WO2020230122A1

WO2020230122A1 - Peptides for the treatment of cancer

Info

Publication number: WO2020230122A1
Application number: PCT/IL2020/050511
Authority: WO
Inventors: Uri Wormser
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Priority date: 2019-05-13
Filing date: 2020-05-12
Publication date: 2020-11-19

Abstract

Compositions and methods for treating cancer are provided, using peptides derived from the C-terminus of protein C inhibitor (PCI) having anti-tumor activity. Further provided are novel peptide conjugates, comprising peptides derived from the C-terminus of PCI conjugated to a biotinyl moiety.

Description

PEPTIDES FOR THE TREATMENT OF CANCER

FIELD OF THE INVENTION

The present invention relates to peptides having anti-tumor activity and their use in the treatment of cancer, particularly solid tumors.

BACKGROUND OF THE INVENTION

Cancer is currently the leading cause of death in most developed countries, and as the average age of the population continues to rise, so do the numbers of diagnosed cases of cancer. Cancer is characterized by uncontrolled proliferation and spread of abnormal cells. Despite advances in cancer treatment over the years, existing therapies still have limitations. Surgery and radiotherapy, for example, may be curative only if the cancer is diagnosed early enough, while current drug therapies seldom offer total eradication and long-term cure. Over the last decade, new strategies to treat cancer patients have emerged. Unlike traditional non-selective cytotoxic therapies, the new strategies are more focused on the tumor itself and its supportive microenvironment. Additional approaches for cancer treatment include preventing angiogenesis, activating the immune system and preventing the tumor and metastases from evading the immune system, for example by using immunotherapies such as cancer vaccines and cell therapies. The main aim is to improve anti-malignant efficacy while reducing treatment-induced toxicity and other adverse effects in treated patients. However, even with new therapies entering the market, there is still an unmet need for new drugs effective as monotherapy or in combination with existing treatments as a first line therapy in various types of cancer, and as second- and third-line therapies for treatment of resistant malignant diseases.

US 8,106,002 discloses anti-cancer agents comprising protein C inhibitor (PCI) or derivatives thereof as an active ingredient. The invention has shown that derivatives containing the heparin-binding domain of PCI, such as derivatives consisting of amino acids 1-354 of the mature PCI, inhibit growth, metastasis and angiogenesis of cancer.

US 5,792,749 discloses methods and compositions for lowering low density lipoprotein cholesterol. Among others, the following peptide may be administered:

S ARLN S QRLVFNRPFLMFI VDNNILFLGKVNRP (SEQ ID NO: 14 of US 5,792,749). Re'hault et al. (2005) Biochimica et Biophysica Acta, 1748:57-65, describes the expression and characterization of recombinant protein C inhibitor (PCI) in a bacterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rPCI for future structure-function studies.

Li et al. (2011) Frontiers in Bioscience, E3, 212-220, screened for serum biomarkers for TACE therapy efficiency of hepatocellular carcinoma. Among others, the peptide S ARLN S QRLVFNRPFLMFI VDNNILFLGKVNRP, corresponding to plasma serine protease inhibitor precursor, was differently expressed between before and after therapy groups.

Zhu et al. (2013) Journal of Cellular Biochemistry, 114:448-455, analyzed serum from colorectal cancer (CRC) patients, healthy controls, and other cancer patients, and identified peptide markers that could aid in the early diagnosis of CRC. Among others, the peptide S ARLN S QRLVFNRPFLMFIVDNNILFLGKVNRP was differentially expressed between CRC and controls.

Meng et al. (2016) RSC Adv., 6:39963, used monodisperse magnetic mesoporous silica microspheres to study gastric cancer-specific peptides in sera. Among others, the peptide SARLNS QRLVFNRPFLMFIVDNNILFLGKVNRP was identified.

He et al. (2013) PLoS One., 8(5):e63724, studied whether the parameters of gender and age influence serum peptide patterns in healthy individuals. The study included 500 serum samples from healthy men and women between 20 and 80 years of age. Samples from breast, lung, and rectal cancer patients were examined as well. Among others, the peptide SARLNS QRLVFNRPFLMFIVDNNILFLGKVNRP was identified.

Hong et al. (2016) Chin J Anal Chem, 44(5), 716-722, analyzed the urine peptidome and its translational modifications using nanoliter liquid chromatography-high resolution time of flight mass spectrometry. Among others, the peptide S ARLN S QRLVFNRPFLMFIVDNNILFLGKVNRP was identified.

US 8,440,409 and Rosenzweig et al. (2009) J Urol., 181(3): 1407—1414, disclose protein-based biomarkers for prostate cancer, including

S ARLN S QRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 9 of US 8,440,409), identified to be a fragment of PCI.

CN 102558329 discloses a group of specific serum polypeptides and their application in preparing reagents for early diagnosis of lung cancer. Among others, the peptide S ARLN S QRLVFNRPFLMFI VDNNILFLGKVNRP (SEQ ID NO: 28 of CN 102558329) is disclosed.

CN 105738631 discloses an autism serum marker, serpin family member 5- A (SERPINA5-A) polypeptide, useful as a target point for serodiagnosing autism by enzyme-linked immunosorbent assay (ELISA), and for manufacturing a medicament for autism. The amino acid sequence of the autism serum marker is SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1 of CN 105738631).

Nowhere is it disclosed or suggested that a fragment from the C-terminal of protein C inhibitor (PCI) possesses anti-tumor activity, or that it can be used in the treatment of cancer.

There remains a need for safe and efficient agents that are useful in treating various types of cancer.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treating cancer comprising peptides derived from the C-terminus of protein C inhibitor (PCI) having anticancer activity.

The peptides of the present invention comprise the sequence SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1), which is a 33-mer fragment from the C-terminus of human PCI. The present invention discloses for the first time that this fragment has marked anti-tumor activities and properties, demonstrated both in vitro on cultured tumor cells of various types and in vivo in a mouse model of metastatic mammary carcinoma. Importantly, no toxic or adverse effects were observed in mice treated with the peptide at a dose that showed significant anti-tumoral activity. Thus, peptides according to the present invention are promising as an efficacious medication for treating a variety of different types of cancer without causing undesired serious adverse effects. In some embodiments, the cancer is a solid tumor selected from the group consisting of breast cancer, prostatic cancer, lung cancer, melanoma and brain tumor.

The present invention further provides novel peptide conjugates in which peptides according to the present invention, comprising the sequence: SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1), are conjugated to a biotinyl moiety. As used herein, the term“biotinyl” refers to the moiety which remains after biotin has been substituted or conjugated to an additional moiety, such as an amino acid or a spacer/linker moiety. The conjugation is made through the carboxyl group of biotin, and thus a biotinyl moiety may be represented by the following formula:

The biotinyl moiety may be conjugated to a peptide according to the present invention directly or via a spacer or a linker. In some embodiments, biotinyl-6- aminohexanoic acid (biotin- Ahx) is conjugated to the peptide. In some embodiments, the peptide conjugates disclosed herein provide an improved anti-tumor activity. In some embodiments, the peptide conjugates disclosed herein provide improved permeability of the peptides through membranal and endothelial barriers, such as the blood-brain-barrier. In some embodiments, the peptide conjugates disclosed herein provide targeting of the peptides to the central-nervous system (CNS), for the treatment of CNS tumors, e.g., brain tumors.

According to one aspect, the present invention provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a peptide of 33-50 amino acids comprising the sequence SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1).

According to a further aspect, the present invention provides a pharmaceutical composition comprising a peptide of 33-50 amino acids comprising the sequence SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1), for use in the treatment of cancer.

In some embodiments, the cancer is a solid tumor.

In some embodiments, the solid tumor is selected from the group consisting of mammary (breast) cancer, prostatic cancer, lung cancer, melanoma and brain tumor. In some embodiments, the solid tumor is a brain tumor. In some particular embodiments, the brain tumor is glioblastoma. In some embodiments, the peptide is 33-40 amino acids in length.

In some embodiments, the peptide consists of the sequence SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1).

In some embodiments, the peptide further comprises at least one modification selected from the group consisting of an amino-terminal modification and a carboxy- terminal modification.

In some embodiments, the amino terminal modification is an amino blocking group selected from the group consisting of an acyl and alkyl.

In some embodiments, the carboxy-terminal modification is a carboxyl blocking group selected from the group consisting of an amide, alcohol and an ester.

In some embodiments, the peptide is represented by the following formula:

R₁ -SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-R₂,

wherein Ri designates a hydrogen of an unmodified amino terminal group or is an amino blocking group selected from the group consisting of an acyl and alkyl; and R2 designates OH of an unmodified carboxy terminal group or is a carboxyl blocking group selected from the group consisting of an amide, alcohol and ester.

In some embodiments, the peptide is:

H-SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-OH .

In some embodiments, the peptide is conjugated to a moiety selected from the group consisting of a permeability-enhancing moiety, a synthetic polymer, a carrier or targeting peptide, a carrier or targeting protein, and a sugar moiety.

In some embodiments, the peptide is conjugated to a permeability-enhancing moiety.

In some embodiments, the peptide is conjugated to a compound selected from the group consisting of biotin, a biotin derivative, and a biotin analog.

In some embodiments, the peptide is conjugated to a biotinyl moiety via the amino terminus of the peptide.

In some embodiments, the biotinyl moiety is biotinyl-6-aminohexanoic acid (Biotin-Ahx).

In some embodiments, the biotinyl-peptide conjugate is according to the following formula:

Biotin- Ahx-ARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-R2, wherein R2 designates OH of an unmodified carboxy terminal group or is a carboxyl blocking group selected from the group consisting of an amide, alcohol and ester.

In some embodiments, the biotinyl-peptide conjugate is:

Biotin- Ahx-ARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-OH

(SEQ ID NO: 5).

In some embodiments, the administering is systemically administering.

According to a further aspect, the present invention provides a conjugate comprising a peptide of 33-50 amino acids comprising the sequence SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1) and a biotinyl moiety.

In some embodiments, the biotinyl moiety is conjugated to the amino terminus of the peptide.

In some embodiments, the peptide is 33-40 amino acid in length.

In some embodiments, the peptide consists of the sequence:

SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1).

In some embodiments, the conjugate is according to the following formula:

Biotin- Ahx-RLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-R2,

wherein R2 designates OH of an unmodified carboxy terminal group or is a carboxyl blocking group selected from the group consisting of an amide, alcohol and ester.

In some embodiments, the biotinyl-peptide conjugate is:

Biotin- Ahx-ARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-OH

(SEQ ID NO: 5).

According to a further aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient a conjugate of the present invention and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is for use in the treatment of cancer.

Other objects, features and advantages of the present invention will become clear from the following description, examples and drawings. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Effect of H-SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-OH (termed“IDO 12”) on lung metastasis in BAFB/c mice inoculated with 4T1 mammary carcinoma cells. (1A) Fung weight; p=0.0001 for comparison between 1mg/kg group and control; p<0.0001 for comparison between 0. lmg/kg group and control; p<0.0001 for comparison between 0.01mg/kg group and control; p<0.0001 for comparison between 0. lmg/kg and lmg/kg group; p<0.0001 for comparison between 0.01mg/kg and lmg/kg group; statistical evaluation was carried out with student’s t test; ( 1B) Number of tumor foci per lung; p=0.0002 for comparison between 1mg/kg group and control; p<0.0001 for comparison between 0. lmg/kg group and control; p<0.0001 for comparison between 0.01mg/kg group and control; p<0.0019 for comparison between 0. lmg/kg and 1mg/kg group; p<0.0592 for comparison between 0.01mg/kg and lmg/kg group; no statistical difference between 0. lmg/kg and 0.01mg/kg; statistical evaluation was carried out with student’s t test; (1C) Histological evaluation. Upper panel: a representative sample from the control group: there are many neoplastic nodules (T) throughout the lung. Fower panel: a representative sample from the ID012-treated group (0. 1mg/kg, s.c. administration): neoplastic cells are not identified. The pulmonary parenchyma is normal; (ID) Average histological score; p=0.0009 for comparison between 1mg/kg group and control; p<0.0001 for comparison between 0. lmg/kg group and control; p<0.0001 for comparison between 0.01mg/kg group and control; p<0.0009 for comparison between 0.01mg/kg and lmg/kg group; p<0.0081 for comparison between 0.01mg/kg and 1mg/kg group; statistical evaluation was carried out with student’s t test.

Figure 2. MTT viability tests of malignant cells incubated with IDO 12 for 24 hours at various concentrations compared to control cells treated with a vehicle. (2A) mouse 4T1 mammary carcinoma cells; ***p<0.0003 for comparison with control cells (student’ s t test); n=4 for 10 mg/ml, n=5 for 0.1 mg/ml and lpg/ml, the rest are hexaplicates as described in Experimental Procedure; (2B) triple-negative human MDA-MB-231 mammary adenocarcinoma cells; **p<0.008 for comparison with control cells (student’s t test); n=5 for O. lng/ml, n=4 for lpg/ml, the rest are in hexaplicates; (2C) human mammary adenocarcinoma MCF-7 cells; ****p<0.0001, ***p=0.0002 for comparison with control (student’s t test); (2D) mouse 3FF Fewis lung carcinoma D122 cells; ****p<0.0001 for comparison with control (student’s t test); (2E) mouse B 16-F10 1elanoma cells; ****p<0.0001 for comparison with control (student’s t test); (2F) human LNCaP prostatic carcinoma cells; ****p<0.0001 for comparison with control (student’s t test).

Figure 3. Trypan blue exclusion assay. Cell counts of triple negative human MDA-MB-231 mammary carcinoma cells treated with ID012 at various concentrations compared to control cells treated with a vehicle; p=0.025 for comparison between 10 mg/ml group and control; p=0.0209 for comparison between 1mg/ml group and control; p=0.0259 for comparison between 100ng/ml group and control; (student’s t test).

Figure 4. Annexin V/Propidium iodide assay of mouse 4T1 mammary carcinoma cells treated with IDO 12 0.0.1 mg/ml (4A) or 0.1 mg/ml (4B) compared to control cells treated with a vehicle. p=0.0158 for 4h- 1 mg/ml vs. 4h-control, p=0.0241 for 24h-0.1 mg/ml and 24h- 0.1 mg/ml vs. 24h-control, p=0.0093 for 48h- 1 mg/ml vs. 48h-control. Results are expressed as mean+SEM using student’s t test for statistical evaluation.

Figure 5. Single- stranded DNA (ssDNA) apoptosis assay of human MDA-MB- 231 mammary carcinoma cells treated with ID012 0.1 mg/ml or 1 mg/ml compared to control cells treated with a vehicle. MD-MB-231 cells were seeded (7.5xl0³/well, 96 well plate, 0.1ml/well). After 24 hours cells were exposed to the peptide at the indicated concentrations (in triplicate) for 4 hours. Control cells (in triplicate) were exposed to the vehicle. Cells were processed according to manufacturer (Millipore, Molshiem, France) instructions for single strand DNA apoptosis; ***p=0.0007 in comparison to control (student’s t test).

Figure 6. Effect of IDO 12 on blood counts in tumor-bearing mice. (6A) WBC counts; (6B) lymphocyte counts; (6C) monocyte counts; (6D) granulocyte counts; (6E) hemoglobin; (6F) platelet counts; (6G) RBC counts.

Figure 7. MTT viability tests of two different types of human-originated glioblastoma cells incubated with IDO 12 and derivative and conjugate thereof for 24 hours at various concentrations compared to cells treated with a vehicle (“control- solvent”) or untreated cells (“control”). (7A) U-87 cells; (2B) U-251 cells.

Figure 8. MTT viability tests of human mammary MDA-MB-231 cells incubated with IDO 12 and the conjugate Biotin- Ahx-IDO 12 for 24 hours at various concentrations compared to cells treated with a vehicle (“control-solvent”) or untreated cells (“control”); ****p<0.0001 for comparison between 10. mg/ml Biotin- Ahx-IDO 12 and 10. mg/ml ID012; p=0.0011 for comparison between 1 mg/ml Biotin- Ahx-IDO 12 and 1 mg/ml ID012; p<0.0001 for comparison between 10. mg/ml Biotin- Ahx-IDO 12 and control equivalent to 10. mg/ml; p<0.0001 for comparison between 10. mg/ml ID012 and control equivalent 10. mg/mll; p<0.0001 for comparison between 10 mg/ml Biotin- Ahx-IDO 12 and control equivalent to 1 mg/ml ; p=0.0009 for comparison between lpg/ml ID012 and control equivalent to 1 mg/ml ; (n=4 for all concentrations, statistical significance calculated by student’s t test).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to peptides derived from the C-terminus of protein C inhibitor, which are useful for the treatment of cancer, particularly solid tumors.

Peptides of the present invention comprise the sequence SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1), which is a 33-mer fragment from the C-terminus of human protein C inhibitor (PCI), also known as SERPINA5. The amino acid sequence of the full-length human protein C inhibitor (including the signal peptide) is set forth below (SEQ ID NO: 2). The amino acid sequence of the mature human protein C inhibitor is also set forth below (SEQ ID NO: 3). The peptides of the present invention comprise a sequence corresponding to amino acids 355-387 of the mature protein C inhibitor (positions 355- 387 of SEQ ID NO: 3, which is the sequence of the mature protein C inhibitor without the signal peptide).

As exemplified herein below, in vivo studies surprisingly demonstrated significant selective anti-tumor effect of a peptide according to the present invention,

H-SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-OH (termed“IDO 12”), using a mouse model of metastatic mammary carcinoma. As further exemplified herein below, in vitro studies demonstrated significant anti-tumor effect of the peptide in several different tumor cell lines. Anti-tumor effect of the peptide was demonstrated in five tumor cell lines of human origin (2 mammary adenocarcinomas, 1 prostatic carcinoma, 2 glioblastoma) and three tumor cell lines of mouse origin (1 mammary carcinoma, 1 lung carcinoma, 1 melanoma). The in vitro studies showed that the peptide affects cell viability, decreases proliferation, and induces apoptosis of tumor cells. Importantly, no toxic effects or adverse effects were observed in the treated mice at a dose that showed significant anti-tumoral activity (unlike“classic” chemotherapy). The peptides of the present invention are therefore promising as safe and effective anticancer agents.

According to certain embodiments, the subject to be treated using the peptides of the present invention is a human subject. According to additional embodiments, the subject to be treated may be a non-human mammal. Treatment according to the present invention may be applicable during the active phase of the cancer, as well as following treatment to prevent relapse or reoccurrence of the cancer, or for treating minimal residual disease (MRD). Each possibility represents a separate embodiment of the present invention.

The term "peptide" as used herein refers to a polymer of amino acid residues linked by peptide bonds. By“peptide” it is meant an amino acid sequence consisting of up to 50 amino acids, for example up to 40 amino acids, up to 35 amino acids, or less. By “polypeptide” or "protein" it is meant an amino acid sequence of more than 50 amino acid residues.

In some embodiments, the peptide comprises an amino-terminal modification. In additional embodiments, the peptide comprises a carboxy-terminal modification. In some embodiments, the peptide comprises both an amino-terminal modification and a carboxy- terminal modification.

In some embodiments, the amino terminal modification is an amino blocking group. In some embodiments, the amino blocking group is selected from the group consisting of an acyl (e.g. acetyl) and alkyl (e.g., methyl). Each possibility represents a separate embodiment of the invention.

In some embodiments, the carboxy-terminal modification is a carboxyl blocking group. In some embodiments, the carboxy-terminal modification is selected from the group consisting of an amide, alcohol and an ester. Each possibility represents a separate embodiment of the invention.

In some particular embodiments, the peptide consists of SEQ ID NO: 1 and is optionally modified with N-terminal and/or C-terminal blocking groups, as follows:

R1-SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-R2,

wherein R₁ designates a hydrogen of an unmodified amino terminal group or is an amino blocking group selected from the group consisting of an acyl and alkyl; and R2 designates OH of an unmodified carboxy terminal group or is a carboxyl blocking group selected from the group consisting of an amide, alcohol and ester. Each possibility represents a separate embodiment of the present invention.

In some particular embodiments, the peptide is:

H-S ARLN S QRL VFNRPFLMFI VDNNILFLGKVNRP-OH .

In some embodiments, the peptide is conjugated to a moiety selected from the group consisting of a permeability-enhancing moiety (e.g. a fatty acid), a synthetic polymer, a carrier or targeting peptide, a carrier or targeting protein, and a sugar moiety. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the peptide is conjugated to a moiety selected from the group consisting of a fatty acid, a synthetic polymer, a carrier or targeting peptide, a carrier or targeting protein, and a sugar moiety. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the peptide consists of SEQ ID NO: 1 and is optionally conjugated to a permeability-enhancing moiety (e.g. a fatty acid), a synthetic polymer, a carrier or targeting peptide, a carrier or targeting protein, and a sugar moiety.

In some embodiments, the peptide is represented by the following formula:

R₁ -S ARENSQRFVFNRPFFMFIVDNNIFFFGKVNRP-R2,

wherein:

R₁ designates a hydrogen of an unmodified amino terminal group or is selected from the group consisting of: (i) an amino blocking group selected from acyl and alkyl, and (ii) a permeability-enhancing or targeting moiety; and

R2 designates OH of an unmodified carboxy terminal group or is selected from the group consisting of: (i) a carboxyl blocking group selected from an amide, alcohol and ester, and (ii) a permeability-enhancing or targeting moiety.

The term "amino acid" refers to any one of the proteinogenic amino acids, including the 20 genetically-encoded amino acids, biosynthetically available amino acids which are not found in proteins (e.g., 4-hydroxy -proline, 5-hydroxy-lysine, citrulline, ornithine, canavanine, djenkolic acid, b-cyanolanine), and also non-natural and/or amino acids that have been chemically modified (synthetic), each amino acid being characterized by having an amino and a carboxy terminus. The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. The amino acids are represented throughout the specification and claims by either one or three-letter codes, as is commonly known in the art. When there is no indication, the L stereoisomer was used. The D isomers are indicated by“D” or "(D)" before the residue abbreviation.

As used herein, an "amino acid residue" means the moiety which remains after the amino acid has been conjugated to additional amino acid(s) to form a peptide, or to a moiety (such as a permeability-enhancing moiety), typically through the alpha-amino and carboxyl of the amino acid.

The terms "peptides of the invention", "synthetic peptides of the invention" and the like all refer to artificial, non-naturally occurring peptides, typically produced by standard peptide synthesis methods known in the art such as solid-phase peptide synthesis (SPPS). In some embodiments, recombinant protein techniques, well known in the art, are used to generate the peptides of the present invention.

According to some embodiments, the peptide of the invention is of 33-50 amino acids, 33-49 amino acids, 33-48 amino acids, 33-47 amino acids, 33-46 amino acids, 33- 45 amino acids, 33-44 amino acids, 33-43 amino acids, 33-42 amino acids, 33-41 amino acids, 33-40 amino acids, 33-39 amino acids, 33-38 amino acids, 33-37 amino acids, 33- 36 amino acids, 33-35 amino acids, 33-34 amino acids, or 33 amino acids. Each possibility represents a separate embodiment of the invention.

Peptides according to the present invention may include chemically modified amino acids. The term“chemically modified", when referring to an amino acid, refers to an amino acid that is modified by one or more chemical modifications, which can be performed by techniques known in the art. Chemical modifications of amino acids encompass, but not limited to, acetylation, acylation, amidation, ADP-ribosylation, glycosylation, glycosaminoglycanation, methylation (e.g. N-methylation), myristoylation, pegylation, prenylation, phosphorylation, ubiquitination, and the like.

Analogs of the peptides are also within the scope of the present invention. As used herein, "analogs" are peptides which have the amino acid sequence according to the invention except for one or more amino acid changes, typically, conservative amino acid substitutions. In some embodiments, an analog has at least about 75% identity to the sequence of the peptide of the invention, for example at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to the sequence of the peptide of the invention. Each possibility represents a separate embodiment of the present invention. Analogs are included in the invention as long as they remain pharmaceutically acceptable and their anti-tumor activity is not damaged.

Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following is an example of classification of the amino acids into six groups, each contains amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K), Histidine (H);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Analogs according to the present invention may comprise also peptidomimetics. “Peptidomimetics” refers to synthetic molecules containing non-pep tidic structural elements that are capable of mimicking the biological action(s) of a natural parent peptide.

Analogs according to the present invention also encompasses peptides in which one or more amino acids has been removed from the sequence, for example, one, two, three, four, five amino acids have been removed. Analogs in which one or more amino acids have been removed from the sequence are included in the invention as long as they remain pharmaceutically acceptable and their anti-tumor activity is not damaged.

Derivatives of the peptides of the invention are also within the scope of the present invention. As used herein, the term“derivatives” cover inter alia derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the pharmacological/biological activity of the peptide, and do not confer significant toxic properties on compositions containing it. These derivatives may include, for example, aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues, e.g., N-acetyl, formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups), or O-acyl derivatives of free hydroxyl group (e.g., that of seryl or threonyl residues) formed by reaction with acyl moieties.

An exemplary derivative of a peptide according to the present invention is a derivative in which the S-methyl thioether group of SEQ ID NO: 1 is in a sulfoxide form, e.g.:

H-SARLNSQRLVFNRPFLM(0)FIVDNNILFLGKVNRP-OH (SEQ ID NO: 4).

The peptides of the present invention are typically utilized in a linear form, although it will be appreciated that in cases where cyclization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized. Cyclization of peptides may take place by any means known in the art, for example through free amino and carboxylic groups present in the peptide sequence, or through amino acids or moieties added for cyclization. Non-limiting examples of cyclization types are: side chain to side chain cyclization, C-to-N terminal cyclization, side chain to terminal cyclization, and any type of backbone cyclization incorporating at least one N₁- -ubstituted amino acid residue/s as described for example in WO 95/33765.

The present invention further provides conjugates comprising a peptide of the invention covalently linked to a moiety selected from the group consisting of a fatty acid, a polymer chain, an amino acid, a carrier or targeting peptide, a carrier or targeting protein, and a sugar moiety. Each possibility represents a separate embodiment of the present invention.

The present invention further provides conjugates comprising a peptide of the invention covalently linked to a compound selected from the group consisting of biotin, a biotin derivative, and a biotin analog.

Biotin (also known as vitamin H or vitamin B7) is a water-soluble vitamin associated with a wide range of metabolic processes in humans and other organisms. Biotin is used extensively in biochemistry and molecular biology for a variety of purposes including macromolecular detection, purification and isolation, and in cytochemical staining. Biotin also has important applications in medicine in the areas of clinical diagnostic assays, tumor imaging and drug delivery, and is used extensively in the field of affinity cytochemistry for the selective labeling of cells, subcellular structures and proteins.

The conjugated biotin (a biotinyl moiety) may be conjugated to the peptide via a direct bond or via a spacer or a linker. In some embodiments, the biotinyl moiety is conjugated to the amino terminus of the peptide. In additional embodiments, the biotinyl moiety is conjugated to a side chain of an amino acid within the peptide.

In some embodiments, biotinyl-6-aminohexanoic acid (biotin-Ahx) is conjugated to the peptide.

Without being limited to any particular mechanism of action, it is contemplated that conjugates of the invention comprising a compound selected from the group consisting of biotin, a biotin derivative, and biotin analog, provide superior permeability through the blood-brain barrier (BBB). The BBB is a highly selective semipermeable layer of endothelial cells and astrocytes that regulates the entry of solutes in the circulating blood from crossing into the extracellular fluid of the central nervous system. The BBB controls the passage of ions (Na+, K+, Ca2+), water, nutrients, metabolites, neurotransmitters, plasma proteins and peptides, cells from the immune system, and drugs in an out of the brain. The BBB can be regarded as an organ that functions to maintain the homeostasis of the brain, protects the brain, and plays a significant role in various neurological and degenerative disorders. The BBB may also obstruct delivery of CNS and brain-affecting drugs to a site of disease in the brain. To effectively treat brain disease, it is critical to develop drugs that can bypass or pass through the BBB into the brain.

The term "conjugate" as used herein, refers to a product comprising the peptide of the invention chemically linked to a moiety e.g., a biotinyl moiety, a fatty acid, an amino acid, a protein, a sugar moiety, or a polymer chain. The moiety conjugated to the peptide of the invention can be attached to the N and/or C terminus of the peptide and/or to a side chain of an amino acid of the peptide directly or via a spacer or a linker. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the fatty acid used in the conjugate of the present invention is selected from the group consisting of saturated, unsaturated, monounsaturated and polyunsaturated fatty acids. According to some embodiments, the fatty acid is selected from the group consisting of decanoic acid, undecanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, trans- hexadecanoic acid, elaidic acid, lactobacillic acid, tuberculostearic acid, and cerebronic acid. Each possibility represents a separate embodiment of the invention. According to additional embodiment the peptide is conjugated to a sphingolipid.

According to some embodiments, the polymer chain used in the conjugate of the present invention is a synthetic polymer selected from the group consisting of polyethylene glycol (PEG), polylactic acid, poly-L-lactic acid, poly-D,L-lactic acid, poly glycolic acid, poly- e-caprolactone, poly-p-dioxanon, tri-methylene carbonate, poly anhydrides, poly ortho ester, poly urethanes, a poly amino acid, poly hydroxy alcanoate, poly phosphazene and poly- b-caprolacto acnied. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the peptide of the present invention as defined above is conjugated to an amino acid, peptide or protein, which functions as a carrier or targeting moiety of the peptide of the invention.

According to some embodiments, the carrier or targeting peptide that is conjugated to the peptide of the invention consists of 2-40 amino acids, for example 10-40 amino acids, or 10 amino acids. According to further embodiments, the carrier or targeting peptide consists of 10-20 amino acids.

According to some embodiments, the carrier or targeting protein that is conjugated to the peptide of the invention is a hormone, a growth factor or a cytokine. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the amino acid, peptide, polypeptide, or protein comprises at least one amino acid in the D configuration.

In some embodiments, the peptide of the invention is covalently conjugated to a compound that improves cell permeability, e.g., a fatty acid. In other embodiments, the conjugate comprises a cell-penetrating peptide.

The term“fatty acid moiety” as used herein refers to a part of a fatty acid that exhibits a particular set of chemical and pharmacologic characteristics similar to the corresponding complete fatty acid origin molecule. The term further refers to any molecular species and/or molecular fragment comprising the acyl component of a fatty (carboxylic) acid.

The term“permeability” as used herein refers to the ability of an agent or substance to penetrate, pervade, or diffuse through a barrier or a membrane. The terms“cell permeability-”,“cell-penetration-” and "permeability-enhancing-" moiety refers to any molecule known in the art which is able to facilitate or enhance penetration of molecules through membranes. Non-limitative examples include: hydrophobic moieties such as lipids, fatty acids, and bulky aromatic or aliphatic compounds; moieties which have cell- membrane receptors or carriers, such as steroids, vitamins, cytokines, growth hormones and the like, and amino acids such as methionine. Conjugation of a peptide of the invention to a permeability-enhancing moiety may be useful, for example, for local dermal administration of the peptide. Such modification may also be useful for systemic intradermal administration of the peptide.

According to some embodiments the hydrophobic moiety is selected from the group consisting of: phospholipids, steroids, sphingosines, ceramides, octyl-glycine, 2- cyclohexylalanine, benzolylphenylalanine, propionoyl (C3); butanoyl (C4); pentanoyl (C5); caproyl (C6); heptanoyl (C7); capryloyl (C8); nonanoyl (C9); capryl (CIO); undecanoyl (Cl l); lauroyl (Cl 2); tridecanoyl (Cl 3); myristoyl (Cl 4); pentadecanoyl (C15); palmitoyl (C16); phtanoyl ((CH3)4); heptadecanoyl (C17); stearoyl (C18); nonadecanoyl (Cl 9); arachidoyl (C20); heniecosanoyl (C21); behenoyl (C22); trucisanoyl (C23); and lignoceroyl (C24).

Synthetic production of peptides is well known in the art. The peptides of the present invention can be synthesized using standard direct peptide synthesis (see, for example, Bodanszky, 1984, Principles of Peptide Synthesis, Springer-Verlag, Heidelberg), such as via solid-phase synthesis (see, for example, Merrifield, 1963, J. Am. Chem. Soc. 85:2149-2154). Examples of solid phase peptide synthesis methods include, but are not limited to, the BOC method, which utilizes tert-butyloxcarbonyl as the a- amino protecting group, and the FMOC method, which utilizes 9- fluorenylmethyloxcarbonyl to protect the a-amino of the amino acid residues, both methods are well-known by those of skill in the art.

Alternatively, the peptides of the present invention can be synthesized using standard solution methods (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer-Verlag, 1984). The present invention also encompasses peptides in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonylamino groups, carbobenzoxyamino groups, t-butyloxycarbonylamino groups, chloroacetylamino groups or formylamino groups. Free carboxyl groups may be derivatized to form, for example, salts, amides, methyl and ethyl esters or other types of esters or hydrazides.

The peptides of the present invention can contain one or more D-isomer forms of the amino acids. Production of retro-inverso D-amino acid peptides where at least one amino acid, and perhaps all amino acids, is D-amino acids is well known in the art. When all of the amino acids in the peptide are D-amino acids, and the N- and C-terminals of the molecule are reversed, the result is a molecule having the same structural groups being at the same positions as in the L-amino acid form of the molecule. However, the molecule is more stable to proteolytic degradation and is therefore useful in many of the applications recited herein.

Full-length human protein C inhibitor (SEQ ID NO: 21:

MQLFLLLCLVLLSPQGASLHRHHPREMKKRVEDLHVGATVAPSSRRDFTFDLY RALASAAPSQSIFFSPVSISMSLAMLSLGAGSSTKMQILEGLGLNLQKSSEKELH RGFQQLLQELNQPRDGFQLSLGNALFTDLVVDLQDTFVSAMKTLYLADTFPTN FRDSAGAMKQINDYVAKQTKGKIVDLLKNLDSNAVVIMVNYIFFKAKWETSFN HKGTQEQDFYVTSETVVRVPMMSREDQYHYLLDRNLSCRVVGVPYQGNATAL FILPSEGKMQQVENGLSEKTLRKWLKMFKKRQLELYLPKFSIEGSYQLEKVLPS LGISNVFTSHADLSGISNHSNIQVSEMVHKAVVEVDESGTRAAAATGTIFTFRSA RLNSQRLVFNRPFLMFIVDNNILFLGKVNRP

Mature human protein C inhibitor (no signal peptide) (SEQ ID NO: 31:

HRHHPREMKKRVEDLHVGATVAPSSRRDFTFDLYRALASAAPSQSIFFSPVSIS MSLAMLSLGAGSSTKMQILEGLGLNLQKSSEKELHRGFQQLLQELNQPRDGFQ LSLGNALFTDLVVDLQDTFVSAMKTLYLADTFPTNFRDSAGAMKQINDYVAK QTKGKIVDLLKNLDSNAVVIMVNYIFFKAKWETSFNHKGTQEQDFYVTSETVV RVPMMSREDQYHYLLDRNLSCRVVGVPYQGNATALFILPSEGKMQQVENGLS EKTLRKWLKMFKKRQLELYLPKFSIEGSYQLEKVLPSLGISNVFTSHADLSGISN HSNIQVSEMVHKAVVEVDESGTRAAAATGTIFTFRSARLNSQRLVFNRPFLMFI VDNNILFLGKVNRP

As noted above, peptides according to the present invention are typically produced by standard peptide synthesis methods known in the art such as solid-phase peptide synthesis (SPPS). Alternatively, recombinant protein techniques may be used to generate the peptides of the present invention. In some embodiments, an isolated polynucleotide is provided, encoding a peptide of the present invention. The term "polynucleotide" refers to a polymer of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a combination thereof, which can be derived from any source, can be single- or double-stranded, and can optionally contain synthetic, non-natural, or altered nucleotides, which are capable of being incorporated into DNA or RNA polymers. An "isolated polynucleotide" refers to a polynucleotide segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to polynucleotides, which have been substantially purified from other components, which naturally accompany the polynucleotide in the cell, e.g., RNA or DNA or proteins. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA, which is part of a hybrid gene encoding additional polypeptide sequence, and RNA such as mRNA. The term "encoding" refers to the inherent property of specific sequences of nucleotides in an isolated polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a peptide or protein if transcription and translation of mRNA corresponding to that gene produces the peptide or protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the peptide or protein or other product of that gene or cDNA. One who is skilled in the art will appreciate that more than one polynucleotide may encode any given peptide or protein in view of the degeneracy of the genetic code and the allowance of exceptions to classical base pairing in the third position of the codon, as given by the so-called "Wobble rules."

It is intended that the present invention encompass polynucleotides that encode a peptide of 33-50 amino acids comprising the sequence set forth in SEQ ID NO: 1 or a peptide consisting of the sequent set forth in SEQ ID NO: 1.

A peptide of the present invention may be produced in a host cells by expressing it as a secreted peptide, where the peptide is isolated from the medium in which the host cell containing the polynucleotide is grown, or as an intracellular peptide by deleting the leader or other peptides, in which case the peptide is isolated from the host cells. The peptide so isolated may then be purified by standard protein purification methods known in the art. The peptide may also be provided to the tissue of interest by transferring an expression vector comprising an isolated polynucleotide encoding the peptide to cells associated with the tissue of interest. The cells produce the peptide such that it is suitably provided to the cells within the tissue to exert a biological activity. The expression vector typically further comprises a promoter, which drives the expression of the peptide within the cells. Many viral promoters are appropriate for use in such an expression vector (e.g., retroviral ITRs, LTRs, immediate early viral promoters (IEp) (such as herpes virus IEp (e.g., ICP4-IEp and ICPO-IEp) and cytomegalovirus (CMV) IEp), and other viral promoters (e.g., late viral promoters, latencyactive promoters (LAPs), Rous Sarcoma Virus (RSV) promoters, and Murine Leukemia Virus (MLV) promoters). Other suitable promoters are eukaryotic promoters, which contain enhancer sequences (e.g., the rabbit b-globin regulatory elements), constitutively active promoters (e.g., the b-actin promoter, etc.), signal and/or tissue specific promoters (e.g., inducible and/or repressible promoters, such as a promoter responsive to TNF or RU486, the metallothionine promoter, etc.), and tumor-specific promoters. Within the expression vector, the polynucleotide encoding the peptide of the present invention are operably linked such that the promoter is able to drive the expression of the polynucleotide. The expression vector may also include other elements, such as splice sites, polyadenylation sequences, transcriptional regulatory elements (e.g., enhancers, silencers, etc.), or other sequences. Expression vectors are introduced into the cells in a manner such that they are capable of expressing the isolated polynucleotide encoding the peptide contained therein. Any suitable vector may be so employed. Examples of such vectors include naked DNA vectors (such as oligonucleotides or plasmids), viral vectors such as adeno-associated viral vectors, adenoviral vectors, herpes virus vectors, packaged amplicons, papilloma virus vectors, picornavirus vectors, polyoma virus vectors, retroviral vectors, SV40 viral vectors, vaccinia virus vectors, and other vectors. Additionally, the vector may also include other genetic elements, such as, for example, genes encoding a selectable marker (e.g., b-gal or a marker conferring resistance to a toxin) or a transcription factor. Methods for manipulating a vector comprising an isolated polynucleotide are known in the art and include direct cloning, site specific recombination using recombinases, homologous recombination, and other suitable methods of constructing a recombinant vector. In this manner, an expression vector may be constructed such that it can be replicated in any desired cell, expressed in any desired cell, and can even become integrated into the genome of any desired cell. The expression vector comprising the polynucleotide of interest is introduced into the cells by any means appropriate for the transfer of DNA into cells. In the case of prokaryotic cells, vector introduction can be accomplished, for example, by electroporation, transformation, transduction, conjugation, or mobilization· For eukaryotic cells, vectors can be introduced through the use of, for example, electroporation, transfection, infection, DNA coated microprojectiles, or protoplast fusion. Examples of eukaryotic cells into which the expression vector can be introduced include, but are not limited to, ovum, stem cells, blactocytes, and the like. Cells into which the polynucleotide has been transferred under the control of an inducible promoter if necessary, can be used as transient transformants. Such cells themselves may then be transferred into a subject for therapeutic benefit therein. Thus, the cells can be transferred to a site in the subject such that the peptide of the invention is expressed therein and secreted therefrom. Alternatively, particularly in the case of cells to which the vector has been added in vitro, the cells can first be subjected to several rounds of clonal selection (facilitated usually by the use of a selectable marker sequence in the vector) to select for stable transformants. Such stable transformants are then transferred to a subject, preferably a human, for therapeutic benefit therein. Within the cells, the polynucleotide Pharding the peptide of the present invention is expressed, and optionally secreted. Successful expression of the polynucleotide can be assessed using standard molecular biology techniques (e.g., Northern hybridization, Western blotting, immunoprecipitation, enzyme immunoassay, etc.)

The present invention provides according to some aspects a pharmaceutical composition comprising the peptide of the invention and a pharmaceutically acceptable carrier, excipient or diluent.

The term“pharmaceutical composition” as used herein refers to any composition comprising at least one pharmaceutically active ingredient, formulated such that it facilitates accessibility of the active ingredient to the target organ.

Carrier(s), excipient(s) and diluent(s) are “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Suitable carriers typically include water, saline, oils, and polyols such as glycerol or propylene glycol.

The peptides may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups) which are formed with inorganic acids such as hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, or from organic bases such as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine and procaine.

The pharmaceutical compositions may be formulated for oral administration or for parenteral administration. For example, the pharmaceutical compositions may be formulated for injection administration, including but not limited to intravenous, intraarticular, intramuscular, subcutaneous or intradermal. Each possibility represents a separate embodiment of the present invention. Other routes of administration include inhalation, sublingual, rectal, dermal, transnasal and intranasal. Each possibility represents a separate embodiment of the present invention.

Thus, according to some embodiments, the pharmaceutical composition is for administration in a route of administration selected from the group consisting of oral, intraperitoneal, intravenous, intramuscular, subcutaneous, topical application, buccal, intradermal, transdermal, inhalation, sublingual, rectal, intravitreal, intravesical, intrathecal, transnasal and intranasal. Each possibility represents a separate embodiment of the invention.

In some embodiments, the pharmaceutical composition is for systemic administration. In some particular embodiments, the pharmaceutical composition is for administering orally. In additional particular embodiments, the pharmaceutical composition is for administering intravenously. In additional particular embodiments, the pharmaceutical composition is for administering intraperitoneally.

In some embodiments, the pharmaceutical composition is for non-systemic administration. In some embodiments, the pharmaceutical composition is for topical administration. In some embodiments, the pharmaceutical composition is for local administration, for example, for administration directly into a tumor, administration into an area from which a tumor or a portion thereof has been surgically resected, and/or into an area surrounding the tumor.

According to some embodiments, the pharmaceutical composition is formulated in a form selected from the group consisting of a solution, an emulsion, a suspension, a powder, a tablet, a capsule, a pill, a lozenge, a paste, a sustained-release formulation and the like. Each possibility represents a separate embodiment of the invention.

The compositions may be suitably formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal administration and comprise sterile aqueous solutions of the peptides, which are preferably isotonic. Such formulations are typically prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. These may be prepared in unit or multi-dose containers, for example, sealed ampoules or vials.

In some embodiments, injectable solutions of the invention are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

The compositions may incorporate a stabilizer, such as for example polyethylene glycol, proteins, saccharides (for example trehalose), amino acids, inorganic acids, metal chelators such as ethylenediamine tetraacetate (EDTA) disodium salt and admixtures thereof. Stabilizers are used in aqueous solutions at the appropriate concentration and pH. The pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8. For oral or sublingual the pH can be reduced. In formulating the peptides, anti-adsorption agent may be used. Other suitable excipients may typically include an antioxidant such as ascorbic acid.

The compositions of the invention may be formulated as controlled release preparations which may be achieved through the use of a polymer to complex or absorb the proteins. Appropriate polymers for controlled release formulations include, for example, polyester, polyamino-acids, polyvinyl polymers such as polyvinylpyrrolidone, polylactic acid (PLA), ethylenevinylacetate, ethylene vinylacetate copolymers, and cellulose derivatives such as methylcellulose. Alternatively, it is possible to entrap the peptides of the invention in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy methylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, macroemulsions, and nanoparticles.

In some embodiments, the compositions of the invention may be formulated for peroral or oral compositions in liquid solutions, emulsions, suspensions, and the like. The pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. Peptides that are orally administered need to be protected as to avoid digestion by the gastrointestinal system.

The peptides of the invention can be coated with enteric coating layer(s) as to protect them from digestion. Enteric coating layer(s) may be applied using standard coating techniques. The enteric coating materials may be dissolved or dispersed in organic or aqueous solvents and may include one or more of the following materials: methacrylic acid copolymers, shellac, hydroxypropylmethcellulose phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose trimellitate, carboxymethylethyl-cellulose, cellulose acetate phthalate or other suitable enteric coating polymer(s). The pH at which the enteric coat will dissolve can be controlled by the polymers, combination and ratio of selected polymers, and/or their side groups. For example, dissolution characteristics of the polymer film can be altered by the ratio of free carboxyl groups to ester groups. Enteric coating layers also contain pharmaceutically acceptable plasticizers such as triethyl citrate, dibutyl phthalate, triacetin, polyethylene glycols, polysorbates or other plasticizers. Additives such as dispersants, colorants, anti-adhering and anti-foaming agents may also be included.

In some embodiments, the preparations described herein are formulated for parenteral administration, e.g., by bolus injection or continuous infusion. In some embodiments, compositions are suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Methods and uses

The present invention provides according to some aspects a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a peptide of the invention and at least one pharmaceutically acceptable carrier, excipient or diluent. In some embodiments, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a peptide conjugate of the invention and at least one pharmaceutically acceptable carrier, excipient or diluent.

In some embodiments, a method of treating a subject with a cancer is provided. In additional embodiments, a method of treating a malignant solid tumor is provided. In additional embodiments, a method for inhibiting the growth of tumor cells is provided. In some embodiments, a method for eradicating tumor cells is provided. In some embodiments, a method for reducing viability of cancer cells is provided.

According to another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a peptide of the invention, for use in the treatment of cancer. According to another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a peptide conjugate of the invention, for use in the treatment of cancer.

According to an additional aspect, the present invention provides the use of a peptide of the invention, or a peptide conjugate of the invention, in the manufacture/preparation of a medicament for treating cancer. As referred to herein, the term "treating" is directed to ameliorating symptoms associated with a disease, and lessening the severity or cure the disease. In some embodiments, treating results in a reduction in tumor size. In additional embodiments, treating results in the decrease of tumor growth, decrease in tumor progression to a more advanced stage and/or decrease or inhibition of tumor metastasis. In some embodiments, treating cancer comprises preventing or reducing tumor metastasis. In some embodiments, metastases are reduced. In additional embodiments, metastases are prevented. In some embodiments, treating cancer comprises increasing the duration of survival of a subject having cancer. In some embodiments, treating cancer comprises increasing the progression-free survival of a subject having cancer. In some embodiments, treating cancer comprises preventing tumor recurrence.

Treatment according to the present invention may also encompass prophylactic treatment. Prophylactic treatment may include prevention of tumor development (e.g., in subjects at risk of developing a particular type of cancer) and also prevention of tumor recurrence following treatment. In some embodiments, the subject to be treated using the methods of the present invention has undergone cancer treatment and displays no symptoms or signs of disease. For example, the subject may have no detectable tumors concomitant with normal levels of the markers of the subject's particular disease. In some embodiments, the subject is a subject with minimal residual disease. According to some embodiments, treatment of cancer according to the present invention includes treatment of pre-cancerous conditions.

The term“therapeutically effective amount” as used herein refers to an amount of the peptide of the present invention that is sufficient to reduce, decrease, and/or inhibit a disease, disorder or condition in an individual.

As used herein, the term“cancer” encompasses precancerous conditions or lesions.

In some embodiments, the cancer is a solid tumor.

In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostatic cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is a brain tumor. In some particular embodiments, the cancer is glioblastoma.

In some embodiments, the cancer is selected from the group consisting of: adrenocortical carcinoma, anal cancer, bladder cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal, pineal tumors, hypothalamic glioma, breast cancer, carcinoid tumor, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing’s family of tumors (pnet), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, brain and spinal cord cancer, hypopharyngeal cancer, islet cell carcinoma, laryngeal cancer, leukemia, acute lymphoblastic leukemia, oral cavity cancer, liver cancer, lung cancer, lymphoma, small cell lymphoma, AIDS -related lymphoma, central nervous system (primary) lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, Merkel cell carcinoma, squamous cell carcinoma, basal cell carcinoma, multiple myeloma, plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, neuroblastoma glioma, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, exocrine pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, salivary gland cancer, Sezary syndrome, skin cancer, cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma, small intestine cancer, soft tissue sarcoma, testicular cancer, thymoma, malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor. Each possibility represents a separate embodiment of the invention.

In some embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer and melanoma. Each possibility represents a separate embodiment of the invention.

The subject to be treated according to the present invention is a mammal, typically a human.

The peptides of the invention may be administered by any suitable administration route, selected from oral, topical, transdermal or parenteral administration. According to some embodiments, the route of administration is via topical application selected from dermal, vaginal, rectal, inhalation, intranasal, ocular, transnasal, auricular, sublingual and buccal. According to some embodiments, the route of administration is via parenteral injection. In various embodiments, the step of administering is carried out by a parenteral route selected from the group consisting of intravenous, intramuscular, subcutaneous, intradermal, topical application to the skin, intraperitoneal, intraarterial, intracerebral, intracerebroventricular, intraosseous and intrathecal. For example, the peptides can be administered systemically, for example, by parenteral routes, such as, intraperitoneal (i.p.), intravenous (i.v.), subcutaneous (s.c.), or intramuscular (i.m.) routes. The peptides of the invention and/or any optional additional agent may be administered systemically, for example, by intranasal and transnasal administration. The peptides of the invention and/or any optional additional agent may be administered locally. For example, the peptides may be administered directly into a tumor, into an area from which a tumor or a portion thereof has been surgically resected and/or into an area surrounding the tumor.

Exemplary effective doses of the peptides of the invention are demonstrated hereinbelow in mice. A person of ordinary skill in the art will be able to determine an equivalent dose in human or other non-human mammals using known standard factors and calculations for converting doses.

According to some embodiments, the pharmaceutical composition is administered at least once a day. According to certain embodiments, the pharmaceutical composition is administered at least twice a week.

According to some embodiments, the pharmaceutical composition is administered every day for at least one week, alternatively the pharmaceutical composition is administered every day for at least one month, or further alternatively the pharmaceutical composition is administered every day until the tumor size is significantly reduced or the tumor is eradicated.

In some embodiments, where the pharmaceutical composition is used for preventing recurrence of cancer, the pharmaceutical composition may be administered regularly for prolonged periods of time, according to instructions from a clinician.

As used herein the term“about” in reference to a numerical value stated herein is to be understood as the stated value +/- 10%, more preferably +/-5%, even more preferably +/-1%, and still more preferably +/-0.1% from the specified value.

In some cases, it may be advantageous to administer a large loading dose followed by periodic (e.g., weekly) maintenance doses over the treatment period. The peptide can also be delivered by slow-release delivery systems, pumps, and other known delivery systems for continuous infusion. Dosing regimens may be varied to provide the desired circulating levels of a particular peptide based on its pharmacokinetics. Thus, doses are calculated so that the desired circulating level of a therapeutic agent is maintained.

Typically, the effective dose is determined by the activity of the peptide and the condition of the subject, as well as the body weight or surface area of the subject to be treated. The dose and the dosing regime are also determined by the existence, nature, and extent of any adverse side effects that accompany the administration of the peptide in the particular subject.

In some embodiments, recombinant cells such as stem cells obtained from the subject to be treated or from a donor may be genetically engineered to constitutively express and secret the peptide. The genetically modified cells may be administered to the subject for cancer treatment or prophylaxis.

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

EXAMPLE 1

Effect of IDQ12 on lung metastasis formation in mice inoculated with

4T1 mammary carcinoma cells

The peptide H-SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-OH, termed “IDO 12”, was synthesized by solid phase synthesis to over 98% purity. Female B ALB/c mice (9-10 weeks old) were i.v. injected (tail vein) with 4T1 mammary carcinoma cells. Each mouse was injected with 2x 10⁴ cells in 0.2 ml culture medium; medium was replaced with fresh medium one day prior to the injection. IDO 12 (dissolved in ImM acetic acid in water and appropriately diluted in saline) was s.c. administered on day 5 after tumor cell inoculation and thereafter 6 days a week, until the end of the experiment. The peptide was injected (0.1 ml) at doses of 0.01, 0.1 and lmg/kg. Controls received saline (vehicle) injections. Each dose group contained 9 mice and the control group contained 10 mice. Additional 4 mice remained naive (no cell injection, no treatment). Mice were sacrificed 15 days after tumor cell inoculation. Lungs were removed, weighed and quantified for number of tumor foci. In addition, part of the lung from each mouse was fixed in formalin and kept for 3 days in 70% ethyl alcohol for histological evaluation. Histological score was evaluated as a semi-quantitative score of the approximate amount of pulmonary tissue occupied by tumors in the section: Score 0: no metastases, Score 1: 1-25%, Score 2: 26-50%, Score 3: 51-75%, Score 4: 76 -100%.

All measured parameters, namely, lung weight, number of tumor foci per lung and histological evaluation, demonstrated significant anti-tumor activity of IDO 12 (Figures 1A-1D).

EXAMPLE 2

Effect of IDQ12 on tumor cells in vitro - MTT viability tests

Six tumor cell lines were tested: three of human origin (MCF-7 mammary carcinoma, MDA-MB-231 mammary carcinoma, LNCaP prostatic carcinoma) and three of mouse origin (D122 3LL Lewis lung carcinoma, B 16-F10 melanoma, 4T1 mammary carcinoma).

Cells were seeded in 96-well plate ( 1x 10⁴/ml) in a total volume of 100ul/well ( 1x 1O³ cells/well). After 24 hours medium was aspirated and the peptide (dissolved in 1mM acetic acid in water and appropriately diluted with culture medium) was added to the cells and the plate was incubated in a CO₂ incubator for 24 hours. Control cells received 0.02mM acetic acid in medium (the highest acetic acid concentration present in the highest peptide concentration namely, 10. mg/ml). At the end of incubation MTT viability test was performed. The results are summarized in Figures 2A-2F. Results are expressed as OD values after subtraction of the average OD value of wells that contained medium only, without cells. As can be seen from the figures, treatment with IDO12 at concentrations 0.1 mg/ml , 1 mg/ml and 10 mg/ml significantly reduced the number of viable cells at the end of the experiment compared to control. EXAMPLE 3

Effect of IDQ12 on triple negative human MDA-MB-231 mammary

carcinoma cells- Trypan blue exclusion

Human MDA-MB-231 mammary carcinoma cells were seeded in a 6-well plate (lxl0⁴/ml) in a total volume of 2ml/well (2x 10⁴ cells/well). After 24 hours the peptide (dissolved in ImM acetic acid in doubled distilled water, mixed well with Vortex for 2 minutes and further diluted in culture medium) was added (volume of 200 ml) to the cells. After incubation of 24 hours with the peptide, cells were detached from the well by trypsinization and viable cells were counted by trypan blue exclusion. Control cells were exposed to diluted acetic acid (0.02mM) diluted in culture medium. n=7 for control, n=4 for lng/ml, n=5 for the rest of peptide concentrations.

As can be seen in Figure 3, treatment with ID012 at concentrations 100ng/ml,1 mg/ml and 10 mg/ml significantly reduced the number of viable cells at the end of the experiment compared to control.

EXAMPLE 4

Apoptotic effect of IDQ12 tested by Annexin V/Propidium iodide assay

4T1 cells were seeded in a 6-well plate (5x 10⁵ cells/well containing 1.5 ml culture medium composed of DMEM medium, 10% FCS, 1% L-glutamine, 1% pen-strep, 1% sodium pyruvate). After overnight incubation ID012 (dissolved in 1mM acetic acid and further diluted in culture medium) was added at the indicated concentrations. Control cells were treated with the vehicle (ImM acetic acid in culture medium). At the end of each indicated incubation time the medium was removed, cells were washed with PBS and treated with trypsin for 5 minutes. Cell suspension was centrifuged and the resulting cell pellet was processed according to manufacturer (eBioscience, San Diego, Ca, USA) instructions. The results have shown an increased percentage of apoptotic cells in wells treated with ID012 compared to control wells (Figures 4A-4B).

EXAMPLE 5

Apoptotic effect of IDQ12 tested by single-stranded DNA (ssDNA) assay

MDA-MB-231 cells were seeded in a 96-well plate (7.5x10³ cells/well, 0.1 ml/well). After 24 hours cells were exposed to ID012 (dissolved in 1mM acetic acid and further diluted in culture medium) at the indicated concentrations (in triplicate) for 4 hours. Control cells (in triplicate) were exposed to the vehicle (ImM acetic acid diluted in culture medium). Cells were processed according to manufacturer (Millipore, Molsheim, France) instructions for ssDNA apoptosis. The results have shown increased apoptosis in wells treated with ID012 compared to control wells (Figure 5).

EXAMPLE 6

Effect of IDQ12 on blood counts in tumor-bearing mice

Female B ALB/c mice (9-10 weeks old) were injected (i.v.) with 4T1 mammary carcinoma cells (2x 10⁴ cells in 0.2 ml culture medium; medium was replaced with fresh medium one day prior to the injection). ID012 O. lmg/kg was s.c. administered on day 5 after tumor cell inoculation and thereafter 6 days a week, until the end of the experiment. Control group received injections (0.1ml) of the vehicle (saline). N=4 for each group. Mice were sacrificed 14 days after tumor cell inoculation. Blood was taken by heart puncture and analyzed by automatic blood cell analyzer.

No toxic effects on blood counts were observed in mice treated with IDO 12 at a dose that showed significant anti-tumoral activity (Figure 6) (unlike “classic” chemotherapy).

EXAMPLE 7

Effect of IDQ12 and derivatives thereof on human glioblastoma

U87 and U251 cells

The structure of N-(+)-Biotinyl-6-aminohexanoic acid (Biotin-Ahx) is as follows:

The peptide was synthesized using a standard biotinylation peptide synthesis procedure. The free carboxyl of Biotin-Ahx was conjugated to the amino terminus of the peptide.

Experimental procedure:

U87 and U251 cells were seeded in 96-well plate ( 1x 10⁴/ml) in a total volume of lOOul/well ( 1x 10³ cells/well). After 24 hours medium was aspirated and each peptide (dissolved in an appropriate formulation and diluted appropriately with saline) was added to the cells at the indicated concentrations and incubated in a CO₂ incubator for 24 hours. The volume of culture medium in the well was 0.1ml.“Control-solvent” wells received the equivalent solvent concentrations.“Control” wells did not receive any addition. At the end of incubation MTT viability test was performed. Each concentration was tested in duplicate, and the control-solvent and control in hexaplicate. The results are summarized in Figures 7A-7B. Results are expressed as OD value subtracted by the value of wells with medium but without cells.

As can be seen from the figures, IDO 12, IDO 12-sulfoxide and Biotin- Ahx-IDO 12 reduced cells’ viability at the end of the experiment compared to control, in a concentration-dependent manner.

EXAMPLE 8

Effect of IDQ12 and its derivative on human mammary MDA-MB-231 cells

IDO 12 and conjugate used in the assay:

B iotin- Ahx-IDO12:

Biotin- Ahx-SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-OH

(SEQ ID NO: 5)

Experimental procedure:

MBA-MD-231 cells were seeded in 96-well plate ( 1x 10⁴/ml) in a total volume of 100ul/well (1x 10³ cells/well). After 24 hours medium was aspirated and the peptides (dissolved in an appropriate formulation and diluted appropriately with saline) was added to the cells at the indicated concentrations and incubated in CO2 incubator for 24 hours. The volume of culture medium in the well was 0. lml. Control-solvent wells received the equivalent solvent concentrations. Control wells did not receive any addition. Saline received the equivalent volume of saline given with the peptides. At the end of incubation MTT viability test was performed. Each peptide concentration and controls were tested in tetraplicate. The results are summarized in Figure 8. Results are expressed as OD value subtracted by the value obtained with medium without cells.

As can be seen from Figure 8, IDO 12 and Biotin-Ahx-IDO12 reduced cells’ viability at the end of the experiment compared to control, in a concentration-dependent manner. Biotin-Ahx-IDO12 was more effective than IDO12 in reducing cells’ viability.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a peptide of 33-50 amino acids comprising the sequence:

SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1).

2. The method of claim 1, wherein the cancer is a solid tumor.

3. The method of claim 2, wherein the solid tumor is selected from the group consisting of mammary (breast) cancer, prostatic cancer, lung cancer, melanoma and brain tumor.

4. The method of claim 2, wherein the solid tumor is a brain tumor.

5. The method of claim 4, wherein the brain tumor is glioblastoma.

6. The method of any one of the preceding claims, wherein the peptide is 33-40 amino acids in length.

7. The method of any one of claims 1-5, wherein the peptide consists of the sequence:

SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1).

8. The method of any one of the preceding claims, wherein the peptide further comprises at least one modification selected from the group consisting of an amino- terminal modification and a carboxy-terminal modification.

9. The method of claim 8, wherein the amino terminal modification is an amino blocking group selected from the group consisting of an acyl and alkyl.

10. The method of claim 8, wherein the carboxy-terminal modification is a carboxyl blocking group selected from the group consisting of an amide, alcohol and an ester.

11. The method of any one of the preceding claims, wherein the peptide is represented by the following formula:

R₁ -SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-R₂,

wherein Ri designates a hydrogen of an unmodified amino terminal group or is an amino blocking group selected from the group consisting of an acyl and alkyl; and R₂ designates OH of an unmodified carboxy terminal group or is a carboxyl blocking group selected from the group consisting of an amide, alcohol and ester.

12. The method of claim 11, wherein the peptide is:

H- SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-OH a synthetic polymer, a carrier or targeting peptide, a carrier or targeting protein, and a sugar moiety.

14. The method of any one of claims 1-12, wherein the peptide is conjugated to a permeability-enhancing moiety.

15. The method of any one of the preceding claims, wherein the peptide is conjugated to a compound selected from the group consisting of biotin, a biotin derivative, and a biotin analog.

16. The method of claim 15, wherein the peptide is conjugated to a biotinyl moiety via the amino terminus of the peptide.

17. The method of claim 16, wherein the biotinyl moiety is biotinyl-6-aminohexanoic acid (Biotin-Ahx).

18. The method of claim 17, wherein the biotinyl-peptide conjugate is according to the following formula:

Biotin- AI1X-ARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-R2,

19. The method of claim 17, wherein the biotinyl-peptide conjugate is:

Biotin- Ahx-ARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-OH (SEQ ID NO: 5).

20. The method of any one of the preceding claims, wherein the administering is systemically administering.

21. A pharmaceutical composition comprising a peptide of 33-50 amino acids comprising the sequence: SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1), for use in the treatment of cancer.

22. The pharmaceutical composition of claim 21, wherein the cancer is a solid tumor.

23. The pharmaceutical composition of claim 22, wherein the solid tumor is selected from the group consisting of mammary (breast) cancer, prostatic cancer, lung cancer, melanoma and brain tumor.

24. The pharmaceutical composition of claim 23, wherein the brain tumor is glioblastoma.

25. The pharmaceutical composition of any one of claims 21-24, wherein the peptide is 33-40 amino acids in length.

26. The pharmaceutical composition of any one of claims 21-24, wherein the peptide consists of the sequence:

(SEQ ID NO: 1).

27. The pharmaceutical composition of any one of claims 21-26, wherein the peptide further comprises at least one modification selected from the group consisting of an amino-terminal modification and a carboxy-terminal modification.

28. The pharmaceutical composition of claim 27, wherein the amino terminal modification is an amino blocking group selected from the group consisting of an acyl and alky.

29. The pharmaceutical composition of claim 27, wherein the carboxy-terminal modification is a carboxyl blocking group selected from the group consisting of an amide, alcohol and an ester.

30. The pharmaceutical composition of any one of claims 21-29, wherein the peptide is represented by the following formula:

31. The pharmaceutical composition of claim 30, wherein the peptide is:

32. The pharmaceutical composition of any one of claims 21-31, wherein the peptide is conjugated to a moiety selected from the group consisting of a permeability enhancing moiety, a synthetic polymer, a carrier or targeting peptide, a carrier or targeting protein, and a sugar moiety.

33. The pharmaceutical composition of claim 32, wherein the peptide is conjugated to a permeability-enhancing moiety.

34. The pharmaceutical composition of any one of claims 21-33, wherein the peptide is conjugated to a compound selected from the group consisting of biotin, a biotin derivative, and a biotin analog.

35. The pharmaceutical composition of claim 34, wherein the peptide is conjugated to a bio tiny 1 moiety via the amino terminus of the peptide.

36. The pharmaceutical composition of claim 35, wherein the biotinyl moiety is biotinyl-6-aminohexanoic acid (Biotin- Ahx).

37. The pharmaceutical composition of claim 36, wherein the biotinyl-peptide conjugate is according to the following formula:

Biotin- Ahx- ARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP-R₂,

wherein R₂ designates OH of an unmodified carboxy terminal group or is a carboxyl blocking group selected from the group consisting of an amide, alcohol and ester.

38. The pharmaceutical composition of claim 37, wherein the biotinyl-peptide conjugate is:

(SEQ ID NO: 5).

39. The pharmaceutical composition of any one of claims 21-38, for use via systemic administration.

40. A conjugate comprising a peptide of 33-50 amino acids comprising the sequence S ARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP (SEQ ID NO: 1) and a bio tiny 1 moiety.

41. The conjugate of claim 40, wherein the biotinyl moiety is conjugated to the amino terminus of the peptide.

42. The conjugate of claim 40, wherein the biotinyl moiety is biotiny1-6-aminohexanoic acid (Biotin-Ahx).

43. The conjugate of any one of claims 40-42, wherein the peptide is 33-40 amino acid in length.

44. The conjugate of any one of claims 40-42, wherein the peptide consists of the sequence S ARENS QRFVFNRPFFMFIVDNNIFFFGKVNRP (SEQ ID NO: 1).

45. The conjugate of claim 40, wherein the conjugate is according to the following formula:

Biotin- Ahx-ARFNSQRFVFNRPFFMFIVDNNIFFFGKVNRP-R₂,

46. The conjugate of claim of claim 40, wherein the conjugate is:

Biotin- Ahx-ARFNSQRFVFNRPFFMFIVDNNIFFFGKVNRP-OH

(SEQ ID NO: 5).

47. A pharmaceutical composition comprising as an active ingredient a conjugate according to any one of claims 40-46 and a pharmaceutically acceptable carrier.

48. The pharmaceutical composition of claim 47, for use in the treatment of cancer.