WO1998020897A1 - Modulation de map kinase phosphatase 1 (mkp-1) - Google Patents

Modulation de map kinase phosphatase 1 (mkp-1) Download PDF

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WO1998020897A1
WO1998020897A1 PCT/US1997/020744 US9720744W WO9820897A1 WO 1998020897 A1 WO1998020897 A1 WO 1998020897A1 US 9720744 W US9720744 W US 9720744W WO 9820897 A1 WO9820897 A1 WO 9820897A1
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mkp
cells
migration
smooth muscle
test compound
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Edgar Haber
Kaihua Lai
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President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to the treatment of vascular conditions .
  • Cellular adhesion and migration characterize many physiological and pathological processes, including embryogenesis, tissue morphogenesis, metastasis and vascular occlusion.
  • Huttenlocher et al . Curr. Opin . Cell . Biol . 7:697-706 (1995); Lauffenburger et al . , Cell 84:359-369 (1996).
  • Vascular conditions such as post- angioplasty restenosis, transplant arteriopathy, graft stenosis following coronary bypass surgery and chronic atherosclerosis are all characterized by the adhesion and migration of smooth muscle cells (SMC) .
  • SMC smooth muscle cells
  • MAP kinase phosphatase 1, MKP-1 has been shown to inhibit the proliferation of SMC in vi tro .
  • MKP-1 was originally identified as an immediate early gene in rat fibroblasts subjected to oxidative stress.
  • Overexpression of MKP-1 has also been shown to inhibit the induction of the mesodermal bud, a process characterized by extensive cellular migration, in the Xenopus embryo. Gotoh et al .
  • MKP-1 is a member of the family of dual specificity tyrosine phosphatases, nuclear proteins which dephosphorylate both phosphotyrosine and phosphothreonine residues on target proteins. Substrates of MKP-1 and other dual specificity tyrosine phosphatases are limited to proteins with adjacent phosphorylated tyrosine and threonine residues. Such proteins include CDC2 , CDK4 and the MAP kinases, all of which are involved in regulating expression of genes involved in cell growth.
  • MKP-1 expression can be influenced by Ca 2+ , angiotensin II, epidermal growth factor (EGF) , platelet derived growth factor (PDGF) and H 2 0 2 . Duff et al . , J “ . Biol . Chem . 268:26037-26040 (1995).
  • MKP-1 homologues have been identified in mouse (3CH134) and human (CL100) .
  • CL100 and 3CH134 are nearly identical (Ishibashi et al . , J “ . Biol . Chem . 269:29897- 29902 (1994) ) , and both dephosphorylate phosphotyrosine, phosphothreonine and phosphoserine on artificial substrates.
  • Oncogene 8:2015-2020 (1993) Charles et al .
  • MKP-1 The rat, mouse and human gene products are collectively referred to as MKP-1, because of their specificity for MAP kinases.
  • the invention is based on the discovery that SMC, e.g., vascular smooth muscle cells, that differ in the level of MKP-1 expression exhibit altered cellular adhesion and migration.
  • Administration of agents which alter MKP-1 expression or activity can therefore be used to treat pathological conditions characterized by SMC adhesion and migration.
  • the invention features a method of inhibiting the migration of vascular smooth muscle cells.
  • the method includes the steps of: identifying a vertebrate suspected of having a condition characterized by the migration of vascular smooth muscle cells; providing an agent that increases expression or activity of MKP-1 in vascular smooth muscle cells; and administering to the vertebrate an amount of the agent sufficient to inhibit migration of the cells.
  • the agent preferably inhibits migration by at least 20%, preferably 50%, more preferably 75% or 85%, and most preferably 95%.
  • the agent is a nucleic acid sequence encoding MKP-1.
  • a naturally occurring nucleic acid sequence may be either genomic DNA or a RNA transcript, such as mRNA.
  • the vertebrate is preferably a mammal, such as a human, a non-human primate, or a dog, cat, cow, pig, horse, sheep, goat, rat, mouse, guinea pig, hamster or rabbit.
  • the condition being treated can be, for example, post-angioplasty restenosis, transplant arteriopathy, graft stenosis, or chronic atherosclerosis.
  • the invention also includes a screening assay for the detection of agents which increase MKP-1 expression or activity.
  • the assay includes the steps of: providing a cell-free preparation of MKP-1, contacting the MKP-1 with a test compound; detecting MKP-1 activity; determining whether the test compound increases MKP-1 activity, wherein an increase in MKP-1 activity in the presence of the test compound indicates that the test compound is potentially useful for inhibiting migration of vascular smooth muscle cells.
  • the test compound inhibits MKP-1 activity by at least 20%, preferably 50%, more preferably 75% or 85%, and most preferably 95%.
  • MKP-1 activity can be detected using a substrate of MKP- l's phosphatase activity.
  • the invention includes a method for identifying a compound which inhibits migration of vascular smooth muscle cells.
  • the method comprises the steps of : providing a cell which expresses MKP-1; contacting the cell with a test compound; detecting MKP-1 expression in the cell; and determining whether the level of MKP-1 expression is increased in the cell in the presence of the test compound, wherein an increase in MKP-1 activity in the presence of the test compound indicates that the test compound is potentially useful for inhibiting migration of vascular smooth muscle cells.
  • the compound inhibits expression by at least 20%, preferably 50%, more preferably 75% or 85%, and most preferably 95%.
  • MKP-1 expression can be detected using an antibody-based assay or a nucleic acid hybridization assay.
  • Fig. 1 is a bar graph showing the number of MKP-1 cells and control smooth muscle cells (SMC) adhering to collagen in an adhesion assay.
  • Fig. 2 is a series of sequential photographs showing cell spreading in individual MKP-1 cells and control SMC in response to collagen.
  • Fig. 3 is a bar graph showing the percentage of MKP-1 cells and control SMC spreading in response to collagen.
  • Fig. 4 is a bar graph showing the number of MKP-1 cells and control SMC migrating in response to collagen, in the presence and absence of platelet derived growth factor (PDGF) .
  • Fig. 5 is a bar graph showing the number of 3T3/MKP-1 cells and control 3T3 cells migrating in response to collagen, in the presence and absence of PDGF.
  • PDGF platelet derived growth factor
  • Fig. 6 is a bar graph showing the effect of antibodies specific for ⁇ 2 l or o;3 / 8l integrins on the migration of MKP-1 cells and control SMC in response to collagen.
  • Fig. 7 is a bar graph showing the number of MKP-1 cells, C258S/MKP-1 cells, and control SMC migrating in response to PDGF.
  • Fig. 8A-8E are Western blots probed with antibodies specific for tyrosine phosphorylated focal adhesion kinase (FAK) (A) ; FAK (B) ; paxillin (C) ; vinculin (D) ; and ⁇ -actin (E) .
  • Fig. 9 is a series of photographs showing focal adhesion complexes in individual MKP-1 cells, C258S/MKP-1 cells and control SMC.
  • Fig. 10 is a series of photographs showing actin structure in individual MKP-1 cells, C258S/MKP-1 cells and control SMC.
  • the invention is based on the finding that MKP-1 regulates cellular adhesion and migration.
  • Overexpression of MKP-1 in smooth muscle cells (SMC) or NIH 3T3 cells results in increased cellular adhesion and migration in the presence of type I collagen.
  • overexpression of MKP-1 in SMC in the absence of type I collagen results in decreased migration of SMC towards platelet derived growth factor (PDGF) .
  • PDGF platelet derived growth factor
  • Administration of agents which alter MKP-1 expression or function can therefore be used to treat pathological conditions characterized by unwanted or aberrant SMC adhesion and migration.
  • Conditions associated with unwanted SMC adhesion and migration for which increased levels of MKP-1 expression or function is desired include post-angioplasty restenosis, transplant arteriopathy, graft stenosis following coronary artery bypass surgery, and chronic atherosclerosis.
  • SMC were transfected with vector pcDNA3 (Invitrogen) (control SMCs) , or with pcDNA3/MKP-l (MKP-1 cells) as described. Lai et al . , J “ . Clin . Invest . 98 : 1560 (1996) . C258S/MKP-1 cells have been transfected with a dominant negative mutant MKP-1 gene C258S, in which the catalytic residue cysteine 258 has been replaced with a serine in the plasmid pcDNA3/MKP-l (Lai et al., J " . Clin . Invest . 98 : 1560-1567 (1996)). Transfections were performed by the Lipofectin (Life Technologies) method according to the manufacturer's instructions .
  • Adhesion Assay Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 0.4% fetal calf serum and 300 ⁇ g/ml G418 for 24 hours. Trypsin-EDTA was added to the cultures, which were then incubated at 37°C for three minutes. The detached cells were washed once in DMEM containing 10% fetal calf serum and three times in phosphate buffered saline (PBS) . After the final wash, the cells were resuspended in DMEM supplemented with 10% fetal calf serum and 300 ⁇ g/ml G418.
  • DMEM Dulbecco's Modified Eagle's Medium
  • PBS phosphate buffered saline
  • the cells were then plated in 6-well tissue culture plates coated with 10 ⁇ g/cm 2 type I collagen for 24 hours.
  • the number of adherent cells was calculated as the initial total plating number (100,000 cells) minus the number of nonadherent cells.
  • Nonadherent cells in the culture medium were removed and combined with nonadherent cells which came off in three PBS washes of the adherent cells, and the number of nonadherent cells was counted on a Coulter counter. Similar results were obtained in two experiments .
  • spreading cells were defined as those cells that were not light reflective, while non-spreading cells were defined as those cells that were light reflective.
  • Each cell in the field was counted, and the percentages of spreading and non-spreading cells were calculated by counting 150-200 cells. The standard deviations were derived from three separate experiments.
  • Migration Assay A migration assay using a modified Boyden chamber was performed as described previously. Bornfeldt et al . , J “ . Clin . Invest . 93:1266-1274 (1994). In order to generate a population of quiescent cells, SMC were incubated in DMEM supplemented with 0.4% calf serum and 300 ⁇ g/ml G418 for 48 hours. The bottom side of a polycarbonate filter (pore size 8 ⁇ m) was coated with 100 ⁇ g/ml type I collagen (Vitrogen 100) 24 hours before use. The bottom of the Boyden Chamber contained DMEM with various concentrations of PDGF, and the top contained
  • the assay was carried out at 37°C in a tissue culture incubator. After four hours, cells adhering to the lower surface of the membrane filter of the Boyden chamber were fixed and stained. Cells which had not migrated were removed by mechanical scraping of the upper chamber. The filter containing the fixed and stained cells (migrated cells) were mounted on glass slides. Cell migration through the polycarbonate filter was assessed by counting the cells at a magnification of 200x.
  • the cells were treated with antibodies specific for either o ⁇ 2 ⁇ l ox ⁇ 3j-?l integrin (Dako Corp.) prior to being loaded into the Boyden chamber.
  • the cells were incubated for twenty minutes at room temperature with the antibodies, which had been diluted 1:50.
  • NIH 3T3 cells were stably transfected with pcDNA vector alone (3T3 control cells) or pcDNA3 with full length human MKP-1 (3T3/MKP-1 cells) . These vectors carry the gene for neomycin resistance. Cells which had taken up the vectors were selected in medium containing G418 (500 ⁇ g/ml) and single cells were cloned. Total RNA was extracted from quiescent cells grown from specific clones, and subjected to electrophoresis and Northern blotting.
  • Quiescent cells were obtained by culturing the cells for 48 hours in quiescent medium (DMEM, 0.4% calf serum, 100 units/ml of penicillin, 100 ⁇ g/ml of streptomycin, 1% L-glutamine) using RNZol B Plus (Tel- Test Inc., TX) . Blots were probed with 32 P-labeled random-primed human MKP-1 probe.
  • quiescent medium DMEM, 0.4% calf serum, 100 units/ml of penicillin, 100 ⁇ g/ml of streptomycin, 1% L-glutamine
  • Confluent cells (control SMCs and MKP-1 cells) were cultured in DMEM supplemented with 0.4% fetal calf serum and 300 ⁇ g/ml G418 for 24 hours. Cells were washed with PBS containing 1 mM sodium vanadate three times, lysed with RIPA buffer at 4°C for 10 min, and the cell lysate was passed through a 22 gauge needle five times. Debris was removed by centrifugation at 12,000 x g for 12 minutes. Supernatants were used for immunoprecipitation and Western blotting.
  • Tyrosine phosphorylated FAK was immunoprecipitated from supernatants (500 ⁇ g protein) using agarose beads conjugated with anti-phosphotyrosine antibody 4G10 (UBI) , and detected using anti-FAK antibodies (Santa Cruz) on ECL (Enhanced
  • Cells with polar focal adhesion complexes were defined as cells having more than 25% of their cell surface edges without focal adhesion complexes, i.e., the focal adhesion complexes were concentrated in certain areas of the cell surface edges.
  • Cells with non-polar focal adhesion complexes were defined as cells having more than 75% of their cell surface edges with focal adhesion complexes .
  • Cells were cultured and permeabilized as above, then stained with 0.25 ⁇ g/ml phalloidin-TRITC (Sigma) to detect actin. Photographs were taken under a fluorescence microscope at a magnification of 200x. Cells with polar actin filaments were defined as cells that had longitudinal actin filaments from one protrusion to another.
  • MKP-1 human MKP-1 was overexpressed in rat pulmonary arterial smooth muscle cells (SMC) .
  • SMC rat pulmonary arterial smooth muscle cells
  • the expression of MKP-1 in transfected SMCs was assessed by Northern blot hybridization analysis. Smooth muscle cells transfected with wild type MKP-1 or C258S had 4-fold (MKP-1) and 2.5-fold (C2583/MKP-1) higher expression than the non-transfected control cells.
  • MKP-1 cells SMC overexpressing MKP-1 (i.e., "MKP-1 cells") exhibited enhanced adhesion to type I collagen coated on tissue culture plates, compared to control SMC. As shown in Fig. 1, thirty minutes after plating, the number of MKP-1 cells adhering to type I collagen was more than twice the number of control SMC adhering to collagen.
  • FIG. 2 shows cells photographed under a phase-contrast microscope at lOOx magnification 30, 60, 120 and 240 minutes after plating on collagen-coated tissue culture plates. Most of the MKP-1 cells were not light reflective 30 minutes after plating, indicating that most of the cells were spreading. In contrast, most of the control SMC were light reflective at this time, indicating that they were not spreading. As shown in
  • the MKP-1 cells reached a flat epithelial-like morphology by 120 minutes after plating.
  • the control SMC took longer to reach this stage, with most of the cells exhibiting a flat morphology 240 min after plating. (Fig. 2) . No significant difference in spreading was observed between MKP-1 cells and control SMC when the cells were plated on fibronectin coated dishes.
  • haptotaxis was measured in MKP-1 cells and control SMC in a modified Boyden chamber assay. Bornfeldt et al . , J “ . Clin . Invest . 93:1266-1274 (1994).
  • the top chamber contained the cells
  • the bottom chamber contained DMEM with 0-10 ng/ml of the chemoattractant, Platelet Derived Growth Factor (PDGF) .
  • PDGF Platelet Derived Growth Factor
  • a filter separating the two chambers was coated on the bottom side with type I collagen.
  • MKP-1 cells showed a marked increase over control SMC in directional migration toward immobilized type I collagen in the lower Boyden chamber, in the presence or absence of PDGF.
  • NIH 3T3 fibroblasts were transfected with DNA encoding an intact MKP-1 gene.
  • the resulting cell line, 3T3/MKP-1 overexpresses MKP-1, as demonstrated by Northern blotting and hybridization to a MKP-1 probe.
  • the 3T3/MKP-1 cell line was tested in the Boyden chamber assay described above. As shown in Fig. 5, 3T3/MKP-1 cells, like MKP-1 cells, demonstrated increased migration toward collagen, compared to control cells.
  • the enhanced migration observed in MKP-1 cells may be due to the interaction of type I collagen with integrins, which are receptors for collagen, on the surface of the cells.
  • the extracellular matrix (ECM) facilitates cellular adhesion and migration through interactions with cell surface receptors such as the integrins.
  • the type I collagen signal can be mediated by the integrins o ⁇ 2 ⁇ l (the collagen receptor) and o;3 ⁇ 1 (the ECM II receptor) .
  • Yoshinaga et al . Melanoma Res . 3:435- 441 (1993); Keely et al . , J. Cell Sci . (1995). Therefore, the role of these integrins in the migration of MKP-1 cells in response to collagen was analyzed.
  • MKP-1 cells and control SMC were preincubated with antibodies specific for the ⁇ 2 l or cx3 ⁇ l integrins as described in Materials and Methods, and then tested in the Boyden chamber assay. The number of migrating cells was then calculated by counting cells in a 3x High Power Field (3x HPF) . As shown in Fig. 6, antibodies specific for either cx2 ⁇ l or c ⁇ 3 ⁇ l inhibited migration of MKP-1 cells by approximately 50% (p ⁇ 0.01), while incubation with antibodies specific for the vitronectin receptor (VNR) had no inhibitory effect on migration. These results indicate that the c.2 l and o ⁇ 3 ⁇ l integrins are involved in migration of MKP-1 cells in response to collagen.
  • 3x HPF 3x High Power Field
  • MKP-1 is involved in both integrin-mediated and PDGF-mediated cellular migration.
  • FAK focal adhesion kinase
  • FIGs. 8A and 8B show the results of experiments in which cells were immunoprecipitated using either anti- phosphotyrosine (c-PY) or anti-FAK ( ⁇ -FAK) antibodies, and Western blotted using anti-FAK antibodies.
  • c-PY anti- phosphotyrosine
  • ⁇ -FAK anti-FAK
  • control SMC and C258S/MKP-1 cells contained considerable amounts of tyrosine phosphorylated FAK
  • MKP-1 cells contained only about 20% of the amount found in control SMC.
  • C258S/MKP-1 cells contained 140% of the amount of tyrosine phosphorylated FAK found in control SMC.
  • Similar results were obtained when anti-FAK antibodies were used for immunoprecipitation and anti- phosphotyrosine antibodies were used on Western blots.
  • the amounts of the cytoskeletal proteins paxillin, vinculin and ⁇ -actin were similar in all of the cell types tested (Figs.
  • a decrease in the amount of tyrosine phosphorylated FAK in MKP-1 cells can therefore be correlated with a decrease in migration of MKP-1 cells, and an increase in migration of C258S/MKP-1 cells, in response to PDGF.
  • a reduction in FAK tyrosine phosphorylation contributes to the alteration of migration in MKP-1 cells.
  • Cell migration has a biphasic relationship with adhesion force; adhesion force at a medial range is optimal for maximal migration speed. Lauffenburger et al . , Cell 84:359-369 (1996); Mitchison et al . , Cell 84:371-379 (1996); DiMilla et al .
  • focal adhesion complexes were examined by immunostaining of the focal adhesion molecule vinculin. As shown in Fig. 9, the focal adhesion complexes in most of the control SMC had a polar distribution (94% of the cells examined) , primarily localized at the edges of the cells. In contrast, the focal adhesion complexes in MKP-1 cells appeared to be stained less intensely and distributed evenly over the edges of the cells (90% of the cells examined) . The focal adhesion complexes in C258S/MKP-1 cells were similar to those observed in control SMC. Similar results were obtained on immunostaining of paxillin, another focal adhesion component.
  • control SMC exhibited a polar distribution of actin filaments.
  • actin filaments were organized in a nonpolar, round to polygonal fashion (95% of the cells examined) .
  • the actin organization observed in C258S/MKP-1 cells was similar to that seen in the control SMC.
  • MKP-1 overexpression may exert its effects by inhibiting MAP kinase activity, thus altering the expression of a tyrosine kinase or phosphatase which regulates the tyrosine phosphorylation of FAK.
  • the alteration of tyrosine phosphorylation of FAK and the altered organization of focal adhesion complexes and actin filaments induced by MKP-1 overexpression may be responsible for changes in SMC adhesion and migration.
  • Screening assays can be used to identify agents which modulates, i.e., increase or decrease the expression or function of MKP-1.
  • a cell-free preparation of MKP-1 is contacted with a test compound.
  • MKP-1 activity is then detected in the preparation, for example, by using a substrate of MKP-l's phosphatase activity.
  • An alteration in MKP-1 activity in the presence of the test compound indicates that the test compound is potentially useful for modulating migration of vascular smooth muscle cells.
  • Cell -based assays can also be used to screen for compounds which modulate migration of vascular smooth muscle cells.
  • a MKP-1-expressing cell is contacted with a test compound.
  • MKP-1 expression is detected in the cell, using, for example, an antibody- based or nucleic acid hybridization assay.
  • An alteration in MKP-1 activity in the presence of the test compound indicates that the test compound is potentially useful for modulating migration of vascular smooth muscle cells.
  • Known inhibitors of MKP-1 can be administered to treat conditions characterized by cellular migration.
  • Other agents which can be used to inhibit MKP-1 function or expression include antibodies specific for MKP-1, ribozymes specific for MKP-1 RNA, and antisense RNA molecules or oligonucleotides which hybridize specifically to the MKP- 1 gene or mRNA.
  • the antibodies used can be polyclonal or monoclonal.
  • Antibody fragments, such as Fab or F(ab) 2 fragments, can also be used. The antibody is preferably humanized.
  • the antisense molecules can be either DNA or RNA. These molecules can be directly introduced into the cells. Alternatively, they can be administered via an expression vector which enters the target smooth muscle cells and expresses the antisense molecules.
  • Ribozymes are RNA molecules that bind to RNA molecules and cleave them. Ribozymes can be used to inhibit MKP-1 expression in order to treat a condition characterized by cellular adhesion and migration.
  • a patient with such a condition is first identified.
  • Conditions characterized by cellular migration include post-angioplasty restenosis, transplant arteriopathy, graft stenosis and chronic atherosclerosis.
  • the inhibitor can be administered as a therapeutic composition including one or more compounds and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline. A therapeutically effective amount of the composition is administered to the patient.
  • a therapeutically effective amount is an amount which is capable of producing a medically desirable result in a treated animal, e.g., inhibition of expression of the target gene, or inhibition of MKP-1 enzymatic activity to the extent necessary to inhibit the target cellular activity, such as adhesion or migration.
  • Parenteral administration such as intravenous, intrapulmonary, intranasal, subcutaneous, intramuscular, and intraperitoneal delivery routes may be used to deliver the compound, with intravenous administration being the preferred route. Dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • Example III Screening Assays for Agents that Increase MKP-1 Activity
  • SMC such as vascular SMC or other cells in which the endogenous MKP-1 gene is present (but the expression of which is down-regulated) can be used as a screening tool to identify compounds or treatment strategies which induce an increase in expression of the MKP-1 gene.
  • Other cell types with intact but unexpressed MKP-1 genes would also be potentially useful in this screening assay.
  • the cells are contacted in vi tro with the candidate compounds, and the amount of MKP-1 expression determined using known methods, e.g., a hybridization assay (e.g., Northern analysis) or an immunoassay to detect MKP-1 polypeptides .
  • a compound effective both in stimulating MKP-1 expression and in preventing SMC migration and adhesion is useful for the treatment of conditions characterized by unwanted SMC migration and adhesion.
  • Example 4 Treatment with Agents that Increase MKP-1 Expression
  • increasing the level of MKP-1 expression in a SMC cell correlates with a decrease in its ability to migrate toward PDGF, a chemoattractant.
  • a useful treatment protocol will therefore be a method such as intravenous injection of the protein in a pharmaceutically acceptable solution in a dosage of 0.001 to 100 mg/kg/day, with the most beneficial range to be determined using routine pharmacological methods.
  • MKP-1 protein or nucleic acid encoding MKP-1 could be incorporated into liposomes or another form of carrier which permits substantial amounts of the protein to pass through the cell membrane. Liposomes would also help protect the protein from proteolytic degradation while in the bloodstream.
  • An expression vector encoding MKP-1 can be introduced into SMC, thereby increasing the production of MKP-1 in the transfected cells, and decreasing the abilities of these cells to migrate.
  • the transfected cells are also shown above to have decreased migrating ability compared to control cells. This evidence indicates that the MKP-1 DNA will be useful for genetic therapy to help control conditions characterized by unwanted migration of SMC. Standard methods of gene therapy may be employed: e.g., as described in Friedmann, Therapy for Genetic Disease, T. Friedman (ed.), Oxford Univ. Press, 1991, pp.105-121.
  • Virus or plasmids containing a copy of the MKD-1 cDNA linked to expression control sequences which permit expression in the target cell would be introduced into the patient, either locally at the site of unwanted SMC migration or systemically. If the transfected DNA encoding MKP-1 is not stably incorporated into the genome of each of the targeted cells, the treatment may have to be repeated periodically.

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Abstract

La présente invention concerne un procédé de modulation de migration de cellules vasculaires de muscles lisses chez un vertébré. Le procédé comporte les étapes consistant à identifier un vertébré soupçonné de présenter un état pathologique caractérisé par la migration de cellules vasculaires d'un muscle lisse, à produire un agent augmentant l'expression de MKP-1 dans les cellules vasculaires de muscles lisses et à administrer une dose d'agent suffisante pour inhiber la migration.
PCT/US1997/020744 1996-11-15 1997-11-14 Modulation de map kinase phosphatase 1 (mkp-1) WO1998020897A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP52282298A JP2001505876A (ja) 1996-11-15 1997-11-14 Ｍａｐキナーゼホスファターゼ１（ｍｐｋ―１）の調節
AU52563/98A AU5256398A (en) 1996-11-15 1997-11-14 Modulation of map kinase phosphatase 1 (mkp-1)

Applications Claiming Priority (2)

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US3092796P 1996-11-15 1996-11-15
US60/030,927 1996-11-15

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WO1998020897A1 true WO1998020897A1 (fr) 1998-05-22

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AU (1) AU5256398A (fr)
WO (1) WO1998020897A1 (fr)

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WO2002062979A2 (fr) * 2001-02-06 2002-08-15 Oxford Biomedica (Uk) Limited Enzyme
EP1274862A1 (fr) * 2000-03-31 2003-01-15 The Regents Of The University Of California Regulation d'un gene induit par des lipides oxydes dans des cellules humaines de paroi arterielle
WO2003003977A2 (fr) * 2001-07-05 2003-01-16 Millennium Pharmaceuticals, Inc. Compositions et procedes de traitement des troubles du poids corporel, y compris l'obesite
WO2005012348A2 (fr) * 2003-08-01 2005-02-10 Adherex Technologies Inc. Composes et procedes pour la modulation de l'adhesion cellulaire
US7250304B2 (en) 2000-03-31 2007-07-31 The Regents Of The University Of California Functional assay of high-density lipoprotein

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* Cited by examiner, † Cited by third party
Title
JONES ET AL: "LIGAND OCCUPANCY OF THE ALPHAVBETA3 INTEGRIN IS NECESSARY FOR SMOOTH MUSCLE CELLS TO MIGRATE IN RESPONSE TO INSULIN-LIKE GROWTH FACTOR I", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES,USA, vol. 93, March 1996 (1996-03-01), pages 2482 - 2487, XP002058895 *
LAI ET AL: "MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE-1 IN RAT ARTERIAL SMOOTH MUSCLE CELL PROLIFERATION", JOURNAL OF CLINICAL INVESTIGATION, vol. 98, no. 7, October 1996 (1996-10-01), pages 1560 - 1567, XP002058893 *
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LAI ET AL: "POTENTIAL ROLES OF TYROSINE PHOSPHATASE MKP-1 IN THE PROLIFERATION OF RAT VASCULAR SMOOTH MUSCLE CELLS", FASEB JOURNAL, vol. 10, no. 6, 30 April 1996 (1996-04-30), pages A1139, XP002058892 *
SKINNER ET AL: "DYNAMIC EXPRESSION OF ALPHA1BETA1 AND ALPHA2BETA1 INTEGRIN RECEPTORS BY HUMAN VASCULAR SMOOTH MUSCLE CELLS", AMERICAN JOURNAL OF PATHOLOGY, vol. 145, 1994, pages 1070 - 1081, XP002058894 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7122623B2 (en) 1996-07-12 2006-10-17 Adherex Technologies, Inc. Compounds and methods for modulating cell adhesion
EP1274862A1 (fr) * 2000-03-31 2003-01-15 The Regents Of The University Of California Regulation d'un gene induit par des lipides oxydes dans des cellules humaines de paroi arterielle
EP1274862A4 (fr) * 2000-03-31 2005-06-22 Univ California Regulation d'un gene induit par des lipides oxydes dans des cellules humaines de paroi arterielle
US7250304B2 (en) 2000-03-31 2007-07-31 The Regents Of The University Of California Functional assay of high-density lipoprotein
WO2002062979A2 (fr) * 2001-02-06 2002-08-15 Oxford Biomedica (Uk) Limited Enzyme
WO2002062979A3 (fr) * 2001-02-06 2002-11-28 Oxford Biomedica Ltd Enzyme
WO2003003977A2 (fr) * 2001-07-05 2003-01-16 Millennium Pharmaceuticals, Inc. Compositions et procedes de traitement des troubles du poids corporel, y compris l'obesite
WO2003003977A3 (fr) * 2001-07-05 2003-11-27 Millennium Pharm Inc Compositions et procedes de traitement des troubles du poids corporel, y compris l'obesite
WO2005012348A2 (fr) * 2003-08-01 2005-02-10 Adherex Technologies Inc. Composes et procedes pour la modulation de l'adhesion cellulaire
WO2005012348A3 (fr) * 2003-08-01 2005-04-28 Adherex Technologies Inc Composes et procedes pour la modulation de l'adhesion cellulaire

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JP2001505876A (ja) 2001-05-08
AU5256398A (en) 1998-06-03

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