FR2817260A1 - New Drosophila immune-induced molecule peptides useful for preventing or treating inflammatory and/or immune disorders - Google Patents

Abstract

Drosophila immune-induced molecule (DIM) peptides are new. Peptides of formula A1A2A3A4A5A6A7A8A9A10A11A12A13A14A15A16A17A18A19A20A21A22A23 (I) and their fragments, are new. A1 = Glu, Gln or pyroglutamic acid (pGlu); A3, A6, A9, A22 = basic amino acids; A2, A4, A11, A13, A18 = hydrophobic amino acids; A5, A7, A8, A10, A12, A14, A15, A16, A17, A21 = negatively charged amino acids, small polar amino acids, large polar amino acids, Gly, Ala or Pro, and A19, A20, A23 = Ala, Gly, Ile, Phe, Ser, Tyr or Val. An Independent claim is also included for polypeptides comprising a DIM peptide and optionally terminal peptide residues necessary for expression and/or targeting of the DIM peptide in a host organism.

Description

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 PEPTIDES HAVING ANTIINFLAMMATORY PROPERTIES, USES THEREOF AND COMPOSITIONS CONTAINING SAME

 The present invention relates to novel peptides having anti-inflammatory properties.

The invention also relates to a composition, in particular pharmaceutical and in particular vaccine containing at least one of said peptides useful for the prevention or treatment of inflammatory and immune disorders.

 Various substances having anti-inflammatory properties for the treatment of inflammatory and immune disorders have been described in the prior art. The most traditional treatments include nonsteroidal anti-inflammatory drugs.

The recently developed anti-inflammatory drugs are based on metalloproteinase inhibitors (Metalloproteinase inhibitors: new opportunities for rheumatoid arthritis and osteoarthritis, Shaw T., Nixon JS, KM Bottomley, Expert Opin Investig Drugs 2000 Jul; 9 (7): 1469 -1478; Therapeutic potential of matrix metalloproteinase inhibitors of atherosclerosis, George SJ, Expert Opin Investig Drugs 2000 May; 9 (5): 993-1007), or on cytokines (WO 9928965; WO 9722289, IL-1 inhibitors: novels agents in the treatment of rheumatoid arthritis, Gabay C., Expert Opin Investig Drugs 2000 Jan; 9 (1): 113-27; Bocci V., Interleukins, Clinical Pharmacokinetics and Practical Applications, Clin Pharmacokinet, 1991,274-284; Elliott JN, Woody and RN Maini, Anti-tumor necrosis factor-alpha therapy of rheumatoid arthritis, Adv Immunol 64, 1997, 283-350).

 Inflammatory and immune disorders are observed in many pathologies, such as arthritis,

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 atherosclerosis, allergic asthma, chronic inflammations of the digestive tract (Crohn's disease, ulcerative colitis) and diabetes.

 In human health, it can be recalled that the incidence of these chronic diseases has increased significantly in recent years.

Inflammation can be caused by many and varied inflammatory agents such as bacteria, viruses and environmental antigens (pollen, dust). The inflammatory response becomes chronic when the antigen is in non-limiting amount and persists thereby causing destruction of the tissue.

 There is therefore a real need to characterize and develop new classes of anti-inflammatory substances.

 Insects have long been known to have developed an effective defense system against microorganisms. This immune response relies to a large extent on the rapid and transient synthesis of several families of antimicrobial peptides with a broad spectrum of activity (Bulet et al., 1999, Dev Comp.

23.329-344). The Applicant has developed a know-how and has expertise in the purification and characterization of substances produced in insects.

 Among the techniques used, MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time Of Flight) mass spectrometry makes it possible to establish precise differential maps of the peptide content of the immunized versus non-immunized Diptera hemipheum Drosophila melanogaster (Uttenweiler-Joseph S. et al., 1998, Differential display of peptides during the immune system of Drosophila: a matrix assisted laser desorption

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ionisation time-of-flight mass spestrometry study, Proc.

ionization time-of-flight mass spermometry study, Proc.

Natl. Acad. Sci. Flight. 95 p. 11342-11347).

 The implementation of this technique made it possible to characterize peptides, some of which correspond to already known antimicrobial molecules, such as drosocine, drosomycin, metchnikowine, defensin, diperericin and cecropin (Bulet P., 1999, Les Drosophila antimicrobial peptides, Medicine / Sciences nl, Vol.15,23-29).

 The Applicant has now isolated, purified and characterized new peptide sequences which show no homology with previously described peptides or peptide fragments. By homology, according to the invention is meant a degree of homology of at least 80% relative to the reference sequence, possibly greater than 90% and up to more than 95%.

Molecules . The subject of the present invention is therefore a new class of peptides which will also be referred to below as DIMs for Drosophila Immune-induced

Molecules.

 The work carried out by the Applicant on the DIMS, allowed him to show that, unexpectedly, they did not exhibit antimicrobial activity as all insect peptides isolated so far, but anti-inflammatory properties.

 Indeed, the peptides of the invention have properties of inhibition of cell adhesion and / or transmigration of leukocytes to the site of inflammation, particularly properties of inhibition of the expression of molecules of cell adhesion involved in inflammatory and immune disorders, and more particularly ICAM-1 expression inhibitory properties.

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 The primary inflammatory response is essentially characterized by the migration of polynuclear leukocytes including neutrophils to sites of inflammation in response to chemotactic factors. This migration occurs in 3 stages involving leukocyte slippage along the endothelial epithelium, leukocyte adhesion to the endothelial epithelium and passage of leukocytes through the endothelial epithelium to the inflamed tissue.

Mayadas-Norton T., pathol Biol, 1998 Mar ; 46 (3) : 164-70). Each of the 3 steps involves a class of specific cellular adhesion molecules (Endothelial adhesion molecules in health and disease, Cotran RS,

Mayadas-Norton T., Pathol Biol, 1998 Mar; 46 (3): 164-70).

Thus, the sliding step is mediated by selectins expressed on the surface of endothelial cells and leukocytes and recognizing the carbohydrates on other cell types and the adhesion and transmigration steps are mediated by the binding between integrins expressed at the surface of leukocytes and platelets and members of the immunoglobulin superfamily, particularly ICAM-1 (expressed on the surface of both endothelial cells and leukocytes).

 The intercellular adhesion molecule ICAM-1 expressed on the surface of endothelial cells therefore plays a key role in the inflammatory response at 2 levels: firstly, its interaction with a ss2 integrin expressed on the surface of leukocytes (Mac-1 for neutrophils, LFA-1 for T lymphocytes) will allow endothelial-leukocyte cell adhesion and in a second stage, transmigration of leukocytes across the vascular endothelium to the site of inflammation (Inflammatory and immune responses are impaired inmice deficient in intracellular adhesion molecule 1, James E. et al., Proc Natl Acad Sci USA, Vol 90,829-8533). The

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 The predominant role that ICAM-1 plays in mediating inflammation has been demonstrated by numerous knockout experiments in mice (Bullett DC & al. Immunol 1996 Oct. 1; 157 (7): 3153-8; Deficiency of inflammatory cell adhesion molecules against atherosclerosis in mice, Nageh et al., Arterioscler Thromb Vase Biol 1997 Aug; 17 (8): 1517-20; Inflammatory and immunized responses are poorly deficient in intracellular adhesion molecule 1, James E. et al., Proc.

Natl. Acad. Sci. USA, Vol. 90.829-8533; Endothelial adhesion molecules in health and disease, Cotran RS,
Mayadas-Norton T., Pathol Biol, 1998 Mar; 46 (3): 164-70).

 Invariably, ICAM-1'mutant mice exhibit a significant reduction in inflammatory response as compared to control wild mice.

ll (D2P3 (1) 4 () 5P 60708P9010 (1) 110 1 2 (b 13 0 14 015 () 16 017 (1) 18CY1 9 (52 0 2 1 P2 2 (y2 3 (i) ou un fragment de celui-ci, dans laquelle : ou un fragment de celui-ci, dans laquelle : (D, 0, (y représentent des résidus d'acide aminé, - l'acide aminé LI est choisi dans le groupe comprenant l'acide glutamique, la glutamine et l'acide pyroglutamique, - les acides aminés ss3, ss6, ss9, ss22, identiques ou différents, sont choisis parmi les acides aminés basiques,

- les acides aminés < , 0, < 11, < , is, identiques ou différents, sont choisis parmi les acides aminés hydrophobes, - les acides aminés 65, 6-,, 9g, 6, 9, 6, 6, 916'917'921'identiques ou différents, sont choisis parmi The subject of the present invention is therefore a peptide of general formula (I):

(D2P3 (1) 4 () 5P 60708P9010 (1) 110 1 2 (b 13 0 14 015 () 16 017 (1) 18CY1 9 (52 0 2 1 P2 2 (y 2 3 (i) or a fragment thereof wherein: or a fragment thereof, wherein: (D, 0, (y are amino acid residues, - the amino acid LI is selected from the group consisting of glutamic acid, glutamine and pyroglutamic acid, the amino acids ss3, ss6, ss9, ss22, which are identical or different, are chosen from among the basic amino acids,

the amino acids <, 0, <11, <, is, identical or different, are chosen from among the hydrophobic amino acids, - the amino acids 65, 6-, 9g, 6, 9, 6, 6, 916'917 '921', whether identical or different, are chosen from

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 the negatively charged amino acids or the small polar amino acids or the broad polar amino acids or the glycine, the alanine, the proline, the amino acids 619, 620, 623, which are identical or different, are chosen from alanine, glycine, isoleucine, phenylalanine, serine, tyrosine or valine.

By convention, in formula (I) above, LI represents the N-terminal amino acid and 023 represents the C-terminal amino acid.
Among the fragments of the peptides of formula (I), the invention more particularly envisages those consisting of at least 17 successive amino acids and preferably 23 successive amino acids of said sequence.

est Cig1 ' ? i9, O20 921 ss22-L'invention envisage plus particulièrement un peptide répondant à l'une des formules générales suivantes : 2486878ss9810118121361466, 7 (II) 2465ss676ss961116113614666, (III) 246667688101161136146156166170190621 (IV) 11 (1) 23 (e4o5P6o7o8P96lO (1) 11012 (1) 13014015016017 (Dl8 (i i i) 111023 (1) 405P60708P9010 (1) 11012 (1) 13014015016017 (l) l8al9cy2Oo2l (IV)
Selon la présente invention, on entend plus particulièrement par, - acides aminés basiques, les acides aminés choisis parmi l'arginine, l'histidine ou la lysine, - acides aminés hydrophobes, les acides aminés choisis parmi l'isoleucine, la leucine, la phénylalanine, le tryptophane, la tyrosine ou la valine, acides aminés chargés négativement, les acides aminés choisis parmi l'acide aspartique ou l'acide glutamique, By way of preferred examples of such fragments, mention may be made of those whose 2 to 6 successive amino acids are deleted at the C-terminal end. These fragments correspond to peptides including the C-terminal amino acid

is Cig1 '? The invention more particularly envisages a peptide corresponding to one of the following general formulas: ## STR13 ## (1) 13014015016017 (D18 (iii) 111023 (1) 405P60708P9010 (1) 11012 (1) 13014015016017 (1) 18al9cy2Oo21 (IV)
According to the present invention, the term "basic amino acids" is understood more particularly to mean the amino acids chosen from arginine, histidine or lysine, hydrophobic amino acids, the amino acids chosen from isoleucine, leucine, phenylalanine, tryptophan, tyrosine or valine, negatively charged amino acids, amino acids selected from aspartic acid or glutamic acid,

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 - Small polar amino acids, amino acids selected from serine or threonine, broad polar amino acids, amino acids selected from asparagine or glutamine.

ceux dans lesquels l'un au moins des acides aminés , 6, 97, 08, 012, 13' 615. is8 < ? 19, Co, 0 : ;23, s'il est présent, à la signification suivante : - est la phénylalanine ou la leucine, - 65 est l'acide glutamique ou l'alanine, - 97 est la proline ou la sérine, - 9g est l'acide aspartique ou la glycine, - 912 est l'acide aspartique ou la thréonine, - #13 est la phénylalanine, la valine ou l'isoleucine, - #15 est l'asparagine ou la sérine, - cog est la phénylalanine ou la valine, - #19 est la sérine, la tyrosine, la valine ou la phénylalanine,

- (Y,, est l'alanine, l'isoleucine ou la valine, - G23 est la phénylalanine, la glycine ou la sérine. Preferred peptides according to the invention, more particularly of formulas (I), (II), (III) or (IV), are

those in which at least one of the amino acids, 6, 97, 08, 012, 13 '615. is8 <? 19, Co, O: 23, if present, to the following meaning: - is phenylalanine or leucine, - 65 is glutamic acid or alanine, - 97 is proline or serine, - 9g is aspartic acid or glycine, - 912 is aspartic acid or threonine, - # 13 is phenylalanine, valine or isoleucine, - # 15 is asparagine or serine, - cog is the phenylalanine or valine, - # 19 is serine, tyrosine, valine or phenylalanine,

- (Y ,, is alanine, isoleucine or valine; - G23 is phenylalanine, glycine or serine.

In the sequences reported below, the amino acids are represented by their one-letter code, but they can also be represented by their three-letter code according to the nomenclature below,
Ala alanine
Cysteine Cysteine
D Asp aspartic acid
E Glu glutamic acid
F Phenylalanine
Gly glycine
H his histidine

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Z F H V E R P G R T V D V G N G G F Y I Q R G - DIM-10 dont la séquence en acides aminés représentée dans la liste de séquence sous le numéro SEQ ID NO : 3 est la suivante :

Z L H V A R P D R T V T I G N G G V Y I Q R S - DIM-8 dont la séquence en acides aminés représentée dans la liste de séquence sous le numéro SEQ ID NO : 4 est la suivante : ZFHVERPDRTVDFGNGGFSAQ 1 Ile isoleucine K Lysine lysine
Leucine leucine
M Met methionine
N Asn asparagine
P Pro proline
Gln glutamine
R Arg arginine
Serine serine
TH threonine
Valine Valley
W tryptophan tr Tyr Tyr
Z pGlu pyrutamic acid
A preferred peptide group according to the invention is defined by the peptides of the following sequence: DIM-13 whose amino acid sequence represented in the sequence list under the number SEQ ID NO: 1 is the following: ZFHVERPDRTVDFGNGGFSAQRF - DIM- 12 whose amino acid sequence represented in the sequence list under the number SEQ ID NO: 2 is the following:

ZFHVERPGRTVDVGNGGFYIQ RG - DIM-10 whose amino acid sequence represented in the sequence listing under the number SEQ ID NO: 3 is the following:

ZLHVARPDRTVTIGNGGVYIQ RS - DIM-8 whose amino acid sequence represented in the sequence listing under the number SEQ ID NO: 4 is the following: ZFHVERPDRTVDFGNGGFSAQ

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DIM-6 whose amino acid sequence represented in the sequence listing under the number SEQ ID NO: 5 is the following: ZFHVERPGRTVDVGNGGF-DIM-5 whose amino acid sequence represented in the sequence listing under the number SEQ ID NO: 6 is the following: ZFHVERPDRTVDFGNGG
The invention also relates to derivatives of the peptides of formula (I) or fragments thereof such as the peptides of formulas (II), (III) and (IV). Such derivatives are those in which the above sequences comprise deletions, additions and / or modifications at the level of one or more amino acids, provided that these deletions, additions and / or modifications do not modify the activity. anti-inflammatory of the peptide from which they are derived. These derivatives can then be considered as functional equivalents of the peptides of the invention.

 As derivatives of the peptides of the invention, mention may be made of those in which the amino acid LI is linked to a protective group, an acyl group, an acetyl group or a methyl group, it being understood that said amino acid II is not not then pyroglutamic acid. Thus, this N-terminal residue may exhibit posttranslational modification and / or chemical modification, particularly acylation, acetylation or methylation, or carry a protecting group. Protective group is understood to mean any group which makes it possible to avoid the degradation of said peptides, in particular the tert-butyl ester for LI corresponding to aspartic acid and in particular the trityl for LI corresponding to asparagine.

 Likewise, the C-terminal residue may exhibit a chemical modification, in particular an amidation. As indicated above, the invention envisages more

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particulièrement les fragments des peptides de formules (I) dont les acides aminés O1, C1g, < 7, 0, O21 ou ss22 constituent l'extrémité C-terminale. Ainsi, l'acide aminé C-terminal 61, Cg1 Cg, < 20, 021, 22, o23 peut être amidé.

particularly the fragments of the peptides of formulas (I) whose amino acids O1, C1g, <7, O, O21 or ss22 constitute the C-terminal end. Thus, the C-terminal amino acid 61, Cg1 Cg, <20, 021, 22, o23 can be amidated.

 The functional equivalents of the peptides of the invention are also said peptides of which one or more amino acids are enantiomers, diastereoisomers, natural amino acids of conformation D, rare amino acids, in particular hydroxyproline, hydroxylysine, methylysine, dimethylysine and preferably pyroglutamic acid, positioned in particular at the Ei Nterminal residue of said peptides and synthetic amino acids including ornithine, norleucine, cyclohexyl-alanine and omega-amino acids.

 The invention also covers retropeptides and retro-inversopeptides, as well as peptides whose side chain of one or more of the amino acids is substituted with moieties which do not alter the anti-inflammatory activity of the peptides of the invention.

 The invention also relates to polypeptides consisting of or comprising a peptide of formula (I) or a fragment thereof. Thus, the invention contemplates a peptide of formula (I), or a fragment thereof, in particular of formulas (II), (III) or (IV), comprising at one and / or both of its ends. , a peptide residue necessary, for example its expression and / or targeting in a host organism. By host organism is meant any organism comprising at least one cell, whether it be microorganisms, in particular yeast or bacteria, or a mammalian cell.

 The DIMs which are the subject of the present invention are remarkable in that they exhibit anti-aging properties.

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 In this respect, they can be used in methods of treatment or prevention in both humans and animals against inflammatory and / or immune disorders.

 The present invention therefore also relates to a composition, preferably pharmaceutical and more particularly vaccine, characterized in that it comprises as active agent at least one peptide as defined above, advantageously associated in said composition with an acceptable vehicle.

 The acceptable vehicle has the first quality of not substantially degrade the peptide or peptides in said composition and not to decrease the anti-inflammatory properties.

 The present invention also relates to the use of at least one peptide for the preparation of a composition for the prevention or treatment of inflammatory and / or immune disorders in humans or animals. Inflammatory and / or immune disorders are the inflammatory and immune disorders observed in many diseases, such as arthritis, atherosclerosis, allergic asthma, chronic inflammations of the digestive tract (Crohn's disease, ulcerative colitis) and diabetes. Inflammation can be caused by many and varied inflammatory agents such as bacteria, viruses and environmental antigens (pollen, dust). The inflammatory response becomes chronic when the antigen is in non-limiting amount and persists thereby causing destruction of the tissue.

 Other advantages and characteristics of the invention will become apparent from the following examples relating to the preparation of DIM-13, DIM-12, DIM-12 peptides.

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 10, DIM-8, DIM-6 and DIM-5 and their anti-inflammatory activity.

 Example 1: Isolation of DIM-13, DIM-12, DIM-10, DIM-8, DIM-6, and DIM-5 from whole body or haemolymph taken from larvae or adults immunized with Diptera Drosophila melanogaster.

1) Induction of the biological synthesis of
DIM-13, DIM-12, DIM-10, DIM-8, DIM-6, and DIM-5 in the hemolymph of Drosophila melanogaster.

 The larvae (3rd stage of development) or adults (male and / or female) of the diptera Drosophila melanogaster were immunized with a 4.6 nl injection of a suspension of a mixture of Micrococcus cells. luteus (Gram-positive) and Escherichia coli 1106 cells (Gram-negative) prepared from cultures made in Luria-Bertani medium for 12 hours at 37 ° C. Immunization can be carried out also from a simple puncture using a suitable needle pre dipped in a pellet of these same bacteria.

The infected animals were kept for 24 hours in a room maintained at 25 ° C.

 2) Preparation of the hemolymph and extraction of said peptides

 The hemolymph (0.1 μl per Drosophila) is removed using a nanoinjector, and transferred into a solution of 0.1% trifluoroacetic acid (0.1% TFA).

A sample prepared from the same quantity of haemolymph or the same number of animals that did not undergo any stimulation of their immune response (so-called control sample) is treated in parallel in order to carry out a differential study (Uttenweiler- Joseph et al., (1998) Proc Natl Acad Sci USA Vol 95 pp. 11342-11347)

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3) Purification of the peptides by high performance liquid chromatography (HPLC) on reverse phase column.

- First stage :
The first purification step is common to all DIMs-13, DIM-12, DIM-10, DIM-8, DIM-6 and DIM-5. A sample corresponding to 180 Drosophila was analyzed by chromatography on a reverse phase microbore column (Aquapore RP-300 C8, Brownlee, 100 x 1 mm, 300) protected by a filter (Ultra-Low Dead Volume Precolumm Filter, Upchurch Scientific ). Elution was performed by a linear gradient of acetonitrile from 2 to 80% in 0.05% TFA for 80 minutes at a constant rate of 80 μl / min. Fractions were collected manually in Low Protein Eppendorf Eppendorf tubes (Poly Labo) following the change in absorbance at 214 nm. The collected fractions were dried under vacuum or in a heating block at 30 ° C, reconstituted with ultra pure water and analyzed by MALDI-TOF (Matrix-Assisted Laser Desorption Ionization-Time of Light) mass spectrometry.

 The following purification steps differ according to the considered DIM. a) Purification of DIM-13.

 Second and final purification step: the second step of purification of the DIM-13 is carried out by chromatography on a reverse phase microbore column (Aquapore RP-300 C8, Brownlee, 100 × 1 mm, 300 λ) protected by a filter ( Ultra-Low Dead-Volume Precolumm Filter, Upchurch Scientific). Elution was carried out by a two-phase linear gradient of acetonitrile from 2 to 22% in 0.05% TFA for 10 minutes and from 22% to 37% acetonitrile for 50 minutes at a constant flow rate of 80 μl / ml. min to 35OC. The fractions were collected

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 manually in Eppendorf tubes with low protein adsorption (Low-Binding Eppendorf, Poly Labo), following the variation of the absorbance at 214 nm. b) Purification of DIM-12.

 Second and final purification step: the second purification step of DIM-12 is carried out by reverse phase chromatography on a reverse phase microbore column (Aquapore RP-300 C8, Brownlee, 100 × 1 mm, 300) protected by a Ultra-Low Dead-Volume Precolumm Filter, Upchurch Scientific. Elution was carried out by a two-phase linear gradient of acetonitrile from 2 to 18% in 0.05% TFA for 10 minutes and from 18% to 28% acetonitrile for 50 minutes at a constant flow rate of 80 μl / ml. min to 35OC. Fractions were collected manually in Low Protein Eppendorf Eppendorf tubes (Poly Labo) following the change in absorbance at 214 nm. c) Purification of DIM-10.

 Second purification step: the second purification step of DIM-10 is carried out by chromatography on a reverse phase microbore column (Aquapore RP-300 C8, Brownlee, 100 × 1 mm, 300) protected by a filter (Ultra-Low Dead-Volume Precolumm Filter, Upchurch Scientific). Elution was carried out by a two-phase linear gradient of acetonitrile from 2 to 17% in 0.05% TFA for 10 minutes and from 17% to 27% acetonitrile for 50 minutes at a constant flow rate of 80 μl / ml. min to 35OC. Fractions were collected manually in Low Protein Eppendorf Eppendorf tubes (Poly Labo) following the change in absorbance at 214 nm.

 Third and final purification step: this last step is carried out under the same conditions as the previously described purification step. The fractions were collected as before and the

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 presence and purity of peptide DIM-10 verified by MALDI mass spectrometry. After this third purification step, the peptide being pure, its structural characterization was performed after enzymatic treatment with pyroglutamate aminopeptidase and sequencing by Edman degradation. d) Purification of DIM-8.

de phase inverse (Aquapore RP-300 C8, Brownlee, 100 x 1 mm, 300 ) protégée par un filtre (Ultra-Low Dead-Volume Precolumm Filter, Upchurch Scientific). L'élution a été réalisée par un gradient linéaire biphasique d'acétonitrile de 2 à 18% dans le TFA 0,05% pendant 10 minutes et de 18% à 28% d'acétonitrile pendant 50 min à un débit constant de 80 ul/min à 35OC. Les fractions ont été collectées manuellement dans des tubes Eppendorf à faible adsorption protéique (Low-Binding Eppendorf, Poly Labo), en suivant la variation de l'absorbance à 214 nm. e) Purification de DIM-6. Second and final purification step: the second purification step of DIM-8 is carried out by reverse phase chromatography on a microbore column

reverse phase (Aquapore RP-300 C8, Brownlee, 100 x 1 mm, 300) protected by a filter (Ultra-Low Dead Volume Precolumm Filter, Upchurch Scientific). Elution was carried out by a two-phase linear gradient of acetonitrile from 2 to 18% in 0.05% TFA for 10 minutes and from 18% to 28% acetonitrile for 50 minutes at a constant flow rate of 80 μl / ml. min to 35OC. Fractions were collected manually in Low Protein Eppendorf Eppendorf tubes (Poly Labo) following the change in absorbance at 214 nm. e) Purification of DIM-6.

 Second purification stage: the second stage of purification of DIM-6 is carried out by chromatography on a reverse phase microbore column (Aquapore RP-300 C8, Brownlee, 100 × 1 mm, 300 Å) protected by a filter (Ultra- Low Dead Volume Precolumm Filter, Upchurch Scientific). Elution was carried out by a two-phase linear gradient of acetonitrile from 2 to 17% in 0.05% TFA for 10 minutes and from 17% to 27% of acetonitrile for 50 minutes at a constant rate of 80 μl / ml. min to 35OC. Fractions were collected manually in Low Protein Eppendorf Eppendorf tubes (Poly Labo) following the change in absorbance at 214 nm.

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 Third and final purification step: this last step is carried out under the same conditions as the previously described purification step. The fractions were collected manually in Eppendorf tubes with low protein adsorpion (Low-Binding Eppendorf, Poly Labo). f) Purification of DIM-5.

 Second and final purification step: the second step of purification of DIM-5 is carried out by reverse phase chromatography on a microbore-filtered column (Aquapore RP-300 C8, Brownlee, 100 × 1 mm, 300 Å) ( Ultra-Low Dead-Volume Precolumm Filter, Upchurch Scientific). Elution was performed by a two-phase linear gradient of acetonitrile from 2 to 13% in 0.05% TFA for 10 minutes and from 13% to 23% acetonitrile in 50 min at a constant flow rate of 80 μl / ml. min to 35OC. Fractions were collected manually in low protein Eppendorf tubes (LowBinding Eppendorf, Poly Labo) following the change in absorbance at 214 nm.

 Example 2: Structural characterization of DIM-13.

 1) Verification of the purity of DIM-13 by MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) mass spectrometry.

de masse Bruker (Bremen, Allemagne) BIFLEX III équipé d'une optique haute résolution SCOUT et d'un réflectron. This study was performed on a spectrometer

Bruker (Bremen, Germany) BIFLEX III with SCOUT high resolution optics and reflectron.

This instrument has a maximum acceleration potential of 20kV and can be used in either linear mode or reflectron mode. Ionization is carried out with a 337 nm beam emitted by a nitrogen laser at a frequency of 3 Hz.

The mass spectra were calibrated externally

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 with a standard mixture of Drosophila antimicrobial peptides of known molecular mass, respectively drosocine-2199.5 Da, metchnikowine-3046.4 Da and drosomycin-4890.5 Da (Bulet P., 1999, The antimicrobial peptides of the Drosophila, Medicine / Sciences nl, Vol.15,23-29).

 The samples to be analyzed by MALDI-TOF mass spectrometry are prepared by the sandwich technique described as follows for the identification of the DIM-13 peptide. The various products to be analyzed (0.5 μl) were deposited on a thin layer of α-cyano-4-hydroxycinnamic acid crystals (4-HCCA, Sigma) obtained by rapid evaporation of a saturated solution in acetone and then covered with 0.5 ul of the second template, saturation 4-HCCA in a solution of 50% acetonitrile in water. After drying under a slight vacuum, the samples were washed with 1.5 μl of 0.1% TFA before being re-dried and introduced into the mass spectrometer.

 2) Edman degradation sequencing.

 Automatic sequencing by Edman degradation of native DIM-13 peptide or proteolysis fragments and detection of phenylthiohydantoin derivatives were performed on an ABI473A sequencer (PEApplied Biosystems division of Perkin Helmer).

 3) Sequencing by tandem mass spectrometry by nanoelectrospray (NanoES-MSn). a) Preparation of DIM-13.

 The DIM-13 peptide is solubilized in an acidic solution and in order to eliminate the TFA, the peptide solution is filtered on a Zip-Tip C18 reverse phase filter (MilliporeTM) and concentrated to 20 J. in a solution of 50% acetonitrile water acidified with 1% formic acid. All spectrometric analyzes of

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masse en tandem sont réalisées avec 2 J. l de DIM-13 concentré et la solution est déposée dans un capillaire de nanonébulisation préalablement rincé. b) Instrumentation.

tandem mass are carried out with 2 J. 1 DIM-13 concentrated and the solution is deposited in a nanospubing capillary previously rinsed. b) Instrumentation.

 The sequencing is carried out on an ESQUIRE-LC ion trap mass spectrometer (Bruker-Franzen Analytik GmbH, Germany) equipped with a source of the nanospray type. The gold / palladium coated capillary is from Protana (Odense, Denmark). The capillary and the counter-electrode are respectively at 750 and 250 V. The voltage applied at the outlet of the metallized glass capillary is at 80 V. The calibration is carried out with the following 5 peptides: Leu-enkephalin, angiotensin, substance -P, bombesin, and ACTH, with the monoisotopic molecular weights expressed in m / z (MH +) following 712.38, 1046.54, 1347.74, 1620.81, and 2465.20.

 4) Proteolytic cleavages. a) Carboxypeptidase-P digestion to determine and confirm the sequence of the DIM-13 C-terminal region.

enzyme substrat de 1 : 20 (poids/poids). Des fractions aliquotes de 0, 5 gl sont déposées sur la cible MALDI-TOF après 30 s, 2 min, 5 min et 10 min d'incubation à 37OC. Three gnoles of DIM-13, solubilized in 25 mM ammonium carbonate buffer at pH 8, are treated with carboxypeptidase-P (Boehringer, Mannheim) at a ratio of

substrate enzyme of 1: 20 (w / w). 0.5 μl aliquots are plated on the MALDI-TOF target after 30 sec, 2 min, 5 min and 10 min incubation at 37 ° C.

After 10 minutes, an equivalent amount of enzyme is added to the reaction medium and identical kinetic analysis is performed. Sequencing of the C-terminal region by carboxypeptidase-P is followed by MALDI-TOF mass spectrometry. The samples are analyzed on the previously described equipment (see Example 2-1) and the samples are prepared using the thin film technique according to the following statement:

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d'acide a-cyano-4-hydroxycinnamique (4-HCCA) à 7 g/l dans l'acétone, est déposé sur la cible MALDI-TOF, 0, 5 jell de TFA à 1% est ajouté à ce lit de matrice cristallisée et 0, 5 J. l de milieu de digestion est ajouté après les différents temps d'incubation. Après séchage sous un vide léger, les sels du milieu réactionnel sont éliminés par lavage (2 pi) avec du TFA à 1%, le tout est séché sous un vide léger et analysé par spectrométrie de masse MALDI-TOF.

of α-cyano-4-hydroxycinnamic acid (4-HCCA) at 7 g / l in acetone is deposited on the MALDI-TOF target, 0.5 μg of 1% TFA is added to this matrix bed. crystallized and 0.5 J. of digestion medium is added after the different incubation times. After drying under a slight vacuum, the salts of the reaction medium are removed by washing (2 μl) with 1% TFA, dried under a slight vacuum and analyzed by MALDI-TOF mass spectrometry.

 The instrumentation is identical to that previously described (see Example 2-2) and the calibration is carried out with the five following peptides: Leuenkephalin, angiotensin, substance-P, bombesin and ACTH, with respective MH + (average and monoisotopic) of: 712.82 / 712, 38; 1047, 20/1046, 54; 1348.66 / 1347, 74; 1621.86 / 1620, 81 and 2466, 71/2465, 20. b) Trypsin digestion for verification of the primary structure of DIM-13.

 Dim-13 is dissolved in 25 mM ammonium carbonate buffer at pH 8, and subjected to trypsin digestion at the enzyme: substrate ratio of 1:20 (w / w).

incubation de 12 heures à 35OC. Les produits de ces deux digestions à 2 et 12 heures ont été analysés par spectrométrie de masse MALDI-TOF comme décrit précédemment (voir exemple 2-2). Two aliquots of 0.5 ju. 1 are analyzed by MALDI-TOF mass spectrometry after incubation for 2 hours and one

incubation for 12 hours at 35OC. The products of these two digests at 2 and 12 hours were analyzed by MALDI-TOF mass spectrometry as previously described (see Example 2-2).

 All of these techniques made it possible to obtain the complete structure of DIM-13. This sequence identity was confirmed by the analysis of the databanks.

 The primary sequence of DIM-13 represented in the sequence list under the number SEQ ID NO: 1 is the following: ZFHVERPDRTVDFGNGGFSAQRF

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Molecular weight measured by MALDI-TOF mass spectrometry: 2649, 4 Da
Molecular mass calculated: 2650.8 Da
Example 3: Structural characterization of DIM-12, DIM-10, DIM-6, DIM-8 and DIM-5.

 1) Verification of the purity of DIM-12, DIM-10, DIM-6, DIM-8 and DIM-5 by MALDITOF mass spectrometry.

 This study was carried out on a Bruker (Bremen, Germany) BIFLEXTM III mass spectrometer equipped with SCOUTS high resolution optics and a reflectron.

This instrument has a maximum acceleration potential of 20kV and can be used in either linear mode or reflectron mode. Ionization is carried out with a 337 nm beam emitted by a nitrogen laser at a frequency of 3 Hz.

The mass spectra were externally calibrated with a standard mixture of Drosophila antimicrobial peptides namely drosocine, metchnikowine and drosomycin of known molecular mass at 2199.5 Da, 3046.4 Da and 4890.5 respectively. da.

 The MALDI-TOF analyzed samples are prepared by the sandwich technique set forth as follows, for the identification of DIM-12, DIM-10, DIM-6, DIM-8 and DIM-5 peptides. The different products to be analyzed (0.5 μl) were deposited on a thin layer of acyano-4-hydroxycinnamic acid crystals (4-HCCA, Sigma) obtained by rapidly evaporating a saturated solution in acetone and then covered with 0.5 ul of the second template, saturation 4-HCCA in a solution of 50% acetonitrile in water. After drying under a slight vacuum, the samples were washed with 1.5 μl of 0.1% TFA before being re-dried and introduced into the mass spectrometer.

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 2) Unblocking the N-terminal region of DIM-12, -10 and -6 by treatment with pyroglutamate aminopeptidase.

 The absence of a signal at Edman degradation sequencing suggests the presence of a cyclic Nterminal amino acid of pyroglutamic acid type (Z or pGlu). As a result, the purified peptides were digested with glutamate aminopeptidase (Boehringer Mannheim) to remove this amino acid pGlu inhibitor from the sequencing reaction by the Edman technique.

heures à 25OC. Les produits des digestions sont purifiés par chromatographie sur une colonne microbore de phase inverse (Aquapore RP-300 C8, Brownlee, 100 x 1 mm, 300 À) protégée par un filtre (Ultra-Low Dead-Volume Precolumm Filter, Upchurch Scientific). L'élution est réalisée par un gradient linéaire biphasique d'acétonitrile de 2 à 18% dans le TFA 0,05% pendant 10 minutes et de 18% à 28% d'acétonitrile pendant 50 min à un débit constant de 80 ul/min à 35 C pour DIM-12 et de 2 à 17 en 10 min et de 17 à 27% d'acétonitrile pendant 50 min pour les DIM-10 et-6. La masse des produits obtenus est vérifiée par spectrométrie de masse MALDI-TOF et les produits sont séquencés par dégradation d'Edman. The peptides are dissolved in the proteolysis buffer consisting of 100 mM sodium phosphate buffer at pH 8 supplemented with 10 mM disodium EDTA, 5 mM dithioerythritol, and 5 mM glycerol in the presence of the enzyme. pyroglutamate aminopeptidase at the enzyme / substrate ratio of 1: 10 (w / w). The incubations are carried out for 18 hours at 4 ° C. After this first incubation, a new enzyme preparation is added and the incubation continues.

hours at 25OC. The products of the digestions are purified by chromatography on a reverse-phase microbore column (Aquapore RP-300 C8, Brownlee, 100 x 1 mm, 300 A) protected by a filter (Ultra-Low Dead Volume Precolumm Filter, Upchurch Scientific). The elution is carried out by a linear two-phase gradient of acetonitrile from 2 to 18% in 0.05% TFA for 10 minutes and from 18% to 28% of acetonitrile for 50 minutes at a constant flow rate of 80 μl / min. at 35 ° C for DIM-12 and from 2 to 17 in 10 minutes and from 17 to 27% acetonitrile for 50 minutes for DIM-10 and -6. The mass of the products obtained is verified by MALDI-TOF mass spectrometry and the products are sequenced by Edman degradation.

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 3) Edman degradation sequencing of DIM-12, -10 and -6 lacking N-terminal pyroglutamic acid. a) Sequencing of DIM-12.

 Automatic sequencing by Edman degradation of DIM-12 peptide and detection of phenylthiohydantoin derivatives were performed on an ABI473A sequencer (PEApplied Biosystems division of Perkin Helmer).

A partial sequence of 10 amino acids was obtained (FHVERPGRTV). This sequence information associated with the mass spectrometry results made it possible to define the complete primary structure of DIM-12.

The primary sequence of DIM-12 represented in the sequence list under the number SEQ ID NO: 2 is the following: ZFHVERPGRTVDVGNGGFYIQ RG
Molecular weight measured by MALDI-TOF mass spectrometry: 2573.1 Da
Molecular mass calculated: 2572.8 Da b) Sequencing of DIM-10.

 Automatic sequencing by Edman degradation of DIM-10 peptide and detection of phenylthiohydantoin derivatives were performed on an ABI473A sequencer (PEApplied Biosystems division of Perkin Helmer).

A partial sequence of 15 amino acids was obtained (LHVARPDRTVTIGNG). This sequence information associated with the mass spectrometry results made it possible to define the complete primary structure of DIM-10.

The primary sequence of DIM-10 represented in the sequence listing under the number SEQ ID NO: 3 is: ZLHVARPDRTVTIGNGGVYIQ RS
Molecular weight measured by MALDI-TOF mass spectrometry: 2521.9 Da
Molecular mass calculated: 2520.9 Da

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 c) Sequencing of DIM-6.

Automatic sequencing by Edman degradation of DIM-6 peptide and detection of phenylthiohydantoin derivatives were performed on an ABI473A sequencer (PEApplied Biosystems division of Perkin Helmer). A partial sequence of 15 amino acids was obtained (FHVERPGRTVDVGNG). This sequence information associated with the mass spectrometry results made it possible to define the complete primary structure of DIM-6. DIM-6 thus corresponds to a truncated form of DIM-12.

The primary sequence of DIM-6 represented in the sequence listing under the number SEQ ID NO: 5 is as follows: ZFHVERPGRTVDVGNGGF
Molecular weight measured by MALDI-TOF mass spectrometry: 1954, 9 Da
Molecular mass calculated: 1955.2 Da
4) Determination of the primary structure of DIM-8 and -5 by trypsinolysis and analysis of trypsinolysis fragments by MALDI-TOF mass spectrometry.

 DIM-8 and -5 are dissolved in 100 mM Tris-HCl buffer at pH 8.5, and subjected to trypsin digestion at the enzyme: substrate ratio of 1: 10 (w / w) for 4 hours. at 37OC. The products of the digestions are purified by chromatography on a reverse-phase microbore column (Aquapore RP-300 C8, Brownlee, 100 x 1 mm, 300 A) protected by a filter (Ultra-Low Dead Volume Precolumm Filter, Upchurch Scientific). Elution was performed by a linear gradient of acetonitrile from 2 to 62% in 0.05% TFA for 80 min at a constant rate of 80 μl / min at 35 ° C for DIM-5 and -8. The masses of the products obtained are verified by MALDITOF mass spectrometry.

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 For DIM-5, a mass of 1166.3 MH + is obtained corresponding to the exact mass of the N-terminal fragment of DIM-13. The C-terminal part of DIM-5 corresponds to the fragment of DIM-13 TVDFGNGG. DIM-5 thus corresponds to a truncated form of DIM-13 or DIM-8 (see below).

The primary sequence of DIM-5 represented in the sequence list under the number SEQ ID NO: 6 is the following: ZFHVERPDRTVDFGNGG
Molecular weight measured by MALDI-TOF mass spectrometry: 1914.3 Da
Molecular mass calculated: 1914.0 Da
For DIM-8, the analysis of trypsinolysis fragments by MALDI-TOF mass spectrometry after purification shows a mass at 1164.9 MH + and another mass at 1199.2 MH +. The molecular weight measured at 1164.9 MH + corresponds exactly to the mass of the N-terminal fragment of DIM-13, ZFHVERPDR. The mass measured at 1199.2 MH + corresponds to the TVDFGNGGFSAQ fragment of DIM-13 or to the C-terminal fragment of DIM-13 lacking the Cterminal Arg-Phe dipeptide. DIM-8 thus corresponds to a truncated form of DIM-13.

The primary sequence of DIM-8 represented in the sequence list under number SEQ ID NO: 4 is as follows: ZFHVERPDRTVDFGNGGFSAQ
Molecular weight measured by MALDI-TOF mass spectrometry: 2346.0 Da
Molecular Weight Calculated: 2346.1 Da
Example 4: ICAM-1 mediated cell adhesion inhibition test

 The inhibitory activity of DIM peptides on ICAM-1 mediated cell adhesion was tested by

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cellules sont lysées et des mesures de spectrofluométrie (Â, 485nm et 530nm) sont effectuées à l'aide d'un cytofluor millipore 2350. measuring the% inhibition of adhesion between blood monocytes (U-937) and human endothelial cells (HUVEC) expressing the intercellular adhesion molecule ICAM-1 after stimulation (Cronstein, BN, et al., 1992, Proct.Natl.Academic Science, 89, 9991-9995: A Mechanism for the Anti-inflammatory Effects of Corticosteroids: The Glucocorticoid Receptor Regules Leukocyte Adhesion Molecule One and Intercellular Adhesion Molecule I). Human monolayer endothelial cells (HUVEC) in 96-well plates are stimulated for 16-18 hours at 37 ° C. in a CO 2 incubator with lipopolysaccharides, said DIM (10) 1M peptide to be tested added at the same time. Then, the stimulating medium containing the lipopolysaccharides is removed and the human monocytes (U-937) labeled with a fluorochrome (BCECF) are added to allow the adhesion of the latter to the human endothelial cells (HUVEC) stimulated for 15 minutes at 37 ° C. in a CO 2 incubator, always in the presence of said DIM peptide to be tested. The

cells are lysed and spectrofluometry measurements (λ, 485nm and 530nm) are performed using a cytofluor millipore 2350.

 A control test was carried out in parallel. The reference inhibitor compound is nordihydroguareaic acid (NDGA). 50% adhesion inhibition (IC50) is obtained for 11 LLM.

 % inhibition measured for peptide DIM-13 (10 RM): 58%

Claims (18)

 the amino acids 0'19'0'20'0'23'identical or different, are chosen from alanine, glycine, isoleucine, phenylalanine, serine, tyrosine or valine.

1) Peptide of general formula (I): ## STR1 ## or a fragment thereof, wherein: or a fragment thereof, wherein: - X, 0, ss, o represent amino acid residues, - the amino acid LI is selected from the group consisting of glutamic acid, glutamine and pyroglutamic acid, the amino acids (, ss6'ss, ss2, which are identical or different, are chosen from the basic amino acids, the amino acids 11) 2 1 (D 4 1 (Dl li (Dl3 1a) 1 8 identical or different, are chosen from hydrophobic amino acids, - the amino acids 9s'97, 9s'910'912'914'91s' 916'917'921'Ithenes or different, are selected from among the negatively charged amino acids or small polar amino acids or broad polar amino acids or glycine, alanine, proline,  2) Peptide according to claim 1, characterized in that it consists of at least 17 successive amino acids and preferably 23 successive amino acids of formula (I).  3) Peptide according to one of claims 1 or

 2, characterized in that the C-terminal amino acid is ig, a19, o0, 021 or ss22. <Desc / Clms Page number 27>

4) Peptide according to any one of the preceding claims, characterized in that it corresponds to one of the following formulas: 12ss3405ss60708ss90101101213014015016017 (II) eee. oiAeiAseis (in) xc, e, p, e, e, e, e, eeee, o ,, oe (iv) 5) Peptide according to any one of the preceding claims, characterized in that the basic amino acids are chosen from arginine, histidine or lysine.  6) Peptide according to any one of the preceding claims, characterized in that the hydrophobic amino acids are chosen from isoleucine, leucine, phenylalanine, tryptophan, tyrosine or valine.  7) Peptide according to any one of the preceding claims, characterized in that the negatively charged amino acids are selected from aspartic acid or glutamic acid.  8) Peptide according to any one of the preceding claims, characterized in that the small polar amino acids are selected from serine or threonine.  9) Peptide according to any one of the preceding claims, characterized in that the broad polar amino acids are selected from asparagine or glutamine.  Peptide according to one of the preceding claims, characterized in that one <Desc / Clms Page number 28>  - CY2 is alanine, isoleucine or valine; - 23 is phenylalanine, glycine or serine.

 less amino acids (D2, 05r 071 08, 012, (1) 131 015, (Dl8l CY191 20'23 where it is present, with the following meaning: - is phenylalanine or leucine, - 95 is glutamic acid or alanine, - 97 is proline or serine, - 9g is aspartic acid or glycine, - 912 is aspartic acid or threonine, - (D1 is phenylalanine, valine or isoleucine - # 15 is asparagine or serine, - cog is phenylalanine or valine, - # 19 is serine, tyrosine, valine or phenylalanine,

 11) A peptide according to any one of the preceding claims, characterized in that it is selected from the group comprising the sequences

 following: - SEQ ID NO: 1: ZFHVERPDRTVDFGNGGFSAQRF - SEQ ID NO: 2: Z F H V E R P G R T V D V G N G G F Y I Q R G - SEQ ID NO: 3:

 ## STR2 ## SEQ ID NO: 5: ZFHVERPGRTVDVGNGGF - SEQ ID NO: 6: ZFHVERPDRTVDFGNGG ## STR2 ## <Desc / Clms Page number 29> 12) A peptide according to any one of the preceding claims, characterized in that the amino acid LI is linked to a protecting group, an acyl group, an acetyl group or a methyl group, it being understood that said amino acid LI is not then pyroglutamic acid.  13. Peptide according to any one of the preceding claims, characterized in that the C-terminal amino acid is chemically modified.  14) A peptide according to claim 13, characterized in that the C-terminal amino acid is amide.  15) A polypeptide characterized in that it comprises a peptide according to any one of claims 1 to 11.  16) Polypeptide according to claim 15, characterized in that it comprises a peptide according to any one of claims 1 to 11, one and / or the other of the ends of said peptide comprises a peptide residue necessary for its expression and / or targeting in a host organism.  17) Pharmaceutical composition, especially vaccine, characterized in that it comprises as active agent at least one peptide according to any one of claims 1 to 14 or a polypeptide according to one of claims 15 or 16, advantageously associated in said composition to an acceptable vehicle.  18) Use of a peptide according to any one of claims 1 to 14 or a polypeptide according to one of claims 15 or <Desc / Clms Page number 30>  16, for the preparation of a pharmaceutical composition useful for the prevention or treatment of inflammatory and / or immune disorders in humans or animals.