WO2022262942A1

WO2022262942A1 - Anti-tumour peptides

Info

Publication number: WO2022262942A1
Application number: PCT/EP2021/066004
Authority: WO
Inventors: Thomas Regnier
Original assignee: Panvir Therapeutics
Priority date: 2021-06-15
Filing date: 2021-06-15
Publication date: 2022-12-22

Abstract

The present invention relates to novel peptides with improved biological property, which are analog to natural buforin peptide, useful again tumor development with an enhanced pro-necrotic, pro-apoptotic, pro-inflammatory and anti-metastatic activities. Compositions and methods disclosed can also be used in the protection of human or animals, including dog, from associated disease.

Description

ANTI-TUMOUR PEPTIDES

FIELD OF THE INVENTION

The present invention relates to novel peptides with improved biological property, which are analog to natural buforin peptide, useful again tumor development with an enhanced necrosis activity. Compositions and methods disclosed can also be used in the protection of human or animals, including dog, from associated disease.

BACKGROUND OF THE INVENTION

Peptides are most versatile tools with huge potential for the development of cancer diagnostics and therapies. A crucial advantage is their size: they are mid-way between full proteins such as antibodies and small molecular biomolecular mimics. Over the years, peptides have been evolved as promising therapeutic agents in the treatment of several diseases, including cancer.

In this regard, a new class of peptides has emerged as an anti-cancer agent and has been termed anti-cancer peptides (ACPs) (Dennison etal, 2006). According to Kurrikoff etal( 2019), ACPs have an intracellular activity in cancer cells.

From a structural point of view, most ACPs have either a-helical or b-sheet conformation but some extended structures have already been mentioned in the literature (Hoskin and Ramamoorthy, 2008; Rodrigues etal, 2009; Wang et al, 2009; Hammami and Fliss, 2010).

According to Gaspar et al (2013), they can be classified into two major groups related to cell targets. The first one contains peptides active against microbial and cancer cells while not being active against healthy mammalian cells, such as cecropins and magainins. The second group includes ACPs that act against all three types of cells: microbial, cancerous and healthy, such as human neutrophil defensins HNP-1 to 3 and the human LL-37 (Papo and Shai, 2005; Hoskin and Ramamoorthy, 2008; Drain etal, 2009; Gaspar etal, 2013).

ACPs are unique compounds with respect to the chemotherapeutic arsenal currently available for cancer treatment and display a variety of modes of action which in some types of cancer appear to co-exist. Natural ACPs, even with a high anticancer activity, have generally 30-40 amino acids in their sequence, which induces an increase of production costs (Felicio et al, 2017).

Properties such as short time-frame of interaction (which decreases the probability of resistance), low theoretical toxicity (which reduces side effects), mode of action, (relative) specificity, good solubility and biocompatibility, low immunogenicity, and finally, good tumor penetration, support ACPs as a future chemotherapeutic agent with high potential (Riedl etal, 2011; Hu etal, 2016; Felicio et al, 2017).

ACPs can exert their anticancer activities through several alternative pathways including cell membrane disruption/lysis/necrosis induction, apoptosis induction, DNA synthesis/replication inhibition, tumor growth and angiogenesis inhibition, essential cell protein targeting, membrane and hormonal receptors, mediated immunity, ROS generation and DNA damage (Gaspar et at, 2013). Finally, different ACPs can act by more than one single mechanism simultaneously (Gaspar et al, 2013).

Among various types of peptides, antimicrobial peptides (AMPs) are a diverse class of naturally occurring molecules. AMPs, also known as host defense peptides, constitute an evolutionarily conserved part of the innate immune defense system (Wang etal, 2013b).

They are short (typically 10 to 50 residues long) and generally positively charged peptides isolated from a wide range of animal, plant, and bacterial species (Mahlapuu etal, 2016; Wang et al 2013b). They display remarkable structural and functional diversity. Indeed, they are structurally diverse in both amino acid natures and secondary structures (a-helix, b-sheets, extended helix, and loop) (Deslouches and Di, 2017).

Besides the ability to kill directly a wide range of pathogens like bacteria, fungi, and viruses and to possess antitumor activities (Wang etal, 2013b), AMPs can act indirectly by modulating the host defense systems (Mahlapuu et al, 2016 ; Hancock et al, 2016). In this regard, some of these peptides are now known to stimulate the immune system while suppressing the inflammatory response (Mahlapuu et al, 2016; Haney and Hancock, 2013 ; Pfalzgraff et al, 2018), which make them especially interesting compounds for the development of novel therapeutics. They can affect epithelial and immune cells modulating, e.g., proliferation, angiogenesis, cytokine release, anti-endotoxin activities, and chemotaxis by binding to cellular receptors at low concentrations and activating signaling pathways (Koszatka et al , 2011; DeZoysa et al, 2009).

There are encouraging examples of AMPs already introduced into the market. Many AMPs have been tested in clinical trials (Fox, 2013; Mahlapuu etal, 2016), which give strong grounds for optimism for introduction of novel AMP-based compounds in several indication areas, especially in oncology.

Among these peptides, Buforin has been described as an effective non-lytic AMP family (Cardoso et al, 2019). The buforin family has complete sequence identity with the N-terminal region of the histone H2A that interacts directly with nucleic acids (Cho etal 2009; Cardoso et al 2019).

A 39-aa peptide, buforin I, was isolated from the stomach tissue of the Asian toad Bufo bufo gargarizan. Compared to other amphibian AMPs including magainin II, buforin I has shown much stronger antimicrobial activities in vitro against a broad spectrum of pathogens (Cho et al, 2009).

A more potent 21 -residue peptide, named buforin II, has been produced from buforin I with a Lys-C endoproteinase (Park et al, 1996). Initially, Buforin II showed antimicrobial activity against a wide range of microorganisms (Park etal, 1996).

Although buforin II bears a structure similar to those of other amphiphatic a-helical AMPs, its mechanism of antimicrobial action appears to differ from those of AMPs that function by membrane permeabilization (Cho et al, 2009).

Buforin II kills bacteria without cell lysis and has a strong affinity for DNA and RNA (Park etal, 2000). This peptide has a-helical-helix-propeller structure, which is amphipathic in hydrophobic environments. The N-terminal extended helix includes residues 5 to 10, and the C-terminal helix comprises residues 12 to 21. The helices are separated by a proline residue located at amino acid position 11 (Yin et al 1996; Park et al, 2000). This two-helix organization and singular mode of action make buforin II a remarkably attractive candidate for decrypting the role of each structural element in providing buforin II with its highly potent antimicrobial activity.

Unlike several other cationic peptides, buforin II does not exhibit cytotoxic activity against normal eukaryotic cells (Cho etal, 2009). These buforin peptides are interesting not only for their antimicrobial activities but also antitumoral properties.

Indeed, buforin lib, also called B2b, is a synthetic analogue of buforin II. Buforin lib is a cationic peptide with good thermal stability (Han et al, 2019). It contains a proline hinge between the two a-helices as well as a model a-helical sequence at the C-terminus (3xRLLR), selectively targets cancer cells (Lee etal, 2008; Cho etal, 2009). The remarkable selectivity of this peptide for cancer cells results widely from the inability of the peptide to penetrate healthy cell membranes.

Buforin lib crosses cancer cell membranes without damaging them and accumulates primarily in the nuclei. Once inside the cells, it induces mitochondria-dependent apoptosis (Lee et al 2008). The suggested mechanisms of action of buforin II against bacteria include DNA- and RNA-binding features after translocation across the lipid bilayer through the action of a proline hinge, without inducing cell lysis (Kobayashi etal, 2000; Xie etal, 2011; Elmore 2012; Cardoso et al, 2019), likely due to the similarity in sequence between buforin II and histone H2A’s N- terminus (Cho et al, 2009; Lopez-Perez et al, 2017). Moreover, Jang et al ( 2015) suggested that the endoplasmic reticulum stress pathway has a significant role in the buforin lib-induced apoptosis in human cervical cancer HeLa cells.

Compared to magainin II, buforin II is more efficient at translocating across the lipid bilayers, without causing lipid flip-flop, suggesting non-membranolytic mechanisms (Kobayashi et al, 2000; Cardoso et al, 2019). It is proposed that buforin II translocates the membranes by the formation of transient toroidal pores with highly short lifespan to act on intracellular targets (Cardoso et a/,2019).

Since buforin II was reported to bind nucleic acids in vitro, it has been hypothesized that this compound kills a microorganism by interacting with its nucleic acids after translocation across the cell membrane (Park et al, 1998 ; Cho et al, 2009). In that way, Uyterhoeven et al (2008) characterized the nucleic acid binding features of buforin II with molecular modeling and a fluorescent intercalator displacement assay. These researchers noticed that, in addition to non-specific electrostatic attractions between nucleic acids and a cationic peptide, specific basic sidechains (Arg 2 and Arg 20) of buforin II form interactions with DNA that are stronger than the nonspecific electrostatic ones. Moreover, disrupting buforin ll-DNA interactions by replacing basic residues of buforin II with alanine generally decreases the antimicrobial activity of buforin II (Cho et al, 2009). This observation supports the claim that buforin II kills bacteria via its interaction with nucleic acids, even though it does not prevent buforin II from having other as yet unidentified intracellular targets.

Buforin lib also displayed selective cytotoxicity against a large number of cancer cell lines by specifically targeting cancer cells via interaction with cell surface gangliosides. Buforin lib has also been shown to overcome multidrug resistance developed in cancer cells (Huang et at, 2014).

Potential antitumor applications of buforin peptides were reported. For instance, they can be used as potential therapeutic drug for prostate cancer (Han et al, 2019), liver cancer (Li and Xu, 2019), breast cancer (Han et al, 2013), solid p53 tumor (Cho et al, 2009).

W099/37664 discloses a peptide that has an altered secondary structure of buforin II. This peptide comprises a random coil (1-4 residue), extended helix (5-10 residue) and normal a- helix (11-21 residue) structures, starting from the N-terminus. This peptide showed a strong antimicrobial activity.

W099/48912 discloses a novel peptide which has a potent antimicrobial activity against a broad spectrum of microorganisms. More particularly, the present invention relates to an antimicrobial peptide, named parasin I, isolated from catfish with the scientific name of Parasilurus asotus and its uses.

US7528227 relates to peptide derivatives and analogs comprising the amino acid sequence of a fragment of mammalian histone H2A and to pharmaceutical compositions comprising same. The compositions of the invention are useful for treating inflammatory, autoimmune and degenerative diseases.

WO 2011/149173 discloses a prophylactic or therapeutic composition for cancer, and more particularly, to a prophylactic or therapeutic composition for cancer comprising a peptide which is represented by an amino acid sequence of the following Formula (I), a method for preventing or treating cancer comprising the step of administering the peptide to a subject, and use of the peptide in the preparation of the prophylactic or therapeutic composition for cancer. BRIEF SUMMARY OF THE INVENTION

It is an object to the present invention to provide novel and more potent biologically active peptides with improved biological property, which are analog to natural buforin peptide, more particularly with improved inflammatory and necrosis activities.

In a first aspect of the present invention, the present invention relates to an isolated or synthetic antitumor peptide having at least 90% sequence identity selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31: SEQ ID NO: 32: SEQ ID NO: 33: SEQ ID NO: 34: SEQ ID NO: 35 or fragment thereof.

In another aspect of the present invention, the present invention relates to an isolated or synthetic antitumor peptide having amino acid sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31: SEQ ID NO: 32: SEQ ID NO: 33: SEQ ID NO: 34: SEQ ID NO: 35 or fragment thereof.

In another aspect of the present invention, the present invention relates to an isolated or synthetic antitumor peptide having at least 90% sequence identity selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11 ; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31 : SEQ ID NO: 32: SEQ ID NO: 33: SEQ ID NO: 34: SEQ ID NO: 35 or fragment thereof and wherein said peptides having the ability to induces necrosis in cancer cells. In another aspect of the present invention, the present invention relates to an isolated or synthetic antitumor peptide having amino acid sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31: SEQ ID NO: 32: SEQ ID NO: 33: SEQ ID NO: 34: SEQ ID NO: 35 or fragment thereof and wherein said peptides having the ability to induces necrosis in cancer cells.

The present invention further relates to an isolated or synthetic antitumor peptide having at least 90% sequence identity selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

The present invention further relates to an isolated or synthetic antitumor peptide selected from the group consisting of an amino acid sequence SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

In another aspect of the present invention, the present invention relates to an isolated or synthetic antitumor peptide selected from the group consisting of an amino acid sequence SEQ ID NO:12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17 or SEQ ID NO: 18.and wherein said peptides having the ability to induces necrosis in cancer cells.

The present invention also relates to an isolated or synthetic peptide having at least 90% sequence identity for of SEQ ID NO: 17 or fragment thereof.

The present invention also relates to an isolated or synthetic peptide consisting of an amino acid sequence as set forth in SEQ ID NO: 17.

The present invention also relates to an isolated or synthetic peptide having at least 90% sequence identity for of SEQ ID NO: 17 or fragment thereof inducing necrosis in cancer cell.

The present invention also relates to an isolated or synthetic peptide having at least 90% sequence identity of SEQ ID NO: 17 or fragment thereof inducing inflammatory response in cancer cell. In another aspect of the present invention, the present invention relates to an isolated or synthetic antitumor peptide consisting of an amino acid sequence SEQ ID NO: 17. and wherein said peptides having the ability to induces necrosis in cancer cells.

More particularly, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 3.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 4.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 5.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 6.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 7.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 8.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 9.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 10.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 11.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 12.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 13.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 14. In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 15.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 16.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 17.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 18.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 19.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 20.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 21.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 22.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 23.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 24.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 25.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 26.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 27. In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 28.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 29. In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 30.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 31.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 32.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 33.

In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 34. In another embodiment, the present invention relates to an isolated or synthetic antitumor peptide according to the invention, consisting of an amino acid sequence of SEQ ID NO: 35.

In a preferred embodiment, the invention relates to a peptide comprising an amino acid sequence selected from the group consisting of:

or a pharmaceutically-acceptable salt thereof.

According to another object, the present invention relates to the use of a peptide according to the invention for the manufacture of an agent for inhibiting the growth of a cell.

Most particularly, according to another object, the present invention relates to the use of a peptide having amino acid sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID

NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20;

SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID

NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31: SEQ ID NO: 32:

SEQ ID NO: 33: SEQ ID NO: 34: SEQ ID NO: 35 for the manufacture of an agent for inhibiting the growth of a cell.

Most particularly, according to another object, the present invention relates to the use of a peptide consisting of SEQ ID NO: 12 for the manufacture of an agent for inhibiting the growth of a cell.

Most particularly, according to another object, the present invention relates to the use of a peptide consisting of SEQ ID NO: 14 for the manufacture of an agent for inhibiting the growth of a cell.

Most particularly, according to another object, the present invention relates to the use of a peptide consisting of SEQ ID NO: 16 for the manufacture of an agent for inhibiting the growth of a cell.

Most particularly, according to another object, the present invention relates to the use of a peptide consisting of SEQ ID NO: 17 for the manufacture of an agent for inhibiting the growth of a cell. Most particularly, according to another object, the present invention relates to the use of a peptide consisting of SEQ ID NO: 18 for the manufacture of an agent for inhibiting the growth of a cell.

According to another object, the present invention relates a peptide according to the invention for use as a pro-inflammatory agent.

Most particularly, according to another object, the present invention relates a peptide having amino acid sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID

NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 22;

SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID

NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31 : SEQ ID NO: 32: SEQ ID NO: 33:

SEQ ID NO: 34: SEQ ID NO: 35 for use as a pro-inflammatory agent.

Most particularly, according to another object, the present invention relates a peptide consisting of SEQ ID NO: 12 for use as a pro-inflammatory agent.

Most particularly, according to another object, the present invention relates a peptide consisting of SEQ ID NO: 14 for use as a pro-inflammatory agent.

Most particularly, according to another object, the present invention relates a peptide consisting of SEQ ID NO: 16 for use as a pro-inflammatory agent.

Most particularly, according to another object, the present invention relates a peptide consisting of SEQ ID NO: 17 for use as a pro-inflammatory agent.

Most particularly, according to another object, the present invention relates a peptide consisting of SEQ ID NO: 18 for use as a pro-inflammatory agent.

The present invention furthermore generally relates to peptide comprising an amino acid sequence according to the invention, the peptide having a pro-inflammatory activity.

Thus, another aspect of the present invention relates to a pharmaceutical composition comprising one or more of the antitumor peptides according to the invention and a pharmaceutically acceptable carrier or diluent. In another embodiment, the present invention relates to an isolated or synthetic peptide according to the invention which is in a pharmaceutically acceptable carrier. In another embodiment, wherein the pharmaceutically acceptable carrier is suitable for intravenous administration.

In a preferred embodiment the present invention relates to a pharmaceutical composition according to the invention for use as a medicament.

In another preferred the invention relates to a pharmaceutical composition according to the invention, for use in inhibiting tumor or metastasis in a subject.

In another preferred the invention relates to a pharmaceutical composition according to the invention, for use in inhibiting a tumor cell in a subject.

Another aspect of the present invention relates to a peptide or a pharmaceutical composition according to the invention, for use inhibiting a tumor cell in a subject, wherein inhibiting comprises inducing apoptosis of said tumor cell and/or necrosis of a tumor tissue comprising said tumor cell.

In another preferred the invention relates to a pharmaceutical composition according to the invention, for use as a cancer promoting cell death agent.

Thus, another aspect of the present invention relates to a peptide or a pharmaceutical composition according to the invention, for use in treating a tumor or cancer in a subject.

More particularly, the invention relates to a peptide or a pharmaceutical composition for use according to the invention, wherein the cancer is one selected from the group consisting of melanoma, colon cancer, lung cancer, renal cancer, breast cancer, brain cancer, head and neck cancer, gastric and intestinal cancer, prostate cancer, liver cancer and glioblastoma.

The present invention furthermore relates to a method of promoting cancer cell death comprising administering to a subject an effective amount of the pharmaceutical composition according to the invention.

In another preferred the invention relates to a method of the invention wherein the peptide inhibits migration and invasion of tumor cells. In a preferred embodiment the present invention relates to a method of inhibiting tumor growth comprising administering to a subject an effective amount of the pharmaceutical composition according to the invention.

The present invention furthermore relates to the use of the peptide according to the invention, or pharmaceutical composition according to the invention, for use in medicine.

In another preferred embodiment, the invention relates to the use of the peptides according to the invention, or pharmaceutical composition according to the invention, for use in promoting cancer cell death in a subject.

In another preferred embodiment, the invention relates to the use of the peptides according to the invention, or pharmaceutical composition according to the invention, in the manufacture of a medicament promoting cancer cell death in a subject.

In another preferred embodiment, the invention relates to peptides according to the invention or pharmaceutical composition for use according to the invention, or the use according to the invention, wherein administration to the subject comprises intravenous, intra-arterial, intra- tumoral, subcutaneous, topical, intraperitoneal, local, regional, systemic, or continual administration.

In another preferred embodiment, the invention relates to peptides according to the invention or pharmaceutical composition for use according to the invention, or the use according to the invention, wherein inhibiting comprises inducing, apoptosis of said tumor cell and/or necrosis of a tumor tissue comprising said tumor cell.

In another preferred embodiment, the invention relates to peptides according to the invention or pharmaceutical composition for use according to the invention, or the use according to the invention, wherein the subject is the recipient of a second anti-cancer therapy, such as surgery, chemotherapy, radiotherapy, hormonal therapy, toxin therapy, immunotherapy, and cryotherapy and, for example, the subject receives the second anticancer therapy prior to, after, or at the same time as, the administration of the peptides according to the invention or pharmaceutical composition according to the invention.

In another preferred embodiment, the invention relates to peptides according to the invention or pharmaceutical composition for use according to the invention, or the use according to the invention, wherein the peptide according to the invention is presented in a pharmacological formulation that includes both the peptide according to the invention and a chemotherapeutic agent, radiotherapeutic agent, immunotherapeutic agent, agent for hormone therapy, or agent for toxin therapy.

In another preferred embodiment, the invention relates to peptides according to the invention or pharmaceutical composition for use according to the invention, or the use according to the invention, wherein said peptide is s administered daily, preferably daily for 7 days, 2 weeks, 3 weeks, 4 weeks, one month, 6 weeks, 8 weeks, two months, 12 weeks, or 3 months.

The present invention relates to the use of the peptide according to the invention for the manufacture of a pharmaceutical composition for anticancer activities in a subject.

The present invention relates to the use of the peptide according to the invention for the manufacture of a pharmaceutical composition for inhibiting tumor or cancer growth in a subject.

The present invention relates to the use of the peptides according to the invention to manufacture a pharmaceutical composition for treating and/or preventing immunological disorders and/or cancer.

In a preferred embodiment, the use according to the invention wherein the peptides are in an effective amount for stimulating the immune response in a human being.

In a preferred embodiment, the use according to the invention wherein the peptides are in an effective amount for stimulating the immune response in an animal being.

In a preferred embodiment, the use according to the invention wherein the peptides are suitable to inhibit cancer metastasis.

DESCRIPTION OF THE FIGURES

FIG. 1A: Histological analysis of B2b analogues on tumor inflammatory infiltration.

FIG. 1 B: Effect of B2b analogues on tumor inflammatory infiltration and scoring calculation.

FIG. 2A: Effect of B2b analogues on tumor Lymphocyte infiltration and scoring calculation. FIG. 2B: Effect of B2b analogues on tumor Lymphocyte infiltration in percent.

FIG. 3A: Effect of B2b analogues on tumor lymphocyte necrosis and scoring calculation.

FIG. 3B: Effect of B2b analogues on tumor lymphocyte necrosis in percent.

FIG. 4A: Cell apoptosis measure by TUNEL immunostaining and scoring calculation.

FIG.4B: Representative TUNEL immunostaining is illustrated i for negative control (A) and PV206 mg/Kg - treated group (B), objective magnification X10.

FIG.5: Digital analysis on necrosis and lymphocyte tumour infiltration. Morphometric analysis was performed on whole slide X40 (A, B) or close up to the whole slide X40 (C, D).

FIG. 6: Effect of B2B analogues on tumour metastatic invasion.

DETAILED DESCRIPTION OF THE INVENTION

In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art.

As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "containing" can be used interchangeably. The expression "of any of claims XX- YY" (wherein XX and YY refer to claim numbers) is intended to provide a multiple dependent claim in the alternative form, and in some embodiments is interchangeable with the expression "as in any one of claims XX- YY."

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the pertinent art.

Whenever a range of values is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. As used herein, ranges specifically include the values provided as endpoint values of the range. For example, a range of 1 to 100 specifically includes the end point values of 1 and 100. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.

As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim element.

As used herein, "consisting of”, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of” and "consisting of” may be optionally replaced with either of the other two terms, thus describing alternative aspects of the scope of the subject matter.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

The following definitions are provided to clarify use of these terms in the context of embodiments of the invention.

When used herein, the term "amino acid" is intended to refer to any natural or unnatural amino acid, whether made naturally or synthetically, including any such in L- or D-configuration. The term can also encompass amino acid analog compounds used in peptidomimetics or in peptoids. The term can include a modified or unusual amino acid or a synthetic derivative of an amino acid, e.g. diamino butyric acid and diamino propionic acid and the like. In embodiments, peptides of the invention comprise amino acids linked together by peptide bonds. The peptides are in general in alpha helical conformation under hydrophobic conditions. Sequences are conventionally given from the amino terminus to the carboxyl terminus. Unless otherwise noted, the amino acids are L-amino acids. When all the amino acids are of L configuration, the peptide is said to be an L-enantiomer. When all the amino acids are of D- configuration, the peptide is said to be a D-enantiomer.

The term "stability" can refer to an ability to resist degradation, to persist in a given environment, and/or to maintain a particular structure. For example, a peptide property of stability can indicate resistance to proteolytic degradation and to maintain an alpha-helical structural conformation.

The following abbreviations are used herein: A, Ala, Alanine; M, Met, Methionine; C, Cys, Cysteine; D, Asp, Aspartic Acid; E, Glu, Glutamic Acid; F, Phe, Phenylalanine; G, Gly, Glycine; H, His, Histidine; I, lie, Isoleucine; K, Lys, Lysine; L, Leu, Leucine; N, Asn, Asparagine; P, Pro, Proline; Q, Glu, Glutamine; R, Arg, Arginine; S, Ser, Serine; T, Thr, Threonine; V, Val, Valine; W, Trp, Tryptophan; Y, Tyr, Tyrosine; RP-HPLC, reversed-phase high performance liquid chromatography; MIC, minimal inhibitory concentration; HC50 hemolytic concentration - 50; CD, circular dichroism spectroscopy; TFE, 2,2,2-trifluoroethanol; TFA, trifluoroacetic acid; RBC, red blood cells; hRBC, human red blood cells.

As used herein, the peptide and/or polypeptide of the invention encompasses derivatives or fragments thereof. According to the invention, the term "derivative thereof" has its general meaning in the art and corresponds to an amino acid sequence or a nucleic acid sequence having at least 90% sequence identity to the referred amino acid sequence or nucleic acid sequence respectively, particularly 95%, and preferably 99%. The term "percentage of identity between two amino acid sequences" or "percentage of identity between two nucleic sequences" refers to the percentage of identic nucleotides or amino acids between two compared sequences, said percentage being obtained with the best alignment of the whole sequence. The term "best alignment" means the alignment that permits to obtain the most elevated identity percentage. It can be realized by using various algorithms and methods well known in the art and computer programs based on said algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA, Genetics Computer Group, 575 Science Dr., Madison, Wl USA). Preferably, the BLAST algorithm is used.

A polypeptide of the invention may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making peptides and proteins are well known to those of skill in the art. A polypeptide of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. A polypeptide of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a MODEL 433A from APPLIED BIOSYSTEMS INC. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides.

The phrase "improved biological property" is meant to indicate that a test peptide exhibits pro- inflammatory activity, or improved pro-inflammatory activity and/or improved pro-necrotic activity, compared to the control peptide (e.g., buforin2b, buforin ...), when tested using the experimental conditions described herein or by any other artknown standard protocols.

“Therapeutically effective amount" as used herein, refers to an amount of formulation, composition, or reagent in a pharmaceutically acceptable carrier or a physiologically acceptable salt of an active compound that is of sufficient quantity to ameliorate the undesirable state of the patient, animal, material, or object so treated. "Ameliorate" refers to a lessening of the detrimental effect of the disease state or disorder, or reduction in contamination, in the receiver of the treatment.

"Pharmaceutical agent or drug" as used herein, refers to a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

"Pharmaceutically acceptable carrier" as used herein, refers to conventional pharmaceutical carriers useful in the methods disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, salts, amino acids, and pH buffering agents and the like, for example sodium or potassium chloride or phosphate, Tween, sodium acetate or sorbitan monolaurate.

The peptides “PV(s)” of the invention exhibit pro-inflammatory activity and/or pro-necrotic activity by themselves or when covalently conjugated or otherwise coupled or associated with another molecule, e.g., polyethylene glycol or a carrier protein such as bovine serum albumin, so long as the peptides are positioned such that they can come into contact with a cell or unit of the target microorganism. These peptides may be modified by methods known in the art provided that the pro-inflammatory and/or pro-necrotic activities are not destroyed or substantially compromised.

In embodiments of the invention, peptide compositions may be isolated or purified. In embodiments, a peptide is synthetic and can be produced by peptide synthesis techniques or by recombinant expression technology as understood in the art.

As used herein, the term "purified" can be understood in embodiments to refer to a state of enrichment or selective enrichment of a particular component relative to an earlier state of crudeness or constituency of another component.

In embodiments, the term can be considered to correspond to a material that is at least partially purified as opposed to a state of absolute purity. For example, in a particular embodiment, a peptide composition can be considered purified even if the composition does not reach a level of one hundred percent purity with respect to other components in the composition.

When employed as pharmaceuticals, especially as antimicrobial agents administered to mammals, the peptides of the invention are administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. Such pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one peptide of the invention.

The pharmaceutical compositions of the present invention contain, as the active ingredient, one or more of the peptides of the invention, associated with pharmaceutically acceptable formulations. In making the compositions of this invention, the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container. An excipient is usually an inert substance that forms a vehicle for a drug. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 30% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. In preparing a formulation, it may be necessary to mill active peptide of the invention to provide the appropriate particle size prior to combining with the other ingredients. If the peptide is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the compound(s) is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, gum Arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulating composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulating compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and the like, each containing a predetermined amount of a compound or compounds of the present invention as an active ingredient. A compound or compounds of the present invention may also be administered as bolus, electuary or paste. In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agaragar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monosterate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may be employed as fillers in soft and hardfilled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form.

The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

Liquid dosage forms for oral administration of the peptide of the invention include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more peptide of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of peptide of the invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically- acceptable carrier, and with any buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active ingredient, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active ingredient, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of peptide of the invention to the body. Such dosage forms can be made by dissolving, dispersing or otherwise incorporating one or more peptide of the invention in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

Pharmaceutical formulations include those suitable for administration by inhalation or insufflation or for nasal or intraocular administration. For administration to the upper (nasal) or lower respiratory tract by inhalation, the peptides of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of one or more peptide of the invention and a suitable powder base, such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler.

For intranasal administration, peptide of the invention may be administered by means of nose drops or a liquid spray, such as by means of a plastic bottle atomizer or metered-dose inhaler.

Drops, such as eye drops or nose drops, may be formulated with an aqueous or nonaqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered by means of a simple eye dropper-capped bottle or by means of a plastic bottle adapted to deliver liquid contents dropwise by means of a specially shaped closure.

Pharmaceutical compositions of this invention suitable for parenteral administrations comprise one or more peptide of the invention in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions.

In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

Suitable alkalinizing agents include alkali metal salts and alkaline earth metal salts. The alkali metal salts include sodium carbonate, sodium hydroxide, sodium silicate, disodium hydrogen orthophosphate, sodium aluminate, and other suitable alkali metal salts or mixtures thereof. Suitable alkaline metal salts include calcium carbonate, calcium hydroxide, magnesium carbonate, magnesium hydroxide, magnesium silicate, magnesium aluminate, aluminum magnesium hydroxide or mixture thereof. More particularly, calcium carbonate, potassium bicarbonate, calcium hydroxide, and/or sodium carbonate may be used as alkalinizing agents to obtain a formulation pH within the desired pH range of pH 8 to pH 13. The concentration of the alkalinizing agent is selected to obtain the desired pH, varying from about 0.1% to about 30%, by weight, and more preferably from about 12.5% to about 30%, by weight, of the total weight of the dosage formulation.

Suitable antioxidants may be selected from amongst one or more pharmaceutically acceptable antioxidants known in the art. Examples of pharmaceutically acceptable antioxidants include butylated hydroxyanisole (BHA), sodium ascorbate, butylated hydroxytoluene (BHT), sodium sulfite, citric acid, malic acid and ascorbic acid. The antioxidants may be present in the dosage formulations of the present invention at a concentration between about 0.001% to about 5%, by weight, of the dosage formulation.

Suitable chelating agents may be selected from amongst one or more chelating agents known in the art. Examples of suitable chelating agents include disodium edetate (EDTA), edetic acid, citric acid and combinations thereof. The chelating agents may be present in a concentration between about 0.001% and about 5%, by weight, of the dosage formulation.

As previously discussed, antimicrobial peptides (AMPs) are ubiquitous components of the innate defense system of many organisms. These natural peptides can selectively kill bacteria, viruses, fungi, or cancer cells. Among them, histone- 2A derived peptides such as Buforins are promising molecules that possess published antibacterial and anticancer activities. Even if several research labs published some mechanistic elements in last years, the exact molecular mechanism of their activity remains poorly understood.

Buforin II (natural, B2) and Buforin lib (B2b) are peptides that contains a proline hinge between two a-helices. B2b is a synthetic analogue that possess a model a-helical sequence at the C- terminus (3xRLLR).

Example 1. Design and preparation of B2b’s analogues.

Different strategies have been pursued to enhance the therapeutic potential of the peptide family such as re-incorporation of the 16 to 18 RVH fragment of natural Buforin II (B2b Vs PV16; PV17 vs PV1), modulation of the global charge of the peptides (BBR2 Vs B2b; PV1 vs PV2), changing the flexibility of the proline hinge by adding a supplementary proline end by modulating the hydrophobicity of the AA at position 6 and 9, changing the CPP (RLLR)3 by other known CPP fragment (KLAKLAK)2.

Table 1. Sequences of reference peptides and peptides of the invention.

All peptides were synthesized at 200 pmole scale using Fmoc-SPPS on a Symphony X instrument (Protein Technologies, Inc., USA).

The standard deprotection-coupling cycle for each residue consisted of six steps:

1. Wash with lO mL of DMF (3 x 30 s);

2. Deprotect Fmoc group with 10 mL of 20% piperidine in DMF (3 x 3 min);

3. Wash with 5 mL of DMF (3 x 30 s);

4. Couple Fmoc-AA (2 x 60 min);

4a. 10 mL of Fmoc-AA dissolved at 200 mM in DMF;

4b. 4 mL of Oxyma pure dissolved at 500 mM in DMF;

4c. 2 mL of DIC dissolved at 1 M in DMF;

5. Capping with 10 mL of 10% Ac20 in DMF (7 min) and finally

6. Wash with 10 mL of DMF (3 x 30 s).

Upon completion of the synthesis, peptide resins were washed 3x with DMF and 3x with DCM. Peptide resins were then cleaved with one of three TFA cleavage cocktails (per 200 pmole scale synthesis): Cocktail: 36 mL TFA, 1000 pL TIS, 1000 mg Phenol, 2000 pL water

After 3 h of TFA cleavage, the cleavage solution was filtered from the resin, and peptides were precipitated by addition to 100 mL of ice-cold ether. After >1 h at -20 °C, ether solutions were centrifuged at 3500 RCF, and the supernatants were decanted. Pellets were washed twice more with ether, and then dried for >3 h in vacuum desiccator prior to dissolution, analytical characterization, and purification.

Purifications of crude peptides were performed with a preparative reversed phase HPLC (Waters Delta Prep 4000) system using a reversed phase column (Vydac Denali prep C-18, 10 pm, 120 A, 50 ^c 300 mm) and an appropriate gradient of increasing concentration of buffer B in buffer A (flow rate of 80 mL/min). The fractions containing the purified target peptide were identified by UV measurement (Waters 2489 UV/Visible detector) at 214 nm and selected fractions were then combined and lyophilized.

The peptides were analyzed by UPLC with an Acquity HCIass equipment and ESI-MS mass spectrometry. The instruments were equipped with BEH C18 (WATERS), 150*2.1mm (150 ^c 2.1 mm) (flow rate: 0.6 mL/min). Solvents A and B were 0.1% T13FA in water and 0.1% TFA in acetonitrile. Elementary analyses were performed on a Flash 2000 THERMOFISHER SCIENTIFIC equipment.

Standards Fmoc-AA reagents, Oxyma Pure and DIC, were purchased from Iris Biotech. DMF, DCM, diethyl ether, and HPLC-grade acetonitrile were purchased from Aldrich.

EXAMPLE 2. Evaluation and optimisation of the anti-tumour activity of B2b’s analogues on human and animal cells

Biological materials

Human cancer cell Lines

SK-Mel-28, Melanoma (ATCC® HTB-72)

HCT116, colorectal carcinoma cells (ECACC® 91091005)

A549, lung carcinoma cells (ATCC® CCL-185™)

MCF-7, breast adenocarcinoma cells (ATCC® HTB-22™)

SH-SY5Y, neuroblastoma cells (ECACC® 94030304)

Hep G2, hepatocellular carcinoma cells (ATCC® HB-8065™)

ACHN, renal cell adenocarcinoma (ATCC® CRL-1611 ™)

A375, melanoma cells (ATCC® CRL-1619™)

U87MG, glioblastoma cells (ECACC™ 89081402)

BxPC-3, pancreatic adenocarcinoma cells (ATCC® CRL-1687™)

LNCaP, prostate carcinoma cells (ATCC® CRL-1740™).

Human primary cells

Primary human melanocytes cells (Clinisciences™, #NB-11-0036). Animal cells

Canine osteosarcoma cells D-17 (ATCC® CCL-183)

Murine pancreatic cancer cells 266-6 (ATCC® CRL-2151).

Cell culture conditions

SK-Mel-28 and D-17 cells were maintained in Gibco™ MEM medium (ThermoFisher Scientific, #11095080) supplemented with 10% Fetal Calf Serum (FCS) and 1% antibiotics (Penicillin 100U/ml_ - Streptomycin 100 pg/mL).

HCT116, A 549, MCF-7, SH-SY5Y, Hep G2, ACHN, A375, U87MG, BxPC-3, LNCaP, were cultured in RPMI 1640-ATCC formulated (Gibco™) supplemented with 10% Fetal Bovine Serum (FBS, Gibco™) and 1% antibiotics;

Primary human melanocytes cells were maintained in Complete Classic Cell Culture Medium Kit with Serum, with CultureBoost™ and without Phenol Red (Clinisciences™, # NB-11-0046).

266-6 were maintained in Gibco™ DMEM high glucose, GlutaMAX medium (ThermoFisher Scientific, #10566016) supplemented with 10% FCS and 1% antibiotics.

Cells were allowed to growth in cell incubator at 37°C and 5% CO2. At 70-90% confluency, cells were split, and cell viability is assessed using the automated cell counter Nucleocounter® NC-200™ (ChemoMetec).

Cell viability assay

Evaluation and optimisation of the peptides on human and animal cancer cell lines, as well as on human primary cells were performed using a cell viability assay (MTS), a colorimetric assay performed in 96-well microplates. The test was performed as previously described in the literature (Buttke, T.M. et al., 1993. J. Immunol. Methods 157, 233).

The assay consists in decomposition of a yellow 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt to the water insoluble purple dye formazan by the enzyme succinate-tetrazolium reductase present in the mitochondria. MTS reduction occurs only in living cells. Cellular concentration was adjusted for each cell line, from 10 000 up to 50 000 cells per well. Cell culture suspension was prepared and 100 pL was applied per well to a 96-well plate. Cells were incubated for 24 h at 37°C in 5% CO2. Medium was removed and replaced with fresh complete medium. Cells were incubated in duplicate conditions with tested peptides at several concentrations or medium alone for additional 24 hours. The medium is ultimately removed and replaced with 100mI_ of complete medium. 20 mI_ of working solution of CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega™ CAT# G3581) is added and after 2 h incubation at 37°C in 5% CO2, the plate is briefly shaken, and optic density (OD) is red at 490nm (reference filter 630nm) using TECAN Sunrise™ reader or Ensight™ plate reader system (Perkin Elmer®). Data acquisition is performed using the Kaleido™ software (version 3.0, Perkin Elmer®).

Each OD490 sample was subtracted to OD490 blank (no peptide) and also subtracted to respective OD well (optical background variation from plastic at 690 nm) before calculation of the cell viability as defined by the formula below:

Cell viability = OD490 sample - OD490 blank - OD₆9o well

In vitro activities on cellular viability of SK-Mel-28 human melanoma cancer cell, canine osteosarcoma cells D-17, and murine pancreatic cancer cells 266-6 are summarized in Table 2. IC50 values, expressed in mM, correspond to peptide concentration which induces 50% of reduction of the number of viable cells, with respect to control cells incubated with medium only. Each experiment represents the average value of at least two independent experiments conducted in duplicate. To calculate IC50, values were transformed into log values, then normalized for each condition and IC50 were eventually calculated using non-linear regression (using GraphPad Prism 5.0) and best fit values are determined according to a sigmoidal curve (X is log of concentration).

Table 2. Inhibitory effect of B2b analogues on cell viability.

Based on the above results, the more potent B2b analogues were selected for evaluation on primary melanocytes cell viability using the same MTS assay and conditions of calculation; Results obtained on both SK-Mel-28 cells and human primary melanocytes, are presented in Table 3. Each experiment represents the average value of at least two independent experiments conducted in duplicates.

Table 3. Comparison of activities on melanoma cancer cells and primary melanocytes. All peptides showed a 2 to 3 fold higher activity on cancer cells as compared to primary cells. The best results were obtained with PV20, which was selected as a lead compound for further experiments, including in vitro testing on a panel of human cancer cell lines of different origins. B2b was used as comparator in the same experiments (see Table 4).

Table 4. Comparison of activities on human and animal cancer cells

As observed with melanoma cells, in most cases PV20 was shown more active than B2b on human and animal cancer cell lines, with IC50 activities ranging from 3,5 to 27,5 mM. Example 3. Evaluation of B2b analogues on cellular cytotoxicity.

LDH assay

The LDH (Lactate Dehydrogenase) enzymatic activity is determined using a two-step assay: In the first step NAD+ is reduced to NADH/H+ by the LDH-catalysed conversion of lactate to pyruvate. In the second step, the catalyst (diaphorase) transfers H/H+ from NADH/H+ to the tetrazolium salt INT which is reduced to formazan. An increase in the amount of dead or plasma membrane-damaged cells results in an increase of the LDH enzyme activity in the culture supernatant. This increase in the amount of enzyme activity in the supernatant directly correlates to the amount of formazan formed during a limited time period. There-fore, the amount of color formed in assay is proportional to the number of lysed cells.

SK-Mel-28 cell suspension was prepared and 100 mI_ (5x10³ cells) was applied per well to a 96-well plate. Cells were incubated for 24 h at 37°C in 5% CO2. Medium was removed and replaced with fresh complete medium. Cells were incubated with peptides at serial concentrations for additional 24 hours.

Using Cytotoxicity detection kit™ (Sigma CAT# 11644793001), 100mI_ reaction mixture (freshly prepared) was added to each well. Plates are incubated up to 30 min at room temperature, the plate is briefly shaken, and absorbance is red at 490 nm (reference filter 630 nm) using TECAN Sunrise™ reader.

EC50 values, expressed in mM, correspond to peptide concentration which induces 50% of cytotoxicity, with respect to control cells incubated with medium only. Each experiment represents the average value of at least two independent experiments conducted in duplicate. T o calculate EC50, values were transformed into log values, then normalized for each condition. EC50 were eventually calculated using non-linear regression (using GraphPad Prism 5.0) and best fit values are determined according to a sigmoidal (X is log of concentration).

Table 5. Comparison of cytotoxicity of B2b analogues on melanoma cancer and primary cells.

The three B2b analogues were shown more active than B2b for inducing cytotoxicity of human melanoma cells, with EC50 closed to 1 mM. In addition, as observed in viability assays, IC50 for B2b analogues were higher against melanoma cells vs primary melanocytes. Example 4. Evaluation of the anticancer activity on SK-Mel-28 tumor derived graft on chorio allantoic membrane.

The study consists in evaluating and comparing the in-ovo efficacy of PV20 and B2b on human cancer cell line SK-MEL-28 tumor derived using chorio-allantoic membrane (CAM) assay. Experimental procedures

Preparation of chicken embryos

Fertilized White Leghorn eggs are incubated at 37.5°C with 50% relative humidity for 9 days. At E9, the CAM is dropped down by drilling a small hole through the eggshell into the air sac, and a 1 cm² window is cut in the eggshell above the CAM. At least 20 eggs (depending on embryo surviving rate after 9 days of development) are grafted for each group.

Amplification and grafting of tumor Cells

SK-MEL-28 cells cultivated in complete RPMI medium are detached on day E9, washed and re-suspended in graft medium. An inoculum of 1.10⁶ cells is added onto the CAM of each egg (E9) and then eggs are randomized into groups. T reatments

Before the first treatment, viability of each egg is checked, and surviving eggs are randomized in groups. All eggs of a group are treated with a volume of 100 pL of the working solution of freshly prepared peptide, resuspended in deionized H2O.

Treatments with peptides were performed at day E11, E13, E15 and E17. Quantitative evaluation of tumor growth

On day E18, the upper portion of the CAM (with tumor) is removed, washed by PBS buffer, and then directly transferred in 4% Paraformaldehyde (PFA). After 48 hours fixation, tumors are carefully cut away from normal CAM tissue and weighed.

Quantitative evaluation of metastatic invasion On day E18, a 1 cm2 portion of the lower CAM is collected to evaluate the number of metastatic cells in 8 samples per group (n=8). Genomic DNA is extracted from the CAM and analysed by qPCR with specific primers for Human Alu sequences.

Calculation of Cq for each sample, mean Cq and relative amounts of metastases for each group are directly managed by the Bio-Rad® CFX Maestro software.

According to the Real-Time PCR Data Markup Language (RDML) data standard (http://www.rdml.org), Cq is defined as the cycle at which the curvature of the amplification curve is maximal (fractional PCR cycles). Cq is taken in the exponential phase where the qPCR curve is linear.

Guantitative Evaluation of Embryonic Tolerability

Embryonic viability or any visible abnormality is checked daily. The number of dead embryos is also counted on E18, to evaluate treatment-induced embryo toxicity.

Statistical analysis and significance

For all quantitative data, a one-way ANOVA (with post-tests between each couple of groups) is done with the specialized computer software Prism® (GraphPad Software). Statistical difference between groups is made visible on graphs by the presence of stars with the following meaning:

- No stars: No statistical different (p value > 0.05);

- One star (*): 0.05 ³ p value > 0.01 ;

- Two stars (**): 0.01 ³ p value > 0.001 ;

- Three stars (***): 0.001 ³ p value.

For Histological analysis (Ki67/TUNEL and morphometry), an unpaired t-test is performed with the specialized computer software Prism® (GraphPad Software).

Histological analysis

Organ / tissue collection and fixation. Organs (tumors) of 25 chicken embryos (5 per group) were harvested, fixed in 4% PFA for 48 hours and then trimmed and put in embedding cassettes. One cassette was prepared per animal. Slide preparation. Paraffin blocks were sectioned at approximately 4 microns thickness. The sections were put on glass slides and stained with Hematoxylin & Eosin (H&E) using standard procedure.

Light microscopy photography. Pictures were taken using Olympus microscope (BX60) at objective magnifications of X110, and microscope's Camera (Olympus DP73).

Digital morphometry: A quantitative analysis of necrosis and cellular inflammatory infiltration of lymphocytes was performed, using a computerized image analysis and the MATLAB software, as follows:

Necrosis - Segmentation of pink areas was performed by setting threshold for the red channel of the RGB color map and for the different channels of the Lab color map. The percent of this area out of the whole tissue area was calculated.

Lymphocytes (%) - Segmentation was performed by setting threshold for the gray color map and for the eosin channel of the Hematoxylin and Eosin (H&E) -Dab color map, and by subtracting large areas with dominant blue values in the RGB color map. The percent of this area out of the whole tissue area was calculated.

Histological evaluation of pathological changes. Microscopically 10 non-overlapping fields (HPF) from each H&E stained section of the tumor were evaluated and graded by a semi- quantitative scoring system for the presence of pathological changes as following :

Scoring for inflammatory infiltration within or at the periphery of the nodule :

- score 0 : Scant or absent inflammatory cells

- score 1 : Inflammatory cells obviously present but markedly less than other cells

- score 2 : Inflammatory cells roughly equal to tumor cells

- score 3 : Predominantly inflammatory cells.

Scoring for lymphocytes infiltration within the tumor :

- score 0: absence of lymphocytes in the tumor

- score 1 : < 5 lymphocytes per HPF

- score 2: >5 and < 20 lymphocytes per HPF

- score 3: > 20 lymphocytes per HPF.

Scoring for Mitotic index was determined by counting the number of mitoses per 10 non overlapping high-power fields (x400): - score 0: no mitotic figures

- score 1 : 1-2 mitotic figures per HPF

- score 2: 2-3 mitotic figures per HPF

- score 3: =>3mitotic figures per HPF.

Scoring for Tumor Necrosis:

- score 0: No necrosis

- score 1 : Small foci of necrosis or widespread single cell necrosis that required careful perusal of the section

- score 2: Obvious presence of necrosis, but in <50% of the field

- score 3: Necrosis in >50% < 75% of the field

- score 4: Necrosis in >75% < 90% of the field

- score 5: Necrosis in 90% of the field.

Scoring for Tumor Matrix was scored according to the ratio between matrix and neoplastic cells:

- score 1 : Relatively less matrix than cells

- score 2: Equal amount of matrix and cells

- score 3: Relatively more matrix than cells.

Scoring for Nuclear pleomorphism (NP) was examined in 10 non-overlapping x400 fields:

- score 0: All nuclei identical.

- score 1 : < 25% NP

- score 2: 25-50% NP

- score 3: 50-75% NP

- score 4: > 75% NP.

TUNEL and Ki-67 staining and scoring :

Immunohistochemical staining was performed on 4pm FFPE sections using the Leica Bond max system (Leica Biosystems Newcastle Ltd, UK). Slides were baked for 30 min at 60° C, dewaxed and pretreated for 20 minutes with epitope-retrieval solution (ER2, Leica Biosystems Newcastle) followed by incubation with primary antibody for 30 minutes (Abeam ab16667 1:200). Detection was performed using the Leica Bond Polymer Refine HRP kit. TUNEL staining was manually performed using the ApopTag Peroxidase in Situ Apoptosis Detection Kit (Merck-Millipore cat. S7100) according to the manufacturer instructions. All slides were counter-stained with Hematoxylin.

Results and analysis.

Quantitative evaluation of embryonic tolerability: no effects of B2b analogues were observed on the number of dead and surviving embryos at the end of the study, nor on the overall survival rate for all groups (n = 13 to 20 per group, data not shown).

Quantitative evaluation of B2b analogues on tumor growth : no statistical differences (n = 13 to 20 in each group) were observed between animal treated with either B2b or B2b analogues and negative control, with mean tumor weight ranging from 147 to 198 mg (data not shown). The histological evaluation of the tumors in the H&E stained sections showed the morphology in the experimental groups treated with increasing doses of PV20 or B2b compared to the control one. Calculation of scoring indexes in each group, according to the procedure section, showed that mitotix indexes, as well as nuclear polymorphism and presence of multi nucleated giant cells, were identical in all groups (see Table 6).

Table 6. Histological analysis of B2b analogues treated tumor grafted The scoring for tumor matrix was calculated according to the ratio between matrix and neoplastic cells in each group (n=5). Tumor matrix area was shown slightly higher than the cells area in PV20-treated animals vs control or B2b-treated groups (Table 6).

In general, all experimental groups showed an inflammatory reaction characterized by infiltration of mono-nuclear cells, edema, and necrosis. The necrosis that was observed in some of the treated samples in the center of the tumor is considered as the natural course of the tumor development. In affected tumors, the necrosis was becoming more peripheral and contained a larger area of the neoplastic cells. Furthermore, there was also loss of tissue in these necrotic areas and many infiltrating heterophils. The number of inflammatory cells was correlated to the grade of necrosis. The scoring indexes in all groups, calculated for inflammation, infiltrating lymphocytes or necrosis, is described in the procedure section.

Figure 1A represent objective magnification X10, H&E staining for tumors at the end of the study, according to the following treatment: negative control (A); B2b 6 mg/Kg (B); PV20 1 mg/Kg (C); PV20 3 mg/Kg (D); PV20 6 mg/Kg (E); A clear and significant increase of tumor inflammatory infiltration is observed in PV20 but not with B2b-treated samples, as compared to negative control.

Scoring for lymphocyte infiltration within or at the periphery of the nodule for each experimental group is illustrated in Figure 2A, which shows significant increases in animals treated with PV20 at 3 or 6 mg/Kg vs control group;

Digital quantitative analysis confirms significant effect (p-value = 0,017) in animals treated with the highest dose of PV20 versus control group (Figure 2B).

Necrosis mean score was highest in the group treated with the high dose of PV20 as compared to B2b-treated or negative control groups (Figure 3A).

Necrosis digital quantification, as detailed in the experimental section, shows a significant (p- value = 0,041) increase of the percent of necrosis area in PV20-treated sample vs control (Figure 3B).

Cell apoptosis was measured by TUNEL immunostaining and scoring calculation. Figure 4A shows a highly significant (p-value = 0,0022) increase in the number of apoptotic cells in samples treated with PV20 vs control. Representative TUNEL immunostaining is illustrated in Figure 4B for negative control (A) and PV206 mg/Kg - treated group (B), objective magnification X10.

Digital analysis on necrosis and lymphocyte tumor infiltration is illustrated in Figure 5.

Morphometric analysis was performed on whole slide X40 (A, B) or close up to the whole slide X40 (C, D). H&E Segmentation of necrosis in the tumor is identified by larger dark areas in 6 mg/Kg PV20-treated samples (B) as compared to control group (A), and localised at the periphery; Segmentation of lymphocytes in the tumor is also more prominent in PV20-treated group (D) vs control group (C).

Many proliferative cells were identified using Ki-67 immunostaining on tumors at the end of the study, but no significant effect was observed following treatment with 6 mg/Kg PV20 vs control group (data not shown).

Evaluation of metastatic invasion measured by qPCR for Alu sequences in the lower CAM showed a strong decrease of relative quantity of metastasis following treatment with either B2b or PV20 (n=8) vs negative control group (n=7) (Figure 6).

Statistical analysis performed using a t-test indicated p-values = 0,0010 for B2b (6 mg/Kg) and 0,0042 for PV20 (1 mg/Kg) when compared to control group.

All together, these results provide strong evidence for in vitro and in ovo functional effects of new B2B analogues for cancer applications. This includes strong effects on tumor cell death (necrosis and apoptosis) as well as recruitment of inflammatory cells and antimetastatic effects.

SEQUENCE LISTING <110> Panvir Therapeutics

<120> BUFORIN IIB ANALOGUES AND THEIR USE AS A NECROSIS AGENT IS TUMOR CELLS

<130> PANB001WO

<160> 35

<170> BiSSAP 1.3.6 <210> 1 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> Buforin 2 <400> 1

Thr Arg Ser Ser Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Val His 1 5 10 15

Arg Leu Leu Arg Lys 20

<210> 2 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> Buforin 2b <400> 2

Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 3 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> B3a sequence <400> 3

Arg Ala Gly Leu Gin Trp Pro lie Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 4 <211> 17 <212> PRT

<213> Artificial Sequence <220>

<223> BR2 sequence <400> 4

Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg

<210> 5 <211> 5 <212> PRT

<213> Artificial Sequence

<220>

<223> PV11 sequence <400> 5

Arg Ala Gly Leu Gin 1 5

<210> 6 <211> 9 <212> PRT

<213> Artificial Sequence

<220>

<223> PV12 sequence <400> 6

Arg Ala Gly Leu Gin Phe Pro Val Gly 1 5

<210> 7 <211> 12 <212> PRT

<213> Artificial Sequence

<220>

<223> PV8 sequence <400> 7

Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg 1 5 10

<210> 8 <211> 12 <212> PRT

<213> Artificial Sequence

<220>

<223> PV7 sequence <400> 8

Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg 1 5 10

<210> 9 <211> 18 <212> PRT

<213> Artificial Sequence

<220>

<223> PV13 sequence <400> 9

Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Lys

<210> 10 <211> 17 <212> PRT

<213> Artificial Sequence

<220>

<223> PV14 sequence <400> 10

Arg Ala Gly Leu Gin Trp Pro lie Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg

<210> 11 <211> 18 <212> PRT

<213> Artificial Sequence

<220>

<223> PV15 sequence <400> 11

Arg Ala Gly Leu Gin Trp Pro lie Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Lys

<210> 12 <211> 24 <212> PRT

<213> Artificial Sequence

<220>

<223> PV16 sequence <400> 12

Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Val His Arg Leu Leu Arg 1 5 10 15

Arg Leu Leu Arg Arg Leu Leu Arg 20

<210> 13 <211> 22 <212> PRT

<213> Artificial Sequence

<220>

<223> PV17 sequence <400> 13

Arg Ala Gly Leu Gin Phe Pro Pro Val Gly Arg Leu Leu Arg Arg Leu 1 5 10 15

Leu Arg Arg Leu Leu Arg 20

<210> 14 <211> 25 <212> PRT

<213> Artificial Sequence

<220>

<223> PV1 sequence <400> 14

Arg Ala Gly Leu Gin Phe Pro Pro Val Gly Arg Val His Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg Arg Leu Leu Arg 20 25

<210> 15 <211> 29 <212> PRT

<213> Artificial Sequence

<220>

<223> PV2 sequence <400> 15

Arg Ala Gly Leu Gin Phe Pro Pro Val Gly Arg Val His Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg 20 25

<210> 16 <211> 29 <212> PRT

<213> Artificial Sequence

<220>

<223> PV19 sequence <400> 16

Arg Val Gly lie Asn Phe Pro Pro Val Gly Arg Val His Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg 20 25

<210> 17 <211> 29 <212> PRT

<213> Artificial Sequence

<220>

<223> PV20 sequence

<400> 17 Arg Ala Gly Leu Gin Trp Pro Pro lie Gly Arg Val His Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg 20 25

<210> 18 <211> 30 <212> PRT <213> Artificial Sequence

<220>

<223> PV21 sequence <400> 18

Arg Ala Gly Leu Gin Trp Pro Pro lie Gly Arg Val His Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg Lys 20 25 30

<210> 19 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> PV22 sequence <400> 19

Arg Ala Gly Leu Gin Phe Pro lie Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 20 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> PV23 sequence <400> 20

Arg Ala Gly Leu Gin Phe Pro Ala Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 21 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> PV24 sequence <400> 21

Arg Ala Gly Leu Gin Phe Pro Leu Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 22 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> PV25 sequence

<400> 22 Arg Ala Gly Leu Gin Trp Pro Ala Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 23 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> PV26 sequence

<400> 23

Arg Ala Gly Leu Gin Trp Pro Leu Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 24 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> PV27 sequence <400> 24

Arg Ala Gly Leu Gin His Pro lie Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 25 <211> 21 <212> PRT

<213> Artificial Sequence

<220>

<223> PV28 sequence

<400> 25 Arg Ala Gly Leu Gin His Pro Ala Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 26 <211> 21 <212> PRT <213> Artificial Sequence

<220>

<223> PV29 sequence

<400> 26

Arg Ala Gly Leu Gin His Pro Leu Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg 20

<210> 27 <211> 24 <212> PRT

<213> Artificial Sequence

<220>

<223> PV04 sequence <400> 27

Arg Ala Gly Leu Gin Phe Pro Pro Val Gly Arg Val Gly His Tyr Gly 1 5 10 15

Arg Lys Lys Arg Arg Gin Arg Arg 20

<210> 28 <211> 23 <212> PRT <213> Artificial Sequence

<220>

<223> PV06 sequence <400> 28

Arg Ala Gly Leu Gin Phe Pro Pro Val Gly Arg Val Gly His Arg Arg 1 5 10 15

Arg Arg Arg Arg Arg Arg Arg 20

<210> 29 <211> 24 <212> PRT

<213> Artificial Sequence

<220>

<223> PV10 sequence <400> 29

Arg Ala Gly Leu Gin Phe Pro Pro Val Gly Lys Leu Ala Lys Leu Ala 1 5 10 15

Lys Lys Leu Ala Lys Leu Ala Lys 20

<210> 30 <211> 24 <212> PRT

<213> Artificial Sequence

<220>

<223> PV30 sequence <400> 30

Arg Val Gly lie Asn Phe Pro Val Gly Arg Val His Arg Leu Leu Arg 1 5 10 15

Arg Leu Leu Arg Arg Leu Leu Arg 20

<210> 31 <211> 24 <212> PRT

<213> Artificial Sequence

<220>

<223> PV31 sequence <400> 31

Arg Val Gly lie Asn Phe Pro Ala Gly Arg Val His Arg Leu Leu Arg 1 5 10 15

Arg Leu Leu Arg Arg Leu Leu Arg 20

<210> 32 <211> 24 <212> PRT

<213> Artificial Sequence

<220>

<223> PV32 sequence <400> 32

Arg Val Gly lie Asn Phe Pro Val Gly Arg Val His Arg Val Val Arg 1 5 10 15

Arg Val Val Arg Arg Val Val Arg 20

<210> 33 <211> 25 <212> PRT

<213> Artificial Sequence

<220>

<223> PV33 sequence <400> 33

Arg Val Gly lie Asn Phe Pro Pro Ala Gly Arg Val His Lys Leu Leu 1 5 10 15

Lys Lys Leu Leu Lys Lys Leu Leu Lys 20 25

<210> 34 <211> 25 <212> PRT

<213> Artificial Sequence

<220>

<223> PV34 sequence <400> 34

Arg Val Gly lie Asn Phe Pro Lys Ala Met Arg Val His Arg Leu Leu 1 5 10 15

Arg Arg Leu Leu Arg Arg Leu Leu Arg 20 25

<210> 35 <211> 24 <212> PRT

<213> Artificial Sequence

<220> <223> PV35 sequence

<400> 35

Arg Val Gly lie Asn Phe Pro Ala Gly Arg Val His Arg Leu Leu Arg 1 5 10 15 Arg Leu Leu Arg Arg Leu Leu Arg

20

Claims

1. An isolated or synthetic antitumor peptide having at least 90% sequence identity selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID

NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID

NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID

NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31: SEQ ID

NO: 32: SEQ ID NO: 33: SEQ ID NO: 34: SEQ ID NO: 35 or fragment thereof.

2. An isolated or synthetic antitumor according to claim 1 , wherein the peptide is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

3. An isolated or synthetic antitumor peptide according to claim 2, consisting of an amino acid sequence of SEQ ID NO: 17.

4. A peptide comprising an amino acid sequence according to claim 3 for use as a pro- inflammatory agent.

5. A peptide comprising an amino acid sequence according to claim 4, which is in a pharmaceutically acceptable carrier.

6. A peptide comprising an amino acid sequence according to claim 5, wherein the pharmaceutically acceptable carrier is suitable for intravenous administration.

7. A pharmaceutical composition comprising a peptide according to claims 6 and a pharmaceutically acceptable carrier or diluent.

8. The pharmaceutical composition according to claim 7 for use as a medicament.

9. A pharmaceutical composition according to claim 8, for use in inhibiting tumor or metastasis in a subject.

10. A pharmaceutical composition according to claims 9, for use in inhibiting a tumor cell in a subject.

11. A peptide or a pharmaceutical composition for use according to any one of claims 1 to

10, wherein inhibiting comprises inducing apoptosis of said tumor cell and/or necrosis of a tumor tissue comprising said tumor cell.

12. A peptide or a pharmaceutical composition for use according to claim 11, wherein the cancer is one selected from the group consisting of melanoma, colon cancer, lung cancer, renal cancer, breast cancer, brain cancer, head and neck cancer, gastric and intestinal cancer, prostate cancer, liver cancer and glioblastoma.

13. A peptide or a pharmaceutical composition for use according to claim 12, wherein administration to the subject comprises intravenous, intra-arterial, intra-tumoral, subcutaneous, topical, intraperitoneal, local, regional, systemic, or continual administration.

14. A method of inhibiting tumor growth comprising administering to a subject an effective amount of the pharmaceutical composition according to any one of claims 7 to 13.

15. Use of the peptide according to claim 1 to 6 for the manufacture of a pharmaceutical composition for inhibiting tumor or cancer growth in a subject.