Note: Descriptions are shown in the official language in which they were submitted.
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PEPTIDES SELECTIVELY LETHAL TO MALIGNANT AND
TRANSFORMED MAMMALIAN CELLS
This application is a continuation-in-part application of Application Serial
No.
09/827,683,_filed April 5, 2001, this application claims the benefit of U.S.
Provisional
- = -
Application No. 60/363,785, filed March 12, 2002, and U.S. Serial No.
09/827,683 claims
the benefit of U.S. Provisional Application Serial No. 60/195,102, filed April
5, 2000.
BACKGROUND OF THE INVENTION
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) C1117.
Op/n. 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.
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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.
Considering that the MDM-2 protein is the expression product of a known
oncogene, it is not surprising that MDM-2 protein is a very important
regulatory protein.
Moreover, 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. More generally, these and other experimental observations have been
interpreted
as suggesting that the anti-tumor effect of the p53 protein might be enhanced
by peptides
capable of interfering with the binding of the MDM-2 protein to the p53
protein. Indeed, a
number of investigators have suggested that the MDM-2/p53 complex might be a
target for
rational drug design. See, e.g., Christine Wasylyk et al., 11p53 Mediated
Death of Cells
Overexpressing MDM2 by an Inhibitor of MDM2 Interaction with p5311, Oncogene,
18,
1921-34 (1999), and U.S. Patent No. 5,770,377 to Picksley et al.
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SUMMARY OF THE INVENTION
The present invention provides a peptide comprising at least about six .
contiguous amino acids of the amino acid sequence: PPLSQETFSDLWKIL (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.
Examples of such peptides include PPLSQETFSDLWKLL (SEQ ED NO:1) or an
analog or derivative thereof, PPLSQETFS (SEQ ID NO:2) or an analog or
derivative
thereof and ETFSDLWKLL (SEQ ID NO:3) or an analog or derivative thereof. In
order to
be transported across a cell membrane and selectively kill a malignant or
transformed cell,
the leader sequence is preferably positioned at the carboxyl terminal end of
the Peptide,
analog, or derivative thereof. Preferably, the leader sequence comprises
predominantly
positively charged amino acid residues. Examples of leader sequences which may
be used
in accordance With the present invention include but are not limited to
penetratin, Arg8,
TAT of H1V1, D-TAT, R-TAT, SV40-NLS, nucleoplasmin-NLS, REV 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. Preferably, the leader
sequence is
the penetratin sequence from antetznapedia protein having the amino acid
sequence
2,0 KKWKNIRRNQFWVICVQRG (SEQ ID NO:4).
Pharmaceutical compositions comprising at least one of the subject peptides
admixed with a pharmaceutically acceptable carrier are also provided. In
addition,
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methods for treating neoplastic disease in a subject i.e., selectively killing
malignant or
neoplastic cells in a subject, are provided. In one embodiment, 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: =
PPLSQEFFSDLANKLL (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. In
another embodiment, 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.
BRIEF DESCRIPTION OF THE DRAWINGS
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.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, it has been discovered that
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
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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.
In accordance with the present invention, there are provided compositions
comprising peptides corresponding to all or a portion of amino acid residues
12-26 of
human 1353. This region is known to contact the mdm-2 protein and adopts an a-
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.
In a first aspect of the invention, there is provided 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. Preferably, the peptide
comprises
from at least about eight (8) to at least about fifteen (15) amino acid
residues. In a
preferred embodiment, 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). In another preferred
embodiment, a peptide comprising from at least about eight (8) to at least
about 15
(fifteen) amino acids of the sequence set forth in SEQ 1D NO:1 has the
following amino
acid sequence: PPLSQETFS (SEQ ID NO:2). In still another preferred embodiment,
a
peptide comprising from at least about eight (8) to at least about fifteen
(15) amino acids of
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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. Preferably, 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. 276,:5836-5840, and 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)16; HIV-1 TAT(47-60) (YGRICKRRQRRRPPQ)(SEQ ID NO:5); D-TAT
(GRKKRRQRRRPPQ) (SEQ ID NO:6); R-TAT G(R)9PPQ(SEQ EI) NO:7);
SV40-NLS (PKKKRKV)(SEQ ID NO:8); nucleoplasrnin-NLS
(KRPAAJKKAGQAKKKK)(SEQ ID NO:9); REV REV (34-50)-
(TRQARRNRRRRWRERQR)(SEQ ID NO:10); FHV (35-49) coat-
(RRRRNRTRRNRRRVR)(SEQ ID NO:11); BMV GAG (7-25)-
(KMTRAQRRAAARRNRWTAR)(SEQ ID NO:12); HTLV-II REX 4-16-
(TRRQRTRRARRNR)(SEQ ID NO:13); CCMV GAG (7-25) -
(KLTRAQ TR)(SEQ ID NO:14); P22 N (14-
30)(NAKTRRHERRRKLAIER)(SEQ ID NO:15); LAMBDA N (1-
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22)(MDAQTRRRERRAEKQAQWKAAN)(SEQ ID NO:16);
Phi N (12-29) (TAKTRYKARRAELIAERR)(SEQ ID NO:17);
YEAST PRP6 (129-124) (TRRNKRNRIQEQLNRK) (SEQ ID NO:18);
HUMAN U2AF (SQMTRQARRLYV)(SEQ ID NO:19); HUMAN C-FOS (139-164)
KRRIRRERNMMAAAKSRNRRRELTDT (SEQ NO:20);
HUMAN C-JUN (252-279) (RLKAERKRMRNRIAASKSRKRKLERIAR)(SEQ ID
NO:21); YEAST GCN4 (KRARNTEAARRSRARKLQR_MKQ)(SEQ ID NO:22);
KLALKLALKALKAALKLA(SEQ ID NO:23); p-vec LLIILRRRIR_KQAKAHSK(SEQ
ID NO:24). Other 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.
Biochern. 267:6043-6049, and Elmquist et al., (2001) Exp. Cell Res. 269:237-
244.
Preferably, 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). Preferably, 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
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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. Examples of 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:
TABLE 1
Suitable residues for amino acid substitutions
Original Residue Exemplary Substitutions
Ala (A) Ser
Arg (R) Lys
Asn (N) Gln; His
Asp (D) Glu
Cys (C) Ser
Gin (Q) Asn
Glu (E) Asp
Gly (G) Pro
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His (H) Asn; Gin
Ile (I) Leu; Val
Leu (L) Ile; Val
Lys (K) Arg; Gin; Glu
Met (M) Leu; Ile
Phe (F) Met; Leu; Tyr . =
Ser (S) Thr
Thr (T) Ser
7 - Trp (W) Tyr
Tyr (Y) Trp; Phe
Val (V) Ile; Leu
When the synthetic peptide is derivatised by amino acid substitution, the
amino =
acids are generally replaced by other amino acids having like properties such
as
hydrophobicity, hydrophilicity, electronegativety, bulky side chains and the
like. As used
herein, 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. For example, 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 may be found in J.
Sturart and
J.S. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford,
Ill.,
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(1984). Peptides may also be synthesized by solution methods as described in
The
Proteins, Vol. II, 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 Chemisny, 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. Likewise, 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.
Additionally, 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.
When applied to cells grown in culture, synthetic 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
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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. When 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
_- -
dosage of 50p,g/m1, the cells were not affected. However, when 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 pg/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 II) 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/mi. 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.
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:
MPFSTGKRIMLGE (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
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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.
When cultured cancer cells were treated with a peptide with the primary
structure
of SEQ ID NO:1 without a leader sequence attached, at a dosage of 100 p.g/rnl,
the cells
were unaffected. Similarly, when cultured cancer cells were treated with
leader sequence
SEQ ID NO:4, the presently preferred leader sequence, at the same dosage, the
cell were
also unaffected. These results indicate that the leader sequence of the
synthetic peptide
allows the synthetic peptide to cross the cellular membranes of treated cells
and that the
effect of the synthetic peptide is necessarily intracellular.
In order to determine whether the synthetic peptides acted by interfering with
the
binding of the p53 protein and the MDM-2 protein, 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. These results indicate that interference with binding
of the p53
protein to the MDM-2 protein may not be the mechanism by which synthetic
peptides
cause selective death of malignant and transformed cells. Although the
synthetic peptides
disclosed herein, their derivatives, analogues, and peptidomimetic molecules
are useful in
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the treatment of neoplastic disease such as cancer, the mechanism for action
on
transformed and malignant cells has not been discovered.
The peptides of the present invention are effective against neoplastic cells
in vivo.
For example, mice having been xenotransplanted with the pancreatic carcinoma
cells
BMRPA1.TUC-3 and having developed tumor size of about 3-6 mm, have the size of
such
_ -
tumors drastically reduced after administration of 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.
Consistent with the observed properties of the peptides of the invention, 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. The term "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.
Preferably, 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.
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Examples of different 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.
- _ =
Administration of 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. When administered
in such
manner, the synthetic peptides of the present invention may be combined with
other
ingredients, such as 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.
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=
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. . .
_ As used herein, 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. In order to obtain a sterile powder, 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. Examples of a
pharmaceutically
effective amount includes peptide concentrations in the range from about at
least about
25ug/m1 to at least about 300 ug/ml.
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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.
In addition, an individual's weight, gender, and overall health must be
considered and will
effect dosage. It can be generally stated, however, that 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.
In accordance with the present invention, there is also provided a method of
treating neoplastic disease. The method 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. Thus for example, in one
embodiment, 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. In
another
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embodiment, 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. For example, 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. In still another embodiment, 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. In accordance with a method of treatment, a mixture
of
synthetic peptides may be administered. Thus, for example, in addition to
administering
one of the peptides, or analogs or derivatives thereof hereinbefore described
in an effective
amount, mixtures of two or more peptides or analogs or derivatives
hereinbefore described
may be administered to a subject.
The following examples further illustrate the invention and are not meant to
limit
the scope thereof.
=
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EXAMPLE I
The following experiment was performed to compare effectiveness of subject
peptides having the leader sequence attached to the amino terminal end. As
described
supra, peptides synthesized with a leader sequence on the carboxyl terminal
promoted a-
helix formation in the peptide, which is the active conformation of the p53
part of this
_ -
peptide when bound to MDM-2. As described supra, subject peptides having the
amino
acid sequences as set forth in SEQ ID NOs:1, 2, and 3 are strongly toxic to a
wide variety
of human cancer cells, including those that are homozygously p53 gene-deleted.
An a-
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 (a) 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. (1987) Biopolymers 26:351-372
and
Vasquez, M., et al., (1987) Biopolymers 26:373-393. Probability profiles
indicated that if
the leader sequence is on the amino terminal end, even though the peptide
still transverses
the cell membrane, the a-helical content is much lower.
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
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and 100 p,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 p,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 p,g/ml. 100% cell death occurred in about 4 days.
These results show that the 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. In Table 2, the leader
sequence is
KKWKMRRNQFWVKVQRG (SEQ ID NO:4).
TABLE 2
NAME p53 seq. PEPTIDE EFFECT
1. PNC 21 12-20 (PPLSQETFS) (SEQ ID NO:2)¨ Cytotoxic
Leader
2. PNC 27 12-26 (PPLSQETFSDLWKLL) (SEQ ID Cytotoxic
NO:1)¨ Leader
3. PNC 28 17-26 (ETFSDLWKLUSEQ ID NO:3) ¨ Cytotoxic
Leader
4. PNC 28' 17-26 Leader (ETFSDLWKLL)(SEQ ID No cell death and
reversion
NO:3)
These results indicate the uniqueness of the subject peptides. i.e., the
leader or
cluster of positively charged residues must be placed at the carboxy terminal
end of any
effector peptide for cancer cell toxicity.
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EXAMPLE H
Nu/Nu mice (Harlan Laboratories, Indianapolis, IN, n=10) and weighing 20-22g,
were xenotransplanted subcutaneously (s.c.) with live pancreatic carcinoma
cells
BMRPA1 .TUC-3 (1 x 106 cells/mouse) in the left hind region. Tumors were
allowed to
develop and grow and during daily examinations it was observed that all mice
developed
tumors with very similar growth rates.
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 20mWmouse. One group of 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:
MPFSTGICREMLGE (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 1.11/ hr into each
mouse. The
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
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and all preceding and post-surgical treatments were therefore performed in a
sterile hood
environment.
As shown clearly in Figure 1, PNC-28 within a 48 to 72hr period of delivery
itito
the mouse effectively arrests tumor growth. In contrast, the control peptide
PNC-29 had
no effect.on normal or tumor cells. In PNC29-treated mice, tumors kept growing
at a
_
continuous rate resulting in tumors of 10 to 16 ram 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.
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EXAMPLE FEE
Using the same methodology of Example II, pumps were started at the same time
as live pancreatic carcinoma cells BMRPALTUC-3 (1 x 106 cells/mouse) were .
xenotransplanted into mice (n---10). Five mice were administered PNC28 and 5
mice were
_ -
not treated at all (sham treated). Results are tabulated below.
TABLE 3
Treatment 7 Days 14 Days 21 Days
Tumor Size
Sham treated 4.8 1.8 11.7 2.3 14.8 3.6
PNC-28 treated 3 + .6 3d .9 4.4 .8
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EXAMPLE IV
Using the same methodology as described in Example II, live pancreatic
carcinoma
cells BMRPA1.TUC-3 (1 x 106 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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The scope of the claims should not be limited by the preferred embodiments set
forth in the examples, but should be given the broadest interpretation
consistent with
the description as a whole.
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