ANTIMICROBIAL PEPTIDES AND USES THEREOF

Abstract

Peptides endowed with an anti-inflammatory, anti-angiogenic and/or antimicrobial activity, a medicament or a pharmaceutical composition including such peptides, and the use thereof in the prevention or treatment of inflammatory diseases, and more particularly inflammatory diseases due to or associated with microbial infections, or in the prevention or treatment of angiogenesis-related diseases.

Claims

1-12. (canceled)

13. A pharmaceutical composition or a medicament comprising: at least one peptide with an anti-inflammatory, anti-angiogenic and/or antimicrobial activity having a sequence of 17 to 100 amino acids comprising the sequence A-L-X1-X2-T-L-X3-K-K-V-G-X4-X5-X6-G-K-A-cp (SEQ ID NO: 29), wherein: X1 is a W or A residue; X2 is a K or D residue; X3 is an L or A residue; X4 is a K, A or P residue; X5 is a V or A residue; X6 is an A, K or D residue; φ is X7-L-N-A (SEQ ID NO: 30), X7-L-N-A-V-T-N-X8-A (SEQ ID NO: 31), X7-L-N-A-V-T-N-X8-A-N-Q-N-X9-X10 (SEQ ID NO: 32), or is absent; and wherein: X7 is a V or P residue; X8 is an M or A residue; X9 represents no residue or is an E residue; X10 represents no residue or is a Q residue; or functional derivatives and pharmaceutically acceptable salts of said peptide, wherein the peptide is not a peptide of sequence SEQ ID NO: 2, or a nucleotide sequence coding for at least one of these peptides; and one or a plurality of pharmaceutically acceptable excipients.

14. The pharmaceutical composition or medicament according to claim 13, wherein the peptide has a sequence of 29 to 100 amino acids comprising the sequence SEQ ID NO: 3, 4, 5, 6, 7 or 8.

15. A peptide with an anti-inflammatory activity and/or antimicrobial activity having a sequence of 17 to 100 amino acids comprising the sequence A-L-X1-X2-T-L-X3-K-K-V-G-X4-X5-X6-G-K-A-cp (SEQ ID NO: 29), wherein: X1 is a W or A residue; X2 is a K or D residue; X3 is an L or A residue; X4 is a K, A or P residue; X5 is a V or A residue; X6 is an A, K or D residue; φ is X7-L-N-A (SEQ ID NO: 30), X7-L-N-A-V-T-N-X8-A (SEQ ID NO: 31), X7-L-N-A-V-T-N-X8-A-N-Q-N-X9-X10 (SEQ ID NO: 32), or is absent; and wherein: X7 is a V or P residue; X8 is an M or A residue; X9 represents no residue or is an E residue; X10 represents no residue or is a Q residue; and functional derivatives and pharmaceutically acceptable salts of said peptide, wherein the peptide is not a peptide of sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

16. The peptide according to claim 15, wherein φ is X7-L-N-A-V-T-N-X8-A-N-Q-N-X9-X10 and X9 and X10 represent no residue.

17. The peptide according to claim 15 having a sequence selected from the group consisting of 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: 21, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and functional derivatives and pharmaceutically acceptable salts of said peptide.

18. A method for preventing or treating an inflammatory disease or an angiogenesis-related disease or for attenuating or suppressing the immune reactions of the organism in a subject in need thereof, said method comprising administering to the subject an effective therapeutic dose of at least one peptide having a sequence of 17 to 100 amino acids comprising the sequence A-L-X1-X2-T-L-X3-K-K-V-G-X4-XS-X6-G-K-A-φ (SEQ ID NO: 29), wherein: X1 is a W or A residue; X2 is a K or D residue; X3 is an L or A residue; X4 is a K, A or P residue; X5 is a V or A residue; X6 is an A, K or D residue; φ is X7-L-N-A (SEQ ID NO: 30), X7-L-N-A-V-T-N-X8-A (SEQ ID NO: 31), X7-L-N-A-V-T-N-X8-A-N-Q-N-X9-X10 (SEQ ID NO: 32), or is absent; and wherein: X7 is a V or P residue; X8 is an M or A residue; X9 represents no residue or is an E residue; X10 represents no residue or is a Q residue; or functional derivatives and pharmaceutically acceptable salts of said peptide.

19. The method according to claim 18, wherein the peptide is not a peptide of SEQ ID NO: 2.

20. The method according to claim 18, wherein the method is for preventing or treating an inflammatory disease.

21. The method according to claim 20, wherein the peptide has a sequence of 29 to 100 amino acids comprising the sequence SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8.

22. The method according to claim 20, wherein the inflammatory disease is selected from the group consisting of rosacea, psoriasis, eczema, contact dermatitis and impetigo.

23. The method according to claim 20, wherein the inflammatory disease is due to an infection.

24. The method according to claim 18, wherein the method is for preventing or treating an angiogenesis-related disease.

25. The method according to claim 24, wherein the peptide has a sequence of 29 to 100 amino acids comprising the sequence SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8.

26. The method according to claim 24, wherein the angiogenesis-related disease is selected from the group consisting of cancers, retinopathy type ophthalmological disorders, atherosclerosis, arthrosis, and rheumatoid arthritis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0836] FIG. 1 shows the survival of mouse and human neutrophils (A), T lymphocytes (B), monocytes (C) and NK cells (D), in the presence of increasing concentrations of DA2N and DA2NEQ, as a percentage with respect to the number of live cells in the absence of peptide.

[0837] FIG. 2 shows the survival of mouse and human non-immune cells (epithelial cells), in the presence of increasing concentrations of DA2N and DA2NEQ peptide, as a percentage with respect to the number of live cells in the absence of peptide.

[0838] FIG. 3 shows the haemolytic activity of DA2N and DA2NEQ at 10 .Math.Mand 50 .Math.M, on human red blood cells, as a percentage with respect to the positive control consisting of Triton X100 (100%).

[0839] FIG. 4 shows the number of immune cells in the peritoneal cavity 36 hours after 3 regular intraperitoneal (i.p.) injections of DA2N (dark grey) or DA2NEQ (light grey) peptide in a mouse model of peritonitis induced by i.p. injection of thioglycolate 3%. The negative control with respect to the peptide consists of injecting 0.15 M NaCl instead of the solution containing the peptide (black). The peritonitis induction control consists of mice non-induced for peritonitis (white); i.p. injection of NaCl.

[0840] FIG. 5 shows the protocol used to test the efficacy of the peptides in a contact eczema model.

[0841] FIG. 6 shows the relative variation in ear thickness in a mouse contact eczema model induced with DNCB 1%, after administering DA2N (A) or DA2NEQ (B). The contact eczema induction control corresponds to mice not treated with DNCB1% (line with triangle symbol) and the negative control corresponds to mice treated with peptide-free DNCB1% (line with square symbol).

[0842] FIG. 7 shows, in a mouse contact eczema model, the effect of administering DA2N on the number of immune cells (A) at the level of the ear, (B) in the systemic circulation and (C) in the spinal cord, after inducing contact eczema by applying DNCB1% (light grey). The contact eczema induction control corresponds to mice not treated with DNCB1% (white) and the negative control corresponds to mice treated with peptide-free DNCB1% (dark grey).

[0843] FIG. 8 shows representative images of the effect of a topical application of DA2N and DA2NEQ peptides in Balb/c mice on psoriatic skin lesions. The images of the left-hand column represent the results on psoriatic skin (skin treated with Imiquimod for 6 days): positive control with no peptide application (A), application of DA2N peptide (B) and application of DA2NEQ peptide (C). The images of the right-hand column represent the results on healthy skin (with no Imiquimod treatment): negative control with no peptide application (A), application of DA2N peptide (B) and application of DA2NEQ peptide (C).

[0844] FIG. 9 represents an enlargement at the level of the treated regions representative of the skin lesions shown in FIG. 8.

[0845] FIG. 10 shows representative images of the effect of a topical application of DA2N and DA2NEQ peptides on angiogenesis conventionally observed on balb/c mice on psoriatic lesions. The images of the left-hand column represent the results on psoriatic skin (skin treated with Imiquimod for 6 days): positive control with no peptide application (A), application of DA2N peptide (B) and application of DA2NEQ peptide (C). The images of the right-hand column represent the results on healthy skin (with no Imiquimod treatment): negative control with no peptide application (A), application of DA2N peptide (B) and application of DA2NEQ peptide (C).

[0846] FIG. 11 shows representative images of aortic rings and of neo-vessel formation after 3 days of treatment with PBS (A), DA2N (B), DA2NEQ (C).

[0847] FIG. 12 shows neo-vessel formation inhibition over time on mouse aortas after adding DA2N and DA2NEQ at the concentration of 12.5 .Math.M, as a percentage with respect to the initial diameter of the aortic rings.

[0848] FIG. 13 shows the survival of mouse bone marrow cells after 2 hours of incubation in the presence of DA2N, DA2NE, N(D), DA2Na, K12A peptides at a concentration of 12.5 .Math.Mor 25 .Math.M, as a percentage with respect to the number of initial live cells. The positive and negative controls consist, respectively, of the incubation of the cells in the presence of DMSO 30% or PBS.

[0849] FIG. 14 shows the survival of mouse non-immune cells (epithelial cells) in the presence of increasing concentrations of DA2N, K12A and DA2Na. DA2N, DA2Na and K12A induce the death of 50% of mouse immune cells originating from bone marrow (LC.sub.50) at a concentration of 5 .Math.M, 3.75 .Math.M and 6.5 .Math.M, respectively.

[0850] FIG. 15 shows the haemolytic activity of DA2Na and K12A peptides at 10 .Math.M and 50 .Math.M on mouse red blood cells, as a percentage with respect to the positive control consisting of Triton X100 (100%).

EXAMPLES

Peptide Synthesis

[0851] The peptides used in the examples were synthesised with the FMOC/tBu strategy on solid substrate by an Activo P11 automatic synthesiser (Activotec). Purification is carried out by HPLC (Waters, C18 column) and peptide identity is verified by MALDI-TOF spectrometry (MS Voyager Applied Biosystems). Due to the presence of a tryptophan residue in the sequence, the concentrations of the solutions are determined by UV spectroscopy (Nanodrop, Labtech. Com).

Example 1: In Vitro Study of the Immunosuppressant Activity of DA2N and DA2NEQ

[0852] The cytotoxic effects of DA2N (SEQ ID NO: 4) and DA2NEQ (SEQ ID NO: 2) were evaluated on mouse and human immune cells. In order to demonstrate that the cytotoxic effects are specific to immune cells, the cytotoxic effects were also evaluated on non-immune cells.

1.1 Materials and Methods

[0853] The mouse immune cells are obtained from C57BL6 mouse bone marrow. The human immune cells were purified from the whole blood of healthy donors (blood bank, IMSS, Cuernavaca, Mexico).

[0854] The following non-immune cells were extracted after sacrificing C57BL6 mice or in a surgical context: epithelial cells, adipocytes, hepatocytes.

[0855] The peptides are incubated at 37° C., 5% CO2 in the presence of 500,000 immune and non-immune cells in RPMI medium at varied concentrations and at varied times. Peptide-free cells are used as the negative control and cells incubated for 5 min at 50° C. or in the presence of DMSO (30%) are used as the positive control. After incubation, the cells are washed then labelled with propidium iodide so as to measure the cell viability. The cell viability is analysed by counting or with flow cytometry in the presence of antibodies coupled with fluorochromes corresponding to characteristic markers of each cell population. The LC.sub.50 is then calculated as being the concentration of DA2N and DA2NEQ peptides inducing the death of 50% of each one of the immune cell populations.

[0856] Cytometry data processing is carried out with FlowJo software.

1.2 Results

[0857] The survival of mouse and human immune cells in the presence of increasing concentrations of DA2N and DA2NEQ is shown in FIG. 1.

[0858] The survival of mouse and human non-immune cells in the presence of increasing concentrations of DA2N and DA2NEQ is shown in FIG. 2.

[0859] By comparing the results shown in FIG. 1 and in FIG. 2, the results obtained demonstrate that DA2N and DA2NEQ, at doses of the micromolar order, induce the death of immune cells but not of non-immune cells.

[0860] Table 1 shows the DA2N and DA2NEQ peptide concentrations inducing the death of 50% of mouse and human immune cells (LC.sub.50).

TABLE-US-00028 LC.sub.50 on immune cells. LC50 (.Math.M) DA2N DA2NEQ Human cells Mouse cells Human cells Mouse cells Neutrophils 7.5 .Math.M 6 .Math.M 7 .Math.M 7.5 .Math.M T lymphocytes 4 .Math.M 3 .Math.M 5 .Math.M 5 .Math.M Monocytes 4 .Math.M 5 .Math.M 4 .Math.M 7 .Math.M NK cells 4 .Math.M 3 .Math.M 4 .Math.M 4 .Math.M

[0861] Thus, these results demonstrate that the peptides according to the invention are endowed with an immunosuppressant activity.

Example 2: In Vitro Study of the Antimicrobial Activity of DA2N and DA2NEQ

2.1 Materials and Methods

Measurement of Antibacterial Activity

[0862] The following strains were used for the antibacterial tests:

[0863] Escherichia coli (ATCC 8739, ATCC ML35p and P7 BLSE), Pseudomonas aeruginosa (ATCC 9027 and ATCC 27853), Klebsiella oxytoca (CIP 7932), Klebsiella pneumoniae (CIP 52.211), Salmonella enterica (CIP 8297), Yersinia ruckeri (ATCC 29473), Vibrio parahemolyticus (IFREMER 01 01/252), Staphylococcus aureus (ATCC 6538, ST 1065 and MRSA), Staphylococcus epidermidis (BM 3302), Listeria monocytogenes (SOR 100), Enterococcus faecalis (CIP A186, CIP 103015)), Kocuria rhizophila (ATCC 9341), Lactococcus garviae (ATCC 43921), Bacillus subtilis (CIP 52.65).

[0864] For each strain, a 5 ml pre-culture in LB medium is prepared by inoculating a colony of the bacterial strain of interest. The pre-culture is incubated at 37° C. under stirring overnight. The culture is subcultured (30 .Math.L) in 5 mL. The culture concentration is evaluated by measuring the optical density at 620 nm. The concentration is adjusted by dilution so as to obtain a suspension with an OD of 0.05 at 620 nm.

Measurement of Minimum Inhibitory Concentration (MIC)

[0865] The MIC is determined using a growth inhibition test in liquid medium. The MIC is defined as being the lowest peptide concentration capable of inhibiting the bacterial strain tested, after incubation for 18 h at 37° C. The test was carried out in a sterile microtitration plate containing 96 wells. A series of increasing concentrations of each peptide (0.2 to 100 .Math.M) was prepared beforehand in sterile MilliQ water. In each well, 10 .Math.L of peptide was incubated with 90 .Math.L of bacterial suspension (10.sup.7 cfu/mL) in yeast extract-free LB medium. The microplate was incubated at 37° C. for 18 h. The bacterial growth was evaluated by measuring the OD at 620 nm using a plate reader.

[0866] The negative growth inhibition control was obtained by replacing the solution containing the peptide by sterile MilliQ water. The positive control suitable for completely inhibiting the growth of the bacterial strains was obtained by replacing the solution containing the peptide by 10 .Math.L of formaldehyde.

[0867] The results are interpreted by calculating the percentage of bacterial growth in each well according to the formula:

[0868] %Growth = (OD well - OD of T.sub.0)/(OD of T.sub.100-OD of T.sub.0)*100 where

[0869] OD of T.sub.100 = OD of growth control (bacterial suspension free from sample under test, 100% growth), [0870] OD of T.sub.0 = OD of sterility control (culture medium + bacterium + 10 .Math.L formaldehyde, 0% growth) and [0871] OD well = OD of well for which it is sought to calculate the growth percentage (bacterial suspension + peptide under test).

Measurement of Minimum Bactericidal Concentration (MBC)

[0872] The MBC is defined as being the minimum peptide concentration leaving no surviving bacteria of the inoculum after incubation at 37° C. for 24 hours. This test consists of smearing the contents of the well wherein the peptide has inhibited bacterial growth on a Petri dish (LB agar), followed by incubation for 24 h at 37° C. The inhibition is referred to as bacteriostatic when the peptide inhibits bacterial growth but does not lyse the bacteria and it is referred to as bacteriolytic when the peptide has completely lysed the bacteria and therefore prevents regrowth of the bacteria in the culture medium after subculturing.

2.2 Results

[0873] The antimicrobial activity of DA2N and DA2NEQ was evaluated on various Gram-positive and Gram-negative reference bacterial strains. The results are shown in Table 2:

TABLE-US-00029 Antimicrobial activities of DA2N and DA2NEQ MIC (.Math.M) MBC (.Math.M) DA2N DA2NEQ DA2N DA2NEQ Gram-negative Escherichia coli ATCC 8739 3.12 3.12 3.12 6.25 Escherichia coli ML35p 3.12 6.25 6.25 6.25 Pseudomonas aeruginosa ATCC 9027 12.5 12.5 12.5 25 Klebsiella oxytoca CIP 7932 3.12 12.5 6.25 >100 Klebsiella pneumoniae CIP 5211 3.12 3.12 Gram-positive Staphylococcus aureus ATCC 6538 6.25 12.5 6.25 12.5 Staphylococcus aureus ST 1065 12.5 >100 12.5 >100 Staphylococcus epidermidis BM 3302 1 >100 12.5 >100 Kocuria rhizophila ATCC 9341 3.12 6.25 3.12 6.25 Bacillus subtilis CIP 52.65 6.25 6.25 6.25 6.25

[0874] These results demonstrate that DA2N and DA2NEQ exhibit an antimicrobial activity on Gram-positive and Gram-negative bacteria. Note that the antimicrobial activity of DA2N is generally greater than that of DA2NEQ, particularly with respect to Escherichia coli P7 (ESBL). This result is of particular interest given that this strain is resistant to most β-lactamases.

Example 3: In Vitro Study of the Haemolytic Activity of DA2N and DA2NEQ

3.1 Materials and Methods

Measurement of Haemolytic Activity

[0875] The haemolytic activity of antimicrobial peptides was demonstrated using mouse or human red blood cells from healthy adult donors. Mouse or donor blood is washed three times in 10 volumes of PBS at 4° C. by centrifugation at 800 g for 10 minutes. The blood sediment is finally diluted to 4% (v/v) in PBS buffer. The test was carried out as follows: either 10 .Math.M, or 50 .Math.M of peptide was added to 100 .Math.L of red blood cell suspension.

[0876] After 1h of incubation at 37° C. followed by centrifugation (800 g, 15 min), the absorbance of the supernatant was measured at 550 nm.

[0877] The negative control for this test (0% haemolysis) contains 5 .Math.L of PBS buffer instead of the solution containing the peptide and the positive control (100% haemolysis) contains 5 .Math.L of 1% Triton X100 instead of the solution containing the peptide.

[0878] Measurement of LC.sub.50

[0879] The mouse red blood cells are obtained from C57BL6 mouse bone marrow.

[0880] The human red blood cells were purified from the whole blood of healthy donors (blood bank, IMSS, Cuernavaca, Mexico).

[0881] The peptides are incubated at 37° C., 5% CO.sub.2 in the presence of 500,000 red blood cells in RPMI medium at varied concentrations and at varied times. Peptide-free cells are used as the negative control and cells incubated for 5 min at 50° C. or in the presence of DMSO (30%) are used as the positive control. The LC.sub.50 is then calculated as being the concentration of DA2N and DA2NEQ peptides inducing the death of 50% of the red blood cells.

3.2 Results

[0882] FIG. 3 shows the percentage of haemolysis of human red blood cells by DA2N and DA2NEQ peptides, with respect to the positive control with reference to 100%. The results show that the DA2N and DA2NEQ peptides do not induce the haemolysis of human (FIG. 3) or mouse (results not shown) red blood cells.

[0883] Table 3 shows the DA2N and DA2NEQ peptide concentrations inducing the death of 50% of human and mouse red blood cells (LC.sub.50).

TABLE-US-00030 LC.sub.50 on red blood cells LC50 (.Math.M) DA2N DA2NEQ Human red blood cells Mouse red blood cells Human red blood cells Mouse red blood cells 750 .Math.M 750 .Math.M 750 .Math.M 750 .Math.M

[0884] Thus, these results demonstrate that the peptides according to the invention have a negligible haemolytic activity.

Example 4: In Vivo Study of the Anti-inflammatory Activity of DA2N and DA2NEQ

4.1 Materials and Methods

[0885] The anti-inflammatory activity of DA2N and DA2NEQ was evaluated in a mouse model of non-infectious peritonitis. Non-infectious peritonitis is induced by intraperitoneal injection of thioglycolate 3%. Six hours after inducing peritonitis, three intraperitoneal injections of peptide (90 .Math.g/injection/mouse) were carried out at regular intervals (every 6 h). At 36 h, the mice are sacrificed in order to observe the immune cell recruitment in the peritoneal cavity. The cells obtained from washing the peritoneal cavity are washed and then labelled with propidium iodide in order to measure the cell viability and in the presence of antibodies coupled with fluorochromes corresponding to characteristic markers of each cell population. The evaluation of the anti-inflammatory potential is based on the measurement of the number of neutrophils, monocytes and macrophages recruited in the mouse peritoneal cavity. The negative control with respect to the peptide for this test consists of 50 .Math.L of 0.15 M NaCl instead of the solution containing the peptide. The peritonitis induction control consists of mice non-induced for peritonitis. Data processing is carried out with FlowJo software.

4.2 Results

[0886] FIG. 4 shows the number of immune cells in the peritoneal cavity in the mouse model of thioglycolate-induced peritonitis, and therefore the effect of DA2N and DA2NEQ in an inflammatory disease model. The results demonstrate that, in vivo, the peptides induce a reduction in neutrophil and monocyte/macrophage recruitment in the peritoneal cavity after inducing peritonitis. The peptides according to the invention therefore have an anti-inflammatory activity.

[0887] In the light of the in vitro results, the inventors consider, without wishing to be bound to any theory, that the reduction in recruitment observed is due to the death of the immune cells under the effect of the DA2N and DA2NEQ peptides.

Example 5: In Vivo Study of DA2N and DA2NEQ Activity in a Contact Eczema Model

5.1 Materials and Methods

[0888] The study is carried out to determine the activity of the peptides DA2N (SEQ ID NO: 4) and DA2NEQ (SEQ ID NO: 2) with respect to contact sensitisation. Mice are sensitised with DNCB 1% for 5 days. After this sensitization phase, 30 .Math.L of a 1:1 olive oil/acetone mixture (vehicle) wherein 50 .Math.g of DA2N or DA2NEQ peptide is dissolved is applied at the level of the ear. Five hours after applying the peptide, the contact reaction is induced by re-exposure to DNCB 1 % at the level of the ear. The intensity of the inflammatory reaction is evaluated locally 24, 48, 72 and 96 hours after re-exposure to DNCB by measuring ear thickness. Moreover, the number of immune cells (dendritic cells, T lymphocytes, neutrophils, monocytes, macrophages) is measured 24 h after re-exposure to DNCB in the ear at the level of the DNCB 1% application site (number of immune cells counted in both ears), in the blood (number of immune cells counted in 50 .Math.L of blood) and in the bone marrow (number of immune cells counted in a tibia). The protocol is represented schematically in FIG. 5.

[0889] In order to count the number of cells, the ears are digested by adding collagenase IV and the number of immune cells is determined by flow cytometry using antibodies coupled with fluorochromes corresponding to characteristic markers of each cell population. The negative contact reaction inhibition control was obtained by replacing the solution containing the peptide with the 1:1 olive oil/acetone solution (indicated as “vehicle” in FIG. 6, and with the symbol “-” in FIG. 7).

5.2 Results

[0890] The results show a reduction in ear thickness (FIGS. 6A and B) after topical application of DA2N or DA2NEQ. Furthermore, FIG. 7 shows a reduction in the number of monocytes, dendritic cells and neutrophils, whether at the level of the ear (FIG. 7A), in the blood (FIG. 7B) or in the bone marrow (FIG. 7C) of the mice after topical application of DA2N.

[0891] The peptides according to the invention therefore have an anti-inflammatory effect in an in vivo inflammatory disease model.

Example 6: In Vivo Study of DA2N and DA2NEQ Activity in a Psoriasis Model

6.1 Materials and Methods

[0892] The DA2N and DA2NEQ peptides were tested in an Imiquimod-induced psoriasis model described by Van der Fits (Van der Fits et al. 2009. Journal of immunology. 182(9):5836-5845) on female balb/c mice, aged 7 to 8 weeks.

[0893] 31 mg of commercial cream containing 5% Imiquimod (Aldara, 3 M Pharmaceuticals) is applied topically on about 2.5 cm of the previously shaven ventral region of the mice for 6 days. The first psoriatic lesions appear on the 3rd day. The peptides resuspended in the cream are applied topically or intravenously (10 .Math.g) for the first 3 days of Imiquimod treatment. Mice with no Imiquimod treatment and/or with no peptide application were used as controls. On the 6th day, the mice are sacrificed and the skin sampled is analysed by microscopy (skin lesions and angiogenesis).

6.2 Results

[0894] FIGS. 8 and 9 (enlargement) show representative images of a topical treatment with DA2N and DA2NEQ at a dose of 10 .Math.g on psoriatic lesions induced by applying Imiquimod for 6 days. Compared to the positive control, DA2N reduces the psoriatic lesions conventionally observed after 6 days of Imiquimod treatment. DA2N and DA2NEQ have no effect on healthy skin.

Example 7: Study of the Anti-angiogenic Activity of DA2N and DA2NEQ

7.1 Materials and Methods

[0895] The DA2N and DA2NEQ peptides were tested on the aortic ring model making it possible to study the influence of various molecules on new blood vessel formation. The aortas of C57BL/6 or Balb/c mice are sampled, washed with PBS then cut into rings. The rings are then deposited in a Matrigel matrix (BD® Matrigel™) in 24-well plates, incubated for 1h at 37° C., 5% CO.sub.2, before adding 1 mL of DMEM (DMEM Advanced 1X, Gibco) supplemented with 5% foetal calf serum (FCS), 2 .Math.M of glutamine, 100 U of penicillin and 50 pg/mL of streptomycin. The rings are then incubated at 37° C., 5% CO.sub.2 for 3 days. The medium is then removed and replaced by medium containing or not the peptides at a concentration of 12.5 .Math.M. The aortic rings are once again incubated for 3 days under the same conditions. Photos of the rings are taken each day to measure neo-vessel formation

[0896] Image processing and the measurement of the area formed by the neo-vessels are carried out using Image J 1.50i software.

7.2 Results

[0897] FIG. 10 shows representative images of a topical treatment with DA2N and DA2NEQ at a dose of 10 .Math.g on angiogenesis, conventionally observed after applying Imiquimod for 6 days. Compared to the positive control, DA2N and DA2NEQ appear to reduce neo-vessel formation after 6 days of Imiquimod treatment. DA3N and DA2NEQ have no effect on the angiogenesis of healthy skin.

[0898] FIG. 11 shows representative images showing neo-vessel formation (angiogenesis) on D0, D1, D2 and D3 from aortic rings, after adding DA2N or DA2NEQ on D0. The control corresponds to neo-vessel formation, after adding only PBS. FIG. 12 shows the neo-vessel formation as a percentage with respect to the initial diameter of the aortic ring. Compared to the control (PBS), adding DA2N or DA2NEQ inhibits neo-vessel formation significantly.

Example 8: In Vitro Study of the Immunosuppressant Activity of DA2NE, DA2N (D), K12A and DANa

[0899] The cytotoxic effects of DA2NE (SEQ ID NO: 3), DA2N (D) (SEQ ID NO: 19), K12A (SEQ ID NO: 11) and DANa (SEQ ID NO: 7) were evaluated on immune cells obtained from mouse bone marrow.

8.1 Materials and Methods

[0900] The mouse immune cells are obtained from C57BL/6 or Balb/c mouse bone marrow. The peptides are incubated at 37° C., 5% CO.sub.2 in the presence of 500,000 immune cells in RPMI medium at varied concentrations for 2 hours. Peptide-free cells are used as the negative control and cells in the presence of DMSO (30%) are used as the positive control. After incubation, the cells are washed then labelled with Trypan blue so as to measure the cell viability by microscope counting. The LC.sub.50 is then calculated as being the concentration of DA2N, K12A and DANa peptides inducing the death of 50% of the immune cells.

8.2 Results

[0901] The survival of the immune cells obtained from mouse bone marrow in the presence of DA2NE, DA2N (D), K12A and DANa peptides at the concentrations of 12.5 and 25 .Math.M is shown in FIG. 13 and compared to DA2N. The DA2NE, DA2N (D), K12A and DANa peptides appear to induce the death of immune cells at a concentration of 12.5 .Math.M with a comparable efficacy to DA2N.

[0902] The survival of mouse non-immune cells in the presence of increasing concentrations of DA2N, K12A and DA2Na is shown in FIG. 14. DA2N, DA2Na and K12A induce the death of 50% of mouse immune cells originating from bone marrow (LC.sub.50) at a concentration of 5 .Math.M, 3.75 .Math.M and 6.5 .Math.M, respectively.

Example 9: In Vitro Study of the Haemolytic Activity of DA2NE, DA2N (D), K12A and DANa

9.1 Materials and Methods

Measurement of Haemolytic Activity

[0903] The haemolytic activity of the antimicrobial peptides was measured as per the protocol described in part 3.1 of Example 3.

9.2 Results

[0904] FIG. 15 shows the percentage of haemolysis of mouse red blood cells after incubation with the peptides for 1 hours, with respect to the positive control with reference to 100%. The results show that the DA2Na and K12A peptides do not exhibit significant haemolytic activity (less than 40% haemolysis) at the concentrations of 10 and 50 .Math.M.

Example 10: In Vitro Study of the Antimicrobial Activity of DA2NE, DA2Na, K4D, L7A, K12A, K12P, V13A, A14D, A14K, M25A, DA2NEQ (D) and DA2N (D)

[0905] The antimicrobial activity of DA2N (SEQ ID NO: 4), DA2NEQ (SEQ ID NO: 2), DA2NE (SEQ ID NO: 3), DA2Na (SEQ ID NO: 7), K4D (SEQ ID NO: 13), L7A (SEQ ID NO: 14), K12A (SEQ ID NO: 11), K12P (SEQ ID NO: 12), V13A (SEQ ID NO: 17), A14D (SEQ ID NO: 10), A14K (SEQ ID NO: 8), M25A (SEQ ID NO: 16), DA2NEQ (D) (SEQ ID NO: 18) and DA2N (D) (SEQ ID NO: 19) was evaluated on various microbial strains.

[0906] ScrL (NLGAKNQVAWEKKAATTKGNVNLKLLQMVAV; SEQ ID NO: 37) and ScrC (KGNVLVKAAMLTKVLAKNKGVQNANALTW; SEQ ID NO: 38) are random sequences of the original peptides used as negative controls.

[0907] Melittin (CAS number: 37231-28-0) and Gramicidin (CAS number: 1405-97-6) are used as positive controls.

10.1 Materials and Methods

Preparation of Bacterial Suspensions

[0908] A 10 mL preculture in Muller-Hinton (MH) medium is prepared by inoculating a colony of the bacterial strain of interest. The preculture is incubated at 37° C. under stirring overnight. The culture is subcultured to 1:100 under the same conditions. The culture concentration is evaluated by measuring the turbidity at 600 nm (OD600 nm 1.0 = 5 × 10.sup.8 CFU/mL for E. coli (Gram-negative bacteria) and 1.5 × 10.sup.8 CFU/mL for S. aureus (Gram-positive bacteria). Once the exponential growth phase (OD600 nm = 0.3 - 0.7) has been achieved, the cultures are diluted in MH medium so as to obtain 10.sup.6 CFU/mL (final inoculum concentration).

Measurement of MIC and MBC

[0909] The MIC and the MBC were determined as per the protocol described in part 2.1 of Example 2.

10.2 Results

[0910] The antimicrobial activity of DA2N and DA2NEQ is shown in Table 4:

TABLE-US-00031 Antimicrobial activities of DA2N and DA2NEQ (ND: not determined) DA2N DA2NEQ Melittin MIC MBC MIC MBC MIC MBC Gram-negative Escherichia coli ATCC 8739 0.8 0.8 1.6 1.6 2.5 2.5 Escherichia coli K12 3.12 6.3 12.5 12.5 10 10 Escherichia coli ML35p 0.8 0.8 1.6 1.6 10 10 Escherichia coli P7 (ESBL) 1.6 12.5 12.5 50 6.25 6.25 Pseudomonas aeruginosa ATCC 9027 3.12 3.12 6.3 6.3 5 5 Pseudomonas aeruginosa ATCC 27853 12.5 50 50 >100 50 >50 Klebsiella pneumoniae CIP 52.211 0.8 0.8 0.8 0.8 10 10 Klebsiella oxytoca CIP 7932 3.12 6.25 12.5 >100 25 50 Salmonella enterica CIP 8297 12.5 25 50 100 50 >50 Yersinia ruckeri ATCC 29473 3.12 12.5 25 >100 6.25 12.5 Vibrio parahemolyticus IFREMER 01/252 >50 >50 >100 >100 >50 >50 Gram-positive Staphylococcus aureus ATCC 6538 1.6 3.12 6.3 12.5 5 5 Staphylococcus aureus ST 1065 6.3 6.3 12.5 25 10 10 Staphylococcus epidermidis BM 3302 6.3 25 12.5 >50 10 10 Staphylococcus aureus MRSA 25 >50 100 >100 6.25 12.5 Listeria monocytogenes SOR 100 25 25 100 >100 1.5 3.12 Enterococcus faecalis CIP A186 50 >50 100 >100 12.5 12.5 Lactococcus garviae ATCC 43921 25 50 100 >100 6.25 25 Enterococcus faecalis CIP 103015 6.3 6.3 12.5 12.5 1.25 1.25 Kocuria rhizophila ATCC 9341 0.8 1.6 1.6 1.6 0.8 0.8 Bacillus subtilis CIP 52.65 6.25 6.25 25 ND ND ND

[0911] These results therefore confirm the results obtained in example 2 (Table 2) which demonstrate that DA2N and DA2NEQ have an antimicrobial activity on Gram-positive and Gram-negative bacteria.

[0912] The antimicrobial activity of DA2NE, DA2Na, K4D, L7A, K12A, K12P, V13A, A14D, A14K, M25A, DA2NEQ (D) and DA2N (D) is shown in Table 5:

TABLE-US-00032 Antimicrobial activities of DA2NE, DA2Na, K4D, L7A, K12A, K12P, V13A, A14D, A14K, M25A, DA2NEQ (D) and DA2N (D) Gram-negative Gram-positive E. coli ATCC 8739 P. aeruginosa ATCC 9027 S. aureus ATCC 6538 E. faecalis CIP 103015 MIC (.Math.M) MBC (.Math.M) MIC (.Math.M) MBC (.Math.M) MIC (.Math.M) MBC (.Math.M) MIC (.Math.M) MBC (.Math.M) DA2NE 0.8 1.6 3.1 3.1 3.1 12.5 12.5 50 DA2Na 0.8 0.8 1.6 1.6 0.8 1.6 3.1 3.1 K4D 12.5 12.5 25 25 12.5 12.5 50 50 L7A 0.8 0.8 1.6 1.6 3.1 3.1 6.3 6.3 K12A 1.6 1.6 6.3 6.3 3.1 6.3 12.5 12.5 K12P 6.3 6.3 12.5 12.5 6.3 6.3 >50 >50 V13A 1.6 1.6 6.3 6.3 6.3 12.5 50 50 A14D 6.3 6.3 50 >50 >50 >50 >50 >50 A14K 1.6 1.6 12.5 12.5 25 50 >50 >50 M25A 0.8 0.8 3.1 3.1 3.1 3.1 12.5 12.5 Scr L >50 >50 >50 >50 >50 >50 >50 >50 Scr C >50 >50 >50 >50 >50 >50 >50 >50 DA2NEQ (D) 0.8 0.8 1.6 3.1 1.6 1.6 12.5 12.5 DA2N (D) 0.4 0.8 0.8 0.8 0.8 0.8 3.1 3.1 Melittin 2.5 2.5 5 5 5 5 1.2 1.2 Gramicidin >100 >100 >100 >100 >50 >50 0.8 0.8

[0913] These results demonstrate that the DA2NE, DA2Na, K4D, L7A, K12A, K12P, V13A, A14D, A14K, M25A, DA2NEQ (D) and DA2N (D) peptides also have an antimicrobial activity on Gram-positive and Gram-negative bacteria, compared to the control peptides Scr L, Scr C, Melittin and Gramicidin.