WO1997003681A1 - Procedes pour tuer selectivement des cellules ou inhiber selectivement la croissance de cellules exprimant le gene waf1 - Google Patents

Procedes pour tuer selectivement des cellules ou inhiber selectivement la croissance de cellules exprimant le gene waf1 Download PDF

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Publication number
WO1997003681A1
WO1997003681A1 PCT/US1996/011886 US9611886W WO9703681A1 WO 1997003681 A1 WO1997003681 A1 WO 1997003681A1 US 9611886 W US9611886 W US 9611886W WO 9703681 A1 WO9703681 A1 WO 9703681A1
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Prior art keywords
wafl
cells
inhibitor
administering
ofthe
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PCT/US1996/011886
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English (en)
Inventor
Alonzo H. Ross
Wojciech Poluha
Dorota K. Poluha
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Worcester Foundation For Biomedical Research, Inc.
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Priority to AU65483/96A priority Critical patent/AU6548396A/en
Priority to EP96925359A priority patent/EP0850066A1/fr
Publication of WO1997003681A1 publication Critical patent/WO1997003681A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors

Definitions

  • the present invention relates to the selective killing or to the selective inhibition ofthe growth of cells. More particularly, the present invention relates to methods and compositions useful in killing or inhibiting the growth of cells expressing the WAFl gene.
  • the present invention has particular utility in the field of cancer therapy.
  • the cell cycle consists of four phases: (1) the mitotic phase, M, in which a cell with duplicated genetic material undergoes mitosis to produce daughter cells, (2) a first gap phase, G Ton during which the cell grows and is, generally, metabolically active, (3) a synthesis phase, S, during which the cell duplicates its genetic material, and (4) a second gap phase, G 2 , in which the cell prepares for mitosis and, perhaps, another cell cycle.
  • M the mitotic phase
  • G Ton a first gap phase
  • S synthesis phase
  • G 2 second gap phase
  • the control or regulation ofthe cell cycle is a complex process involving dozens of intracellular and extracellular signals which appear to act at particular "checkpoints" at the transitions between different stages ofthe cell cycle.
  • CDKs cyclin-dependent kinases
  • CDIs cyclin-dependent kinase inhibitors
  • This gene appears to play a role in the normal cellular response to DNA damage in mammalian cells such that, when DNA damage has occurred, the p53 gene is expressed and the cell either arrests at the G, stage ofthe cell cycle or enters the apoptotic cell death pathway.
  • Heritable defects in p53 are believed to be causally involved with oncogenesis either by allowing cells with damaged DNA to replicate and pass on the damaged genetic material or by preventing damaged cells from undergoing apoptosis. See, e.g., Canman, et ai. (1995) Genes & Devel. 9:600-611.
  • the p53 protein is now known to be a transcriptional regulator and several genes have been identified with putative p53-binding sites (El-Deiry et al. (1993) CeU 75:817-825).
  • WAFl is a CDI which appears to be involved in the arrest ofthe cell cycle at a checkpoint in G,. Because ofthe varying means by which it has been identified, this CDI has been known by a variety of names in the recent literature: WAFl (Wild-type p53 activated fragment 1; El-Deiry, et al. (1993) CeU 75:817-825), Cipl (CDK-interacting protein 1; Harper, et al. (1993) £eli 75:805-816), SDH (Senescent cell-derived inhibitor 1; Noda, et al. (1994) Exp. Cell Res.
  • Sheikh et al. investigated the regulation of WAFl expression in human breast carcinomas (Sheikh et al. (1994) Oncogene 9:3407-3415).
  • Cells expressing wild-type p53 were found to constitutively express WAFl at levels 26-33 fold higher than p53 mutants.
  • exogenous mutant p53 (Val-143) counteracted the positive transcriptional effect of endogenous wild-type p53.
  • WAFl could be induced both by p53- independent as well as p53-dependent signaling pathways.
  • the DNA-damaging agent etoposide and serum starvation could induce both WAFl expression and growth arrest even in p53 mutant cell lines.
  • Nakanishi et al. investigated the effect of WAFl inhibition on normal human fibroblasts grown to G 0 arrest in vitro (Nakanishi et al. (1995) Proc. Natl. Acad. Sci. (TJSA ⁇ ) 92:4352-4356). These authors found higher levels of WAFl protein in cells arrested in G 0 than in mitogen-stimulated cells in early S phase. In addition, Nakanishi et al. found that expression of WAFl -antisense RNA caused cells arrested in G 0 to resume the cell cycle and proliferate.
  • WAFl expression in adult mice was found to be localized to terminally differentiated tissues and to be unaltered in p53-knockouts. Thus, these authors concluded that WAFl has a role in normal terminal differentiation and embryogenesis but that WAFl induction is not dependent upon p53 induction.
  • WAFl may be induced either by a p53-dependent pathway activated by DNA damage or by a p53 -independent pathway activated by mitogens.
  • the fact that WAFl is induced by mitogens is curious in light of its apparent role in G] arrest and suggests that WAFl may have an unknown function independent of G, arrest.
  • WAFl is an important element in regulating the cell cycle transition from G, to S phase and that WAFl can be induced by a p53 -dependent pathway activated by DNA damage as well as p53- independent pathways activated by cell differentiation signals.
  • p53 -independent inducers of WAFl have now been shown to include transforming growth factor ⁇ (TGF ⁇ ) in keratinocytes (Datto et al. (1995) Proc. Natl. Acad. Sci.
  • TPA 12-0-tetradecanoyl ⁇ horbol-13-acetate
  • Vit D3 l,25-dihydroxyvitamin D3
  • RA retinoic acid
  • DMSO dimethyl-sulfoxide
  • the present invention provides methods of killing or inhibiting the growth of cells in which the WAFl gene is being expressed.
  • the methods comprise the administration a WAFl inhibitor to cells in which a WAFl -dependent pathway has been induced and wherein said inhibitor is administered in an amount sufficient to kill or inhibit the growth of said cells.
  • the inhibitor comprises a WAFl -antisense oligonucleotide.
  • the oligonucleotide may be a modified oligonucleotide in which the backbone linkages, termini or bases have been modified to increase resistance to degradation or to increase binding affinity.
  • Particularly preferred modified oligonucleotides are those containing a plurality of phosphorothioate linkages.
  • the oligonucleotide is selected from the group consisting of (a) oligonucleotides comprising at least 10 consecutive bases from the WAFl sequence disclosed as SEQ ID NO.: 1 ; and (b) oligonucleotides capable of hybridizing to the complements ofthe oligonucleotides of (a) under physiological conditions.
  • the inhibitor is a vector which expresses a WAFl -antisense oligonucleotide.
  • the inhibitor is an inhibitor of WAFl gene transcription.
  • the inhibitor is an inhibitor of WAFl protein activity such as an intracellular anti- WAFl antibody or a fragment of either WAFl or a CDK which competitively inhibits the formation of complexes between the WAFl protein and endogenous CDKs.
  • WAFl protein activity such as an intracellular anti- WAFl antibody or a fragment of either WAFl or a CDK which competitively inhibits the formation of complexes between the WAFl protein and endogenous CDKs.
  • the method may include subjecting the cells to a treatment which induces a WAFl -dependent pathway.
  • This pathway may be a p53-dependent pathway and the treatment may be one which induces p53 gene expression.
  • treatments may include X- irradiation, ⁇ -irradiation, UV-irradiation, administering to the cells an alkylating agent , administering to the cells cisplatin, administering to the cells bleomycin, doxorubicin, administering to the cells 5-fluorouracil, administering to the cells genistein, administering to the cells hydrogen peroxide, or administering to the cells methylmethane sulfonate.
  • the pathway may be a p53 -independent pathway.
  • the treatment may include administering to the cells differentiation-inducing agents or inhibitors of DNA synthesis.
  • Such treatments may include administering to the cells a pharmaceutical composition selected from the group consisting of PDGF, FGF, EGF, NGF, ⁇ -interferon, TGF ⁇ , TPA, Vit D3, RA, DMSO, MyoD, IL2, rapamycin, aphidicolin, etoposide, methotrexate, cytosine arabinoside, 6- thioguanine, 6-mercaptopurine.
  • the method is particularly intended for use with cells which abnormally proliferate, and in a particularly important embodiment with cancer cells in a human host.
  • Preferred cancer cells include neuroblastoma, melanoma, epithelioma, fibroblastoma, carcinoma, leukemia and myeloma cells.
  • the present invention provides a method of treating a human patient having cancerous cells in which a WAFl -dependent pathway has been induced comprising administering a WAFl inhibitor to the patient in an amount sufficient to kill or inhibit the growth ofthe cells.
  • the WAFl inhibitor may be any of those described above.
  • the treatment may also include subjecting the patient to a treatment which induces a WAF1- dependent pathway.
  • This additional treatment may include any of those described above.
  • the additional treatment may include radiation or chemotherapy therapy which induces DNA damage in the cells or which induces growth arrest or differentiation ofthe cells.
  • the preferred WAFl inhibitor a WAFl -antisense oligonucleotide and, preferably, a modified WAFl -antisense oligonucleotide with a plurality of phosphorothioate linkages.
  • the invention further involves use ofthe foregoing compositions in the preparation of medicaments and in particular the preparation of medicaments for treating abnormal cell proliferation such as cancer.
  • the present invention depends, in part, upon the surprising discovery that the selective inhibition ofthe expression of WAFl in cells in which a WAFl -dependent pathway has been induced does not lead to cell proliferation but, rather, to cell death.
  • WAFl is believed to play a role in the G, arrest and/or terminal differentiation of cells and, therefore, it has previously been proposed that inducing the WAFl gene or otherwise increasing the levels ofthe WAFl protein might be a useful means of controlling the growth of cell lines in vitro and, more important, tumor cells in vivo.
  • the present invention in contrast, provides methods of killing or inhibiting the growth of cells by inhibiting transcription ofthe WAFl gene, translation ofthe WAFl mRNA transcript, or activity ofthe WAFl protein.
  • WAFl is only one component in these "WAFl -dependent" pathways, it appears essential. Therefore, when WAFl is inhibited after induction of a WAFl- dependent pathway, it is believed that the cells cannot complete the Gj arrest or differentiation pathways and, instead, initiate programmed cell death or apoptosis.
  • the present invention is useful only with cells in which the WAFl gene is being expressed.
  • the target cells ofthe invention may, without prior treatment, already be expressing WAF 1.
  • the methods of the invention include an additional treatment which induces the expression of a WAFl -dependent pathway.
  • additional treatment will typically comprise radiation or chemotherapies.
  • WAFl WAFl.
  • WAFl human cyclin-dependent kinase inhibitor gene described in the various references cited herein and denoted as “WAFl,” “Cipl,” “CIPl,” “SDIl,” or “p21.”
  • a cDNA to one allele of WAFl was disclosed in Harper et al. (1993) and El-Deiry et al. (1993).
  • one WAFl allele and the corresponding protein are disclosed herein as SEQ. ID NO.: 1 and SEQ ID NO.: 2, respectively.
  • WAFl -dependent pathway means a biochemical pathway in human cells in which expression ofthe WAFl gene is induced. Such a pathway may require induction of WAFl expression by the p53 tumor suppressor protein, in which case the pathway is said to be “p53-dependent.” Alternatively, the pathway may not require induction of WAFl expression by the p53 tumor suppressor protein, in which case the pathway is said to be "p53 -independent.”
  • WAFl inhibitor means a compound which, when present in a cell, inhibits the transcription ofthe WAFl gene, translation ofthe WAFl mRNA transcript, or activity ofthe WAFl protein product.
  • WAFl -antisense antibodies or fragments of antibodies which act intracellularly against the WAFl protein or WAFl-cyclin-CDK complexes, fragments of CDKs which would act as competitive inhibitors of WAF1 interaction with endogenous CDKs, small molecule inhibitors such as OK- 1035 [Biochemical and Biophysical Research Communications 221, 207-212 (1996)] and ribozymes which inhibit WAFl expression.
  • WAFl -antisense oligonucleotide As used herein, the term "WAFl -antisense oligonucleotide” or “WAFl -antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide or a modified oligonucleotide which hybridizes under physiological conditions to a WAFl mRNA transcript or to WAFl DNA and, thereby, acts as a WAFl inhibitor.
  • the antisense molecule is constructed and arranged so as to interfere with transcription or translation of WAFl upon hybridization with the target.
  • the exact length ofthe antisense oligonucleotide and its degree of complementarity will depend upon the specific target selected, including the sequence ofthe target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be selected so as to hybridize selectively with the target under physiological conditions, i.e., to hybridize substantially more with the target sequence than with any other sequence in the target cell under physiological conditions.
  • the p53 protein is a potent inducer of WAFl expression. Therefore, p53 or inducers of p53 may be used to induce WAFl -dependent pathways.
  • p53 inducers are known in the art and, because of p53's activity as a tumor suppressor, are already in use in the field of cancer therapy.
  • Preferred p53 inducers are DNA-damaging radiation (e.g., X-rays, ⁇ -rays, UV), DNA-damaging compounds (e.g., alkylating agents such as nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, busulfan, and nitrosureas (BCNU, CCNU, methyl-CCNU); as well as etoposide, cisplatin, bleomycin, doxorubicin, 5 -fluorouracil, genistein, hydrogen peroxide, and methy lmethane sulfonate).
  • WAFl has been shown to be induced by p53 -independent pathways.
  • Additional preferred inducers of WAFl therefore include WAFl -inducing growth factors and differentiation factors such as PDGF, FGF, EGF, nerve growth factor (NGF), ⁇ - interferon, TGF ⁇ , TPA, Vit D3, RA, DMSO, MyoD and IL2, rapamycin and inhibitors of DNA synthesis such as aphidicolin, methotrexate, cytosine arabinoside, 6-thioguanine, 6- mercaptopurine.
  • WAFl -inducing growth factors and differentiation factors such as PDGF, FGF, EGF, nerve growth factor (NGF), ⁇ - interferon, TGF ⁇ , TPA, Vit D3, RA, DMSO, MyoD and IL2, rapamycin and inhibitors of DNA synthesis such as aphidicolin, methotrexate, cytosine arabinoside, 6-thioguanine, 6- mercaptopurine.
  • the present invention depends, in part, upon the discovery that the selective inhibition of the expression of WAFl in cells in which a WAFl -dependent pathway has been induced leads to the inhibition of cell growth and/or cell death.
  • the present invention requires that the targeted cells be subject to conflicting conditions: conditions inducing WAFl -dependent G, arrest or differentiation and conditions under which WAFl expression is inhibited.
  • the WAFl -inhibiting conditions comprise treatment with WAFl -antisense oligonucleotides.
  • WAFl -antisense oligonucleotides Based upon SEQ. ID NO.:l, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules.
  • WAFl -antisense oligonucleotides should comprise at least 10 bases and, more preferably, at least 15 bases.
  • antisense molecules are between 15 and 32 bases.
  • the antisense oligonucleotides comprise 18-20 bases.
  • oligonucleotides may be chosen which are antisense to any region ofthe WAFl gene or mRNA transcript, in preferred embodiments the antisense oligonucleotides correspond to the N-terminal or, more preferably, translation initiation region of the WAFl mRNA or to mRNA splicing sites.
  • WAFl antisense may, preferably, be targeted to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al. (1994) Cell. Mol. Neurobiol. 14(5):439-457) and at which proteins are not expected to bind.
  • the WAFl -inhibiting antisense oligonucleotides ofthe present invention need not be perfectly complementary to the WAFl gene or mRNA transcript in order to be effective. Rather, some degree of mismatches will be acceptable if the antisense oligonucleotide is of sufficient length. In all cases, however, the oligonucleotides should have sufficient length and complementarity so as to selectively hybridize to a WAFl transcript under physiological conditions. Preferably, of course, mismatches are absent or minimal.
  • the WAFl -antisense oligonucleotides may have one or more non-complementary sequences of bases inserted into an otherwise complementary WAFl -antisense oligonucleotide sequence.
  • Such non-complementary sequences may "loop" out of a duplex formed by a WAFl transcript and the bases flanking the non-complementary region. Therefore, the entire oligonucleotide may retain an inhibitory effect despite an apparently low percentage of complementarity.
  • the WAFl -antisense oligonucleotides ofthe invention may be composed of deoxyribonucleotides, ribonucleotides, or any combination thereof.
  • the 5' end of one nucleotide and the 3' end of another nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleotide linkage.
  • These oligonucleotides may be prepared by art recognized methods such as phosphoramidate, H-phosphonate chemistry, or methylphosphoramidate chemistry (see, e.g., Uhlmann et al. (1990) Chem. Rev. 90:543-584; Agrawal (ed.) Meth. Mol. Biol..
  • the WAFl -antisense oligonucleotides ofthe invention also may include modified oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not compromise their ability to hybridize to nucleotide sequences contained within the transcription initiation region or coding region ofthe WAFl gene.
  • modified oligonucleotide as used herein describes an oligonucleotide in which at least two of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide.
  • the most preferred synthetic linkages are phosphorothioate linkages. Additional preferred synthetic linkages include alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. Oligonucleotides with these linkages or other modifications can be prepared according to known methods (see, e.g., Agrawal and Goodchild (1987) Tetrahedron Lett. 28:3539-3542; Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083; Uhlmann et al. (1990) Chem.
  • modified oligonucleotide also encompasses oligonucleotides with a modified base and/or sugar.
  • modified oligonucleotides include oligonucleotides having the sugars at the most 3' and/or most 5' positions attached to chemical groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • Other modified ribonucleotide-containing oligonucleotides may include a 2'-O-alkylated ribose group such as a 2'-O-methylated ribose, or oligonucleotides with arabinose instead of ribose.
  • unoxidized or partially oxidized oligonucleotides having a substitution in one nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides.
  • modifications may be at some or all ofthe internucleoside linkages, at either or both ends ofthe oligonucleotide, and/or in the interior ofthe molecule (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10:152-158 and Agrawal (ed.) Meth. Mol. Biol.. Humana Press, Totowa, NJ (1993) Vol. 20).
  • modified oligonucleotides are oligonucleotides having nuclease resistance-conferring bulky substituents at their 3' and/or 5' end(s) and/or various other structural modifications not found in vivo without human intervention.
  • Other modifications include additions to the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose.
  • the inhibition of WAF 1 expression need not be accomplished by means of a WAF 1 - antisense oligonucleotide. Rather, inhibitors of WAFl transcription or WAFl protein activity also may be employed to the same effect.
  • antibodies or fragments of antibodies which act intracellularly against the WAFl protein or WAFl-cyclin-CDK complexes fragments of CDKs or recombinant genes encoding fragments of CDK which would act as competitive inhibitors of WAFl interaction with endogenous CDKs, and ribozymes which inhibit WAFl expression.
  • WAFl is only one component in these "WAFl -dependent" pathways, it appears essential.
  • WAFl is inhibited, it is believed that the cells cannot complete the G, arrest or differentiation pathways and, instead, initiate programmed cell death.
  • the WAFl -inhibiting conditions must be more specific to WAFl than the WAFl -inducing conditions so that the WAFl -dependent pathway is induced while WAFl itself is inhibited.
  • the WAFl -inducing conditions and WAFl -inhibiting conditions should not act at the same point or level in the WAFl -dependent pathway.
  • simultaneous administration ofthe p53 protein (as the WAFl -inducing condition) and administration of anti-p53 antibodies (as the WAFl -inhibiting condition) would be ineffective because the conditions would largely counteract each other at the same level.
  • the methods ofthe present invention are particularly well suited for use in the field of cancer therapy. Because current radiation and chemotherapy methods typically involve treatments which cause DNA damage and/or induce terminal differentiation of tumor cells and/or inhibit proliferation of tumor cells, these treatments, in many cases, already induce expression of the WAFl gene. Therefore, by combining these treatments with administration of a WAF1- inhibitor, a more effective means of killing or inhibiting the growth of tumor cells is provided.
  • a cancer patient is treated with ionizing radiation (e.g., X-rays or ⁇ -rays) or an agent (e.g.,doxorubicin) which causes DNA damage, induces p53 expression and, thereby, induces a p53-dependent, WAFl -dependent pathway toward cell cycle arrest.
  • ionizing radiation e.g., X-rays or ⁇ -rays
  • an agent e.g.,doxorubicin
  • a WAFl inhibitor is administered to the patient.
  • This administration may be oral, intravenous, parenteral, cutaneous or subcutaneous.
  • the administration also may be localized to the region ofthe tumor by injection to or perfusion ofthe tumor site.
  • the WAFl inhibitor is WAFl -antisense administered in a pharmaceutically acceptable carrier or a recombinant vector with a WAF 1 -antisense gene which expresses a WAFl -antisense oligonucleotide.
  • a cancer patient is treated with compounds which induce expression of a p53-independent, WAFl -dependent pathway.
  • WAFl -dependent pathway compounds which induce expression of a p53-independent, WAFl -dependent pathway.
  • these embodiments may be of particular importance because p53 mutations are associated with many cancers and mutant p53 proteins may fail to induce WAFl expression.
  • the patient has been treated with an agent that induces p53 -independent G, arrest or differentiation ofthe tumor cells and at the same time, or shortly thereafter, is administered a WAFl inhibitor.
  • Appropriate non-proliferation and differentiation agents are well known in the art and vary according to tumor type.
  • ⁇ -interferon and mezerein may be administered to melanoma patients, or TGF ⁇ may be administered to any of a number of different types of cancer patients in order to induce a WAFl -dependent pathway.
  • a WAFl inhibitor, as described above, may be simultaneously or subsequently administered to kill or further inhibit the growth ofthe tumor cells.
  • the cancer may be virtually any cancer, including, but not limited to, brain cancer including glioblastoma and medulloblastoma, breast cancer, cervical cancer, colon cancer, endometrial cancer, liver cancer, lung cancer, oral cancer, prostate cancer, sarcomas, skin cancer, and renal cancer.
  • WAF1 -antisense oligonucleotides or other WAFl inhibitors may be administered as part of a pharmaceutical composition.
  • Such a pharmaceutical composition may include the WAFl inhibitor in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness ofthe biological activity ofthe active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • the characteristics ofthe carrier will depend on the route of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • the pharmaceutical composition ofthe invention may also contain other active factors and/or agents which inhibit WAFl expression or otherwise inhibit cell growth or increase cell death. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect or to minimize side-effects caused by the WAFl inhibitor ofthe invention.
  • the pharmaceutical composition ofthe invention may be in the form of a liposome in which WAFl -antisense oligonucleotides are combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids wliich exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which are in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323.
  • the pharmaceutical composition ofthe invention may further include compounds such as cyclodextrins and the like which enhance delivery of oligonucleotides into cells, as described by Zhao et al. (in press).
  • cationic detergents e.g. Lipofectin
  • the inhibitor When a therapeutically effective amount of a WAFl inhibitor is administered orally, the inhibitor will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition ofthe invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder may contain from about 5 to 95% of a WAFl -antisense oligonucleotide and preferably from about 25 to 90% ofthe oligonucleotide.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, sesame oil, or synthetic oils may be added.
  • the liquid form ofthe pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition may contain from about 0.5 to 90% by weight of a WAFl -antisense oligonucleotide and preferably from about 1 to 50% ofthe oligonucleotide.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the WAFl inhibitor, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or another vehicle as known in the art.
  • the pharmaceutical composition ofthe present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • administration of WAF 1 -antisense oligonucleotides is localized to the region of the targeted cells in order to maximize the delivery ofthe WAFl -antisense, minimize WAFl inhibition in non-target cells, and minimize the amount of WAFl -antisense needed per treatment.
  • administration is by direct injection at or perfusion ofthe site ofthe targeted cells, such as a tumor.
  • the WAFl -antisense oligonucleotides may be adhered to small particles (e.g.,microscopic gold beads) which are impelled through the membranes ofthe target cells (see, e.g., U.S. Pat. No. 5,149,655).
  • a recombinant gene is constructed which encodes a WAFl -antisense oligonucleotide and this gene is introduced within the targeted cells on a vector.
  • a WAFl -antisense gene may, for example, consist ofthe normal WAFl sequence, or a subset ofthe normal WAFl sequence, operably joined in reverse orientation to a promoter region.
  • An operable WAFl -antisense gene may be introduced on an integration vector or may be introduced on an expression vector. In order to be most effective, it is preferred that the WAFl- antisense sequences be operably joined to a strong eukaryotic promoter which is inducible or constitutively expressed.
  • the WAFl inhibitors are administered in therapeutically effective amounts.
  • these treatments are also administered in therapeutically effective amounts.
  • therapeutically effective amount means the total amount of each active component ofthe pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., the killing or inhibition ofthe growth ofthe target cells.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts ofthe active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • the amount of WAFl inhibitor in the pharmaceutical composition ofthe present invention will depend not only upon the potency ofthe inhibitor but also upon the nature and severity ofthe condition being treated, and on the nature of prior treatments which the patent has undergone. Ultimately, the attending physician will decide the amount of WAFl inhibitor with wliich to treat each individual patient. Initially, the attending physician will administer low doses ofthe inhibitor and observe the patient's response. Larger doses of a WAFl inhibitor may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
  • the WAFl inhibitor is a WAF1- antisense oligonucleotide and it is contemplated that the various pharmaceutical compositions used to practice the method ofthe present invention should contain about 1.0 ⁇ g to about 100 mg of oligonucleotide per kg body weight.
  • the duration of intravenous therapy using the pharmaceutical composition ofthe present invention will vary, depending on the severity ofthe disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of a WAFl -antisense oligonucleotide will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition ofthe present invention.
  • the efficacy ofthe present invention was demonstrated in vitro on a neuroblastoma cell line using conditions known to inhibit cell proliferation and to induce differentiation as the WAFl -inducing conditions and using treatment with WAFl -antisense oligonucleotides as WAFl -inhibiting conditions. As described above, this technique did not lead to cell proliferation but, on the contrary, increased cell death.
  • SH-SY5Y (Biedler, J. et al. (1978) Cancer Res. 38:3751-3757), a neuroblastoma cell line, was used as a model for neuronal terminal differentiation (LoPresti P. et al., (1992) Cell Growth Diff. 3:627-635; Poluha, W.
  • aphidicolin + NGF is a WAF1- inducing treatment for these cells.
  • the differentiated cells require NGF for survival and, in the presence of NGF, are stable for 4-6 weeks. These cells express neuronal markers and resemble sympathetic neurons.
  • NGF alone does not stop cell proliferation and induces only slight neurite extension (Chen, J. et al., (1990) Cell Growth Diff. 1:79-85; Sonnenfeld and Ishii, (1982) J. Neurosci. Res. 8:375-391). Treatment with aphidicolin does not induce neurite extension, and the cells resume proliferation, following removal of aphidicolin.
  • Cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine and 100 ⁇ g/ml of gentamicin.
  • fetal bovine serum 10% heat-inactivated fetal bovine serum
  • 2 mM glutamine 100 ⁇ g/ml of gentamicin.
  • gentamicin 100 ⁇ g/ml of gentamicin.
  • cells were plated on Primaria (Falcon Plastics) dishes or flasks and were treated with 100 ng/ml of NGF (2.5 S; Bioproducts for Science) and/or 0.3 ⁇ M of aphidicolin. Fresh (aphidicolin+NGF)-containing medium was added every 2-3 days.
  • WAFl To verify the role of WAFl in neuroblastic differentiation, expression ofthe mRNA for WAFl was assessed by Northern blotting. Expression ofthe WAFl mRNA transcript was up ⁇ regulated following treatment for 1 hr with aphidicolin+NGF and further increased as the aphidicolin+NGF treatment progressed. On day 6, the cells were changed from aphidicolin+NGF medium to NGF-containing medium. Despite the removal of aphidicolin from the medium, expression of WAFl transcripts was slightly greater on day 14 than on day 6. Treatment with aphidicolin alone also induced WAFl expression, but following removal on day 6 of aphidicolin from the medium, expression greatly declined. Treatment with NGF alone did not induce expression of WAFl.
  • an antisense oligonucleotide with phosphorothioate linkages was employed as a WAFl -inhibitor.
  • the cationic detergent Lipofectin was used (Quattrone, A. et al., (1995) Biochemica 1:25-29; Wagner, R. (1994) Nature 372:333-335). The low concentration of detergent used in the experiments had no effect on cell viability or differentiation.
  • Phosphorothioate oligonucleotides 100 ⁇ M
  • Lipofectin 1 mg/ml, a 1:1 (w/w) mixture of N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride and dioleoyl phosphatdylethanolamine (Gibco)) were incubated at 37 °C for 15 min.
  • the oligonucleotide- detergent mix was diluted with serum-containing medium and added to the SH-S Y5 Y cells. In most cases, the dilution was 1:100 giving a final oligonucleotide concentration of 1 ⁇ M.
  • Fresh oligonucleotide-containing medium was added to the cells each day.
  • a WAFl -antisense oligonucleotide was employed which is complementary to the region around the translational start site (5'-TCC CCA GCC GGT TCT GAC AT-3' from Oligos, Etc.).
  • an 18-mer Control- 1 (5'-TGG ATC CGA CAT GTC AGA-3') and an antisense oligonucleotide directed against M. tuberculosis (5'-CGC TTC ATC CTG CCG TGT CGG-3'), Control-2, were employed.
  • ADDRESSEE WOLF, GREENFIELD & SACKS, P.C.
  • CTGCCCCCCA GCCTCTGGCA TTAGAATTAT TTAAACAAAA ACTAGGCGGT TGAATGAGAG 777
  • GAAGGGCACC CTAGTTCTAC CTCAGGCAGC TCAAGCAGCG ACCGCCCCCT CCTCTAGCTG 1557

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Abstract

La présente invention se rapporte à des procédés pour tuer sélectivement des cellules ou inhiber la croissance de cellules. L'invention consiste à administrer un inhibiteur de WAF1 à des cellules ayant induit un processus dépendant de WAF1. La quantité d'inhibiteur WAF1 administrée aux cellules est suffisante pour inhiber leur croissance ou même tuer les cellules. Le procédé peut consister à soumettre les cellules à un traitement qui induira un processus dépendant de WAF1.
PCT/US1996/011886 1995-07-20 1996-07-19 Procedes pour tuer selectivement des cellules ou inhiber selectivement la croissance de cellules exprimant le gene waf1 WO1997003681A1 (fr)

Priority Applications (2)

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AU65483/96A AU6548396A (en) 1995-07-20 1996-07-19 Methods for selectively killing or inhibiting the growth of cells expressing the waf1 gene
EP96925359A EP0850066A1 (fr) 1995-07-20 1996-07-19 Procedes pour tuer selectivement des cellules ou inhiber selectivement la croissance de cellules exprimant le gene waf1

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US124895P 1995-07-20 1995-07-20
US60/001,248 1995-07-20

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0941124A1 (fr) * 1996-11-21 1999-09-15 Board Of Regents Of The University Of Nebraska Compositions d'oligonucleotides antisens pour tuer selectivement les cellules cancereuses
US6242201B1 (en) 1994-11-03 2001-06-05 Cyclacel Limited Identification of the P21WaF1-PCNA interaction site and therapeutic applications thereof
US6455593B1 (en) * 1995-06-27 2002-09-24 The Henry Jackson Foundation For The Advancement Of Military Medicine Method of dynamic retardation of cell cycle kinetics to potentiate cell damage
US6962792B1 (en) 1996-05-08 2005-11-08 Cyclacel Limited Methods and means for inhibition of Cdk4 activity
US8809350B2 (en) 2004-08-27 2014-08-19 Cyclacel Limited Purine and pyrimidine CDK inhibitors and their use for the treatment of autoimmune diseases

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5302706A (en) * 1991-12-16 1994-04-12 Baylor College Of Medicine Senescent cell derived inhibitors of DNA synthesis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5302706A (en) * 1991-12-16 1994-04-12 Baylor College Of Medicine Senescent cell derived inhibitors of DNA synthesis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MOLECULAR AND CELLULAR DIFFERENTIATION, 1996, Vol. 4, No. 1, MANFREDI et al., "The Cyclin-Dependent Kinase Inhibitor p21 as a Target for Differentiation Therapy", pages 33-45. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6242201B1 (en) 1994-11-03 2001-06-05 Cyclacel Limited Identification of the P21WaF1-PCNA interaction site and therapeutic applications thereof
US6455593B1 (en) * 1995-06-27 2002-09-24 The Henry Jackson Foundation For The Advancement Of Military Medicine Method of dynamic retardation of cell cycle kinetics to potentiate cell damage
US6962792B1 (en) 1996-05-08 2005-11-08 Cyclacel Limited Methods and means for inhibition of Cdk4 activity
EP0941124A1 (fr) * 1996-11-21 1999-09-15 Board Of Regents Of The University Of Nebraska Compositions d'oligonucleotides antisens pour tuer selectivement les cellules cancereuses
US8809350B2 (en) 2004-08-27 2014-08-19 Cyclacel Limited Purine and pyrimidine CDK inhibitors and their use for the treatment of autoimmune diseases

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AU6548396A (en) 1997-02-18
EP0850066A1 (fr) 1998-07-01

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