Gliosis is a common denominator for disorders of the neuroglia, the supporting tissues of the neuronal tissue in the central nervous system comprising the brain, spinal cord and ganglia. Astrogliosis defines gliosis of astrocytes. Trauma or injury and other pathological events in the CNS cause gliosis, in particular astrocytic scar reaction. This event has many negative effects such as prevention of axonal regrowth, damage to the blood brain barrier, and destabilization of synapses (Mathewson and Berry, 1985; Eng, 1988). Three observations characterize this astrogliosis : hypertrophy, hyperplasia and the accumulation of gliofilaments. It has been shown that thrombin can induce the similar effects on cultured astrocytes (Beecher et al., 1994; Grabham and Cunningham, 1995; Pindon et al., 1998).

Our working hypothesis was that a high level of thrombin at the site of injury might initially induce an astrocyte reaction and later increase the expression of its specific inhibitor, thrombomodulin. Thrombomodulin could then stabilize the astroglial scar through its adhesive properties.

Thrombin is a multifunctional serine protease. It has three specific G protein coupled receptors, proteolytic activated receptor-1,3 and 4 (PAR-1, PAR-3 and PAR-4; Vu et al., 1991; Ishihara et al., 1997; Xu et al., 1998). This protease activates a wide range of cells, such as neurons (Gurwitz and Cunningham, 1988), inducing cell differentiation or growth, so that some authors view thrombin as a growth factor (Grand et al., 1996).

Thrombomodulin is a transmembrane glycoprotein inhibitor of thrombin (Esmon, 1987), present and functionally active on primary astrocyte cultures (Pindon et al., 1997). The lectin-like domain of thrombomodulin might involve the molecule in a new type of cell-cell adhesion (Imada et al., 1990), which is also suggested by its inhibitory effect on cancer metastasis and by its role in embryological development.

We have recently demonstrated that thrombomodulin is a new marker of astrogliosis in vivo after lesion, and that it is up regulated in astrocyte primary cultures after thrombin administration (Pindon et al., submitted). Unexpectedly, it has now been found that thrombin is implicated in the etiology of gliosis, and that antagonists of the thrombin receptor are effective in blocking the cascade of events leading to gliosis. Targetting the receptor rather than thrombin itself has the advantage that one can inhibit cellular effects of thrombin without increasing the risk of bleeding, inhibiting fibrin formation, or altering hemostasis.

The present invention is thus concerned with the use of a thrombin receptor antagonist for the manufacture of a medicament for treating or preventing gliosis.

The invention enables one to treat and prevent lesions of the CNS with antagonists of thrombin receptors, to decrease astrogliosis scar formation, and to impact on the regeneration process. Particular indications which now can be treated or prevented are the following: astrogliosis caused by irradiation, myelopathy, head trauma or spinal cord injury.

A-Decrease irradiation side effect Irradiation of the CNS as a cancer treatment induces astrogliosis in white matter, cognition and memories deficiency, and in long term astroglioblastomas. For these reasons irradiation is avoided for children under 11 years and very carefully used for adults. Thrombin receptor antagonists could now be administered prophylactically to a patient undergoing irradiation treatment so as to prevent radiation-induced astrogliosis.

B-Myelopathy Myelopathy is a common disorder. It is encountered when the spinal cord is being compressed by an external mass such as bone calcification or a tumor. Although no direct neurological damage can be observed, the compression induces the formation of astrogliosis and edema. Thrombin receptor antagonists could now be administered therapeutically to a patient suffering from myelopathy C-Astroglial scar in head trauma and spinal cord injury Astroglial scar formation in spinal cord injuries or after spinal surgery is a blockage of axonal regeneration and prevents repeated operations. It does not seem afflict patient

recovery except for triggered epilepsy. Thrombin receptor antagonists could now be administered prophylactically to a patient undergoing spinal cord injury so as to prevent surgery-induced astrogliosis.

Depending on the nature of the thrombin receptor antagonist, in particular its bioavailability, the antagonists may be administered topically or parenterally, e. g. by injection, and preferably orally.

Experimental Part Example 1 : In vitro Screening for modulators of the thrombin receptor C6 glioma cells were seeded and cultured in 96 well plates with DMEM containing 10% of dialyzed calf serum, under 5% C02.

When cells were confluent, they were loaded during one hour in DMEM containing 1 mM Fluo-3AM, 5 mM probenecid, 20 mM Hepes. Cells were washed 2 times with the assay buffer (1.19 g/1 Hepes, 1.25mM CaC12, 8.182 g/l NaCI, 0.203g/I MgC12, 0.373 g/l KCI, 1.982 g/l glucose, pH 7.4). Compounds, agonist and antagonist, were diluted in assay buffer at different concentrations and added to the cells using the Fluorometric Imaging Plate Reader (FLIPR) system. Cells were pre-incubated with the antagonist for 20 minutes at 37 C before being stimulated by thrombin at 1 IU/ml, or by thrombin receptor activating protein (TRAP-6, SFLLRN at 50 uM) Release of intracellular calcium triggered by the stimulation of thrombin receptor was measured during 2 minutes, by the fluorescence emitted by the complex Fluo-3AM-calcium using the FLIPR.

Controls: 1-All compounds were tested as antagonist to endothelin receptors, to check their specificity to the thrombin receptor and not downstream in the general signal transduction pathway. The same protocol as for thrombin receptor was used, with stimulation by endothelin at 10-7 M instead of thrombin.

2-All compounds were tested in their agonist activity to check whether the inhibition of thrombin response was not due to a previous activation followed by desensitization of the receptor.

As a result of screening a library of proprietary compounds, four compounds were identified as thrombin receptor antagonists. Of these four one is known previously from EP-0,156,433 as an antiviral agent, viz. 1- (6-chloro-3-pyridazinyl)-4- (2- naphthalenyl)-4-piperidinol which had a pIC50 = 6.9 (against TRAP-6). The assay also showed that the known antagonist FR-171113 (Fujisawa) was less potent having a pIC50 = 6.3 (against TRAP-6).

Example 2: Treatment of spinal cord injury as a model of astrogliosis Animal model of spinal cord in jurv : The dorsal funiculus of the spinal cord is crushed at the level of T8 by forceps. The surgical approach is standard through a partial laminectomy, dura and arachnid are incised, and the point of forceps separated to the medial margins of the dorsal root entry zone along the dorso-lateral sulcus, and lowered to a depth of 2mm. Approximation of the tips crushes the dorsal columns including all the axons in the ascending gracile tracts and the descending corticospinal tracts bilaterally. The pia remains intact and the patency of the overlying vessels is preserved.

Treatments Scramble agonist peptide of PAR1, PAR3 and PAR4, will be infused on the site of the lesion at different time after lesion (Ohr, 4hr, 8hr, 24hr).

Astrovliosis evaluation Will be performed by an evaluation of the glial filament acidic protein (GFAP) as a marker of astrogliosis, on cryosections from the lesion site.

Morphological studies will include immunostaining and in situ hybridization.

PARI Knock out mice Astrogliosis induced by lesion will be studied in these mice.

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