Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
  • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/04—Antipruritics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Atherothrombosis a world-leading life-threatening disease, is linked to inappropriate activation of blood leukocytes and platelets leading to a destructive inflammatory response within the vascular wall and thrombotic occlusion and/or rupture due the fibrinolytic cascade. Consequently, a restoration of the physiologic cell regulation at the interface between the blood and the vessel wall would represent an innovative therapeutic option to fight atherothrombosis.
• Autoantigens may become immunogenic because they are altered chemically, physically, or biologically. Certain chemicals couple with body proteins, making them immunogenic (as in contact dermatitis). Drugs can produce several autoimmune reactions by binding covalently to serum or tissue proteins (see below). Photosensitivity exemplifies physically induced autoallergy: Ultraviolet light alters skin protein, to which the patient becomes allergic. In animal models, persistent infection with an RNA virus that combines with host tissues alters autoantigens biologically, resulting in an autoallergic disorder resembling SLE.
• CD31 (PECAM- 1)
• CD31 is a single chain, homophilic transmembrane receptor exclusively and constitutively present on all endothelial cells, platelets and leukocytes.
• CD31 consists of a single chain 130-kDa glycoprotein comprising six Ig-like extracellular domains, a short transmembrane segment and a cytoplasmic tail.
• the cytoplasmic tail contains two important tyrosine- based motifs (around Y663 and Y686) that form specific docking sites for SH2-containing adaptor molecules that preferentially associate with phosphatases and act as ImmunoTyrosine-based Inhibitory Motif (ITIM)s.
• ITIM ImmunoTyrosine-based Inhibitory Motif
• the intracellular CD31 ITIMs are not phosphorylated in resting conditions because CD31 does not possess an intrinsic kinase activity.
• CD31 molecules bind to each other via a trans-homophilic liaison of the Ig-like domains 1 -2 between interacting cells. This trans- homophilic binding is required to trigger the clustering of the CD31 molecules on the membrane plane, which in turn requires a cis-homophilic juxtamembrane sequence.
• the phosphorylation of the CD31 intracellular ITIMs becomes then possible because its ITIMs can be exposed to the activity of the tyrosine kinases that are carried close by other cluster-associated membrane receptors (such for instance the T cell receptor).
• CD31 is “lost” on certain circulating lymphocytes. Its loss is observed upon lymphocyte activation and it has been recently shown that the absence of lymphocyte CD31 signalling, in turn, heightens the pathologic immune responses involved in the development of atherothrombosis.
• the inventors have previously reported that the assumed loss of CD31 on activated/memory T lymphocytes is actually incomplete and results from shedding of CD31 between the 5 th and the 6 th extracellular Ig-like domains (described in international patent publication WO2010/000741 ).
• the shed extracellular domain of CD31 (further referred to as "shed CD31 ") is then released into the circulation, where it is present together with a soluble splice variant of CD31 .
• a high risk of atherothrombosis is linked with the increase in shed CD31 and decrease in splice variant CD31 in the circulation, and not with the total level of circulating CD31 .
• CD31 is not lost on blood lymphocytes but only cleaved provided a unique opportunity to rescue its physiological immunoregulatory function by targeting the residual portion of the molecule.
• the inventors have identified a specific 8 amino acid peptide within the membrane juxta-proximal part of extracellular CD31 , which is of particular utility in inhibiting platelet and leukocyte activation and in treatment of a thrombotic or an inflammatory disorders.
• This sequence is soluble in water is an advantage on the pharmacological point of view.
• They have further shown that peptide sequences corresponding to this 8 amino-acid fragment comprising inversions, and/or unnatural aminoacids, such as D- enantiomers, also retain these activities or demonstrate improved activity. Incorporation of unnatural aminoacids in peptides intended for therapeutic use is of utility in increasing the stability of the peptide, in particular in vivo stability.
• the peptide of the invention is soluble in an organic or nonorganic solvent, such as water.
• the CD31 peptide of the invention may comprise a chirality change such as e.g. replacement of one or more naturally occurring amino acids (L enantiomer) with the corresponding D-enantiomers.
• D-enantiomers of amino acids are referred to by the same letter as their corresponding L-enantiomer, but in lower case.
• the L- enantiomer of arginine is referred to as 'R'
• the D-enantiomer is referred to as 'r'.
• 1 , 2, 3, 4, 5, 6, 7 or 8 of the amino acids in the peptide may be in the D- enantiomer form.
• the peptides may comprise ther modified or non-natural amino acids, as described below.
• the CD31 peptide of the invention may comprise an inverted sequence, namely an inversion of the amino acid chain (from the C-terminal end to the N-terminal end).
• the entire amino acid sequence of the peptide may be inverted, or a portion of the amino acid sequence may be inverted.
• a consecutive sequence of 2, 3, 4, 5, 6, 7 or 8 amino acids may be inverted.
• Reference herein to 'inverted' amino acids refers to inversion of the sequence of consecutive amino acids in the sequence.
• the peptide of the invention may thus have the sequence:
• H-RVFLAPWK-OH (SEQ ID NO 5), corresponding to the amino acid sequence of amino acids 582 to 589 of SEQ ID NO: 2; (P8F)
• H-kwpalivr-OH (SEQ ID NO 8), corresponding to the inverted sequence of amino acids 593-600 of SEQ ID NO: 1 in D-enantiomer form, namely a retro-inversion of said sequence.
• the peptide of the invention commences with the motif
• isolated nucleic acids encoding the peptides of the invention, and pharmaceutical compositions comprising said peptides, as described below.
• the peptide may optionally comprise sequences heterologous to CD31 .
• These heterologous sequences may e.g. correspond to a carrier molecule such as the Keyhole Limpet Hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), thyroglobulin (THY) or the multiple antigenic peptide (MAP).
• KLH Keyhole Limpet Hemocyanin
• BSA bovine serum albumin
• OVA ovalbumin
• THY thyroglobulin
• MAP multiple antigenic peptide
• the sequence of CD31 peptides according to the invention is preferably derived from the sequence of human or murine CD31 .
• the sequence of CD31 may be derived from any non-human mammalian CD31 sequence.
• Figure 1 shows an alignment between the human, murine, bovine and pig CD31 sequences. The person skilled in the art can easily identify the corresponding sequence in another non-human mammalian CD31 protein by performing a sequence alignment with the sequences shown in Figure 1 .
• sequence alignment and determination of sequence identity are well known in the art, for example using publicly available computer software such as BioPerl, BLAST, BLAST-2, CS-BLAST, FASTA, ALIGN, ALIGN-2, LALIGN, Jaligner, matcher or Megalign (DNASTAR) software and alignment algorithms such as the Needleman-Wunsch and Smith-Waterman algorithms.
• publicly available computer software such as BioPerl, BLAST, BLAST-2, CS-BLAST, FASTA, ALIGN, ALIGN-2, LALIGN, Jaligner, matcher or Megalign (DNASTAR) software and alignment algorithms such as the Needleman-Wunsch and Smith-Waterman algorithms.
• CD31 peptides according to the invention may have the biological activity of exerting a dose-dependent inhibition of T-cell proliferation in vitro and/or of inhibiting the mixed- lymphocyte reaction (MLR; inhibition of platelet aggregation; inhibition of platelet activatin; inhibition of thrombin generation by platelets and/or inhibition of VCAM-1 expression of endothelial cells.
• MLR mixed- lymphocyte reaction
• Their biological activity may for example be measured as described in Example 1 , 2, 3 or 4 or in Zehnder et al. 1995, Blood. 85(5):1282-8, Fornasa et al. 2010, J Immunol 184: 6585-6591 ; Fornasa et al, 2012, Cardiovascular Research 94: 30-37.
• the T-cell proliferation assay may comprise comparing the radioactivity incorporated into T-cells cultured either in the presence or in the absence of the compound to be tested. This assay may for example be performed as follows:
• peripheral blood mononuclear cells or spleen cells, or lymph node cells
• the leukocyte activation may be assessed by comparing expression levels of the early activation marker CD69 leukocytes (blood mononuclear cells or spleen cells, or lymph node cells) cultured either in the presence or in the absence of the compound to be tested.
• This assay may for example be performed as follows:
• an appropriate stimulus anti-CD3 purified antibodies and bone marrow derived dendritic cells; or LPS or a lectin such as ConA,
• N-terminal and/or C-terminal ends of the peptides such as e.g. N- terminal acylation (preferably acetylation) or desamination, or modification of the C- terminal carboxyl group into an amide or an alcohol group;
• Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, it will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching.
• an “isolated” peptide it is intended that the peptide is not present within a living organism, e.g. within human body.
• the isolated peptide may be part of a composition or a kit.
• the isolated peptide is preferably purified.
• the compounds of the invention may be produced by any well-known procedure in the art, including chemical synthesis technologies and recombinant technologies.
• Examples of chemical synthesis technologies are solid phase synthesis and liquid phase synthesis.
• a solid phase synthesis for example, the amino acid corresponding to the C-terminus of the peptide to be synthesized is bound to a support which is insoluble in organic solvents, and by alternate repetition of reactions, one wherein amino acids with their amino groups and side chain functional groups protected with appropriate protective groups are condensed one by one in order from the C-terminus to the N- terminus, and one where the amino acids bound to the resin or the protective group of the amino groups of the peptides are released, the peptide chain is thus extended in this manner.
• Solid phase synthesis methods are largely classified by the tBoc method and the Fmoc method, depending on the type of protective group used.
• protective groups include tBoe (t-butoxycarbonyl), Cl-Z (2-chlorobenzyloxycarbonyl), Br-Z (2- bromobenzyloyycarbonyl), Bzl (benzyl), Fmoc (9-fluorenylmcthoxycarbonyl), Mbh (4, 4'- dimethoxydibenzhydryl), Mtr (4-methoxy-2, 3, 6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl), Z (benzyloxycarbonyl) and Clz-Bzl (2, 6-dichlorobenzyl) for the amino groups; N02 (nitro) and Pmc (2,2, 5,7, 8-pentamethylchromane-6-sulphonyl) for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups).
• the peptide may be synthesized using recombinant techniques.
• a nucleic acid encoding a peptide according to the invention (further referred to as "a nucleic acid according to the invention") is cloned into an expression vector.
• the nucleic acid of the invention is preferably placed under the control of expression signals (e.g. a promoter, a terminator and/or an enhancer) allowing its expression.
• the expression vector is then transfected into a host cell (e.g. a human, CHO, mouse, monkey, fungal or bacterial host cell), and the transfected host cell is cultivated under conditions suitable for the expression of the peptide.
• the method of producing the peptide may optionally comprise the steps of purifying said peptide, chemically modifying said peptide, and/or formulating said peptide into a pharmaceutical composition.
• CD31 peptides for the treatment of thrombotic and inflammatory disorders
• CD31 peptides according to the invention are capable of activating CD31 -mediated signaling, even in CD31 " (i.e. CD31 shed ) T lymphocytes.
• CD31 shed i.e. CD31 shed
• such peptides are capable of preventing disease progression and aneurysm formation in a mouse model for atherosclerosis, and improving clinical score in a mouse model of multiple sclerosis.
• the invention therefore also provides a peptide of the invention for use in activating CD31 -mediated signaling.
• These peptides preferably exert a dose-dependent inhibition of T-cell proliferation in vitro.
• the activation of CD31 -mediated signaling may be an in vitro or an in vivo activation.
• the in vivo method may be a method of treatment as defined below.
• CD31 -mediated signaling refers to a signaling pathway in which CD31 is involved.
• Such pathways are well known in the art and include those described e.g. in Newman and Newman (2003 Arterioscler Thromb Vase Biol 23:953-964) and in Newton-Nash and Newman (1999. J Immunol 163:682-688).
• the invention therefore also provides a peptide of the invention for use in the treatment of a thrombotic or an inflammatory disorder.
• These peptides preferably exert a dose-dependent inhibition of T-cell proliferation in vitro.
• the activation of CD31 -mediated signaling may be an in vitro or an in vivo activation.
• methods of treatment of a thrombotic or an inflammatory disorder using the peptides of the invention preferably comprising administration of said peptides to an individual in need thereof.
• thrombotic disorder includes but is not limited to atherothrombosis, atherosclerosis, acute coronary syndrome, ischemic stroke, peripheral arterial disease and abdominal aortic aneurysm.
• the term "inflammatory disorder” includes but is not limited to chronic inflammatory diseases such as inflammatory bowel disease, psoriasis, atopic dermatitis, cerebral amyloid angiopathy, vasculitis.
• the term also includes autoimmune disorders, including but not limited to rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD), systemic lupus erythematodes (SLE), Graves' disease and diabetes mellitus.
• Other conditions such as acute and chronic grant rejection including graft versus host disease (GVHD) and septic shock may be encompassed by the term.
• the peptides of the invention may be used to treat septic shock (optionally in combination with antibiotic therapy, e.g. large spectrum antibiotic therapy) and in transplantation such as bone marrow, kidney, heart, liver or lung transplantation.
• said thrombotic or inflammatory disorder is associated with a loss of CD31 + T lymphocyte phenotype.
• CD31 peptides restore CD31 signaling even in individuals with a CD31 T lymphocytes phenotype. Therefore, in the context of the present invention, CD31 peptides are preferably used to treat a subgroup of individuals and/or patients having a CD31 T lymphocytes phenotype.
• CD31 T lymphocyte phenotype is used interchangeably with the term “CD31 shed T lymphocyte phenotype”. These terms refer to the phenotype of an individual having apparently lost CD31 on its circulating T cells when conventional prior art methods for detecting CD31 , e.g. such as those described in Stockinger et al. (Immunology, 1992, 75(1 ):53-8), Demeure et al. (Immunology, 1996, 88(1 ):1 10-5), Caligiuri et al. (Arterioscler Thromb Vase Biol, 2005, 25(8): 1659-64) or Caligiuri et al.
• the antibody used for detecting CD31 binds to an epitope located on any one of the 1 st to the 5 th extracellular Ig-like domains.
• individuals having a CD31 " T lymphocyte phenotype meaning that at least 50%, 60%, 65%, 70%, 75%, 80%, 90% or 95% of their circulating T lymphocytes are CD31 shed lymphocytes.
• Either the plasma concentration of T -cell-derived truncated CD31 or the frequency of CD31 " T lymphocytes, compared to CD31 + T lymphocytes, may be measured.
• the invention is also directed to a method of treating or preventing a thrombotic or an inflammatory disorder comprising the step of administering an effective amount of a peptide as described herein, or a nucleic coding therefore, to an individual in need thereof.
• Said individual in need thereof preferably suffers from or is at risk of suffering from a thrombotic or an inflammatory disorder.
• said individual has a CD31 " T lymphocytes phenotype.
• the individuals to be treated in the frame of the invention are preferably human individuals. However, the veterinary use of CD31 peptides for treating other mammals is also contemplated by the present invention.
• compositions comprising at least one peptide of the invention include all compositions wherein the peptide(s) are contained in an amount effective to achieve the intended purpose.
• the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
• suitable pharmaceutically acceptable vehicles are well known in the art and are described for example in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, USA, 1985)., which is a standard reference text in this field. Pharmaceutically acceptable vehicles can be routinely selected in accordance with the mode of administration, solubility and stability of the peptides.
• formulations for intravenous administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
• the peptides of the present invention may be administered by any means that achieve the intended purpose.
• administration may be achieved by a number of different routes including, but not limited to subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intracerebral, intrathecal, intranasal, oral, rectal, transdermal, buccal, topical, local, inhalant or subcutaneous use.
• endovascular prostheses such as tubes and stents, artificial heart valves, bone and dental prostheses
• Dosages to be administered depend on individual needs (which may be quantified by measuring cell-specific truncated CD31 in the plasma with using our bead-based method), on the desired effect and the chosen route of administration. It is understood that the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The total dose required for each treatment may be administered by multiple doses or in a single dose.
• the compositions are presented in unit dosage forms to facilitate accurate dosing.
• unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a pre-determined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
• Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.
• the compound of the invention is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
• physiologically acceptable is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which is administered.
• the above active ingredients may be formulated in unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
• the invention also contemplates a pharmaceutical composition
• a pharmaceutical composition comprising a nucleic acid encoding the peptide of the invention in the frame of e.g. a treatment by gene therapy.
• the nucleic acid is preferably present on a vector, on which the sequence coding for the peptide is placed under the control of expression signals (e.g. a promoter, a terminator and/or an enhancer) allowing its expression.
• expression signals e.g. a promoter, a terminator and/or an enhancer
• the vector may for example correspond to a viral vector such as an adenoviral or a lentiviral vector.
• kits comprising a pharmaceutical composition comprising a CD31 peptide of the invention and instructions regarding the mode of administration. These instructions may e.g. indicate the medical indication, and/or the route of administration, and/or the dosage, and/or the group of patients to be treated.
• 'Treatment' includes both therapeutic treatment and prophylactic or preventative treatment, wherein the object is to prevent or slow down the targeted pathologic condition or disorder.
• Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
• the terms 'therapy', 'therapeutic', 'treatment' or 'treating' include reducing, alleviating or inhibiting or eliminating the symptoms or progress of a disease, as well as treatment intended to reduce, alleviate, inhibit or eliminate said symptoms or progress.
• Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
• methods and compositions of the invention are used to delay development of a disease or disorder or to slow the progression of a disease or disorder.
• an effective amount preferably a therapeutically effective amount of the protein or vector of the invention is administered.
• An 'effective amount' refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
• the effective amount may vary according to the drug or prodrug with which the protein or vector is co-administered.
• a 'therapeutically effective amount' of a peptide of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein, to elicit a desired therapeutic result.
• a therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the protein are outweighed by the therapeutically beneficial effects.
• a therapeutically effective amount also encompasses an amount sufficient to confer benefit, e.g., clinical benefit.
• FIG. 2 shows the effect of P8F (SEQ ID NO 5)and P8R1 (SEQ ID NO 6) peptides on T-cell activation.
• Jurkat T cells were preincubated with the fluo 3-AM calcium probe and stimulated by cross-linking the TCR with anti-CD3 antibodies and secondary antibody F(ab')2 fragments.
• the figure shows the effect of 60 ⁇ g/ml of each peptide on calcium mobilization following TCR stimulation as follows: No peptide control (squares), P8F (SEQ ID NO 5) peptide (circles) and P8RI (SEQ ID NO 6) peptide (triangles).
• Figure 3 shows that immobilised P8RI onto aminated solid surfaces inhibits leukocyte adherence and activation.
• Peripheral blood mononuclear cells (5x10 5 cells/ml) were transferred in P8RI-coated and control Immulon® wells and stimulated with 1 ⁇ g/ml LPS for 60'.
• Control wells were coated with acetic acid (with EDC/S-NHS) as irrelevant source of COOH groups (Control) or left uncoated (Bare). Left, after 3 washes with PBS, Control wells contained several adherent leukocytes at variance with P8RI wells in which leukocytes were virtually absent.
• Figures 4-6 show that immobilised P8RI prevents platelet activation.
• Thrombin generation was measured at 37°C by calibrated automated thrombogram in platelet rich plasma in the presence of Tissue Factor (0.5 pM) in aminated polystyrene wells. P8Rlwas covalently bound onto the wells.
• a scramble peptide was used as negative control
• Fig 4 , Representative thrombograms
• Fig 5 Velocity index of thrombin activity, calculated as Peak / Time to peak - LagTime, was significantly reduced in P8RI vs Bare and Control wells.
• Fig 6 The concentration of soluble P-selectin (sCD62P), released by platelets, was significantly reduced in the supernatant of P8RI-coated wells. Data are from 8 independent experiments, * p ⁇ 0.01
• Figure 8 demonstrates a therapeutic effect of P8RI peptide in experimental autoimmune encephalitis, a mouse model of multiple sclerosis.
• a curative assay was performed in the EAE mouse model by starting the administration of P8RI at 2mg/kg/day subcutaneously after the onset of ascending paralysis (score ⁇ 1 ), using prednisone as a pharmaceutical control.
• the figure shows ascending paralysis (clinical score) linked to active encephalitis in mice treated with vehicle (squares), prednisone at 2mg/kg/day (circles) or P8RI at 2mg/kg/day (triangles).
• Figure 9 demonstrates a therapeutic effect of P8F peptide in a mouse model of acture thrombosis.
• Atherosclerotic mice were treated with P8F at 2.5, 5 or 10 mg/kg/day, with aspirin as a positive control and vehicle as a negative control.
• Figure 10 shows a Cochran-Mantel-Haenszel test (Chi Square) analysis of the data presented in Figure 9.
• SEQ ID NO: 1 corresponds to the sequence of human CD31 .
• SEQ ID NO: 2 corresponds to the sequence of murine CD31 .
• SEQ ID NO: 4 corresponds to the sequence of pig CD31 .
• SEQ ID NO: 5 corresponds to the P8F peptide.
• SEQ ID NO: 6 corresponds to the P8RI peptide.
• SEQ ID NO: 7 corresponds to the human equivalent of the P8F peptide.
• SEQ ID NO: 8 corresponds to the human equivalent of the P8RI peptide H-kwpalivr-
• Fig 2 shows continuous flow cytometry acquisition before and after additionof the stimulus ⁇ the peptides.
• P8RI H-kwpalfvr-OH
• P8F H-RVFLAPWK-OH
• the microcapillaries of Vena8 Fluoro+TM biochip (400 ⁇ width ⁇ ⁇ ⁇ depth x20 mm length, Cellix) were coated with 2.5 mg/ml insoluble horse type I collagen overnight at 4 ⁇ ⁇ and then blocked with 0.1 % HSA for 1 hour at room temperature.
• P-PACK a direct thrombin inhibitor, non- chelating agent
• Example 3 Effect of the immobilised peptide on platelet activation and thrombin generation.
• Thrombin generation was measured at 37 ⁇ ⁇ by calibrated automated thrombogram (Hemker et al. Thromb Haemost. 2000; 83(4): 589-91 ) in platelet rich plasma (1 .5 x 10 8 platelets/ml) in the presence of Tissue Factor (0.5 pM) in aminated polystyrene wells (Covalink, Immunon® 2HB, Stago).
• SEQ ID NO 8 (P8RI, H-kwpalfvr-OH, MW 1016.26, TFA salt replaced by HCI salt, purity 100%, dissolved in water at 50 ⁇ , pH 4-4.5) was pre-treated with EDC/S-NHS (10:1 molar ratio) and covalently bound onto the Immulon® wells.
• a scramble peptide was used as control peptide
• Figs 4-6 show that thrombin activity, calculated as Peak / Time to peak - LagTime, was significantly reduced in P8RI vs Bare and Control wells, and that the concentration of soluble P-selectin (sCD62P), released by platelets, was significantly reduced in the supernatant of P8RI-coated wells.
• the inventors evaluated the effect of P8RI on the expression of VCAM-1 by immunofluorescence on the immortalized human brain endothelial cell Line HCMEC/D3 submitted to overnight stimulation with TNFa (50ng/ml) and IFNg (100ng/ml). Although the increase in VCAM-1 expression was not impressive, the presence of the peptide prevented it (data not shown). Quantitative analysis by FACS of the same experimental conditions ( Figure 7) showed that the reduction of VCAM-1 expression observed in the "peptide" wells was statistically significant (n 3 wells/condition). The effect was observed already at 6.25 ⁇ g/ml and did not change with higher doses of the peptide (doses were up to 50 ⁇ g/ml).
• P8RI H-kwpalfvr-OH (SEQ ID NO 6) MW 1016.26, TFA salt replaced by HCI salt, purity 100%, dissolved in water at 50 ⁇ , pH 4-4.5
• EDC/S-NHS 10:1 molar ratio
• Control wells were coated with acetic acid (with EDC/S-NHS) as irrelevant source of COOH groups.
• Wells were blocked with endothelial cell culture medium supplemented with 3% FCS prior to seeding primary Human umbilical endothelial cells (HUVECs, 5x10 5 cells/ml).
• a curative assay was performed in the EAE mouse model (experimental autoimmune encephalitis, a mouse model of multiple sclerosis), with prednisone used as a reference drug.
• P8RI was administered at 2mg/kg/day, s.c.
• the reference drug was used at the same dosing schedule.
• the CD31 peptide was even more rapid and effective than prednisone in reducing the extent of the ascending paralysis (clinical score) linked to active encephalitis in treated mice.
• the peptide was effective in preventing the appearance of an occlusive thrombus, at all the tested doses.
• the dose of 5mg/Kg/d showed the same extent of antithrombotic effect as the reference drug (aspirin, given subcutaneously at 150mg/kg/d).
• the number of sections showing an occlusive thrombus, out of 1000 sections/sample, was significantly lowered in P8F-treated mice as compared to vehicle (Figs 9 and 10). No occlusive thrombus could be found in either the aspirin or the 5mg/ml group.
• ED 50 the minimal dose producing 50 percent of the maximum obtainable inhibition of intracellular calcium mobilization in Jurkat cells stimulated with antiCD3+Fab antibodies.
• Stability was determined by subjecting the peptides to repeated freeze-thaw cycles and determining the number of cycles after which ED 50 was increased and/or biological activity abolished. The activity of the peptides was diminished or abolished after the number of freeze-thaw cycles indicated in Table 1

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