Background of the Invention Multiple sclerosis (MS) is a common heterogeneous, inflammatory autoimmune disease of the central nervous system (CNS), resulting in the slowing of electrical conduction along the nerve. It is a progressively debilitating disease that manifests itself in the prime of peoples'lives, usually between the ages of 20 and 40. Although not fatal, the disease is unpredictable and can lead to blurred vision, loss of balance, poor coordination, slurred speech, tremors, numbness, extreme fatigue, even paralysis and blindness. It is estimated that close to a third of a million people in the United States have Multiple Sclerosis. It is more prevalent in northern latitudes where it affects roughly 140 per 100,000 people (0.14%). Twice as many women as men have multiple sclerosis. The cause of multiple sclerosis is unknown, although genetic and environmental factors are associated with higher risk. Multiple sclerosis results from inflammation and breakdown in the myelin surrounding the nerve fibres of the central nervous system. Once the myelin is destroyed, it is replaced by sclerotic patches or plaques which can appear in multiple locations within the central nervous system, hence the name"multiple sclerosis".

The disease is further characterized by an increase in the infiltration of inflammatory cells, loss of oligodendrocytes, and increased gliosis. MS is associated in Northern European Caucasoid MS patients with the HLA- <BR> <BR> DR2 (DRB1*1501) haplotype [Jersild, C. et al. , Lancet 2: 1221-1225 (1973); Spielman, R. S. and Nathenson, N. Epidemiol. Rev. 4: 45-65 (1982); Olerup, <BR> <BR> O. and Hillert, J. Tissue Antigens. 38: 1-15 (1991) ]. Its causes and the factors that contribute to its heterogeneity are unknown. The animal model used for the study of MS, is induced with experimental autoimmune encephalomyelitis (EAE), which is a T cell mediated autoimmune disease that can be induced by immunization with peptides derived from myelin components such as myelin basic protein (MBP) [Zamvil, S. S. et al., Nature 324: 258-260 (1986); Kono, D. H. et al. J. Exp. Med. 168: 213-227 <BR> <BR> (1988) ; Madsen, L. S. et al. Nat. Genet. 23: 343-347 (1999) ], proteolipid<BR> protein (PLP) [Tuohy, V. K. et al. , J. Immunol. 142: 1523-1527 (1989);<BR> Greer, J. M. et al. , J. Immunol. 149: 783-788 (1992) ], or myelin<BR> oligodendrocyte glycoprotein (MOG) [Mendel, I. et al. , Eur. J. Immunol.<BR> <P>25: 1951-1959 (1995) ]. In the course of EAE, autoreactive CD4+ T cells recognize self-antigens presented by murine class II MHC molecules (e. g., H-2s), ultimately leading to pathological changes that can be monitored as clinical signs of disease. EAE provides a well-studied system for testing the efficacy of therapeutic compounds to suppress the disease. These have included treatment with cytokines [Santambrogio, L. et al. , J.<BR> <P>Neuroimmunol. 58 : 211-222 (1995); Leonard, J. P. et al. , Ann. NY Acad. Sci.<BR> <P>795: 216-226 (1996) ], peptide antigens that induce anergy [Gaur, A. et al.,<BR> Science 258: 1491-1494 (1992) ], oral tolerance [Whitacre, C. C. et al. , Clin.<BR> <P>Immunol. Immunopathol. 80 : S31-S39 (1996); Kennedy, K. J. et al. , J.

Immunol. 159: 1036-1044 (1997); Weiner, H. L. Immunol. Today 18: 335-343 (1997) ], or altered peptide ligands [Evavold, B. D. and Allen, P. M. Science<BR> 252: 1308-1310 (1991); Karin, N. et al. , J. Exp. Med. 180 : 2227-2237 (1994);<BR> Pfeiffer, C. et al. , J. Exp. Med. 181: 1569-1574 (1995); Nicholson, L. B. et al., Proc. Natl. Acad. Sci. USA 94: 9279-9284 (1997); Wingerchuk, D. M. et al., Lab. Invest. 81: 263-281 (2001)].

Infiltration of inflammatory cells into the CNS requires their transmigration through the blood-brain barrier, a process involving upregulation of cell adhesion molecules and proteolytic activity of extracellular matrix (ECM) and plasminogen activating enzymes <BR> <BR> [Rosenberg, G. A. et al. , Brain Res. 576: 203-207 (1992) ; Johnatty, R. N. et<BR> al. , J. Immunol. 158: 2327-2333 (1997) ]. Matrix metaloproteases (MMPs) have recently been implicated in the pathogenesis of MS [Kieseier, B. et <BR> <BR> al. , Brain 121: 159-166 (1998); Maeda, A. and Sobel, R. A. J. Neuropathol<BR> Exp Neurol. 55: 300-309 (1996); Cuzner, M. I. et al. , Exp Neurol. 55: 1194-<BR> 1204 (1996); Galboiz, Y. et al. , Ann. Neurol. 50: 443-451 (2001); Rosenberg,<BR> G. A. Ann. Neurol. 50: 431-433 (2001); Teesalu, T. et al. , Am. J. Pathol.

159: 2227-2237 (2001); Balabanov, R. et al., Clin. Diag. Lab. Immunol. <BR> <BR> <P>8 : 1196-1203 (2001) ] ; their roles include the disruption of the BBB, immune cell transmigration into the CNS, and myelin degradation. uPA could affect the development of inflammatory response by three different functions; The first is proteolysis of vascular ECM and basement membranes [Ossowski, L. J. Cell Biol. 107: 2437-2445 (1988) ; Jankun, J. et <BR> <BR> al. , Cancer Res. 57: 559-563 (1997) ]. The second is by modulating the adhesion of cells to the vasculature, an effect that is mediated through its cellular receptor, uPAR. The third is by changing the blood vessels vasoactivity that would change their permeability [Higazi, A. A-R. FASEB J. 14: 1411-1422 (2000), Higazi, A. A-R. J. Biol. Chem. 277: 40499-40504 <BR> <BR> (2002) ]. Binding of scuPA (single chain uPA) to uPAR promotes cell adhesion to vitrinectin and modulates integrin function [Ossowski, L. et <BR> <BR> al. , Cancer Res. 51: 274-281 (1991); Wei, Y. et al. , Science 273: 1551-1555<BR> (1996); Kanse, S. M. et al. , Exp. Cell Res. 224: 344-353 (1996) ]. Indeed it has been previously suggested that the plasminogen activators play an important role in the development of several neurological diseases including MS [Teesalu (2001) ibid. ; Akassoglou, K. and Stricland, S. Biol.

Chem. 383: 37-45 (2002)].

Over the last decade there has been a broad approach to multiple sclerosis treatments. These include antigen-specific immunsuppressive compounds, general immunosuppressive compounds, anti-proliferative compounds, cytokine compounds, re-myelination compounds and compounds to <BR> <BR> improve nerve conduction [For review see Polman, et al. , J. M. J. 321: 490-<BR> 494 (2000); Tselis, et al. , Arch. Neuro. 56: 277-280 (1999); Rudick, et al., N. E. J. M. 337: 1604-11 (1997)].

The principal medications in the past included steroids possessing antiinflammatory activity, including adrenocorticotropic hormone, prednisone, prednisolone, methylprednisolone, betamethasone and dexamethasone. Although these steroids reduce the severity and duration of the attacks in some patients, the drawback is that they do not affect the course of the disease over time.

More recently, the major drugs used for the treatment of multiple sclerosis include the beta-interferon compounds: Avonex, Betaseronllm and RebifrM. These treatments have been shown to reduce the number of exacerbations and formation of plaques, and to slow the progression of the <BR> <BR> physical disability [Yong, et al. , Neuro. 51: 682-89 (1998); Chofflon, et al.,<BR> Eur. J. Neur. 7: 369 (2000); Weinstock-Guttman, et al. , Drug 59: 401-10<BR> (2000) ]. Interferon-alpha has also been considered for the treatment of<BR> multiple sclerosis [Myhe, et al. , Neuro. 23: 52 (5): 1049-56 (1999) ]. Although the mechanism of action in uncertain, it is thought that the interferons suppress the immune system. However, at the dosages used and the length of time required for treatment, the interferon compounds produce adverse effects. These include local injection reactions, flu-like syndrome and depression. In some patients reduction in dosage or discontinuation of the drug may be required. Moreover, up to 40% of the patients develop neutralizing antibodies to interferon.

An additional agent on the market is Copaxone, a synthetic copolymer with some immunological similarities to myelin basic protein that has been shown to suppress the progression of the disease by inhibiting the <BR> <BR> immune system [Johnson, et al. , Neuro. 45 : 1268-76; (1995); Johnson, et<BR> al. , Neuro. 50: 701-8 (1998) ]. Again, because of the dosages required and the length of treatment, many adverse reactions are manifested. The side effects of this compound are similar to those observed with interferon therapies.

There are a number of emerging compounds for treating multiple sclerosis which are either in clinical trials or in early research development. These include the development of vaccines for multiple sclerosis using myelin proteins, or treating with myelin basic protein or fragments of this protein to decrease the attack of the immune system on myelin [U. S. Pat. No.

6,036, 957; WO 99/13904; U. S. Pat. Nos. 5, 858, 980; 5,948, 764 and WO 98/45327]. Other compounds include the use of antibodies to cytokines such as anti-TNF (tumor necrosis factor) antibodies which inhibit inflammatory responses as well as tissue destruction [U. S. Pat. Nos.

5, 888, 511; 5,958, 409; 6,143, 866]. Interleukin-4 is also capable of inhibiting de-myelination and improving the clinical course of disease in mouse models of multiple sclerosis [Xu, et al., Clin. Exp. Immunol. 118: 115-21 <BR> <BR> (1999) ]. Although these compounds are promising, they have multiple effects on the immune system, such as increased number of activated T cells and enhanced proliferation and cytokine secretion. Therefore, they may be problematic in treating established autoimmune diseases such as multiple sclerosis.

Due to the nature of this disease, patients undergo long term treatments and often experience negative effects. There is therefore a need for the development of superior treatments for multiple sclerosis.

Urokinase plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes including: Fibrinolysis : uPA activates the proenzyme plasminogen to the proteolytically active enzyme, plasmin, which in turn mediates fibrinolysis. Transgenic mice with a targeted disruption in the uPA gene (uPA-/-) are prone to form thrombi when exposed to endotoxin [Carmeliet, P. et al. , Nature 369: 419-424 (1994) ] and hypoxia [Carmeliet, P. and<BR> Collen, D. Haemostasis 26 Suppl 4: 132-153 (1996); Pinsky, D. J. et al. , J.<BR> <P>Clin. Invest. 102: 919-928 (1998) ] or when the uPA gene is disrupted in otherwise healthy tPA-/-mice [Carmeliet (1994) ibid. ; Bugge, T. H. et al., Proc. Nat. Acad. Sci. USA. 93: 5899-5904 (1996) ]. Furthermore, the present inventors have previously reported that clearance of pulmonary microemboli is delayed in uPA-/-mice despite the presence of an intact tPA system (tissue type PA) [Bdeir, K. et al. , Blood 96: 1820-1826 (2000)].

Tumor invasiveness and formation of metastases : uPA participates in formation of tumor metastases [Foekens, J. A. et al. , Cancer Res. 52: 6101-<BR> 6105 (1992); Grondahl-Hansen, J. et al. , Cancer Res. 53: 2513-2521 (1993);<BR> Nekarda, H. et al. , Cancer Res. 54: 2900-2907 (1994); de Vries, T. J. et al.,<BR> Am. J. Pathol. 144: 70-81 (1994) ]. The propensity of certain tumors to metastasize in humans is associated with the level of uPA and its receptor (uPAR) [Xing, R. H. and Rabbani, S. A. Int. J. Cancer. 67: 423-429 (1996)].

Tumor progression is impeded by uPA inhibitors [Ossowski, L. J. Cell Biol. <BR> <BR> <P>107: 2437-2445 (1988); Jankun, J. et al. , Cancer Res. 57: 559-563 (1997)],<BR> antagonists to uPAR [Min, H. Y. et al. , Cancer Res. 56: 2428-2433 (1996);<BR> Cohen, R. L. et al. , Blood 78: 479-487 (1991); Crowley, C. W. et al. , Proc.<BR> <P>Nat. Acad. Sci. U. S. A. 90: 5021-5025 (1993) ], soluble uPAR [Kruger, A. et<BR> al. , Cancer Gene Ther. 7: 292-299 (2000) ] and anti-sense uPAR mRNA<BR> [Kook, Y. H. et al. , EMBO J. 13: 3983-3991 (1994); Yu, W. et al. , J. Cell<BR> Biol. 137: 767-777 (1997) ] and certain tumors are less prone to metastasize<BR> in uPA--mice [Bajou, K. et al. , Nat. Med. 4: 923-928 (1998) ]. The mechanism by which uPA promotes metastasis has been ascribed to two functions. The first is proteolysis of vascular basement membranes, a concept that is supported by the finding that receptor occupancy by enzymatically inactive uPA inhibits metastases. The second is by modulating the adhesion of tumor cells to the vasculature, an effect that is mediated through its cellular receptor, uPAR. Binding of scuPA to uPAR promotes cell adhesion to vitronectin and modulates integrin function.

PAI-1 competes with uPAR for binding to vitronectin and thereby interferes with cell adhesion in vitro.

Angiogenesis wound healing, neointima : uPA has also been implicated in <BR> <BR> angiogenesis [Odekon, L. E. et al. , J. Cell. Physiol. 150: 258-263 (1992);<BR> Bachrach, E. et al. , Proc. Nat. Acad. Sci. USA. 89: 10686-10690 (1992);<BR> Pepper, M. S. et al. , J. Cell Biol. 111: 743-755 (1990); Van Hinsbergh, V. W.<BR> <P>M. et al. , In Regulation of angiogenesis. Goldberg, I. D. and Rosen, E. M.<BR> editors. Birkhauser, Verlag, Basel. 391-411 (1997) ] and wound healing [for review Jensen, P. J. and Lavker, R. M. Cell Growth Differ. 7: 1793-1804 <BR> <BR> (1996) ]. These processes are characterized by cell adhesion and migration through physical barriers, such as fibrin or extracellular matrices. UPA and uPAR are expressed in a temporally and spatially restricted manner by migrating cells [Pepper, M. S. et al. , J. Cell Biol. 122: 673-684 (1993);<BR> Okada, S. S. et al. , Exp. Cell Res. 217: 180-187 (1995); Strickland, S. et al.,<BR> Cell 9: 231-240 (1976) ] and this may explain why uPA-/-mice show<BR> impaired wound healing [Carmeliet, P.. et al. , J. Clin. Invest. 99: 200-208 (1997); Carmeliet, P. and Collen, D. Fibrinolysis 8, Suppl. 1: 269-276 (1994)].

Pulmonary inflammation and repair : The plasminogen activation system and uPA specifically have been implicated in the development of pulmonary fibrosis and certain types of infections. UPA-/-mice are more susceptible to lethal pulmonary infection [Gyetko, M. et al. , J. Clin. Invest.<BR> <P>97: 1818-1826 (1996) ] and to the development of pulmonary fibrosis<BR> [Sisson, T. H. et al. , Hum. Gene Ther. 10: 2315-2323 (1999) ]. In contrast, PAI-1-/-mice develop less pulmonary fibrosis in this model [Eitzman, D. T. et al. , J. Clin. Invest. 97: 232-237 (1996)].

Regulation of blood vessel tone : The inventors have previously found that uPA is involved in the regulation of blood vessels contractility [Haj-Yejia, A. et al. , FASEB J 14: 1411-1422 (2000) ]. More recently the inventors have identified the mechanism of the uPA effect, including a novel signal transducing cellular receptor pathway involved in the regulation of vascular contractility and the present inventors have reported for the first time that uPA-/-mice have a significantly lower mean arterial blood pressure than wild type animals [Nasser, T. et al. , J. Biol. Chem. 277: 40499- 40504 (2003)].

Although uPA is clearly a multifunctional protein, the structural basis for each of these activities is not well resolved and may or may not reside in the same portion of the molecule. uPA is composed of two major subunits: an amino-terminal fragment (ATF; amino acids 1-143, as referred to the amino acid sequence of uPA as denoted by GenBank Accession No. P00749 and a catalytic subunit (low molecular weight uPA; amino acids 144-411) [Ploug, M. et al. , Semin Thromb Hemost 17: 183-193 (1991)].

The ATF is composed of two domains: the growth factor domain (GFD; amino acid residues 1-46, in the mature protein and residues 21-66 as referred to the sequence as denoted by the GenBank Accession No.

P00749) and the kringle domain (amino acids 47-135, in the mature protein and 67-155 in the sequence of GenBank Accession No. P00749). It is widely accepted that the GFD domain mediates the binding of uPA to its receptor (uPAR). However, although the kringle domain of uPA has been shown to bind heparin in vitro, its physiological role is yet to be determined.

Between the kringle and the protease domain is a region designated the "connecting peptide" (amino acid residues 136-158 as in the mature protein, which refers to amino acid residues 156-178 as denoted by GenBank Accession No. P00749). Within this region is an eight amino acid sequence (136-143, KPSSPPEE in single letter code, also denoted by SEQ ID NO: 1, as referred to by the mature protein and residues 156-163 as in the sequence of GenBank Accession No. P00749) that can be generated by the combined activities of tcuPA (two chain uPA), that cleaves uPA between K135 and Kl36 and matrix metalloproteinases, such as matrilysin (MMP-7) and stromolysin (MMP-3), that digest between Ei43 and L144. uPA is synthesized and secreted as a single chain molecule (scuPA).

ScuPA has generally been considered to be a proenzyme with very low intrinsic activity in terms of cleaving plasminogen [Ellis, V. et al. , J. Biol.<BR> <P>Chem. 262: 14998-15003 (1987); Urano, T. et al. , J. Arch. Biochem.

Biophys. 264: 222-230 (1988); Gurewich, V. and Pannell, R. Semin.

Thromb. Hemost. 13: 146-151 (1987); Husain, S. S. Biochemistry 30: 5707- <BR> <BR> 5805 (1991) ]. ScuPA is activated to a two chain molecule (tcuPA) through the cleavage of a single peptide bond (Lysl58-Ilels9) or, as the present inventors have previously reported, when it binds as a single chain molecule to its receptor [Kasai, S. et al. , J. Biol. Chem. 260: 12382-12389<BR> (1985); Higazi, A A-R. et al. , J. Biol. Chem. 270: 17375-17380 (1995) ]. The inventors have also reported that the two active forms of uPA, tcuPA and the scuPA/uPAR complex, differ in their intrinsic activity [Higazi, A. A-R. et al. , (1995) ibid.] and in their susceptibility to inhibition by PAI-<BR> l [Higazi, A. A-R. et al. , Blood 87: 3545-3549 (1996) ]. How these two forms differ in the non-catalytic functions of uPA is less well understood. uPA interacts with several other cellular proteins besides uPAR, albeit with lower affinity. One of these, the low-density lipoprotein receptor/a2-macrogrobulin receptor (hereafter designated as LRP for simplicity) mediates the internalization and degradation of uPA [Zhang, L. et al. , J. Biol. Chem. 272: 27053-27057 (1997); Nykjaer, A. et al. , J. Biol.<BR> <P>Chem. 269: 25668-25676 (1994); Higazi, A. A-R. et al. , Blood 88 : 542-551<BR> (1996) ]. The interaction of uPA with LRP is subject to regulation by several ligands that bind to uPA. For example, the present inventors and others have previously reported that binding of uPA to soluble uPAR (suPAR) inhibits its binding to LRP, whereas the interaction of uPA with PAI-1 increases the affinity of the resultant complex for LRP [Nykjaer (1994) ibid. ; Higazi (1996) ibid.]. The interaction between uPA and LRP has been considered primarily as the mechanism by which inactive uPA- PAI-1 complexes are cleared from the cell surface and unoccupied uPAR returns, ready for additional encounters with uPA. There is limited information about the determinants in uPA that are recognized by LRP and less concerning the manner by which LRP may regulate the non- catalytic activity of uPA. The potential role of uPA in uPA-mediated signal transduction has also received little attention.

The concept that scuPA is a proenzyme must be reconsidered in view of the many newly described functions of urokinase, including the induction <BR> <BR> of intracellular signaling events [Li, C. et al. , J. Biol. Chem. 270: 30282-<BR> 30285 (1995); Franco, P. et al. , J. Cell Biol. 137: 779-791 (1997); Franco, P.<BR> et al. , J. Biol. Chem. 273: 27734-27740 (1998)] that lead to cell adhesion,<BR> migration, vascular contraction and differentiation [Yebra, M. et al. , J.<BR> <P>Biol. Chem. 271: 29393-29399 (1996); Okada, S. A. et al. , Arterioscler.<BR> <P>Thromb. Vasc. Biol.; 16: 1269-1276 (1996); Stoppelli, M. P. et al. , Proc. Nat.

Acad. Sci. U. S. A. 82 : 4939-4943 (1985) ; Nusrat, A. R. and Chapman, Jr. H. <BR> <BR> <P>A. J. Clin. Invest.; 87: 1091-1097 (1991) ]. It is now clear that several of these events do not require uPA catalytic activity. Rather, there is evidence to suggest that at least part of uPA-induced signaling is mediated through uPAR. Several groups have reported that binding of the isolated ATF or its sub-fragment GFD, to uPAR leads to signaling and cell adhesion. This observation has created the impression that the GFD- uPAR interaction is solely responsible or is required for signal transduction events. More recently, it has been reported that phosphorylation of Seras (which lies within the connecting peptide) or its mutation to glutamic acid abolishes uPA-induced adhesion and motility of myelomonocytic cells without affecting receptor binding [Franco (1997) ibid. ; Franco (1998) ibid.].

As indicated above, uPA and its receptor (uPAR) have been shown to play a critical role in the development of the immune response and in the process of cancer invasion and metastasis. Activation of lymphocytes and their migration plays an essential role in the development of multiple sclerosis (MS). Therefore, the inventors have studied the role of uPA in the development of MS and in the animal model used for the study of this disease, i. e. experimental autoimmune encephalomyelitis (EAE). In this experimental model, as in the human disease, inhibition of myelin antigen-specific T-cells is an important step in treatment. The present invention shows that the clinical presentation of the disease in uPA-/-and uPAR--mice is more severe than in WT (wild type) animals. These observations suggest that uPA and uPAR play a protective role in the development of the disease. In order to determine the portion of uPA responsible for the protective effect, the present inventors have examined the effect of different portions of uPA on the development of MS and found that an 8 amino acid peptide (octapeptide of SEQ ID NO: 1) derived from uPA inhibits the response of lymphocytes to myelin antigens.

It is therefore an object of the invention to use the octapeptide of the invention that derives from the connecting peptide of uPA or any functional derivatives thereof, in the preparation of a pharmaceutical composition for the treatment of MS. Yet another object of the invention is to provide methods and compositions for the treatment of MS. These and other objects of the present invention will become apparent as the description proceeds.

Summary of the Invention In a first aspect, the present invention relates to the use of a peptidic compound derived from urokinase plasminogen activator (uPA), in the preparation of a pharmaceutical composition for the treatment of an autoimmune disorder associated with myelin injury in the central nervous system (CNS). More particularly, the invention relates to the use of an octapeptide derived from the connecting peptide of uPA which connects between the kringle and the protease catalytic domains. The octapeptide used by the invention comprises the amino acid sequence Lys-Pro-Ser-Ser- <BR> <BR> Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, and any variant or derivative thereof.

In a preferred embodiment, the invention relates to the use of the octapeptide in the preparation of a pharmaceutical composition for the treatment of an autoimmune disorder such as multiple sclerosis (MS).

The invention further provides for the use of a peptidic compound derived from urokinase plasminogen activator (uPA) comprising the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu also denoted by SEQ ID NO : 1, and any variant or derivative thereof in the preparation of an inhibitory composition for inhibiting the activation of lymphocytes by a myelin- derived antigen.

According to one preferred embodiment, variant or derivative of the octapeptide used by the invention may be any one of a substitution variant, an addition variant, a chemical derivative, a cyclic octapeptide and a peptidomimetic agent of said peptidic compound.

A specifically preferred peptide derivative is the octapeptide of the invention capped on both sides, C and N terminals, Ac-KPSSPPEE-Am, also denoted by SEQ ID NO: 2.

In yet another preferred embodiment the preferred octapeptide derivative used by the invention is a cyclized form of the octapeptide of SEQ ID NO : 1 or 2.

In a second aspect, the invention relates to a method for inhibiting the activation of lympocytes by a myelin-derived antigen. This method comprises the step of contacting the lymphocytes under suitable conditions, with an inhibitory sufficient amount of a peptidic compound derived from urokinase plasminogen activator (uPA) or of a composition comprising the same. This peptidic compound comprises the octapeptide carrying the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu which is also denoted by SEQ ID NO: 1, or any variant or derivative thereof.

Still further, the invention relates to a method for inhibiting the activation of lympocytes by a myelin-derived antigen, in a subject suffering of an autoimmune disorder associated with myelin injury in the CNS. According to this embodiment, this method comprises the step of administering to said subject an inhibitory sufficient amount of a peptidic compound derived from urokinase plasminogen activator (uPA) or of a composition comprising the same, which compound comprises the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, or any variant or derivative thereof.

The invention further provides for a method of treatment of an autoimmune disorder associated with myelin injury in the CNS in a mammalian subject in need of such treatment, preferably, multiple sclerosis (MS). The method of the invention comprises the step of administering to such subject a therapeutically effective amount of a peptidic compound derived from urokinase plasminogen activator (uPA) or of a composition comprising the same. The peptidic compound comprises the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, or any variant or derivative thereof.

According to a particularly preferred embodiment, a variant or derivative of the peptide used by the methods of the invention may be any one of a substitution variant, an addition variant chemical derivative, a cyclic octapeptide and a peptidomimetic agent of said peptidic compound.

A specifically preferred peptide derivative is the octapeptide of the invention capped on both sides, C and N terminals, Ac-KPSSPPEE-Am, also denoted by SEQ ID NO: 2.

In another specifically preferred embodiment, a preferred derivative is a cyclic form of the octapeptide.

In a further aspect, the invention relates to an inhibitory composition for inhibiting activation of lympocytes by a myelin derived antigen. The composition of the invention comprises as an active ingredient an inhibitory sufficient amount of peptidic compound derived from urokinase plasminogen activator (uPA) comprising the amino acid sequence Lys-Pro- Ser-Ser-Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, or any variant or derivative thereof, which composition optionally further comprises pharmaceutically acceptable carrier, diluent, adjuvant and/or excipient.

Still further, the present invention provides a pharmaceutical composition for the treatment of an autoimmune disorder associated with myelin injury in the CNS, preferably, multiple sclerosis (MS), in a mammalian subject in need of such treatment. The composition of the invention comprises as an active ingredient a therapeutically effective amount of a peptidic compound derived from urokinase plasminogen activator (uPA) comprising the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, or any variant or derivative thereof, which composition optionally further comprises pharmaceutically acceptable carrier, diluent, adjuvant and/or excipient.

According to one preferred embodiment, the peptide used for the compositions of the invention may be variant or derivative thereof such as any one of a substitution variant, an addition variant chemical derivative, a cyclic octapeptide and a peptidomimetic agent of said peptidic compound.

A specifically preferred peptide derivative is the octapeptide of the invention capped on both sides, C and N terminals, Ac-KPSSPPEE-Am, also denoted by SEQ ID NO: 2.

Another specifically preferred peptide derivative is a cyclized octapeptide according to the invention.

Brief Description of the Figures Figure 1 shows that compared to WT mice, the disease in uPA-/-animals reach a higher clinical score which indicates that they developed more severe clinical symptoms (n=14 in each group). Abbreviations: Me. Dis. Sc.

(mean disease score), D af. Imm. (days after immunization).

Figure 2 shows that the absence of uPA receptor (UPAR) leads to the same effect seen in Figure 1. The uPAR-/-mice develop more severe clinical symptoms than WT mice, (n=7 in each group). Abbreviations: Me.

Dis. Sc. (mean disease score), D af. Imm. (days after immunization).

Figure 3 shows that the absence of tPA (tissue type PA) leads to the same effect seen in Figure 1. The tPA-/-mice develop more severe clinical symptoms than WT mice, (n=7 in each group). Abbreviations: Me. Dis. Sc.

(mean disease score), D af. Imm. (days after immunization), WT (wild type).

Figure 4A-4D Axonal loss, axonal injury and cellular infiltrates in the CNS of mice with EAE. The figure shows brain and spinal cord sections stained with Bielschowsky and cresyl violet for simultaneous evaluation of axonal loss (AL), axonal injury (AI) and inflammatory mononuclear infiltrates (inf). The axons are stained in brown and mononuclear cells nuclei in blue.

Fig. 4A. Brain hemisphere section removed 30 days after disease induction, showing injured axons and many infiltrating inflammatory cells.

Fig. 4B. Spinal cord section removed 30 days after disease induction, showing massive infiltration of mononuclear inflammatory cells in a white matter area with cells penetrating into the area of the axons, causing axonal injury.

Fig. 4C. Spinal cord section as above, with pronounced axonal injury and damaged dystrophic and spheroid axons.

Fig. 4D. the same section as in B with higher magnification.

Figure 5A-5D shows histograms summarizing scoring of from 0 to 2 of the following three parameters: axonal loss (AL) (Fig. 5A), axonal injury (AI) (Fig. 5B) infiltration (inf.) (Fig. 5C) and amount of cell per infiltrate (am. Ce. Inf.) (Fig. 5D), preformed in female WT, tPA-/-, uPA-/-or uPAR-/- C57BL mice (n=7) immunized with MOG. In the case of AL the score was: 0-normal, 1-mild to moderate axonal loss, 2-severe axonal loss. AI scoring was: 0-normal, 1-scattered, injured axons, 2-mild to moderate axonal injury, 3-severe axonal injury. The Inf scoring was: 0-no infiltrates, 1-10- 20 cells per 40x optical field, 2-20-60 cells per 40x optical field, 3-60-100 cells per 40x optical field.

Figure 6 A histogram representing activation of lymphocytes by the MOG peptide in the presence of different concentrations of the octapeptide of the invention (SEQ ID NO: 2). The results show that the presence of the octapeptide KPSSPPEE that corresponds to the connecting peptide (amino acids 136-143, in the mature protein and residues 156-163 as referred to GenBank Accession No. P00749) inhibited the activation of lymphocytes.

Abbreviations: Lym. Act. (lymphocyte activation), pep. (peptide).

Detailed Description of the Invention As used herein, the following terms have the following definitions: "Nervous system"means neurons, glia, and other supporting cells (e. g., Schwann, satellite) and the connective tissue layerings and coverings of neurons, glia, and other supporting cells.

"Central nervous system"or"CNS"means the nervous system of the brain and spinal cord.

"Peripheral nervous system"or"PNS"is the nervous system excluding the CNS.

"Blood brain barrier"or"BBB"is a selectively permeable, functional and physical barrier to the movement of cells and molecules into and out of the CNS via blood vessels.

"About"in reference to a numerical value means approximately +/-10% of the numerical value, e. g. ,"about 10%"means approximately 9 to 11%.

As will be shown in the following Examples, the present inventors have discovered the surprising therapeutic activity of the octapeptide (SEQ ID NO: 1) on MS. The present invention presents the observation that the said uPA-derived octapeptide has intrinsic biological activity, particularly in mediating lymphocyte response to the MS antigen. The present inventors have reported previously that this uPA-derived peptide inhibits the response of blood vessels to phenylephrine both in vitro and in vivo, and that this peptide induces apoptosis in cancer cells [Haj-Yehjia (2000) ibid.]. Both responses may contribute to its inhibitory effect in MS. The peptide could inhibit the response of lymphocytes and/or induce apoptosis of lymphocytes that invade the brain; the apoptosis of these cells is critical for termination of episodes of MS.

Based on the data of the invention, it seems feasible that the said uPA- derived octapeptide will inhibit the development of the disease in vivo, as it did in vitro (see Example 2).

Thus, in a first aspect, the invention relates to the use of a peptidic compound derived from urokinase plasminogen activator (uPA), in the preparation of a pharmaceutical composition for the treatment of an autoimmune disorder associated with myelin injury in the central nervous system (CNS). More particularly, the invention relates to the use of an octapeptide derived from the connecting peptide of uPA which connects between the kringle and the protease catalytic domains. The octapeptide used by the invention comprises the amino acid sequence Lys-Pro-Ser-Ser- Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, and any variant or derivative thereof.

Acute and chronic diseases associated with myelin injury in the CNS include multiple sclerosis (which is characterized at various stages as acute, relapsing-remitting, primary-progressive, secondary-progressive), neuromyelitis optica, optic neuritis, acute encephalomyelitis (which can be post-infectious or post-exanthem) and cervical myelopathy (which can be associated with infectious, connective tissue disease, transverse myelitis or autoimmune etiologies). Other diseases associated with CNS myelin degeneration including the leukodystrophies and progressive multifocal leukoencephalop athy.

Acute and chronic diseases associated with myelin injury in the peripheral nervous system (PNS) include acute inflammatory polyneuropathy, acute autoimmune neuropathy, Guillain Barre syndrome (GBS), recurrent and relapsing polyneuropathy, chronic inflammatory demyelinating polyneuropathy (CIDP), paraneoplastic syndromes, diabetes mellitus, <BR> <BR> connective tissue disease (e. g. , vasculitis, systemic lupus erythematosus), and neuropathies associated with autoimmune diseases, cancer and infections caused by retroviruses, viruses and other infectious agents.

There are other inflammatory (presumably autoimmune) disorders affecting both the CNS and PNS which are associated with perivascular lesions with mononuclear inflammatory infiltrates and abnormalities of white matter (myelinated nerve fibers). For example, Sjogren's syndrome (SS) is an autoimmune disorder which affects approximately 3% of the adult population. Conservatively, approximately 25% of SS patients develop neurologic complications affecting the CNS and the PNS (i. e., dorsal root ganglia, spinal nerve roots and peripheral nerves of the sensory, motor and autonomic systems). SS is an example of an inflammatory neurological disorder in which the blood brain and blood nerve barrier of small vessels are compromised and are unable to prevent trafficking of mononuclear cells across the vascular endothelium into the perivascular space and nervous system tissue.

Mononuclear infiltrates of the small blood vessels of the central nervous system are a prominent and ubiquitous feature of SS. Although the organization and function of the white matter (myelinated fibers) is abnormal in SS, frank demyelination (with plaque formation) of the histopathologic type observed in Multiple Sclerosis (MS) is not present.

The perivascular, mononuclear cells synthesize cytokines and excitatory neurotoxins which damage the surrounding nervous system. Perivascular inflammatory infiltrates containing mononuclear cells also occur in idiopathic polymyositis, a disorder of the musculoskeletal system which is thought to be immune-mediated.

In a preferred embodiment, the invention relates to the use of the said octapeptide in the preparation of a pharmaceutical composition for the treatment of an autoimmune disorder such as multiple sclerosis (MS).

MS, which is limited to the CNS, has all of the foregoing clinical and histopathological manifestations of SS but is also characterized by demyelination and gliosis (scarring). MS affects 350,000 Americans and is, with the exception of trauma, the most frequent cause of neurologic disability in early to middle adulthood. Indirect evidence supports an autoimmune etiology for MS, perhaps triggered by a viral infection in a genetically susceptible host. As in other chronic inflammatory disorders, the manifestations of MS are variable and range from a benign illness to a rapidly evolving and incapacitating disease. Complications of MS may affect multiple body systems and may require profound adjustments in lifestyle and goals for patients and their families.

MS derives its name from the multiple scarred areas visible on macroscopic examination of the brain. These demyelinating lesions, termed plaques, are well-demarcated gray or pink areas easily distinguished from surrounding white matter. Demyelinating lesions are historical evidence of the occurrence of or the continued presence of perivascular lesions. Occasionally, plaques are also present in gray matter (neuron cell bodies). Plaques vary in size from 1 or 2 millimeters to several centimeters. The MS lesion is defined as including both perivascular and demyelinating lesions. The acute MS lesion, occasionally found on autopsy, is characterized by increased permeability of the blood brain barrier, perivascular cuffing and tissue infiltration by mononuclear cells, predominantly T lymphocytes and macrophages, and by demyelination. B cells and plasma cells are rarely found. The inflammatory infiltrates appear to mediate the loss of myelin sheaths that surround axon cylinders.

As the lesion progresses, large numbers of macrophages and microglial cells, (specialized CNS phagocytes of bone marrow origin) scavenge the myelin debris, and proliferation of astrocytes (gliosis) occurs. Proliferation of oligodendrocytes is also present initially, but these cells appear to be destroyed as the infiltration and gliosis progress. Gliosis is more severe in MS lesions than in most other neuropathologic conditions. In chronic MS lesions, complete or nearly complete demyelination, dense gliosis, and loss of oligodendrocytes are present.

The invention further provides for the use of a peptidic compound derived from urokinase plasminogen activator (uPA) comprising the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, and any variant or derivative thereof, in the preparation of an inhibitory composition for inhibiting the activation of lymphocytes by a myelin derived antigen.

The octapeptide and derivatives thereof used by the invention may be prepared using recombinant DNA technology. However, given their length, they are preferably prepared using solid-phase synthesis, such as that generally described by Merrifield, J. [Amer. Chem. Soc. 85: 2149-54 (1963)], although other equivalent chemical syntheses known in the art are also useful. Solid-phase peptide synthesis may be initiated from the C- terminus of the peptide by coupling a protected a-amino acid to a suitable resin. Such a starting material can be prepared by attaching an a-amino- protected amino acid by an ester linkage to a chloromethylated resin or to a hydroxyrriethyl resin, or by an amide bond to a BHA resin or MBHA resin.

The amino acids can be coupled to the growing peptide chain using techniques well known in the art for the formation of peptide bonds. For example, one method involves converting the amino acid to a derivative that will render the carboxyl group of the amino acid more susceptible to reaction with the free N-terminal amino group of the growing peptide chain. Specifically, the C-terminal of the protected amino acid can be converted to a mixed anhydride by the reaction of the C-terminal with ethylchloroformate, phenyl chloroformate, sec-butyl chloroformate, isobutyl chlorofonnate, or pivaloyl chloride or the like acid chlorides.

Alternatively, the C-terminal of the amino acid can be converted to an active ester, such as a 2,4, 5-trichlorophenyl ester, a pentachlorophenyl ester, a pentafluorophenyl ester, a p-nitrophenyl ester, or a N- hydroxysuccinimide ester. Another coupling method involves the use of a suitable coupling agent, such as N, N'-dicyclohexylcarbodiimide or N, N'- diisopropylcarbodiimide.

The a-amino group of each amino acid employed in the peptide synthesis must be protected during the coupling reaction to prevent side reactions involving their active a-amino function. Certain amino acids contain <BR> <BR> reactive side-chain functional groups (e. g. , sulfhydryl, amino, carboxyl, and hydroxyl) and such functional groups must also be protected with suitable protecting groups to prevent a chemical reaction from occurring at either (1) the a-amino group site or (2) a reactive side chain site during both the initial and subsequent coupling steps.

In the selection of a particular protecting group to be used in synthesizing the peptides, the following general rules are typically followed.

Specifically, an a-amino protecting group (1) should render the a-amino function inert under the conditions employed in the coupling reaction; and (2) should be readily removable after the coupling reaction under conditions that will not remove side-chain protecting groups and will not alter the structure of the peptide fragment.

On the other hand, a side-chain protecting group (1) should render the side chain functional group inert under the conditions employed in the coupling reaction (2) should be stable under the conditions employed in removing the a-amino protecting group, and (3) should be readily removable from the desired fully-assembled peptide under reaction conditions that will not alter the structure of the peptide chain.

It will be apparent to those skilled in the art that the protecting groups known to be useful for peptide synthesis vary in reactivity with the agents employed for their removal. For example, certain protecting groups, such as triphenylmethyl and 2- (p-biphenyl) isopropyloxycarbonyl, are very labile and can be cleaved under mild acid conditions. Other protecting groups, such as t-butyloxycarbonyl (BOC), t-amyloxycarbonyl, adamantyl- oxycarbonyl, and p-methoxybenzyloxycarbonyl, are less labile and require moderately strong acids for their removal, such as trifluoroacetic, hydrochloric, or boron trifluoride in acetic acid.

Among the classes of amino acid protecting groups useful for protecting the a-amino group or for protecting a side chain group include the following: 1. For an a-amino group, three typical classes of protecting groups are: (a) aromatic urethane-type protecting groups, such as fluorenylmethyloxycarbonyl (FMOC), CBZ, and substituted CBZ, such as, p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzy loxycarbonyl, and p-methoxybenzyloxycarbonyl, o-chlorobenzyloxy carbonyl, 2, 4-dichlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxy carbonyl, and the like; (b) aliphatic urethane-type protecting groups, such as BOC, t- amyloxycarbonyl, isopropyloxycarbonyl, 2- (p-biphenyl) isopropyloxy carbonyl, allyloxycarbonyl and the like; and (c) cycloalkyl urethane-type protecting groups, such ascyclopentyloxy carbonyl, adamantyloxycarbonyl, andcyclohexyloxycarbonyl.

Preferred a-amino protecting groups may be BOC and FMOC.

(2) For the side chain amino group present in Lys, protection may be by any of the groups mentioned above in (1) such as BOC, 2- chlorobenzyloxycarbonyl and the like.

(3) For the guanidino group of Arg, protection may be provided by nitro, tosyl, CBZ, adamantyloxycarbonyl, 2,2, 5,7, 8- pentamethylchromansulfonyl, 2,3, 6-trimethyl methoxyphenyisulfonyl, or BOC groups.

(4) For the hydroxyl group of Ser or Thr, protection may be for example, by t-butyl; benzyl (BZL); or substituted BZL, such as p- methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and 2,6- dichlorobenzyl.

(5) For the carboxyl group of Asp or Glu, protection may be, for example, by esterification using such groups as BZL, t-butyl, cyclohexyl, cyclopentyl, and the like.

(6) For the imidazole nitrogen of His, the benzyloxymethyl (BOM) or tosyl moietyis suitably employed as a protecting group.

(7) For the phenolic hydroxyl group of Tyr, a protecting group such astetrahydropyranyl, tert-butyl, trityl, BZL, chlorobenzyl, 4-bromobenzyl, and 2,6-dichlorobenzyl are suitably employed. The preferred protecting group is bromobenzyloxycarbonyl.

(8) For the side chain amino group of Asn or Gln, xanthyl (Xan) is preferably employed.

(9) For Met, the amino acid is preferably left unprotected.

(10) For the thio group of Cys, p-methoxybenzyl is typically employed. <BR> <BR> <P>The first C-terminal amino acid of the growing peptide chain, e. g. , Glu, is typically protected at the a-amino position by an appropriately selected protecting group such as BOC. Following the coupling of the BOC- protected amino acid to the resin support, the a-amino protecting group is usually removed, typically by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone.

Other standard a-amino group de-protecting reagents, such as HCl in dioxane, and conditions for the removal of specific a-amino protecting groups are within the skill of those working in the art. Following the removal of the a-amino protecting group, the unprotected a-amino group, generally still side-chain protected, can be coupled in a stepwise manner in the intended sequence.

An alternative to the stepwise approach is the fragment condensation method in which pre-formed peptides of short length, each representing part of the desired sequence, are coupled to a growing chain of amino acids bound to a solid phase support. For this stepwise approach, a particularly suitable coupling reagent is N, N'-dicyclohexylcarbodiimide or diisopropylearbodlimide. Also, for the fragment approach, the selection of the coupling reagent, as well as the choice of the fragmentation pattern needed to couple fragments of the desired nature and size are important for success and are known to those skilled in the art.

Each protected amino acid or amino acid sequence is usually introduced into the solid-phase reactor in amounts in excess of stoichiorrietric quantities, and the coupling is suitably carried out in an organic solvent such as dimethylformamide (DMF). If incomplete coupling occurs, the coupling procedure is customarily repeated before removal of the N-amino protecting group in preparation for coupling to the next amino acid.

Following the removal of the a-amino protecting group, the remaining a- amino and side-chain-protected amino acids can be coupled in a stepwise manner in the intended sequence. The success of the coupling reaction at each stage of the synthesis may be monitored.

Upon completion of the desired peptide sequence, the protected peptide must be cleaved from the resin support, and all protecting groups must be removed. The cleavage reaction and removal of the protecting groups is suitably accomplished concomitantly or consecutively with de-protection reactions. When the bond anchoring the peptide to the resin is an ester bond, it can be cleaved by any reagent that is capable of breaking an ester linkage and of penetrating the resin matrix. One especially useful method is by treatment with liquid anhydrous hydrogen fluoride.

This reagent will usually not only cleave the peptide from the resin, but will also remove all acid-labile protecting groups and, thus, will directly provide the fully de-protected peptide. When additional protecting groups that are not acid-labile are present, additional de-protection steps must be carried out. These steps can be performed either before or after the hydrogen fluoride treatment described above.

When it is desired to cleave the peptide without removing protecting groups, the protected peptide-resin can be subjected to methanolysis, thus yielding a protected peptide in which the C-terminal carboxyl group is methylated. This methyl ester can be subsequently hydrolyzed under mild alkaline conditions to give the free C-terminalcarboxyl group. The protecting groups on the peptide chain can then be removed by treatment with a strong acid, such as liquid hydrogen fluoride.

Purification of the peptides of the invention is typically achieved using chromatographic techniques, such as preparative HPLC (including reverse phase HPLC), gel permeation, ion exchange, partition chromatography, affinity chromatography (including monoclonal antibody columns), and the like, or other conventional techniques such as countercurrent distribution or the like.

The invention further includes any functional derivatives and functional fragments of the octapeptide used by the invention. The terms functional derivatives and functional fragments used herein mean the peptide or any fragment thereof, with any insertions, deletions, substitutions and modifications, that inhibits the activation of lymphocytes by myelin derived antigen.

A specifically preferred peptide derivative is the octapeptide of the invention capped on both C and N terminals ends, Ac-KPSSPPEE-Am, also denoted by SEQ ID NO : 2.

Another specifically preferred peptide derivative is a cyclic octapeptide.

To date, there have been limited therapeutic applications involving peptides, due in considerable part to lack of oral bioavailability and to proteolytic degradation. Typically, for example, peptides are rapidly degraded in vivo by exo-and endopeptidases, resulting in generally very short biological half-lives. Degradation of the peptides by proteolytic enzymes in the gastrointestinal tract is likely an important contributing factor. The design of peptide mimics which are resistant to degradation by proteolytic enzymes has become of increasing interest to peptide chemists.

A primary goal has been to reduce the susceptibility of mimics to cleavage and inactivation by peptidases. In one approach, such as disclosed by Sherman and Spatola [J. Am. Chem. Soc. 112: 433 (1990) ], one or more amide bonds have been replaced in an essentially isosteric manner by a variety of chemical functional groups. This stepwise approach has met with some success in that active analogs have been obtained. In some instances, these analogs have been shown to possess longer biological half- lives than their naturally-occurring counterparts.

In another approach, a variety of un-coded or modified amino acids such as D-amino acids and N-methyl amino acids have been used to modify mammalian peptides. Alternatively, a presumed bioactive conformation has been stabilized by a covalent modification, such as cyclization or by incorporation of-lactam or other types of bridges. See, e. g. , Veber and<BR> Hirschmann, et al. , [Proc. Natl. Acad. Sci. USA, 75: 2636 (1978); and<BR> Thorsett, et al. , Biochem Biophys. Res. Comm. 111: 166 (1983) ]. The primary purpose of such manipulations has not been to avoid metabolism or to enhance oral bioavailability but rather to constrain a bioactive conformation to enhance potency or to induce greater specificity. Another approach, disclosed by Rich, D. H. [in Protease Inhibitors, Barrett and Selveson, eds. , Elsevier, p. 179-217 (1986) ], has been to design peptide mimics through the application of the transition state analog concept in enzyme inhibitor design.

Nicolaou and Hirschmann, et al. , describe the design and synthesis of a peptidomimetic employing ! 3-D-glucose for scaffolding [in Peptides, Chemistry, Structure and Biology: Proceedings of the llth American Peptide Symposium, Rivier and Marshall, eds. , ESCOM (1990), p. 881- 884].

Further peptidomimetics are compounds that appear to be unrelated to the original peptide, but contain functional groups positioned on a non- peptide scaffold that serve as topographical mimics. This type of peptidomimetics is referred to herein as a"non-peptidyl analogue."Such peptidomimetics may be identified using library screens of large chemical databases.

Peptidomimetics (both peptide and non-peptidyl analogues) may have improved properties (e. g. , decreased proteolysis, increased retention or increased bioavailability). Peptidomimetics generally have improved oral availability, which makes them especially suited to treatment of different pathological conditions. It should be noted that peptidomimetics may or may not have similar two-dimensional chemical structures, but share common three-dimensional structural features and geometry. Each peptidomimetic may further have one or more unique additional binding elements.

As discussed above, the lack of structure of linear peptides renders them vulnerable to proteases in human serum and acts to reduce their affinity for target sites, because only few of the possible conformations may be active. Therefore, it is desirable to optimize the peptide structure, for example by creating different derivatives of the various peptides of the invention.

Thus, the peptide of the invention or any fragment, analog or derivatives thereof may be in the form of a dimer, a multimer or in a spatially constrained conformation. The octapeptide used by the invention may be conformationally constrained by internal bridges, short-range cyclization, extension or other chemical modification.

In order to improve peptide structure, the octapeptides used by the invention can be coupled through their N-terminus to a lauryl-cysteine (LC) residue and/or through their C-terminus to a cysteine (C) residue, or to other residue/s suitable for linking the peptide to adjuvant/s.

The peptides of the invention, as well as derivatives thereof may all be positively charged, negatively charged or neutral.

Further, the peptides may be in the form of a dimer, a multimer or in a constrained conformation, in which the constrained conformation is obtained by internal bridges, short-range cyclizations, extension or other chemical modification.

As indicated above, the peptide of the invention may be further modified to improve its function, affinity, or stability. For instance, cyclization may be preferably used to impart greater stability and/or overall improved performance upon the peptide. A number of different cyclization methods have been developed, including side chain cyclization and backbone cyclization. These methods are well documented in the prior art [see e. g., <BR> <BR> Yu et al. , Bioorg. Med. Chem. 7: 161-75 (1999); Patel, et al. , J. Pept. Res.<BR> <P>53: 68-74 (1999); Valero, et al. , J. Pept. Res. 53 : 56-67 (1999); Romanovskis,<BR> et al. , J. Pept. Res. 52: 356-74 (1998); Crozet, et al. , Mol. Divers. 3: 261-76<BR> (1998); Rivier, et al. , J. Med. Chem. 41: 5012-9 (1998); Giblin et al. , Proc.<BR> <P>Natl. Acad. Sci. USA 95: 12814-8 (1998); Limal, et al. , J. Pept. Res. 52: 121- 9,1998, and US Patent No. 5,444, 150].

A preferred method of cyclization involves stabilization of an amphipathic alpha-helix by using para-substituted amino acid derivatives of a benzene ring, see [Yu (1999) ibid.]. Another preferred method of cyclization is backbone cyclization, as disclosed in Reissmann, et al. [Biomed. Pept. <BR> <BR> <P>Proteins Nucleic Acids 1: 51-6 (1994-95) ], and in references therein. A relatively new method of cyclization which involves backbone-to side chain connections may also be used [Reissmann (1994-5) ].

It should be appreciated that cyclization via lactam bond is also within the scope of the invention. Peptides comprise a lactam bridge may be constructed via a single amide bond formed directly between the two functional groups of the appropriate residues or by the insertion of a linker, comprising one a, ß, or y amino acid. It should be further noted that using this method is another way of controlling the ring size.

Still further, the peptides of the invention may be extended at the N- terminus and/or C-terminus thereof with various identical or different amino acid residues. As an example for such extension, the peptide may be extended at the N-terminus and/or C-terminus thereof with identical or different hydrophobic amino acid residue/s which may be naturally occurring or synthetic amino acid residue/s. A preferred synthetic amino acid residue may be D-alanine.

An additional example for such an extension may be provided by peptides extended both at the N-terminus and/or C-terminus thereof with a cysteine residue. Naturally, such an extension may lead to a constrained conformation due to Cys-Cys cyclization resulting from the formation of a disulfide bond.

Another example is the incorporation of an N-terminal lysyl-fatty acyl tail, the lysine serving as linker and the fatty acid as a hydrophobic anchor. A suitable fatty acid may be palmitic acid.

In addition the peptides may be extended by aromatic amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s. A preferred aromatic amino acid residue may be for example, tryptophan.

Further, according to the invention, the peptides of the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different organic moieties which are not naturally occurring or synthetic amino acids. As an example for such extension, the peptide may be extended at the N-terminus and/or C-terminus thereof with an N-acetyl group.

The peptides or any fragments, analogs or derivatives thereof used by the invention and comprised as an active ingredient within the compositions of the invention, may be any peptides according to the invention, e. g. , in the form of a monomer, dimer, a multimer or in a constrained conformation, as well as the other modifications described above.

According to one preferred embodiment, variant or derivative of the octapeptide used by the invention may be any one of a substitution variant, an addition variant, a chemical derivative and a peptidomimetic agent of said peptidic compound.

Amino acid substitution and addition variants are also included in this invention are peptides in which at least one amino acid residue and preferably, only one, has been removed and a different residue inserted in its place. For a detailed description of protein chemistry and structure, see <BR> <BR> Schulz, G. E. et al. , Principles of Protein Structure, Springer-Verlag, New York, (1979) and Creighton, T. E. Proteins-Structure and Molecular Principles, W. H. Freeman &Co., San Francisco, (1984), which are hereby incorporated by reference. The types of substitutions which may be made in the peptide molecule used by the present invention are conservative substitutions and are defined herein as exchanges within one of the following groups. <BR> <BR> <P>Small aliphatic, non-polar or slightly polar residues: e. g. , Ala, Ser, Thr,<BR> Gly. Polar, negatively charged residues and their amides: e. g. , Asp, Asn,<BR> Glu, Gln. Polar, positively charged residues: e. g. , His, Arg, Lys and Pro, because of its unusual geometry, tightly constrains the chain. Substantial changes in functional properties are made by selecting substitutions that are less conservative, such as between, rather than within, the above groups (or two other amino acid groups not shown above), which will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Most substitutions according to the present invention are those which do not produce radical changes in the characteristics of the peptide molecule. Even when it is difficult to predict the exact effect of a substitution in advance of doing so, one skilled in the art will appreciate that the effect can be evaluated by routine screening assays, preferably the biological assays described in the examples. Modifications of peptide properties including redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan.

Also included in this invention are addition variants wherein two or more residues are added to the octapeptide, for example, residues may be added to the C-terminus after Glu (or after any of its above substituents) in SEQ ID NO : 1.

Also included in this invention are addition variants wherein one or more residues is/are added to the N-terminus before Lys (or any of its above substituents) in SEQ ID NO : 1.

"Chemical derivatives"of the octapeptide KPSSPPEE used by the invention [SEQ ID NO : 1], contain additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chain terminal residues.

Examples of chemical derivatives of the peptide may be added as follow.

Lysinyl and amino terminal residues are derivatized with succinic or other carboxylic acid anhydrides. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge of the lysinyl residues.

Other suitable reagents for derivatizing amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal- chloroborohydride, 0-methylisourea; 2,4-pentanedione ; and transaminase- catalyzed reaction with glyoxylate. <BR> <BR> <P>Carboxyl side groups, aspartyl or glutamyl. , may be selectively modified by reaction with carbodilmides (R-N=C=N-R').

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, ethylation of the amino group of lysine. acetylation of the N- terminalamine, and amidation of the C-terminal carboxyl groups.

For every single peptide sequence used by the invention and disclosed herein, this invention includes the corresponding retro-inverso sequence wherein the direction of the peptide chain has been inverted and wherein all the amino acids belong to the D-series. For example the retro-inverso analogue of the natural L-series peptide KPSSPPEE is EEPPSSPK which is composed of D-series amino acids and in which E is the N-terminus and K is the C-terminus.

In a preferred embodiment, the peptide of the invention is capped on both C and N terminal ends. Thus, for example the retro-inverso analogue of the natural L-series capped peptide Ac-KPSSPPEE-Am is Ac-EEPPSSPK- Am which is composed of D-series amino acids and in which the N- terminal E is acetylated (Ac) and the C-terminal K is amidated (Am). The complete range of N-terminal capping groups and the complete range C- terminal capping groups specified for the L-series peptides are also intended for the D-series peptides. Also included are peptides wherein one or more D-amino acids has been substituted for one or more L-amino acids. Additionally, modified amino acids or chemical derivatives of amino acids may be provided such that the peptide contains additional chemical moieties or modified amino acids not normally a part of a natural protein. Such derivatized moieties may improve the solubility, absorption, biological half life, and the like.

Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed. , Mack Publishing Co., Easton, PA (1980).

It is to be appreciated that the present invention also includes longer peptides in which the basic peptidic sequence (the octapeptide of SEQ ID NO: 1, or the capped octapeptude of SEQ ID NO : 2) is repeated from about two to about 100 times.

In a second aspect, the invention relates to a method for inhibiting the activation of lympocytes by a myelin derived antigen. This method comprises the step of contacting the lymphocytes under suitable conditions, with an inhibitory sufficient amount of a peptidic compound derived from urokinase plasminogen activator (uPA) or of a composition comprising the same. This peptidic compound comprises the octapeptide carrying the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu which is also denoted by SEQ ID NO : 1, or any variant or derivative thereof. It should be noted that as used herein, a myelin-derived antigen in the EAE model maybe for example MBP (myelin basic protein), PLP (proteolipid protein) and MOG (myelin oligodendrocyte glycoprotein).

Still further, the invention relates to a method for inhibiting the activation of lympocytes by a myelin-derived antigen, in a subject suffering of an autoimmune disorder associated with myelin injury in the CNS. According to this embodiment, this method comprises the step of administering to said subject an inhibitory sufficient amount of a peptidic compound derived from urokinase plasminogen activator (uPA) or of a composition comprising the same, which compound comprises the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, or any variant or derivative thereof. A specifically preferred peptide derivative is the octapeptide of the invention capped on both sides, C and N terminals, Ac-KPSSPPEE-Am, also denoted by SEQ ID NO: 2.

It should be noted that another preferred derivative is a cyclic form of said octapeptide, preferably, a cyclized form of the peptide of SEQ ID NOs. 1 or 2.

The invention further provides for a method of treatment of an autoimmune disorder associated with myelin injury in the CNS in a mammalian subject in need of such treatment, preferably, multiple sclerosis (MS). The method of the invention comprises the step of administering to such subject a therapeutically effective amount of a peptidic compound derived from urokinase plasminogen activator (uPA) or of a composition comprising the same. The peptidic compound comprises the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, or any variant or derivative thereof. A preferred derivative may be the peptide of SEQ ID NO: 2. Another derivative is the cyclyzed peptide, preferably, a cyclized form of the peptides of SEQ ID NOs. 1 or 2..

The'therapeutically effective amount', for purposes herein, is determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art. The doses may be single doses or multiple doses over a period of several days, but single doses are preferred. It should be noted that referred dose ranges between 1 to 1000O,ug/kg/day, more preferably, between 10 to 700, ug/kg/day, most preferably, 50 to 500 gg/kg/day.

According to a particularly preferred embodiment, a variant or derivative of the peptide used by the methods of the invention may be any one of a substitution variant, an addition variant chemical derivative, cyclic peptide and a peptidomimetic agent of said peptidic compound.

The magnitude of therapeutic dose of the peptides used by the invention or of any composition thereof will of course vary with the group of patients (age, sex, etc. ), the nature of the condition to be treated and with the route administration and will be determined by the attending physician.

Although the method of the invention is particularly intended for the treatment of disorders associated with autoimmune disorders associated with myelin injury in the CNS in humans, other mammals are included.

By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, rodents such as mice and rats, and pigs.

The peptides used by the invention or any compositions thereof can be administered and dosed by the methods of the invention, in accordance with good medical practice, systemically, for example by parenteral, e. g. intravenous, intraperitoneal or intramuscular injection. In another example, the pharmaceutical composition can be introduced to a site by any suitable route including intravenous, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e. g. oral, intranasal, or intraocular administration.

Local administration to the area in need of treatment may be achieved by, for example, local infusion during surgery, topical application or direct injection into the inflamed joint, directly onto the eye, etc.

For oral administration, the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or in solid form as tablets, capsules and the like. For administration by inhalation, the compositions are conveniently delivered in the form of drops or aerosol sprays. For administration by injection, the formulations may be presented in unit dosage form, e. g. in ampoules or in multidose containers with an added preservative.

The peptides used by the invention can also be delivered in a vesicle, for example, in liposomes. In another embodiment, the peptides can be delivered in a controlled release system.

As mentioned, the amount of the therapeutic or pharmaceutical composition of the invention which is effective in the treatment of a particular disease, condition or disorder will depend on the nature of the disease, condition or disorder and can be determined by standard clinical techniques. In addition, in vitro assays as well in vivo experiments may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, condition or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In a further aspect, the invention relates to an inhibitory composition for inhibiting activation of lymphocytes by a myelin derived antigen. The composition of the invention comprises as an active ingredient an inhibitory sufficient amount of peptidic compound derived from urokinase plasminogen activator (uPA) comprising the amino acid sequence Lys-Pro-Ser-Ser-Pro- Pro-Glu-Glu also denoted by SEQ ID NO: 1, or any variant or derivative thereof, which composition optionally further comprises pharmaceutically acceptable carrier, diluent, adjuvant and/or excipient.

Still further, the present invention provides a pharmaceutical composition for the treatment of an autoimmune disorder associated with myelin injury in the CNS, preferably, multiple sclerosis (MS), in a mammalian subject in need of such treatment. The composition of the invention comprises as an active ingredient a therapeutically effective amount of a peptidic compound derived from urokinase plasminogen activator (uPA) comprising the amino acid sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu also denoted by SEQ ID NO: 1, or any variant or derivative thereof, which composition optionally further comprises pharmaceutically acceptable carrier, diluent, adjuvant and/or excipient.

According to one preferred embodiment, the peptide used for the compositions of the invention may be variant or derivative thereof such as any one of a substitution variant, an addition variant chemical derivative, cyclic peptide and a peptidomimetic agent of said peptidic compound.

A specifically preferred peptide derivative is the octapeptide of the invention capped on both sides, C and N terminals, Ac-KPSSPPEE-Am, also denoted by SEQ ID NO: 2.

Another specifically preferred peptide derivative is a cyclic peptide.

Preferably, a cyclized form of the peptide of any one of SEQ ID NOs. 1 or 2.

The peptidic compound or the pharmaceutical composition of the invention can be administered in various ways and may comprise, in addition to the active ingredient, pharmaceutically acceptable carriers, diluents, adjuvants, preserving agents and vehicles. According to one embodiment, the pharmaceutical composition of the invention is in a dosage unit form.

The pharmaceutical compositions can be administered subcutaneously or parenterally including intravenous, intra-arterial, intramuscular, and intraperitoneal administration, as well as intratheccal techniques.

Implants of the pharmaceutical preparations may also be useful. The pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents, or encapsulating material not reacting with the active ingredients of the invention.

When administering the peptidic compound or the pharmaceutical composition of the invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions and sterile powders for reconstitution into sterile injectable solutions. The carrier can be any physiologically acceptable suitable carrier, for example, water, or aqueous buffer solutions.

Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In addition, various additives which enhance the stability, sterility and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, cheating agents and buffers can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and the like. In many cases it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, any vehicle, diluent, or additive used would have to be compatible with the compositions. Considering that the active agent is prone to proteolysis, the composition of the invention may contain as additive pharmaceutically acceptable protein inhibitors.

Sterile injectable solutions can be prepared by incorporating the compositions utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.

Nonetheless, the composition disclosed herein in detail can be administered orally to the patient. Conventional forms such as administering the composition as tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques which deliver it orally or intravenously and retain the biological activity are preferred.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms"a","an"and"the"include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word"comprise", and variations such as"comprises"and"comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

The invention will now be described in more detail on hand of the following Examples, which are illustrative only and do not limit the scope of the invention, which is defined by the appended claims.

Examples Experimental procedures Preparation of the Ac-KPSSPPEE-Am derivatiue The capped peptide (Ac-KPSSPPEE-Am, also denoted by SEQ ID NO: 2) was synthesized by standard solid-phase methodology using p-methyl- benzhydrylamine resin and L-amino acids protected with t- butyloxycarbonyl (BOC) groups. Removal of the BOC groups was accomplished with 50% trifloroacetic acid in dichloromethane. Coupling was achieved with 1-hydroxybenotriazole and dicyclohexylcarbodiimide.

Side-chain protection was with 2-chorobenzyloxycarbonyl for lysine, benzyl for serine, and cyclohexyl for glutamic acid. The amino-terminal lysine was capped by treatment with acetic anhydride. Deprotection and detachment of the completed peptide from the resin were accomplished with anhydrous hydrofluoric acid in the presence of anisole. The peptide was purified on high performance liquid chromatography (HPLC) on a Waters C18 preparative column using 0-40% linear gradient of 1.0% aqueous triethylamine phosphate into CH3CN. The column was washed with 3 column volumes of 1.0% aqueous acetic acid and then eluted with a 0-50% linear gradient of 1.0% aqueous acetic acid into CH3CN. Fractions containing the peptide were reapplied to the column, eluted in the same manner, and lyophilized. The final product was > 99% pure, colorless, and readily soluble in water to > 500 mM.

EAE: Induction and evaluation of clinical disease EAE was induced in 8-week-old female C57BL mice (purchased from Jackson Laboratories, USA) by injection of 100-125, ug of MOG36 s6 peptide (Myelin Oligodendrocyte Glycoprotein), emulsified in complete Freund's adjuvant (CFA) containing 4mg/ml heat killed Mycobacterium tuberculosis into the hind footpads. Immediately thereafter, and, again, at 48h, the mice were inoculated with 0.1 ml of pertussis toxin (200-400ng).

Additional injection of MOG3s s6 peptide in CFA was given 7 days later.

All animals were examined daily and evaluated for clinical signs of disease. The first clinical signs appear on day 12-18 post immunization.

The clinical status of the mice was graded as follows: 0, without clinical disease; 1, tail weakness; 2, hind limb weakness sufficient to impair righting; 3, one limb plagic; 4, paraplegia with forelimb weakness; 5, quadriplegia; 6, death.

Mice uPA depleted mice (uPA-\-), and urokinase receptor deficient mice (uPAR- /-) on C57 blank background, and littermate controls were purchased from Jackson Immuno-research Laboratories, West Grove, PA. All mice weighed 20-30 g at the time of the study.

Histopathology following treatment Brains and spinal cords were removed, fixed in 4% buffered formalin, sectioned, and routinely processed for paraffin embedding. Sections prepared from the same level were stained with luxol-fast-blue- hematoxylin and eosin. Inflammatory foci containing at least 20 perivascular mononuclear cells were counted in each section [Brenner, T. <BR> <BR> et al. , J. Immunol 158: 2940-2946 (1997); Brenner, T. et al. , Exper. Neurol.

154: 489-498 (1998)].

Lymphocyte proliferation assay Pooled lymph node cells (LNCs) were prepared from inguinal, axillary and mesenteric lymph nodes from mice that had been s. c. inoculated 9 days earlier with MOGss-ss peptide in CFA. The in vitro response of the LNCs was assayed in triplicate wells of 96-well flat-bottom microtiter plates. A quantity of 400,000 LNCs, suspended in 0.2 ml RPMI supplemented with 5% fetal calf serum (FCS), was added to each well with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 lOjug/ml, or Concanavalin A (Con A), lug/ml or PHA (Sigma, U. S. A. ). At 72 h after seeding, IjnCi 3Rthymidine (Amersham Pharmacia Biotech, UK) was added to each well and the plates were incubated for an additional 18 h. The plates were then harvested with a semiautomated harvester onto a glass fiber filter, and the radioactivity was determined by liquid scintillation Brenner (1997) ibid. ; Brenner (1998) ibid.].

Antigen presentation assay LNCs were prepared from mice inoculated 9 days earlier with MOG 35-55 in CFA. The cells were seeded in a 24 well plate in the presence of MOG 35-55 and refreshed with IL-2 (20 u/ml) 4 days later. The cells were grown for additional 7 days and plated together with APC (spleen cells). The spleen cells were prepared from spleens that had been extracted from C57BL/6JOlaHsd mice following irradiation (300 Rad). The lymphocyte response to MOG presentation was determined by thymidine incorporation as indicated above.

Histopathology assay Brain and spinal cord sections were stained with Bielschowsky and cresyl violet for simultaneous evaluation of axonal loss (AL), axonal injury (AI) and infiltration (inf). AL scoring: 0-normal, 1-mild to moderate axonal loss, 2-severe axonal loss. AI scoring: 0-normal, 1-scattered, injured axons, 2- mild to moderate axonal injury, 3-severe axonal injury. Inf scoring: 0-no infiltrates, 1-10-20 cells per 40x optical field, 2-20-60 cells per 40x optical field, 3-60-100 cells per 40x optical field.

Example 1 Involvement of uPA and uPAR in the course of MS In order to examine the possible involvement of uPA and uPAR in the development of MS, the EAE model was used. As a first step, the inventors examined the effect of uPA on the development of EAE as described in the experimental procedures. Briefly, EAE was induced in uPA-/-as well as control mice by injection of 100ig of MOG35-55 peptide. The EAE mice were then evaluated for clinical signs of the disease. The data presented in Figure 1 show that compared to WT mice, the disease in uPA- -animals reach a higher clinical score which indicate that they developed more severe clinical symptoms, (n=14 in each group).

The inventors next similarly examined the involvement of the urokinase receptor as well as the tPA (tissue type PA) (Figure 2 and Figure 3, respectively). As shown by Figure 2, the absence of uPA receptor (UPAR) leads to the same effect seen in Figure 1. The uPAR-/-mice develop more severe clinical symptoms than WT mice, (n=7 in each group). Similarly, the absence of tPA resulted in sever symptoms (Figure 3).

Table 1 summarizes the examination of clinical parameters affected by different components of the PSA system, as shown by Figures 1-3.

Table 1-Clinical parameters of EAE Severity Incidence CS ut1. 70. 2 90.9% 29. 1 + 4. 4 tPA-/-4.1 0.3* 100.0% 81. 0 + 7. 8* uPAR-/-2. 5 + 0. 4 92. 3% 35. 1 1 3. 9 Severity Incidence CS wt 1. 1 + 0. 1 56.2% 13. 2 : b 2. 5 1 uPA-/-1 1. 8 0. 0* 87. 5% 24. 53. 6 Table 2 shows that in addition to the severity of the disease that was more than double in the uPA-/-mice (compared to WT), the duration of the clinical symptoms was also doubled in uPA-/-mice.

Table 2 Duration/Day uPA+/+ 12 3 uPA-/-23 3 The data presented herein indicate that different components of the PA play an important role in the course of MS.

In order to further analyze the role of PA components on EAE, the inventors have next have examined axonal loss, injury and cell infiltration in the CNS as result of the absence of PA components. Twenty days after induction of EAE in female WT, tPA-/-, uPA-/-or uPAR-/-C57BL mice (n=7) by immunization with the MOG36-55 peptide, brain tisue and spinal cords were extracted for pathology study. After fixation brains and spinal cords were dissected and the sections were stained with Bielschowsky and cresyl violet for simultaneous evaluation of axonal loss (AL), axonal injury (AI) and infiltration (inf). Figure 4 represent an example of such analysis performed in PA-/-mice. As shown by this figure there is an increased neurological damage, as well as inflammatory response in uPA-/- compared to the wild type.

These three parameters were scored from 0 to 2: In the case of AL the score was: 0-normal, 1-mild to moderate axonal loss, 2-severe axonal loss.

AI scoring was: 0-normal, 1-scattered, injured axons, 2-mild to moderate axonal injury, 3-severe axonal injury. The Inf scoring was: 0-no infiltrates, 1-10-20 cells per 40x optical field, 2-20-60 cells per 40x optical field, 3- 60-100 cells per 40x optical field. The scores of the three parameters in the 4 groups were analyzed and depicted in Figure 5A-D. Similarly, as shown by these histograms, the absence of the different components of PA in the different knockout mice resulted in increased sensitivity to axonal loss and injury which reflects demyaelination and increased inflammation. Thus, tPA, uPA, and uPAR may play a protective role in the development of EAE.

Example 2 An octapeptide derived from the connecting peptide inhibits the activation of lymphocytes by the MOG33-55peptide In an attempt to establish which portion of uPA is critical for the development of the disease, the inventors next examined in vitro the effect of different portions of uPA on the response of lymphocytes to the MOGss- 55 peptide (the antigen used to induce MS in vivo). The response of lymphocytes is representative of in vivo first step of the development of MS.

Lymphocyte proliferation assay was performed in the presence of different peptides portions of uPA including the catalytic domain (amino acid residues 144-411 in the mature protein, or residues 164-431, as denoted in GenBank Accession No. P00749), ATF domain (amino acids 1-135, in the mature protein or 21-155 as denoted in GenBank Accession No.

P00749) and the peptide that connects both domains of uPA (amino acids 136-144, in the mature protein or, 156-164, referred to the amino acid sequence as denoted in GenBank Accession No. P00749). Figure 6 shows that the presence of the octapeptide Ac-KPSSPPEE-Am (also denoted by SEQ ID NO: 2), which is a derivative of the KPSSPPEE (SEQ ID NO : 1) that corresponds to the connecting peptide (amino acids 136-143 in the mature protein, or residues 156-163, as referred to the amino acid sequence as denoted in GenBank Accession No. P00749), inhibited the activation of lymphocytes. In contrast, other uPA portions had no effect. n=7 in each group.

Thus, the data of the present invention support the conclusions that: a) uPA regulates the lymphocyte response to the MS antigen. b) uPA plays a central protective role in the development of the disease in vivo. c) The active portion in uPA is the octapeptide that connects the two major domains of the molecule.

Example 3 The octapeptide of the invention preuents MS symptoms more efficiently then CopaxoneTM (Teva) The inventors have next compared in vitro the effect of the uPA derived peptide used by the invention, to CopaxoneTM, which represents a well known efficient medicament for MS. Both compositions (the octapeptide of the invention and Copaxon) were analyzed on the response of lymphocytes to the MOG3s-s5 peptide (the antigen used to induce MS in vivo), as described above. Lymphocyte proliferation assay performed in the presence of the octapeptide of the invention reveled inhibitory effect stronger then that of Copaxone. Therefore, the peptide used by the invention may represent a novel efficient therapeutic approach for the treatment of MS.