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Title:  Glycopeptides, their preparation and use in the diagnosis or therapeutic treatment of multiple sclerosis
United States Patent: 
7,037,893

Issued:  May 2, 2006
Inventors:  Papini; Anna Maria (Florence, IT); Chelli; Mario (Incisa Val d'Arno, IT); Rovero; Paolo (Florence, IT); Lolli; Francesco (Florence, IT)
Assignee:
 Universita' Degli Studi Di Firenze (Florence, IT)
Appl. No.:
 482044
Filed:
 June 19, 2002
PCT Filed:
 June 19, 2002
PCT NO:
 PCT/EP02/06767
371 Date:
 December 22, 2003
102(e) Date:
 December 22, 2003
PCT PUB.NO.:
 WO03/000733
PCT PUB. Date:
 January 3, 2003


 

Patheon


Abstract

Glycopeptides capable of identifying multiple sclerosis antibodies, and therefore useful as diagnostic tools or for the treatment of said pathology are described.

Description of the Invention

FIELD OF INVENTION

The present invention refers to glycopeptides formed of 11-24 aminoacids capable of identifying multiple sclerosis antibodies and therefore useful as diagnostic tools or for the treatment of said pathology.

STATE OF THE ART

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system highly invalidating and therefore of high social impact. It causes the degradation of the central nervous system white matter (myelin) bringing about serious damage in the transmission of nerve signals.

The etiopathogenesis of the disease has not yet been clearly defined, but it is hypothesised that an autoimmune process directed against the principal myelin proteins is an important pathogenic mechanism of the disease. The myelin damage is most probably due to the synergic action of the T cell response and the antibody response against myelin proteins and glycoproteins. In particular the autoantibodies can play a key role in the activation of macrophages, in demyelinization, and in the blocking of nervous conduction.

In a previous study [A. M. Papini et al., Proceedings of the 10th International Congress of Immunology, Monduzzi Editore, International Proceeding Division Bologna, Italy (1998), pp. 1239-1244] the possibility of identifying MS specific antibodies through a glycosylated peptide constituted of a sequence of 21 aminoacids of the myelin oligodendrocytic glycoprotein (MOG) (from positions 30 to 50), and indicated by the formula Asn31(Glc)hMOG(30-50) has been demonstrated.

The interest in developing new glycosylated peptides capable of carrying out the function of identifying said antibodies with greater efficiency and therefore useful both for the diagnosis and for the treatment of multiple sclerosis is evident.

SUMMARY OF THE INVENTION

The present invention refers to glycopeptides of 11-24 aminoacids containing a tetrapeptide of formula (I):

X-Asn(G)-Y-Z  (I)

in which:

  • X=aminoacid carrying an amino or carboxylic group on the side chain;
  • Y=Pro, Gly;
  • G is a sugar
  • Z=Ala, Val, Ile, His
  • DETAILED DESCRIPTION OF THE INVENTION

    It has now been surprisingly found, and is a subject of the present invention, that short glycopeptides, constituted of 11-24 aminoacids, containing the above-defined tetrapeptide play a very efficient role in the recognition of the antibodies typical of MS and are therefore useful in its diagnosis or its therapeutic treatment.

    According to the present invention, glycopeptides of formula (II) are preferred:

    A-B-X-Asn(G)-Y-Z-C-D  (II)

    in which:
  • Y and G are as defined above;
  • A=2-5 aminoacids or absent
  • B and C=trifunctional aminoacids forming a lactam bridge between each other by means of the respective side chains, or absent;
  • D=5-15 aminoacids;
  • X=Glu, Asp, Lys, Arg, Orn, Dap;
  • Z=Ala, Val, Ile, His.

    For trifunctional aminoacids forming a lactam bridge between each other as defined above, is meant, for example, the pair Dap-Asp or Asp-Dap, Dab-Glu or Glu-Dab, Orn-Asp or Asp-Orn, and the pair formed by other aminoacids, for example non-natural aminoacids, having analogous characteristics.

    For sugar is preferably meant: mono and disaccharides of type Glc, GlcNAc, β-D-Glcp-(1→4)-D-Glc (cellobiose), etc.

    For aminoacids, when not otherwise defined, is meant natural or non-natural aminoacids.

    Obviously residues A, if present, and D may contain an appropriate alkyl spacer to lengthen the chain, where for alkyl spacer, in the sense used herein, is meant ω-aminoacids with linear alkyl chains (H2N—(CH2)n—CO2H where n is 2-6.

    The presence of the formula (I) tetrapeptide as defined above, can induce a folding in the peptide conformation that can, for this reason, assume a "hook like" form (when the tetrapeptide is present in the terminal portion of the peptide) or "a hairpin like" form (when the tetrapeptide is present in the central portion of the peptide). These conformations allow an optimal binding of the patients' autoantibodies; this fact is essential for the unexpected properties of the peptides according to the invention.

    Particularly preferred, according to the invention are the peptides represented by the following sequences: H-Thr-Pro-Arg-Val-Glu-Arg-Asn(Glc)-Gly-His-Ser-Val-Phe-Leu-Ala-
    Pro-Tyr-Gly-Trp-Met-Val-Lys-OH (SEQ ID NO: 1) H-Thr-Pro-Arg-Val-cyclic[Dap-Arg-
    Asn(Glc)-Gly-His-Asp]-Val-Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-OH (SEQ ID NO: 2) H-Thr-Pro-Arg-Val-cyclic[Asp-Arg-Asn(Glc)-Gly-His-Orn]-Val-Phe-Leu-Ala-Pro-Tyr-Gly-
    Trp-Met-Val-Lys-OH (SEQ ID NO: 3)

    H-Ala-Lys-Thr-Ala-Lys-Asn(Glc)-Gly-His-Val-Glu-Ala-Ser-Gly-OH (SEQ ID NO: 4)

    H-Glu-Asn(Glc)-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro-OH (SEQ ID NO: 5)

    H-Asp-Asn(Glc)-Pro-Val-Glu-Ala-Phe-Lys-Gly-Ile-Ser-OH (SEQ ID NO: 6) H-Thr-Pro-Arg-
    Val-Glu-Arg-Asn(Glc)-Gly-His-Ser-HN-(CH2)6-CO-Val-Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-
    Val-Lys-OH (SEQ ID NO: 7) H-Asp-Asn(Glc)-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-
    (βAla)3-OH (SEQ ID NO: 8)

    The peptides as defined above can also contain an —HN—(CH2)n—CO2H group in the C-terminus (where n=2-6) so as to allow the attachment to a resin as requested for their practical use in diagnostics or in therapeutic treatments.

    The peptides according to the invention can be prepared according to known methods for solid or liquid phase synthesis.

    The solid phase method is particularly useful, well known to experts in the field, the basis of which is that the C-terminal tesidue is covalently bound to an appropriate solid support, for example polystyrene (Wang's ® resin), polystyrene-polyoxyethylene (TentaGel® resin or PEG-PS) or polyethylene glycol and polyacrylaminde co-polymers (PEGA resin), and the successive aminoacids are added sequentially, through acylation of the amino group of the residue bound to the resin, for example through the symmetric anhydride of the following aminoacid, appropriately protected, where necessary, on the side chain. Upon completion of the synthesis the crude peptide is obtained by treating the resin with an appropriate acid, for example hydrofluoric acid or trifluoracetic acid, and separated by precipitation in ethyl ether and successive lyophilisation. The peptide is finally purified using chromatographic techniques, such as for example preparative HPLC. It is also possible to maintain the synthetic peptide bound to the solid support (for example polystyrene-polyoxyethylene TentaGel® resin or PEG-PS), carrying out the selective deprotection of the side chains with an appropriate reagent.

    Alternatively, still according to known techniques, the attachment of the peptide to the appropriate support is achieved so as to form the corresponding conjugates, useful in diagnostics or in therapy. The preferred supports for this purpose include resins, insoluble in water, and completely compatible with organic liquids, such as: silica gel, cellulose, polyacrylate, sepharose and analogues, as well as the same resins normally used by experts in the field for the preparation of synthetic peptides, as for example Wang's resin, polystyrene-polyoxyethylene (TentaGel® resin or PEG-PS) or polyethylene glycol and polyacrylamide copolymers (completely compatible with water) such as PEGA resin and more stable analogue resins such as POEPS (polyoxyethylene-polystyrene), POEPOP (polyoxyethylene-polyoxypropylene), as well as macroporous resins described for their interest for the solid phase glycosylation of peptides, such as SPOCC (PEG substituted with oxethane) [Rademann, J; Grøtli, M; Meldal, M; and Bock, K. J. Am. Chem. Soc. 1999, 121, 5459-5466] or derivatives thereof like EXPO3000 (copolymer with tetrakis-[4-(3-methyl-oxethane-3-ylmethyl)-phenyl]-silane) [Tornøe, C. W.,; and Meldal M. In: Peptides 2000, J. Martinez and J. A. Fehrentz (Eds.) EDK, Paris, France 2001].
     

  • Claim 1 of 18 Claims

    1. Glycopeptides constituted of 11 to 24 amino acids containing a formula (I) tetrapeptide

    X-Asn(G)-Y-Z  (I)


    in which:

    X=amino acid carrying an amino or carboxylic group on the side chain;

    Y=Pro, Gly;

    G=sugar; and

    Z=Ala, Val, Ile, His.
     

    ____________________________________________
    If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

     

     

         
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