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WO2001074381A2 - Inhibiteurs de calpaines pour le traitement du cancer - Google Patents

Inhibiteurs de calpaines pour le traitement du cancer Download PDF

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Publication number
WO2001074381A2
WO2001074381A2 PCT/US2001/010521 US0110521W WO0174381A2 WO 2001074381 A2 WO2001074381 A2 WO 2001074381A2 US 0110521 W US0110521 W US 0110521W WO 0174381 A2 WO0174381 A2 WO 0174381A2
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cells
calpain
cpi
cell
inhibitor
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WO2001074381A3 (fr
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Fatih M. Uckun
De-Min Zhu
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Parker Hughes Institute
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Parker Hughes Institute
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to inhibition of calpain for treatment of cancer and more particularly to the induction of apoptosis in human cancer cells by inhibitors of calpain.
  • Apoptosis also referred to as programmed cell death, is a form of cell death characterized by cell membrane blebbing, cytoplasmic shrinkage, nuclear chromatin condensation and DNA fragmentation. Willie (1980) Int. Rev. Cytol. , 68: 251-306. Many genes are involved in the apoptotic process. In general, the products of these genes are classified as either inducers or inhibitors of apoptosis. Apoptotic signaling pathways involve substantial interactions between the products of the apoptotic inducers and the apoptotic inhibitors. Oltvai et al. (1993) Cell, 80: 293-299.
  • Calpains a group of calcium activated cytosolic proteases, have been implicated in calcium dependent death (Squier et al. (1997) J Irnmonol. , 158: 3690-3697; Villa et al. (1998) J Cell Science, 111: 713-722; Nafh et al. (1996) Neutropharmcol. Neurotoxicol., 8:249-255; Nath et al. (1996) Biochem J, 319: 683-690; Gressner et al. (1997) Biochem. Biophys. Res. Commun., 231: 457-462; McGinnis et al. (1998) J Biol. Chem., 273: 19993-20000; Jordan et al.
  • calpain inhibitors are generally considered inhibitors of calpain-dependent apoptosis. Nath et al. (1996) Neutropharmcol. Neurotoxicol., 8:249-255; Nath et al. (1996) Biochem J, 319:
  • the present invention involves the surprising and unexpected discovery that calpain inhibitors and methods of use for calpain inhibitors trigger caspase dependent apoptosis in cancer cells and in cells having aberrant levels of calpain.
  • One aspect of the present invention is a method for inhibiting the growth of tumor cells in a subject by administering to the subject a calpain inhibitor. The method can include inducing the tumor cells to become apoptotic. Preferable types of tumor cells in the context of the present invention include acute lymphoblastic leukemia and non-Hodgkin's lymphoma.
  • Another aspect of the invention is a method of treating cancer cells in a subject by administering a therapeutically effective amount of calpain inhibitor to the subject.
  • the cancer cells can be solid cancer cells, for example squamous cell carcinoma, but are more preferably white blood cells, for example acute lymphoblastic leukemia and non-Hodgkin's lymphoma.
  • Another aspect of the invention is a method for inhibiting inflammatory disease in a subject by administering to the subject a calpain inhibitor.
  • the inflammatory disease can be rheumatoid arthritis.
  • Another aspect of the invention is a method for inducing cytotoxicity in a cell by administering a cytotoxic dose of calpain inhibitor to the cell.
  • Another aspect of the invention is a pharmaceutical composition having a substantially purified calpain inhibitor and a pharaceutically acceptable carrier.
  • Another aspect of the invention is a method for inhibiting growth of a tumor cell by making a recombinant vector that expresses a calpain inhibitor and administering the recombinant vector to the tumor cell.
  • Another aspect of the invention is a method for inducing apoptosis in a cell by expressing a heterologous nucleic acid sequence encoding the calpain inhibitory peptide in a host cell having enhanced calpain activity as compared to control host cells.
  • Figure 1 is a microscopic image of tumor cells stained with anti-calpain monoclonal antibodies and FITC-labeled goat anti-mouse IgG.
  • Figure 2 shows a representative FACS-correlated display of CPI-2 treated human ALL and NHL cells. The percentages indicate the fraction of cells at an early stage of apoptosis, as measured by MC540 fluorescence, and the fraction of cells at an advanced stage of apoptosis, as measured by dual MC540/PI fluorescence.
  • Figure 3 illustrates that myeloid leukemia cells are resistant to apoptosis as induced by CPI-2 treatment.
  • the percentage of apoptotic cells is the sum of the percentages of the cells at early and advanced apoptotic stages.
  • Figure 4 illustrates that solid tumor cell lines are generally resistant to apoptosis as induced by CPI-2 treatment. Only the SQ-20B cell line showed CPI- 2 induced apoptosis. The percentages indicate the fraction of cells at an early stage of apoptosis, as measured by MC540 fluorescence, and the fraction of cells at an advanced stage of apoptosis, as measured by dual MC540/PI fluorescence.
  • Figure 5 shows a representative FACS-correlated display of CPI-2 treated
  • FIG. 6 shows a representative FACS-correlated display of CPI-2 treated human MOLT-3 and SQ-20B cells in the presence and absence of caspase inhibitor I.
  • apoptosis refers to the genetically programmed process of cell death characterized by cell membrane blebbing, cytoplasmic shrinkage, nuclear chromatin condensation and DNA fragmentation.
  • Apoptotic regulatory genes include the p53 tumor suppresser gene, the Bcl-2 gene family, and the caspase family of genes.
  • cell line refers to cells established in ex vivo culture. It is a characteristic of host cells discussed in the present disclosure that they be capable of expressing calpain and calpain inhibitors.
  • suitable host cells include insect and mammalian cells. Specific examples of such cells include SF9 insect cells (Summers and Smith (1987) Texas Agriculture Experiment Station Bulletin, 1555), human primary embyonal kidney cells (293 cells), Chinese hamster ovary (CHO) cells (Puck et al. (1958) Proc. Natl. Acad. Sci.
  • HELA human cervical carcinoma cells
  • SQ-20B human breast cancer cells
  • BT-20 human breast cancer cells
  • U373 human breast cancer cells
  • K-562 glioblastoma cells
  • RS4;11 highly radiation-resistant MILL-AF4 fusion transcript positive t(4;ll) pre-pre B ALL cell line
  • ALL-1 multidrug resistant BCR-ABL fusion transcript positive t(9;22) pro-B ALL cell line
  • RAMOS highly radiation-resistant and p53 deficient Burkitt's lymphoma cell line
  • DAUDI Burkitt's leukemia/lymphoma cell line
  • JURKAT and MOLT-3 T-lineage ALL/NHL cells
  • NALM-6 pre-B ALL cell line
  • LYN deficient clone and BTK deficient clone of DT-40 cells Uckun et al. (1996) Science, 273: 1096-1100), etc.
  • calpain inhibitor refers to chemical compositions, polypeptides, polynucleotides, etc. that inhibit the enzymatic activity of calpain family members.
  • calpain inhibitor II N- Ac-Leu-Leu-Met
  • CPI-2 may be purchased from Calbiochem, La Jolla, California.
  • cancer refers to or describe the physiologic condition in mammals that is typically characterized by the loss of responsiveness to normal growth controls.
  • cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. Cancers can originate from among other things epithelial cells like a carcinoma, or can originate from mesenchymal cells like cancers of the blood cells. Examples of carcinomas include but are not limited to squamous cell, adenocarcinoma and melanoma and examples of cancers of the blood cells are leukemia and lymphoma.
  • solid tumors are tumors that are not of blood cell origin.
  • cDNA refers to recombinant DNA formed from the rnRNA of the target protein, in this case a calpain inhibitor rriRNA. cDNA molecules can be inserted into vectors that favor their expression in host cells.
  • control host cell refers to a cell that has been cultured in parallel with a cell treated under the specified experimental condition but unlike the treated cell, the host cell has not undergone the specified experimental condition. Control cells represent a baseline from which comparisons are made.
  • inflammatory disease or “disorder” refers to a fundamental pathogenic process consisting of a dynamic complex of cytologic and histologic reactions that occur in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by physical, chemical, or biologic agent. Examples of inflammatory disease within the context of the present invention include, rheumatoid arthritis, osteoarthritis, etc.
  • nucleic acid sequence refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides deterrnines the order of amino acids along the polypeptide chain. The deoxyribonucleotide sequence thus codes for the amino acid sequence.
  • pharmaceutically acceptable salt thereof includes an acid addition salt or a base salt.
  • pharmaceutically acceptable carrier includes any material which, when combined with a compound of the invention, allows the compound to retain biological activity, such as the ability to induce apoptosis of leukemia or breast tumor cells, and is non-reactive with the subject's immune system.
  • compositions comprising such carriers are formulated by well known conventional methods
  • polynucleotide refers to a linear sequence of nucleotides.
  • the nucleotides are either a linear sequence of polyribonucleotides or poly deoxyribonucleotides, or a mixture of both.
  • Examples of polynucleotides in the context of the present invention include - single and double stranded DNA, single and double stranded RNA, and hybrid molecules that have both mixtures of single and double stranded DNA and RNA.
  • the polynucleotides of the present invention may have one or more modified nucleotides.
  • subject in the context of this invention means a mammal, i.e. , any class of higher vertebrates that nourish their young with milk secreted by mammary glands.
  • treating means the prevention or reduction of severity symptoms or effect of a pathological condition, including prolonging life expectancy.
  • treatment includes prevention, of tumor growth, reduction of tumor size, enhanced tumor cell death, and increased apoptosis.
  • tumor cell within the context of the present invention is used synonymously with cancer cell and means a cell that has lost, in some manner, its ability to respond to normal growth signals, i.e., is undergoing abnormally regulated growth.
  • vector refers to a first piece of DNA, usually double-stranded, which may have inserted into it a second piece of DNA, for example a piece of foreign DNA like the cDNA of CPI-2.
  • Foreign DNA is defined as heterologous DNA, which is DNA that may or may not be naturally found in the host cell and includes additional copies of nucleic acid sequences naturally present in the host genome.
  • the vector transports the foreign DNA into a suitable host cell. Once in the host cell the vector may be capable of integrating into the host cell chromosomes.
  • the vector may also contain the necessary elements to select cells containing the integrated DNA as well as elements to promote transcription of mRNA from the transfected DNA.
  • vectors within the scope of the present invention include, but are not limited to, plasmids, bacteriophages, cosmids, retro viruses, and artificial chromosomes.
  • ALL Acute Lymphoblastic Leukemia
  • NHL non-Hodgkin's Lymphoma
  • BTK Bruton's tyrosine kinase
  • CPI-2 Propidium Iodide
  • the present invention includes novel and unexpected methods of use for calpain inhibitors in the selective triggering of caspase dependent apoptosis in cancer cells having aberrant levels of calpain as well as in non-cancerous cells having aberrant levels of calpain.
  • Calpain inhibitors for example CPI-2, may be useful agents in the treatment of cancer, for example by inhibitting tumor growth or by killing tumor cells.
  • Preferable cancers for treatment with embodiments of the present invention include ALL and NHL.
  • Calpain inhibitors may also be of use in the treatment of inflammatory disease or any other disease state where the cells show aberrant calpain expression.
  • the calpain inhibitors of the invention are effective cytotoxic agents, for example, against tumor cells such as acute lymphoblastic leukemia and non- Hodgkin's lymphoma.
  • the cytotoxic effects of calpain inhibitors are achieved by treating, such as tumor cells, with micromolar amounts of the inhibitory compound.
  • a particularly useful anti-tumor agent is CPI-2 as shown in the Examples below.
  • Other calpain inhibitors may be known to those of skill in the art.
  • the present invention also includes polynucleotide encoding calpain inhibitors including fragments, analogs and derivatives of CPI-2 as cytotoxic compounds.
  • a fragment, analog or derivative may be made by mutagenesis techniques or other methods known to the art.
  • the polynucleotide fragments, analogs and derivatives may include substitutions, deletions or additions that involve one or more nucleotides.
  • Another embodiment of the present invention are polynucleotides that are at least 75 % identical to the polynucleotide sequence for CPI-2 as used as cytotoxic compounds. Further, preferred embodiments are between 80 and 95% identical to the CPI-2 cDNA and highly preferred embodiments are between 95 and 99% identical.
  • the calpain inhibitors of the invention can also be used in methods of tumor treatment, for example, by administering to a subject a calpain inhibitor in order to achieve an inhibition of tumor cell growth, a killing of tumor cells, induced apoptosis, and/or increased patient survival time.
  • the present invention also includes polynucleotide fragments, analogs and derivatives of CPI-2 as used in tumor treatment.
  • a fragment, analog or derivative may be made by mutagenesis techniques or other methods known to the art.
  • the polynucleotide fragments, analogs and derivatives may include substitutions, deletions or additions that involve one or more nucleotides.
  • polynucleotides that are at least 75% identical to the polynucleotide sequence for CPI-2 as used in tumor treatment. Further, preferred embodiments are between 80 and 95% identical to the CPI-2 cDNA and highly preferred embodiments are between 95 and 99% identical.
  • calpain inhibitors of the invention are suitable for use in mammals.
  • mammals means any class of higher vertebrates that nourish their young with milk secreted by mammary glands, including, for example, humans, rabbits, and monkeys.
  • the calpain inhibitors of the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, including a human patient, in a variety of forms adapted to the chosen route of administration.
  • the compounds are preferably administered in combination with a pharmaceutically acceptable carrier, and may be combined with or conjugated to specific delivery agents, including targeting antibodies and/or cytokines.
  • the calpain inhibitors can be administered by known techniques, such as orally, parentally (including subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques), by inhalation spray, topically, by absorption through a mucous membrane, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles.
  • Pharmaceutical compositions of the invention can be in the form of suspensions or tablets suitable for oral administration, nasal sprays, creams, sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions or suppositories.
  • the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation.
  • the compositions can contain microcrystallme cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents.
  • the compositions can contain microcrystallme cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
  • compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation.
  • the compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons or other solubilizing or dispersing agents known in the art.
  • the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable dispersing or wetting and suspending agents such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • the compositions can be prepared by mixing with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.
  • Preferred administration routes include orally, parenterally, as well as intravenous, intramuscular or subcutaneous routes. More preferably, the compounds of the present invention are administered parenterally, i.e., intravenously or intraperitoneally, by infusion or injection. In one embodiment of the invention, the compounds may be administered directly to a tumor by tumor injection; or by systemic delivery by intravenous injection. Solutions or suspensions of the compounds can be prepared in water, isotonic saline (PBS) and optionally mixed with a nontoxic surfactant. Dispersions may also be prepared in glycerol, liquid polyethylene, glycols, DNA, vegetable oils, triacetin and mixtures thereof. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • PBS isotonic saline
  • Dispersions may also be prepared in glycerol, liquid polyethylene, glycols, DNA, vegetable oils, triacetin and mixtures thereof. Under ordinary conditions of storage
  • the pharmaceutical dosage form suitable for injection or infusion use can include sterile, aqueous solutions or dispersions or sterile powders comprising an active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol such as glycerol, propylene glycol, or liquid polyethylene glycols and the like, vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size, in the case of dispersion, or by the use of nontoxic surfactants.
  • the prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, buffers, or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents delaying absorption-- for example, aluminum monosterate hydrogels and gelatin.
  • Sterile injectable solutions are prepared by incorporating the compounds in the required amount in the appropriate solvent with various other ingredients as enumerated above and, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • novel polynucleotides substantially similar to the CPI-2 polynucleotide sequence are subcloned into an extra- chromosomal vector.
  • the subcloned polynucleotide(s) may be joined to a vector having a cis-acting or regulatory element for increased propagation in a host cell
  • trans-acting factors involved are supplied to the host, supplied by a second vector or supplied by the vector itself upon introduction into the host).
  • This aspect of the invention allows for the in vivo and in vitro expression of the CPI-2 polynucleotide, thus permitting an analysis of cathepsin activity and function.
  • vectors can be used in the context of this invention, including:
  • PcDNA3 vector (Invitrogen), vectors having the T3 and T7 polymerase promoters, vectors having the SV40 promoter or the CMV promoter, or any other promoter that either can direct expression of a polypeptide off a polynucleotide, or that one wishes to test for the ability to direct expression of a polypeptide off a polynucleotide.
  • the present invention also includes polynucleotide fragments, analogs and derivatives of CPI-2 that are subcloned into extra-chromosomal vecotors.
  • a fragment, analog or derivative may be made by mutagenesis techniques or other methods known to the art.
  • the polynucleotide fragments, analogs and derivatives may include substitutions, deletions or additions that involve one or more nucleotides.
  • polynucleotides that are at least 75 % identical to the polynucleotide sequence for CPI-2 as subcloned into extra-chromosomal vectors. Further, preferred embodiments are between 80 and 95 % identical to the CPI-2 cDNA and highly preferred embodiments are between 95 and 99% identical.
  • host cells can be genetically engineered to incorporate the polynucleotides encoding calpain inhibitors of the present invention and to express the polypeptides of the present invention.
  • Techniques required for this aspect of the invention are well known in the art (Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Press, 1989) and can include calcium phosphate transfection, dextran sulfate transfection, electroporation, lipofection and viral infection (see Graham and van der Eb (1978) Virology, 52, 456-457; Chisholm et al. (1995) DNA
  • the host cells of the present invention may be of any type, including, but limited to, non-eukaryotic and eukaryotic cells. Host cells are cultured using standard tissue culture techniques in conventional media as is well known in the art. The level of expression of the CPI-2 cDNA introduced into a host cell of the invention depends on multiple factors, including gene copy number, efficiency of transcription, messenger RNA processing, stability, and translation efficiency.
  • high level expression of a desired CPI-2 polypeptide according to the present invention will typically involve optimizing one or more of those factors.
  • the experiments of the present invention were conducted on several different acute lymphoblastic leukemia, non-Hodgkin's lymphoma, myeloid leukemia and solid tumor cell lines.
  • the acute lymphoblastic leukemia, non- Hodgkin's lymphoma and myeloid leukemia were all cultured in RPMI 1640 (Gibco BRL, Grand Island, NY) supplemented with 10% head-inactivated fetal bovine serum (Summit Biotech, Ft. Collins, CO), 100 U/ml penicillin and 100 ⁇ g/ml streptomycin (Gibco BRL, Grand Island, NY).
  • the solid tumor lines HeLa and U373, were cultured in MEM supplemented with non-essential amino acids, Earl's BSS, 1 mM sodium pyruvate and 10% fetal bovine serum.
  • the solid tumor line SQ-20B was grown in DMEM supplemented with 20% fetal bovine serum and the line PC-3 cells was cultured in Ham's F12K medium supplemented with 10% fetal bovine serum.
  • wild type and LYN and BTK deficient, chicken lymphoma B-cell DT-40 clones (Uckon et al.
  • Quantitative flow cytometic apoptosis detection assays and TUNEL assays were performed to determine if calpain inhibitor-2 (CPI-2) treated ALL, NHL, myeloid lymphoma cells and solid tumor cells undergo apoptosis.
  • CPI-2 calpain inhibitor-2
  • Quantitative flow cytometric apoptosis detection assays were performed on ALL cells (ALL-1, RS4;11, JURKAT), NHL cells (RAMOS, DAUDI), myeloid leukemia cells (K562, HL-60, U937) and solid tumor cells (SQ20B, HeLa, U373, PC-3). Cells were treated with CPI-2 at varying concentrations for 24 hours. Just prior to the assay, one mg/ml stock solutions of MC540 and PI were filtered through a 0.22 ⁇ m filter and stored at 4°C in the dark.
  • Each cell line was harvested, and lxlO 6 cells suspended and stained with 5 ⁇ g/ml MC540 and 10 ⁇ g/ml propidium iodide (PI) (Uckun et al. (1996) Science, 273: 1096-1100) at 4°C in the dark for 24 hours. Stained cells were analyzed with a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA) using the 488-nm excitation from an argon laser.
  • PI propidium iodide
  • MC540 and PI emissions were split with a 600-nm short pass diachronic mirror; a 575-nm band pass filter was placed in front of one photomultiplier tube to measure MC540 emission, and 635-nm band pass filter was used for PI emission.
  • approximately 10,000 cells were analyzed by FACS and the percentage of cells at early (MC540 fluorescence only) and advanced (dual MC540 plus PI fluorescence) stages of apoptosis obtained.
  • DNA cleavage as an indicator of apoptosis was determined using the in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling method (TUNEL assay) of Boehringer Mannheim (Cell Death Detection Kit) as described in Zhu et al. (1999) Clin. Can. Res., 5:355-360. Briefly, CPI-2 treated cells (50 or 100 ⁇ M) and control cells were harvested and resuspended in phosphate-buffered saline (PBS) at a density of 5 x 10 6 cells/ml.
  • PBS phosphate-buffered saline
  • Permeabilized cells were washed 3 times with PBS and incubated for one hour at
  • Calpain Inhibitor-2 Induced Apoptosis is LYN and BTK-Independent
  • Example III The materials and methods discussed in Example III were used to determine whether the protein tyrosine kinases LYN or BTK (Uckun et al. (1995) Science, 267: 886-891); Wang et al. (1996) J Exp. Med., 184: 831-838; Vassilev et al. (1996) J Biol. Chem., 274: 1646-1656) were required for CPI-2 induced apoptosis in CPI-2 sensitive cells. Wild type DT-40, LYN deficient and BTK deficient DT-40 lymphoid cells (Uckun et al. (1996) Science, 273: 1096-1100) were examined for susceptibility to CPI-2 induced apoptosis after treatment with 50 ⁇ M CPI-2. Results:
  • Example III The materials and methods discussed in Example III were used to determine whether the family of caspases (Zuo et al. (1997) Cell, 90: 405-413; Li et al. (1997) Cell, 91: 479-489; Schlegel et al. (1996) J Biol. Chem., 271: 1841- 1844; Thornberry et al. (1998) Science, 281: 1312-1316; Green et al. (1998) Science, 281:1309-1312; Henkart (1996) Immunity, 4: 195-201) were required for CPI-2 induced apoptosis in CPI-2 sensitive cells.
  • caspase inhibitor I inhibitited CPI-2-induced apoptosis.
  • This data provides evidence that an apoptosis-promoting caspase system is activated following calpain inhibition with CPI-2.
  • This data also illustrates a previously unknown cross-talk between the caspase mediators of apoptosis and calpain in neoplastic lymphoid cells and likely cross-talk between the two family members in the normal regulation of cells.
  • Calcium/calmodulin-dependent protein kinase IV is cleaved by capsase-3 and calpain in SH-SY5Y human neuroblastoma cells undergoing apoptosis. J Biol Chem 273: 19993-20000.
  • Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bcl-2-independent regulator of apoptosis in human B-lineage lymphoma cells. Proc Natl Acad Sci U S A 92: 9575-9579.
  • CPP32/apopain is a key interleukin 1 deta converting enzyme-like protease involved in Fas-mediated apoptosis. J Biol Chem 271: 1841-1844.

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Abstract

Les modes de réalisation de la présente invention concernent des méthodes permettant d'inhiber la croissance des cellules tumorales ou de traiter le cancer chez un sujet par l'administration d'un ou de plusieurs inhibiteurs de calpaïnes. L'invention concerne également des méthodes permettant d'inhiber les cellules de maladies inflammatoires ainsi que d'autres cellules exprimant la calpaïne chez un sujet à l'aide d'un ou de plusieurs inhibiteurs de calpaïnes. L'invention concerne en outre l'induction de cytotoxicité ou d'apoptose dans des cellules par l'administration à la cellule d'une dose cytotoxique ou apoptotique d'un ou de plusieurs inhibiteurs de calpaïnes. L'invention concerne enfin l'administration de vecteurs exprimant la calpaïne à des cellules tumorales afin d'inhiber la croissance des cellules tumorales.
PCT/US2001/010521 2000-03-31 2001-03-30 Inhibiteurs de calpaines pour le traitement du cancer Ceased WO2001074381A2 (fr)

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