WO2000064939A2

WO2000064939A2 - Peptides derived from vasostatin i and the use thereof as anti-fungal agents

Info

Publication number: WO2000064939A2
Application number: PCT/FR2000/000839
Authority: WO
Inventors: Dominique Aunis; Marie-Hélène METZ-BOUTIGUE; Karine Lugardon
Original assignee: Institut National De La Sante Et De La Recherche Medicale
Priority date: 1999-04-26
Filing date: 2000-04-04
Publication date: 2000-11-02
Also published as: WO2000064939A3; FR2792638A1

Abstract

Peptides representing a fragment of the vasostatin I sequence, functional equivalents and use thereof as anti-fungal agents.

Description

PEPTIDES DERIVED FROM VASOSTATIN I AND THEIR USE

AS ANTIFUNGALS

The present invention relates to peptides derived from vasostatin I and to their use as antifungals. Vasostatin I is a natural peptide derived from chromogranin A

(CGA), which belongs to the family of acid secretory glycophosphoproteins, proteins widely expressed in many endocrine, neuroendocrine cells and in neurons (Simon, JP et al. (1989) Biochem. J. 262, 1-13, Helle, KB (1990) Neurochem, fnt. 17, 165-175, Huttner, WB et al. (1991) Trends Biochem. Sci. 16, 27-30, Winkler, H. et al. (1992) Neuroscience 49, 497-528 ). Chromogranins, in particular chromogranins A and B, are considered to be precursors which are degraded into peptides inside the secretory granules of chromoma cells of the adrenal medulla (Simon, JP et al. (1989) already cited; Dillen, L. et al. (1993) Neurochem. fnt. 22, 315-352; Metz-Boutigue, MH et al. (1993) Eur. J. Biochem. 217, 247-257; Strub JM et al. (1995) Eur. J. Biochem. 229, 356-368; Soszynski, D. et al. (1993) J. Neuroendocrinol. 5, 655-662).

Vasostatin I, highly conserved during evolution, is composed of 76 residues which correspond to the N-terminal fragment of CGA and has a disulfide bridge between the cysteines C _I7 and C ₃₈ . This fragment corresponds to CGA ,. ₇₆ (SΕQ ID NO: 1 to SΕQ ID NO: 4).

CGA, _.76 exhibits great stability against proteolytic enzymes of the intragranular matrix such as, for example, pro-hormone convertases, trypsin and carboxypeptidases.

It is mainly localized in the matrix of secretion granules of the chromaffin cells of the adrenal medulla and released with catecholamines in the extracellular medium after stimulation (Metz-Boutigue, MH, et al. (1993) already cited; Helle, K. B ,. et al. (1993) J. Neuroendocrinol. 5, 413-420). It is also released from nerve endings in response to stimulation (Liang, F., et al. (1995) Acta Physiol. Scand. 1S5, 23-30), suggesting that vasostatin I may play a role. important in vivo.

As early as 1992, it was established that vasostatin I exerts a relaxing effect on the saphenous vein by inhibiting vasoconstriction induced by endothelin-1 (Aardal, S. et al. (1992) Regul. Pept. 41, 9-18; Aardal, S. et al. (1993) J. Neuroendocrinol. 5, 405-412).

Different studies have then shown that vasostatin I has other biological activities; thus it is capable of inhibiting the secretion of the parathyroid hormone (Russel, J., et al. (1994) Endocrinology 133, 337-342), of regulating the adhesion of cells (Gaspam, A., et al. (1997) J. Biol. Chem. 272, 20835-20843) and induce the neurotoxicity observed in the cocultures of neurons and microglial cells (Ciesielski-Treska, J. et al. (1998) J. Biol. Chem. 273, 14339-14346). Synthetic peptides, derived from chromogranin A, are also capable of inhibiting the secretion of parathyroid hormone (US Patents 5,747,454 and US 5,514,775).

More recently, the inventors have shown that vasostatin I and other natural peptides derived from chromogranins A and B, which are released during secretion, have a powerful antibacterial activity against Gram-positive and Gram-bacteria. -negative (Strub, JM, et al. (1995) Eur. J. Biochem. 229, 356-368; Strub, j. M., et al. (1996) FEBS Lett. 379, 273-278; Strub, j M., et al. (1996) J. Biol. Chem. 271, 28533-28540; Metz-Boutigue, MH, et al. (1998) Ce / 7. Mol. Neurobiol. 18, 249-266). Mycoses are still an important cause of morbidity and mortality in humans and animals, despite the presence in their bodies of natural substances capable of developing defense mechanisms and despite the existence of numerous synthetic substances with antifungal activity. The incidence of fungal infections has increased sharply in recent years, on the one hand by the increase in deep visceral and septicemic mycotic localizations and on the other hand by the frequent importation of exotic visceral mycoses.

The first are due to fungi very widespread in nature, usually saprophytes, becoming pathogenic only under certain conditions: increasing use of broad spectrum antibiotics, corticosteroid therapy, chemotherapy, immunosuppressive therapies, AIDS patients, transplants and surroundings cardiovascular, we speak of mycoses latrogenes. They are caused by opportunistic fungi which are either levuπforms (Candida, Cryptococcus, Torulopsis) or filamentous (Aspergillus, Cephalosporum, Mucor)

The second are due to pathogenic fungi which develop in hot countries and which are imported through tourism and the mixing of populations. These exotic mycoses are histoplasmosis, coccidioidomycosis, blastomycosis and mycetomas.

If the treatment of superficial mycoses cutaneous or touching the mucous membranes does not present real difficulties, it is quite different from the deep mycoses, these always present a character of gravity and are quickly progressive because they occur on a weakened or immunodépπmé ground.

Also in immunodeficient patients. we often see either a failure of antifungal treatment, or the appearance of strains resistant to the various antifungals used, in particular Candida albicans These therapeutic failures have recently given renewed interest in research in the field of antifungals.

Many natural antifungal peptides have been found in plants (Tailor R .H et al (1997), J Biol. Chem., 272, 24480-24487), insects (especially cecropms, Boman H. G et al (1972 ) Nature, 237, 232-235, Boman H. G et al (1994) m Phylogenetic perspectives in îmmunity ^• the insect host defense Hoffmann JA, Janeway CA, Natoπ S Eds., 3-17, R G. Landes Company, Austin ) and invertebrates (WO 98/07440).

The development of antifungal agents comes up against three obstacles ^: - low activity in vivo with regard to m vitro activity, - poor diffusion through the wall of fungi which is made up of chitin, phospho pides and sterols; these constituents, absent from the wall of bacteria, explain why most of the antibacterials are not antifungals; in fact, in order to be able to penetrate the membrane, the antifungals must have a high hydrophobicity and - significant toxicity, obstacles which make it necessary to develop new antifungal peptides which are active in vivo, capable of acting effectively on the cells and little toxic. Also the In \ enteurs have given themselves the goal of providing antifungal peptides which are active in vivo, able to act effectively on the cells and little toxic.

The present invention relates to peptides, caractéπsés in that they consist of a fragment of vasostatin I and respond

- either to the sequence of formula (I) a- (SEQ ID NO 14) -b (I) in which

* a is zero or represents the sequence SEQ ID NO 15 of formula ^•

X, LPVNSPMX ₂ KGDTX ₃ VMKCX EVISDX ₃ LSKPSPMPVSX ₆ ECX, ETLX ₈ GDE where X, is zero or represents a sequence comprising from 1 to 10 amino acids, X ₂ represents N or T, X ₃ represents E or K, X ₄ represents IV or VL, X ₅ represents T or S, X ₆ represents K, P or Q, X ₇ represents F or L, X ₈ represents R or Q, and where the two cysteine residues (C) are optionally connected by a disulfide bridge, or

. a fragment of SEQ ID NO: 15 comprising at least one C-terminal amino acid of said sequence, preferably at least its 6 C-terminal amino acids (TLRGDE)

* the sequence SEQ ID NO: 14 represents RX ₉ LSILRHQNLLKE, where X ₉ represents I or V, and * b is zero, or represents ^• the sequence SEQ ID NO 16 of formula LQDLALQGAKERX ₁₀ X ,, QQX ₁₂ where X _l0 represents T , A or S, X ,, represents H or Q and X ₁₂ is zero or represents KK or polyQ, or

. a fragment of SEQ ID NO: 16 comprising at least one N-term amino acid of said sequence, preferably at least between 5 and 16 N-term amino acids of said sequence, provided that a- (SEQ ID NO: 14) -b is different from SEQ ID NO 'l, from SEQ ID NO 2, from

SEQ ID NO'3, SEQ ID NO'4 and SEQ ID NO: 5,

- either to the sequence of formula (II) ^• c- (SEQ ID NO '17) -d (II) in which * c is zero, or represents: . the sequence SEQ ID NO: 18 of formula, X, LPVNSPMX, KGDTX ₃ where X ,, X ₂ and X ₃ are as defined above, or

. a fragment of the sequence SEQ ID NO: 18 comprising at least one C-terminal amino acid of said sequence * the sequence SEQ ID NO: 17 represents

VMKCX ₄ EVISDX ₅ LSKPSPMPVSX ₆ ECX ₇ E, where X ₄ , X ₅ , X ₆ and X. are as defined above and where the two cysteine residues (C) are optionally linked by a disulfide bridge, and * d is zero or represented . SEQ ID NO: 19 of formula

TLX ₈ GDERX ₉ LSILRHQNLLKELQDLALQGAKERX, ₀ X,, QQX ₁₂ where X ₈ , X _Q , X ₁₀ , X „and X _I2 are as defined above, or

. a fragment of the sequence SEQ ID NO: 19 comprising at least one N-terminal amino acid of said sequence, preferably at least between 5 and 10 N-terminal amino acids of said sequence, provided that c- (SEQ ID NO: 17 ) -d is different from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or does not represent the fragment 1-40 of the sequence SEQ ID NO: 1, as well as their functional equivalents. The inventors have shown, surprisingly, that vasostatin I and peptides derived from vasostatin I exhibit remarkable antifungal activity, in the absence of lyric activity on erythrocytes.

Also within the meaning of the present invention, the term peptides representing a fragment of the sequence of vasostatin I, both the natural fragments obtained by enzymatic digestion or by chemical cleavage of vasostatin I, as well as the synthetic fragments obtained by conventional techniques peptide synthesis, and in particular by the solid phase synthesis technique described by Merrifield RB (1963), J. Am. Chem. Soc, 85, 2149-2154 or by recombinant DNA techniques (Corti, A., et al. (1997) Eur. J. Biochem. 248, 692-699) or by a combination of these two types of techniques.

For the purposes of the present invention, the term “functional equivalents” is intended to mean amino acid sequences comprising deletions or additions, modifications or conservative substitutions or a combination thereof, at the level of one or more amino acids, so that the tertiary structure and the antifungal activity are not altered

The modifications include in particular those resulting from post-translational processes (phosphorylation, glycosylation, sulfation) or chemical modifications carried out by conventional techniques known to those skilled in the art (acylation, fixing of lipids, nucleotides, cychsation.)

The modifications also include those resulting from a coupling of the peptides, by covalent, ionic or weak bonds to a molecule capable of increasing their affinity for a particular cell type (with a conventional antibiotic molecule), in particular couplings with peptides vectors.

Conservative substitutions well known to those skilled in the art, include in particular the substitution of an ammoacid by another amino acid having the same charge (amino acids Asp and Glu; basic amino acids: Lys, Arg and ornithm; neutral polar amino acids ^• Gly, Ser , Thr, Cys, Tyr, Asn, Gin, His and Trp), same size, same hydrophilicity (hydrophobic amino acids: Ala, Val, Leu Ile, Pro, Phe and Met), same aromaticity (Phe, Trp and Tyr) and / or even the ability to form equivalent secondary structures. It is understood that the functional equivalents also include, as amino acids, those which are enantiomers and diastereoisomers of natural amino acids of conformation D, rare amino acids, in particular hydroxyproline, methyllysme and dimethyllysine and synthetic amino acids, in particular ornithine, norleucme, cyclohexylalanine and omega-amino acids. Functional equivalents also cover retropeptides.

According to a preferred embodiment of the invention, the peptides of formula (I) and (II) consist of non-oxidized amino acids.

The invention includes in particular the following peptides ^•. peptides corresponding to formula I - - SEQ ID NO-14 (a and b are zero) ^• CGA _47.60 ,

- SEQ ID NO: 15 + SEQ ID NO: 14: CGA ,. ₆₀

- SEQ ID NO: 14 + SEQ ID NO: 16 - CGA _{47 76} /

- SEQ ID NO 14- fragment comprising the 10 N-terminal acids of SEQ ID NO 16 CGA ₄₇ .. ₀

- fragment comprising the 5 C-term amino acids of SEQ ID NO 1 + SEQ ID NO 14 - fragment comprising the 10 N-terminal acids of SEQ ID NO: 16 CGA _{4 70}

- fragment comprising at least one C-terminal amino acid of SEQ ID NO 15+ SEQ ID NO: 14

- fragment comprising the 5 C-term amino acids of SEQ ID NO- 15 ^ SEQ ID NO- 14 ^• CGA ₄₁ . ₆₀ - fragment comprising at least one C-terminal amino acid of the

SEQ ID NO- 15+ SEQ ID NO '14+ fragment comprising at least one N-terminal amino acid of SEQ ID NO: 16

SEQ ID NO '14+ fragment comprising at least one N-terminal amino acid of SEQ ID NO' 16, for example the first six N-terminal amino acids of the sequence SEQ ID NO: 16: CGA ₄₇ . ₆₆ . peptides corresponding to formula II: - SEQ ID NO: 17: CGA _I4 ^ ₀ - SEQ ID NO '17+ SEQ ID NO: 19 CGA ₁₄ . ₇₆

- fragment comprising at least one C-terminal amino acid of SEQ ID NO: 18+ SEQ ID NO '17

- SEQ ID NO: 18+ SEQ ID NO '17 CGA _M0 (with the exception of the fragment from the sequence SEQ ID NO: 1)

- SEQ ID NO: 17+ fragment comprising at least one N-terminal amino acid of SEQ ID NO: 19 - fragment comprising at least one C-terminal amino acid of SEQ

ID NO: 18+ SEQ ID NO '17 + fragment comprising at least one N-terminal amino acid of SEQ ID NO: 19.

According to another preferred embodiment of the invention, the peptides are selected from the group consisting of the following peptides: SEQ ID NO: 7 which corresponds to the CGA ₄₇ fragment. ₆₀ of bovine, human and porcine origin, SEQ ID NO: 8 which corresponds to the CGA ₄₇ fragment. ₆₀ of rat, SEQ ID NO 9 which corresponds to the CGA ₄₁ fragment. ₇₀ of bovine, human and porcine origin, SEQ ID NO: 10 which coπes- lays the CGA ₄ fragment, _{^ 0} of rat, SEQ ID NO 1 1 which corresponds to the CGA fragment, ₄₀ of bovine πgme, SEQ ID NO 12 which corresponds to the CGA fragment ₁₄ ^ ₀ of human and porcine origin, SEQ ID NO 13 which corresponds to the fragment CGA _I4 ^ ₀ of rat, SEQ ID NO 20, which corresponds to the fragment _{41 60} CGA bovine, porcine or human, SEQ ID NO 21, which coπespond CGA fragment ₄₇ ₀ bovine oπgine , porcine or human, SEQ ID NO 22, which corresponds to the CGA ₄ - _6o fragment of bovine, porcine or human origin

The present invention also relates to antifungal pharmaceutical compositions, characterized in that they comprise at least one peptide deviated from vasostatin I, of formula (I) or of formula (II), as defined above, associated with at least one pharmaceutically acceptable excipient

In a preferred embodiment of the invention, the pharmaceutical compositions according to the invention comprise at least one peptide whose sequence is chosen from the group consisting of the sequences SEQ ID NO 7, SEQ ID NO-8, SEQ ID NO. 9, SEQ ID NO '10, SEQ ID NO ll, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 20, SEQ ID NO'21 and SEQ ID NO 22

The present invention also relates to the use of a peptide whose sequence is chosen from the group consisting of the sequences SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO'6 and fragment 1-40 of the sequence SEQ ID NO 1, for the preparation of a medicament useful as an antifungal

The pharmaceutical compositions according to the invention are suitable for systemic administration, in particular by the intravenous, subcutaneous, intramuscular or intraraponeal route, for oral administration in particular in the form of tablets, capsules and capsules, for topical administration. , especially in the form of a gel, lotion and cream, for rectal administration, especially in the form of suppositories or ointment, for vaginal administration, especially in the form of a capsule or jelly, for pulmonary administration, especially in aerosol form, for nasal administration, especially in the form of drops or nasal ointment, for ocular administration, especially in the form of eye drops or ophthalmic ointment and for oral administration, especially in the form of mouth or toothpaste. The peptides according to the invention (SEQ ID NO 7-13 and SEQ ID NO 20-22), vasostatin I (SEQ ID NO 1-4), the peptides of SEQ ID NO 5 and SEQ ID NO 6 as well as the fragment 1-40 of SEQ ID NO 1 can be used in the treatment or prevention of diseases caused by different types of filamentous fungi, in particular by Neurospora crassa, Aspergillus sp, in particular Aspergûlus fumigatus, Alternana sp, in particular Alternaria brassicola, Trichoderma sp , in particular Trichoderma viride, Nectria haematococca, Fusarium culmorum and Fusanum oxyporum or by yeasts, in particular by Candida sp and by Saccharo- myces cerevisiae Also in accordance with the invention

- in the treatment or prevention of diseases caused by different types of filamentous fungi or by yeasts, the peptides of SEQ ID NO 1 to 4 will preferably be used

- in the treatment or prevention of superficial mycoses with cutaneous localization, of cutaneous-mucosal mycoses that they are cutaneous, oropharyngeal or digestive and deep mycoses, in particular in the cases of candi- doses disseminated at the ocular, osteoarticular, cardiac, uπnary level, genital, pulmonary, cerebral or septicemic, and in all cases of yeast infections at the ocular, osteoarticular, cardiac, uπnary, pulmonary, cerebral or septicemic level, peptides of SEQ ID NO 1 to 4 will also preferably be used

The peptides according to the invention (SEQ ID NO 7-13 and SEQ ID NO.20-22), vasostatin I (SEQ ID NO 1-4), the peptides of SEQ ID NO 5 and SEQ ID NO 6 as well as fragment 1-40 of SEQ ID NO 1 can be used advantageously in immunosuppressed patients, in particular in AIDS patients or in patients treated with immunotherapy or chemotherapy or in patients subjected to prolonged antibiotic therapy as well as '' in neonatology

These different compounds can advantageously be used alone or in combination with other active ingredients, in particular with antibiotics.

The peptides according to the invention can also be used in the context of targeted treatment, in particular when they have been modified beforehand by coupling, by covalent, ionic or weak bonds to a molecule capable of increasing their affinity for a type. of particular cell The peptides according to the invention can also be used in the context of gene therapy, in particular by injection of a cell previously modified to express a peptide according to the invention in vivo

The present invention also relates to the nucleic acids coding for the peptides of formula (I) and of formula (II), as defined above.

According to a preferred embodiment of said nucleic acids, they code for the sequences chosen from the group constituted by the sequences SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 1 1, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO'20, SEQ ID NO 21 and SEQ ID NO 22

Other characteristics and advantages of the invention appear in the following description and examples illustrated by the figures in which

- Figure 1 illustrates the purification by high pressure liquid chromatography (HPLC) of the natural peptide CGA, ₇₆ from bovine (SEQ ID NO '1). The peptides are isolated according to the technique described in Example 1 The solvent system used comprises 0.1% fluoroacetic acid in water (solvent A) and 0.1% tfluoroacetic acid in acetonitrile (solvent B) The absorption is measured at 214 nm and the elution is carried out according to the linear gradient represented on the right scale. The arrow indicates the fraction containing the bovine vasostatin I. - Figure 2 illustrates the analysis in mass spectrometπe according to the MALDf-TOF technique (measurement of the time of flight after laser desorption assisted by matπce) of peptides deviated from N-terminal fragments. (A) CGA ,. ₇₆ of cattle; (B) human recombinant VS-1 (SEQ ID NO: 5), (C) synthetic peptide CGA _7.57 from rat (SEQ ID NO: 6), EXAMPLE 1: Equipment and methods used for the purification and identification of the peptides

1 Puπfication of the natural peptide vasostatin I of bovm (SEQ ID NO 'l)

The secretory granules are isolated from the bovine adrenal gland according to the technique described by Smith, A. D., et al (1967) Biochem J 103, 483-492.

The soluble proteins are separated after lysis of the membranes and centrifugation, according to the technique determined by Aums, O, et al (1977) J Neurochem. 29, 439-447 CGA, _.76 is purified by high pressure liquid chromatography, on a Macherey Nagel Nucleosil 300-5C18 column (4x250 mm, particle size 5 μm and pore size 100 nm) using the Applied Biosystems HPLC 140 B system The absorption is measured at 214 nm and the solvent system used comprises 0.1% trifluoroacetic acid (TFA) in water (solvent A) and 0.1% trifluoroacetic acid in acetomethyl (solvent B) L elution is carried out at a speed of 0.7 ml / mm using successively a 0-30% gradient of B in A for 15 minutes, then a 30-50% gradient for 40 minutes then finally a 50-100% gradient for 10 minutes The fractions are collected manually and evaporated to dryness 2 Synthesis of the human recombinant peptide VS-1 (SEQ ID NO'5)

The human recombinant peptide VS-1 is cloned, purified and characterized according to the technique determined by Corti, A, et al (1997) Eur J Biochem 248, 692-699 This peptide corresponds to the human CGA, ₇₈ sequence and comprises the tπpeptide STA at the N-terminus. 3 Peptide synthesis ^• a) general procedure

Synthetic peptides are prepared according to the technique determined by Atherton, Ε et al (1989) in "Solid phase peptide synthesis a practical approach" (Rickwood, D and Hames, BD, eds) IRL press using an ABI peptide synthesizer 431 A (Applied Biosystems) b) formation of disulfide bridges

The formation of the disulfide bridges between the cysteine residues is obtained in the presence of N-ethyl-dnsopropylamine at pH 8.5, leaving the reaction mixture for 24 hours in the open air, until the reaction, measured according to the Ellman test decπt by Deakm, H. et al (1963) Biochem J 89, 296-304 be complete c) alkylation of cysteine residues

The alkylation (S-pyridylatwή) of the cysteine residues is obtained by treating the peptides with 4-vιnylpyπdιne, according to the technique decπte by Tarr, G. E (1986) in "Methods ofprotein microcharactemation" 162, Shively, JE ed ( Humana press, Clifton, ΝJ), after reduction of the disulfide bridge in the presence of guanidine-HCl 8 M, in Tπs-HCl buffer at pH 8.5 containing 4 mM EDTA followed by elimination of excess reagents, by high pressure liquid chromatography with a gradient such as that described in example 1 paragraph 1 d) oxidation of peptides

To carry out the oxidation of vasostatin I from bo \ ιn and that of the human recombinant peptide VS-1, an oxidizing agent is prepared by adding formic acid to hydrogen peroxide at 30% (v / v, 9 / 1) ^* followed by stirring the mixture for 45 minutes at room temperature the resulting performic acid is cooled on ice to 0 ° C. 100 .mu.l oxidized agent thus prepared, are added to the protein to treat the reaction medium is left for 30 minutes at 0 ° C, diluted with 500 μl of water, evaporated and concentrated to dryness This washing step is carried out three times in succession, in order to remove the excess of the reactants

4 Sequence analysis

The peptide sequences are determined by Edman degradation using an Applied Biosystems 473A sequencer. The samples previously puπfiés by high pressure liquid chromatography are analyzed according to the technique described by Metz-Boutigue, MU, et al (1993) Eur. J Biochem 217, 247-257

5. Analysis by mass spectrometry

It is performed according to the MALDf-TOF technique (measurement of the flight time after laser desorption assisted by matπce) as determined by Vorm, O et al (1994) J Am Soc Mass Spec 5, 955-958 and Vorm, O et al (1994) Anal Chem 66, 3281-3287, using a Bruker BIFLEX ™ mass spectrograph EXAMPLE 2: Equipment and methods used to measure antifungal activity

1 Culture of filamentous mushrooms

They are cultivated on a culture medium comprising a medium with five cereals and the spores are collected according to the technique determined by Broekaert, W F et al (1990) FEMS Microbiol Lett 69, 55-60

The following strains were used Alternana brassicola, (MUCL 20297 deposited at the Mycotheque of the Catholic University of Louvain), Trichoderma vinde, (MUCL 29724 deposited at the Mycotheque of the Catholic University of Louvain), Trichophyton mentagrophytes, Nectna haematococca, (160.2.2), Fusanum culmorum (MUCL 30162), Fusanum oxyporum, (MUCL 909 deposited at the Mycotheque of the Catholic University of Louvain), Botrytis cinerea (MUCL 30158 deposited at the Mycotheque of the Catholic University of Louvain), Neurospora crassa (CBS 327-54 deposited at the Centraalbureau vooi Schimmelcultures) and Aspergillus fumigatus

2 Yeast cultures The cells are precultured on a Sabouraud medium

The following strains have been used Saccharomyces cerevisiae (TGY481 pJM600) and Candida albicans

3 Measurement of antifungal activity

The spores are suspended so as to have a final concentration of 10 ⁴ spores / ml, in a culture medium containing potato dextrose

The yeasts are suspended in a Sabouraud medium, at a concentration such that an absorption of 0.001 at 620 nm is obtained.

Tetracyclme (10 μg / ml) and cefotaxime (0.1 μg / ml) are added to these culture media.

The growth of the fungi is evaluated after an appropriate incubation time, specific to each fungus (24 to 48 hours at 30 ° C)

Aliquots of 20 μl of the peptides puπfies are incubated on microtiter plates with 80 μl of spores of fungi or yeasts. The final concentrations of peptides are between 1 μM and 10 μM and correspond to the concentration of peptide which inhibits 100%. growth of fungi (CMI, ₀₀ )

EXAMPLE 3 Structural and biological characterization of the antifungal activity of vasostatin I (SEQ ID NO: 1) 1 puπfication and characterization of vasostatin I (SEQ ID NO 1) a) puπfication

After separation by high pressure liquid chromatography according to the technique described in Example 1, there is a peak eluted with a solvent containing 47% of solvent B (Figure 1) b) caractéπsation

The sequencing of the peptide contained in this fraction according to the technique described in Example 1 reveals a unique sequence which corresponds to the N-terminal sequence of bovine chromogram A

Analysis of this fragment according to the MALDI-TOF technique determined in Example 1, makes it possible to define 3 peaks corresponding to masses of 8583.9 Da, 4289.8 Da and 2860.3 Da (Figure 2A)

These results are in agreement with the theoretical mass of the CGA peptide, - ₆ equal to 8584.9 Da

2 Antifungal activity of vasostatin I (SEQ ID NO 1) and its alkylated (S-pyridylated form) and oxide forms

TABLE I

These results show in particular that

- vasostatin I has no effect on Trichophyton mentagrophytes under the conditions of experience - the presence of the disulfide bridge is not compulsory for antifungal activity and.

- oxidation with performic acid induces a significant decrease in antifungal activity.

In addition, vasostatin I is also active with a MIC, ₀₀ of 10 μM, against Botrytis cinerea EXAMPLE 4 Structural and biological characterization of the antifungal activity of the human recombinant peptide VS-1 (SEQ ID NO: 5)

1. Characterization of the human recombinant peptide VS-1 (SEQ ID NO: 5)

Analysis of this fragment according to the MALDI-TOF technique described in Example 1, makes it possible to define 3 peaks corresponding to masses of 9069.7 Da, 4536.1 Da and 3024.1 Da (Figure 2B).

These results are in agreement with the theoretical mass of the peptide equal to 9069.5 Da.

2. Antifungal activity of the human recombinant peptide VS-1 and of its alkylated and oxidized forms.

TABLE II

These results show in particular that:

- the peptide VS-1 has no effect on Trichoderma viride, Aspergillus fumigatus, Fusarium oxyporum and Trichophyton mntagrophytes under the conditions of the experiment and it neither inhibits the growth of Saccharomyces cerevisiae, nor that of Candida albicans under the conditions of l 'experience. - the presence of the disulfide bridge is not compulsory for antifungal activity.

- oxidation with performic acid decreases antifungal activity. EXAMPLE 5 Structural and biological characterization of the antifungal activity of the synthetic peptide CGA _7.57 from rats (SEQ ID NO: 6)

1 Characterization of the synthetic peptide CGA- .- from rats

The analysis of this fragment according to the XfALDI-TOF technique determined in Example 1, makes it possible to define 2 peaks corresponding to masses of 5654.4 Da, and 2827.1 Da (Figure 2C).

These results are in agreement with the theoretical mass of the peptide equal to 5655.7 Da.

2. Antifungal Activity of the Synthetic Rat CGA ₇ Peptide Before a Disulfide Bridge and Its Reduced Form

TABLE III

These results show in particular that: the peptide with disulfide bridge has no effect on Aspergillus fumigatus, Fusarium oxyporum and on Trichophyton mentagrophytes under the conditions of the experiment and does not inhibit either the growth of Saccharomyces cerevisiae, or that of Candida albicans in the conditions of the experience.

- the presence of the disulfide bridge is not compulsory for antifungal activity. EXAMPLE 6 Measurement of the antifungal activity of the CGA ₄₇ peptide. ₆₀

TABLE IV

EXAMPLE 7 Measurement of the antifungal activity of the CGA _4I peptide. ₇₀

TABLE V

U

EXAMPLE 8 Measurement of the antifungal activity of the peptides CGA _41.60 , CGA ₄₇ . ₇₀ and CGA - ^• , 47-66

TABLE VI

Comparison of the activity of these peptides with that of the peptide

CGA ₄₇ . ₆₀ shows that:

(i) the peptide CGA ₄₁ . ₆₀ has weak activity, suggesting that the tripeptide R ₄₃ G ₄₄ D ₄₅ may hinder antifungal activity;

(ii) the CGA ₄₁ peptide. ₇₀ makes it possible to kill numerous fungi, suggesting the importance of the C-terminal end of this fragment, which neutralizes the inhibitory effect of the tripeptide R ₄₃ G ₄₄ D ₄₅ ;

(iii) the CGA ₄₇ fragments. ₆₀ and CGA ₇ . ₇₀ are active against filamentous fungi but the increase in their activity seems to be linked to the length of the peptide chain. (iv) unlike natural vasostatin I (CGA, _.76 ), these synthetic peptides corresponding to the C-terminal end of this protein are inactive vis-à-vis yeasts, with the exception of peptides CGA ₄₇ . ₇₀ and CGA ₄₇ . ₆₆ , which are active at 200 μM and 50 μM respectively against Candida albicans.

All of these results confirm that the peptides according to the invention could be useful as antifungal agents in the treatment or prevention of deep or superficial yeast infections caused by filamentous fungi and yeasts. As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly, on the contrary it embraces all of them. variants which may come to the espπt of the technician in the field, without departing from the scope, or the scope, of the present invention.

Claims

1. Peptides, characterized in that they consist of a fragment of the vasostatin I sequence and respond:

- either to the sequence of formula (I) a- (SEQ ID NO: 14) -b (I) in which:

* a is zero or represents:

. the sequence SEQ ID NO: 15 of formula:

X, LPVNSPMX ₂ KGDTX ₃ VMKCX ₄ EVISDX ₅ LSKPSPMPVSX ₆ ECX ₇ ETLX „GDE where X, is zero or represents a sequence comprising from 1 to 10 amino acids, X ₂ represents N or T, X ₃ represents E or K, X ₄ represents IV or VL, X ₅ represents T or S, X ₆ represents K, P or Q, X ₇ represents F or L, X _s represents R or Q, and where the two cysteine residues (C) are optionally linked by a bridge disulfide, or. a fragment of SEQ ID NO: 15 comprising at least one C-terminal amino acid of said sequence, preferably at least its 6 C-terminal amino acids

* the sequence SEQ ID NO: 14 represents RX ₉ LSILRHQNLLKE, where X ₉ represents I or V, and

* b is zero, or represents:

. the sequence SEQ ID NO: 16 of formula LQDLALQGAKERX _I0 X ,, QQX, ₂ where X, ₀ represents T, A or S, X ,, represents H or Q and X, ₂ is zero or represents KK or polyQ, or

. a fragment of SEQ ID NO: 16 comprising at least one N-terminal amino acid of said sequence, preferably at least between 5 and 16 N-terminal amino acids of said sequence, provided that a- (SEQ ID NO: 14) -b is different from SEQ ID NO: l, from SEQ ID NO: 2, from

SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5,

- either to the sequence of formula (II): c- (SEQ ID NO: 17) -d (II) in which: * c is zero, or represents:

. the sequence SEQ ID NO: 18 of formula, X, LPVNSPMX ₂ KGDTX ₃ where X „X ₂ and X ₃ are as defined above, or

. a fragment of the sequence SEQ ID NO 18 comprising at least one C-terminal amino acid of the said sequence

* the sequence SEQ ID NO: 17 represents VMKCX ₄ EVISDX ₅ LSKPSPMPVSX ₆ ECX, E, where X ₄ , X ₅ , X ₆ and X, are as defined above and where the two cysteine residues (C) are optionally linked by a disulfide bridge, and

* d is zero or represents

. SEQ ID NO T 9 of formula TLX ₈ GDERX ₉ LSILRHQNLLKELQDLALQGAKERX, ₀ X,, QQX _[2 where X ₈ , X ₉ , X ₁₀ , X,, and X, ₂ are as defined above, or

. a fragment of the sequence SEQ ID NO: 19 comprising at least one N-terminal amino acid of the said sequence, preferably at least between 5 and 10 N-terminal amino acids of the said sequence, provided that c- (SEQ ID NOT7) - d is different from SEQ ID NO 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or does not represent the 1-40 fragment of the sequence SEQ ID NO: l, as well as their functional equivalents.

2. Peptides according to claim 1, characterized in that they consist of non-oxidized amino acids.

3. Peptides according to any one of claims 1 and 2, characterized in that they are selected from the group consisting of the peptides of the following sequences: the sequences SEQ ID NO'7, SEQ ID NO: 8, SEQ ID NO; 9, SEQ ID NOT0, SEQ ID NO: ll, SEQ ID NO: 12, and SEQ ID NO: 13, SEQ ID NO: 20 and SEQ ID NO: 21 and 22.

4. Antifungal pharmaceutical compositions, caractéπsées in that they comprise at least one peptide according to any one of claims 1 to 3, associated with at least one pharmaceutically acceptable excipient.

5. Use of a peptide whose sequence is chosen from the group consisting of the sequences SEQ ID NO: 1, SEQ ID NO.2, SEQ ID NO: 3, SEQ ID NO'4, SEQ ID NO'5 and SEQ ID NO: 6 and fragment 1-40 of the sequence SEQ ID NO: 1, for the preparation of a medicament useful as an antifungal. ~> 7

6. Nucleic acids characterized in that they code for the peptides according to any one of claims 1 to 3.