WO2005025557A1 - Agent traitant les etats associes a l'activation du plasminogene et a la metalloproteinase matricielle, et ses procedes d'utilisation - Google Patents

Agent traitant les etats associes a l'activation du plasminogene et a la metalloproteinase matricielle, et ses procedes d'utilisation Download PDF

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WO2005025557A1
WO2005025557A1 PCT/AU2004/001259 AU2004001259W WO2005025557A1 WO 2005025557 A1 WO2005025557 A1 WO 2005025557A1 AU 2004001259 W AU2004001259 W AU 2004001259W WO 2005025557 A1 WO2005025557 A1 WO 2005025557A1
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alkyl
substituted
oxamflatin
mmp
aryl
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PCT/AU2004/001259
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Anthony Edwin Dear
Robert Lindsay Medcalf
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Monash University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/61—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a ring other than a six-membered aromatic ring of the carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/01—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms
    • C07C311/02—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C311/08—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/15—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C311/21—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C333/00—Derivatives of thiocarbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C333/02—Monothiocarbamic acids; Derivatives thereof
    • C07C333/12—Monothiocarbamic acids; Derivatives thereof having nitrogen atoms of thiocarbamic groups bound to other hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/40—Nitrogen atoms, not forming part of a nitro radical, e.g. isatin semicarbazone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/32—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated

Definitions

  • the present invention relates to an agent for plasminogen-activation and matrix metalloproteinase associated conditions namely oxamflatin and derivatives thereof, as well as compositions containing oxamflatin and derivatives thereof for use as agents for plasminogen-activation and matrix metalloproteinase associated conditions.
  • the invention also includes methods of using oxamflatin and derivatives thereof for preventing and treating plasminogen activation and matrix metalloproteinase associated conditions, such as metastatic cancer spread, leukaemia, lymphoma, myeloma and associated haemoncological conditions.
  • 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.
  • MMP matrix metalloproteinase
  • ECM extracellular matrix
  • 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.
  • 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.
  • u-PA urokinase
  • 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
  • PAI-2 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.
  • u-PA bound to its cell surface receptor u-PAR is responsible for mitogenic and cell adhesion events also involved in the metastatic phenotype.
  • MMP Matrix Metalloproteinase
  • 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.
  • 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.
  • present methods of eradicating the primary tumour do not alleviate metastatic spread.
  • 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.
  • 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.
  • a method of modulating a plasminogen-activation/MMP associated condition involving a plasminogen activating/MMP system comprising administering an effective amount of oxamflatin or an oxamflatin derivative to a patient in need thereof.
  • a method of preventing and treating a plasminogen-activation/MMP associated condition involving a plasminogen activating/MMP system comprising administering an effective amount of oxamflatin or an oxamflatin derivative to a patient in need thereof.
  • plasminogen activation/MMP associated conditions such as, but not limited to extracellular matrix degradation, metastatic cancer spread, leukaemia, lymphoma, myeloma and associated haemoncological conditions.
  • a method of modulating extracellular matrix degradation comprising administering to a patient in need, an effective amount of oxamflatin or an oxamflatin derivative in conjunction with a cytotoxic agent.
  • compositions for use in the modulation of a plasminogen-activation/MMP associated condition 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.
  • oxamflatin derivatives that behave in a similar manner to oxamflatin, that may be more potent for inhibiting metastatic spread and cell mobility.
  • MCT-1 a compound having the following structure:
  • Figure 1 shows that Oxamflatin treatment induces PAI-2 mRNA expression in U-
  • the filter was hybridised with a labelled cDNA probe complementary to PAI-2 mRNA. The same filter was reprobed for ⁇ -Actin mRNA.
  • Figure 2 shows that Oxamflatin treatment potentiates PMA-mediated induction of endogenous PAI-2 mRNA expression in HT-1080 cells.
  • the filter was hybridised with a labelled cDNA probe complementary to PAI-2 mRNA.
  • the same filter was reprobed for ⁇ -Actin mRNA (Lane 1 ) Untreated cells
  • Figure 3 shows that Oxamflatin treatment inhibits u-PA mRNA expression in U- 937 and HT-1080 cells.
  • 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 ⁇ -Actin (Lane 1 ) Untreated cells.
  • 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 ⁇ -Actin. (Lane 1 ) Untreated
  • 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 5 ⁇ M oxamflatin, 25 nM PMA or a combination of 5 ⁇ M 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-1 a 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 1 ⁇ M 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-1 a 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
  • Lanes 10-18 use the PAI-2 CRE oligonucleotide as a probe.
  • Lane 10. Control + no antibody Lane.11.
  • Figure 6 shows that Oxamflatin treatment of HT-1080 cells is associated with reduced u-PA-mediated proteolytic activity in zymography studies.
  • Conditioned medium from HT-1080 cells untreated (0), or treated with 1 , 2.5, 5.0 or 7.5 ⁇ M 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 7.5 ⁇ M 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.
  • 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.
  • 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 ⁇ M 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 placental 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 5 ⁇ M 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 5 ⁇ M 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 5 ⁇ M 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.
  • 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 ⁇ M 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 ⁇ M Ox (B) or 0.1 ⁇ M 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.
  • a method of modulating a plasminogen-activation/MMP associated condition involving a plasminogen activating/MMP enzyme system comprising administering an effective amount of oxamflatin or an oxamflatin derivative to a patient in need thereof.
  • 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, lymphoma, myeloma and associated haemoncological conditions, and extracellular matrix degradation,.
  • a method of preventing and treating a plasminogen-activation/MMP associated condition involving a plasminogen activating/MMP system 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.
  • PAI-2 plasminogen activator inhibitor Type 2
  • modulation of the plasminogen activation/MMP associated condition may be via modulation of PAI-2.
  • plasminogen activation may be inhibited to prevent or treat a plasminogen activation/MMP associated condition when production of PAI-2/TIMPs is increased.
  • u-PA urokinase
  • ECM extracellular matrix
  • MMP matrix metalloproteinase
  • hydroxamic acid derivative oxamflatin previously noted to revert the malignant phenotype in K-ras transformed NIH-3T3 cells
  • 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.
  • 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.
  • modulation of the plasminogen activating/MMP system involves upregulating PAI-2/TIMPs gene expression and down regulating u-PA/MMP gene expression or activity.
  • modulating a plasminogen-activation/MMP associated condition may include inhibiting the condition or enhancing or stimulating the condition.
  • inhibiting a plasminogen activation/MMP associated condition may include inhibiting the processes involved in plasminogen/MMP activation.
  • Urokinase u-PA
  • u-PA Urokinase
  • Plasminogen activation inhibitor type 2 PAI-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.
  • 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:
  • 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.
  • an oxamflatin derivative having a hydroxamic acid and/or a sulphonamide held spatially apart by an unsaturated carbon skeleton.
  • the carbon skeleton contains a benzene ring the groups are orientated meta to each other.
  • 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.
  • 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:
  • 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:
  • oxamflatin derivatives may include those where the central benzene ring is replaced with an indole. Examples may include:
  • 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.
  • a method of modulating extracellular matrix degradation 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.
  • PAI-2 serine protease inhibitor
  • 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, lymphoma, 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.
  • a method of reducing malignant metastatic spread comprising administering to a patient in need, an effective amount of oxamflatin or an oxamflatin derivative.
  • DV., Virology 303:345-63 2002 may also be regulated by Oxamflatin and/or
  • Applicants have commenced a cDNA microarray analysis using a 10.8 K human array and both MCT-1 and Oxamflatin.
  • 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.
  • 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.
  • 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.
  • 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.
  • a method of preventing and treating metastatic spread 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.
  • 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, lymphoma 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 lymphoma cell. More preferably the cell is a U937 histiocytic lymphoma 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.
  • u-PA activity is also regulated by PAI-2.
  • 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.
  • ECM degradation may be prevented or reduced.
  • this invention 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, lymphoma, myeloma and associated haemoncological conditions. Leukaemia, lymphoma 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.
  • 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.
  • 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.
  • oxamflatin or derivatives thereof may prevent further metastatic spread and treat the spread that has already occurred.
  • identification of a primary tumour may be too late, in which case, some ECM degradation may have already occurred.
  • oxamflatin and its derivatives described above will be useful to prevent further metastatic spread and to treat the spread which has already occurred.
  • a method of preventing and treating vascular disease involving a plasminogen activating/MMP system comprising administering an effective amount of oxamflatin or an oxamflatin 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.
  • carrier molecules may include antibodies or biological compounds having cellular receptors such as cytokines targeted to their respective receptors.
  • 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.
  • 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.
  • 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.
  • a method of modulating extracellular matrix degradation comprising administering to a patient in need, an effective amount of oxamflatin or an oxamflatin derivative in conjunction with a cytotoxic agent.
  • a method of reducing malignant metastatic spread 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, 5Fluorouracil, etoposide, cyclophosphamide, and capecitabine.
  • a use of oxamflatin and oxamflatin derivatives in the preparation of a medicament for the modulation of a plasminogen-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.
  • compositions for use in the modulation of a plasminogen/MMP-activation associated condition comprising oxamflatin or an oxamflatin derivative and a carrier or diluent.
  • the plasminogen/MMP-activation associated condition is ECM degradation. More preferably, it is malignant metastatic spread.
  • the carrier or diluent may be a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • treatment by use of oxamflatin or derivative is preferably aimed at reducing or inhibiting metastatic spread.
  • a method of preparing a composition for use in the modulation of a plasminogen/MMP- activation associated condition 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.
  • the plasminogen/MMP-activation associated condition is ECM degradation. More preferably, the plasminogen/MMP-activation associated condition is metastatic spread.
  • the present invention provides a compound having a formula selected from the group consisting of
  • composition comprising a compound disclosed above.
  • 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.
  • 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 glutamine and 10% heat-inactivated foetal calf serum (HI-FCS, Gibco BRL, Australia).
  • DMEM Dulbecco's modified Eagles Medium
  • HI-FCS heat-inactivated foetal calf serum
  • 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.
  • RNA from HT-1080 cells was isolated by the method of Chomczynski and Sacchi (1987) Anal. Biochem, 162, 156-159. 10 ⁇ g 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 32 P-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.
  • Example 2 Oxamflatin treatment suppresses u-PA mRNA expression and PMA-mediated induction of u-PA mRNA.
  • Figure 3 Panel A lane 5 also suggests a degree of inhibition of PMA-mediated induction of u-PA mRNA by oxamflatin treatment.
  • 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).
  • 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.
  • ⁇ -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.
  • 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 ) Nucl.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.
  • CRE Cyclic AMP Response Element
  • AP-1 Activation Protein-1
  • 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 nd 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 ⁇ g/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 5 ⁇ M 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 ⁇ M or 5 ⁇ M), 25 nM PMA, or a combination of both agents.
  • Cytoplasmic extracts of transfected cells (usually between 10-20 ⁇ g in 40 ⁇ l of 250 mM Tris-HCI, pH 7.4) were incubated with 5 ⁇ l of 4.4 mM acetyl coenzyme A (Boehringer Mannheim) and 1 ⁇ l 14 C-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 14 C- chloramphenicol to its acetylated products was quantified by phosphorimaging using a Fujix BAS 1000 phosphorimager.
  • Example 4 The effects of oxamflatin treatment on the binding of nuclear proteins to the CRE and AP-1 a binding sites in the PAI-2 gene promoter.
  • 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. 2 nd 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).
  • oligonucleotides used for this study were: 5'-GATTCAATGACTCACGGCTGTG-3'. (AP-1 a 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-1 a 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).
  • HT-1080 cells were treated with oxamflatin (1 ⁇ M) 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.
  • PMA treatment used as a positive control in this experiment.
  • 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.
  • 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. 5x10 3 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 ⁇ l of supernatant samples were run through an SDS-PAGE gel and zymographic analysis undertaken.
  • 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
  • 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 (O ⁇ M), 1 ⁇ M and 5 ⁇ M oxamflatin were tested and proteolytic activity measured.
  • Figure 9 shows reduced u-PA-mediated proteolytic activity in conditioned medium when treated with oxamflatin.
  • 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 (O ⁇ M), 1 ⁇ M and 5 ⁇ M oxamflatin were tested and proteolytic activity measured. Figure 10 shows reduced u-PA-mediated proteolytic activity in conditioned medium when treated with oxamflatin.
  • 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.
  • Example 11 Effect of oxamflatin and MCT-1 on growth, viability and invasion of PC3, HT1080 and MDA-MB21 cells.
  • the Invasion assay was carried out as follows: a) Matrigel Invasion Assay
  • HT1080 cells (source) were suspended to a concentration of 2.5X10 6 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 ⁇ l distributed to pre-chilled cell culture inserts with 8.0 ⁇ m pores (Becton Dickenson). Coated inserts were allowed to gel for 30 minutes at 37°. They were then placed in companion wells with 600 ⁇ l SFM containing the correct concentrations of drug, and were overlayed with 100 ⁇ l of the same medium.
  • 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.
  • Example 13 Oxamflatin and MCT-1 inhibit histone deactylase activity.
  • 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
  • primary human placental fibroblasts were cultured to 70% confluence in Dulbecco's modified Eagles Medium (DMEM) supplemented with 2 mM glutamine, 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) CO 2 and 95% (w/v) air mixture.
  • DMEM Dulbecco's modified Eagles Medium
  • FCS heat-inactivated foetal calf serum
  • CM complete medium
  • 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.
  • DMSO dimethyl sulfoxide
  • the compounds were added to serum free (SF) DMEM medium to give final concentrations ranging between 0.1-100 ⁇ M. The final DMSO concentration did not exceed 0.1% and DMSO was added to control samples at the same final concentration.
  • 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.
  • HT-1080, MDA-MB-231 , and PC-3 cells were suspended at a concentration of 2.5x10 6 /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 ⁇ l distributed to pre-chilled cell culture inserts with 8.0 ⁇ M 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 ⁇ l SFM with various concentrations of either Ox or MCT-1 and overlaid with 100 ⁇ l of the same medium.
  • 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.
  • Ox and MCT-1 regulate expression of components of the plasminogen activating system in HT-1080, MDA-MB-231 and PC-3 cells.
  • 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).
  • Example 16 Oxamflatin and MCT-1 inhibit MMP-2 and MMP-9 expression in HT-1080 and MDA-MB-231 cells.
  • 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 5 ⁇ M 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 5 ⁇ M treatment (Fig 20B).
  • 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

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Abstract

L'invention porte sur des procédés et des composés de traitement d'états tels que la diffusion des métastases cancéreuses et l'athérosclérose utilisant l'oxamflatine et ses dérivés. On a en effet découvert que l'oxamflatine et ses dérivés peuvent moduler le système 'activation du plasminogène/ métalloprotéinase matricielle' et permettent par-là de réguler la dégradation des protéines matricielles extracellulaires.
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US9890172B2 (en) 2011-04-28 2018-02-13 The Broad Institute, Inc. Inhibitors of histone deacetylase
US10662199B2 (en) 2011-04-28 2020-05-26 The Broad Institute, Inc. Inhibitors of histone deacetylase
US11572368B2 (en) 2011-04-28 2023-02-07 The General Hospital Corporation Inhibitors of histone deacetylase
US11377423B2 (en) 2012-07-27 2022-07-05 The Broad Institute, Inc. Inhibitors of histone deacetylase
US9914717B2 (en) * 2012-12-20 2018-03-13 The Broad Institute, Inc. Cycloalkenyl hydroxamic acid derivatives and their use as histone deacetylase inhibitors
US10793538B2 (en) 2012-12-20 2020-10-06 The Broad Institute, Inc. Cycloalkenyl hydroxamic acid derivatives and their use as histone deacetylase inhibitors
US9745253B2 (en) 2015-03-13 2017-08-29 Forma Therapeutics, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors
US10266487B2 (en) 2015-03-13 2019-04-23 Forma Therapeutics, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors
US10508077B2 (en) 2015-03-13 2019-12-17 Forma Therapeutics, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors
US10988441B2 (en) 2015-03-13 2021-04-27 Valo Early Discovery, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors
US11919839B2 (en) 2015-03-13 2024-03-05 Valo Health, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors
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