WO2003105880A1 - Peptides selectivement letaux pour des cellules de mammiferes transformees et malignes - Google Patents

Peptides selectivement letaux pour des cellules de mammiferes transformees et malignes Download PDF

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WO2003105880A1
WO2003105880A1 PCT/US2003/007687 US0307687W WO03105880A1 WO 2003105880 A1 WO2003105880 A1 WO 2003105880A1 US 0307687 W US0307687 W US 0307687W WO 03105880 A1 WO03105880 A1 WO 03105880A1
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peptide
derivative
analog
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PCT/US2003/007687
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The Research Foundation Of The State University Of New York
Pincus, Matthew, R.
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Priority to AU2003277292A priority Critical patent/AU2003277292A1/en
Priority to EP03760194A priority patent/EP1490089A4/fr
Priority to CA2477878A priority patent/CA2477878C/fr
Priority to JP2004512781A priority patent/JP2005520866A/ja
Publication of WO2003105880A1 publication Critical patent/WO2003105880A1/fr

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    • 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
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • This invention relates to therapeutic modalities for treatment of neoplastic disease. More specifically, this invention involves synthetic peptides that selectively destroy malignant and transformed cells, and a method for treatment of neoplastic disease based thereon.
  • the p53 protein is a vital regulator of the cell cycle. It blocks the oncogenic effects of a number of oncogene proteins that induce mitosis, in part by blocking transcription of proteins that induce mitosis and by inducing the transcription of proteins that block mitosis, and promote apoptosis. Absence of the p53 protein is associated with cell transformation and malignant disease. Haffner, R & Oren, M. (1995) Cwr. Opin. Genet. Dev. 5: 84-90.
  • the p53 protein molecule consists of 393 amino acids. It includes domains that bind to specific sequences of DNA in a DNA-binding domain that consists of residues 93- 313. The crystal structure of this region has been determined by x-ray crystallography. Residues 312-393 are involved in the formation of homotetramers of the p53 protein. Residues 1-93 are involved in regulation of the activity and half life of the p53 protein.
  • the p53 protein binds to another important regulatory protein, the MDM-2 protein.
  • the MDM-gene that encodes the MDM-2 protein is a known oncogene.
  • the MDM-2 protein forms a complex with the p53 protein, which results in the degradation of the p53 protein by a ubiquination pathway.
  • the p53 protein binds to MDM-2 protein using an amino acid sequence that includes residues 14-22 of the p53 protein, which are invariant.
  • the entire MDM-2 protein binding domain of the p53 protein spans residues 12-26. Hafther, R & Oren, M. (1995) Curr. Opin. Genet. Dev. 5: 84-90.
  • MDM-2 protein is the expression product of a known oncogene
  • MDM-2 protein is a very important regulatory protein.
  • overexpression or amplification of MDM-2 protein has been found in 40-60% of human malignancies, including 50% of human breast tumors. It has been suggested that formation of a complex between the p53 protein and the MDM-2 protein may result in the inhibition of transcription activity of the p53 protein, and thus the anti-tumor effect of the molecule by blocking of an activation domain of the p53 protein, or of a DNA binding site within it.
  • the present invention provides a peptide comprising at least about six . contiguous amino acids of the amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO:l), or an analog or derivative thereof, wherein said peptide or analog or derivative thereof is fused to a membrane-penetrating leader sequence and is selectively lethal to malignant or transformed cells.
  • the leader sequence is preferably positioned at the carboxyl terminal end of the peptide, analog, or derivative thereof.
  • the leader sequence comprises predominantly positively charged amino acid residues.
  • leader sequences which may be used in accordance with the present invention include but are not limited to penetratin, Arg 8 , TAT of HIV1, D-TAT, R-TAT, SV40-NLS, nucleoplasmin-NLS, HIV REV (34-50), FHV coat (35-49), BMV GAG (7-25), HTLV-II REX (4-16), CCMV GAG (7-25), P22N (14- 30), Lambda N (1-22), Delta N (12-29), yeast PRP6, human U2AF, human C-FOS (139- 164), human C-JUN (252-279), yeast GCN4, and p-vec.
  • the leader sequence is the penetratin sequence from antennapedia protein having the amino acid sequence KKWKMRRNQFWVKVQRG (SEQ ID NO:4).
  • compositions comprising at least one of the subject peptides admixed with a pharmaceutically acceptable carrier are also provided.
  • methods for treating neoplastic disease in a subject i.e., selectively killing malignant or neoplastic cells in a subject, are provided.
  • the method comprises administering to the subject, a therapeutically effective amount of a peptide comprising at least about six contiguous amino acids of the amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO: 1), or an analog or derivative thereof, wherein said peptide or analog or derivative thereof is fused at its carboxy terminal end to a membrane- penetrating leader sequence and is selectively lethal to malignant or transformed cells.
  • the method comprises administering to the subject, a therapeutically effective amount of at least one peptide having the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:3, or an analog or derivative thereof, wherein a membrane- penetrating leader sequence is fused to the carboxy terminal end of the peptide, analog, or derivative thereof.
  • Figure 1 graphically depicts the in vivo tumor-inhibiting effect of PNC-28 (SEQ ID NO:3 fused at its carboxy terminal end to SEQ ID NO:4) in homozygous NU/NU mice xenotransplanted with pancreatic carcinoma cells.
  • the arrow with a star indicates the time of s.c. pump implantation on day 13 (precisely 13.5) during the tumor growth period.
  • malignant and transformed cells are selectively destroyed by administration of a synthetic peptide comprising a sequence of amino acids within the p53 protein and a leader sequence as a single continuous polypeptide chain.
  • the mechanism of action appears to be independent of the p53 protein binding to the MDM-2 protein, as the p53 peptide selectively kills transformed cells that do not produce the p53 protein at all.
  • the p53 peptide also selectively kills malignant and transformed cells that express normal or elevated levels of the p53 protein without killing normal cells.
  • compositions comprising peptides corresponding to all or a portion of amino acid residues 12-26 of human p53. This region is known to contact the mdm-2 protein and adopts an ⁇ -helical conformation when bound to mdm-2. When fused on the carboxy-terminal end with a membrane-penetrating leader sequence, the subject peptides selectively kill malignant and transformed human cells.
  • a peptide comprising at least . about six contiguous amino acids of the following amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO: 1), wherein the peptide comprising at least about six contiguous amino acids is fused to a leader sequence.
  • the peptide comprises from at least about eight (8) to at least about fifteen (15) amino acid residues.
  • a peptide comprising from at least about eight (8) to at least about 15 (fifteen) amino acids of the sequence set forth in SEQ ID NO:l has the following amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO:l).
  • a peptide comprising from at least about eight (8) to at least about 15 (fifteen) amino acids of the sequence set forth in SEQ ID NO: 1 has the following amino acid sequence: PPLSQETFS (SEQ ID NO:2).
  • a peptide comprising from at least about eight (8) to at least about fifteen (15) amino acids of the sequence set forth in SEQ ID NO:l has the following amino acid sequence: ETFSDLWKLL (SEQ ID NO:3).
  • Leader sequences which function to import the peptides of the invention into a cell may be derived from a variety of sources.
  • the leader sequence comprises predominantly positively charged amino acid residues since a positively charged leader sequence stabilizes the alpha helix of a subject peptide.
  • Examples of leader sequences which may be linked to the peptides of the present invention are described in Futaki, S. et al (2001) Arginine-Rich Peptides, J. Biol Chem.
  • leader sequences include but are not limited to the following membrane-penetrating leader sequences (numbering of the amino acid residues making up the leader sequence of the protein is indicated parenthetically immediately after the name of the protein in many cases): penetratin (KKWKMRRNQFWVKVQRG)(SEQ ID NO:4); (Arg) 8 or any poly-R from (R) 4 -(R) ⁇ 6 ; fflV-1 TAT(47-60) (YGRKKRRQRRRPPQ)(SEQ ID NO:5); D-TAT (GRKKRRQRRRPPQ) (SEQ ID NO:6); R-TAT G(R) 9 PPQ(SEQ ID NO:7); SV40-NLS (PKKKRKV)(SEQ ID NO:8); nucleoplasmin-NLS (KRPAAIKKAGQAKKKK)(SEQ ID NO:9); HIV REV (34-50)- (TRQARRNRRRRWRERQR)(SEQ ID NO:4);
  • HUMAN U2AF (SQMTRQARRLYV)(SEQ ID NO: 19); HUMAN C-FOS (139-164) KRRIRRERNKMAAAKSRNRRRELTDT (SEQ ED NO :20);
  • membrane penetrating leader sequences may also be used. Such sequences are widely available and are described e.g., in Scheller et al. (2000) Eur. J.
  • the positively charged leader sequence of the penetratin leader sequence of antennapedia protein is used.
  • This leader sequence has the following amino acid sequence: KKWKMRRNQFWVKVQRG (SEQ ID NO:4).
  • the leader sequence is attached to the carboxyl terminal end of the p53 peptide to enable the synthetic peptide to kill transformed and malignant cells.
  • Structurally related amino acid sequences may be substituted for the disclosed sequences set forth in SEQ ID NOs: 1, 2, 3, or 4 in practicing the present invention. Any of the sequences set forth in SEQ ID NOs: 1, 2 or 3, including analogues or derivatives thereof, when joined with a leader sequence, including, but not limited to the sequence set forth in SEQ ID NO: 4, will be referred to herein as either a "synthetic peptide" or
  • Synthetic peptides Rigid molecules that mimic the three dimensional structure of these synthetic peptides are called peptidomimetics and are also included within the scope- of this invention.
  • Alpha helix stabilizing amino acid residues at either or both the amino or carboxyl terminal ends of the p53 peptide may be added to stabilize the alpha helical conformation which is known to be the conformation of this region of the p53 protein when it binds to the MDM-2 protein.
  • alpha helical stabilizing amino acids include Leu, Glu (especially on the amino terminal of the helix), Met and Phe.
  • Amino acid insertional derivatives of the peptides of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in a subject peptide although, random insertion is also possible with suitable screening of the resulting product. Deletional variants may be made by removing one or more amino acids from the sequence of a subject peptide.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with the following Table 1 :
  • the amino acids are generally replaced by other amino acids having like properties such as hydrophobicity, hydrophilicity, electronegativety, bulky side chains and the like.
  • the terms "derivative”, “analogue”, “fragment”, “portion” and “like molecule” refer to a subject peptide having the amino acid sequence as set forth in SEQ ID ⁇ Os:l, 2, 3, or 4, having an amino acid substitution, insertion, addition, or deletion, as long as said derivative, analogue, fragment, portion, or like molecule retains the ability to enter and selectively kill transformed or neoplastic cells.
  • the synthetic peptides of the present invention may be synthesized by a number of known techniques.
  • the peptides may be prepared using the solid-phase technique initially described by Merrifield (1963) in J. Am. Chem. Soc. 55:2149-2154.
  • Other peptide synthesis techniques may be found in M. Bodanszky et al. Peptide Synthesis, John Wiley and Sons, 2d Ed., (1976) and other references readily available to those skilled in the art.
  • a summary of polypeptide synthesis techniques may be found in J. Sturart and J.S. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, 111., (1984).
  • Peptides may also be synthesized by solution methods as described in The Proteins, Nol. II, 3d Ed., eurath, H.
  • peptides of the present invention may also be prepared by chemical or enzymatic cleavage from larger portions of the p53 protein or from the full length p53 protein.
  • leader sequences for use in the synthetic peptides of the present invention may be prepared by chemical or enzymatic cleavage from larger portions or the full length proteins from which such leader sequences are derived.
  • the peptides of the present invention may also be prepared by recombinant DNA techniques. For most amino acids used to build proteins, more than one coding nucleotide triplet (codon) can code for a particular amino acid residue. This property of the genetic code is known as redundancy. Therefore, a number of different nucleotide sequences may code for a particular subject peptide selectively lethal to malignant and transformed mammalian cells.
  • the present invention also contemplates a deoxyribonucleic acid (DNA) molecule that defines a gene coding for, i.e., capable of expressing a subject peptide or a chimeric peptide from which a peptide of the present invention may be enzymatically or chemically cleaved.
  • DNA deoxyribonucleic acid
  • s ⁇ mthetic peptides When applied to cells grown in culture, s ⁇ mthetic peptides are selectively lethal to malignant or transformed cells, resulting in dose dependent reduction in cell number. The effect is observable generally within two to three and at most 48 hours.
  • a line of rat pancreatic acinar cells (BMRPA.430) grown in culture was transformed with K-ras.
  • the normal cell line displays the architecture typical of pancreatic acinar cells; the transformed cells (TUC-3) lack the differentiated morphology of acinar cells, appearing as typical pancreatic ' cancer cells.
  • TUC-3 pancreatic acinar cells
  • BMRPA.430 cells were treated with a synthetic peptide with the primary structure of SEQ ID NO: 1 coupled to leader sequence SEQ ID NO:4, at a
  • TUC-3 cells were not affected.
  • TUC-3 cells were treated with a peptide with the primary structure of SEQ ID NO:l coupled to leader sequence SEQ ID NO:4, at a dosage of 100 ⁇ g/ml, they died within three to four days. Similar results
  • SEQ ID NO: 1 was substituted with either SEQ ID NO:2, or SEQ ID NO:3. Additionally, transformed and malignant cell death was observed in human breast carcinoma cell lines and Melanoma and HeLa cells treated with a synthetic peptide with the primary structure of SEQ ID NO: 1 coupled to leader sequence SEQ ID NO:4, at a dosage of 100 ⁇ g/ml. In contrast, the same synthetic peptide at the same dosage had no effect on non-malignant and non-transformed human breast or fibroblast cell lines.
  • leader sequence set forth in SEQ ID NO:4 When the leader sequence set forth in SEQ ID NO:4 was positioned at the carboxy terminal end of PNC29, a control protein having the following amino acid sequence: -VIPFSTGK-RIMLGE (SEQ ID NO: 25), there was no effect on malignant or normal cells. Additionally, the peptide having the amino acid sequence as set forth in SEQ ID NO:3 fused at the carboxy terminal end to the leader peptide set forth in SEQ ID NO:4, has no effect on the ability of human stem cells to differentiate into hematopoietic cell lines in the presence of growth factors. This indicates that this peptide will not be injurious to bone marrow cells when administered as a chemotherapeutic agent. See Kanovsky et al., (October 23, 2001) Proc. Nat. Acad. Sci. USA 98(22); 12438- 12443, the disclosure of which is incorporated by reference herein as if * fully set forth.
  • the synthetic peptides were tested on transformed colorectal adenocarcinoma cells that had been rendered incapable of making the p53 protein by homozygous deletion. Surprisingly, the synthetic peptides selectively killed the transformed cells, but had no effect on the normal cells. These results indicate that the mechanism of action appears to be independent of the p53 protein binding to the MDM-2 protein, as the p53 peptide selectively kills transformed cells that do not produce the p53 protein at all.
  • a subject synthetic peptide e.g., a peptide having the amino acid sequence as set forth in SEQ ID NO:3 fused to a leader sequence at the carboxy terminal end.
  • the subject peptides may be used to selectively kill neoplastic or malignant cells, i.e.., cancer cells in animals, preferentially humans.
  • the synthetic peptides of the present invention are thus administered in an effective amount to kill neoplastic cells in a subject animal or human.
  • the synthetic peptides of the present invention may be administered preferably to a human patient as a pharmaceutical composition containing a therapeutically effective dose of at least one synthetic peptide according to the present invention together with a pharmaceutical acceptable carrier.
  • a pharmaceutical composition containing a therapeutically effective dose of at least one synthetic peptide according to the present invention together with a pharmaceutical acceptable carrier.
  • therapeutically effective amount or “pharmaceutically effective amount” means the dose needed to produce in an individual, suppressed growth including selective killing of neoplastic or malignant cells, i.e., cancer cells.
  • compositions containing one or more of the synthetic peptides of the present invention are administered intravenously for the purpose of selectively killing neoplastic cells, and therefore, treating neoplastic or malignant disease such as cancer.
  • cancers which may be effectively treated using one or more the peptides of the present invention include but are not limited to: breast cancer, prostate cancer, lung cancer, cervical cancer, colon cancer, melanoma, pancreatic cancer and all solid tissue tumors (epithelial cell tumors) and cancers of the blood including but not limited to lymphomas and leukemias.
  • the synthetic peptides of the present invention may be by oral, intravenous, intranasal, suppository, intraperitoneal, intramuscular, intradermal or subcutaneous administration or by infusion or implantation.
  • the synthetic peptides of the present invention may be combined with other ingredients, such as carriers and/or adjuvants.
  • other ingredients such as carriers and/or adjuvants.
  • carriers and/or adjuvants There are no limitations on the nature of the other ingredients, except that they must . be pharmaceutically acceptable, efficacious for their intended administration, cannot degrade the activity of the active ingredients of the compositions, and cannot impede importation of a subject peptide into a cell.
  • the peptide compositions may also be impregnated into transdermal patches, or contained in subcutaneous inserts, preferably in a liquid or semi-liquid form which patch or insert time- releases therapeutically effective amounts of one or more of the subject synthetic peptides.
  • the pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The ultimate solution form in all cases must be sterile and fluid.
  • Typical carriers include a solvent or dispersion medium containing, e.g., water buffered aqueous solutions, i.e., biocompatible buffers, ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils. Sterilization may be accomplished utilizing any art-recognized technique, including but not limited to filtration or addition of antibacterial or antifungal agents. Examples of such agents include paraben, chlorbutanol, phenol, sorbic acid or thimerosal. Isotonic agents such as sugars or sodium chloride may also be incorporated into the subject compositions. .
  • a "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and the like. The use of such media and agents are well-known in the art.
  • sterile injectable solutions containing the subject synthetic peptides is accomplished by incorporating one or more of the subject synthetic peptides described hereinabove in the required amount in the appropriate solvent with one or more of the various ingredients enumerated above, as required, followed by sterilization, preferably filter sterilization.
  • sterilization preferably filter sterilization.
  • the above solutions are vacuum- dried or freeze-dried as necessary.
  • Inert diluents and/or assimilable edible carriers and the like may be part of the pharmaceutical compositions when the peptides are administered orally.
  • the pharmaceutical compositions may be in hard or soft shell gelatin capsules, be compressed into tablets, or may be in an elixir, suspension, syrup or the like.
  • the subject synthetic peptides are thus compounded for convenient and effective administration in pharmaceutically effective amounts with a suitable pharmaceutically acceptable carrier in a therapeutically effective dosage.
  • a pharmaceutically effective amount includes peptide concentrations in the range from about at least about 25ug/ml to at least about 300 ug/ml.
  • a precise therapeutically effective amount of synthetic peptide to be used in the methods of the invention applied to humans cannot be stated due to variations in stage of neoplastic disease, tumor size and aggressiveness, the presence or extent of metastasis, etc.
  • an individual's weight, gender, and overall health must be considered and will effect dosage.
  • the synthetic peptides of the present invention be administered in an amount of at least about 10 mg per dose, more preferably in an amount up to about 1000 mg per dose. Since the peptide compositions of the present invention will eventually be cleared from the bloodstream, re-administration of the pharmaceutical compositions is indicated and preferred.
  • the synthetic peptides of the present invention may be administered in a manner compatible with the dosage formulation and in such an amount as will be therapeutically effective. Systemic dosages depend on the age, weight, and condition of the patient and the administration route.
  • An exemplary suitable dose for the administration to adult humans ranges from about 0.1 to about 20 mg per kilogram of body weight. Preferably, the dose is from about 0.1 to about 10 mg per kilogram of body weight.
  • a method of treating neoplastic disease comprises administering to a subject in need of such treatment, a therapeutically effective amount of a synthetic peptide hereinbefore described, including analogs and derivatives thereof.
  • a therapeutically effective amount of a synthetic peptide hereinbefore described including analogs and derivatives thereof.
  • an effective amount of a peptide comprising at least about six contiguous amino acids as set forth in SEQ ID NO:l or an analog or derivative thereof fused on its carboxy terminal end to a leader sequence may be administered to a subject.
  • an effective amount of a peptide comprising at least from about eight (8) to at least about ten (10) contiguous amino acids as set forth in SEQ ID NO:l or an analog or derivative thereof, fused on its carboxy terminal end to a leader sequence, may be administered to a subject.
  • an effective amount of a peptide having the amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof, fused on its carboxy terminal end to a leader sequence may be administered to a subject.
  • An effective amount of a peptide having the amino acid sequence as set forth in SEQ ID NO:2 or an analog or derivative thereof, fused on its carboxy terminal end to a leader sequence may also be administered to a subject.
  • an effective amount of a peptide having the amino acid sequence set forth in SEQ ID NO:3 or an analog or derivative thereof, fused on its carboxy terminal end to a leader sequence may be administered to a subject.
  • a mixture of synthetic peptides may be administered.
  • mixtures of two or more peptides or analogs or derivatives hereinbefore described may be administered to a subject.
  • the peptide having the sequence set forth in SEQ ID NO:3 was synthesized by solid phase synthesis with the leader sequence attached to the amino terminal end.
  • This peptide is labeled PNC28 1 in Table 2 below.
  • the PNC28' peptide was incubated with transformed pancreatic cancer (TUC-3) cells at three different concentrations, i.e., 25, 50 and 100 ⁇ g/ml. After two weeks of incubation, at the highest dose of peptide, there was no cell death, and approximately half of the cells were seen to form acini and exhibited the untransformed morphological phenotype. The same phenomena were observed at 50 ⁇ g/ml, and at 25 ⁇ g/ml significantly fewer cells were seen to revert. In contrast, when the leader sequence was attached to the carboxyl terminal end of the peptide (PNC28 in Table 2), at dosages of 50 and 100 ⁇ g/ml. 100% cell death occurred in about 4 days.
  • leader sequence is preferentially added to the carboxyl terminal end of the MDM-2 portion of the p53 peptide to enable the peptide to cross the cell membrane and specifically kill malignant cells.
  • the leader sequence is KKWKMRRNQFWVKVQRG (SEQ ID NO:4).
  • mice After 12 days the tumors had reached sizes of 3 to 6 mm diameter and the mice were separated into two groups of 5 mice each. Each group was implanted s.c. with Alzet ® osmotic pumps to deliver in a constant rate and over a defined period of 14 days a total volume of 0.095 ml volume of normal saline containing the respective peptide at a concentration of 20mg/mouse.
  • mice received PNC-28 (the peptide having the amino acid set forth in SEQ ID NO:3) fused at its carboxy terminal end to the penetratin leader sequence (SEQ ID NO:4) and the other group of mice received PNC-29, a control peptide of similar size, having the following amino acid sequence: MPFSTGKR-M GE (SEQ ID NO: 25).
  • the pumps were filled according to the manufacturers guidelines and under sterile conditions The pumps were implanted s.c. on the left flank of the anaesthetized mice by creating a pocket underneath the mouse skin into which the tiny pumps were inserted. Each pocket was. closed with a simple suture. From their inside chamber the pumps delivered continuously 0.25 ⁇ l / hr into each mouse.
  • mice were observed until they had recovered from the surgery when they were returned to the isolation ward of the animal facility. Since the animals were Nu/Nu mice and, thus, immuno-compromised they are highly susceptible when exposed to pathogens. Surgery and all preceding and post-surgical treatments were therefore performed in a sterile hood environment.
  • PNC-28 within a 48 to 72hr period of delivery iftto the mouse effectively arrests tumor growth.
  • the control peptide PNC-29 had no effect .on normal or tumor cells.
  • PNC29-treated mice tumors kept growing at a continuous rate resulting in tumors of 10 to 16 mm diameter over the 2 - week treatment and follow-up period when the pumps cease to release any more peptide solution.
  • Statistical analyses of the measurement of tumor size in both groups of mice has produced a significance between them of p ⁇ 0.001.
  • TUC-3 (1 x 10 6 cells/mouse) were transplanted to the peritoneal cavity of five mice. Pumps were placed in the right shoulder region at the same time of tumor cell transplantation. In all five mice, there were no visible tumors after three weeks.

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  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
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  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des peptides correspondant à tous ou à une partie des résidus d'acides aminés 12-26 de la protéine p53 humaine, lesquels peptides étant létaux pour des cellules transformées ou malignes, lorsqu'elles sont fusionnées à une séquence de tête de pénétration membranaire. Les peptides de l'invention sont ainsi utiles dans le traitement d'une maladie néoplastique chez l'animal, de préférence chez l'humain. L'invention concerne des compositions pharmaceutiques contenant lesdits peptides mélangés à un support pharmaceutiquement acceptable. L'invention concerne des méthodes de traitement d'une maladie néoplastique chez un patient, lesquelles méthodes consistant à administrer un peptide fusionné à son extrémité carboxy terminale à une séquence de tête de pénétration membranaire.
PCT/US2003/007687 2002-03-12 2003-03-12 Peptides selectivement letaux pour des cellules de mammiferes transformees et malignes WO2003105880A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2003277292A AU2003277292A1 (en) 2002-03-12 2003-03-12 Peptides selectively lethal to malignant and transformed mammalian cells
EP03760194A EP1490089A4 (fr) 2002-03-12 2003-03-12 Peptides selectivement letaux pour des cellules de mammiferes transformees et malignes
CA2477878A CA2477878C (fr) 2002-03-12 2003-03-12 Peptides selectivement letaux pour des cellules de mammiferes transformees et malignes
JP2004512781A JP2005520866A (ja) 2002-03-12 2003-03-12 哺乳動物の悪性細胞および形質転換細胞に対する選択的致死性ペプチド

Applications Claiming Priority (2)

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US36378502P 2002-03-12 2002-03-12
US60/363,785 2002-03-12

Publications (1)

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WO2003105880A1 true WO2003105880A1 (fr) 2003-12-24

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EP (1) EP1490089A4 (fr)
JP (1) JP2005520866A (fr)
AU (1) AU2003277292A1 (fr)
CA (1) CA2477878C (fr)
WO (1) WO2003105880A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1608409A2 (fr) * 2003-03-12 2005-12-28 The Research Foundation of the State University of New York Peptides selectivement letaux pour les cellules malignes et transformees
WO2006006954A2 (fr) * 2004-01-13 2006-01-19 The Research Foundation Of State University Of New York Peptides selectivement letaux pour des cellules mammiferes transformees et malignes
WO2010040051A2 (fr) * 2008-10-03 2010-04-08 The Research Foundation Of State University Of New York Un peptide membranaire résident dans des peptides anticancéreux cause la nécrose de cellules tumorales plutôt que l’apoptose des cellules cancéreuses
US7772367B2 (en) * 2004-01-30 2010-08-10 The Trustees Of Columbia University In The City Of New York C-terminal p53 palindromic peptide that induces apoptosis of cells with aberrant p53 and uses thereof
WO2011005219A1 (fr) * 2009-07-07 2011-01-13 Agency For Science, Technology And Research Nouveaux peptides de liaison à mdm2 et leurs utilisations
US9539327B2 (en) 2007-11-26 2017-01-10 The Research Foundation For The State University Of New York Small molecule cancer treatments that cause necrosis in cancer cells but do not affect normal cells

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2008081812A1 (ja) * 2006-12-28 2010-04-30 東亞合成株式会社 抗腫瘍ペプチド及びその利用

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US4215102A (en) * 1979-01-05 1980-07-29 Lee Sin H Cytochemical agents and methods for the detection of steroid hormone receptors in human tissues
US5369012A (en) * 1992-03-26 1994-11-29 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Method of making a membrane having hydrophilic and hydrophobic surfaces for adhering cells or antibodies by using atomic oxygen or hydroxyl radicals

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US5770377A (en) * 1994-07-20 1998-06-23 University Of Dundee Interruption of binding of MDM2 and P53 protein and therapeutic application thereof
US20040038902A1 (en) * 2000-04-05 2004-02-26 Pincus Matthew R. Peptides selectively lethal to malignant and transformed mammalian cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4215102A (en) * 1979-01-05 1980-07-29 Lee Sin H Cytochemical agents and methods for the detection of steroid hormone receptors in human tissues
US5369012A (en) * 1992-03-26 1994-11-29 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Method of making a membrane having hydrophilic and hydrophobic surfaces for adhering cells or antibodies by using atomic oxygen or hydroxyl radicals

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1490089A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1608409A2 (fr) * 2003-03-12 2005-12-28 The Research Foundation of the State University of New York Peptides selectivement letaux pour les cellules malignes et transformees
EP1608409A4 (fr) * 2003-03-12 2009-09-16 Univ New York State Res Found Peptides selectivement letaux pour les cellules malignes et transformees
WO2006006954A2 (fr) * 2004-01-13 2006-01-19 The Research Foundation Of State University Of New York Peptides selectivement letaux pour des cellules mammiferes transformees et malignes
WO2006006954A3 (fr) * 2004-01-13 2006-07-13 Univ New York State Res Found Peptides selectivement letaux pour des cellules mammiferes transformees et malignes
US7772367B2 (en) * 2004-01-30 2010-08-10 The Trustees Of Columbia University In The City Of New York C-terminal p53 palindromic peptide that induces apoptosis of cells with aberrant p53 and uses thereof
US9539327B2 (en) 2007-11-26 2017-01-10 The Research Foundation For The State University Of New York Small molecule cancer treatments that cause necrosis in cancer cells but do not affect normal cells
WO2010040051A2 (fr) * 2008-10-03 2010-04-08 The Research Foundation Of State University Of New York Un peptide membranaire résident dans des peptides anticancéreux cause la nécrose de cellules tumorales plutôt que l’apoptose des cellules cancéreuses
WO2010040051A3 (fr) * 2008-10-03 2010-05-27 The Research Foundation Of State University Of New York Un peptide membranaire résident dans des peptides anticancéreux cause la nécrose de cellules tumorales plutôt que l’apoptose des cellules cancéreuses
US8822419B2 (en) 2008-10-03 2014-09-02 The Research Foundation Of State University Of New York Membrane resident peptide in anti-cancer peptides causes tumor cell necrosis rather than apoptosis of cancer cells
WO2011005219A1 (fr) * 2009-07-07 2011-01-13 Agency For Science, Technology And Research Nouveaux peptides de liaison à mdm2 et leurs utilisations

Also Published As

Publication number Publication date
JP2005520866A (ja) 2005-07-14
EP1490089A1 (fr) 2004-12-29
AU2003277292A1 (en) 2003-12-31
EP1490089A4 (fr) 2006-08-30
CA2477878A1 (fr) 2003-12-24
CA2477878C (fr) 2013-05-14

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