WO2012130999A1

WO2012130999A1 - Peptides isolated and purified from fruit bat testicles

Info

Publication number: WO2012130999A1
Application number: PCT/EP2012/055724
Authority: WO
Inventors: Pierrïck AUVRAY; Elise DUVAL; Amandine BARON; Cécile CHAUVIN; Vincent HESRY; Déborah JOUQUAN; Mélanie MARTIN; Emma REDON; Pascal SOURDAINE; Frédéric BOURGEON; Nathalie MELAINE; Charles Pineau; Astrid Rouillon; Adrien BOSSEBOEUF
Original assignee: Kelia
Priority date: 2011-04-01
Filing date: 2012-03-29
Publication date: 2012-10-04
Also published as: WO2012130999A9; FR2973380B1; FR2973380A1

Abstract

The present invention relates to bioactive peptides that have been isolated and purified from fruit bat testicles, to the use thereof as a drug, in particular to the use thereof for preventing and treating cancer.

Description

Peptides isolated and purified from testicles of fruit bats

The present invention relates to peptides isolated and purified from testes of fruit bats, their use as a medicament, in particular their application in the field of prevention and / or treatment of cancer as well as in the field of prevention and / or or the treatment of metabolic diseases such as diabetes.

Cancer treatment today rests mainly on four axes, often combined with each other: surgery, radiotherapy, chemotherapy and hormone therapy.

Chemotherapy, for example, aims to stop or slow the uncontrolled proliferation of cancer cells in the body. This technique is widely used nowadays. Unfortunately, there are many side effects in patients with chemotherapy. Indeed, the majority of the anticancer products used are toxic for the blood cells and lead for some a fall in the number of these cells and the appearance of infections and haemorrhages. The skin and mucous membranes are also likely to be affected. Some substances are still extremely toxic to the digestive tract and to kidneys. In addition, hair loss and cardiac arrhythmias are very common in treated patients.

Given the relatively modest efficacy / toxicity ratio, as well as the side effects caused by the usual treatments, in particular by chemotherapy, we are now looking for new anti-cancer strategies including therapies based on the use of products of natural origin. .

For example, various studies have demonstrated antiangiogenic effects of shark cartilage extracts (Cho and Kim, 2002). However, these treatments were not very effective in phase III trials (Escudier et al, 2004, Lu et al, 2007 and 2010). It is further known that shark liver oil is an oil rich in compounds such as squalene, squalamine or alkylglycerols. These compounds are compounds of interest known for their antiangiogenic and / or antitumor properties. Finally, the epigonal tissue of the dogfish (Squalus acanthias) also seems to contain a compound called EGIF (Epigonal Growth-Inhibitory Factor) capable of inhibiting the proliferation of pre-meiotic germ cells (Pieffer et al, 1995).

Thus, it is clear from all these data that sharks belong to an interesting species for the characterization of new anticancer molecules. Sharks are selacean fish belonging to the class Chondrichthyes and the subclass of Elasmobranchs. Within this species is a fish commonly known as a dogfish.

The dogfish has testicles of large size (12 to 15 cm, 8 to 13 g) and having a particular anatomical organization. Indeed, as in other selacians, the testicles of flying fox are composed of closed spermatocysts (cysts) in which the spermatogenesis is synchronous. These cysts consist of spermatoblasts arranged around a central cavity. Each spermatoblast consists of a somatic cell, or cystic cell or Sertoli, associated with a clone of germ cells. During spermatogenesis, the succession of cysts in the germinal zone, where they are formed, to the area of spermiation results in a zonation of the testis in four zones: the germinal zone of spermatogonia cysts (zone A), the zone to be spermatocytes (zone B), the zone with round spermatids (zone C) and the zone with elongated spermatids (zone D) (Sourdaine et al, 1989, Loir et al, 1995). The choice of the testicular model of dogfish is therefore based both on the study of spermatogenesis as a biological process of proliferation, cell differentiation, apoptosis and on the peculiarities of the organization. anatomical testicle which allows easy separation and in large amounts of tissue corresponding to different stages of spermatogenesis (Loir et al, 1995).

Diabetes is now one of the top five causes of death in many Western countries. There are two main forms of diabetes. Insulin-dependent diabetes mellitus or type-I diabetes mellitus occurs either in infancy, adolescence or young adults, and non-insulin-dependent diabetes or type II diabetes occurs much later, usually after the age of 40 . There are currently active antidiabetic drugs active orally with hypoglycemic action such as biguanides, sulphonamides hypoglycemiants, inhibitors of α-glucosidases and insulin secretors. In case of failure of these drugs, insulin treatment is necessary. In this area also there is a need to identify and characterize new natural and effective compounds.

The applicant company has undertaken to research and characterize new molecules useful in the field of health and has thus been able to isolate and purify new active peptides having, in particular, a significant anti-tumor activity and antidiabetic activity.

The subject of the present invention is thus one or more new peptides, isolated and purified from the genital genital tract belonging to the Selacian class, for their use as medicaments, in particular for their use in the prevention and / or the treatment of cancers as well as in the prevention and / or treatment of metabolic diseases such as diabetes.

The peptides according to the invention were obtained from the genital genitalia previously dissected and separated according to the testicular zones as described above. The proteins were extracted from the samples taken and then purified, fractionated and selected according to their size. The isolated and purified peptides were then evaluated for their functional properties and were sequenced.

In particular, the invention relates to peptides isolated and purified from the genital tract of fruit bat for their use in the prevention and / or treatment of colon cancer, breast cancer, preferentially hormone-dependent breast cancer, promyelocytic acute leukemia, ovarian cancer, melanoma, lung cancer and prostate cancer. According to one embodiment of the invention, the peptides have a size less than 100 amino acids, preferably less than or equal to 20 amino acids and / or have a molecular mass less than or equal to 10 KDa.

The peptide extracts from the samples taken from the testicles of the dogfish are fractionated using, for example, microconcentrators with a cutoff threshold of 10 KDa.

According to one embodiment of the invention, the peptides comprise, consist of or are derived from, an amino acid sequence selected from the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

According to a particular embodiment, the invention relates to a peptide comprising, derived from or consisting of the amino acid sequence SEQ ID NO: 1 for its use in the prevention and / or treatment of hormone-dependent human breast carcinoma, promyelocytic acute leukemia or prostate cancer, preferentially hormone-dependent human breast carcinoma and for its use in the prevention and / or treatment of diabetes.

According to another particular embodiment, the invention relates to a peptide comprising, derived from or consisting of, the amino acid sequence SEQ ID NO: 2 for its use in the prevention and / or treatment of human adenocarcinoma of ovarian, human melanoma, prostate cancer, acute promyelocytic leukemia, hormone-dependent human breast carcinoma or human small cell lung carcinoma, preferentially human ovarian adenocarcinoma and cancer of the ovary prostate ; and for its use in the prevention and / or treatment of diabetes.

According to another particular embodiment, the invention relates to a peptide comprising, derived from, or consisting of, the amino acid sequence SEQ ID NO: 3 for its use in the prevention and / or treatment of human melanoma, Small-cell human lung carcinoma or promyelocytic acute leukemia, preferentially human melanoma.

According to another particular embodiment, the invention relates to a peptide comprising, derived from or consisting of, the amino acid sequence SEQ ID NO: 4 for its use in the prevention and / or treatment of human adenocarcinoma colon, acute promyelocytic leukemia or small-cell human lung carcinoma, preferentially human adenocarcinoma of the colon. According to a preferred embodiment of the invention, the dogfish belongs to the species Scyliorhinus canicula.

The invention also relates to a delivery vector comprising at least one peptide as defined in one of the preceding claims.

For the purposes of the invention, the term "delivery vector" is intended to mean any vector capable of conveying or transporting a peptide according to the invention inside the human or animal body.

By way of example, vectors which are particularly suitable for the delivery of peptides according to the invention are archeosomes such as those described in patent application PCT / EP2005 / 05655. These delivery vectors are liposomes and generally consist of tetraether bipolar lipids isolated from methanogenic or thermoacidophilic archaebacteria or synthetic analogues. Any other delivery vector known to those skilled in the art is of course envisaged. As such it is important that said delivery vector can cross biological barriers depending on its mode of administration.

The invention further relates to a pharmaceutical composition comprising at least one peptide isolated and purified from the genital tract of the dogfish, preferably associated with a vehicle, a adjuvant and / or a pharmaceutically acceptable diluent.

By "vehicle" within the meaning of the invention is meant any substance present within the composition containing a peptide of the invention and intended to facilitate its transport, ensure its protection against any degradation in the composition and preserve its properties. A pharmaceutically acceptable carrier will be chosen depending on the route of administration of the composition containing the peptide (s) according to the invention.

In particular, the invention relates to such a pharmaceutical composition for its use for the prevention and / or treatment of cancer, preferentially colon cancer, hormone-dependent breast cancer, promyelocytic acute leukemia, cancer of the ovarian, melanoma, small cell lung cancer and prostate cancer and for its use in the prevention and / or treatment of diabetes.

According to a particular embodiment, the invention relates to a pharmaceutical composition which comprises at least one peptide comprising, derived from, or consisting of, the sequence SEQ ID NO: 1 for the treatment and / or prevention of hormone-dependent human breast carcinoma, acute promyelocytic leukemia or prostate cancer, preferentially hormone-dependent human breast carcinoma, and for its use in the prevention and / or treatment of diabetes.

According to another particular embodiment, the invention relates to a pharmaceutical composition which comprises at least one peptide comprising, derived from, or consisting of, the sequence SEQ ID NO: 2 for the treatment and / or prevention of human adenocarcinoma ovarian, human melanoma, prostate cancer, acute promyelocytic leukemia, hormone-dependent human breast carcinoma or human small cell lung carcinoma, preferentially human ovarian adenocarcinoma and cancer of the prostate, and for its use in the prevention and / or treatment of diabetes.

According to yet another particular embodiment, the invention relates to a pharmaceutical composition which comprises at least one peptide comprising, derived from, or consisting of, the sequence SEQ ID NO: 3 for the treatment and / or prevention of human melanoma, small-cell human lung carcinoma or acute promyelocytic leukemia, preferentially human melanoma.

According to a still further particular embodiment of the invention, a pharmaceutical composition which comprises at least one peptide comprising, derived from, or consisting of, the sequence SEQ ID NO: 4 for the treatment and / or prevention of human adenocarcinoma of the colon, acute promyelocytic leukemia or small-cell human lung carcinoma, preferentially, human adenocarcinoma of the colon.

The compositions according to the invention will for example be in solid form, such as a tablet, a capsule or a powder, a liquid form, such as a solution, a suspension, an emulsion, a syrup, or in the form of a cream or gel, depending on the intended mode of administration.

In the composition according to the invention, said peptide may be in the form of a salt.

A pharmaceutical composition according to the invention may, in addition, contain one or more additional active ingredients.

The invention further relates to the use of a peptide as defined above for the preparation of a medicament. The invention further relates to a method of treating cancer, preferably colon cancer, hormone-dependent breast cancer, promyelocytic acute leukemia, ovarian cancer, melanoma, small cell lung cancer or prostate cancer, and / or a method of treating diabetes in which a peptide isolated and purified from testes of dogfish or derived from testes of dogfish is administered to a patient.

The invention also relates to a peptide comprising, derived from, or consisting of, a sequence, amino acids selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, as that such.

Table 1 below indicates the peptide sequences SEQ ID NO: 1, SEQ

ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 according to the invention:

Table 1

Sequence Code to 1 letter

SEQ ID NO: 1 NFDTDEQALEDVFSKYG

3-letter code

SEQ ID NO: 1 Asn Phe Asp Asp Asp Glu Gin Ala Leu Glu Asp Val Phe Ser Lys Tyr Gly Denomination K092A

Sequence Code to 1 letter

SEQ ID NO: 2 E APPE A AEEDE W

Sequence Code with 3 letters

SEQ ID NO: 2 Glu Ala Pro Pro Glu Ala Glu Glu Asp Glu Trp

denomination K092B

Sequence Code to 1 letter

SEQ ID NO: 3 CNRRRV

Sequence Code with 3 letters

SEQ ID NO: 3 Cys Asn Arg Arg Arg Val

denomination K092C

Sequence Code to 1 letter

SEQ ID NO: 4 QLTPEALADEEEMNALAAR

Within the meaning of the invention, a peptide comprising, derived from, or consisting of, a sequence chosen from the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 may be a natural or synthetic peptide.

By "derived" peptide is meant a peptide having between 70 and 99%, preferably between 75 and 95%, more preferably between 85 and 95% identity with one of the sequences SEQ ID NO: 1 to 4, and that is, peptides having undergone at least one addition and / or at least one substitution and / or at least one deletion of an amino acid.

"Derivatives" within the meaning of the invention also include peptides having a post-translational modification and / or a chemical modification such as glycosylation, amidation, acylation, acetylation, methylation or peptides comprising a protective group provided to prevent degradation of said peptide. Among the derivatives of the peptides of the invention are still included those in which one or more amino acids are natural amino acids of conformation D, enantiomers, diastereoisomers, rare amino acids such as hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylsilyl, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid and synthetic amino acids, especially ornithine, norleucine, norvaline, cyclohexyl-alanine and omega-amino acids.

The invention also relates to a nucleic acid comprising a nucleic sequence coding for a peptide according to the invention, in particular for a peptide comprising, derived from or consisting of an amino acid sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. The invention also relates to a nucleic sequence complementary to such a coding nucleic sequence.

The term "nucleic acid" according to the present invention, a nucleic acid sequence of DNA or RNA. The sequence of such a nucleic acid can be deduced using the genetic code by a person skilled in the art from the acid sequence amino acids of a peptide according to the invention. Such nucleic acid can then be isolated by screening nucleic acid libraries using one or more oligonucleotides of the deduced sequence.

The invention also relates to an expression cassette comprising at least one sequence of such a nucleic acid as well as an expression and / or cloning vector comprising at least one sequence of such nucleic acid or one such cassette. 'expression.

Preferably, an expression cassette, or a cloning and / or expression vector, comprises an appropriate promoter, that is to say, a constitutive or inducible promoter, as well as a terminator.

A cloning and / or expression vector according to the invention may be a plasmid, a cosmid, a bacteriophage or a virus and advantageously comprises elements allowing its maintenance, its replication and / or its selection in a host organism. Such vectors are well known to those skilled in the art.

The present invention is now described in more detail in connection with the following exemplary embodiments as well as the figures in which:

Figs. 1 to 6 illustrate the anti-proliferative effects of a peptide consisting of the sequence SEQ ID NO: 4, in comparison with a control, on human cancer lines HT-29 (FIGS. 1 and 2), NCI H69 (FIGS. 3 and 4) and EMC CCRF (FIGS. 5 and 6), as a function of the log of the concentration of said peptide expressed in μg / μl. The results are expressed as IC50 (μ§ / μϊ) or IC75 (μ§ / μΐ).

Figs. 7 to 14 illustrate the anti-proliferative effects of a peptide consisting of the sequence SEQ ID NO: 1, in comparison with a control, on the human cancer lines MCF-7 (FIGS. 7 and 8), ZR-75-1 (Figs 9 and 10), CCRF CEM (Figs 1 1 and 12) and PC3 (Figs 13 and 14), as a function of the log of the concentration of said peptide expressed in μg / μl. The results are expressed in IC50 (μ§ / μΐ).

Figs. Figures 15 to 26 illustrate the anti-proliferative effects of a peptide containing SEQ ID NO: 2, in comparison with a control, on human cancer lines NCI H69 (Figs 15 and 16), SK-OV-3 (Figs. 17 and 18) and A375 (Figs 19 and 20), MDA Pca2b (Figs 21 and 22), CCRF-CEM (Figs 23 and 24), ZR-75-1 (Figs 25 and 26), according to log of the concentration of said peptide expressed in μg / μl. The results are expressed in IC50 (μ§ / μΐ).

Figs. 27 to 32 illustrate the anti-proliferative effects of a peptide containing SEQ ID NO: 3, in comparison with a control, on cancer lines A375 (Figs 27 and 28), NCI H69 (Figs 29 and 30) and CCRF CEM (Figs 31 and 32), as a function of the log of the concentration of said peptide expressed in μg / μl. The results are expressed in IC50 (μ§ / μΐ).

In Figs. 1 to 32, the antiproliferative effects of the positive controls corresponding to each cell line are also represented as a function of the log of the concentration of said control expressed in molar (M). The results are expressed as IC50 (M).

Fig. 33 is a graph representing the average tumor volume between T0 and T22 measured during an in vivo test of a peptide consisting of the sequence SEQ ID NO: 4.

Fig. 34 is a graph representing the values of the ratios T / C% (median tumor volume treated group / median tumoral volume negative control group) between T0 and T22 found during an in vivo test of a peptide consisting of the sequence SEQ ID NO : 4.

Fig. 35 is a graph showing the average tumor volume between T0 and

T10 measured during an in vivo test of a peptide consisting of the sequence SEQ ID NO: 3.

Fig. 36 is a graph representing the values of the ratios T / C% (median tumor volume treated group / median tumor volume negative control group) between T0 and T10 found during an in vivo test of a peptide consisting of the sequence SEQ ID NO : 3.

Fig. 37 is a graph representing the average tumor volume between T0 and TU measured during an in vivo test of a peptide consisting of the sequence SEQ ID NO: 2.

Fig. 38 is a graph showing the values of the ratios T / C% (median tumor volume treated group / median tumor volume negative control group) between T0 and TU found during an in vivo test of a peptide consisting of the sequence SEQ ID NO : 2.

Figs. 39 to 40 are graphs representing the secretion of insulin (as a percentage) as a function of the concentration of a peptide consisting of the sequence SEQ ID NO: 1 or of the sequence SEQ ID NO: 2, in μg / μl ( Fig. 39), or induced by a positive control (Fig. 40), on the RIN-5F line. 1) PREPARATION OF PEPTIDE FRACTIONS AND PEPTIDES a) Collection of biological samples

Adult fruit bats belonging to the species Scyliorhinus canicula were fished and maintained in natural seawater. The fruit bats are demedulated and decerebrated and the testes are quickly removed and cut in cross sections of 2 mm. The different testicular zones A, B, C and D, as defined above, as well as the lymphopoietic epigonal tissue which borders one of the faces of the testicle are dissected. Samples from at least 6 fruit bats are grouped into a single sample to avoid individual variations.

These tissues, cut into pieces, are crushed and homogenized in lysis buffer (10 mM HEPES pH 7.5, 1 mM EDTA, 0.5 mM DTT) and a mixture of protease inhibitors. Five sonications of 10 seconds are carried out then the lysate is purified by ultracentrifugation (15000g, 30 min at 4 ° C and then 105000g, lh at 4 ° C).

b) Separation of peptides according to their size

After ultracentrifugation, the biological extracts were fractionated by Amicon Ultra 4 membrane filtration (30,000 MWL, Millipore) with a cut-off threshold of 10 Kda, in order to eliminate proteins larger than 10 kDa. c) Reverse phase HPLC fractionation

Peptide samples smaller than 10 KDa were fractionated by reverse-phase high performance liquid chromatography (RP-HPLC) on a Breeze ™ system (Waters Company) using a Vydac Cl 8 reverse phase column (218TP54). ).

d) Screening of the anti-cancer activity in the fractions purified by RP-HPLC

The different fractions obtained by RP-HPLC were then tested by cytotoxicity tests to determine their potential anti-cancer activity on cell lines listed in Table 2 (see Example 1).

e) Identification of the active peptides in the active fractions by mass spectrometry.

Peptide extracts showing interesting activities were analyzed by ion trap mass spectrometry (Esquire HCT Ultra®, Bruker Daltonics) coupled with nano-liquid chromatography (nanoLC Ultimate 3000®, Dionex). The set of raw data obtained after analysis of the mass spectra (MS / MS fragmentation) is interpreted by de novo analysis (PEAKS © Studio, B SI: "Protein identification" module, having the advantage of being able to work in native peptide mode (no enzyme) and allowing the exploitation of the mass lists resulting from MS / MS fragmentations; "Spider" module, allowing a search on a blast-like mode; Module "Inchorus", allowing a grouping of the results Protein identification and Spider). A filter is applied taking into account the confidence scores obtained on the proposed de novo sequences. Several peptides were identified including the peptides of sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 and the corresponding sequences then served as primers for the PCR screening of the libraries. complementary DNA.

f) Construction of genebanks

Three complementary DNA libraries (cDNA) were constructed using a kit provided for this purpose (Stratagene pBluescript II XR CDNA library construction kit) from 5 μg of mRNA extracted from the different testicular zones. For each library, 5 μg mRNA was retranscribed to [α 32 P] GTP-labeled double-stranded cDNA. EcoRI and XhoI adapters were then bound to the ends of the cDNAs. These cDNAs were fractionated according to their size on sepharose resin and then cloned into the EcoRI and XhoI sites of plasmid pBluescript II SK (+).

g) Sequence Search by Oligonucleotide Screening of the cDNA Banks

From the peptide sequences obtained by the mass spectrometry analyzes, the nucleotide sequences were drawn. Thus, using the Codehop program, primers having a 5 'non-degenerate portion (whose nucleotide sequence is fixed in agreement with the codon usage of Squalus acanthias) and a degenerate 3' portion are drawn. These degenerate sequences have been defined in order to screen gene banks. The screens were carried out for each tag by PCR on the cDNA libraries with the degenerate primer (s) corresponding to the peptide tag and a primer defined on the pBluescript II SK (+) cloning vector of the bank. After cloning and sequencing the isolated sequence by the primary screen, confirmatory PCR is performed to obtain the exact sequence of the target clone. PRODUCTIONS OF PEPTIDES

The peptides of the invention can be obtained by extraction from testis or fruit bats or synthesized by any method known to those skilled in the art. The peptides of the invention can thus be obtained by chemical synthesis. A recombinant peptide may also be produced by a method in which a vector containing a nucleic acid encoding a peptide of interest is transferred into a host cell which is cultured under conditions permitting the expression of the corresponding peptide. The product peptide can then be recovered and purified by purification methods known to those skilled in the art. The nucleotide sequences of the messenger RNAs capable of coding for each peptide SEQ ID NO: 1 to SEQ ID NO: 4 can be deduced from the peptide sequences by a person skilled in the art who knows the codons coding for each amino acid. A stop codon is added at the end of the mRNA sequence.

EXAMPLE 1 Characterization of the Antiproliferative Efficacy of the Peptides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4

The anti-tumor efficacy of the peptides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 was evaluated on a panel of different cell lines representative of the main cancers encountered in humans. whose characteristics are summarized in Table 2 below.

Table 2

CELLULAR LINES ORIGIN REFERENCE

A375 Human melanoma (Giard et al., 1973)

CCRF ΟΕΜ ^φ) Acute promyelocytic leukemia (Foley et al., 1965)

NCI H69 ^(a) Human lung carcinoma to small (Carney et al., 1985) cells

HT-29 ^(a) Human colon colon adenocarcinoma (Zupi et al., 1988)

II

SK-OV-3 Human Adenocarcinoma of the Ovary (Fogh et al., 1977)

ZR-75-l ^(a) Hormonal human breast carcinoma (Engel et al., 1978) dependent MCF-7 Human Hormonal Carcinoma Hormone (Sugarman et al., 1985 Dependent)

PC-3 Human Prostate Adenocarcinoma (Alimirah et al., 2006) Hormone-independent

(Navone et al., 1997)

MDA PCa2b ^(b) bone metastasis from a

human adenocarcinoma of the prostate

hormone-dependent

(Fogh et al., 1977)

Calu-6 ^(a) Large human lung carcinoma

cell

(Yunis e / a /., 1977)

MIA Paca-2 ^ Human pancreatic carcinoma

(Beckman et al, 1971)

U-87-MG * 'Human glioblastoma

(a): ECACC, Sigma, France; (b) ATCC, LGC Promochem, France.

Culture of these cell lines is performed in a suitable medium described in Table 3 below.

Table 3

CULTURE MIDDLE LINES

CONDITIONS OF CELLULAR CULTURE

A375 DMEM (Ref.BE12-602F / Ul, Lonza,

France) + 2mM L-glutamine (BE17-605E, 37 ° C, 5% C02 Lonza) + 15% FCS (DE14-801F, Lonza)

CCRF CEM RPMI 1640 (Ref BE17-702F, Lonza) +

10% FCS (DE14-801F, Lonza) + HEPES 37 ° C, 5% CO 2 mM (Ref BE17-737E, Lonza) + Glucose 4.5

g / L (Ref G7528, Sigma, France) + NaHCO3

1.5 g / L (S5761 Ref., Sigma) + Sodium

Pyruvate 1 mM (Ref DE14-801F, Lonza)

NCI H69 RPMI 1640 + 10% FCS + 2 mM Glutamine

37 ° C, 5% C02

HT-29 McCOY's 5A (Ref BE12-688F, Lonza) +

10% FCS 37 ° C, 5% C02

SK-OV-3 Me Coy's 5A + 15% SVF + 2mM L-

37 ° C, 5% C02

glutamine

ZR-75.1 RPMI 1640 + 10% FCS + 2 mM L-

37 ° C, 5% C02

Glutamine + 1 mM Sodium Pyruvate +

10 nM beta-estradiol (Catalog E2758, Sigma)

The cells are amplified, collected, counted and their viability is assessed by the Trypan Blue exclusion method. The presence of mycoplasmas is evaluated using the PCR detection kit for mycoplasmas (Ref 6601, Takara, France). The cells are then seeded in a 96-well plate at 500 to 15000 cells / well as indicated in Table 4 below. They are incubated at 37 ° C. under a 5% CO 2 atmosphere for 24 hours.

Table 4

Cell lines Number of cells to implant Positive control

A375 500 CDDP

CCRF CEM 5000 Doxorubicin

HT-29 2500 CDDP

MCF-7 5000 Tamoxifen

NCI H69 15000 CDDP

SK-OV-3 850 Paclitaxel

ZR-75.1 1000 Paclitaxel

PC-3 1000 CDDP

MDA PCa2b 1000 Paclitaxel

Calu-6 2500 CDDP

MIA Paca-2 5000 Gemcitabine

U-87-MG 2500 CDDP The lines are implanted in order to test different concentrations of peptides between 0.005 μg / μl and 2.5 μg / μl at the rate of three wells per concentration.

For this purpose, 24 hours after incubation, the cells are again incubated at 37 ° C. for 96 h in the presence of the peptides at concentrations of between 0.005 μg / μl and 2.5 μg / μl or in the presence of a positive control (cf. Table 4). At the end of the treatment, the cells are incubated at 37 ° C. for 2 to 4 hours (depending on the cell lines) with 10 μl of solution WST-1 (tetrazolium salts, reference 1644807, Roche, France) to evaluate cell viability (formation of formazan by mitochondrial succinate dehydrogenase). The reading of the absorbance at 450 nm and the analysis relative to the controls makes it possible to determine the anticancer efficacy of the peptides on these cellular models in vitro. Thus after this incubation, the 96-well plates are shaken dry with the Multiskan® EX (Thermo Labsystems, France). The absorbance is then measured against a blank control using the Multiskan® EX microplate reader at 450 nm, the reference wavelength being 620 nm. The results are shown in Figs. 1, 2, 3 and 4 for the peptides comprising the sequences SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

IC 50 and IC 75, indicating the amount of peptide needed to inhibit 50%, 25%, of cell multiplication, for cell lines are shown below:

- SEQ ID NO: 1: IC50 at 1.09 μg / μl (0.55 mM) on MCF7 (hormone-dependent human breast carcinoma) (Fig. 7), IC50 at 1.22 μg / μl (0.62 mM) ) on ZR-75-1 (hormone-dependent human breast carcinoma) (Fig. 9), IC50 at 0.96 μg / μl (0.49 mM) on CCRF CEM (acute promyelocytic leukemia) (Fig. 1 1), IC50 at 1.7μ§ / μ1 (0.86 mM) on PC3 (human adenocarcinoma of hormone-independent prostate) (Fig.

13);

- SEQ ID NO: 2: IC50 at 1, 13 μg / μl (0.82 mM) on NCI H69 (human small cell lung carcinoma) (Fig. 15); IC50 at 1, 16 μg / μl (0.85 mM) on SK-OV3 (Human Ovarian Adenocarcinoma) (Fig. 17), IC50 at 1.25 μg / μl (0.91 mM) on A375 (Melanoma) human) (Fig. 19); IC50 at 1.3 μg / μl (0.95 mM) on MDA Pca2b (hormone-dependent human adenocarcinoma of the prostate) (Fig. 21); IC50 at 2.2 μg / μl (1.6 mM) on CCRF-CEM (acute promyelocytic leukemia) (Fig. 23); IC50 at 2.4 μ / μ1 (1.75 mM) on ZR-75-1 (hormone-dependent human breast carcinoma) (Fig. 25);

- SEQ ID NO: 3: IC50 at 1.8 μg / μl (2.2 mM) on A375 (Fig. 27), IC50 at 1.8 μ ^ μΐ (2.2 mM) on NCI H69 (Fig. 29) ) and IC50 at 1.53 μg / μl (1.9 mM) on CCRF CEM (Fig. 31);

- SEQ ID NO: 4: IC50 at 1.79 μg / μl (0.86 mM) on HT-29 (human colon adenocarcinoma of grade II) (Fig. 1), IC50 at 2.24 μg / μl (1 1 mM) on CCRF CEM (Fig. 5) and IC75 at 1.249 μg / μl (0.6 mM) on NCI H69 (Fig. 3). Example 2 Peptide mutagenicity test comprising the sequence SEQ

ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 (Ames test)

This study was conducted according to the principles of Good Laboratory Practice (GLP) as described in OECD Principles of Good Laboratory Practice (revised 1997), ENV / MC / CHEM (98) 17; Directive 2004/10 / EC of the European Parliament and of the Council of 11/02/2004 on the harmonization of laws, regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for testing of chemical substances (OJ No. L50 of 20.2.2004); French Good Laboratory Practice (Official Journal of 23/03/00, Order of 14 March 2000).

The aim is to evaluate the mutagenic activity of peptides on five modified Salmonella typhimurium strains (TA98, TA1535a, TA97a, TA100, TA102) according to the Ames test. The principle of such a test is that the strains grow in a medium lacking histidine if the molecule tested is mutagenic. The bacterial strains Salmonella typhimurium (Biogenic, France) whose genotypes are summarized in Table 5 below are cultured overnight at 37 ° C. before treatment.

Table 5

Target Strain Antibiotic Resistance

Mutation

TA98 hisD3052 GC Ampicillin

TA1535 hisG46 GGG

TA97a hisC3076 CCC Ampicillin

TA100 hisG46 GGG Ampicillin TA102 TAA Ampicillin and hisG428

tetracycline

The peptides are dissolved in order to obtain the following concentrations: 15.5; 5; 1.5; 0.5; 0, 15; 0.05 and 0.015 mg / ml. Finally, the bacteria are then treated with 5000; 1550; 500; 155; 50; 15.5; 5 and 1.55 μg per box. The bacteria are also treated with a solvent control and six positive controls (2-aminoanthracene at 2 μg / box, 2-nitrofluorene at 1 μg / box, 4 nitroquinoline N-oxide at 0.125 μg / box, sodium azide at 650 μg / box, acridine mutagen dihydrochloride 15 μg / box and cumene Hydroperoxide 30 μg / box) are also prepared. After an incubation of 72h at 37 ° C, the bacteria are then counted and the results are analyzed using software such as XenoMatrix ™ software.

The mutagenic activity (expressed as mutagenicity induction factor) is thus determined:

Mutagenic ratio = (Average number of mutated colonies following treatment with the test substance) / (Average number of mutated colonies following treatment with the control solvent)

The results are expressed as a ratio to negative controls (DMSO for positive controls and sterile water for molecules). According to this test, a molecule is mutagenic if the ratio observed is greater than 2 and a dose-dependent effect is observed. The set of results demonstrated that, according to the regulatory Ames test, the peptides comprising, respectively, the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 and their Metabolites have no mutagenic effect on TA97a, TA98, TA100, TA102 and TA1535 at concentrations tested according to GLP standards.

Example 3 Study of the Acute Toxicity of the Peptides Comprising, respectively, the Sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 This study was carried out according to the principles of good practice laboratory

(GLP) previously cited. The purpose of this test is to determine the maximum tolerated peptide dose. This dose should be used for in vivo efficacy studies. Depending on their solubility, the peptides are solubilized in 0.9% NaCl or ammonium bicarbonate for the peptide comprising SEQ ID NO: 1, in order to obtain a maximum concentration and to make doses ranging from 2000. mg / kg maximum at 5 mg / kg. Female Swiss Nude mice are randomized to T0 (first day) with 5 mice per group: one control group (solvent) and one group for each dose / peptide. One mouse per group is treated at T0 and then observed for 24 hours. Depending on the signs of toxicity, the other mice are treated. Viability, mouse weight and behavior are noted once daily for 14 days. The weight loss is evaluated in relation to the weight at T0 of the mice. During the study, animals are sacrificed if any of these elements occur: Signs of suffering (cachexia, weakness, difficulty moving or eating), Product toxicity (curling, convulsions), 25% weight loss over a few days. An autopsy is performed in each of these cases. The mice are sacrificed at T13 (or before if toxicity is observed) by inhalation of C0 ₂ . A macroscopic autopsy of the organs is then performed for each of the mice. Tolerance to the peptides tested is determined by a comparative analysis of the change in mean body weight, mortality, and macroscopic observation of organs in different groups. Clinical signs are noted.

The statistical analysis of the mean body weight change is performed according to the Bonferroni / Dunn test. All groups are compared to each other. A value p <0.05 is considered significant.

The acute toxicity of the peptides comprising, respectively, the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 was evaluated according to OECD regulation No. 420 with the modifications. following:

the chosen route of administration was the intravenous route;

the solubility of the peptide of sequence SEQ ID NO: 4 being 50 mg / ml, the maximum dose tested was 400 mg / kg (instead of 2000 mg / kg recommended).

the solubility of the peptide of sequence SEQ ID NO: 1 being 25 mg / ml, the maximum dose tested was 200 mg / kg.

Four mice per group (4 doses per test substance) were treated by intravenous (IV) injection with peptides or negative control. These mice were then observed for 14 consecutive days, weighed twice a week and their behavioral analysis was performed once a week. On D0, one mouse per group was treated with the treatment doses determined during the study. orientation (dose of 5, 50, 300 and 400 mg / kg for the peptide incorporating the sequence SEQ ID NO: 4) Dose of 5, 50 and 300 and 2000 mg / kg for the peptides incorporating the sequence SEQ ID NO: And SEQ ID NO: 3. Dose of 5, 50, 100 and 200 for the peptide incorporating the sequence SEQ ID NO: 1).

The set of results demonstrated that the peptides comprising, respectively, the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, administered intravenously in the Nude mouse were proved non-toxic respectively up to the dose of 200 mg / kg, 300 mg / kg, 300 mg / kg and 400 mg / kg.

Example 4: Study of the in vivo anti-tumor efficacy of the peptides comprising, respectively, the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 The purpose of this study is to evaluate the anti-tumor activity of the 4 SEQ peptides

ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 in the athymic rodent on the most sensitive types of cancer, determined according to the results of the in vitro study.

The peptide comprising the sequence SEQ ID NO: 4 is tested on the HT-29 cell line, the peptide comprising the sequence SEQ ID NO: 3 is tested on the A375 cell line and the peptide comprising the sequence SEQ ID NO: 2 is tested. on the SK-0 V-3 cell line.

The different cell lines are amplified in order to obtain 10 million cells by subcutaneous induction. Prior to tumor induction, the mice are anesthetized with isoflurane. The tumors are then induced by injection of 200 μΐ of cells contained in medium without serum. After inoculation, the mice are observed 2 hours post-injection. When the tumor volume reaches the desired volume (volume measured with a vernier caliper according to the formula (lxL2) / 2, the delay varies between 10 and 60 days depending on the model), the mice are treated according to the chosen treatment regimen.

The viability, the behavior of the mice as well as the volume of the tumor are recorded twice a week. During the experiment, the animals are sacrificed if one of these cases occurs: signs of suffering, toxicity of the product, 25% of weight loss over a few days, tumor growth of 10% of the body weight, tumor ulcerating and remaining open, position of the tumor interfering with movements. An autopsy is performed in each case according to the ethical principles of animal experimentation. Surviving mice are sacrificed when the tumors reach a maximum volume of 2000 mm ³ . Each tumor is collected immediately, included in reagent TissueTek OCT fixative reagent (Ref 4583, Bayer, France) and frozen in isopentane (2-methylbutane, Ref.27034-2, Aldrich, France) and nitrogen liquid. The tumor samples are stored at -80 ° C.

For all animals, the size of the tumor and measured twice a week and the individual curves are plotted. Are calculated:

- Tumor growth curves (plotted against average tumor volumes) (VTM)

- Relative mean tumor volume curves (VTMR).

- The tumor doubling time (TD) (defined as the period necessary to obtain an average tumor volume of 200%)

- Inhibition of tumor growth (T / C%) (determined as the ratio of the tumor volumes of the treated animals versus the control group).

- The specific growth period (SGD) calculated according to the following formula:

(TD - TD solvent)

SGD =

(TD solvent)

- The time required to reach a defined volume V (TTRV)

Animals with a tumor smaller than the pre-defined limit are considered tumor implant failure and are excluded from the study on the day of treatment.

All statistical analyzes are performed using the VisualStat® Professional software (Visualstat Computing, USA).

Statistical analyzes of mean body weight change, tumor volumes, doubling time, relative tumor volume, and inhibition of tumor growth are performed using the Bonferroni / Dun test. All groups are compared to each other. a) In vivo anti-tumor efficacy of a peptide comprising the sequence SEQ ID NO: 4 (peptide named K092D)

In order to evaluate the antitumor efficacy of the peptide incorporating the sequence SEQ ID NO: 4 against colon tumors, the HT29 tumor cells were injected with QOx10 ⁶ cells / 200 μl) on the right flank of the Nude mice. Four days later, the mice were randomized to tumor volume (56-58 mm ³ ) and treated for 5 consecutive days with this peptide at doses of 10, 60 and 130 mg / kg by intravenous (IV) injection (one injection per day for 5 days (Q1D * 5)). A control group was treated with CPT-11, a camptothecin analogue, a specific topoisomerase I inhibitor, which has been shown to be effective in the treatment of colorectal cancers. The tumor volume of each animal was measured twice a week for 25 days. The activity of the peptide is evaluated by the mean tumor volume (MTV) (Fig. 33) and by the ratio T / C% (median tumor volume treated group / median tumoral volume negative control group) (Fig. 34). According to the criteria of the National Cancer Institute (NCI), a molecule is a very good anticancer candidate when the ratio T / C% is less than 42%.

The peptide-treated mice incorporating the sequence SEQ ID NO: 4 (IV) and the CPT-11 positive control group (an intraperitoneal injection (IP) per week for 5 weeks) are compared to the control group (solvent group). model was validated with CPT-11 positive control which helped highlight a beginner antitumor activity J5 (5 ^th day) (% T / C = 86%) and s' accentuating until J52 (T / C % = 47%) (Fig. 34).

At day 10, day 14, day 22 and day 38, the VTM and VTMR of the untreated groups and solvents were higher than those of the groups treated with the peptide incorporating the sequence SEQ ID NO: 4 at 10, 60 and 130 mg / kg (Fig. 33). ).

The T / C% of the groups treated with the peptide incorporating the sequence SEQ ID NO: 4 decreased between J0 and J22 (70.29%) for the group treated at 10 mg / kg and 52.05%) for the treated group. at 60 mg / kg on D22) and remain low until D38 (69.68%) (10 mg / kg) to 78.64% (130 mg / kg)). Analysis of the T / C%> parameter therefore makes it possible to conclude that the peptide incorporating the sequence SEQ ID NO: 4 has antitumor activity with a dose-dependent effect. In addition, we note that these effects were observed several weeks after the end of treatments. As of D5, the peptide incorporating the sequence SEQ ID NO: 4 at a dose of 60 mg / kg (30 μηιοΐ / kg) showed an onset of activity (T / C = 88%), comparable to CPT-11 (40 μιηοΐ / kg). At D22, antitumor activity was observed for this peptide (T / C = 52% at 60 mg / kg, ie 30 μιηοΐ / kg) comparable to that of CPT-11 (T / C = 48%) (40 μιηοΐ / kg). These results are particularly interesting because the peptide incorporating the sequence SEQ ID NO: 4 is more active than the CPT-11 if one reasons in terms of moles. In addition, the treatment regimen must also be taken into account knowing that the peptide was injected only from D0 to D4 for one administration per day while the CPT-11 was administered once a week for 5 weeks.

Therefore, the peptide incorporating the sequence SEQ ID NO: 4 showed a dose-dependent antitumor activity against the HT-29 model of colon cancer. This activity has been demonstrated by the different parameters VTM, VTMR, T / C and TTRV. b) In vivo anti-tumor efficacy of a peptide comprising the sequence

SEQ ID NO: 3 (peptide named K092C)

To evaluate the antitumor efficacy of the peptide incorporating the sequence SEQ ID NO: 3 against melanoma tumors, A375 tumor cells were injected on the right flank of nude mice at a rate of ^e 10x10 cells / 200 μΐ. When the tumor volumes reached an average of 800 mm ³ , the mice were randomized and treated for 4 consecutive days with 50 μΐ for 800 mm ³ , a solution of this peptide at 12.5 mg / ml by intratumoral injection ( IT), a dose of 25 mg / kg. A control group received the solvent only. The tumor volume of each animal was measured twice a week for 10 days. The activity of the peptide is evaluated by the mean tumor volume (VTM) (Fig. 35) and the ratio T / C% (median tumor volume treated group / median tumor volume negative control group) (Fig. 36).

The mice treated with the peptide incorporating the sequence SEQ ID NO: 3 are compared with the control group (solvent) whose pH was adjusted to that of the peptide in solution in 0.9% NaCl (IT).

A J10 ^(10th day), the VTM was 2274.93 ± 286.82 for the control group and 1812.97 ± 335.20 for the treated group the peptide incorporating the sequence SEQ ID NO: 3 to 25 mg / kg. The corresponding VTMRs (J0-J10) were 2.98 ± 0.48 for the group treated with the peptide incorporating the sequence SEQ ID NO: 3 at 25 mg / kg and 2.24 ± 0.49 mm ³ for the control group. These values are significantly different.

The T / C% of the group treated with the peptide incorporating the sequence SEQ ID NO: 3 decreased from 86.73% to 69.82% between J6 and J10 confirming that this peptide has antitumor activity.

In addition, the doubling time (TD), ie the time required for a double volume tumor, was measured during the exponential growth phase of tumors between 850 and 1750 mm ³ . The TD values were 5.77 ± 2.20 days for the control group and 9.81 ± 2.91 days for the peptide-treated group incorporating the sequence SEQ ID NO: 3 at 25 mg / kg. These values are significantly different and show an effect of the peptide incorporating the sequence SEQ ID NO: 3.

The SGD values are also significantly different with an SGD value of 0.00 ± 0.38 for the control group and 0.70 ± 0.50 for the group treated with the peptide incorporating the sequence SEQ ID NO: 3.

Consequently, the analysis of the T / C, VTM, VTMR, TD, SGD parameters, whose values are significantly different between the groups, made it possible to demonstrate a significant antitumor activity of the peptide incorporating the sequence SEQ ID NO: 3 administered by intra-tumoral injection (IT) at 25 mg / kg. In addition, it is important to note that this peptide has proved very effective despite the relatively large tumor volumes (approximately 800 mm ³ ) at the time of treatment.

c) In vivo anti-tumor efficacy of a peptide comprising the sequence

SEQ ID NO: 2 (peptide named K092B)

To evaluate the antitumor efficacy of the peptide incorporating the sequence SEQ ID NO: 2 against ovarian tumors, tumor cells SK-OV3 were injected at a rate of ^e 10x10 cells / 200 μΐ, on the right flank of mice Nude. When the tumor volumes reached an average of 325 mm ³ , the mice were randomized and treated 4 times with one treatment every 2 days at 25 mg / kg and 130 mg / kg intratumoral injection (IT). The tumor volume of each animal was measured twice a week for 11 days. The activity of the peptide is evaluated by the mean tumor volume (VTM) (Fig. 37) and the ratio T / C% (median tumor volume treated group / median tumor volume negative control group) (Fig. 38). The mice treated with the peptide incorporating the sequence SEQ ID NO: 2 (in IT) are compared with a control group receiving solvent whose pH has been adjusted to that of this peptide in solution in 0.9% NaCl (IT).

During the study, two tumors of mice treated with the peptide incorporating the sequence SEQ ID NO: 2 disappeared, including a tumor of a mouse treated with the peptide injected at 25 mg / kg and a tumor treated with the peptide injected at 130 mg / kg.

At pH1, the control group VTMR and peptide-treated groups incorporating SEQ ID NO: 2 at 130 mg / kg and 25 mg / kg were 2.23 ± 0.25, 1 , 52 ± 0.61 and 1.69 ± 0.91 mm ³ , respectively.

At the same date, the T / C% parameter of the group treated with 25 mg / kg of peptide incorporating the sequence SEQ ID NO: 2 was 107.03% and that of the group treated with 130 mg / kg of this same peptide was of 72.90%. This analysis confirms the antitumor activity of this peptide injected at a rate of 130 mg / kg.

In addition, the doubling time (TD) was measured during the exponential growth phase of the tumors between 500 and 1000 mm ³ . These TD values of the control groups, peptide at 130 mg / kg and peptide at 25 mg / kg were 10.50 ± 3.56, 13.04 ± 2.85 and 8.54 ± 1.97 days, respectively.

SGD values were 0.00 ± 0.34 for the control group, 0.24 ± 0.27 for the peptide group at 130 mg / kg and -0.19 ± 0.19 for the peptide group at 25 mg / kg.

The TTRV (600 mm ³ ) values were 9.20 ± 6.72 days for the control group, 11.75 ± 5.91 days for the peptide group at 130 mg / kg and 7.25 ± 5, 19 days for the peptide group at 25 mg / kg.

Therefore, even if no difference was significant, the study of the parameters TD, VTM, VTMR, T / C% and TTRV made it possible to demonstrate an antitumor activity of the peptide incorporating the sequence SEQ ID NO: 2 administered. by intratumoral injection at a dose of 130 mg / kg. This conclusion is reinforced by the fact that the tumor of a mouse treated at a dose of 130 mg / kg (IT) with the peptide incorporating the sequence SEQ ID NO: 2 disappeared completely during the course of the study.

EXAMPLE 5 Characterization of the Antidiabetic Efficacy of the Peptides SEQ ID NO: 1 (peptide named K092A) and SEQ ID NO: 2 (peptide named K092B)

The RIN-5F cell line is derived from rat pancreatic beta islets. This cellular model, capable of producing and secreting insulin, is used to study the anti-diabetic effects of molecules. The RIN-5F cells are inoculated into 24-well plates at a rate of 2x10 ⁵ / well / 1000 μl of culture medium. The cells are then incubated at 37 ° C. under a humid atmosphere at 5% CO ₂ for 96 hours. After 96h, the wells are rinsed with KRB buffer and the cells are incubated for 40 minutes at 37 ° C. After 40 minutes, the cells are incubated for 20 minutes at 37 ° C. in the presence of KRB buffer and in the presence or absence of the test substances. At the end of the incubation, the supernatants are recovered and then centrifuged at 1100 rpm for 5 min to pellet the cells present. The insulin assay in the supernatants is performed using the "ELIT® rat insulin" kit based on an ELISA assay.

Figs. 39 to 40 illustrate the effects on the secretion of insulin of the peptides consisting of the sequence SEQ ID NO: 1 or of the sequence SEQ ID NO: 2, in comparison with a positive control; repaglinide on the RIN-5F line as a function of the concentration of said peptide expressed in μg / μl. The results are expressed as a percentage.

The effects of peptides on insulin secretion by RIN-5F are shown below:

- SEQ ID NO: 0.5 and 2.5 μg / μL concentrations induce insulin secretion in a dose-dependent manner

- SEQ ID NO: 2: only the concentration 2.5 μg / μL induces the secretion of insulin SEQUENCE LISTING

<110> KELIA

<120> peptides isolated and purified from dogfish testicles

<130> 16425 AM / OC / RT

<150> FR 11/52786

<151> 201 1-04-01

<160> 4

<170> Patentln version 3.3

<210> 1

<21 1> 17

<212> PRT

<213> Scyliorhinus canicula

<220>

<221> PEPTIDE

<222> (1) .. (17)

<400> 1

Asn Phe Asp Asp Asp Glu Gin Ala Leu Asp Glu Val Phe Ser Lys Tyr

1 5 10 15

Gly

<210> 2

<211> 12

<212> PRT

<213> Scyliorhinus canicula

<220>

<221> PEPTIDE

<222> (1) .. (12)

<400> 2

Glu Ala Pro Glu Pro Ala Glu Glu Glu Asp Glu Trp

1 5 10

<210> 3

<211> 6

<212> PRT

<213> Scyliorhinus canicula <220>

<221> PEPTIDE

<222> (1) .. (6)

<400> 3

Cys Asn Arg Arg Arg Val

1 5

<210> 4

<21 1> 19

<212> PRT

<213> Scyliorhinus canicula

<220>

<221> PEPTIDE

<222> (1) .. (19)

<400> 4

Gin Leu Thr Pro Glu Ala Leu Ala Asp Glu Glu Glu Met Asn Ala Leu 1 5 10 15

Ala Ala Arg

Claims

1) Peptide isolated and purified for use as a medicament, said peptide having a size of less than 100 amino acids, being obtained by a method of extraction, purification and fractionation according to the size of the testicle protein of flying foxes belonging to the Selacian class.

2) Peptide according to claim 1 for its use in the prevention and / or treatment of cancer.

3) Peptide according to claim 1 for its use in the prevention and / or treatment of metabolic diseases such as diabetes.

4) Peptide according to claim 2, for its use in the prevention and / or treatment of colon cancer, hormone-dependent breast cancer, acute promyelocytic leukemia, ovarian cancer, melanoma, cancer. lung, prostate cancer.

5) Peptide according to one of claims 1 to 4, characterized in that it has a size less than or equal to 20 amino acids and / or a molecular mass less than or equal to 10 KDa.

6) Peptide according to one of claims 1 to 5, characterized in that it comprises, consists of or is derived from, an amino acid sequence selected from the sequences SEQ ID NO: 1, SEQ ID NO: 2 , SEQ ID NO: 3 or SEQ ID NO: 4.

7) Peptide according to claim 6, characterized in that it comprises, is derived from, or consists of the amino acid sequence SEQ ID NO: 1 for its use in the prevention and / or treatment of hormonal human breast carcinoma -dependent, acute promyelocytic leukemia or prostate cancer, preferentially hormone-dependent human breast carcinoma and in the prevention and / or treatment of metabolic diseases such as diabetes.

8) Peptide according to claim 6, characterized in that it comprises, is derived from, or consists of the amino acid sequence SEQ ID NO: 2 for its use in the prevention and / or treatment of human adenocarcinoma ovarian, human melanoma, prostate cancer, acute promyelocytic leukemia, human hormone-dependent breast carcinoma or human lung carcinoma, preferentially human adenocarcinoma ovarian and prostate cancer and in the prevention and / or treatment of metabolic diseases such as diabetes.

9) Peptide according to claim 6, characterized in that it comprises, is derived from, or consists of the amino acid sequence SEQ ID NO: 3 for its use in the prevention and / or treatment of human melanoma, human small cell lung carcinoma or acute promyelocytic leukemia, preferentially human melanoma.

10) Peptide according to claim 6, characterized in that it comprises, is derived from, or consists of the amino acid sequence SEQ ID NO: 4 for its use in the prevention and / or treatment of human adenocarcinoma of the colon, acute promyelocytic leukemia or small-cell human lung carcinoma, preferentially human adenocarcinoma of the colon.

11) Peptide according to one of the preceding claims, characterized in that the fruit bat is Scyliorhinus canicula.

12) A delivery vector comprising at least one peptide as defined in one of the preceding claims.

13) A pharmaceutical composition comprising, in a pharmaceutically acceptable carrier, at least one peptide as defined in one of claims 1 to 11.

14) Pharmaceutical composition according to claim 13, characterized in that it comprises at least one peptide comprising, derived from, or consisting of the sequence SEQ ID NO: 1 for the prevention and / or treatment of hormone-dependent human breast carcinoma , promyelocytic acute leukemia or prostate cancer, preferentially hormone-dependent human breast carcinoma, or in the prevention and / or treatment of metabolic diseases such as diabetes.

15) Pharmaceutical composition according to claim 13, characterized in that it comprises at least one peptide comprising, derived from, or consisting of, the sequence SEQ ID NO: 2 for the prevention and / or treatment of human adenocarcinoma of ovarian, human melanoma, prostate cancer, acute promyelocytic leukemia, hormone-dependent human breast carcinoma or human lung carcinoma, preferentially human ovarian adenocarcinoma and prostate cancer, or in the prevention and / or treatment of metabolic diseases such as diabetes. 16) Pharmaceutical composition according to claim 13, characterized in that it comprises at least one peptide comprising, derived from, or consisting of, the sequence SEQ ID NO: 3 for the treatment and / or prevention of human melanoma, carcinoma human small cell lung or acute promyelocytic leukemia, preferentially human melanoma.

17) Pharmaceutical composition according to claim 13, characterized in that it comprises at least one peptide comprising, derived from, or consisting of, the sequence SEQ ID NO: 4 for the treatment and / or prevention of human adenocarcinoma of the colon, acute promyelocytic leukemia or small-cell human lung carcinoma, preferentially, human colon adenocarcinoma.

18) Peptide, characterized in that it comprises, consists of or is derived from, an amino acid sequence selected from the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

19) A nucleic acid comprising a nucleic acid sequence encoding a peptide comprising, derived from, or consisting of an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleic sequence complementary to said coding sequence.

20) expression cassette, comprising at least one nucleic acid sequence according to claim 19.

21) A cloning and / or expression vector comprising at least one nucleic acid sequence according to claim 19.