mg (1. 16 mmol ; yield, 59%) desired compound 9 (MCT-1).

Example 10-Oxamflatin and MCT-1 have no effect on MMP-mediated proteolytic activity.

Human placental fibroblasts were exposed to varying concentrations of oxamflatin and MCT-1 (Panel A). The results were quantitated (Panel B) showing that MMP-mediated proteolytic activity is unaffected by oxamflatin and MTC-1 at concentrations of 5 uM.

Example 11-Effect of oxamflatin and MCT-1 on growth, viability and invasion of PC3, HT1080 and MDA-MB21 cells.

All cell lines were cultured under normal conditions as suggested by the suppliers. Medium was supplemented with 0. 1 M of oxamflatin or MCT-1 for a period of 40 hours. Control cells were not exposed to either compound. At the end of 40 hours cells were rated for growth, viability and ability to invade (see Figure 21).

The Invasion assay was carried out as follows : a) Matrigel Invasion Assay HT1080 cells (source) were suspended to a concentration of 2. 5X106 in SFM (serum-free medium: alpha-MEM with pen/strep and 0. 1% BSA) with or without drug. Cell suspensions were chilled and diluted 1: 1 in Matrigel (Becton Dickinson), and 80 NI distributed to pre-chilled cell culture inserts with 8. 0 urn pores (Becton Dickenson). Coated inserts were allowed to gel for 30 minutes at 37°. They were then placed in companion wells with 600pI SFM containing the correct concentrations of drug, and were overlayed with 100 NI of the same medium. They were pre-incubated for 6 hours before being moved to wells with

600pI of 2% FBS in media containing the correct drug dilutions. Invasion took place over a 24 hour incubation at 37°. To visualize invading cells, the inner sides of the membranes were first wiped using cotton wool. Membranes were removed from inserts using forceps, and carefully inverted on a drop of solubilization buffer (0. 01% Triton X, 0. 01% Na Acetate in phosphate buffered saline). After 10 minutes membranes were stained for 20 seconds in. 05% methylene blue, washed three times in water, and allowed to dry before being mounted on slides. Invading cells were counted by visualizing their stained nuclei, averaging over 5 fields per membrane.

The results are shown in Figure 14. Panel A shows that oxamflatin has a substantial effect on the ability of cells to invade. HT1080 cells were the most affected cell line. Panel B shows that MCT-1 has a dramatic effect on the ability of all three cell lines to invade. Growth and viability is essentially retained for both drugs, but invasion is strongly inhibited.

Example 12-Effect of oxamflatin and MCT-1 on invasion of 4T1. 13con2 mouse mammary cancer cells through Matrigel.

Cells were cultured according to the suppliers directions and exposed to either oxamflatin (0. 1 M) or MCT-1 (0. 1 M) and then assessed by microscopy for the ability to migrate through matrigel (see Figure 15). Matrigel invasion assays are well known to the skilled person. Methods have been described in literature by Knutson et al for example (Mol. Biol. Cell 7: 383-396,1996). 4T1. 13con2 cells are to be utilised in vivo models of cancer cell metastasis.

Example 13: Oxamflatin and MCT-1 inhibit histone deactylase activity.

The effect of varying concentrations of oxamflatin and MCT-1 on histone deactylase activity was investigated. The results shown in Figure 16 demonstrate inhibition at a concentration of 1.0 uM.

Example 14: Materials and Methods The following materials and methods were used for Examples 15 to 18.

Ce ! ! Culture and Materials.

Human HT-1080 fibrosarcoma (American Type Tissue Culture Collection (ATCC) ), MDA-MB-231 breast cancer (Dr J Price, MD Anderson Cancer Center), PC-3 prostate cancer cells (Walter and Eliza Hall Institute, Melbourne, Australia) and primary human placental fibroblasts were cultured to 70% confluence in Dulbecco's modified Eagles Medium (DMEM) supplemented with 2 mM glutamin, 10% heat-inactivated foetal calf serum (FCS) (Gibco BRL) together with 50u/ml penicillin and 50ug/ml streptomycin, called complete medium (CM). All cells were incubated at 37 °C with 5% (w/v) C02 and 95% (w/v) air mixture.

Oxamflatin (Ox) and Metacept-1 (MCT-1) were synthesized in the Department of Chemistry, Monash University, Melbourne, Victoria, Australia. MCT-1 was generated by methyl substitution of the phenyl group of Ox. Prior to use, the powdered form of each compound was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 10 mM. The compounds were added to serum free (SF) DMEM medium to give final concentrations ranging between 0. 1-100uM.

The final DMSO concentration did not exceed 0. 1% and DMSO was added to control samples at the same final concentration.

Northern Blot Analysis RNA was isolated using the method of Chomczynski and Sacchi (Chomczynski P, Sacchi N,. Anal. Biochem. 162: 156-159 (1987)). 10 g of RNA was loaded in each lane and electrophoresed through a 1% agarose gel containing 20% formaldehyde before being transferred to Hybond-N+ membrane (Amersham).

Filters were hybridized overnight at 42°C in a standard 50% formamide hybridization buffer (Sambrook J, Fritsch EF, Maniatis T. Molecular cloning : a laboratory manual. 2nd edn, Cold Spring Harbour Laboratory. Cold Spring Harbour, New York, (1989) ) containing 32P-labeled cDNA inserts. The cDNA probes used for this procedure included the 1.9 kb Eco R1 fragment of the plasmid pJ7 containing the near-full length PAI-2 cDNA (Schleuning WD, Medcalf RL, Hession C, Rothenbuhler R, Shaw A, Kruithof EKO,. Mol. Cell.

Biol. 53 (1987) 4564-67), the 2.5 kb Eco-R1 fragment of u-PA (Medcalf RL, Richards RR, Crawford RJ, Hamilton JA, EMBO J. 5: 2217-2222 (1986) ) and ß- actin (Medcalf RL, Van Den Berg E, Schleuning WD, J. Cell. Biol 106 971-78

(1988) ), the 2.0 kb Xho-1 fragment of MMP-2, the 2.0 kb Not 1 and Kpn 1 fragment of MMP-9 (MMP-2 and MMP-9 fragments, kind gift of Professor Erik Thompson, St Vincent's Institute of Medical Research, Melbourne, Victoria, Australia) and the 1.1 kb Pst 1 fragment of GAPDH (34). After hybridization, the membranes were washed by standard techniques and exposed to Kodak BioMax film (Eastman Kodak, Rochester, NY) at-80°C with an intensifying screen. Signal intensity was quantified utilizing the Gel-Pro Analyser Version 2.0. Results were analysed using Prism Software by analysis of variance (ANOVA).

RT-PCR Analysis Real time quantitative reverse transcriptase PCR was performed on cDNA from Trizol RNA extracts and detected by SYBR green using the Applied Biosystems 7000 Sequence Detection System. Primers for u-PA, u-PAR, PAI-2, PAI-1and GAPDH were utilized. Transcript abundance relative to GAPDH was determined. Treatment with Ox or MCT-1 did not alter the abundance of the GAPDH transcript (data not shown).

SDS-PAGE and Zymography Studies HT-1080, MDA-MB-231 or PC-3 cells at 5x105/dish were cultured in complete DMEM medium. The following day, the cells were rinsed with phosphate buffered saline (PBS), the medium was replaced with 2ml serum free (SF) DMEM, and cells were treated with Ox or MCT-1 for 16 hrs. An aliquot of conditioned medium from cells treated with Ox or MCT, together with untreated control cells was electrophoresed through a 10% acrylamide SDS-PAGE gel under non-denaturing conditions. Fibrin and gelatin zymographic analysis were performed on SDS-PAGE gels as previously described (Granelli-Piperno, E Reich E. , J. Exp. Med 148: 223-234 (1978) ) (Fridman R, Toth M, Pena D,<BR> Mobashery S. , Cancer Res 55: 2548-2555 (1995).

Matrigel Invasion Assay HT-1080, MDA-MB-231, and PC-3 cells were suspended at a concentration of 2. 5x106/ml in serum free medium (SFM) supplemented with 0. 1% BSA with or without Ox or MCT-1. Cell suspensions were chilled and diluted 1: 1 in Matrigel

(Becton Dickinson), and 80 pi distributed to pre-chilled cell culture inserts with 8.0 uM pores (Becton Dickinson). Coated inserts were allowed to gel for 30 minutes at 37°C before being inserted into wells of a plate containing 600 NI SFM with various concentrations of either Ox or MCT-1 and overlaid with 100 NI of the same medium. They were pre-incubated for 16 hours before being moved to wells with 600 ul of 2% FBS in medium containing the same concentrations of Ox and MCT-1. After 24-hrs at 37°C, the membrane was fixed in 10% buffered formalin and the upper side of the membrane was wiped free of cells using cotton wool. Cells remaining on the lower side of the membrane were permeabilised with 0. 1% Tween-20 (5min) and stained with DAPI (0. 51ug/ml, 15 min). Membranes were rinsed, dried and mounted on glass slides. Five fields were counted per membrane. Results are presented as the average of five fields per membrane.

Growth Curves Cells were suspended in CM and 500 cells per well were distributed in 100 pi volumes into 4 x 96-well plates and left overnight at 37°C. A further 100 ul of CM, containing various drug concentrations, was added. At intervals over the next 4 days, cell content was assayed using a Sulphorhodamine B (Aldrich) colorimetric assay (SRB assay).

Statistical Analysis Results from Northern Blot analysis and Zymography were analysed using Prism Software by analysis of variance (ANOVA) and Students t-test.

Statistical significance was reached when *P<0.05 or **P<0. 01.

Example 15: Oxamflatin and MCT-1 regulate expression of components of the plasminogen activating system in HT-1080, MDA-MB-231 and PC-3 cells.

To determine the effects of Ox and MCT-1 on u-PA mRNA expression, cells were subjected to 5 pM Ox or MCT-1 for 16 hours (hrs) prior to RNA extraction. u-PA levels were measured by northern blot analysis. Ox induced a statistically significant reduction in u-PA mRNA levels in all three cell lines. The reduction caused by MCT-1 was not as marked (Figure 17). The effects were

observed in the absence of significant reduction in cell viability over this 16 hr period as determined by trypan blue staining (data not shown). Treatment of MDA-MB-231 metastatic breast cancer cells with a combination of Ox and MCT-1 at 5 uM resulted in additive inhibition of u-PA mRNA expression (data not shown). In contrast to the effects of Ox and MCT-1 on u-PA mRNA expression in metastatic cancer cell lines neither compound significantly affected u-PA mRNA expression in primary placental fibroblasts (data not shown).

Expression of u-PA and other components of the plasminogen activating system were analysed further by real-time quantitative RT-PCR after 0.1 uM or 5 uM Ox or MCT-1 treatment for 16 hrs (Figure 18). u-PA expression in PC-3 and HT-1080 cells was greatly diminished after 5uM treatment consistent with our observations using northern blotting techniques (Figure 17). Ox was a more potent inhibitor of u-PA expression than MCT-1 in PC-3 (Figure 18A) cells but both Oxamflatin and MCT-1 were equipotent in HT-1080 cells (Figure 18B).

PAI-1 and PAI-2 transcript levels were increased in HT-1080 cells (Figure 18B) treated with 5, uM Oxamflatin. Induction of PAI-2, and to a lesser extent PAI-1, was noted with 5pM MCT-1 treatment (Figure 18B). MCT-1 inhibited PAI-1 and PAI-2 expression in PC-3 cells whilst an increase was noted with Oxamflatin treatment (Figure 18A). No changes in PAI-2, u-PA, and uPAR expression levels in PC-3 and MDA-MB-231 cells were detected by drug treatment at the lower concentration of 0. 1, uM (data not shown).

Example 16: Oxamflatin and MCT-1 inhibit MMP-2 and MMP-9 expression in HT-1080 and MDA-MB-231 cells.

Aberrant, excessive expression of MMP-2 and MMP-9 at the in vitro, in vivo and clinical levels have been associated with progression of the metastatic <BR> <BR> phenotype (Brinckerhoff C. E. and Matrisian L. M. , Nature Reviews: Molecular<BR> Cell Biology, 3: 207-214 (2002) ), (Campo, E. , Merino, M. J., Tavassoli, F. A., Charonis, A. S., Stetler-Stevenson, W. G. and Liotta, L. A., Am. J. Surg. Pathol 16: 500-507 (1992) (Talvensaari-Mattila, A. , Paakko, P. , Hoyhtya, M., Blanco-<BR> Sequeiros, G. and Turpeenniemi-Hujanen, T. , Cancer 83: 1153-1162 (1998).

Since MMPs are clearly important in the progression of metastatic cancer, we were interested to determine the effects of Oxamflatin and MCT-1 on their expression. HT-1080 and MDA-MB-231 cells were treated for 16hrs with 5uM Ox or MCT-1. Treatment resulted in significant inhibition of MMP-2 and MMP-9 mRNA expression in HT-1080 and MDA-MB-231 cells with Oxamflatin again being the more effective of the two agents (Figure 19). MMP-2 expression was inhibited to a greater extent than MMP-9.

Example 17: Oxamflatin and MCT-1 inhibit u-PA and MMP-mediated proteolysis in HT-1080, MDA-MB-231 and PC-3 cells. u-PA and MMP-mediated proteolysis was assessed by fibrin and gelatin zymography following treatment with Oxamflatin or MCT-1 for 16 hrs (Figure 20). Both Oxamflatin and MCT-1 were able to inhibit u-PA activity at concentrations as low as 100 nM in HT-1080 cells (20A and B) with MCT-1 being more potent than Oxamflatin. The effects of Oxamflatin and MCT-1 treatment on MMP-9 mediated proteolysis in HT-1080 cells were less marked although statistically significant inhibition of proteolytic activity was achieved with 5p. M treatment (Fig 20B).

The effects of both Ox and MCT-1 on u-PA-mediated proteolysis in MDA-MB- 231 cells were profound with complete inhibition of proteolytic activity observed at 5 uM (Figure 20C). A partial inhibition of MMP-9 activity was achieved at 5 s1M with MCT-1 being the more effective of the two agents.

Treatment of PC-3 cells with either Ox or MCT-1 inhibited u-PA and MMP- mediated proteolysis (Figure 20D) to a statistically significant extent. Ox was more effective at inhibiting u-PA-mediated proteolysis whilst both Ox and MCT- 1 were equipotent at inhibiting MMP-mediated proteolytic activity.

The effects of both Ox and MCT-1 on the inhibition of u-PA and MMP mediated proteolysis were not attributable to direct inhibition of u-PA or MMP-mediated catalytic activity as incubation of recombinant u-PA, MMP-2 or MMP-9 with either Ox or MCT-1 at up to 100pM did not have any effect on u-PA or MMP- mediated proteolytic activity in zymography experiments (data not shown).

Example 18: Oxamflatin and MCT-1 inhibit invasion of HT-1080 cells without significant loss of cell viability or effect on cell growth.

Having determined that both Ox and MCT-1 were capable of inhibiting u-PA and MMP expression at low micro molar concentrations we were interested in assessing the anti-invasive effects of our agents in an in vitro model of cell invasion. Reduced expression of these proteases has been previously demonstrated to correlate with reduced cellular invasion and reduced in vivo cancer cell metastasis. HT-1080 cells were left untreated or treated with 5 uM Ox or MCT-1 for 16 hrs and invasion through matrigel was assessed over a 24 hr period. Both Ox and MCT-1 significantly reduced HT-1080 cell invasion by up to 80% compared to untreated cells. Cell viability was determined by trypan blue staining and revealed no significant reduction with either Ox or MCT-1 treatment over the 40 hr period (Figure 21A).

Given the profound inhibition of invasion after treatment with 5 uM concentrations of either drug, lower concentrations were tested. With concentrations as low as 0. 1 uM, significant inhibition of invasion was detected in all three cell lines without significant loss of viability or proliferative capacity.

MCT-1 was more effective than Ox in PC-3 and MDA-MB-231 cells (Figure 21 B and C).

Example 19: Anti-invasive/metastatic potential of Ox and MCT-1 using an orthotopic model of breast cancer Figure 22 shows the in vivo anti-invasive/metastatic potential of Ox and MCT-1 using an orthotopic model of breast cancer whereby mouse mammary cancer cells harbouring the neomycin gene (4T1. 2) are injected into the mammary fat pad of BALB C nude mice. The tumour subsequently disseminates and tumour burden and metastasis in treated and untreated animals were quantitated using real time PCR Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.