Classifications

• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

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) Curr. 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. Haffner, 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.
• Retro- isomer Linear modified retro-peptide structures appear in the literature and the term "retro- isomer” was designated to include an isomer in which the direction of the sequence is reversed compared with the parent peptide. See, e.g., Goodman, M., et al., "On the Concept of Linear Modified Retro-Peptide Structures", Ace. ofChem. Res., 12(1), 1-7 (1979) and U.S. Patent Application No. 10/399,127 to Bonny. Retro-inverso isomers in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted also appear in the literature. Recently, Jameson et al.
• the present invention provides 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 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 (KKWKMRRNQFWVKVQRG)(SEQ ID NO:4); (Arg) 8 (SEQ ID NO:26) or any poly-R from (R) 4 -(R) -6 (SEQ ID NO:27); HIV-I TAT(47-60)
• HUMAN C-JUN 252-279 (RECAERKRMRNRIAASKSRKRKLERIAR)(SEQ E) NO:21); YEAST GCN4 (KRARNTEAARRSRARKLQRMKQ)(SEQ E) NO:22);
• the positively charged leader sequence of the penetration leader sequence of antennapedia protein is used.
• any of the subject peptides described hereinabove may have specific alterations made thereto, which alterations render the peptides less susceptible to proteolysis.
• a subject peptide may have one or more peptide bonds replaced with an isostere pseudopeptide bond or a retro-inverso pseudopeptide bond.
• a subject peptide may be a directional peptide isomer of the corresponding portion of the naturally occurring p53 protein.
• enantio, retro-inverso, and partially modified retro-inverso peptides are particularly contemplated.
• the present invention provides a subject peptide, analog or derivative thereof, hereinbefore described, fused to a membrane-penetrating leader sequence, and selectively lethal to malignant or transformed cells, which comprises one or more D-amino acids, or which has at least one peptide bond substituted with an isostere pseudopeptide bond or a retro-inverso pseudopeptide bond.
• a D-amino acid may be positioned at the N-terminus of a subject peptide.
• the presence of an N-terminal D-amino acid increases the stability of a peptide since exopeptidases acting on the N-terminal residue cannot utilize a D-amino acid as substrate.
• a D-amino acid may be positioned at the C-terminus of a subject peptide. The presence of a C-terminal D-amino acid also stabilizes the peptide since exopeptidases acting on the C-terminal residue cannot utilize a D-amino acid as a substrate.
• a peptide comprising at least 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 to a membrane-penetrating leader sequence, is selectively lethal to malignant or transformed cells and wherein at least one amino acid is in the D form or wherein one or more peptide bonds are replace with an isostere pseudopeptide bond or a retro-inverso pseudopeptide bond.
• Examples include peptides comprising the amino acid sequence: PPLSQETFS (SEQ ID NO: 2), ETFSDLWKLL (SEQ ID NO: 3), or an analog or derivative thereof.
• a subject peptide, analog or derivative thereof, fused to a membrane-penetrating leader sequence and selectively lethal to malignant or transformed cells comprises all D-amino acids assembled in reverse order to the natural sequence found in the p53 protein.
• Such peptide is referred to herein as a retro-inverso (RI) peptide.
• RI retro-inverso peptide comprising at least six contiguous D-amino acids assembled in the reverse order of the amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO: 1), or an analog or derivative thereof, fused to a membrane-penetrating leader sequence, and selectively lethal to malignant or transformed cells.
• the peptide comprises at least six contiguous D-amino acids assembled in the reverse order of the amino acid sequence PPLSQETFS (SEQ ID NO: 2) or ETFSDLWKLL (SEQ ID NO: 3), or an analog or derivative thereof.
• a partially modified retro-inverso (PMRI) peptide is also provided wherein only a portion of the p53 sequence is retro-inverted.
• a peptide comprising at least six amino acids having only a portion of amino acids in the D form and assembled in reverse order of the amino acid sequence set forth in SEQ ID NO: 1.
• a portion of the at least six D-amino acids are assembled in reverse order of the sequence PPLSQETFS (SEQ ID NO: 2) or ETFSDLWKLL (SEQ ID NO: 3), or an analog or derivative thereof.
• the membrane-penetrating leader sequence may also comprise one or more D-amino acids or one or more isostere pseudopeptide bonds or inverso pseudopeptide bonds.
• the membrane penetrating leader sequences are themselves retro-inverso, or partially modified retro-inverso peptide isomers.
• a membrane- penetrating leader sequence in a retro-inverso form comprises all D-amino acids assembled in reverse order to any of the sequences set forth in SEQ ID NOs: 4-24 or SEQ ID NOs:26-27.
• a membrane penetrating leader sequence in a partially modified retro-inverso form has only a portion of the amino acid residues in a D-form and in reverse order to any of the sequences set forth in SEQ ID NOs: 4-24 or SEQ ID NOs:26-27.
• compositions comprising at least one of the subject peptides admixed with a pharmaceutically acceptable carrier are also provided. Such pharmaceutical compositions may also include any of the subject peptides comprising one or more D-amino acids or one or more isostere pseudopeptide bonds or retro-inverso pseudopeptide bonds, and may also include any of the subject retro-inverso, and partially modified retro-inverso peptides.
• 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.
• 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.
• Further embodiments of the method comprise administering to a subject a therapeutically effective amount of a subject peptide comprising one or more D-amino acids or one or more isostere pseudopeptide bonds or retro-inverso pseudopeptide bonds, or a retro-inverso, or partially modified retro-inverso peptide, 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. Also provided are methods of assessing the effectiveness of a subject peptide in killing malignant, neoplastic, or transformed cells in vitro.
• the method comprises the steps of contacting malignant, transformed or neoplastic cells in vitro with at least one subject peptide or an analog or derivative thereof, and assessing the level of effectiveness of the peptide based on the ratio or percentage of dead cells compared to live cells and its effect on the growth of untransformed (normal) cells in culture.
• 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.
• 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: 1 has the following amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO:1).
• 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: PPLSQETFSDLWKLL (SEQ ID NO:1).
• a peptide comprising from at least about eight (8) to at least about 15 has the following amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO:1).
• a peptide comprising from at least about eight (8) to at least about 15 has the following amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO:1).
• 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: 1 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, /. Biol. Chem.
• YEAST PRP6 (129-124) (TRRNKRNRIQEQLNRK) (SEQ ID NO: 18); HUMAN U2AF (SQMTRQARRLYV)(SEQ ID NO: 19); HUMAN C-FOS (139-164) KRRIRRERNKMAAAKSRNRRRELTDT (SEQ ID NO:20); HUMAN C-JUN (252-279) (RIKAERKRMRNRIAASKSRKRKLERIAR)(SEQ ID N0:21); YEAST GCN4 (KRARNTEAARRSRARKLQRMKQ)(SEQ ID NO:22); KLALKLALKALKAALKLA(SEQ ID NO:23); p-vec LLHLRRRIRKQAKAHSK(SEQ ID NO:24).
• 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. Biochem. 2(57:6043-6049, and Elmquist et al., (2001) Exp. Cell Res. 269:237-244.
• the positively charged leader sequence of the penetration 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 .
• a subject peptide hereinbefore described i.e., any of SEQ ID NOs.: 1-3 (p53 peptides) or SEQ ID NOs: 4-25 or 26-27 (membrane-penetrating leader sequences) may comprise one or more amino acids in the D-form and/or may comprise one or more isostere pseudopeptide bonds or one or more retro-inverso pseudopeptide bonds.
• a D-amino acid is positioned at the N-terminus of a subject peptide.
• a D- amino acid may also be positioned at the C-terminus of the membrane-penetrating leader sequence, which sequence is fused to the p53 peptide at its carboxy terminus. Such positioning of a D-amino acid helps stabilize the peptide since exopeptidases acting on C-terminal residues cannot utilize D-amino acids as a substrate.
• a subject peptide of the present invention can be synthesized as a retro- inverso peptide (RI) comprising all D-amino acids as well as a reversed sequence.
• the peptide comprises both reversed sequence and inverted stereochemistry at all chiral centers.
• a subject peptide may be synthesized as a partially modified retro-inverso peptide (PMRI) wherein only a portion of the p53 sequence or membrane- penetrating leader sequence is retro-inverted.
• PMRI retro-inverso peptide
• a peptide comprising at least six amino acids having only a portion of amino acids in the D form and assembled in reverse order.
• a peptide comprising at least 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 to a membrane-penetrating leader sequence, is selectively lethal to malignant or transformed cells and wherein at least one amino acid is in the D form or wherein one or more peptide bonds are replace with an isostere pseudopeptide bond or a retro-inverso pseudopeptide bond.
• Examples include peptides comprising the amino acid sequence: PPLSQETFS (SEQ ID NO: 2), ETFSDLWKLL (SEQ ID NO: 3), or an analog or derivative thereof.
• a subject peptide, analog or derivative thereof, fused to a membrane-penetrating leader sequence and selectively lethal to malignant or transformed cells comprises all D-amino acids assembled in reverse order to the natural sequence found in the p53 protein.
• Such peptide is referred to herein as a retro-inverso (RI) peptide.
• RI retro-inverso peptide comprising at least six contiguous D-amino acids assembled in the reverse order of the amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO: 1), or an analog or derivative thereof, fused to a membrane-penetrating leader sequence, and selectively lethal to malignant or transformed cells.
• the peptide comprises at least about six contiguous D-amino acids assembled in the reverse order of the amino acid sequence PPLSQETFS (SEQ K) NO: 2) or ETFSDLWKLL (SEQ ID NO: 3), or an analog or derivative thereof.
• a partially modified retro-inverso (PMPvI) peptide is also provided wherein only a portion of the p53 sequence is retro-inverted.
• a peptide comprising at least six amino acids having only a portion of amino acids in the D form and assembled in reverse order of the amino acid sequence set forth in SEQ ID NO: 1.
• a portion of the at least six D-amino acids are assembled in reverse order of the sequence PPLSQETFS (SEQ ID NO: 2) or ETFSDLWKLL (SEQ ID NO: 3), or an analog or derivative thereof.
• the membrane-penetrating leader sequence is located at the carboxyl terminal end of the peptide, analog or derivative thereof in order to cross the cell membrane and specifically kill malignant, transformed, or neoplastic cells.
• peptides having a membrane-penetrating leader sequence at the N-terminal end of the p53 peptide are useful as control peptides in various experiments such as the in vitro experiments described herein.
• the membrane-penetrating leader sequence may also comprise one or more D-amino acids or one or more isostere pseudopeptide bonds or inverso pseudopeptide bonds.
• the membrane penetrating leader sequences are themselves retro-inverso, or partially modified retro-inverso peptide isomers.
• a membrane- penetrating leader sequence in a retro-inverso form comprises all D-amino acids assembled in reverse order to any of the sequences set forth in SEQ ID NO: 4-24 or SEQ ID NO:26-27.
• a membrane penetrating leader sequence in a partially modified retro-inverso form has only a portion of the amino acid residues in a D-form and in reverse order to any of the sequences set forth in SEQ ID NO: 4-24 or SEQ ID NO:26-27.
• RI and PMRI peptides results in the introduction of two non-amino acid residues into the newly formed isomers, the gem-diaminoalkyl (gXaa) and the 2-alkylmalonyl (mXaa) residues at the amino and carboxy sides of the transformed sequence, respectively.
• gXaa gem-diaminoalkyl
• mXaa 2-alkylmalonyl
• 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.
• 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, GIu (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 NOs: 1, 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. 85:2149-2154.
• 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 maybe found in J. Sturart and J.S. Young, Solid Phase Peptide
• Peptides may also be synthesized by solution methods as described in The Proteins, Vol. ⁇ , 3d Ed., Neurath, H. et al., Eds., pp. 105- 237, Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in different peptide syntheses are described in the texts listed above as well as in J.F.W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973).
• the 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
• a subject peptide having one or more amino acids in the D-form may be synthesized by incorporating into the peptide chain, one or more D-amino acids instead of the naturally occurring L-amino acids.
• the synthesis of linear D-form peptides may be prepared using conventional protocols of peptide synthesis including synthesis by automated procedure. See, e.g., Scheibler, L. and Chorev, J. (2003) In Synthesis of Peptides and Peptidomimetrics (Houben-Weyl Methods of Organic Chemistry, 4 th Ed., Vol. 22C)(Goodman, M., ed), pp 528-551, Thieme, Stuttgart.
• the reduced isostere pseudopeptide bond is a pseudopeptide bond which enhances stability to proteolytic cleavage with little or no loss of biological activity.
• a subject peptide may be identical to an L-amino acid peptide having the amino acid sequences set forth in any of
• SEQ ID NOs: 1-3 p53 peptides
• SEQ ID NOs: 4-24 or 26-27 membrane penetrating leader sequences
• one or more peptide bonds are replaced by an isostere pseudopeptide bond.
• Methods of synthesizing peptides with one or more reduced isostere pseudopeptide bonds are well known in the art. See Couder et al. (1993) Int. J. Peptide Protein Res. 42:181-184, incorporated by reference herein as if fully set forth.
• Peptide bonds may also be replaced with retro-in verso pseudopeptide bonds.
• a subject peptide may be identical to the L-amino acid peptides having the amino acid sequences set forth in any of SEQ ID NOs: 1-3 (p53 peptides) or any of SEQ ID NOs: 4-24 or 26-27 (membrane penetrating leader sequences) except that one or more retro-inverso pseudopeptide bonds are substituted for peptide bonds.
• Procedures for synthesizing peptides with one or more retro- inverso pseudopeptide bonds are available in the literature extant; see e.g., Dalpozzo, et al. (1993) In. J. Peptide Protein Res.
• a subject RI peptide can be synthesized using D-amino acids and attaching the amino acids in a peptide chain such that the sequence of amino acids in the retro-inverso peptide analog is the exact opposite of that in the selected peptide which serves as the model.
• the retro- inverso peptide of the peptide set forth in SEQ ID NO: 1 comprises all D-amino acids assembled in the following sequence: LLKWLDSFTEQSLPP (SEQ ID NO:28).
• the retro-inverso peptide of the peptide set forth in SEQ ID NO:2 comprises all D-amino acids assembled in the following sequence: SFTEQSLPP (SEQ ID NO:29).
• SFTEQSLPP SEQ ID NO:29.
• SEQ ED NO:3 comprises all D-amino acids assembled in the following sequence: LLKWLDSFTE (SEQ ID NO:30).
• the retro-inverso peptide may be synthesized by Fmoc chemistry on a Fmoc-2,4- dimethyloxy-4'(carboxymethyloxy)-benzhydrylamine resin. See, e.g., Briand, J.P., et al., (1995) "Retro-inverso peptidomimetics as a new immunological probe: validation and application to the detection of autoantibodies in rheumatic diseases". /. Biol Chem. 270, 11921-11926, which is incorporated by reference herein as if fully set forth.
• the NH 2 -termini of the retro-inverso peptides can be acetylated.
• the crude peptides can be purified by standard methods such as on a column chromatography using a preparative HPLC apparatus.
• the purity of the retro-inverso peptides can be determined by analytical HPLC or other well-known methods known in the art.
• Solution-based methodologies are preferred.
• Solution-based chemistry can generate suitably protected gem-diaminoalkyl and 2- alkylmalonyl moieties needed for the synthesis of PMRI-peptides by a variety of well-known reactions.
• the generated crude building blocks and the pseudopeptide units may then be subjected to purification and characterization.
• precursors such as HO-Ala-(RS)-mPhe-(R)-Lys(N 2 -Boc)-NH 2 or HO-mGly-(R)-Phe-NH 2
• ⁇ pseudopeptide bond
• TUC-3 cells were treated with a 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, they died within three to four days. Similar results were obtained when the same experiment was performed but 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.
• the same synthetic peptide at the same dosage had no effect on non-malignant and non- transformed human breast or fibroblast cell lines.
• 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: MPFSTGKRIMLGE (SEQ ID NO: 25), there was no effect on malignant or normal cells.
• 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. When cultured cancer cells were treated with a peptide with the primary structure of SEQ
• 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.
• 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.
• 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 sdlutior ⁇ 'torm 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.
• 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
• 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.
• Production of 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.
• 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
• 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.
• an effective amount of a peptide comprising at least about six contiguous amino acids 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 comprising at least from about eight (8) to at least about ten (10) contiguous amino acids 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: 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 ammo acid sequence as set forth in SEQ ID NO: 2 or an analog or derivative thereof, fused on its carboxy tenninal 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.
• any of the subject peptides comprising one or more D-amino acids, isostere pseudopeptide or retro-inverso pseudopeptide bonds, or any of the RI or PMRI peptides hereinbefore described and fused at the carboxy terminal end to a membrane-penetrating leader sequence, may also be used in a method of killing malignant or neoplastic cells in a patient.
• 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.
• Also provided by the present invention is a method of assessing the level of effectiveness of a peptide in selectively killing malignant, neoplastic, or transformed cells in vitro.
• the method comprises the steps of contacting malignant, transformed, or neoplastic cells with any of the peptides hereinbefore described, assessing the level of effectiveness based on the ratio or percentage of dead cells compared to live cells and evaluating the effects of the peptide on the growth of untransformed (normal) cells in culture.
• those peptides which kill malignant, transformed or neoplastic cells in vitro while exerting no negative effects on untransformed or normal cells in culture would be considered valuable candidates for use in treating patients suffering from neoplastic disease.
• ⁇ -helix probability profile for each peptide having the sequences set forth in SEQ ID NOs: 1-3 was performed using two different methods, one using helix probabilities from the protein database (Karplus, K. et al., (1998) Bioinformatics 14:846- 856), and the other using the Ising model based on helix nucleation ( ⁇ ) and growth (s), equilibrium constants determined experimentally from block copolymers for each of the twenty naturally occurring L amino acids, modified by inclusion of the effects of charges on these parameters as described in Vasquez, M., et al.
• 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' 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), afa ⁇ sages 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 ' i a control peptide of similar size, having the following amino acid sequence: MPFSTGKRIMLGE (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 into 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.
• BMRPA l.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.
• a peptide having an amino acid sequence as set forth in SEQ ID NO: 2 or 3 is synthesized with one or more amino acids in the D-form by solid phase synthesis with a membrane-penetrating leader sequence attached to the carboxy terminal end.
• the solid-phase peptide synthesis methodology involves coupling each protected amino acid residue to a resin support, preferably a 4-methyl-benzhydrylamine resin, by activation with dicyclohexylcarbodimide to yield a peptide with a C-terminal amide.
• the protected peptide-resin is treated with hydrogen fluoride to cleave the peptide from the resin, as well as deprotect the side chain functional groups. Crude product can be further purified by reverse phase HPLC.
• the peptide may be incubated with malignant, transformed or neoplastic cells such as pancreatic cancer cells (TUC3) at three different concentrations, i.e., 25, 50, and 100 ⁇ l/ml, in order to assess the level of effectiveness in killing such cells at these concentrations.
• TUC3 pancreatic cancer cells
• a retro-inverso (RI) peptide having all D-amino acids assembled in the reverse order of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 is synthesized using D-amino acids.
• the retro-inverso form is synthesized by standard Fmoc chemistry on an ABI 433A Peptide Synthesizer (Applied Biosystems, Foster City, California, United States). See, Ben-Yedida, et al., (2002) Molecular Immunology, 39:323-331. Crude product is further purified by reverse-phase HPLC over a C18 preparatory column (Varian, Palo Alto, California, United States). The identity of the peptides is confirmed by mass spectrometry.
• the peptide is fused to a membrane-penetrating leader sequence at its carboxy terminal end and may be incubated with malignant, transformed or neoplastic cells such as pancreatic cancer cells (TUC3) at three different concentrations, i.e., 25, 50, and 100 ⁇ l/ml, in order to assess the level of effectiveness in killing such cells at these concentrations.
• malignant, transformed or neoplastic cells such as pancreatic cancer cells (TUC3) at three different concentrations, i.e., 25, 50, and 100 ⁇ l/ml, in order to assess the level of effectiveness in killing such cells at these concentrations.
• a partially modified retro-inverso (PMRI) peptide having a portion but not all of the amino acids in D form and in reverse order to the sequence set forth in SEQ ID Nos:2 or 3 is synthesized using solution-based chemistry to generate suitably protected gem-diaminoalkyl and 2-alkylmalonyl moieties needed for the synthesis of PMRI-pep tides.
• PMRI retro-inverso

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