WO2005030933A2 - Proteine morphogenetique osseuse 7 (bmp-7) utilisee dans le diagnostic et le traitement du cancer - Google Patents

Proteine morphogenetique osseuse 7 (bmp-7) utilisee dans le diagnostic et le traitement du cancer Download PDF

Info

Publication number
WO2005030933A2
WO2005030933A2 PCT/US2004/031468 US2004031468W WO2005030933A2 WO 2005030933 A2 WO2005030933 A2 WO 2005030933A2 US 2004031468 W US2004031468 W US 2004031468W WO 2005030933 A2 WO2005030933 A2 WO 2005030933A2
Authority
WO
WIPO (PCT)
Prior art keywords
bmp
cells
cancer
individual
expression
Prior art date
Application number
PCT/US2004/031468
Other languages
English (en)
Other versions
WO2005030933A3 (fr
Inventor
Marco G. Cecchini
Gabriel Van Der Pluijm
Slobodan Vukicevic
Clemens W. G. M. Lowik
Original Assignee
Genera Doo
Leiden University Medical Center
Universitat Bern
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genera Doo, Leiden University Medical Center, Universitat Bern filed Critical Genera Doo
Publication of WO2005030933A2 publication Critical patent/WO2005030933A2/fr
Publication of WO2005030933A3 publication Critical patent/WO2005030933A3/fr

Links

Classifications

    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • BMP-7 Bone Morphogenetic Protein
  • This invention relates generally to the field of cancer.
  • the invention relates to the activity of bone morphogenetic protein-7 (BMP-7) for inhibiting or reversing the progression of a cancer cell to a more malignant phenotype.
  • BMP-7 bone morphogenetic protein-7
  • Cancer is a disease caused by multiple mutations in genes of somatic cells at some location in the body such that there is a loss of the normal orderly control over growth, multiplication, and differentiation of the cells.
  • a cell in which there is a breakdown of normal control over cell growth typically multiplies and forms a mass of clonal cells referred to as a tumor.
  • Some tumors may remain localized and contained in a tissue or organ, e.g., warts, and do not pose much, if any, of a risk to the immediate tissue or organ or to the overall health of an individual.
  • cancer cells that have lost normal control over cell growth such that they continue to divide and grow more rapidly than the surrounding normal cells.
  • the cancer cells from a primary tumor may invade neighboring tissues and organs and may even spread progeny cancer cells beyond neighboring tissues and organs, e.g., via the body's blood or lymph, to other organs and tissues and establish secondary malignant tumors in a process known as metastasis.
  • the progressive process whereby cancer cells proliferate rapidly and become increasingly invasive and/or metastatic with respect to other tissues and organs in the body is referred to as "the acquisition of a more malignant phenotype".
  • Such cancer cells and tumors comprising cancer cells are referred to as "malignant" and, without medical intervention, become increasingly more malignant.
  • both primary and secondary tumors may also produce growth factors that induce the formation of blood and lymphatic vessels (i.e., angiogenesis, lymphangiogenesis) to provide an adequate supply of blood to the tumors to sustain continued growth.
  • angiogenesis lymphangiogenesis
  • lymphatic vessels i.e., angiogenesis, lymphangiogenesis
  • angiogenesis lymphangiogenesis
  • lymphangiogenesis lymphatic vessels
  • cancers have been identified for most of the 300 types of cells in the human body.
  • the cells of malignant tumors usually retain some characteristics from the normal cell type from which they arose and also may express certain marker molecules that are unique to the particular cancer, e.g., breast or prostate cancer specific markers (Lodish et al., In Molecular Cell Biology, fourth edition. (W.H. Freeman & Co., New York, 2000), pp. 1054-1057).
  • Cancer is diagnosed in more than 1 million people in the United States alone and, in all its various malignant forms, remains a major cause of suffering and death throughout the world.
  • BMP-7 bone morphogenetic protein-7
  • the invention provides a method of diagnosing cancer in an individual comprising measuring the level of expression of BMP-7 in a sample of cells obtained from the individual, wherein a decrease or loss of the detectable level of expression of BMP-7 in the cells relative to the level in normal healthy cells indicates that the cells are cancer cells, i.e., that the individual has cancer.
  • Detection and measurement of levels of expression of BMP-7 maybe carried out by any of a variety of assays for expression of a protein, including but not limited to, immunoassays (e.g., ELISA, immunoblots) and methods of detecting and measuring BMP-7 specific mRNA transcripts in cells.
  • immunoassays e.g., ELISA, immunoblots
  • Diagnostic methods of the invention may further comprise one or more steps of detecting the level of expression of an epithelial phenotypic markers, e.g., E-cadherin or a cytokeratin, and a mesenchymal phenotypic marker, e.g., vimentin.
  • the invention provides a method of inhibiting progression of cancer cells to a more malignant state comprising contacting the cancer cells with BMP-7.
  • the invention also provides a method of reversing progression of cancer cells to a more malignant state comprising contacting the cancer cells with BMP-7.
  • Such a reversal in the progression of malignancy may be indicated by one or more detectable indices, including but not limited to, a decrease in the level of expression of a mesenchymal phenotypic marker (e.g., vimentin), an increase in expression of an epithelial phenotypic marker (e.g., E-cadherin, a cytokeratin), and/or a decrease in tumor size.
  • a mesenchymal phenotypic marker e.g., vimentin
  • an epithelial phenotypic marker e.g., E-cadherin, a cytokeratin
  • the invention provides a method of treating cancer in an individual comprising administering to the individual an effective amount of BMP-7.
  • the invention also provide a method of treating a primary malignant tumor in an individual comprising administering to the individual an effective amount of BMP-7.
  • the invention provides a method of treating a metastatic tumor in an individual comprising administering to the individual an effective amount of BMP-7.
  • BMP-7 may be used to treat cancer, to inhibit the acquisition of a more malignant phenotype by cancer cells, to reverse progression of cancer cells to a more malignant state, and to prepare a medicament to treat cancer.
  • a composition comprising a BMP-7 may be administered according to the invention by any of a variety of enteral or parenteral routes.
  • a composition comprising BMP-7 is administered to an individual intravenously (i.v.).
  • BMP-7 in its homodimer form comprising two processed (mature) monomers is the preferred protein useful in compositions and methods of the invention.
  • BMP- 7 heterodimers e.g., comprising a mature BMP-7 monomer and an unprocessed BMP-7 monomer or comprising a BMP-7 monomer and a monomer of another BMP species, may also be used in the invention.
  • Figure 1 shows levels of expression of epithelial marker E-cadherin mRNA and of mesenchymal marker vimentin mRNA as determined by real-time PCR in prostate cancer cell lines. Values were normalized to level of expression of ⁇ -actin mRNA.
  • the prostate cancer cell lines in Figure 1 are listed in order (from left to right) of increasing tumorigenic and metastatic potential: LNCaP, C4-2, C4-2B4, PC-3, and PC-3M-Pro4. See text for additional details.
  • Figure 2 shows the level of BMP-7 mRNA expression as determined by real-time PCR in prostate cell lines LNCaP, C4-2, C4-2B4, PC-3, and PC-3M-Pro4. Values were normalized to level of expression of ⁇ -actin mRNA. See text for additional details.
  • Figure 3 shows levels of expression of epithelial marker E-cadherin mRNA and of mesenchymal marker vimentin mRNA as determined by real-time PCR in breast cancer cell lines. Values were normalized to level of expression of ⁇ -actin mRNA.
  • the breast cancer cell lines in Figure 3 are listed in order (from left to right) of increasing tumorigenic and metastatic potential: ZR-75- 1 , T-47D, MDA-MD-231 , and MDA-231 -B. See text for additional details.
  • Figure 4 shows the level of expression of BMP-7 mRNA as determined by real-time PCR in breast cell lines ZR-75-1, T-47D, MDA-MB-231, and MDA-231-B. Values were normalized to level of expression of ⁇ -actin mRNA. See text for additional details.
  • Asterisk indicates statistical significance (p ⁇ 0.05) for difference between data point of mice receiving BMP-7 at a dose of 100 ⁇ g/kg/d and that of control (vehicle only) mice at end of treatment period.
  • Total intensity refers to the signal emitted by growing cancer cells (i.e., tumor growth in mice). See text for additional details.
  • FIG. 6 shows bioluminescent reporter images (BLI) of representative mice as described in Figure 5 after 10 days of treatment, i.e., control (vehicle only, no BMP-7), BMP-7 at 10 ⁇ g/kg/d, or BMP-7 at 100 ⁇ g/kg/d.
  • control vehicle only, no BMP-7
  • BMP-7 at 10 ⁇ g/kg/d
  • BMP-7 at 100 ⁇ g/kg/d.
  • Significant inhibition of tumor growth and spreading is evident in animals treated with BMP-7 at a dose of 100 ⁇ g/kg/d compared to control.
  • Double asterisks indicate statistical significance (p ⁇ 0.01) for the difference between data point of mice receiving 100 ⁇ g/kg/d relative and that of control (vehicle only) mice at the end of the treatment period.
  • Total intensity refers to the signal emitted by growing cancer cells (i.e., tumor growth in mice). See text for additional details.
  • FIG 8 shows bioluminescent reporter images (BLI) of representative mice as described in Figure 7 after 3 weeks of treatment, i.e., control (vehicle only, no BMP-7) or BMP-7 at 100 ⁇ g/kg/d. Significant inhibition of tumor growth and spreading is evident in the animal receiving BMP-7 treatment compared to the control animal. See text for additional details.
  • BMP-7 bioluminescent reporter images
  • FIG 10 shows bioluminescent reporter images (BLI) of representative mice as described in Figure 9 after 3 weeks of treatment, i.e., control (vehicle only, no BMP-7) or BMP-7 treated (100 ⁇ g/kg/d).
  • Vehicle-treated control animals show progressive growth of breast cancer cells in both inoculated mammary fatpads and progressive invasion of subcutaneous tissue of the abdomen. Significant inhibition of tumor growth and spreading is evident in the animal receiving BMP-7 treatment compared to the control animal. Tumor growth was also limited to only one of the two inoculation sites in the BMP-7 treated animal. See text for additional details.
  • Figures 11 A-l 1C show results of semi-quantitative PCR analysis of steady-state levels of BMP-7 mRNA expressed in uveal melanoma cancer cells grown in vivo and in vitro. Under PCR protocol employed, expression of BMP-7 mRNA is indicated by appearance of a cDNA fragment of 316 base pairs (bp) (shown in lane labeled "+" for positive control).
  • FIGS 11A and 1 IB show results of PCR analysis of steady state BMP-7 mRNA levels in uveal melanoma tissue samples from human patients 1 -30. BMP-7 expression was not detected in the majority of the patients. Only patients 2 and 12 expressed detectable, but low levels of BMP-7 mRNA. See text for additional details.
  • Figure 11C shows results of PCR analysis for expression of BMP-7 mRNA in cells often malignant cell lines grown in culture as compared to normal (control, non-cancerous) melanocytes.
  • Numbered lanes correspond to uveal melanoma cell lines as follows: lane 1 is cell line OMM-1, lane 2 is cell line OCM-3, lane 3 is cell line OCM-8, lane 4 is cell line Mel-202, lane 5 is OCM-1, lane 6 is OMM-1.5, lane 7 is 9201, lane 7 is Mel-285, lane 9 is OMM-1.3, and lane 10 is Mel-290.
  • a low detectable level of BMP-7 was expressed in OCM-3 cells (lane 2). See text for additional details.
  • Figure 12 shows bar graphs of the levels of expression of BMP-7 protein by cells of stably transfected, uveal melanoma cell line OCM1-FRT/BMP-7 and cell line OCM1- FRT/mock (no BMP-7 cDNA gene) as detected by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Figure 13 shows bar graphs of the growth of cells of stably transfected, uveal melanoma cell line OCMl -FRT/BMP-7 (expressing BMP-7) and of cells of stably transfected cell line OCMl-FRT/mock (no significant BMP-7 expression) as described for Figure 12. Growth of cells (relative cell number) is shown for cultures after 4 and 7 days of growth. Asterisk indicates p ⁇ 0.05 for difference between growth of cells of OCMl - FRT/BMP-7 and that of cells of OCMl-FRT/mock.
  • Figure 14 shows bar graphs of the tumor burden (% total area of tumor growth) in eyes of mice that were orthotopically implanted with uveal melanoma cells of transfected cell lines OCMl-FRT/mock (no transfected BMP-7 gene, "Mock") and OCMl -FRT/BMP-7 (expressing BMP-7). Values are expressed as means +/- sem.
  • Figures 15A and 15B show diagrams of photomicrographs of tumor growth in cross- sections (4 x magnification) of eyes of animals orthotopically implanted (intra-ocular inoculation) with cells of uveal melanoma cell lines OCMl-FRT/mock (no transfected
  • BMP-7 gene ( Figure 15 A) and OCMl -FRT/BMP-7 (expressing BMP-7 gene) ( Figure 15B) as described above for Figure 14.
  • Tumor growth was assessed 3 weeks after intra-ocular inoculation. "T” indicates tumor tissue, “L” indicates lens, and dashed line delineates approximate outline of tumor area in cross-section. See text for additional details.
  • This invention is based on the discovery that the expression of bone morphogenetic protein-7 (BMP-7) is absent or substantially decreased in cells of a malignant tumor, i.e., in cancer cells, and the discovery that BMP-7 may inhibit or reverse the progression of cancer cells to a more malignant state.
  • BMP-7 inhibits the transition (or
  • transformation of cancer cells from an epithelial phenotype to a mesenchymal phenotype.
  • the progression of cancer cells through this epithelial-mesenchymal transition or transformation is the pattern of changing cellular phenotype that occurs as cancer cells of a tumor become increasingly more malignant.
  • cancer cells originate with an epithelial phenotype (e.g., as evidenced by expression of one or more epithelial markers, such as E-cadherin or cytokeratin) at a primary site in the body of an individual and begin to proliferate more rapidly than surrounding normal cells.
  • the rapidly proliferating cancer cells eventually take on a mesenchymal phenotype (e.g., as evidenced by increasing expression of mesenchymal phenotypic markers, such as vimentin) and invade surrounding tissues and organs and, with time, will even spread to distal sites in the body (metastasis) via transport of some of the cancer cells through the vasculature (blood and/or lymph) to colonize and establish malignant secondary tumors that threaten other tissues and organs of the body.
  • mesenchymal phenotypic markers such as vimentin
  • the invention provides a method of diagnosing cancer based on the detection of a decrease or absence of a detectable level of BMP-7 expression in cells relative to the level of expression in normal healthy cells.
  • the invention also provides a method of inhibiting or reversing progression of cancer cells through the epithelial-mesenchymal transition (EMT) by contacting cancer cells with an effective amount of BMP-7.
  • Therapeutic methods of the invention include a method of treating cancer in an individual comprising administering to the individual an effective amount of BMP-7.
  • Antibody or “antibody molecule”, as used and understood herein, refers to a specific binding member that is a protein molecule or portion thereof or any other peptide containing molecule, whether produced naturally, synthetically, or semi-synthetically, which possesses an antigenic binding domain formed by an immunoglobulin variable light chain region or domain (V L ), or portion thereof, and/or an immunoglobulin variable heavy chain region or domain (VH), or portion thereof.
  • V L immunoglobulin variable light chain region or domain
  • VH immunoglobulin variable heavy chain region or domain
  • antibody also covers any polypeptide or protein molecule that has an antigen-binding domain that is identical, or homologous to, an antigen-binding domain of an immunoglobulin.
  • Antibodies maybe "polyclonal”, i.e., population of antigen-binding molecules that bind to different sites on the antigen or "monoclonal”, i.e., a population of antigen-binding molecules that bind to only one site on an antigen.
  • an antibody molecule examples include any of the well known classes of immunoglobulins (e.g., IgG, IgM, IgA, IgE, IgD) and their isotypes; fragments of immunoglobulins that comprise an antigen binding domain, such as Fab or F(ab') molecules; single chain antibody (scFv) molecules; double scFv molecules; single domain antibody (dAb) molecules; Fd molecules; and diabody molecules.
  • immunoglobulins e.g., IgG, IgM, IgA, IgE, IgD
  • fragments of immunoglobulins that comprise an antigen binding domain such as Fab or F(ab') molecules
  • scFv single chain antibody
  • dAb single domain antibody
  • Diabodies are formed by association of two diabody monomers, which form a dimer that contains two complete antigen binding domains wherein each binding domain is itself formed by the intermolecular association of a region from each of the two monomers (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)).
  • Antibodies useful in the invention may be linked to a detectable label or labeling system that permits detection and/or quantification of complexes formed between BMP-7 molecules and an anti-BMP-7 antibody.
  • An "individual” or “patient” is a human or other mammal that has, is suspected to have, or is being diagnosed as having cancer.
  • metastasis and “metastatic” have the meanings understood in the fields of medicine and oncology.
  • the spread of tumor cells from an initial or primary site in the body, e.g., via the blood and/or lymph, and the colonization and establishment of (secondary) tumors at other (secondary) sites in the body is referred to as metastasis.
  • metastasis is characteristic of a higher state of malignancy for cancer cells and tumors composed of cancer cells.
  • cancer cells and tumors comprising cancer cells are referred to as "malignant”.
  • tumors and cancer cells become increasingly more malignant in the body of an individual.
  • BMP transforming growth factor- ⁇
  • morphogen any member of a particular subclass of the transforming growth factor- ⁇ (TGF- ⁇ ) super family of proteins (see, e.g., Hoffmann et al., Appl. Microbiol. Biotechnol., 57: 294-308 (2001); Reddi, J. Bone Joint Surg., 83-A(Supp. 1): S1-S6 (2001); US Patent Nos. 4,968,590; 5,011,691; 5,674,844; 6,333,312). All BMPs have a signal peptide, prodomain, and a carboxy-terminal (mature) domain.
  • TGF- ⁇ transforming growth factor- ⁇
  • the carboxy-terminal domain is the mature form of the BMP monomer and contains a highly conserved region characterized by seven cysteines that form a cysteine knot (see, Griffith et al., Proc. Natl. Acad. Sci. US A., 93: 878-883 (1996)).
  • BMPs were originally isolated from mammalian bone using protein purification methods (see, e.g., Urist et al., Proc. Soc. Exp. Biol. Med., 173: 194-199 (1983); Urist et al., Proc. Natl. Acad. Sci. USA, 81: 371-375 (1984); Sampath et al., Proc. Natl. Acad. Sci.
  • BMPs have also been detected in or isolated from other mammalian tissues and organ including kidney, liver, lung, brain, muscle, teeth, and gut. BMPs may also be produced using standard in vitro recombinant DNA technology for expression in prokaryotic or eukaryotic cell cultures (see, e.g., Wang et al., Proc. Natl. Acad. Sci. USA, 87: 2220-2224 (1990); Wozney et al, Science, 242: 1528-1534 (1988)). Some BMPs are commercially available for local use as well (e.g., BMP-7 is manufactured and distributed for long bone non-unions by Stryker-Biotech
  • BMP-2 is manufactured and distributed for long bone acute fractures by Wyeth (Madison, NJ), and also for spinal fusions by Medtronic, Inc., Minneapolis, MN).
  • BMPs normally exist as dimers of the same monomeric polypeptides (homodimers) held together by hydrophobic interactions and at least one interchain (between monomers) disulfide bond.
  • BMPs may also form heterodimers, e.g., by combining two monomers that have different degrees (lengths) of processing (e.g., a full-length, unprocessed monomer associated with a processed, mature monomer) or by combining monomers of different BMPs (e.g., a BMP-7 monomer with a BMP-6 monomer).
  • a BMP dimer of unprocessed monomers or a BMP heterodimer of one processed BMP monomer and one unprocessed BMP monomer are typically soluble in aqueous solutions, whereas a BMP homo- or heterodimer of two processed (mature) monomers is only soluble in an aqueous solution at a low pH (e.g., acetate buffer, pH 4.5) (see, e.g., Jones et al., Growth Factors, 11: 215-225 (1994)).
  • a low pH e.g., acetate buffer, pH 4.5
  • BMP-7 which is useful in the methods and composition described herein, also has an "osteoinductive” or “osteogenic” activity, i.e., the ability to stimulate bone formation in a standard osteoinductive assay.
  • osteoinductive assays include ectopic bone formation assays in which a carrier matrix comprising collagen and a BMP are implanted at an ectopic site in rodent, and the implant then monitored for bone formation (Sampath and Reddi, Proc. Natl. Acad. Sci. USA, 78: 7599-7603 (1981)).
  • the matrix may be implanted at an ectopic site and the BMP administered to the site, e.g., by intravenous injection into the rodent.
  • Another way to assay for BMP osteoinductive activity is to incubate cultured fibroblast progenitor cells with a BMP and then monitor the cells for differentiation into chondrocytes and/or osteoblasts (Asahina et al., Exp. Cell. Res. 222: 38- 47 (1996)).
  • a number of BMPs have osteoinductive activity including, but not limited to, BMP-7, BMP-6, BMP-2, BMP-4, BMP-9, BMP-12, BMP-13, BMP-14, and heterodimers thereof, whether purified from a natural source, produced recombinantly, or produced in whole or in part by in vitro protein synthesis.
  • a BMP that has an osteoinductive activity may also possess one or more other beneficial pharmacological activities such as the ability to restore or regenerate damaged soft tissues or organs, e.g., ischemic kidneys (Vukicevic et al., J. Clin.
  • BMP-7 epithelial to mesenchymal transition
  • a “drug” refers to any compound or composition that has a pharmacological activity.
  • a “therapeutic drug” is a compound or composition that can be administered to an individual to provide a desired pharmacological activity.
  • a “prophylactic drug” is a compound or composition that can be administered to an individual to prevent or provide protection from the development in an individual of an undesired or harmful disease.
  • a drug may have therapeutic as well as prophylactic uses.
  • composition composition
  • formulation formulation
  • preparation preparation
  • pharmaceutically acceptable any compound or mixture that is not biologically, chemically, or in any other way, incompatible with body chemistry and metabolism and also does not adversely affect the desired, effective activity of a bone morphogenetic protein or any other component in a composition that may be administered to an individual to effectively inhibit or reverse the EMT of a cancer cell or to inhibit or reverse tumor growth in an individual.
  • oral refers to a route or mode for administering an effective amount of a compound, such BMP-7, or composition thereof, to an individual anywhere along the alimentary canal of the individual.
  • enteral routes of administration include, without, limitation, from the mouth, e.g., swallowing a solid (e.g., pill, tablet, capsule) or liquid (e.g., syrup, elixir) composition; sub-lingual (absorption under the tongue); nasojejunal or gastrostomy tubes (into the stomach); intraduodenal administration; and rectal (e.g., using suppositories for release and absorption of a compound or composition in the lower intestinal tract of the alimentary canal).
  • a solid e.g., pill, tablet, capsule
  • liquid e.g., syrup, elixir
  • sub-lingual e.g., syrup, elixir
  • nasojejunal or gastrostomy tubes into the stomach
  • intraduodenal administration e.g., using suppositories for release and absorption of a compound or composition in the lower intestinal tract of the alimentary canal.
  • rectal e.g.
  • oral formulations are the same as “enteral” formulations and broadly encompass formulations that maybe swallowed from the mouth as well as those that permit administration of the BMP anywhere along the alimentary canal.
  • parenteral and “parenterally” refer to routes or modes of administration of a compound such as BMP-7, or composition thereof, to an individual other than along the alimentary canal.
  • parenteral routes of administration include, without limitation, intravenous (i.v.), intramuscular (i.m.), intra-arterial (i.a.), intraperitoneal (i.p.), subcutaneous (s.c), transdermal (absorption through the skin or dermal layer), nasal or pulmonary (e.g., via inhalation or nebulization, for absorption through the respiratory mucosa or lungs), direct injections or infusions into body cavities or organs, as well as by implantation of any of a variety of devices into the body that permit active or passive release of a compound or composition into the body.
  • a decrease in or loss of detectable levels of expression of BMP-7 in cells taken from a particular tissue or organ of an individual as compared to the level normally found in healthy cells from that same tissue or organ (from the same individual and/or from other individuals of the same species) indicates that the cells are cancer cells, i.e., from a malignant tumor.
  • the level of BMP-7 expression in cells from an individual may be monitored by assaying for BMP-7 expression in samples of cells taken over a period of time, e.g., by biopsy or blood sample, from a tissue or organ suspected of having a malignant tumor.
  • the level of BMP-7 may be compared to a catalogue or library of known standard ranges of expression levels of BMP-7 for normal healthy cells from a particular tissue or organ of healthy individuals of the same species.
  • Some cancers, such as prostate cancer are multifocal in which case the level of expression of BMP-7 in samples from different locations in an organ (e.g., prostate) can be assayed, e.g., by needle biopsy or other precision tissue sampling method.
  • This type of assaying of "patient-matched" tissue samples provides a highly reliable basis for distinguishing cancer cells from normal cells in an individual.
  • there is some prior basis for a suspicion of a malignant tumor either based on clinical symptoms or prior histological examination.
  • BMP-7 e.g., as measured by BMP-7 protein or mRNA
  • a finding that the level of expression of BMP-7 in cells from the tissue or organ is essentially absent, e.g., below the level of detection of most immunoassays or RT-PCR assays for BMP-7 detection, or is significantly lower than, e.g., less than 50% of, the level of expression in normal healthy cells of the tissue or organ indicates that the cells are cancer cells and, thus, that the tissue or organ from which the cells were taken has a malignant tumor.
  • Levels of BMP-7 expression in cells may be measured by any of a variety of assay techniques and formats available in the art. For example, various procedures are available in the art that are readily adapted for use in the diagnostic methods for detecting cancer cells from an in vitro or in vivo source (e.g., a malignant tumor in an individual), such as those that are based on nucleic acid detection assays that employ one or more primer molecules or nucleic acid probes for detecting and/or measuring (quantitative analysis for) levels of BMP-7 specific mRNA transcripts expressed in cells.
  • an in vitro or in vivo source e.g., a malignant tumor in an individual
  • nucleic acid detection assays that employ one or more primer molecules or nucleic acid probes for detecting and/or measuring (quantitative analysis for) levels of BMP-7 specific mRNA transcripts expressed in cells.
  • Such procedures include, without limitation, Northern blots of mRNA that employ BMP-7 specific nucleic acid probes and the quantitative detection of levels of BMP-7 specific mRNA transcripts from cells using a reverse transcriptase (RT) to generate BMP-7 specific cDNA and polymerase chain reaction (PCR) protocols that employ BMP-7 specific oligonucleotide primer molecules.
  • RT reverse transcriptase
  • PCR polymerase chain reaction
  • the level of BMP-7 protein may also be readily detected by any of a variety of methods that permit detection of a known protein. Such methods include any of a variety of immunodetection assays that employ one or more antibodies specific for BMP-7, including (without limitation), Western immunoblots, enzyme linked immunosorbent assays (ELISAs), immunoaffinity chromatography, immunoprecipitation protocols, and the like. Antibodies to BMP-7 may be obtained commercially or produced using any of a variety of standard protocols known in the art for obtaining polyclonal or monoclonal antibodies to a known protein, such as BMP-7.
  • a sample of cells is obtained from a culture or an individual and then analyzed for the level of expression of BMP-7 protein.
  • an extract of the cells is contacted with a BMP-7 binding partner, such as an anti-BMP - 7 antibody, under conditions suitable for the formation of an anti-BMP-7 antibody/BMP-7 binding complex.
  • a BMP-7 binding partner such as an anti-BMP - 7 antibody
  • Immunoassays useful in the diagnostic methods of the invention preferably detect the complex that forms between an anti-BMP-7 antibody and a BMP-7 molecule in an ' extract of cells or conditioned medium.
  • Some immunoassays may be sandwich-type assays, e.g., in which a complex that is formed between a "primary" anti-BMP-7 antibody and a BMP-7 molecule is detected by a "secondary” or “detection” antibody that binds to the anti- BMP antibody.
  • a detectable label or system such as, a fluoresceinated tag, a bioluminescent tag, a colorimetric tag, a colored bead, detectable metal atom, streptavidin, biotin, or other label.
  • detectable tags and detection systems provide a signal that may be readily observed and in some cases quantified, e.g., by fluorimetry, epifluorescence microscopy, confocal scanning laser microscopy, luminometer, or colorimetric assay.
  • a BMP-7 binding partner may be a polypeptide other than an antibody or a non-peptide molecule, e.g., a nucleotide-based molecule (aptamer), that may be substituted for an anti-BMP-7 antibody molecule in an immunoassay protocol to detect the level of expression of BMP-7 in cells for diagnosing cancer.
  • a nucleotide-based molecule e.g., a nucleotide-based molecule (aptamer)
  • kits for diagnosing cancer by detecting loss or decrease in the level of BMP-7 protein expressed in cells from a culture (in vitro) or from an individual (in vivo).
  • a preferred kit of the invention comprises a first (capture) BMP-7 binding partner (e.g., an anti-BMP-7 antibody) and instructions that indicate how to use the kit to detect or measure BMP-7 levels in samples of cells to diagnose cancer.
  • a diagnostic method for cancer as described herein may further comprise the step of assaying the cells obtained from an individual for the expression (or lack thereof) of an epithelial phenotypic marker (e.g., a cytokeratin, E-cadherin) and/or a mesenchymal marker (e.g., vimentin).
  • an epithelial phenotypic marker e.g., a cytokeratin, E-cadherin
  • a mesenchymal marker e.g., vimentin.
  • Cancer cells expressing epithelial markers are at an early stage of malignancy, while cancer cells that exhibit extensive expression of mesenchymal markers are at a relatively advanced stage of malignancy (i.e., highly proliferative, invasive, and/or metastatic).
  • assays for detecting epithelial and mesenchymal phenotypic markers include any of the variety of protocols for detecting the specific protein markers, including, but not limited to, immunoassays, immunohistological staining, and assays for marker-specific mRNA transcripts (e.g., nucleic acid blots probed with marker specific nucleic acid probes or RT-PCR protocols using marker specific primers). It is understood that such assays for epithelial and mesenchymal phenotypic markers maybe carried out prior to, concurrently, or subsequently to the steps of measuring levels of BMP-7 expression in cells taken from the individual.
  • the levels of expression of at least one epithelial marker and at least one mesenchymal marker are determined for the cells taken from an individual and then compared with the levels of expression (or a ratio of the levels of expression) of the same epithelial and mesenchymal phenotypic markers in normal cells.
  • BMP-7 can inhibit the progression a cancer cell from a less malignant to a more malignant state.
  • BMP-7 can inhibit or even reverse progression of cancer cells through the epithelial-mesenchymal transition (EMT) wherein cancer cells become more invasive and metastatic.
  • EMT epithelial-mesenchymal transition
  • Data that support this invention indicate that cancer cells that are made to express BMP-7, e.g., as demonstrated by transfection with a BMP-7 expression system, or that are contacted with BMP-7, either in vitro or in vivo, will usually maintain or regain epithelial phenotypic characteristics of a reduced malignant state and may also lose or not acquire expression of a mesenchymal phenotypic marker (e.g., vimentin) that is characteristic of cancer cells in an advanced stage of malignancy, such as highly invasive (e.g., spreading to neighboring tissues and organs) and metastatic cancer cells.
  • a mesenchymal phenotypic marker e.g., vimentin
  • BMP-7 to inhibit progression of cancer cells to a more malignant state is evident in the in vivo prevention of tumor growth and metastasis for human cancers that are among the most widely occurring and aggressive known, such as prostate cancer, breast cancer, and, most notably, the highly aggressive uveal melanomas (see, e.g. Figures 6, 8, and 10).
  • a highly malignant tumor such as one comprising cancer cells that have acquired a mesenchymal phenotype
  • the cancer cells must reverse progress through the EMT process to acquire a more epithelial phenotype and less malignant state.
  • Methods for treating cancer in an individual according to the invention comprise administering an effective amount of BMP-7 or a composition thereof to the individual.
  • BMP-7 a composition thereof
  • the therapeutic methods described herein are not to be interpreted, restricted to, or otherwise limited to a "cure” for cancer, but rather to the use of BMP-7 to "treat" a cancer, i.e., to effect a desirable or beneficial change in the health of an individual who has cancer.
  • Such benefits are recognized by skilled healthcare providers who treat cancer patients and include, but are not limited to, a decrease in tumor size (tumor regression), an improvement in vital functions (e.g., improved function of cancerous tissues or organs), a decrease or inhibition of further metastasis, a decrease in opportunistic infections, an increased survivability, a decrease in pain, improved motor function, improved cognitive function, improved feeling of energy (vitality, decreased malaise), improved feeling of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof.
  • regression of a particular tumor in an individual e.g., as the result of
  • BMP-7 treatment may also be assessed by taking samples of cancer cells from the site of the tumor (e.g., over the course of treatment) and testing the cancer cells for the level of epithelial marker (e.g., E-cadherin, cytokeratin) and mesenchymal marker (e.g., vimentin) expression to monitor the EMT status of the cancer cells to verify at the molecular level the regression of the cancer cells to a less malignant phenotype.
  • epithelial marker e.g., E-cadherin, cytokeratin
  • mesenchymal marker e.g., vimentin
  • a BMP-7- induced regression of cancer cells to a less malignant phenotype would be indicated by finding a higher ratio of the level of expression of epithelial to mesenchymal markers (e.g., E-cadherin/vimentin) in cancer cells from an individual during the course of or at the end of BMP-7 treatment compared to that of cancer cells in a more malignant state, e.g., prior to or earlier in the course of BMP-7 treatment.
  • epithelial to mesenchymal markers e.g., E-cadherin/vimentin
  • BMP-7 Administration of BMP-7 to a patient may be achieved by intravenous (i.v.) injection of a solution comprising BMP-7 or by another appropriate route (see, below). Desirable blood levels may be maintained by a continuous infusion or by intermittent infusions, e.g., by using pumps or slow release technologies available in the art.
  • BMP-7 may be administered as the raw chemical
  • BMP-7 will be administered to a cancer patient as an active ingredient in a pharmaceutical composition.
  • Standard methods of preparing dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition. (Alfonso R. Gennaro, ed.) (Mack Publishing Co., Easton, PA 1990)).
  • the invention thus further provides a pharmaceutical composition
  • a pharmaceutical composition comprising BMP- 7, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers and, optionally, one or more other therapeutic or beneficial agents, such as, another anti-cancer drug, an antibiotic, an antiviral compound, an anti-fungal drug, a vitamin, a trace metal supplement, or ions to restore or maintain proper ionic balance in blood or other tissues.
  • agents may be administered to an individual together with or separately from BMP-7.
  • the combination therapies described herein are merely exemplary and are not meant to limit possibilities for other combination treatments or co- administration regimens comprising BMP-7.
  • a pharmaceutically acceptable carrier used in a pharmaceutical composition of the invention must be "acceptable" in the sense of being compatible with the physiology of the patient and also non-deleterious to the anti-cancer activity of BMP-7 or of the beneficial property or activity of any other ingredient that may be present in a composition that is to be administered to a patient.
  • compositions comprising BMP-7 for use in the invention may include those suitable for administration by a parenteral or enteral (along the alimentary canal) route, including (without limitation), an intravenous (i.v.), subcutaneous (s.c), oral (swallowing by mouth), sub-lingual (absorption under the tongue), rectal (e.g., suppositories), nasal (e.g., inhalation or insufflation), auricular (ear), ocular, topical, transdermal, or vaginal route.
  • a parenteral or enteral (along the alimentary canal) route including (without limitation), an intravenous (i.v.), subcutaneous (s.c), oral (swallowing by mouth), sub-lingual (absorption under the tongue), rectal (e.g., suppositories), nasal (e.g., inhalation or insufflation), auricular (ear), ocular, topical, transdermal, or vaginal route.
  • a pharmaceutical composition may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. Such methods may include the step of bringing BMP-7 into association with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired composition.
  • BMP-7 may be formulated for parenteral administration and may be presented in unit dose form in ampoules, pre-filled syringes, a small volume infusion, or in multi-dose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing, and/or dispersing agents.
  • BMP-7 may be prepared and supplied in a crystallized, lyophilized, or other solid form (e.g., as obtained by aseptic isolation of sterile solid or by lyophilization from solution) for constitution with a suitable aqueous vehicle, e.g., sterile, pyrogen-free water or sterile physiological buffer, prior to parenteral administration.
  • a suitable aqueous vehicle e.g., sterile, pyrogen-free water or sterile physiological buffer
  • Pharmaceutical compositions suitable for oral administration of BMP-7 may conveniently be presented as discrete units such as capsules, cachets, or tablets containing a predetermined amount of a compound of the invention in a powder or granule form, in a solution, in a suspension, or as an emulsion.
  • a formulation comprising BMP-7 may also be presented as a bolus, electuary, or paste.
  • Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents.
  • the tablets may be coated according to methods well known in the art.
  • Oral liquid preparations comprising BMP-7 may be in the form of, by way of example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.
  • BMP-7 may be formulated in ointments, creams, gels, jellies, or lotions. Alternatively, BMP-7 may also be incorporated into a transdermal patch. Such transdermal patches may contain penetration enhancers such as lanolin, linalool, carvacrol, thymol, citral, menthol, t- anethole, and the like. Ointments and creams may, e.g., be formulated with an aqueous or oily base comprising one or more suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • compositions suitable for administration of BMP-7 via the mouth include, without limitation, lozenges comprising BMP-7, optionally, in a flavored base, and comprising sucrose, acacia, and/or tragacanth; pastilles comprising BMP-7 in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient (BMP-7) in a suitable liquid carrier.
  • compositions suitable for rectal administration may comprise BMP-
  • Suitable carriers include cocoa butter and other materials commonly used in the art, where the suppository may be conveniently formed by admixture of BMP-7 with the softened or melted carrier(s) followed by chilling and shaping in molds.
  • compositions comprising BMP-7 that are suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or sprays and further may comprise one or more carriers as are known in the art to be appropriate.
  • a composition comprising BMP-7 may be used as a liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one more dispersing agents, solubilizing agents, or suspending agents. Liquid sprays may conveniently be delivered from pressurized packs. For administration to the lungs by inhalation, BMP-7 may be delivered from an insufflator, nebulizer, a pressurized pack, or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • BMP-7 may be incorporated into a dry powder composition, e.g., in combination with a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, e.g., capsules or cartridges, or, e.g., in gelatin or blister packs from which the powder mixture comprising BMP-7 may be administered with the aid of an inhalator or insufflator.
  • BMP-7 may also be formulated into a pharmaceutical composition for treating cancer of the eye or ear.
  • Eye and ear cancers may be treated by administering BMP-7 to a patient by any of the various routes described above or by direct administration to the affected eye or ear.
  • pharmaceutical compositions comprising BMP-7 for use according to the invention for treating an eye or ear cancer may be a liquid or lotion that may be administered directly into or on the eye or ear.
  • the above-described compositions may be adapted to give a sustained or time-delayed release of BMP-7 using any of the sustained or time-delayed formats available in art.
  • BMP-7 and one or more other pharmaceutically acceptable compounds may be administered to an individual either sequentially or simultaneously, in separate or combined pharmaceutical compositions.
  • the dose of each compound maybe either the same as or differ from that when the compound is used alone.
  • Appropriate doses will be readily appreciated by those skilled in the art.
  • the ratio between BMP-7 and a second therapeutic compound for co-administration to a patient will be readily appreciated by those skilled in the art. For example, one may use a ratio in the range from about 1 : 1 to about 1: 50 (by weight) of BMP-7 : second therapeutic compound or, vice versa, i.e., of the second therapeutic compound:BMP-7.
  • the ranges of ratios that may be used in preparing a composition for co- administration of BMP-7 with a second therapeutic compound include, without limitation: about 1:1 to about 1:30 (by weight), about 1:1 to about 1: 20 (by weight), about 1:1 to about 1:15 (by weight), about 1:1 to about 1:10 (by weight), about 1:1 to about 1:5 (by weight), and about 1 : 1 to about 1 :3 (by weight), or vice versa. If yet a further therapeutic compound(s) is added, ratios are adjusted accordingly.
  • BMP-7 may be intravenously (i.v.) administered to an individual to provide a dose in the range of from 0.05 mg/day to 5 mg/day in the methods described herein.
  • BMP-7 may be administered periodically or cyclically to an individual, e.g., administration to an individual for a period of time, discontinued for a period for time, and then re-initiated, at the discretion of the skilled healthcare provider.
  • the limitation on a course of dosing or repetition of dosing typically will be based on whether the attending healthcare provider believes such dosing or repetition may or may not provide further benefit to a particular individual and/or whether there is any evidence of acute or chronic side effects that would limit the use of a particular dose or duration of administering BMP-7 to the individual.
  • doses of pharmacologically active compounds such as a BMP-7
  • pharmacologically active compounds such as a BMP-7
  • mass e.g., milligrams, mg
  • mg mg per kilogram
  • mg per unit volume of formulation e.g., mg per unit volume of formulation, and the like.
  • dosages of BMP-7 in terms of mg/day refer to mg of BMP-7 per patient per day and are based on the commonly used standard of a 70 kg male human patient.
  • dosing in terms of mg of BMP-7 per kg of body weight (mass) assume a 70 kg male human being.
  • doses may have to be modified for a particular individual or population of individuals. For example, this is particularly relevant in the case of patients having advanced cancers or who have undergone extensive chemotherapy as many of these are likely to weigh less than 70 kg and may be relatively frail compare to a healthy individual (male or female). Hence, it is understood that when treating an individual that is more or less than 70 kg, a dose may be appropriately modified in accordance with standard pharmacological adjustments. Accordingly, various examples of doses described herein are readily converted by persons skilled in the art to various other dosing units (and vice versa) required for treating specific individuals or populations of individuals with a particular pharmaceutically acceptable formulation comprising BMP-7 as described herein.
  • Example 1 Levels of BMP in malignant prostate and breast cancer cells. This study was carried out to assess the level of expression of BMP-7 in various prostate cancer (CaP) cells.
  • Prostate tissue specimens from prostate cancer patients undergoing radical prostatectomy were obtained from prostate cancer patients at the time of surgery at the
  • RNA/ ter stabilization reagent Qiagen GmbH, Heiden, Germany.
  • the adjacent tissue was processed for paraffin embedding to serve as histological control. Histological diagnosis and pattern definition for Gleason score were performed by a certified pathologist.
  • Total RNA was extracted using an RNeasy Midi extraction kit (Qiagen).
  • Gleason scoring method which is the current standard of the histo-pathological assessment of prostate cancer malignancy. This method has a widely accepted prognostic value.
  • Gleason pattern defines, in a scale from 1 to 5, progressive loss of acinar architecture and epithelial differentiation.
  • Gleason score defines, in a scale from 2 to 10, the sum of two most prominent patterns represented in a prostate cancer specimen.
  • Reverse transcription was performed with random primers in the presence of RNase inhibitor (Roche Diagnostics, Rotsville, Switzerland).
  • Real-time polymerase chain reaction (real-time PCR) was performed where indicated using commercially obtained exon-specific primers for BMP-7, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), E-cadherin, vimentin, and ⁇ -actin (primer catalogue numbers: Hs 002333477 ml (BMP-7), Hs 99999905 ml (GAPDH), Hs 00170423 ml (E-cadherin), Hs 00185584 ml (vimentin), and Hs 99999903 ml ( ⁇ -actin), Applied Biosystems, Rotnch, Switzerland) on an ABI Prism 7700 sequence detection system (Applied Biosystems).
  • the resulting values for BMP-7 expression were normalized to GAPDH expression in the case of patient specimens or to ⁇ - actin expression in the case
  • Prostate cancer in vivo is multifocal, i.e., scattered tumor nodules among the prostate gland provides cancer foci separated by normal prostate cells.
  • LCM laser capture microdissection
  • both normal epithelial prostate cells and cancer epithelial (or mesenchymal in advanced malignancy) cells from tissue of the same patient, i.e., this type of comparison provides highly accurate patient-matched data.
  • a reduction in the level of expression of expression of BMP-7 in various cancer cell lines was evident by comparing levels of expression of less malignant cell lines with the level in the most malignant cell lines.
  • Prostate cancer in vivo is multi-focal, i.e., scattered tumor nodules among the prostate gland provides cancer foci separated by normal prostate acini and stroma:
  • LCM laser capture microdissection
  • the human prostate cancer cell lines PC-3 (ATCC Number CRL-1435, ATCC-LGC Promochem, Molsheim Cedex, France) and PC-3M-Pro4 (see, description in Example 2, below) were grown in DMEM supplemented with 10% fetal calf serum (FCS) (BioWittaker, Venders, Belgium).
  • the human prostate cancer cell line LNCaP and LNCaP-derived cell lines C4-2 and C4-2B4 (provided by Dr. L. Chung, Winship Cancer Center, Emory University, Atlanta, Georgia, USA) were grown in T-Medium.
  • Mammary tumor cell line ZR-75-1 was originally established from malignant effusion of a 63 year old female Caucasian with infiltrating ductal carcinoma of the breast.
  • Cells of cell line ZR-75-1 have receptors for estrogen and other steroid hormones.
  • the cell line was obtained from the American Type Culture Collection (ATCC), Manassas, Virginia, 20108 USA (ATCC Number: CRL-1500). See, also Engel et al., Cancer Res., 38(10): 3352-3364 (1978).
  • the cell line T-47 was originally isolated by I. Keydar from a pleural effusion obtained from a 54 year old female patient with an infiltrating ductal carcinoma of the breast. A differentiated epithelial substrain, designated T-47D, was found to contain cytoplasmic junctions and receptors to 17- ⁇ -estradiol, other steroids, and calcitonin.
  • the T- 47D cell line used in this study was obtained from the American Type Culture Collection (ATCC Number HTB-133). See also, Keydar et al., Eur. J. Cancer, 15(5): 659-670 (1979). Cells at 70%-80% confluence were used for RNA extraction using an RNeasy Midi RNA extraction kit (Qiagen).
  • BMP-7 mRNA was invariably under-expressed in cancer cells from all patients (mean decrease 85% +/- 21% SD, p ⁇ 0.005, Wilkoxon test). All the prostate cancer cell specimens examined attained at least a 60% reduction in the BMP-7 mRNA levels, as compared to patient-matched, normal epithelial cell specimens. The loss of BMP-7 mRNA expression was significant only when the analysis was performed in pure cancer cells isolated by laser capture, but not in bulk tissue samples.
  • the prostate cell lines examined in this study have progressively greater tumorigenic and metastatic potential when arranged in the following order: LNCaP, C4-2, C4-2B4, PC- 3, PC-3M-Pro4, where PC-3 and its derivative PC-3M-Pro-4 are highly tumorigenic and metastatic.
  • This arrangement of the cell lines also coincides with the range from most epithelial phenotype (highest expression of E-caderin epithelial marker) to least epithelial phenotype, i.e., where the cancer cells display the mesenchymal phenotype of highly malignant and metastatic tumor cells.
  • Vimentin is a characteristic marker for the mesenchymal phenotype of cancer cells. See, Figure 1.
  • BMP-7 expression correlates with malignancy and degree of EMT.
  • less malignant cells had a higher ratio of expression of epithelial to mesenchymal markers (i.e., E- cadherin/vimentin; low EMT degree) and a higher level of expression of BMP-7 as compared to higher malignant cells that had a lower E-cadherin/vimentin ratio (high EMT degree) and lower level of BMP-7 expression.
  • the osteolytic and highly tumorigenic and metastatic human prostate cancer cell line PC-3 and its variant PC-3M-Pro4 did not show detectable BMP-7 mRNA expression, either constitutively or after TGF- ⁇ or BMP -2 stimulation.
  • BMP-7 was expressed in the cytoplasm facing the acinar lumen of apical cells of normal prostate acini, and in capillary endothelial cells. Staining was also evident in the stroma (stromal cells and intercellular matrix). In prostate cancer tissue, cancer cells lacked BMP-7 immunoreactivity, and only capillary endothelial cells showed BMP-7 expression.
  • a comparative study with breast cancer cell lines yielded results similar to those obtained with the prostate cancer cell lines described above. The following breast cancer cell lines (in order of increasing degree of malignancy) were studied: ZR-75-1, T-47D, MDA-MB-231, and MDA-231-B.
  • Cancer cells of the less malignant cell lines ZR-75-1 and T-47D expressed relatively high levels of the epithelial phenotypic marker E-cadherin mRNA and relatively low levels of the mesenchymal phenotypic marker vimentin mRNA, whereas cancer cells of the more malignant cell lines MDA-MB-231 and MDA-231-B expressed relatively low levels of E-cadherin mRNA but relatively high levels of the mesenchymal phenotypic marker vimentin mRNA. See, Figure 3.
  • breast cancer cells of the less malignant cell lines e.g., ZR-75-1
  • breast cancer cells of the other more malignant cell lines also expressed a higher level of detectable BMP-7 mRNA compared to breast cancer cells of the other more malignant cell lines. See, Figure 4.
  • BMP-7 expression correlates (inversely) with the degree of malignancy and the degree of progression along the epithelial-mesenchymal transition
  • EMT epithelial to mesenchymal markers
  • E-cadherin/vimentin low degree of progression through the EMT
  • BMP-7 BMP-7 expression
  • Example 2 Experimental treatment of breast and prostate cancer with BMP-7 in vivo.
  • prostate or breast cancer cells were implanted in the bone marrow (intra-osseous innoculation) or breast cancer cells were implanted into mammary fatpads (orthotopic innoculation), to provide in situ physiological environments conducive to both the proliferation of the implanted cancer cells as well as the progression of the cancer cells to metastasis resulting in additional tumors in other locations in the mice.
  • MDA-MB-231 is an estrogen independent, human breast, epithelial cancer cell line that was obtained from the American Type Culture Collection (ATCC Number HTB-26; ATCC, Manassas, Virginia, USA). The origin of MDA-MB-231 was a metastatic human breast tumor (pleural effusion adenocarcinoma) (see, Cailleau et al., J. Natl. Cancer Inst., 53(3): 661-674 (1974)).
  • a subclone of cell line MDA-MB-231 (bone-seeking clone MDA- 231 -B) that invariably induces bone metastases after intracardiac inoculation, was established by four consecutive sequential cycles of intracardiac inoculation of cells of MDA-MB-231 in vivo and subsequent expansion in vitro of the cell population recovered from the resulting bone metastases as described previously (Wetterwald et al., Am. J. Pathol, 160: 1143-1153 (2002)).
  • MDA-231-B cells were stably transfected with a CMV- promotor driven mammalian expression vector for luciferase (CMV-luc), and one clone (MDA-23 l-B/luc + ) with the highest expression of luciferase activity was successfully used for implantation into bone marrow and in vivo whole body bioluminescent reporter imaging (BLI) as previously described by Wetterweld et al. (2002).
  • CMV-luc CMV- promotor driven mammalian expression vector for luciferase
  • MDA-23 l-B/luc + clone with the highest expression of luciferase activity was successfully used for implantation into bone marrow and in vivo whole body bioluminescent reporter imaging (BLI) as previously described by Wetterweld et al. (2002).
  • MDA-23 l-B/luc + cells were cultured in Dulbecco's modified Eagle's Medium (DMEM, Gibco-BRL, Breda, The Netherlands) containing 4.5 g of glucose/L and supplemented with 10% fetal calf serum (Gibco-BRL, Breda, The Netherlands), and 800 ⁇ g/ml geneticyne/G418 (Gibco-BRL, Breda, the Netherlands). Cells were regularly certified free of mycoplasma contamination.
  • DMEM Dulbecco's modified Eagle's Medium
  • MDA-23 l-B/luc+ cell cultures were harvested at sub-confluence after being re- fed with fresh medium 24 hours before inoculation preparation.
  • Cell suspensions of MDA- 23 l-B/luc + (1 x 10 5 cells/10 ⁇ L phosphate buffered saline (PBS)) for intra-osseous or orthotopic inoculation were prepared as previously described (Wetterweld et al., 2002; van der Pluijm et al., Am. J. Pathol, 159: 971-982 (2001); van der Pluijm et al., J. Bone Miner. Res., 16: 1077-1091 (2001)).
  • Human prostate cancer cell line PC-3 was obtained from the American Type Culture
  • PC-3 cell line was initiated from a bone metastasis of grade IV prostatic adenocarcinoma in a 62 year old male Caucasian (see, Kaiglin et al., invest. Urol, 17: 16-23 (1979)).
  • Prostate cancer cell lines PC-3M-Pro4 and PC-3M-LN4 giftss from F.C. Hamdy, University of Sheffield, United Kingdom) were originally derived from PC-3 cells. Briefly, PC-3 cells were injected into the prostates of athymic mice. Tumors from the prostate or lymph nodes were harvested, and cells were re-injected into the prostate as described previously (Pettawy et al., Clin. Cancer Res., 2(9): 1627-1636 (1996)). This cycle was repeated four times to yield cell lines PC- 3M-Pro4 (derived from prostate) and PC-3M-LN4 (derived from lymph nodes).
  • PC-3M-Pro4 prostate cancer cells were stably transfected with a CMV-luc mammalian expression vector for luciferase as described above, and one clone (PC-3M-Pro4/luc + ) with the highest expression of luciferase activity was successfully used for in vivo whole body bioluminescent reporter imaging (BLI) by the method of Wetterwald et al. (2002).
  • PC-3M-Pro4/luc + cells were cultured in Dulbecco's modified Eagle's Medium (DMEM, Gibco-BRL, Breda, The Netherlands) containing 4.5 g of glucose/L and supplemented with 10% fetal calf serum (Gibco-BRL, Breda, The Netherlands), and 800 ⁇ g/ml geneticyne/G418 (Gibco- BRL, Breda, The Netherlands). Cells were regularly certified free of mycoplasma contamination.
  • DMEM Dulbecco's modified Eagle's Medium
  • Gibco-BRL Gibco-BRL, Breda, The Netherlands
  • fetal calf serum Gibco-BRL, Breda, The Netherlands
  • 800 ⁇ g/ml geneticyne/G418 Gibco- BRL, Breda, The Netherlands
  • PC-3M-Pro4/luc + cell cultures were harvested at sub-confluence after being re-fed with fresh medium 24 hours before inoculation preparation.
  • Cell suspensions of PC-3M-Pro4/luc + (1 x 10 5 cells/10 ⁇ L PBS for intra-osseous or orthotopic inoculation) were prepared as described above.
  • Animals Female and male nude (Balb/c nu/nu) mice were purchased from Charles River (L' Arbresle,
  • mice were housed in individual ventilated cages under sterile condition according to the Dutch guidelines for the care and use of laboratory animals (DEC). Sterile food and water were provided ad libitum. Mice were 6 weeks old when used for the orthotopic and intra-osseous inoculation of cancer cells.
  • mice were anesthetized by intraperitoneal injection of a 50 ⁇ L 1 :1 :1 mixture; ketamine HC1 (Stock solution of 100 mg/ml Nimatek; Vetimex Animal Health B.V., Bladel, The Netherlands.) + xylazine (2 % Rompun, Bayer AG, Leverkusen, Germany) + PBS (pH 6.8).
  • Intracardiac inoculation of cancer cells was performed under Isofluorane anesthesia (0.8 L/min, Isofluorane, Air Products, Waddinxveen, The Netherlands) using the Vapex3 system (VetTech Solutions Ltd, United Kingdom). At the end of the experimental period animals were sacrificed by cervical dislocation.
  • a single cell suspension of MDA-23 l-B/luc + or PC-3M-Pro4/luc + cells was injected into the right tibiae.
  • two holes at a distance of 4 mm to 5 mm from each other, and each with a diameter of approximately 0.35 mm, were drilled through the bone cortex of the upper right tibia with the aid of a dental drill. Space in the bone marrow was created by flushing out the bone marrow from the proximal end of the shaft.
  • the upper hole was sealed by surgical wax and 1 x 10 5 MDA-23 l-B/luc + or PC-3M-Pro4/luc + cells/10 ⁇ L PBS were slowly inoculated via a 30-gauge needle through the lower hole.
  • a single cell suspension of MDA-23 l-B/luc + cells was injected into mammary fatpads of 6- week-old Balb C nu/nu.
  • MDA-23 l-B/luc + cells (1 x 10 6 ) in 10 ⁇ L PBS were surgically inoculated into the mammary fatpads of anesthesized animals.
  • the cutaneous wound was sutured.
  • BMP-7 100 ⁇ g/kg/d
  • BMP-7 was freshly dissolved and prepared as a stock solution (1 mg/mL in 20 mM acetate buffer with 5% mannitol, pH 4.5). BMP-7 (20 ⁇ L) or vehicle solution (20 ⁇ L, 20 mM acetate buffer with 5% mannitol) was administered daily into the tail veins of mice for 21-23 days (10 ⁇ g/kg/day and/or 100 ⁇ g/kg/d human BMP-7). Control animals received injection of vehicle alone (no BMP-7). Tumor growth detected by whole body bioluminescent reporter imaging (BLI) and quantification of bioluminescent signal of cancer cells in vivo
  • Dissected tissues were fixed in 4% paraformaldehyde (pH 6.8), decalcified (only bones) as described previously and processed for paraffin embedding (van der Pluijm et al., Am. J. Pathol., 159: 971-982 (2001); van der Pluijm et al., J. Bone Miner. Res., 16: 1077-1091 (2001)).
  • Longitudinal sections (5 ⁇ m) were cut through the sagittal plane of tissues containing tumors. Sections were either submitted to hematoxylin-eosin (HE) staining or immunohistochemical staining, as described previously (van der Pluijm et al., Am. J.
  • BMP-7 The dose-dependent, anti-cancer activity of BMP-7 is also shown vividly in representative BLI photographs in Figure 6 that compare tumor growth (seen as imaging signal) for two control mice, two mice treated with BMP-7 at a dose of 10 ⁇ g/kg/d, and two mice treated with BMP-7 at a dose of 100 ⁇ g/kg/d.
  • Example 3 BMP-7 expression in highly malignant uveal melanoma cells and cell lines. Uveal melanomas of the eye are among the most malignant of all cancer cells.
  • the level of BMP-7 expression was determined in 30 uveal melanoma tissue samples from actual uveal melanoma patients and also from selected uveal melanoma cell lines using semi-quantitative PCR analysis for steady state BMP-7 mRNA levels. Tumors of enucleated eyes of patients with uveal melanoma were included in this study, which conformed to the requirements of the Declaration of Helsinki.
  • tumor fragments were snap-frozen in liquid nitrogen and stored at -80°C for use in PCR procedures and immunohistochemistry.
  • Species-specific semi-quantitative PCR was used for the evaluation of BMP-7 expression in cancer cells in vitro and in vivo as described previously by van der Pluijm et al. (J. Bone Miner. Res., 16(6): 1077-1091 (2001)).
  • cDNA was prepared by reverse transcription of mRNA and then co-amplified with human specific BMP-7 primers over 32- 38 cycles in 25 mL of reaction volume containing reaction buffer (75 mM Tris-HCl, pH 9.0; 20 mM (NH 4 ) 2 SO 4 , 0.01% (w/v) Tween 20), 2.0 mM MgCl 2 , 200 mM dNTPs, 0.25 mM sense (S) and antisense (AS) primer (Eurogentec, Seraing, Belgium), 0.125 U Goldstar DNA polymerase (Eurogentec). Negative controls, in which cDNA or cDNA and internal standard were omitted, were run in parallel in each experiment.
  • the primers used to detect human BMP-7 (hBMP-7) steady-state levels'of BMP-7 specific mRNA by semi-quantitative PCR in melanoma cancer cells had the following sequences (although other primers specific for BMP-7 exon sequences may also be used): CAT GCT GGA CCT GTA CAA CG (SEQ ID NO:l), for the hBMP-7 "sense" primer, and
  • expression of BMP-7 was below the level of detection in cells of melanoma cell lines OMM-1 (lane 1), OCM-8 (lane 3), Mel-202 (lane 4), OCM-1 (lane 5), OMM-1.5 (lane 6), 92-1 (lane 7), Mel- 285 (lane 8), OMM-1.3 (lane 9), and Mel-290 (lane 10) as compared to normal melanocytes, whereas cells of melanoma cell line OCM-3 (lane 2) expressed a detectable, but low, level of BMP-7.
  • the data are consistent with the findings for other malignant forms of cancer, such as breast and prostate cancer (above), and indicate that a reduction or loss of expression of BMP-7 is diagnostic of malignant tumors.
  • Example 4 Stable transfections for expression of BMP-7 in melanoma cancer cells.
  • Stable transfectants of OCMl uveal melanoma cell lines expressing BMP-7 were prepared using a Flp-InTM Flp recombinase-mediated site-specific integration and expression system following the manufacturer's protocol (Invitrogen, Breda, The Netherlands; see, also Sauer, Curr. Opin. Biotech., 5: 521-527 (1994); O'Gorman et al., Nature Biotechnology, 14: 315-319 (1996)).
  • This type of expression system employed Flp-InTM host OCMl cell lines that have a single Flp recombinase target (FRT) site located at a transcriptionally active genomic locus.
  • FRT Flp recombinase target
  • Flp-InTM host cell lines were generated by transfection with the Flp-InTM target site vector pFRT/lacBlasticidine and selection on Blasticidine. Transfected clones are screened by Southern blotting and by assaying for ⁇ -galactosidase activity to identify those clones that have a single FRT integrated at a transcriptionally active locus. Upon transfection and recombination with the pcDNA5/FRT plasmid vector containing the gene of interest, i.e., encoding BMP-7, hygromycin resistant cell clones that stably express BMP- 7 were obtained. Mock-transfected control cells were designated OCMl-FRT/mock and BMP-7 expressing stable transfectants were designated OCMl -FRT/BMP-7.
  • OCM1-FRT/MOCK and OCMl -FRT/BMP-7 cells were seeded in a 6-well plate (120,000 cells/mL). After 4 days of culture, conditioned medium from both cultures was analyzed by ELISA for BMP-7 expression. As shown in Figure 12, OCMl -FRT/BMP-7 cells expressed significant levels of BMP-7. OCMl-FRT/mock (control) cancer cells grew in a comparable manner to cells of the original OCMl melanoma cell line, and morphological features of OCMl-FRT/mock cells were also comparable to the original OCMl cells. In contrasts, cellular characteristics of the BMP-7expressing OCM1- FRT/BMP-7 were different.
  • Proliferation in vitro was strongly and significantly (p ⁇ 0.05) inhibited in the BMP-7 expressing OCMl -FRT/BMP-7 cells when compared to OCMl- FRT/mock cancer cells (see, Figure 13).
  • cellular morphology of OCMl - FRT/BMP-7 cells differed from the OCMl-FRT/mock cells in that the BMP-7 expressing cells exhibited more epithelial characteristics, e.g., expression of vimentin, a marker for the mesenchymal phenotype for advanced malignancy and metastasis, was strongly down regulated in OCMl -FRT/BMP-7 cells as compared to OCMl-FRT/mock cells.
  • OCMl-FRT/mock cancer cells and OCMl -FRT/BMP-7 cancer cells were also orthotopically implanted into the eyes of mice, and growth of uveal melanoma tumors assessed after 3 weeks. Tumor growth of the orthotopically implanted melanoma cells was strongly and significantly decreased in the case of the BMP-7 expressing cancer cells compared to the highly malignant mock cells.
  • the percent total area of implanted eyes occupied by melanoma tumor i.e., tumor burden
  • Example 5 Survey of BMP expression in stably transfected prostate and breast cancer cell lines grown in vitro and in vivo.
  • human PC-3M-Pro4 prostate cancer cells implanted into the tibiae of nude mice did not express BMP-7 mRNA, whereas expression of mRNA for other major BMPs was detected. Consistent with the above data, BMP-7 mRNA was not detected in cells of human breast cancer cell line MDA-MB-231. However, among various breast cancer cell lines tested, cells of the mouse mammary carcinoma cell line NF9006 were found to express BMP-7 mRNA, although expression was strongly down-regulated by TGF- ⁇ .
  • NF9006 is a known mouse cell line derived from transgenic mice that carry an activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV) (see, Muller et al., Cell, 54(1): 105-115 (1988)). Such transgenic mice that uniformly express the MMTV/c-neu gene develop mammary adenocarcinomas that involve the entire epithelium in each mammary gland.
  • NF9006 cells were a gift of Dr. P. Leder (Harvard Medical School, Boston, Massachusetts). BMP-7 protein expression was also detected in various cell lines by ELISA.
  • BMP-7 protein expression was detected in cells of BMP-7 expressing, stably transfected prostate cell line PC-3M-Pro4/BMP-7 and breast cancer cell line OCMl -FRT/BMP-7, described above, but not in cells of other human prostate and breast cancer cell lines.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne des méthodes de diagnostic et de traitement du cancer basées sur la propriété de la protéine morphogénétique 7 (BMP-7) permettant d'inhiber ou d'inverser la progression d'une cellule cancéreuse vers un état plus malin.
PCT/US2004/031468 2003-09-24 2004-09-24 Proteine morphogenetique osseuse 7 (bmp-7) utilisee dans le diagnostic et le traitement du cancer WO2005030933A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US50555203P 2003-09-24 2003-09-24
US60/505,552 2003-09-24

Publications (2)

Publication Number Publication Date
WO2005030933A2 true WO2005030933A2 (fr) 2005-04-07
WO2005030933A3 WO2005030933A3 (fr) 2006-05-26

Family

ID=34393035

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/031468 WO2005030933A2 (fr) 2003-09-24 2004-09-24 Proteine morphogenetique osseuse 7 (bmp-7) utilisee dans le diagnostic et le traitement du cancer

Country Status (1)

Country Link
WO (1) WO2005030933A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017046226A2 (fr) * 2015-09-17 2017-03-23 Histide Ag Association pharmaceutique pour convertir une cellule néoplasique en cellule non-néoplasique et ses utilisations
WO2019191115A1 (fr) 2018-03-26 2019-10-03 Icahn School Of Medicine At Mount Sinai Procédés de traitement d'un cancer résiduel minimal
EP3906932A3 (fr) * 2015-09-17 2021-12-01 Histide AG Association pharmaceutique pour la conversion d'une cellule neoplasique en cellule non-neoplasique et ses utilisations
EP3949975A1 (fr) * 2015-09-17 2022-02-09 Histide AG Association pharmaceutique d'agoniste de recepteur de facteur de croissance et d'inhibiteur de proteine d'adhesion pour convertir une cellule neoplastique en cellule non-neoplastique, et ses utilisations
US11578110B2 (en) 2015-08-25 2023-02-14 Histide Ag Compounds for inducing tissue formation and uses thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANDREWS P.W. ET AL: 'Inhibition of proliferation and Induction of differentiation of Pluripotent Human Embryonal carcinoma Cells by Osteogenic Protein-1 (Or Bone Morphogenetic protein-7)' LAB. INVEST. vol. 71, no. 2, 1994, pages 243 - 251, XP000929102 *
CARICASOLE A. ET AL: 'Bone Morphogenetic Proteins (BMPs) induce epithelial differentiation of NT2D1 human embryonal carcinoma cells' INT.J.DEV.BIOL. vol. 44, 2000, pages 443 - 450, XP002998558 *
FRANZEN A. ET AL: 'BMP-7 Induced Cell Cycle Arrest of Anaplastic Thyroid Carcinoma Cells via p21 and p27' BIOCHEM. BIOPHYS. RES. COMMUN. vol. 285, 2001, pages 773 - 781, XP002998557 *
SCHWALBE M. ET AL: 'Differential expression and regulation of bone morphogenetic protein 7 in breast cancer' INT. J. ONCOL. vol. 23, 2003, pages 89 - 95, XP002998555 *
SULZBACHER I. ET AL: 'The expression of bone morphogenetic proteins in osteosarcoma and its relevance as a prognostic parameter' J. CLIN. PATHOL. vol. 55, 2002, pages 381 - 385, XP002998556 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11578110B2 (en) 2015-08-25 2023-02-14 Histide Ag Compounds for inducing tissue formation and uses thereof
WO2017046226A2 (fr) * 2015-09-17 2017-03-23 Histide Ag Association pharmaceutique pour convertir une cellule néoplasique en cellule non-néoplasique et ses utilisations
WO2017046226A3 (fr) * 2015-09-17 2017-06-29 Histide Ag Association pharmaceutique pour convertir une cellule néoplasique en cellule non-néoplasique et ses utilisations
EP3906932A3 (fr) * 2015-09-17 2021-12-01 Histide AG Association pharmaceutique pour la conversion d'une cellule neoplasique en cellule non-neoplasique et ses utilisations
EP3949975A1 (fr) * 2015-09-17 2022-02-09 Histide AG Association pharmaceutique d'agoniste de recepteur de facteur de croissance et d'inhibiteur de proteine d'adhesion pour convertir une cellule neoplastique en cellule non-neoplastique, et ses utilisations
WO2019191115A1 (fr) 2018-03-26 2019-10-03 Icahn School Of Medicine At Mount Sinai Procédés de traitement d'un cancer résiduel minimal
CN112166187A (zh) * 2018-03-26 2021-01-01 西奈山伊坎医学院 治疗微小残留癌的方法
US20210121527A1 (en) * 2018-03-26 2021-04-29 Icahn School Of Medicine At Mount Sinai Methods of treating minimal residual cancer
EP3775171A4 (fr) * 2018-03-26 2022-06-15 Icahn School of Medicine at Mount Sinai Procédés de traitement d'un cancer résiduel minimal

Also Published As

Publication number Publication date
WO2005030933A3 (fr) 2006-05-26

Similar Documents

Publication Publication Date Title
ES2234818T3 (es) Procedimiento para tratar canceres que expresan el factor de crecimiento endotelial d.
JP4457169B2 (ja) 非機能性ｐ２ｘ７レセプター抗体、癌及びその他の容態の診断及び処理
ES2575508T3 (es) CAFs de duocarmicina que presentan una mejora en la actividad antitumoral in vivo
Lango et al. Gastrin-releasing peptide receptor-mediated autocrine growth in squamous cell carcinoma of the head and neck
JP2014515759A (ja) 扁平上皮がんを治療する方法関連出願
Tang et al. Piceatannol inhibits the IL-1β-induced inflammatory response in human osteoarthritic chondrocytes and ameliorates osteoarthritis in mice by activating Nrf2
JP2005533001A5 (fr)
CA2813101C (fr) Expression du recepteur eph dans les cellules souches tumorales
WO2008141438A1 (fr) Modulateurs gabaergiques destinés au traitement d'affections des voies respiratoires
ES2431040T3 (es) Detección y modulación de la angiogénesis mediada por Slit y Roundabout (Robo), y sus usos
JP2015514062A5 (fr)
Assaggaf et al. Prevention of phenytoin-induced gingival overgrowth by lovastatin in mice
WO2005030933A2 (fr) Proteine morphogenetique osseuse 7 (bmp-7) utilisee dans le diagnostic et le traitement du cancer
ES2324818T3 (es) Procedimiento de dosificacion de ngf para el diagnostico in vitro de cancer de mama y utilizacion terapeutica.
ITRM20090081A1 (it) Uso di inibitori della proteina 90k per la preparazione di un medicamento per la cura o prevenzione dei tumori.
US20080145369A1 (en) Autocrine Growth Factor Receptors and Methods
JP2022514087A (ja) ＩＬ－１β結合抗体の使用
US20030166503A1 (en) Method for recognizing and determining GnRH receptors and use of GnRH agonist for decreasing the replication of malignant cells bearing GnRH receptors of tumors orginating in the nervous system and/or meninges and/or of Kaposi sarcoma
EP3815709A1 (fr) Compositions pharmaceutiques et leur utilisation pour soulager la tolérance cancérostatique et l'amélioration de la sensibilité de médicaments anti-cancer
Grandis et al. The role of peptide growth factors in head and neck carcinoma
ES2277461T3 (es) Terapia de hcg para el tratamiento de cancer de mama metastatico.
EP3443986A1 (fr) Amélioration de l'efficacité d'une chimiothérapie par la sphingosine-1-phosphate
US20040105860A1 (en) Cell modulation using a cytoskeletal protein
JP2022510606A (ja) 子宮内膜症を処置するための組成物および方法
TWI842966B (zh) 一種具緩解抗癌藥物抗藥性及增加抗癌藥物敏感性之醫藥組合物及其用途

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
122 Ep: pct application non-entry in european phase