WO2013026157A1

WO2013026157A1 - Anti-cancer peptides and uses thereof

Info

Publication number: WO2013026157A1
Application number: PCT/CA2012/050569
Authority: WO
Inventors: Kenneth Sokoll
Original assignee: Pharmagap Inc.
Priority date: 2011-08-24
Filing date: 2012-08-20
Publication date: 2013-02-28

Abstract

A class of peptidic compounds of general formula (I) and the retro-inverso form thereof (I-R) that are useful in the treatment of cancer. N_xB_y(A/N)_xB_yN_y-J(M)-N_yB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-HY (I) HY-HB2-(HY-HB)₂ - (linker) - B_xN_yB_yA_xB_zN_y-J(M)-N_yB_y(A/N)_xB_yN_x (I-R).

Description

ANTI-CANCER PEPTIDES AND USES THEREOF

FIELD OF THE INVENTION

[001] The present invention pertains to the field of cancer therapeutics and, in particular, to peptide and peptide analogues for the treatment of cancer.

BACKGROUND OF THE INVENTION

[002] Peptides are attracting increasing attention as drug candidates. Over 50 therapeutic peptides have received approval from at least one regulatory agency for various indications from HIV therapy to antimicrobials, and many more are in clinical or preclinical development (see "Development Trends for Peptide Therapeutics: 2010 Report Summary, " Peptide Therapeutics Foundation, San Diego, CA). Insulin, vancomycin, oxytocin, cyclosporine, Fuzeon® (enfuvirtide) and Integrilin® (eptifibatide) are a few examples of approved peptide-based drugs. Peptide therapeutics have a number of advantages including their small size, specificity and lower side-effects than traditional small molecule drugs. Candidate peptide therapeutics can be quickly investigated for therapeutic potential.

[003] International Patent Application Nos. PCT/CA06/00521 (Publication No. WO2006/108270) and PCT/CA06/01298 (Publication No. WO2007/016777) describe peptide- based inhibitors of protein kinases that are useful as therapeutics, for example, in the treatment of cancer.

[004] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. SUMMARY OF THE INVENTION

[005] An object of the present invention is to provide anti-cancer peptides and uses thereof. In accordance with an aspect of the invention there is provided a peptide between about 12 and about 40 amino acid residues in length having a sequence of general formula (I) or the retro- inverso form thereof (I-R):

N_xB_y(A/N)_xB_yN J(M)-N_yB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-HY (I)

HY-HB2-(HY-HB)₂ - (linker) - B_xN_yB_yA_xB_zN_y-J(M)-N_yB_y(A/N)_xB_yN_x (I-R) wherein:

J is 1 -2 Lys residues;

M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0-1 ;

each y is independently 0-2;

z = 0-l ;

(linker) is 3 to 9 amino acid residues selected from the group of: glycine, D-glycine, alanine and D-alanine;

each HY is 1 to 4 amino acid residues selected from the group of: Ala, Gly, He and Leu;

each HB is 1 to 3 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser,

wherein the sequences -N_yB_zA_xB_yN_yB_x and - B_xN_yB_yA_xB_zN_y are 2 or more amino acids in length, and

with the proviso that the peptide is other than:

Compound 1 [SEQ ID NO:l] Compound 2 [SEQ ID NO:2] and

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

[007] In accordance with another aspect of the present invention, there is provided a method of treating malignant ascites in a subject in need thereof comprising administering to the subject an effective amount of a peptide between about 12 and about 40 amino acid residues in length having a sequence of general formula (I) or the retro-inverso form thereof (I-R):

N_xB_y(A/N)xB_yNy-J(M)-NyB_zA_xB_yNyBx - (linker) - (HB-HY)₂-HB2-HY (I)

J is 1-2 Lys residues; M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues; each N is independently Ala, He, Leu, Val or Gly; each B is independently Arg or Lys; each A is independently Phe, His or Trp; each x is independently 0-1 ; each y is independently 0-2; 2 = 0-1;

(linker) is 3 to 9 amino acid residues selected from the group of: glycine, alanine and D-alanine; each HY is 1 to 4 amino acid residues selected from the group of: Ala, Gly, He and Leu; each HB is 1 to 3 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, wherein the sequences -N_yB_zA_xB_yN_yB_x and - B_xN_yB_yA_xB_zN_y are 2 or more amino acids in length.

[008] In accordance with another aspect of the present invention, there is provided a method of treating or preventing metastasis of a cancer in a subject in need thereof comprising administering to the subject an effective amount of a peptide between about 12 and about 40 amino acid residues in length having a sequence of general formula (I) or the retro-inverso form thereof (I-R):

N_xBy(A N)_xByN_y-J(M)-NyB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-IIY (I) HY-HB2-(HY-HB)₂ - (linker) - B_xN_yB_yA_xB_zN_y-J(M)-N_yB_y(A/N)_xB_vN_x (I-R) wherein:

J is 1-2 Lys residues;

M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues; each N is independently Ala, He, Leu, Val or Gly; each B is independently Arg or Lys; each A is independently Phe, His or Trp; each x is independently 0-1 ; each y is independently 0-2; z = 0-l ;

[009] In accordance with another aspect of the invention, there is provided a method of treating a drug- or hormone-resistant cancer in a subject in need thereof comprising administering to the subject an effective amount of a peptide between about 12 and about 40 amino acid residues in length having a sequence of general formula (I) or the retro-inverso form thereof (I-R):

N_xB_y(A/N)_xB_yN_y-J(M)-NyB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-HY (I)

HY-HB2-(HY-HB)₂ - (linker) - B_xN_yB_yA_xB_zN_y-J(M)-N_yB_y(A/N)_xByN_x (I-R) wherein:

J is 1-2 Lys residues;

M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues; each N is independently Ala, He, Leu, Val or Gly; each B is independently Arg or Lys; each A is independently Phe, His or Tip; each x is independently 0-1 ; each y is independently 0-2; z = 0-l ;

HD2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, wherein the sequences -N_yB_zA_xB_yN_yB_x and - B_xN_yB_yA_xB_zN_y are 2 or more amino acids in length.

BRIEF DESCRIPTION OF THE DRAWINGS

[010] These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings.

[Oil J Figure 1 presents a bar chart summarizing the effect of compounds 4, 9, 16 and 17 on the proliferation of OCC-1 human ovarian cancer cells in vitro. Cells were treated with 25 μΜ peptide for 24 hours. All groups were found to have a statistically significant difference from control (p = <0.001 ). "x" denotes groups not statistically different from each other.

[012] Figure 2 presents a bar chart summarizing the effect of compounds 4 and 9 on the proliferation of RT4, TCCSUP and T24 human bladder cancer cells in vitro. Cells were treated with 25 μΜ compound 4 or 12 μΜ compound 9 for 24 hours. *** p = <0.001 after a t-test.

[013] Figure 3 presents a bar chart summarizing the effect of compounds 4 and 9 on the proliferation of LS513 human colon cancer cells, and A2780cp and ES-2 human ovarian cancer cells in vitro. Cells were treated with 25 μιΜ of either compound 4 or compound 9 for 48 hours. *** p = <0.001 after a t-tcst.

[014] Figure 4 presents (A) a bar chart summarizing the mean tumour burden expressed as mean total tumour weight from control (n-6), compound 9 ("D-isomer") treated mice (n=6) and compound 4 ("L-isomcr") treated mice (n=5 *). No significant difference in total tumour burden between the any of the groups was observed (p>0.05; unpaired t-test). *Tumour burden for one mouse was not recorded due to experimental error. (B) A bar chart summarizing the mean total ascites volume from control (n=6), compound 9 ("D-isomer") treated mice (n=6) and compound 4 ("L-isomer") treated mice (n=6). A significant difference in total volume of ascites between the compound 9 treated mice and the control mice was observed (p<0.05; unpaired t-test). There was no significant difference in ascites volume between control mice and those treated with compound 4.

[015] Figure 5 depicts a Western Blot analysis of pAKT status in A2780cp cells lhr after treatment with compound 4. Data is from one independent experiment.

[016] Figure 6 depicts a Western Blot analysis of pAKT status in A2780cp cells at various time intervals after treatment with compound 4. Three replicates A, B, C are shown. The 30 hour time point probing for pAKT was performed twice.

DETAILED DESCRIPTION OF THE INVENTION

[017] The present invention relates to a class of peptidic compounds of general formula (I) and the retro-inverso form thereof (I-R) that are useful in the treatment of cancer. In one embodiment of the invention, the peptides demonstrate activity in inhibiting the formation of malignant ascites and thus are useful in the treatment of malignant ascites, as well as advanced and/or metastatic cancers and cancers with a high potential for metastasis and/or development of malignant ascites.

[018] In one embodiment of the invention, the peptides are capable of inhibiting expression of Akt. Accordingly, in certain embodiments, the present invention provides for the use of the peptides to inhibit Akt expression. As is known in the art, Akt has been implicated in certain cancer functions, including cell motility and invasion, hormone independence, and chemotherapy and radiation resistance. Abnormalities in Akt are associated with certain breast cancers, pancreatic cancers, colorectal cancers, gastric cancers and ovarian cancers. Accordingly, in certain embodiments, methods of treating hormone-resistant cancers (for example, hormone- resistant breast or prostate cancer) and chemotherapy or radiation resistant cancers with the peptides are provided. In other embodiments, methods of treating breast cancer, pancreatic cancer, colorectal cancer, gastric cancer and/or ovarian cancer are provided.

[019] Certain embodiments of the invention provide for peptidic compounds that are variants of compound 1 below, which have one or more improved properties over compound 1 and thus provide alternative options for effective anti-cancer therapeutics:

Compound 1 [SEQ ID NO: l]

Definitions

[020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[021] The term "naturally-occurring," as used herein with reference to an object, such as a protein, peptide or amino acid, indicates that the object can be found in nature. For example, a protein, peptide or amino acid that is present in an organism or that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is considered to be naturally-occurring.

[022] The term "amino acid residue," as used herein, encompasses both naturally-occurring amino acids and non-naturally-occurring amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, D-amino acids (i. e. an amino acid of an opposite chirality to the naturally-occurring form), N-a-methyl amino acids, C-a-methyl amino acids, β- methyl amino acids and D- or Ι,-β-amino acids. Other non-naturally occurring amino acids include, for example, β-alanine (β-Ala), norleucine (Nle), norvaline (Nva), homoarginine (Har), 4-aminobutyric acid (γ-Abu), 2-aminoisobutyric acid (Aib), 6-aminohexanoic acid (ε-Ahx), ornithine (orn), sarcosine, a-amino isobutyric acid, 3-aminopropionic acid, 2,3-diaminopropionic acid (2,3-diaP), D- or L-phenylglycine, D-(trifluoromethyl)-phenylalanine, and D-p- fluorophenylalanine.

[023] The term "retro-inverso sequence" or "retro-inverso peptide," as used herein, refers to a sequence of amino acids that has been altered with respect to a reference amino acid sequence in that the amino acid sequence has been reversed and all L-amino acids have been replaced with D-amino acids. Compared to the reference peptide, a retro-inverso peptide has a reversed backbone while retaining substantially the original spatial conformation of the side chains, resulting in an isomer with a topology that closely resembles the reference peptide.

[024] The terms "therapy" and "treatment," as used interchangeably herein, refer to an intervention performed with the intention of improving a subject's status. The improvement can be subjective or objective and is related to ameliorating the symptoms associated with, preventing the development of, or altering the pathology of a disease or disorder being treated. Thus, the terms therapy and treatment are used in the broadest sense, and include the prevention (prophylaxis), moderation, reduction, and curing of a disease or disorder at various stages. Preventing deterioration of a subject's status is also encompassed by the term. Subjects in need of therapy/treatment thus include those already having the disease or disorder as well as those prone to, or at risk of developing, the disease or disorder and those in whom the disease or disorder is to be prevented.

[025] The term "ameliorate" includes the arrest, prevention, decrease, or improvement in one or more of the symptoms, signs, and features of the disease or disorder being treated, either temporarily or in the long-term.

[026] The term "subject" or "patient" as used herein refers to an animal, such as a mammal or a human, in need of treatment.

[027] As used herein, the term "about" refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[028] Naturally-occurring amino acids are identified throughout by the conventional three-letter or one-letter abbreviations indicated below, which are as generally accepted in the peptide art and are recommended by the IUPAC-IUB commission in biochemical nomenclature:

Table 1. Amino Acid Codes

Name -Vleltcr 1 -letter Nil me ί -letter 1 -letter

code code code code

Alanine Ala A Leucine Leu L

Arginine Arg R Lysine Lys K

Asparagine Asn N Methionine Met M

Aspartic Asp D Phenylalanine Phe F

Cysteine Cys C Proline Pro P

Glutamic acid Glu E Serine Ser S

Glutamine Gin Q Threonine Thr T

Glycine Gly G Tryptophan Trp w

Histidine His H Tyrosine Tyr Y

Isoleucine He I Valine Val V [029] The peptide sequences set out herein are written according to the generally accepted convention whereby the N-terminal amino acid is on the left and the C-terminal amino acid is on the right. By convention also, L-amino acids are represented by upper case letters and D-amino acids by lower case letters.

PEPTIDES

[030] In general, the peptides of the present invention are between about 12 and about 40 amino acid residues in length and have a sequence of general formula (I) or the retro-inverso form thereof (I-R):

N_xB_y(A/N)_xB_yN_y-J(M)-N_yB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-HY (I)

J is 1-2 Lys residues;

each N is independently Ala,Tle, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0-1 ;

each y is independently 0-2;

z = 0-l ;

(linker) is 3 to 9 amino acid residues selected from the group of: glycine, , alanine and D-alanine;

each HB is 1 to 3 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser,

wherein the sequences -N_yB_zA_xB_yN_yB_x and - B_xN_yB_yA_xB_zN_y are 2 or more amino acids in length.

[031] In certain embodiments, the peptides in accordance with the present invention are variants of compound 1 [SEQ ID NO: l] (shown below), for example, variants comprising one or more D-amino acids, variants in which the PNA moiety is absent, and variants comprising a protein translocation domain (PTD) peptide, which variants may also include one or more D- amino acids and/or lack the the PNA moiety. LIGISI-NH₂

Compound 1 [SEQ ID NO: 1]

[032] In accordance with this embodiment, the peptide is between about 12 and about 40 amino acid residues in length and has a sequence of general formula (I) or the retro-inverso form thereof (I-R), as described above, with the proviso that the peptide is other than:

Compound 1 [SEQ ID NO: l] Compound 2 [SEQ ID NO:2] and

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

[033] In another embodiment of the present invention, the peptide is between about 12 and about 40 amino acid residues in length and has a sequence of general formula (I) or the retro- inverso form thereof (I-R):

N_xB_y(A/N)_xB_yN_y-J(M)-N_yB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-HY (I)

HY-HB2-(HY-HB)₂ - (linker) - B_xNyB_yA_xB_zN_rJ(M)-NyBy(A/N)_xB_yN_x (I-R) wherein:

J is 1-2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0-1 ; each y is independently 0-2;

z = 0-l ;

(linker) is 3 to 9 amino acid residues selected from the group of: glycine, alanine and D-alanine;

wherein the sequences -N_yB_zA_xB_yN_yB_x and - B_xN_yB_yA_xB_zN_y are 2 or more amino acids in length,

wherein when the peptide has a sequence of Formula (I), the peptide further comprises a protein translocation domain (PTD) peptide, and

with the proviso that the peptide is other than:

NH₂ and NH₂

Compound 4 [SEQ ID NO:4] Compound 5 [SEQ ID NO:5] 4] In one embodiment of the present invention, the peptide is between about 12 and about amino acid residues in length and has a sequence of general formula (I-R): HY-HB2-(HY-HB)₂ - (linker) - B_xN_yB_yA_xB_zN_y-J(M)-N_yB_y(A/N)_xB_yN_x (T-R) wherein:

J is 1-2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0- 1 ;

each y is independently 0-2;

z = 0-l ;

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and

wherein the sequence -B_xN_yB_yA_xB_zN_y is 2 or more amino acids in length. 5] In one embodiment of the present invention, the peptide is between about 12 and about amino acid residues in length and has a sequence of general formula (I): N_xB_y(A/N)_xB_yN_y-J(M)-N_yB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-HY (I) wherein:

J is 1 -2 Lys residues;

M is absent;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0- 1 ;

each y is independently 0-2;

z = 0-l ;

wherein the sequence -N_yB_zA_xB_yN_yB_x is 2 or more amino acids in length, and with the proviso that the peptide is other than:

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3] [036] In one embodiment of the present invention, the peptide has a sequence of general formula (II) or the retro-inverso form thereof (II-R):

B_y(A N)_xB_yN_y-J(M)-N_yB_zA_xB_yN_y - (linker) - (HB-HY)₂-HB2-HY (II)

HY-HB2-(HY-HB)₂ - (linker) - N_yB_yA_xB_zN_y-J(M)-N_yB_y(A/N)_xB_y (II-R) wherein:

J is 1-2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

z = 0-l ;

(linker) is 3 to 9 amino acid residues selected from the group of: glycine, alanine, valine, leucine and isoleucine;

HB2 represents 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser,

wherein the sequences -N_yB_zA_xB_yN_y and - N_yB_yA_xB_zN_y are 2 or more amino acids in length.

[037] In one embodiment of the present invention, the peptide has a sequence of general formula (II) or the retro-inverso form thereof (II-R), as described above, with the proviso that the peptide is other than:

Compound 1 [SEQ ID NO:l] Compound 2 [SEQ ID NO: 2] and

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

[038] In one embodiment of the present invention, the peptide has a sequence of general formula (II) or the retro-inverso form thereof (II-R):

By(A/N)_xB_yN_y-J(M)-N_yB_zA_xB_yN_y - (linker) - (HB-HY)₂-HB2-HY (II)

J is 1-2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly; each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

z = 0-l ;

wherein the sequences -N_yB_zA_xB_yNy and - N_yB_yA_xB_zNy are 2 or more amino acids in length,

wherein when the peptide has a sequence of Formula (II), the peptide further comprises a protein translocation domain (PTD) peptide, and

with the proviso that the peptide is other than:

Compound 4 [SEQ ID NO:4] Compound 5 [SEQ ID NO:5] [039] In one embodiment of the present invention, the peptide has a sequence of general formula (II-R):

J is 1-2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

z = 0-l ;

HB2 represents 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and wherein the sequence - N_yB_yA_xB_zN_y is 2 or more amino acids in length.

[040] In one embodiment of the present invention, the peptide has a sequence of general formula (11):

B_y(A N)_xByN_y-J(M)-N_yB_zA_xB_yNy - (linker) - (HB-HY)₂-HB2-HY (II) wherein:

J is 1-2 Lys residues;

M is absent;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2; z = 0-l ;

wherein the sequence -N_yB_zA_xB_yN_y is 2 or more amino acids in length, and with the provise that the peptide is other than:

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

[041] In one embodiment of the present invention, the peptide has a sequence of general formula (III) or the retro-inverso form thereof (III-R):

(A/N)_xB_yN_y-J(M)-N_yA_xB_yN_y - (linker) - (HB-HY)₂-HB2-HY (III)

HY-HB2-(HY-HB)₂ - (linker) - N_yB_yA_xN_y-J(M)-N_yB_y(A/N)_x (III-R) wherein:

J is 1-2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 glycine residues;

each HB is 1 to 3 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser;

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser;

wherein the sequences -N_yA_xB_yN_y and - N_yB_yA_xN_y are 2 or more amino acids in length.

[042] In one embodiment of the present invention, the peptide has a sequence of general formula (III) or the retro-inverso form thereof (III-R), as described above, with the proviso that the compound is other than:

Compound 1 [SEQ ID NO:l] Compound 2 [SEQ ID NO:2] and

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

[043] In one embodiment of the present invention, the peptide has a sequence of formula (III) or the retro-inverso form thereof (III-R):

(A/N)_xB_yNy-J(M)-N_yA_xB_yNy - (linker) - (HB-HY)₂-HB2-HY (III)

HY-HB2-(HY-HB)₂ - (linker) - N_yB_yA_xN_y-J(M)-N_yB_y(A N)_x (III-R) wherein:

J is 1 -2 Lys residues; M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 glycine residues;

wherein the sequences -N_yA_xB_yN_y and - N_yB_yA_xN_y are 2 or more amino acids in length,

wherein when the peptide has a sequence of Formula (HI), the peptide further comprises a protein translocation domain (PTD) peptide, and

with the proviso that the peptide is other than:

Compound 4 [SEQ ID NO:4] Compound 5 [SEQ ID NO:5]

[044] In one embodiment of the present invention, the peptide has a sequence of general formula (III-R):

HY-HB2-(HY-HB)₂ - (linker) - N_yB_yA_xN_y-J(M)-N_yB_y(A/N)_x (III-R) wherein:

J is 1-2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 glycine residues;

wherein the sequence - N_yB_yA_xN_y is 2 or more amino acids in length.

[045] In one embodiment of the present invention, the peptide has a sequence of general formula (III):

(A/N)_xB_yN_y-J(M)-N_yA_xB_yN_y - (linker) - (HB-HY)₂-HB2-HY (III) wherein:

J is 1-2 Lys residues;

M is absent;

each N is independently Ala, lie, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 glycine residues;

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser; wherein the sequence -N_yA_xB_yN_y is 2 or more amino acids in length, and with the proviso that the peptide is other than:

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

[046] In one embodiment, in general formulae (III) and (III-R): J is Lys;

M is absent or is an ATP mimetic moiety attached to J via the side chain of the Lys residue;

each N is independently He or Leu;

each B is independently Arg or Lys;

each A is independently Phe;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 glycine residues;

each HB is 1 to 2 amino acid residues selected from the group of: Asn, Asp, Gin and Lys, and

HB2 is 1 amino acid residue selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser.

[047] In one embodiment of the present invention, the peptide has a sequence of general formula (IV) or the retro-inverso form thereof (IV-R): A_xB_y-J(M)-A_xB_yN_y - (linker) - (HB-HY)₂-HB2-HY (IV)

HY-HB2-(HY-HB)₂ - (linker) - N_yB_yA_x-J(M)-B_yA_x (IV-R) wherein:

J is 1 -2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 glycine residues;

wherein the sequences -A_xB_yN_y and - N_yB_yA_x are 2 or more amino acids in length..

[048] In one embodiment of the present invention, the peptide has a sequence of general formula (IV) or the retro-inverso form thereof (IV-R), as described above, with the proviso that the peptide is other than:

Compound 1 [SEQ ID ΝΟ:1] Compound 2 [SEQ ID NO:2] and

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

[049] In one embodiment of the present invention, the peptide has a sequence of general formula (IV) or the retro-inverso form thereof (IV-R):

A_xB_y-J(M)-A_xB_yN_y - (linker) - (HB-HY)₂-HB2-HY (IV)

HY-HB2-(HY-HB)₂ - (linker) - N_yB_yA_x-J(M)-B_yA_x (IV-R) wherein:

J is 1-2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2; (linker) is 3 to 9 glycine residues;

wherein the sequences -A_xB_yN_y and - N_yB_yA_x are 2 or more amino acids in length, and

wherein when the peptide has a sequence of Formula (IV), the peptide further comprises a protein translocation domain (PTD) peptide, and

with the proviso that the peptide is other than:

Compound 4 [SEQ ID NO:4] Compound 5 [SEQ ID NO:5]

[050] In one embodiment of the present invention, the peptide has a sequence of general formula (IV-R):

HY-HB2-(HY-HB)₂ - (linker) - N_yB_yA_x-J(M)-B_yA_x (IV-R) wherein:

J is 1-2 Lys residues; M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 glycine residues;

wherein the sequence - N_yB_yA_x is 2 or more amino acids in length.

[051] In one embodiment of the present invention, the peptide has a sequence of general formula (IV):

A_xBy-J(M)-A_xB_yN_y - (linker) - (HB-HY)₂-HB2-HY (IV) wherein:

J is 1 -2 Lys residues;

M is absent;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 glycine residues;

with the proviso that the peptide is other than:

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3] In one embodiment, in general formulae (IV) and (I V-R) : J is Lys;

each N is independently He or Leu;

each B is independently Arg or Lys; each A is independently Phe;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 Gly residues;

[053] In one embodiment of the invention, the peptide has a sequence of general formula (V) or the retro-inverso form thereof (V-R):

X-(lmker)-Y (V)

[Y-R] -(linker)- [X-R] (V-R)

wherein:

X is an amino acid sequence comprising at least 5 consecutive residues of the sequence: FRRKFRL [SEQ ID NO: 6], wherein the Lys residue is optionally attached to an ATP mimetic moiety (M);

Y is an amino acid sequence comprising at least 8 consecutive residues of the sequence: KDAQNLIGISI [SEQ ID NO:7];

[X-R] is an amino acid sequence comprising at least 5 consecutive residues of the sequence: lrfkrrf [SEQ ID NO:8], wherein the Lys residue is optionally attached to an ATP mimetic moiety (M);

[Y-R] is an amino acid sequence comprising at least 8 consecutive residues of the sequence: isigilnqadk [SEQ ID N0:9], and

(linker) is 3 to 9 amino acid residues selected from the group of: glycine, alanine and D-alanine.

[054] In one embodiment of the invention, the peptide has a sequence of general formula (V) or the retro-inverso form thereof (V-R), as described above, with the proviso that the peptide is other than:

Compound 1 [SEQ ID NO: 1] Compound 2 [SEQ ID NO:2] and

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

[055] In one embodiment of the invention, the peptide has a sequence of general formula (V) or the retro-inverso form thereof (V-R):

X-(linker)-Y (V)

[Y-R] -(linker)- [X-R] (V-R)

wherein:

X is an amino acid sequence comprising at least 5 consecutive residues of the sequence: FRRKFRL [SEQ ID NO:6], wherein the Lys residue is optionally attached to an ATP mimetic moiety (M);

[X-R] is an amino acid sequence comprising at least 5 consecutive residues of the sequence: Irfkrrf [SEQ ID NO:8], wherein the Lys residue is optionally attached to an ATP mimetic moiety (M);

[Y-R] is an amino acid sequence comprising at least 8 consecutive residues of the sequence: isigilnqadk [SEQ ID NO:9],

(linker) is 3 to 9 amino acid residues selected from the group of: glycine, alanine and D-alanine, and

wherein when the peptide has a sequence of Formula (V), the peptide further comprises a protein translocation domain (PTD) peptide, and

with the proviso that the peptide is other than:

Compound 4 [SEQ ID NO:4] Compound 5 [SEQ ID NO:5]

[056] In one embodiment of the invention, the peptide has a sequence of general formula (V- R):

[Y-R]-(linker)-[X-R] (V-R)

wherein:

[X-R] is an amino acid sequence comprising at least 5 consecutive residues of the sequence: lrfkrrf [SEQ ID N0:8], wherein the Lys residue is optionally attached to an ATP mimetic moiety (M);

[Y-R] is an amino acid sequence comprising at least 8 consecutive residues of the sequence: isigilnqadk [SEQ ID NO:9], and

[057] In one embodiment of the invention, the peptide has a sequence of general formula (V):

X-(linker)-Y (V)

wherein:

X is an amino acid sequence comprising at least 5 consecutive residues of the sequence: FRRKFRL [SEQ ID O:6];

with the proviso that the peptide is other than:

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO: 3]

[058] In one embodiment, the peptide is a fragment of the peptide of general formula (I) or (I- R) having a length of about 5 to about 12 amino acids and a sequence of general formula (VI) or the retro-inverso form thereof (VI-R):

N_xB_y(A/N)_xB_yN J(M)-N_yB_zA_xB_yN_yB_x (VI) B_xN_yB_yA_XB_ZN_y-J(M)-NyB_y(A/N)_XByN_X (VI-R) wherein J, M, N, B, A, x, y and z are as described above for general formula (I).

[059] In one embodiment, the peptide is a fragment of the peptide of general formula (I) or (I- R) having a length of about 5 to about 12 amino acids and a sequence of general formula (VI) or the retro-inverso form thereof (VI-R), as described above, with the proviso that the peptide is other than:

Compound 6 [SEQ ID NO: 10] Compound 7 [SEQ ID NO: l 1] and

Compound 8 [SEQ ID NO: 12]

[060] In one embodiment, the peptide is a fragment of the peptide of general formula (I) or (I- R) having a length of about 5 to about 12 amino acids and a sequence of general formula (VI) or the retro-inverso form thereof (VI-R): N_xB_y(A/N)_xB_yN_y-J(M)-N_yB_zA_xB_yN_yB_x (VI)

B_xNyB_yA_xB_zN_y-J(M)-N_yB_y(A/N)_xB_yN_x (VI-R) wherein J, M, N, B, A, x, y and z are as described above for general formula (I), and wherein when the peptide has a sequence of Formula (VI), the peptide further comprises a protein translocation domain (PTD) peptide.

[061] In one embodiment, the peptide is a fragment of the peptide of general formula (I) having a length of about 5 to about 12 amino acids and a sequence of general formula (VI) or the retro- inverso form thereof (VI-R):

N_xB_y(A N)_xB_yN_y-J(M)-N_yB_zA_xB_yN_yB_x (VI)

B_xNyB_yA_xB_zN_y-J(M)-N_yB_y(A/N)_xB_yN_x (VI-R) wherein J, N, B, A, x, y and z are as described above for general formula (I), and M is absent.

[062] In one embodiment, in general formulae (VI) and (VI-R):

J is Lys;

M is absent or is an ATP mimetic moiety attached to J via the side chain of the Lys residue; each N is independently He or Leu; each B is independently Arg or Lys; each A is independently Phe; each x is 1 , and each y is independently 0-2.

[063] In one embodiment of the present invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R) (V) and (V-R) is between about 12 and about 35 amino acid residues in length. In another embodiment, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R) (V) and (V-R) is between about 12 and about 30 amino acid residues in length. In other embodiments, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R) (V) and (V-R) is between about 12 and about 29 amino acid residues in length, between about 12 and about 28 amino acid residues in length, between about 12 and about 27 amino acid residues in length; between about 12 and about 26 amino acid residues in length and between about 12 and about 25 amino acid residues in length.

[064] In certain embodiments of the present invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R) (V), (V-R), (VI) and (VI-R) comprises an ATP mimetic moiety (M). In the context of the present invention, an ATP mimetic is a compound that comprises adenine or a derivative of adenine. A "derivative of adenine," as used herein, refers to a compound that retains the hetero aromatic ring structure of adenine (shown below) but which may contain additional, fewer or different substituents attached to the ring structure and/or additional, fewer or different heteroatoms within the ring structure when compared to adenine.

[065] The term "derivative of adenine" also encompasses molecules that are isosteric with adenine. In the context of the present invention, a molecule that is isosteric with adenine (an "adenine isostere") is a molecule that has a similarity of structure and spatial orientation to adenine and a resulting similarity of properties, in particular with respect to three-dimensional space-filling properties.

[066] Suitable adenine derivatives are known in the art and include, but are not limited to, 1 - deazaadenine; 3-deazaadenine; 7-deazaadenine; 7-deaza-8-azaadenine; 1 -methyladenine; 2- aminoadenine; 2-propyl and other 2-alkyl derivatives of adenine; 2-aminopropyladenine; 8- amino, 8-aza, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines; 8-oxo-N⁶- methyladenine; N⁶-methyladenine; N⁶-isopentenyladenine; 2-aminopurine; 2,6-diaminopurine; 2-amino-6-chloropurine; 6-thio-2-aminopurine; hypoxanthine; inosine; xanthine; 8-aza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza-8-aza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6- chloropurine, hypoxanthine, inosine and xanthine; 1-deaza derivatives of 2-aminopurine, 2,6- diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; and 3-deaza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; and adenine isosteres, such as 4-methylindole.

[067] Examples of ATP mimetics that can be incorporated into the peptides include adenine peptide nucleic acid (PNA). As used herein, the term "adenine PNA" includes PNAs comprising adenine or an adenine derivative, such as those described above, as the adenine moiety. In one embodiment of the invention, the peptide of any one of general formulae (I), (I-R), (II), (H-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R) comprises an ATP mimetic moiety (M) that is an adenine PNA.

[068] In certain embodiments of the present invention, in the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (Vl-R), the ATP mimetic moiety (M) is absent.

[069] In one embodiment of the present invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R) further comprises a protein translocation domain (PTD) peptide. The PTD can be attached to the N- or C-terminus, or to the side chain of one of the constituent amino acids of the peptide, for example, to the side chain of a lysine, arginine, glutamate, aspartate, asparagine or glutamine residue. In those embodiments in which the peptide includes an ATP mimetic moiety, the P l^'D may be attached to the ATP mimetic. In one embodiment, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R) comprises a PTD attached to the side chain of one of the constituent amino acids of the peptide. In another embodiment, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R) comprises a lysine residue and a PTD attached to the side chain of the lysine residue.

[070] Examples of PTDs are provided in Table 2. Fragments (for example, of at least 5 amino acids in length) of these sequences could also be used as PTDs.

Table 2: Peptides with Translocation Activity

[071] In one embodiment of the present invention, the peptide of any one of general formulae

(I) , (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R) further comprises a PTD having a sequence selected from the sequences provided in Table 2 or a fragment thereof. In another embodiment, the peptide of any one of general formulae (I), (I-R),

(II) , (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R) further comprises a PTD having the sequence: KRRQRRKKR [SEQ ID NO: 19] or rkkrrqrrk [SEQ ID NO:20] or a fragment of one of these sequences.

[072] In one embodiment of the present invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R) comprises a modified N- and/or C-terminus. Examples of chemical substituent groups suitable for modifying the N-terminus and/or C-terminus of peptides are known in the art and include, but are not limited to, alkyl, alkenyl, alkynyl, amino, aryl, aralkyl, heteroalkyl, hydroxy, alkoxy, aralkyloxy, aryloxy, carboxy, acyl, aroyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, dialkylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, alkylthio, aralkylthio, arylthio, alkylene, and NZiZ₂ where Zi and Z₂ are independently hydrogen, alkyl, aryl, or aralkyl, and the like. Blocking groups such as Fmoc (fluorenylmethyl-O-CO-), carbobenzoxy (benzyl-O-CO), monomethoxysuccinyl, naphthyl-NH-CO-, acetylamino-caproyl and adamantyl-NH-CO-, can also be used. Other modifications contemplated by the present invention include C-terminal amidation, esterification, hydroxymethyl modification and O- modification (for example, C-terminal hydroxymethyl benzyl ether), as well as N-terminal modifications such as substituted amides, for example alkylamides and hydrazides.

[073] In one embodiment of the present invention, in the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R), the N- terminus of the peptide is modified with an acyl group. Non-limiting examples of suitable acyl groups are benzoyl, acetyl, /-butylacetyl, 7-phenylbenzoyl, trifluoroacetyl, cyclohexylcarbonyl, phenylacetyl, 4-phenylbutanoyl, 3,3-diphenylpropanoyl, 4-biphenylacetyl, diphenylacetyl, 2- naphthylacetyl, 3-phenylbutanoyl, a-phenyl-or Tzo-toluoyl, indole-3 -acetyl, 3-indolepropanoyl, 3-indolebutanoyl, 4-(4-methoxyphenyl)butanoyl, and the like. In another embodiment, the N- terminus of the peptide is modified with an acetyl group. In another embodiment, in the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R), the C-terminus of the peptide is modified with an amino group.

[074] In one embodiment of the invention, the peptide is selected from the group of:

Ac-isigilnqadkggggggglrfkrrf-NH_j

Compound 9

Ac-isigilnqadkggggggglrfkrrf-NH₂

Compound 10 Compound 1 1 [SEQ ID NO:27]

rkkrrqrrk-Ac

Compound 12 [SEQ ID O:28] Compound 13

Compound 14 Compound 15 [SEQ ID NO:29]

Ac-FRR-HN

Compound 16 [SEQ ID NO:30] Compound 17

0

Ac-FRR-HN

^" FRLGGGGGGGKDAQNLIGISI-NH₂

/

Ac— rkkrrqrrk— HN

Compound 18 [SEQ ID NO:31]

O

Ac— isigilnqadkggggggglrf— HN„ , .J-L

J rrf-NH₂

HN— KRRQRRKR-Ac

Compound 19

N— - c c-krrqrrkkr-HN Compound 20 [SEQ ID NO:32] Compound 21

O

Ac— Irf-HN

Ac-FRR-HN , n f-NH₂

FRL-NH₂

/ HN-KRRQRRKR-Ac

Ac— krrqrrkkr— HN

Compound 22 [SEQ ID NO:33] Compound 23

[075] In one embodiment of the invention, the peptide is selected from the group of:

Compound 9 Compound 1 1 [SEQ ID NO:27]

-rkkrrqrrk-Ac

Compound 12 [SEQ ID O:28] Compound 13

-HN

Compound 16 [SEQ ID NO:30] Compound 17

Ac-FRR— HN

FRLGGGGGGGKDAQNLIGISI-NH2

Ac— rkkrrqrrk— HN

Compound ] 8 [SEQ ID NO:31]

O o

Ac-FRR-HN . A Ac— Irf— HN ,

RL-NH₂ ^{^}rrf-NH,

HN— RRQRRKKR-Ac Ac-krrqrrkkr-HN Compound 20 [SEQ ID NO:32] Compound 21 In another embodiment of the invention, the peptide is selected from the group of:

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Ac-isigilnqadkgggggggirfkr rf-NH, [ _N „_rkkrrqrrk._Ac

N ■ „ rkkrrcrrk-Ac O I ^' -" N

I NH₂

N H₂ ²

Compound 9 Compound 1 1 [SEQ ID NO:27]

o

FRR-HN , „, „ „ , Ac— isigilnqadkggggggglrf- FRLGGGGGGGKDAQNLIGISI-NH₂ saaaaaa

/

HN-KRRQRRKKR-Ac Ac-krrqrrkkr- Compound 16 [SEQ ID NO:30] Compound 17

O

Ac-FRR-HN ,

FRLGGGGGGGKDAQNLIGISI-NH2

Ac— rkkrrqrrk— HN

Compound 18 [SEQ ID NO:31 ]

PREPARA TION OF THE PEPTIDES

[077] The peptides of the present invention can be prepared, for example, by standard peptide synthesis techniques known in the art, such as by standard solution, suspension or solid phase techniques, including exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis.

[078] In one embodiment of the present invention, solid phase techniques are employed to prepare the peptides. The principles of solid phase chemical synthesis of peptides are well known in the art and may be found in general texts in the area such as Pennington, M.W. and Dunn, B.M., Methods in Molecular Biology, Vol. 35 (Humana Press, 1994); Dugas, H. and Penney, C, Bioorganic Chemistry (1981 ) Springer-Verlag, New York, pgs. 54-92; Merrifield, J. M., Chem. Soc, 85 :2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis, pp. 24-66, Freeman (San Francisco, 1969).

[079] In general, an insoluble polymer support (or resin) is used to prepare the starting material by attaching a protected version of the required a-amino acid to the resin. The resin acts to anchor the peptide chain as each additional a-amino acid is attached and is composed of particles (generally between about 20-50 μηι diameter) that are chemically inert to the reagents and solvents used in solid phase peptide synthesis. These particles swell extensively in solvents, which makes the linker arms more accessible. Examples of resins used in solid phase peptide synthesis include chloromethylated resins, hydroxymethyl resins, benzhydrylamine resins, and the like. Various resins suitable for solid phase peptide synthesis applications are available commercially, for example, phenylacetamidomethyl (PAM) resin, hydroxymethyl polystyrene- vinylbenzene copolymer, polyamide, p-benzyloxybenzyl alcohol resin (Wang resin) and modified versions thereof, 4-hydroxymethylphenoxymethyl-copoly(styrene- 1 % divinylbenzene), and 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl and [5-(4-Fmoc- aminomethyl-3,5-dimethoxyphenoxy)valeric acid]-polyethylene glycol-polystyrene resins (which are commercially available from Applied Biosystems, Foster City, CA) and can be used in the preparation of the peptides.

[080] The a-amino acid is coupled to the resin using a standard coupling reagent such as Ν,Ν'- dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC) or O-benzotriazol- 1 -yl- Ν,Ν,Ν',Ν'-tetramethyluronium-hexafluoi phosphate (HBTU), with or without 4- dimethylaminopyridine (DMAP), 1 -hydroxybenzotriazole (HOBT), benzotriazol-l -yloxy- tris(dimethylamino)phosphonium-hexafluorophosphate (BOP) or bis(2-oxo-3- oxazolidinyl)phosphine chloride (BOPC1). The coupling generally takes place in a solvent such as dichloromethane, DMF or NMP.

[081] After the initial coupling, the a-amino protecting group is removed using a standard reagent, such as a solution of trifluoroacetic acid (TFA), hydrochloric acid in an organic solvent or 20% piperidine in DMF solvent.

[082] Suitable a-amino protecting groups are known in the art of and include, for example, acyl type protecting groups (such as, formyl, trifluoroacetyl, acetyl), aromatic urethane type protecting groups (such as, benzyloxycarboyl (Cbz) and substituted Cbz), aliphatic urethane protecting groups (such as, t-butyloxycarbonyl (Boc), isopropyloxycarbonyl and cyclohexyloxycarbonyl), alkyl type protecting groups (such as, benzyl and triphenylmethyl) and 9-fluorenylmethoxy carbonyl (Fmoc).

[083] Side chain protecting groups, when used, remain intact during coupling and typically are not removed during the deprotection of the amino-terminus protecting group or during coupling. Side chain protecting groups are generally selected such that they are removable upon the completion of the synthesis of the final peptide and under reaction conditions that will not alter the peptide. Examples of side chain protecting groups include, but arc not limited to, benzyl, 2,6- dichlorobenzyl, methyl, ethyl, and cyclohexyl for Asp; acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and Cbz for Ser; nitro, Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), 2,2,4, 6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) or Boc for Arg and Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), and 2-bromobenzyloxycarbonyl (2-BrCbz), allyloxycarbonyl (alloc), ivDde, Tos, or Boc for Lys. Other examples are known in the art.

[084] After removal of the a-amino protecting group, the remaining protected amino acids are coupled in the desired order to the peptide chain in a stepwise manner. An excess of each protected amino acid is generally used with an appropriate carboxyl group activator, such as dicyclohexylcarbodiimide (DCC) in methylene chloride and/or dimethyl formamide (DMF), N- [(dimethylamino)-lH-l ,2,3 riazolo[4,5-¾]pyridin-l-ylmethylene]-N-methylmethanam hexafluorophosphate N-oxide (HATU), N-[lH-benzotriazol-l -yl)-(dimethylamino)methylene]- N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), and (benzotriazol-l -yl-N- oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP).

[085] Once the desired amino acid sequence has been synthesized, the stable blocking groups are removed and the peptide is decoupled from the resin support by treatment with a suitable reagent, such as Reagent K, which includes TFA (82.5%), thioanisole (5%), phenol (5%), H₂0 (5%), 1 ,2-ethanedithiol (EDT, 2.5%). The decoupling reagent may simultaneously cleave any side chain protecting groups. Alternatively, the side chain protecting groups can be cleaved off using a separate reagent, for example, 20% piperidine in DMF for Fmoc groups or 2% hydrazine in DMF for ivDde groups.

[086J The above techniques can also be used to synthesize peptides which include one or more non-naturally occurring amino acids. Covalent modifications can be introduced, for example, by reacting targeted amino acid residues with an organic derivatising agent that is capable of reacting with selected amino acid side chains or with the terminal residue(s) as is known in the art. Selection of appropriate derivatising agent(s) can be readily accomplished by a worker skilled in the art.

[087] Methods of synthesizing peptides having one or more modified peptide bonds are known in the art (see, for example, "Solid Phase Peptide Synthesis" Methods in Enzymology (ed. Fields, G.B. (1997) Academic Press, San Diego).

[088] The peptides can also be prepared in their salt form. The peptides may be sufficiently acidic or sufficiently basic to react with a number of inorganic bases, inorganic acids or organic acids, to form a salt. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulphonic acid, methanesulphonic acid, oxalic acid, p-bromophenyl-sulphonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.

[089J Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Examples of bases useful in preparing the salts include, but are not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.

[090] Standard conjugation techniques known in the art can be employed to conjugate the peptide to one or more additional components, such as those described above (see, for example, Morrison and Boyd, Organic Chemistry, 6th Ed. (Prentice Hall, 1992); J. March, Advanced Organic Chemistry, 4^th Ed. (Wiley 1992); G. T. Harmanson, Bioconjugate Techniques, (Academic Press, Inc. 1995), and S. S. Wong, Chemistry of Protein Conjugation and Cross- Linking, (CRC Press, Inc. 1991 )).

[091] A variety of chemical groups can be subject to conjugation reactions. For example, hydroxyl groups (-OH) can be used to conjugate a second component through reaction with alkyl halides (R-Cl, R-Br), acyl anhydrides, acyl halides, aldehydes (-CHO), hydrazides (R-CO-NH- NH₂), and the like. Primary amino groups (-NH₂) can be used to conjugate a second component through reaction with alkyl halides (R-Cl, R-Br, R-I), aryl azides, acyl anhydrides, acyl halides, acyl esters, carboxylates activated with carbodiimides, aldehydes (-CHO), and the like. Carboxylic groups (-COOII) can also be used to conjugate a second component after the group has been activated. Suitable activation agents include, for example, organic or inorganic acid halides (for example pivaloyl chloride, ethyl chloroformate, thionyl chloride, PCI5), carbodiimides (R-CO-OH+R'-N=C=N-R", for example EDC, DCC), benzotriazolyl uronium or phosphonium salts (TBTU, BOP, PyBOP, HTBU), diacyl chlorides, diisocyanates, and the like.

[092] Once synthesized, the peptides can be submitted to one or more purification procedures, if desired. Purification methods are well known in the art (see, for example, T. Hanai, HPLC: A Practical Guide, RSC Press, UK 1999; L.M. Harwood, C.J. Moody and J.M. Percy, Experimental Organic Chemistry: Standard and Microscale, Blackwell Scientific Publishing, 1998; Current Protocols in Protein Science, Coligan, J.E., et al. (eds.), John Wiley & Sons, (2001 & updates)) and can include one or more chromatographic steps, for example, ion exchange chromatography, hydrophobic adsorption/interaction chromatography, silica gel adsorption chromatography, and various forms of high performance liquid chromatography (HPLC), such as reverse-phase HPLC.

TESTING OF THE PEPTIDES

[093] The peptides can be tested for their suitability as anti-cancer agents in vitro and/or in vivo by standard tests known in the art. For example, the peptides may be tested for their ability to inhibit proliferation of cancer cells, to inhibit tumour growth, to inhibit ascites formation and/or to inhibit metastasis. Exemplary methods of testing the peptides in this regard are provided below and in the Examples. Other methods of testing are well known to those of skill in the art.

[094] In certain embodiments, the peptides inhibit Akt. The activity of the peptides in this regard can be readily tested by standard methods, such as, Western blot analysis, real time PCR analysis, microarray analysis, and the like (see, for example, Ausubel et al. (1994 & updates) Current Protocols in Molecular Biology, John Wiley & Sons, New York). i) In vitro Testing

[095] Initial determinations of the efficacy of the peptides may be made using one or more standard in vitro assays.

[096] For example, the cytotoxicity of the peptides can be assayed in vitro using a suitable cell cancer line. In general, cells of the selected test cell line are grown to an appropriate density and the candidate compound is added. After an appropriate incubation time (for example, about 48 to 72 hours), cell survival is assessed. Methods of determining cell survival are well known in the art and include, but are not limited to, the resaxurin reduction lest (see Fields & Lancaster (1993) Am. Biotechnol. Lab. 1 1 :48-50; O'Brien et al., (2000) Eur. J. Biochem. 267:5421 - 5426 and U.S. Patent No. 5,501 ,959), the sulforhodamine assay (Rubinstein et al, (1990) J. Natl. Cancer Inst. 82: 1 13-1 18) or the neutral red dye test (Kitano et al., (1991 ) Euro. J. Clin. Investg. 21 :53-58; West et al, (1992) J. Investigative Derm. 99:95-100). Cytotoxicity is determined by comparison of cell survival in the treated culture with cell survival in one or more control cultures, for example, untreated cultures and/or cultures pre-treated with a control compound (typically a known therapeutic).

[097] The ability of the peptides to inhibit proliferation of cancer cells in vitro can be assessed by culturing cells of a cancer cell line of interest in a suitable medium. After an appropriate incubation time, the cells can be treated with the candidate peptide and incubated for a further period of time. Cells are then counted and compared to an appropriate control. Suitable controls include, for example, cells treated with a standard chemotherapeutic and/or untreated cells.

[098] Alternatively, the peptides can be tested in vitro by determining their ability to inhibit anchorage-independent growth of tumour cells. Anchorage-independent growth is known in the art to be a good indicator of tumourigenicity. In general, anchorage-independent growth is assessed by plating cells from a selected cancer cell-line onto soft agar and determining the number of colonies formed after an appropriate incubation period. Growth of cells treated with the candidate peptide can then be compared with that of control cells (as described above).

[099] A variety of cancer cell-lines suitable for testing the candidate compounds are known in the art and many are commercially available (for example, from the American Type Culture Collection, Manassas, VA), including a wide variety of human cancer cell lines. Examples include, but are not limited to, mesothelial cell lines MSTO-21 1H, NCI-H2052 and NCI-H28; ovarian cancer cell lines OV90, SK-OV-3, OCC, OVCA 8 and A2780cp; breast cancer cell lines MCF-7 and MDA-MB-231 ; colon cancer cell lines CaCo, HCT1 16, LS513 and HT29; cervical cancer cell line HeLa; non-small cell lung carcinoma cell lines A549 and H1299; pancreatic cancer cell lines MIA-PaCa-2 and AsPC- 1 ; prostatic cancer cell line PC-3; bladder cancer cell lines T24, RT4 and TCCSUP; liver cancer cell line HepG2; brain cancer cell line U- 87 MG; melanoma cell line A2058, and lung cancer cell line NCI-H460. Other examples of suitable cell lines are known in the art.

[0100] If necessary, the toxicity of the peptides can also be initially assessed in vitro using standard techniques. For example, human primary fibroblasts can be transfected in vitro with the candidate peptide and then tested at different time points following treatment for their viability using a standard viability assay, such as the assays described above, or the trypan-blue exclusion assay. Cells can also be assayed for their ability to synthesize DNA, for example, using a thymidine incorporation assay, and for changes in cell cycle dynamics, for example, using a standard cell sorting assay in conjunction with a fluorocytometer cell sorter (FACS). ii) In vivo Testing

[0101] The ability of the peptides to inhibit tumour growth, cancer cell proliferation, ascites formation and/or metastasis in vivo can be determined in an appropriate animal model using standard techniques known in the art (see, for example, Enna, et al, Current Protocols in Pharmacology, J. Wiley & Sons, Inc., New York, NY).

[0102] Current animal models for screening anti-tumour compounds include xenograft models, in which a human tumour has been implanted into an animal. Examples of xenograft models of human cancer include, but are not limited to, human solid tumour xenografts, implanted by subcutaneous injection or implantation and used in tumour growth assays; human solid tumour isografts, implanted by fat pad injection and used in tumour growth assays; human solid tumour orthotopic xenografts, implanted directly into the relevant tissue and used in tumour growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice. In addition to the implanted human tumour cells, the xenograft models can further comprise transplanted human peripheral blood leukocytes, which allow for evaluation of the anti-cancer immune response.

[0103] Alternatively, murine cancer models can be used for screening anti-tumour compounds. Examples of appropriate murine cancer models are known in the art and include, but are not limited to, implantation models in which murine cancer cells are implanted by intravenous, subcutaneous, fat pad or orthotopic injection; murine metastasis models; transgenic mouse models; and knockout mouse models. [0104] For example, the peptides can be tested in vivo on solid tumours using mice that are subcutaneously grafted bilaterally with an appropriate amount of a tumour fragment, or implanted with an appropriate number of cancer cells, on day 0. The animals bearing tumours are mixed before being subjected to the various treatments and controls. In the case of treatment of advanced tumours, tumours are allowed to develop to the desired size, animals having insufficiently developed tumours being eliminated. The selected animals are distributed at random to undergo the treatments and controls. Animals not bearing tumours may also be subjected to the same treatments as the tumour-bearing animals in order to be able to dissociate the toxic effect from the specific effect on the tumour. Chemotherapy generally begins from 3 to 22 days after grafting, depending on the type of tumour, and the animals are monitored daily. The candidate peptide can be administered to the animals, for example, by i.p. injection or bolus infusion. The different animal groups are weighed about 3 or 4 times a week until the maximum weight loss is attained, after which the groups are weighed at least once a week until the end of the trial.

[0105] The tumours are measured after a pre-determined time period, or they can be monitored continuously by measuring about 2 or 3 times a week until the tumour reaches a pre-determined size and / or weight, or until the animal dies if this occurs before the tumour reaches the predetermined size / weight. The animals are then sacrificed and the tissue histology, size and / or proliferation of the tumour assessed.

[0106] Orthotopic xenograft models are an alternative to subcutaneous models. In this model, tumour cells are implanted at the site of the organ of origin and develop internally. Daily evaluation of the size of the tumours is thus more difficult than in a subcutaneous model. A recently developed technique using green fluorescent protein (GFP) expressing tumours in noninvasive whole-body imaging can help to address this issue (Yang and al, Proc. Nat. Aca. Sci, (2000), pp 1206- 121 1). This technique utilises human or murine tumours that stably express very high levels of the Aqueora vitoria green fluorescent protein. The GFP expressing tumours can be visualised by means of externally placed video detectors, allowing for monitoring of details of tumour growth, angiogenesis and metastatic spread. Angiogenesis can be measured over time by monitoring the blood vessel density within the tumour(s). The use of this model thus allows for simultaneous monitoring of several features associated with tumour progression.

[0107] For the study of the effect of the peptides on leukaemias, for example, the animals are grafted with a particular number of cells, and the anti-tumour activity is determined by the increase in the survival time of the treated mice relative to the controls.

[0108] To study the effect of the peptides on tumour metastasis, tumour cells are typically treated with the candidate peptide ex vivo and then injected into a suitable test animal. The spread of the tumour cells from the site of injection is then monitored over a suitable period of time.

[0109] To study the effect of the peptides on ascites development, animals can be injected intraperitoneally with tumour cells, for example, human ovarian cancer cells. Animals are distributed at random to undergo treatment or act as controls. Animals not bearing tumours may also be subjected to the same treatments as the tumour-bearing animals in order to be able to dissociate the toxic effect from the specific effect on the tumour. After a sufficient time to allow tumour initiation, treatment is started and the animals are observed daily. After an appropriate period of the time, the animals are euthanized and tumour tissues and ascites fluid are collected and analyzed.

[0110] In vivo toxic effects of the peptides can be evaluated, for example, by measuring their effect on animal body weight during treatment and by performing haematological profiles and liver enzyme analysis after the animal has been sacrificed.

Table 3: Examples of Xenograft Models of Human Cancer

Cancer Model Cell Type

Tumour Growth Assay Prostate (PC-3, DU145)

Human solid tumour xenografts in mice (subBreast (MD A-MB-231 , VB-9)

cutaneous injection)

Colon (HT-29)

Lung (NCI-H460, NCI-H209)

Pancreatic (ASPC-1 , SU86.86)

Pancreatic: drug resistant (BxPC-3)

Skin (A2058, C8161 )

Cervical (SIHA, HeLa-S3)

Cervical: drug resistant (HeLa S3-HU- resistance)

Liver (HepG2)

Brain (U87-MG)

Renal (Caki-1, A498)

Ovary (S -OV-3)

Tumour Growth Assay Breast: drug resistant (MDA-CDDP-S4, MDA-

MB435-To. l)

Human solid tumour isografts in mice (fat pad

injection)

Survival Assay Human: Burkitts lymphoma (Non-Hodgkin's)

Experimental model of lymphoma and (raji)

leukaemia in mice Murine: erylhroleukemia (CB7 Friend retrovirus-induced)

Experimental model of lung metastasis in mice Human: melanoma (C8161 )

Murine: fibrosarcoma (R3)

[0111] Therapeutic efficacy and toxicity can of the peptide be determined by standard pharmaceutical procedures such as, for example, by determination of the median effective dose, or ED50 (i. e. the dose therapeutically effective in 50% of the population) and the median lethal dose, or LD₅₀ (i.e. the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is known as the "therapeutic index," which can be expressed as the ratio, LD50/ED50. The data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for human or animal use. The dosage contained in such compositions is usually within a range of concentrations that include the ED50 and demonstrate little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the subject, and the route of administration and the like.

PHARMACEUTICAL COMPOSITIONS

[0112] For administration to a subject, the present invention provides for pharmaceutical compositions comprising a peptide of the invention and one or more non-toxic pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants. If desired, other active agents may be included in the compositions.

[0113] The pharmaceutical compositions may comprise, for example, from about 1% to about 95% of a peptide of the invention. Compositions formulated for administration in a single dose form may comprise, for example, about 20% to about 90% of the peptide, whereas compositions that are not in a single dose form may comprise, for example, from about 5% to about 20% of the peptide. Non-limiting examples of unit dose forms include dragees, tablets, ampoules, vials, suppositories and capsules.

[0114] The pharmaceutical compositions can be formulated for administration by a variety of routes. For example, the compositions can be formulated for oral, topical, rectal or parenteral administration or for administration by inhalation or spray. The term "parenteral" as used herein includes subcutaneous injection, intraperitoneal injection, intravenous, intramuscular, intrathecal, intrasternal injection or infusion techniques. Intra-tumoral and intravesical administration is also contemplated for the treatment of cancer.

[0115] In certain embodiments of the invention, the pharmaceutical compositions may comprise a peptide of the invention formulated in an appropriate vehicle, such as an artificial membrane vesicle (including a liposome, lipid micelle and the like), microparticle or microcapsule. In some embodiments, the pharmaceutical compositions comprise a peptide of the invention formulated as a liposomal formulation. In one embodiment, the pharmaceutical compositions comprise a peptide of the invention formulated as a liposomal formulation in which the peptide is associated with anionic liposomes. In this context, an "anionic liposome" is a liposome having a net negative charge at neutral pH. This net negative charge is provided by one or more anionic lipids included in the liposome. In certain embodiments, the pharmaceutical compositions comprise a liposomal formulation of the peptide in which the anionic lipids and peptide are present in appropriate amounts to provide a lipid:peptide charge ratio of between about 1.1 and about 30.0.

[0116] Pharmaceutical compositions for oral use can be formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs. Such compositions can be prepared according to standard methods known to the art for the manufacture of pharmaceutical compositions and may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the peptide in admixture with suitable non-toxic pharmaceutically acceptable excipients including, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid; binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they may be coated by known techniques in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

[0117] Pharmaceutical compositions for oral use can also be presented as hard gelatine capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

[0118] Pharmaceutical compositions formulated as aqueous suspensions contain the peptide in admixture with one or more suitable excipients, for example, with suspending agents, such as sodium carboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, hydroxypropyl- -cyclodextrin, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethyene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or ^-propyl jp-hydroxy-benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.

[0119] Pharmaceutical compositions can be formulated as oily suspensions by suspending the peptide in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cctyl alcohol. Sweetening agents such as those set forth above, and/or flavouring agents may be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0120] The pharmaceutical compositions can be formulated as a dispersible powder or granules, which can subsequently be used to prepare an aqueous suspension by the addition of water. Such dispersible powders or granules provide the peptide in admixture wilh one or more dispersing or wetting agents, suspending agents and/or preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavouring and colouring agents, can also be included in these compositions.

[0121] Pharmaceutical compositions of the invention can also be formulated as oil-in-water emulsions. The oil phase can be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or it may be a mixture of these oils. Suitable emulsifying agents for inclusion in these compositions include naturally-occurring gums, for example, gum acacia or gum tragacanth; naturally-occurring phosphatides, for example, soy bean, lecithin; or esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monoleate. The emulsions can also optionally contain sweetening and flavouring agents.

[0122] Pharmaceutical compositions can be formulated as a syrup or elixir by combining the peptide with one or more sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations can also optionally contain one or more demulcents, preservatives, flavouring agents and/or colouring agents.

[0123] The pharmaceutical compositions can be formulated as a sterile injectable aqueous or oleaginous suspension according to methods known in the art and using suitable one or more dispersing or wetting agents and/or suspending agents, such as those mentioned above. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be employed include, but are not limited to, water, Ringer's solution, lactated Ringer's solution and isotonic sodium chloride solution. Other examples include, sterile, fixed oils, which are conventionally employed as a solvent or suspending medium, and a variety of bland fixed oils including, for example, synthetic mono- or diglycerides. Fatty acids such as oleic acid can also be used in the preparation of injectables.

[0124] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philidelphia, PA (2000). USES OF THE PEPTIDES

[0125] The peptides in accordance with the present invention arc useful in the treatment of cancer. In this context, the peptides may, for example, exert either a cytotoxic or cytostatic effect resulting in a reduction in the size of a tumour, the slowing or prevention of an increase in the size of a tumour, an increase in the disease-free survival time between the disappearance or removal of a tumour and its reappearance, prevention of an initial or subsequent occurrence of a tumour (e.g. metastasis or ascites formation), an increase in the time to progression, reduction of one or more adverse symptom associated with a tumour, or an increase in the overall survival time of a subject having cancer.

[0126] Examples of cancers which may be may be treated or stabilized in accordance with certain embodiments of the present invention include, but are not limited to, haematologic neoplasms, including leukaemias, myelomas and lymphomas; carcinomas, including adenocarcinomas and squamous cell carcinomas; melanomas and sarcomas. Carcinomas and sarcomas are also frequently referred to as "solid tumours," examples of commonly occurring solid tumours include, but are not limited to, cancer of the brain, breast, cervix, colon, head and neck, kidney, lung, ovary, pancreas, prostate, stomach and uterus, non-small cell lung cancer and colorectal cancer. Various forms of lymphoma also may result in the formation of a solid tumour and, therefore, are also often considered to be solid tumours.

[0127] The term "leukaemia" refers broadly to progressive, malignant diseases of the blood- forming organs. Leukaemia is typically characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow but can also refer to malignant diseases of other blood cells such as erythroleukaemia, which affects immature red blood cells. Leukaemia is generally clinically classified on the basis of (1 ) the duration and character of the disease - acute or chronic; (2) the type of cell involved — myeloid (myelogenous), lymphoid (lymphogenous) or monocytic, and (3) the increase or non-increase in the number of abnormal cells in the blood - leukaemic or aleukaemic (subleukaemic). Leukaemia includes, for example, acute nonlymphocytic leukaemia, chronic lymphocytic leukaemia, acute granulocytic leukaemia, chronic granulocytic leukaemia, acute promyelocytic leukaemia, adult T-cell leukaemia, aleukaemic leukaemia, aleukocythemic leukaemia, basophylic leukaemia, blast cell leukaemia, bovine leukaemia, chronic myelocytic leukaemia, leukaemia cutis, embryonal leukaemia, eosinophilic leukaemia, Gross' leukaemia, hairy-cell leukaemia, hemoblastic leukaemia, hemocytoblastic leukaemia, histiocytic leukaemia, stem cell leukaemia, acute monocytic leukaemia, leukopenic leukaemia, lymphatic leukaemia, lymphoblastic leukaemia, lymphocytic leukaemia, lymphogenous leukaemia, lymphoid leukaemia, lymphosarcoma cell leukaemia, mast cell leukaemia, megakaryocytic leukaemia, micromyeloblastic leukaemia, monocytic leukaemia, myeloblastic leukaemia, myelocytic leukaemia, myeloid granulocytic leukaemia, myelomonocytic leukaemia, Naegeli leukaemia, plasma cell leukaemia, plasmacytic leukaemia, promyelocytic leukaemia, Rieder cell leukaemia, Schilling's leukaemia, stem cell leukaemia, subleukaemic leukaemia, and undifferentiated cell leukaemia.

[0128] The term "lymphoma" generally refers to a malignant neoplasm of the lymphatic system, including cancer of the lymphatic system. The two main types of lymphoma are Hodgkin's disease (HD or HL) and non-Hodgkin's lymphoma (NHL). Abnormal cells appear as congregations which enlarge the lymph nodes, form solid tumours in the body, or more rarely, like leukemia, circulate in the blood. Hodgkin's disease lymphomas, include nodular lymphocyte predominance Hodgkin's lymphoma; classical Hodgkin's lymphoma; nodular sclerosis Hodgkin's lymphoma; lymphocyte-rich classical Hodgkin's lymphoma; mixed cellularity Hodgkin's lymphoma; lymphocyte depletion Hodgkin's lymphoma. Non-Hodgkin's lymphomas include small lymphocytic NHL, follicular NHL; mantle cell NHL; mucosa-associated lymphoid tissue (MALT) NHL; diffuse large cell B-cell NHL; mediastinal large B-cell NHL; precursor T lymphoblastic NHL; cutaneous T-cell NHL; T-cell and natural killer cell NHL; mature (peripheral) T-cell NHL; Burkitt's lymphoma; mycosis fungoides; Sezary Syndrome; precursor B-lymophoblastic lymphoma; B-cell small lymphocytic lymphoma; lymphoplasmacytic lymphoma; spenic marginal zome B-cell lymphoma; nodal marginal zome lymphoma; plasma cell mycloma/plasmacytoma; intravascular large B-cell NHL; primary effusion lymphoma; blastic natural killer cell lymphoma; enteropathy-type T-cell lymphoma; hepatosplenic gamma- delta T-cell lymphoma; subcutaneous panniculitis-like T-cell lymphoma; angioimmunoblastic T- cell lymphoma; and primary systemic anaplastic large T/null cell lymphoma.

[0129] The term "sarcoma" generally refers to a tumor which originates in connective tissue, such as muscle, bone, cartilage or fat, and is made up of a substance like embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include soft tissue sarcomas, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented haemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

[0130] The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.

[0131] The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colorectal carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, haematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, non-small cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

[0132] The term "carcinoma" also encompasses adenocarcinomas. Adenocarcinomas are carcinomas that originate in cells that make organs which have glandular (secretory) properties or that originate in cells that line hollow viscera, such as the gastrointestinal tract or bronchial epithelia. Examples include, but are not limited to, adenocarcinomas of the breast, lung, pancreas and prostate. [0133] One embodiment of the present invention provides for the use of the peptides in the treatment of a solid tumour or a melanoma.

[0134] Another embodiment of the invention provides for the use of the peptides in the treatment of malignant ascites, advanced and/or metastatic cancers, or cancers with a high potential for metastasis and/or development malignant ascites. In some embodiments, the invention provides for the use of the peptides in the treatment of a cancer known to be associated with the development of ascites, such as, colon cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, breast cancer, lymphoma, pulmonary cancer, cervical cancer, uterine cancer or ovarian cancer.

[0135] In one embodiment, the invention provides for the use of the peptides in the treatment of a cancer associated with an abnormality in Akt, for example, breast cancer, pancreatic cancer, colorectal cancer, gastric cancer and/or ovarian cancer.

[0136] In some embodiments, the present invention provides for the use of the peptides in the treatment of early stage cancers including early neoplasias that may be small, slow growing, localized and/or nonaggressive, for example, with the intent of curing the disease, causing regression of the cancer or reducing the spread of the disease (metastasis).

[0137] In other embodiments, the present invention provides for the use of the peptides in the treatment of intermediate or advanced cancers, for example, indolent cancers, recurrent cancers including locally recurrent, distantly recurrent and/or refractory cancers (i. e. cancers that have not responded to treatment), metastatic cancers, locally advanced cancers and aggressive cancers. One skilled in the art will appreciate that many of these categories may overlap, for example, aggressive cancers are typically also metastatic.

[0138] Certain embodiments of the invention provide methods of preventing, reducing or halting metastasis of a cancer. In accordance with these embodiments, the cancer may be an early stage cancer or it may be an intermediate or late stage cancer. [0139] In some embodiments, the invention provides for the use of the peptides to treat refractory cancers, for example chemotherapy or radiation resistant cancers. In certain embodiments, the invention provides for the use of the peptides to treat hormone-resistant cancers (for example, hormone-resistant breast or prostate cancer).

[0140] In some embodiments, the present invention provides for the use of the peptides as "sensitizing agents," which selectively inhibit the growth of cancer cells. In this case, the peptide alone has a weak inhibitory and/or non-cytotoxic effect on the cancer cell, but provides a means of weakening the cancer cells, and thereby facilitates the benefit from conventional anti-cancer therapeutics.

[0141] In some embodiments, the present invention provides for the use of the peptides for the treatment of cancers, such as those outlined above, in combination with one or more standard anti-cancer therapeutics. The peptides can be administered, for example, before, during or after treatment with the anti-cancer therapeutic. An "anti-cancer therapeutic" as used herein refers to a compound, composition or treatment that prevents or delays the growth and/or metastasis of cancer cells. Such anti-cancer therapeutics include, but are not limited to, chemotherapeutic drug treatment, radiation, gene therapy, hormonal manipulation, immunotherapy and antisense oligonucleotide therapy. Examples of useful chemotherapeutic drugs include, but are not limited to, actinomycin D, adriamycin, alkeran, ara-C, arabinoside, arsenic trioxide (Trisenox™), avastin, bleomycin, busulphan, carboplatin, cisplatin, chlorambucil, cyclophosphamide, cytosine, danorubicin, docetaxel, doxorubicin, DTIC, erlotinib, epirubicin, etoposide, fludarabine, 5-FU, gemcitabine, herceptin, hydroxyurea, idarubicin, ifosphamide, irinotecan, leustatin, melphalan, methotrexate, mitomycin C, mitoxantrone, Navelbine® (vinorelbine), neocarcinostatin, paclitaxel, suramin, taxol, taxotere, teniposide, vincristine, vinblastine, xeloda and the like.

THERAPEUTIC PA CKS AND KITS

[0142] The present invention additionally provides for therapeutic kits or packs containing a peptide or a pharmaceutical composition comprising the peptide for use in the treatment of cancer. Individual components of the kit can be packaged in separate containers, associated with which, when applicable, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human or animal administration. The kit can optionally further contain one or more other therapeutic agents for use in combination with the peptide. The kit may optionally contain instructions or directions outlining the method of use or dosing regimen for the peptide and/or additional therapeutic agents.

[0143] When one or more components of the kit are provided as solutions, for example an aqueous solution, or a sterile aqueous solution, the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the solution may be administered to a subject or applied to and mixed with the other components of the kit.

[0144] The components of the kit may also be provided in dried or lyophilised form and the kit can additionally contain a suitable solvent for reconstitution of the lyophilised components. Irrespective of the number or type of containers, the kits of the invention also may comprise an instrument for assisting with the administration of the composition to a patient. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or similar medically approved delivery vehicle.

[0145] To gain a better understanding of the invention described herein, the following examples are set forth. It will be understood that these examples are intended to describe illustrative embodiments of the invention and are not intended to limit the scope of the invention in any way.

EXAMPLES

EXAMPLE 1: PREPARATION OF COMPOUND 9

[0146] The following provides an example of a method for preparing a peptide comprising all D- amino acids, a PNA moiety coupled to the side chain of a constituent lysine residue, and a PTD coupled to the PNA moiety.

[0147] Peptide Sequence

Ac-D-Ile^{1 1} -D-Ser ¹²-D-Ile ¹³-Gly ¹⁴-D-Ile' ⁵-D-Leu' ⁶-D-Asn^{] 7}-D-Gln' ⁸-D-Ala^{] 9}-D-Asp²⁰-D-Ly s²¹ - Gly²²-Gly²³-Gly²⁴-Gly²⁵-Gly²⁶-Gly²⁷-Gly²⁸-D-Leu²⁹-D-Arg³⁰-D-Phe³¹-D-Lys³²(PNA-A¹⁰-D- Arg⁹-D-Lys⁸-D-Lys⁷-D-Arg⁶-D-Arg⁵-D-Gln⁴-D-Arg³-D-Arg²-D-Lys⁾-Ac)-D-Arg³³-D-Arg³⁴-D- Phe ⁵-NH₂

[0148] Empirical Formula: Ci₈iH₃io ₇₄0₄₄ [0149] Molecular Weight: 4226.87 Da

[0150] In brief, the peptide was assembled in linear fashion on Rink amide AM resin using standard Fmoc (N-9-fluorenylmethoxycarbonyl) chemistry with DIC/ HOBt as the coupling reagents for coupling of the D-phenylalanine through acetylation of the N-terminal D-isoleucine. Side chain, D-Lys³² (Alloc) was then deprotected with Pd (PPh₃)₄/AcOH/piperidine in DCM. The peptide nucleic acid (PNA monomer) and the remaining amino acids were coupled through the side chain of the D-lysine³² residue using the same coupling procedure for the other amino acids. Acetylation of the N-terminal of the D-lysine¹ was conducted after deprotection of its Ν^α- Fmoc group. The assembled peptide was cleaved and deprotected, then precipitated as a crude solid. The crude was purified and salt-exchanged by standard RP-HPLC methods and isolated by lyophilization.

Key Raw Materials and Solutions

[0151] Resin: 0.44mmol/g substituted Fmoc-Rink Amide AM Resin Table 4: Protected Amino Acids and PNA monomer

Fmoc-Gly-OH 29022-1 1-5

Fmoc-D-Leu-OH 1 14360-54-2

Fmoc-D-Lys(Boc)-OH 92122-45-7

Fraoc-D-Asp(OtBu)-OH 1 12883-39-3

Fmoc-D-Ala-OH 79990-15-1

Fmoc-D-Gln(Trt)-OH 200623-62-7

Fmoc-L-Arg(Pbf)-OII 154445-77-9

Fmoc-D-Asn(Trt)-OH 180570-71 -2

Fmoc-D-Ile-OH 143688-83-9

Fmoc-PNA-A(Bhoc)-OH N/A

Table 5: Coupling Reagents

[0152] Fmoc Deprotection Solution: 1%DBU (l ,8-Diazabicyclo[5.4.0]undec-7-ene) / 5%Piperdine / l %HOBt in DMF (dimethylformamide).

[0153] Alloc Deprotection Solution: 0.25eq. of Pd (PPh₃)₄ [Tetrakis (triphenylphosphine) palladium]/ 37.5 eq. of AcOH (acetic acid) /37.5 eq. of piperidine in DCM (dichloromethane).

[0154] Cleavage Solution: 85% Trifluoroacetic Acid (TFA) / 10% Thioanisole / 5% EDT. Amino Acid Coupling Conditions

[0155] The N-terminal Fmoc protecting group was removed by treatment of the peptide resin with the Fmoc Deprotection Solution two (2) times each prior to sequential amino acid coupling. Coupling of the protected amino acid to the growing peptide chain was accomplished using DIC/HOBt in DMF as the coupling reagent. The Fmoc deprotection reaction was confirmed using the Ninhydrin (Kaiser) test. Completion of coupling reaction was also controlled after each amino acid coupling by the ninhydrin test (Kaiser et al, Analytical Biochem., 1975, 39, 305) on primary amines. Semi-quantitative color test is based on revealing the unreacted amines. Alloc Deprotection

[0156] The Alloc group was removed by treatment of the peptide resin with [(C₆H₅)₃P]₄Pd (0.25eq), AcOH (37.5 eq) and piperidine (37.5 eq) in DCM under nitrogen for two hours. The residue palladium on the resin was eliminated by washes of the resin three times with 40mM of diethyldithiocarbamic acid sodium in DMF.

Peptide Resin Drying and Synthesis Yield

[0157] After deprotection of the last Fmoc group, the peptide resin was washed with DMF and MTBE, and then dried in an oven under vacuum at room temperature. The yield of the solid phase synthesis assembly was calculated using the ratio of the weight increase on the resin to the theoretical weight increase as calculated from the initial loading substitution of the resin.

Cleavage/ Deprotection

[0158] The peptide was simultaneously cleaved from the resin and amino acid side-chains deprotected using TFA/ Thioanisole/ EDT (1 , 2-Ethanedithiol) [85/10/5 (v/v/v)]. The deprotected peptide solution was then precipitated in cold methyl tert-butyl ether (MTBE), filtered, washed in MTBE and then dried under vacuum to a constant weight to yield the crude peptide.

Peptid e Assembling Yield, Cleavage Yield ami Purity o f the C rude Peptid e

[0159] A 6.0-mmol starting synthesis scale from 10.6 gm of initial Rink amide AM resin yielded 45.0 gm of peptide resin, which afforded 10.9 gm of crude peptide, TFA salt after cleavage.

[0160] The yield of the solid phase assembling was calculated by the ratio of the weight increase on the resin to the theoretical weight increase as calculated from the initial loading of the resin.

[0161] The quality of the crude peptide, TFA salt was checked by HPLC. The identity of the crude peptide was confirmed by mass spectral analysis. Results

[0162] Peptide Assembling Yield = (45.0- 10.6)/(6.0^χ7428.02/1000) ^χ 100% = 77.2%

[0163] Peptide Cleavage Yield = 10.9^χ45/(45^χ6^χ4227.85^χ77.2%/1000) * 100% = 55.7%

[0164] Crude peptide Purity: According to the RP-HPLC area %, the purity of crude peptide was 34.7%.

[0165] Mass spectral analysis: The deconvolution of the ESI⁺ spectrum showed the molecular ion was M=4227.00. This is in agreement with the expected molecular ion (M/Z= 4227.85 +/- 1 ) for the proposed chemical structure.

EXAMPLE 2: PREPARATION OF COMPOUND 4

[0166] The following provides an example of a method for preparing a peptide comprising all L- amino acids, a PNA moiety coupled to the side chain of a constituent lysine residue, and a PTD coupled to the PNA moiety.

[0167] Peptide Sequence

Ac-Phe¹¹-Arg^{I 2}-Arg¹³-Lys¹⁴(PNA-A^{1 0}-Lys⁹-Arg⁸-Arg⁷-Gln⁶-Arg⁵-Arg⁴-Lys³-Lys²-Arg¹-Ac)- Phe¹⁵-Arg¹⁶-Leu¹⁷-Gly^{1 8}-Gly¹⁹-Gly²⁰-GIy²¹ -Gly²²-Gly²³-Gly²⁴-Lys²⁵-Asp²⁶- Ala²⁷-Gln²⁸-Asn²⁹- Leu³⁰-Ile³¹-Gly³ -Ile³³-Ser³⁴-Ile³⁵-NH₂

[0168] Empirical Formula: Ci₈] H309N73C>45

[0169] Molecular Weight: 4227.85 Da

[0170] In brief, the peptide was assembled following the same protocol as outlined in Example 1 with the exceptions that side chain, Lys¹⁴ (Alloc) was deprotected with Pd (PPh₃)₄/AcOH/piperidine in DCM and the peptide nucleic acid (PNA monomer) and the remaining amino acids were coupled through the side chain of the lysine¹⁴ residue using the same coupling procedure for the other amino acids. ev Raw Materials and Solutions

[0171] Resin: 0.44mmol/g substituted Fmoc-Rink Amide AM Resin Table 6: Protected Amino Acids and PNA monomer

[0172] Coupling Reagents, Fmoc Deprotection Solution, Alloc Deprotection Solution and Cleavage Solution: As for Example 1.

[0173] The Amino Acid Coupling Conditions, Alloc Deprotection, Peptide Resin Drying and Synthesis Yield and Cleavage/ Deprotection steps were as described in Example 1 .

Results

[0174] A 28.5-mmol starting synthesis scale from 64.6 gm of initial Rink amide AM resin yielded 210.0 gm of peptide resin, which afforded 100.33 gm of crude peptide, I^'FA salt after cleavage.

[0175] Peptide Assembling Yield = (210.0-64.6)/(28.5 x7428.02/1000) ^χ 100% [0176] Peptide Cleavage Yield = 100.33 ^χ210/(210^χ28.5 ^χ4227.85^χ68.7%/1000) x 100% = 121.2%

[0177] Crude peptide Purity: According to the RP-HPLC area %, the purity of crude peptide was 39.7%.

[0178] Mass spectral analysis: The deconvolution of the ESI⁺ spectrum showed the molecular ion was M=4227.60. This is in agreement with the expected molecular ion (M/Z= 4227.85 +/- 1) for the proposed chemical structure.

EXAMPLE 3: INHIBITION OF PROLIFERATION OF OVARIAN CANCER CELLS IN VITRO

[0179] The effect of compounds 9, 16 and 17 (below) on the proliferation of ovarian cancer cells in vitro was investigated and compared to the effect of compound 4. Compound 4 is identical to compound 1 , but includes a PTD attached to the PNA moiety (see below). Compound 9 is the retro-inverso form of compound 4. Compound 16 comprises the same base amino acid sequence as compound 4, but lacks the PNA moiety. Compound 17 is the retro-inverso form of compound

16.

Ac-isigilnqadkgggggg

NH₂

Compound 9

O

FRR-HN JL _{FRLGGGGGGGKDAQNL|G|S|}__N Ac-isigilnqadkggggggglrf-

/

HN-KRRQRRKKR-Ac Ac-krrqrrkkr- Compound 16 [SEQ ID NO:30] Compound 17

Compound 4 [SEQ ID NO:4]

Methods

[0180] Cultures: OCC-1 cells (ovarian clear cell carcinoma cells; a gift from Dr. Barbara Vanderhyden, Ottawa Hospital Research Institute, Ottawa, Ontario) were seeded at 4000 cells per well in a 96-well plate (BD Falcon; 35307) in MEM Alpha 5% FBS (HyClone SH30265.01 ; SH30396.03) and allowed to adhere for 2 hours at 37°C in a water-saturated atmosphere with 5% C0₂. Sterile prepared formulations of the peptides (ImM in H₂0) were added to cultures to a final concentration of 25uM. Cells were incubated for 24 hours at 37°C in a water-saturated atmosphere with 5% C0₂, at which point media was removed by quick inversion of the plate, and plates were stored at -80°C until ready to be assayed.

[0181] Proliferation Assay: Cell Proliferation was measured using the CyQUANT® GR cell proliferation assay kit (Invitrogen; C7026) according to the manufacturer's protocol, without the use of a standard curve. Briefly, CyQUANT lysis buffer and reagent were added to nuclease free dd¾0 (Ambron; 9932) at 1 :20 and 1 :400 respectively. Care was taken to protect from light. Plates to be read were thawed at room temperature. The prepared reagent was added to each sample well (200ul/well) and incubated (2-5min, RT, light protected). Plates were read and sample fluorescence measured using a FLUOStar Galaxy Microplate Reader (BMG Technologies) with filters at 485nm excitation and 520 nm emission. Proliferation was determined by subtracting background from all samples. Results were normalized by making untreated cells equal to 100% proliferation.

[0182] Statistics: All statistical significances were determined using SigmaPlot®. Results

[0183] The results are shown in Figure 1 and demonstrate treatment with all of the four tested compounds inhibited cancer cell proliferation to a statistically significant extent (p = <0.001 over control). Compounds 4 and 16 (containing L-amino acids) inhibited OCC-1 cancer cell proliferation to a lesser extent than compounds 9 and 17 (containing D-amino acids). When administered at equivalent dose (25 μΜ) compounds 4 and 16 moderately inhibited OCC-1 cancer cell proliferation to a similar extent (33% and 38% respectively), whereas compounds 9 and 17 both proved more potent inhibiting cell proliferation by 81 % and 87% respectively.

EXAMPLE 4: INHIBITION OF PROLIFERATION OF OVARIAN, BLADDER AND COLON CANCER CELLS IN VITRO

[0184] The effect of compound 9 on the proliferation of cancer cells in vitro was investigated using ovarian, bladder and colon cancer cells, and compared to the effect of compound 4.

Methods

[0185] The methods outlined in Example 3 were followed with the following exceptions:

[0186] RT4, TCCSUP and T24 bladder cancer and LS 13 colon cancer cells were cultured using RPMI (SH30027.01) medium, A2780 ovarian cancer cells were cultured using DMEM (SH30243.01 ) medium and ES-2 ovarian cancer cells were cultured using DMEM (SH30243.01 ) medium.

[0187] RT4, TCCSUP and T24 bladder cancer cells were treated with 25 μΜ compound 4 or 12 μΜ compound 9 for 24 hours. LS513 colon cancer cells, and A2780 and ES-2 ovarian cancer cells were treated with 25 μΜ compound 4 or 25 μΜ compound 9 for 48 hours.

Results

[0188] The results for the RT4, TCCSUP and T24 bladder cancer cells are shown in Figure 2 and demonstrate that compound 4 (all L-amino acids) administered at 25 μΜ decreased proliferation to 52% in RT4 cells, to 50% in TCCSUP cells, and to 54% in T24 cells as compared with control untreated cells. Compound 9 (all D-amino acids) administered at 12 μΜ was found to decrease proliferation to 45% in RT4 cells, to 29% in TCCSUP cells, and to 55% in T24 cells as compared with control untreated cells, demonstrating a 2X fold improvement in potency of compound 9 relative to compound 4 when administered to bladder cancer cells

[0189] The results for the LS513 colon cancer cells, and A2780 and ES-2 ovarian cancer cells are shown in Figure 3 and demonstrate that compound 4 decreased proliferation to 34% in LS513 cells, to 29% in A2780cp cells and to 74% in ES2 cells (known to be a resistant cell line) as compared with control untreated cells. In contrast, compound 9 was found to decrease proliferation to 8% in LS513 cells, to 8% in A2780cp cells, and to 26% in ES2 cells as compared with control untreated cells. At equivalent doses (25 μΜ) compound 9 proved to be more potent that compound 4 with a net decrease in cell proliferation of 26% for the colon cancer cell line LS513 and 21% and 48% for the A2780cp and ES-2 ovarian cancer cell lines respectively.

EXAMPLE 5: PEPTIDE STABILITY IN PLASMA/URINE Methods

[0190] Test compounds were incubated in mouse plasma for 0, 15, 30, 60 and 120 minutes at 37°C. At the end of incubation at each of the time points, the samples were diluted with methanol, centrifuged and prepared for analysis using solid-phase extraction. Samples were analyzed by HPLC-MS/MS and peak areas were recorded for each analyte. The area of precursor compound remaining after each of the time points relative to the amount remaining at time zero, expressed as percent, was calculate. Subsequently, the half-life was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) versus time, assuming first order kinetics.

Results

[0191] Stability of peptides in mouse plasma (ex vivo): [0192] Compound 4: approximately 3 minutes [0193] Compound 1 : approximately 120 minutes. [0194] Compound 9: approximately 27 minutes

EXAMPLE 6: DETERMINATION OF MAXIMUM TOLERATED DOSE IN VIVO

[0195] This example and Example 7 investigate the effect of compounds 4 and 9 on small tumours by testing the efficacy of these peptides in a xenograft model based on the intraperitoneal (IP) injection of the OCCl ovarian cancer cell line. When OCCl cells are injected IP into CD-I nude mice (1 x 10⁷ cells), they develop small tumour nodules (<0.5cm³) and large volumes of ascites, with a period of survival of 15-20 days.

Test Peptides

[0196] Compound 4 in powder form was dissolved to a stock concentration of 20m in ddH₂0. The fresh 20mM stock was then divided into aliquots for each experiment, frozen and stored at - 20° C until needed. [0197] Compound 9 in powder form and was subsequently dissolved to a stock concentration of 3.25mM in ddH₂0. The fresh 3.25mM stock was then divided into aliquots for each experiment, frozen and stored at -20° C until needed.

Mice

[0198] 34 female Fox Chase SCID mice (C.B.-17 SCID from Charles Rivers Laboratories, ages 6-8 weeks) were used in this experiment. The mice were allowed to accommodate in the animal facility for five days before injection of the test peptides. Seventeen mice were used to determine the MTD of compound 4 and the other 17 mice were used to determine the MTD of compound 9. The mice in each treatment group were divided into 5 groups with 3 or 4 mice per group (Table 7).

Table 7: Planned Schedule of Injections of SCID Mice

Injections

[0199] Injections were planned for each group of mice using twice daily IP injections for 7 days with the following doses of compounds 4 and 9: 5, 10, 20, 40 and 80 mg/kg. Each dose of compounds 4 and 9 was prepared and diluted in PBS to a 500μΙ, volume. In order to avoid unnecessary toxicity and waste of peptide, the initiation of the 7-day time course of injections was staggered to allow higher doses to be administered only after the animals treated at the lower dose were shown to tolerate their treatment. The mice were euthanized and tissues (peritoneum, kidney, spleen, heart, brain, ovary, liver and lung) were removed 24 hours after the last treatment of each group, or earlier in the case of acute toxicity. Tissues for all mice except SCID 27 and 28 were fixed in formalin and paraffin-embedded and archived at room temperature. No tissues were collected from SCID 14, 29 or 30 since they were found dead during the study. Tissues from SCID 27 and 28 were frozen in O.C.T. by indirect exposure to isopentane chilled in liquid nitrogen and stored at -80°C.

Results

[0200] Tables 8 and 9 describe the health status of mice treated with each dose of compounds 4 or 9. The highest dose of compound 4 administered that did not require premature euthanization was 40mg/kg. The highest dose of compound 9 administered that did not require premature euthanization was 20mg/kg. For this reason, the twice daily IP injection of 40mg/kg was determined to be the maximum tolerated dose of compound 4 and the twice daily IP injection of 20mg/kg was determined to be the maximum tolerated dose of compound 9 for future xenograft studies in SCID mice.

Table 8: The Health Status of Mice Receiving Daily Injections of Compound 4

Group (Dose) Status

Group 5 (80mg/kg) Beginning on day 1 of the injection schedule, these mice exhibited the usual characteristics as mentioned above after the first injection and were struggling to survive the second dose given in the afternoon that day. One of the 4 mice in this group did not survive the night. The remaining mice were not well enough to receive their injections following day and were euthanized on day 3 of the injection schedule.

Table 9: The Health Status of Mice Receiving Daily Injections of Compound 9

EXAMPLE 7: EFFICACY IN OVARIAN CANCER 7iV VIVO

[0201] In this experiment, the phenotype of the OCCl xenograft model was first verified (Part A), then the efficacy of compounds 4 and 9 in this model was determined (Part B).

PART A: Verification of the phenotype of the OCCl xenograft model

Mice

[0202] 5 female Fox Chase SCID mice (CB-17 SCID from Charles Rivers Laboratories, ages 6-8 weeks) were employed. The mice were allowed to accommodate in the animal facility for five days before injection of the OCCl cancer cells.

Cell line maintenance and injection

[0203] OCCl human ovarian cancer cells were grown in Alpha Modified Eagle Medium supplemented with 5% fetal bovine serum. Five SCID mice were injected IP with 1 mL of PBS buffer containing 10⁷ OCC l human ovarian cancer cells and monitored for loss of wellness and examined at endpoint to verify the phenotype previously reported for this xenograft model. Mice were monitored using Wellness and Endpoints as per standard protocol at the testing facility. The timing of symptomology was noted and the total tumour burden and volume of ascites were measured at necropsy performed at endpoint.

PART B: Efficacy of compounds 4 and 9 in the OCCl xenograft model

Mice

[0204] 18 female Fox Chase SCID mice (CB-17 SCID from Charles Rivers Laboratories, ages 6- 8 weeks) were used. The mice were allowed to accommodate in the animal facility for five days before injection of the OCCl cancer cells.

Cell line maintenance and injection

[0205] OCCl human ovarian cancer cells were grown in Alpha Modified Eagle Medium supplemented with 5% fetal bovine serum. Eighteen SCID mice were injected IP with 1 mL of PBS buffer containing 10⁷ OCCl human ovarian cancer cells. After 7 days to allow tumour initiation, the mice were randomly divided into three groups:

[0206] Control group: 6 mice, vehicle, injected TP twice daily over 2 weeks (5 days on and 2 days off).

[0207] Compound 9 group: 6 mice, 20mg/kg of compound 9, injected IP twice daily over 2 weeks (5 days on and 2 days off).

[0208] Compound 4 group: 6 mice, 40mg/kg of compound 4, injected IP twice daily over 2 weeks (5 days on and 2 days off).

[0209] All injections consisted of vehicle or peptides diluted with PBS to a final volume of 0.5 mL.

Monitoring of tumour progression and necropsy

[0210] Animals were monitored daily for disease progression, and were euthanized 13 days after treatment began. At necropsy, peritoneal ascites fluid was collected and quantified from each mouse and stored for future analyses. All tumour tissues were retrieved and weighed. Samples of tumour, liver, kidney, spleen and peritoneum for each mouse were frozen in O.C.T. by indirect exposure to isopcntane chilled in liquid nitrogen. Frozen samples were then stored at -80°C for future molecular analysis.

Statistical analysis

[0211] Statistical analysis was performed using the Graphpad Prism Version 3.02 software using the unpaired t-tcst.

Results

PART A: Verification of the phenotype of the OCC1 xenograft model

[0212] Four of the 5 SCID mice injected IP with OCC 1 cells developed small tumour nodules (5-10mm) near the site of injection by day 14 post injection. Abdominal distention was apparent in 4 of the 5 animals by day 15-16 and all animals had some form of abdominal distention by necropsy day, day 18. Ascites fluid was observed (~l mL to ~1.5mL) in each animal. There were numerous small (l-2mm) nodules adhered to the intestinal mesentery while there were a few other tumours (5- 10mm) present in the peritoneal cavity of most animals.

[0213] Having confirmed the phenotype of the OCC1 xenografts in the pilot study, the efficacy study was then performed (Part B).

PART B: Efficacy of compounds 4 and 9 in the OCC1 xenograft model

Toxicity during Treatment

[0214] During the first few days of the treatment schedule, mice treated with either peptide did not exhibit signs of abnormal behaviour or overt toxicity immediately after injection of the peptide. As the treatments progressed, many of the treated mice in both compound 4 and 9 treated groups developed a mild hunched posture during daily wellness checks, indicating they were experiencing mild pain. In general, the mice in the control arm of the experiment did not exhibit this mild hunched posture.

[0215] As predicted from the pilot OCC1 study described in Part A, nearly all the mice developed some form of abdominal distention by day 16, which caused the mice to deteriorate until autopsy day (day 19). Near the end of the treatment schedule, most of the mice exhibited more of a hunched posture and some mice showed signs of respiratory distress, which were likely caused by tumours associated with their diaphragms.

Tumour Burden and Ascites Volume

[0216] As shown in Figure 4A, there was no significant difference in the mean total tumour burden of the mice treated with compound 4 or compound 9 compared with mice in the control group. However, there appeared to be a trend of decreased mean tumour burden in the compound 9 treated animals compared with control animals (p=0.19). Due to the nature of the tumour burden (numerous small nodules in the intestinal mesentery, pancreas and diaphragm), it was not feasible to micro-dissect the tumour nodules from any remnants of normal mesentery, pancreas or diaphragm. As a result, measurements of tumour weight by necessity include the weights of the mesentery, pancreas and diaphragm that are associated with the tumour tissue. Ascites volumes were collected using a l mL syringe and an 18G1 ^{1 2} gauge needle. Volumes were estimated by comparison to control tubes containing 0-2mL of water in O. lmL increments. Figure 4B shows that there was a significant difference in the volume of ascites observed between mice treated with vehicle and mice treated with compound 9 (p<0.05, unpaired t-test), while a slight trend towards decreased total ascites volume was present in mice treated with compound 4 (p=0.63). The amount of ascites in control animals was similar in amount to the ascites volume estimated in the OCC 1 pilot study animals used for Part A.

Summary/Conclusions

[0217] Compound 4 treatment (at MTD of 40 mg/kg) of mice bearing OCC1 xenografts did not lead to significant therapeutic benefit based either on tumour burden or ascites production. In contrast, treatment with the maximally tolerated dose of 20 mg/kg of compound 9 in this same model led to a significant decrease in the accumulation of ascites with a modest, but not significant decrease in tumour burden. The reduction of ascites may have clinical significance due to the high morbidity and poor prognosis associated with the development of ascites in women with ovarian cancer.

EXAMPLE 8: EFFECT OF PEPTIDES ON AKT EXPRESSION

[0218] The following example shows the effect of compound 4 on Akt expression. Other compounds of general formula (I) and (I-R) are expected to show similar activity in this regard.

[0219] A2780cp ovarian cancer cells were treated with 0.1 μΜ, 1 μΜ or 6.25 μΜ compound 4. After 1 hour, the cells were lysed and 40 μg of total cell extracts of untreated and treated samples were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with an antibody to phospho-AKT (pAKT). GAPDH was used as a loading control. The results of are shown in Figure 5 and show a significant decrease in pAkt in cells treated with as little as 1 μΜ compound 4.

[0220] A2780cp cells were treated with 25 μΜ compound 4 and collected at 1 , 4, 8, 14, and 30 hour time intervals. Cells were lysed and 40 μg of total cell extracts of untreated and treated samples were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with an antibody to phospho-AKT (pA T). GAPDH was used as a loading control. The results are shown in Figure 6.

[0221] The disclosure of all patents, publications, including published patent applications, and database entries referenced in this specification are expressly incorporated by reference in their entirety to the same extent as if each such individual patent, publication, and database entry were expressly and individually indicated to be incorporated by reference.

[0222] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WE CLAIM:

1. A peptide between about 12 and about 40 amino acid residues in length having a sequence of general formula (I) or the retro-inverso form thereof (I-R):

N_xBy(A/N)xByNy-J(M)-N_yB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-HY (I)

J is 1 -2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0-1 ;

each y is independently 0-2;

z = 0-l ;

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, wherein the sequences -N_yB_zA_xByN_yB_x and

amino acids in length, and

with the proviso that the peptide is other than: SI-NH_j

Compound 1 [SEQ ID NO:l] Compound 2 [SEQ ID NO:2] and

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO: 3]

2. The peptide according to claim 1 , wherein the peptide is a peptide of formula (I) and further comprises a protein translocation domain (PTD) peptide, with the proviso that the peptide is other than:

NLIGISI-NH₂ RKKRRQR

Compound 4 [SEQ ID NO:4] Compound 5 [SEQ ID NO:5]

3. The peptide according to claim 2, wherein the PTD peptide has the sequence KRRQRRKKR [SEQ ID NO: 19] or rkkrrqrrk [SEQ ID NO:20] or a fragment of one of these sequences.

4. The peptide according to claim 1 , wherein the peptide has a sequence of general formula (I-R).

5. The peptide according to any one of claims 1 to 3, wherein the peptide has a sequence of general formula (I), and wherein M is absent, with the proviso that the compound is other than:

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

6. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 5 and a pharmaceutically acceptable dliuent or carrier.

7. The pharmaceutical composition according to claim 6, wherein the peptide is formulated as a liposomal formulation.

8. A method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a peptide according to any one of claims 1 to 5.

9. The method according to claim 8, wherein the peptide is formulated as a liposomal formulation.

10. The method according to claim 8 or 9, wherein the cancer is colon cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, breast cancer, lymphoma, pulmonary cancer, cervical cancer, uterine cancer or ovarian cancer.

1 1. A method of treating malignant ascites in a subject in need thereof comprising administering to the subject an effective amount of a peptide between about 12 and about 40 amino acid residues in length having a sequence of general formula (I) or the retro-inverso form thereof (I-R):

N_xB_y(A/N)_xB_yN_y-J(M)-N_yB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-HY (I)

J is 1 -2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0-1 ;

each y is independently 0-2;

z = 0-l ;

wherein the sequences -N_yB_zA_xByNyBx and - B_xN_yB_yA_xB_zNy are 2 or more amino acids in length.

12. The method according to claim 1 1 , wherein the malignant ascites are associated with colon cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, breast cancer, lymphoma, pulmonary cancer, cervical cancer, uterine cancer or ovarian cancer.

13. A method of treating or preventing metastasis of a cancer in a subject in need thereof comprising administering to the subject an effective amount of a peptide between about 12 and about 40 amino acid residues in length having a sequence of general formula (I) or the retro- inverso form thereof (I-R):

N_xB_y(A/N)_xB_yN_y-J(M)-N_yB_zA_xB_yNyB_x - (linker) - (HB-HY)₂-HB2-HY (I)

J is 1 -2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0- 1 ;

each y is independently 0-2;

z = 0-l ;

14. A method of treating a drug- or hormone-resistant cancer in a subject in need thereof comprising administering to the subject an effective amount of a peptide between about 12 and about 40 amino acid residues in length having a sequence of general formula (I) or the retro- inverso form thereof (I-R):

N_xB_y(A N)_xByN_y-J(M)-N_yB_zA_xB_yN_yB_x - (linker) - (HB-HY)₂-HB2-HY (I)

J is 1 -2 Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0- 1 ;

each y is independently 0-2;

z = 0-l ;

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, wherein the sequences -N_yB_zA_xByNyB_x and - B_xNyB_yA_xB_zN_y are 2 or more amino acids in length.

15. The method according to any one of claims 1 1 to 14, wherein the peptide is other than:

Ac-FRR FRLGGGGGGGKDAQNLIGISI-NH₂ Ac-FRRKFRLGGGGGGGRDAQNLiG1SI-NH₂

Compound 1 [SEQ ID NO:l ] Compound 2 [SEQ ID NO:2] and

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂ Compound 3 [SEQ ID NO:3]

16. The method according to any one of claims 1 1 to 14, wherein when the peptide is a peptide of formula (I) and further comprises a protein translocation domain (PTD) peptide, with the proviso that the peptide is other than:

Ac-FRR F

Compound 4 [SEQ ID NO:4] Compound 5 [SEQ ID NO:5]

17. The method according to claim 16, wherein the PTD peptide has the sequence KRRQRRKKR [SEQ ID NO: 19] or rkkrrqrrk [SEQ ID NO:20] or a fragment of one of these sequences.

18. The method according to any one of claims 1 1 to 14, wherein the peptide has a sequence of general formula (I- ).

19. The method according to any one of claims 1 1 to 14, wherein the peptide has a sequence of general formula (I), and wherein M is absent, with the proviso that the compound is other than:

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH₂

Compound 3 [SEQ ID NO:3]

20. The method according to any one of claims 1 1 to 19, wherein the peptide is formulated as a liposomal formulation.

21. The peptide according to any one of claims 1 to 3, wherein the peptide is selected from the group consisting of:

O O

Ac-FRR-HN ,11. Ac— isigilnqadkggggggglrf— HN

FRLGGGGGGGKDAQNLIGISI-NH₂ y^" rrf-NH₂

/^' / HN-KRRQRRKKR-Ac Ac-krrqrrkkr-HN

Compound 16 [SEQ ID NO:30] Compound 17

22. The method according to any one of claims 8 to 20, wherein the peptide is selected from the group consisting of:

O

FRR-HN JL Ac— isigilnqadkggggggglrf- FRLGGGGGGGKDAQNLiGisi-NH₂ ^{a yyyyyyy}

HN-KRRQRRKKR-Ac Ac-krrqrrkkr- HN

Compound 16 [SEQ ID NO:30] Compound 17

23. The pharmaceutical composition according to claim 6, wherein the peptide is selected from the group consisting of:

O O

-^HN r^{J L} FRLGGGGGGGKDAQNLIGISI-NH₂ Ac-isigilnqadkggggggglrf-HN

H 'N,-KRRQRRKKR-Ac Ac-krrqrrkkr-HN

Compound 16 [SEQ ID NO:30] Compound 17