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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P13/00—Drugs for disorders of the urinary system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• TGFß TGFß
• the effect of TGFß on the tumor cell results in cooperation with (i) the expression of oncogenic Ras, with (ii) the overexpression of normal Ras or receptor tyrosine kinases that activate the Ras signaling pathway or with (iii) others in the tumor cell activated oncogenes to convert epithelial cells into fibroblastoid cells with invasive potential.
• TGFßl is primarily produced by infiltrating cells of the tumor stroma, such as fibrocytes, endothelial cells, lymphocytes and macrophages.
• the interaction of the tumor cells with the different cell types of the tumor stroma should trigger the efficient production and / or activation of TGFßl. This in turn should induce the epithelial tumor cells to transform into the fibroblastoid and invasive phenotype.
• TGFßl fibroblastoid cells
• autocrine loop autocrine loop
• Other mutations or selective mechanisms are likely to cause some of these invasively growing cells to migrate in and out of blood vessels and eventually form secondary tumors at distant sites.
• This model is consistent with findings that show that increased TGFß1 expression is also involved in the progression to malignancy in a prostate cancer mouse model (Thompson, et al., 1992; 1993).
• TßRII-dn the dominant-negative TGFß receptor
• the cells form smaller or larger compact clumps in the collagen gel and grow on plastic as epithelioid cells, which form hemicysts (domes) and large amounts of E -cadherin and ZO-1 express.
• the TßRII-dn apparently transformed the cells back into cells with an epithelial phenotype (fibroblastoid-epithelial conversion, FEC).
• TGFß itself as a detection system.
• This assay principle is based on the knowledge gained in the context of the present invention that the activation of the TGFß receptor system in oncogene expressing cells by the ligand TGFß causes the autocrine production of TGFß, which has an autocrine loop effect on the cells.
• Inhibitors of the TGF ⁇ receptor found in one of the test systems described in the primary screen are expediently tested for their specificity in secondary screens. This can be done primarily by direct inhibition of the TGFß-dependent EF conversion of EpRas cells in collagen gels. Another possibility is the incubation of converted EpRas cells (eg from mouse tumors) sown on plastic dishes in sparse density with the inhibitors of the TGF ⁇ receptor found. Effective substances should trigger the conversion of fibroblastoids into epithelial cells even in the presence of TGFß. The same substances should cause re-epithelialization (FE conversion) in CT26 cells.
• Ras inhibitors in the sense of the definition of the present invention either inhibit Ras directly by inhibiting the activation / function of Ras itself or by inhibiting the activation / function of a Ras effector molecule which acts in the Ras signal transmission path below Ras.
• Examples are inhibitors of Raf, such as Raf antisense oligonucleotides (Monia et al., 1996).
• Ras activation can also be brought about by inhibiting these receptors.
• genes coding for mutations of the Ras proteins H-Ras, K-Ras or N-Ras which lead to a constitutive activation of Ras are introduced into mammalian cells, e.g. using retroviral vectors, and the selective cytotoxic effect of test substances on the ras ⁇ transformed cells is determined.
• a suitable method for identifying ras inhibitors is e.g. described in EP-A 604 181.
• TGFß receptor
• TGFß receptor
• most tumors constantly produce TGFß see below
• TGFß which is released into the environment and has an immunosuppressive effect there, i.e. inhibits the function of cytotoxic T lymphocytes and other cells of the immune system. If the conversion of invasive tumor cells into non-invasive, more epithelial cells is effected by the TGFß receptor inhibitor, these should switch off the TGFß secretion and thus be more easily attacked and lysed by cytotoxic T cells.
• the cells obtained from the tumors were grown for the first 5 days in the presence of G418 or hygromycin.
• the collagen gels were similarly digested with collagenase to isolate the cells for subsequent tissue culture.
• Ras-transformed mouse breast epithelial cells show two completely different cell phenotypes. When grown on plastic substrates, these cells grow as ordered, dome-forming monolayers (hemicysts), indicating a polarized epithelial phenotype (Fig. 1A, B). However, after injection into mice, these same polarized cells formed tumors consisting of depolarized spindle-shaped cells capable of invasive growth (Fig. 1A, C). In order to obtain further information about the mechanisms underlying this phenotypic plasticity, a combination of in vivo and in vitro was used experimental approaches investigated the observed cell conversion in detail.
• the EpH4 cells When injected subcutaneously, the EpH4 cells developed into layers of epithelial cells that sometimes formed lumens and expressed cytokeratins but did not express vimentin (Fig. 2D). After a long time these cells necrotized and were reabsorbed by the surrounding stroma.
• Fig. 3A Ep4H cells form channels and "end-bud” similar swellings in serum-free collagen gels. On plastic, these cells formed a regular epithelial monolayer and crests (Hemicysts, domes).
• 6A-D Clones of fibroblastoid cells isolated from a tumor (ex-tumor cells) are gradually converted into clones consisting of epithelial cells. To generate the clones, 500 cells per 100 mm dish were sown in medium containing 1% FCS. The medium was changed daily to dilute any autocrine factors. The same typical cell clone was photographed on day 1 (A), day 3 (B), day 5 (C) and day 10 (D) after plating. The gradual conversion of fibroblastoid cells to cells with an epithelial morphology is clearly visible.
• F Fibroblastoids, EpRas cells isolated from a tumor were selected in G418 for 5 days (to remove cells from the recipient animal) and then sown in serum-free collagen gels. This was done either in the absence (E) or in the presence (F) of TGFßl neutralizing antibodies. It can be seen that the tumor cells develop into lumen-shaped structures in the presence of a TGFßl neutralizing antibody, while in the absence of the antibody they form the expected disordered cell strands.
• TGFß1 In contrast, higher concentrations of TGFß1 (> 0.25 ng / ml) caused the normal epithelial cells to stop growing and die by programmed cell death (apoptosis) (Fig. 5C). This is an important difference between the normal epithelial cells and the cells containing Ha-Ras. While the latter are not induced to apoptose even at 20 times higher concentrations of TGFßl (5 ng / ml) and undergo EFC without exception, the TGFßl concentration is , which regulates morphogenetic processes in normal breast epithelial cells, strictly specified. An aberrant morphogenesis due to excessive TGFßl concentrations may be avoided by instead inducing growth inhibition and apoptosis in the cells.
• TßRII-dn TGFß receptor type II
• a cDNA of this TßRII-dn was expressed with the help of retroviral vectors in Ras-transformed EpH4 cells (Ep-Ras).
• EpH4 cells Ras-transformed EpH4 cells
• the clones obtained grew very slowly and required medium with a high (20%) serum content in order to be able to expand.
• CT26-TßRII-dn can also prevent CT26 cells that are already in circulation from settling in the lungs.
• CT26 control cells grew into large reticular and strand-like structures consisting of spindle-shaped, fibroblastoid cells (left panels)
• CT26-TßRII-dn type 1 cells formed compact cell clumps with very few cells growing out of the gel (middle panels).
• the CT26-TßRII-dn type 2 clones form only tiny compact cell groups without any ability to grow into the collagen gel (right panels).
• the diagram in the upper part of the picture shows the course of the experiment.
• Syngeneic Balb-C mice (3 per cell type and cell amount) were intravenously injected with CT26 control cells and several CT26-TßRII-dn clones (tail vein).
• CT26 control cells
• the figure shows that all three mice treated with 5,000 and 50,000 control cells (CT26) had died of lung metastases after 28 and 14 days (+), while all animals injected with CT26-TßRII-dn clones were still alive and free after 40 days of lung metastases were (-).
• TGF ⁇ -induced PAI-I expression is correlated in transient transfection tests with the tumor or metastasis formation of the corresponding cells.
• CT26 control cells and 5 CT26-TßRII-dn clones which had already been tested in mice (see Example 11, FIG. 16) were constructed with the 3TP-lux PAI-1 reporter gene construct (Wrana et al., 1992) transfected, stimulated with TGFß or left untreated and tested for PAI-1 expression (measurement of luciferase activity).
• TßRI T204D

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• 1996
  • 1996-04-05 DE DE19613691A patent/DE19613691A1/de not_active Ceased
• 1997
  • 1997-04-04 US US09/155,716 patent/US6383733B1/en not_active Expired - Fee Related
  • 1997-04-04 AU AU25085/97A patent/AU2508597A/en not_active Abandoned
  • 1997-04-04 EP EP97916436A patent/EP0896543A1/de not_active Withdrawn
  • 1997-04-04 WO PCT/EP1997/001699 patent/WO1997037678A1/de not_active Application Discontinuation
  • 1997-04-04 JP JP9535833A patent/JP2000509024A/ja active Pending
  • 1997-04-04 CA CA002250839A patent/CA2250839A1/en not_active Abandoned
• 2002
  • 2002-03-01 US US10/086,073 patent/US20020127542A1/en not_active Abandoned

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