Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
  • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
  • A61K31/355—Tocopherols, e.g. vitamin E
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
  • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

• the invention relates to methods for inhibiting the Notch signalling pathway. More particularly, the invention relates to the use of Notch inhibitors for the treatment of a disease associated with an up-regulated Notch signalling pathway activity.
• Notch signalling pathway has a simple framework that is highly conserved throughout the animal kingdom (Artavanis-Tsakonas et al., 1999; Schweisguth, F., 2004; Radtke et al. 2003).
• Both the Notch receptor and its ligands are transmembrane proteins with large extracellular domains that consist primariiy of epidermal growth factor (EGF)-like repeats. Ligand binding promotes two proteolytic cleavage events in the Notch receptor. The first cleavage is catalysed by ADAM-family metalloproteases, whereas the second is mediated by gamma-secretase, an enzyme complex that contains presenilin, nicastrin, PEN2 and APH1 (Fortini et al., 2002).
• the second cleavage releases the Notch intracellular domain (Nicd), which then translocates to the nucleus and cooperates with the DNA- binding protein CSL (named after CBF1 , Su(H) and LAG-1 ) and its co-activator Mastermind (Mam) to promote transcription.
• Notch intracellular domain Nicd
• CSL DNA- binding protein
• Notch paralogues differ between species. For example, there are four Notch receptors in mammals (Notchi to 4), two in Caenorhabditis elegans (LIN-12 and GLP-1 ) and one in Drosophila melanogaster (Notch), but the basic paradigm is common throughout evolution (Artavanis et al., 1999; Schweisguth, 2004; Radtke et al., 2003).
• Notch signalling pathway represents a promising tool for treating different kinds of diseases and disorders, such as Alzheimer disease (Beher and Graham, 2005); multiple sclerosis (John et 2002; Nat, Med. 8:1115-1121 ); brain tumours (Miele et al., 2006), and autoimmune disorders (Briend E, . 2005).
• Notch ligand expression plays a role in cancer. Indeed, up regulated Notch ligand expression has been observed in some tumour cells.
• By downregulating Notch signalling pathway in vivo in T cells it is possible to prevent tumour cells from inducing immunotolerance in those T cells that recognise tumour- specific antigens. In turn, this allows the T cells to mount an immune response against the tumour cells (see document WO01/35990).
• Document WO2006052128 discloses the fact that inhibitors of Notch pathway activation (for example gamma-secretase inhibitors) are extremely useful in the treatment of intestinal adenoma and/or adenocarcinoma
• Document WO03102242 describes the use of Notch inhibitors for inducing apoptosis for the prevention or treatment of diseases and conditions associated with insufficient apoptosis.
• the invention relates to the use of a compound of formula (I) for inhibiting the Notch signalling pathway
• R1 , R2, R3 and R4 are identical or not, and are each independently a hydrogen, a hydroxyl, a C1-C6 alkyl, a C1-C6 alkoxy or a C1-C6 alkanoyloxy
• R5 is a hydrogen or a C1-C6 alkyl
• - m is an integer from 0 to 2, preferably 1 or 2, even more preferably 1 n is an integer from 8 to 25, preferably from 8 to 20, even more preferably between 8 and 16 or between 8 and 14.
• the invention also relates a compound of formula (I) for the treatment of a disease associated with an up-regulated Notch signalling pathway activity.
• the invention also relates to the use of a compound of formula (I) for the manufacture of a medicament for the treatment of a disease associated with an up- regulated Notch signalling pathway activity.
• the invention also relates to a method of treatment of a disease associated with an up-regulated Notch signalling pathway activity, said method comprising administrating a compound of formula (I) to a patient in need thereof.
• Another object of the invention is the non therapeutic use of compound of formula (I) for inhibition of the Notch signalling pathway.
• Another object of the invention is a non therapeutic method for inhibiting the Notch signalling pathway comprising the step of adding compound of formula (I) to a cell culture.
• alkyl refers to a branched or unbranched saturated hydrocarbon group.
• C1-C6 alkyl refers to a straight chain or branched hydrocarbon moiety having from 1 to 6 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert- butyl, pentyl, neopentyl, n-hexyl...
• alkoxy refers to an alkyl group optionally substituted that is bonded through an oxygen atom.
• C1- C6 alkoxy includes the groups methoxy, ethoxy, isopopoxy, ter-butoxy...
• alkanoyloxy refers to -OCO-alkyl groups, where the term alkyl is as defined above.
• C1-C6 alkanoyloxy includes acetate, propionate, butylate, pentanoate or hexanoate.
• salts embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases.
• the nature of the salt is not critical, provided that it is pharmaceutically acceptable.
• “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
• a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• Notch signalling pathway refers to the biological activity associated with activation or inhibition of the Notch signalling pathway, including any of the downstream biological effects otherwise resulting from the binding Notch receptors of its natural ligand.
• the term "disease associated with an up-regulated Notch signalling pathway activity” includes any disease which results from the activation or the up-regulated activity of the Notch signalling pathway or is associated with the activation or the up- regulated activity of the Notch signalling pathway.
• Assays for the activation of the up- regulated activity of the Notch signalling pathway can include the quantification of the expression of the genes downstream of Notch, such as Hes1 and Hes5, eg by quantitative RT-PCR, northern-blot and immunological assays
• the term “treating” or “treatment”, as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
• “Therapeutically effective amount” means an amount of a compound/ medicament according to the present invention effective in producing the desired therapeutic effect.
• the term "patient”, or “patient in need thereof, is intended for a human or non-human mammal affected or likely to be affected with a disorder.
• the patient is a human.
• the present invention provides the first demonstration that compounds of formula (I) are Notch signalling pathway inhibitors.
• the present invention relates to compounds of formula (I) for the treatment of a disease associated with an up-regulated Notch signalling pathway activity.
• R1 , R2, R3 and R4 are identical or not, and are each independently a hydrogen, a hydroxyl, a C1-C6 alkyl, a C1-C6 alkoxy or a C1-C6 alkylcarbonyl,
• R5 is a hydrogen or a C1-C6 alkyl
• m is an integer from 0 to 2, preferably 1 or 2, even more preferably 1
• n is an integer from 8 to 25, preferably from 8 to 20, even more preferably between 8 and 16 or between 8 and 14.
• the compounds of the present invention contain an asymmetric carbon atom (the one bearing R5), and, therefore, the instant invention may also include the individual diastereomers and enantiomers, which may be prepared or isolated by methods known to those skilled in the art. It will be appreciated that the compounds of the invention may be isolated in optically active or racemic forms.
• n is 1 and the compounds are named Tocopherol Fatty Alcohols (TFA-n), where n is defined as above.
• TFA-n Tocopherol Fatty Alcohols
• Compounds for which n is 10, 11 , 12, 13, 14, 15 or 16 are particularly suitable for the invention.
• preferred compounds are selected from the group consisting of TFA-12, TFA-14, TFA-15, TFA-16 and TFA-18.
• the preferred compound is TFA-12 which has the formula:
• Diseases associated with an up-regulated Notch signalling pathway activity include but are not limited to cancer, central nervous system diseases such as multiple sclerosis, Alzheimer and more generally CNS diseases associated with inflammation.
• the disease is cancer, In another embodiment, the disease is multiple sclerosis. In another embodiment the disease is a disease associated with an up- regulated Notch signalling pathway activity excepting nervous system diseases affecting oligodendrocytes or other cells of the nervous system, inflammation of the nervous system, degenerative neuropathies, demyelinating diseases, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Creutfeldt-Jakob's disease, strokes or any other lesion of the nervous system.
• nervous system diseases affecting oligodendrocytes or other cells of the nervous system, inflammation of the nervous system, degenerative neuropathies, demyelinating diseases, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Creutfeldt-Jakob's disease, strokes or any other lesion of the nervous system.
• Another object of the invention relates to a method for treating a disease associated with an up-regulated Notch signalling pathway activity comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I). It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
• the daily dosage of the products may be varied over a wide range from 0.01 to 1 ,000 mg per adult per day.
• the compositions contain 0.01 , 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
• a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
• An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
• the compound of formula (I) may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
• the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
• Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
• the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the compounds of formula (I) can be formulated into a composition in a neutral or salt form.
• Suitable pharmaceutically-acceptable acid addition salts of compounds of Formula I may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.
• organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, propanoic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicyclic, salicyclic, p- hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic) , methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, eyclohexylaminosulfonic, stearic, algenic, ⁇ -
• Suitable pharmaceutically-acceptable base addition salts of compounds of formula I include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N 1 N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding compound of formula I by reacting, for example, the appropriate acid or base with the compound of formula I.
• the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
• the 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.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
• solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
• aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
• one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
• the compounds of formula (I) may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0,1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
• other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration ; liposomal formulations ; time release capsules ; and any other form currently used.
• Non therapeutic compounds, methods and uses are:
• the invention also relates to compounds of formula (I) for non-therapeutical use and methods comprising administering compounds of formula (I) to cell cultures.
• Fig. 1 TFA-12 inhibits astroglial and microglial activation in vitro.
• RNA extraction was performed using Trizol reagent. TFA-12 inhibits both astrogliosis and inflammatory gene response, (b): RT-PCR gene expression analysis in MMGT12 microglial cell line. RNA extraction was performed after 6 hours of incubation with TFA-12 (10 ⁇ 5 M), with or without LPS activation (0.01 ⁇ g/ml). Expression of NOS-II,
• TFA-12 inhibits both TNF- ⁇ and IL-1 ⁇ secretion.
• Fig. 2 TFA-12 improves the clinical course of the EAE disease.
• Fig.3 Inflammation, astrogliosis and demyelination are reduced by TFA-12 treatment.
• (a,b,c) CD45 immunohistochemistry on spinal cord sections of vehicle- (a) and TFA- 12- treated (b) mice reveals immune foci (dashed lines) in the dorsal funiculus,
• (c) The number of CD45+ cells is decreased by 2.8 fold in TFA-12-treated mice compared to controls.
• d,e,f In vehicle treated mice, demyelinated lesions are characterised by the absence of MBP staining (d), whereas demyelination is barely visible in TFA-12-treated groups.
• Fig.4 TFA-12 induces oligodendrocyte differentiation and reduces axonal damage in EAE.
• a-d Spinal coronal sections stained with the 2H3 anti-neurofilament antibody (b,d) and counterstained with the nuclear dye Hoechst 33342 (a,c). Intense axonal swelling (arrows) is detected in white matter lesions of vehicle-treated spinal cords (b), while very few degenerating axons are evidenced in TFA-12-treated spinal cord lesions (d).
• e Axonal degeneration is 3.3 fold lower in TFA-12-treated mice than controls.
• f,g CC1 + oligodendrocytes are more abundant at the periphery of spinal cord lesions in TFA-12-treated mice (g) compared to controls (f).
• Fig 5 TFA-12 promotes OPC differentiation in vitro.
• the CG4 oligodendroglial cells were differentiated in N1 medium (a, d, g) or in N1 supplemented with 5.10 "7 M of TFA-12 (b, e, h), during 48 hours.
• the number of 04+ oligodendrocytes (arrows) is increased by TFA-12 treatment (b) compared to untreated cultures (a),
• Proliferating Ki67+ cells are reduced in N1 medium plus TFA-12 (h) compared to N1 alone (g).
• Wilcockson test performed on ⁇ Ct was used to confirm significance between TFA-12 and control groups (p ⁇ 0.05). Student t test: * p ⁇ 0.05, ** p ⁇ 0.001. Scale bars, 50 ⁇ m (a.b.g.h); 25 ⁇ m (d, e).
• Fig 6 Quantification of the number of GaIC+ differentiated oligodendrocytes in CG4 culture, after 5 days of differentiation in N1 medium alone or N1 supplemented with 5.10 "7 M of TFA-12. TFA-12 treatment significantly enhances the generation of GaIC+ oligodendrocytes compared to untreated cultures. Mann-Whitney test, * p ⁇ 0.05.
• Fig 7 Rat oligodendrocyte precursors were cultured during 2 days in the absence or the presence of 5x1 u '7 M of TF12. C; control; T12: TFA-12-treated cells. The number of GaIC+ cells was counted and expressed as percentage of total cells. Values are mean ⁇ SEM for each group. * *p ⁇ 0,001 versus control group of two independent experiments.
• Fig 8 Rat oligodendrocyte precursors were cultured during 2 days in the absence or the presence of 5x10 "7 M of TF12. C: control; T12: TFA12-treated cells. The number of 04+ (a), MBP+ (b) and myelin-like sheaths exhibiting cells (c and d) was counted and expressed as percentage of total cells. Values are mean ⁇ SEM for each group. **p ⁇ 0.001 versus control group of two independent experiments.
• Fig 9 Mouse P2 brain slices were cultured during 6 days in the absence or the presence of 5x10 "7 M of TFA-12.
• Fig 10 Human neurospheres were cultured as adherent cells during 5 days in the absence or the presence of 2x10 "7 M TFA-12.
• C control
• T TFA-12-treated cells.
• TFA-12 acts as an antagonist of the Notch signalling pathway.
• CG4 oligodendrocyte precursor cell line was cultured on polyomithine-laminine and expanded in N1/B104 (70v/30v) medium, as previously described (Louis et al., 1992).
• N1/B104 was replaced by N1 containing TFA- 12 at 5.10 "7 M or by N 1 alone. Media were changed daily and differentiation was carried out during 48 hours.
• Primary astrocyte cultures were prepared from newborn Wistar rat pups, as previously reported (Morga et al., 2000).
• the murine microglial cell line MMGT12 (gift from Dr.
• Vanmechelen Innogenetics, Gent, Belgium was cultured in DMEM/Ham's F12 (1/1 ), supplemented with 2% fetal calf serum (FCS, Invitrogen), 1 % Insulin/Transferrin/Selenium (ITS, Invitrogen) and 15% filtered conditioned medium of WEHI cells (WEHI3, WEHI 3B and WEHI 3D, ATCC).
• FCS fetal calf serum
• ITS Insulin/Transferrin/Selenium
• WEHI3B and WEHI 3D filtered conditioned medium of WEHI cells
• TFA-12 treatment (10 ⁇ 6 M) was performed during 24 hours, with or without LPS (1 ⁇ g/ml), MMGT12 cultures were treated with TFA-12 (10 "5 M) with or without LPS stimulation (0.01 ⁇ g/ml), during 6 hours for RT-PCR and 24 hours for ELISA experiments.
• the synthesis of the tocopherol long chain fatty alcohol TFA-12 was performed as previously described (Muller et al., 2004; 2006).
• mice EAE induction and TFA-12 treatment Twelve weeks-old C57BL/6 female mice (Elevage Janvier) were immunized sub- cuteanously with an emulsion consisting of 200 ⁇ g of synthetic MOG35-55 peptides (NeoMPS) in complete Freund's adjuvant (CFA, Difco), supplemented with 500 ⁇ g of heat-inactivated Mycobacterium tuberculosis (strain H37Ra, Difco). Mice were injected intravenously with Pertussis toxin (List Biological Laboratories) on the day of the immunization and 48 hours later, and then divided in two groups.
• Pertussis toxin List Biological Laboratories
• Sections were stained with either Luxol fast blue (LFB; to visualize myelin, from Sigma) or Oil Red O (ORO; to visualize macrophages containing myelin debris, from Sigma).
• LFB Luxol fast blue
• ORO Oil Red O
• sections were dehydrated and then incubated in a 0.1% LFB solution at 37 0 C overnight. Slides were then cooled at 4°C, incubated in a 0.05% lithium carbonate solution, and cleared in 70% ethanol. Slides were next stained with Cresyl violet, rinsed twice, dehydrated and mounted in Eukitt (O.Kindler Gmbh).
• Sections were pre-incubated in 90% ethanol solution (5 min) for MOG and MBP staining. They were incubated overnight with primary antisera at 4°C, rinsed, and incubated with the appropriate secondary antibodies for 1 hour at 2O 0 C. Sections were counterstained with bisbenzimide (Hoescht 33342, Sigma), washed, and mounted with Fluoromount (Clinisciences). For in vitro experiments, cells were incubated with O4 primary antibody (1 :1 ; ATCC) for 30 min at 20°C. For Ki67 staining (1 :100; BD Pharming ⁇ n), cells were fixed with ice cold 2% PFA, rinsed twice with PBS, and then incubated with the primary antibody.
• cell counts were established on 3 separates experiments. Quantifications are mean + SEM values of the data collected from at least 3 coverslips for each experiment. In order to avoid subjective selection, microscopic fields were randomly selected and at least 500 cells were counted for each coverslip. For in vivo experiments, cell counts were established on a minimum of 3 animals. For each animal, staining and cell counts were made on at least 3 slides containing 15 sections of 12 ⁇ m thickness and covering the cervical, thoracic and lumbar levels of the spinal cord. To limit experimental bias, all slides were stained at the same time and all images were taken with the same acquisition parameters.
• Measurements were performed in the lesions, on images taken at a magnification of 4OX and processed with ImageJ 1.34S, except for NG2 and CC1 immuno-positive cells, which were counted in the margins of the lesions (area comprised between the limits of the lesion and 200 ⁇ m apart). Lesions were identified by the presence of inflammatory foci and by the lack of MBP immunolabelling.
• TFA-12 inhibits astroglial and microglial activation in vitro
• TFA-12 As a first screen of the biological activity of TFA-12, we tested whether this molecule could modulate astroglial and microglial activation induced by LPS treatment (1 ⁇ g/ml and 0.01 ⁇ g/ml for astroglial and microglial cultures, respectively).
• LPS treatment (1 ⁇ g/ml and 0.01 ⁇ g/ml for astroglial and microglial cultures, respectively).
• PCR experiments for GFAP, vimentin and N-Cadherin indicated that TFA-12 reduces the expression of astroglial genes, even in response to LPS activation (Fig. 1a).
• NOSII and TNF ⁇ gene expression were also significantly down- regulated in astrocyte cultures treated with TFA-12.
• TFA-12 is an effective inhibitor of astroglial and microglial activation, and therefore may prevent inflammatory insults in the CNS.
• TFA-12 enhances clinical recovery in experimental autoimmune encephalomyelitis (EAE)
• TFA-12 could have therapeutic effects on the clinical course of MOG-induced EAE in mouse, a chronic inflammatory demyelinating model that mimics important pathological hallmarks of MS lesions.
• inflammation and demyelination occur mainly in the spinal cord, leading to chronic lesions with axonal loss (Hobom et al., 2004).
• mice treated with the vehicle alone showed a clinical course typical of MOG-induced EAE (Fig. 2a).
• Vehicle-treated EAE mice presented clinical scores increasing from day 12 to day 21 post immunization, reaching a mean clinical score of 2.5.
• TFA- 12-treated mice displayed a significant reduction of the EAE disease, as assessed by the mean clinical score which never reached a score greater than 1.5.
• Differences in clinical recovery between vehicle- and TFA-12-treated groups became significant 21 days post-immunization (p ⁇ 0.05) and were reproduced in 3 independent EAE experiments.
• TFA-12 reduces inflammation, astrogliosis and demyelination in EAE disease
• TFA-12-treated mice showed much smaller demyelinated lesions (FIg. 3e) compared to controls. Quantification of the demyelinated lesions, determined as the percentage of MBP- negative areas in the dorsal funiculus, revealed that TFA-12 reduced the extent of demyelination by 4.2 fold (Fig. 3f).
• TFA-12 inhibits both astroglial and microglial activation in vitro.
• spinal cords of both groups for astrocytes and activated microglial cells, using immunohistochemistry for GFAP and F4/80 respectively.
• astrocytes expressed lower levels of GFAP in spinal cord lesions of TFA-12-treated mice compared to controls (Fig. 3g,h).
• Quantification of GFAP immunostaining revealed a 2.4 fold reduction of the reactive astrogliosis, after TFA-12 treatment (Fig. 3i).
• Microglial activation was also strongly attenuated in TFA-12-treated mouse spinal cords, as compared to controls.
• TFA-12 reduces axonal damage and induces oligodendrocyte differentiation in EAE lesions
• TFA-12 could promote remyelination in EAE lesions.
• TFA-12 treatment increased by 1.6 fold the number of mature NG2- /CC1 + oligodendrocytes compared to vehicle-treated mice (Fig. 4f,g,h).
• the number of NG2+/CC1- OPCs, detected in the periphery of lesions was decreased by 1.5 fold in the TFA-12-treated group (Fig. 4i,j,k), attesting that this molecule had a pro-differentiation effect on oligodendroglial cells.
• TFA-12 promotes OPC differentiation through the inhibition of Notch signalling.
• TFA-12 (5.10 "7 M) on CG4 cells in basal medium, using immunocytochemistry for A2B5, a marker of OPCs and 04 which labels pre-oligodendrocytes and mature oligodendrocytes.
• 48 hours treatment with TFA-12 increased by 2.5 fold the number of 04+ oligodendrocytes compared to control cultures (Fig. 5a,b,c).
• the number of GaIC+ differentiated oligodendrocytes was also enhanced in TFA-12 experiments (data not shown).
• CG4 cells were significantly more branched compared to vehicle-treated cells (Fig, 5d,e,f), confirming that this molecule promotes OPC differentiation. As differentiation is often correlated with cell cycle exit, we used Ki67 labelling to quantify proliferation in the CG4 cell cultures. Under TFA- 12 treatment, the number of Ki67+ cells was reduced by 11 fold compared to the controls (Fig. 5g,h,i).
• Notch signalling pathway plays a crucial role in the control of the oligodendrocyte development (Park et al., 2005; Gaiano et al., 2002). Activation of Notch receptors in OPCs was shown to maintain OPCs at a proliferating stage and inhibit their differentiation. Notch activation induces the expression of the downstream effectors Hes1 and Hes5, which down-regulate gene transcription of b-HLH transcription factors such as Mashi . Therefore, we tested whether this compound could modulate Notch signalling in oligodendrocytes.
• MS Multiple sclerosis
• TFA-12 treatment decreases the extent of several deleterious components of EAE, including inflammation, astrogliosis, demyelination and axonal damage. Furthermore, we demonstrate both in vitro and in vivo that TFA- 12 promotes oligodendrocyte precursor differentiation through the inhibition of the Notch pathway. Altogether, these results indicate that TFA-12 has a relevant therapeutic potential for the treatment of diseases associated with an up-regulated Notch signalling pathway activity such as inflammatory demyelinating CNS disorders, MS in particular.
• CG4 oligodendrocyte precursor cell line was cultured on polyornithine- laminine coated dishes and expanded in N1/B104 (70v/30v) medium, as described above (Louis et al., 1992).
• N1/B104 was replaced N1 supplemented with TFA-12 at 5.1 CT 7 M or N1 alone, and with or without the soluble form of the Notch agonist Jaggedi (recombinant rat Jagged1/Fc chimera, R&D Systems Inc). Media were changed daily and differentiation was carried out during 3 days.
• Dose-response curve of the effects of Jagged 1 on CG4 cells was first established in N1 medium with increasing concentration of Jagged 1 /Fc (ranging from 0 to 8 ⁇ g/ml). Jagged 1 /Fc concentration of 4 ⁇ g/ml was found to inhibit the generation of 04+ oligodendrocytes by 5 fold compared to control cells, after 3 days in differentiation condition. This concentration was therefore used in these experiments.
• oligodendrocytes Primary cultures of oligodendrocytes were performed basically as described by Mc Carthy and de Vellis (1980). Briefly, cerebral hemispheres were dissected out from newborn rats, freed of meninges, and dissociated by gentle repetitive pipetting in a mixture of DMEM:F12 (1 :1 ) containing 1 % streptomycin/penicillin, supplemented with 10% fetal calf serum (FCS). The cell suspension was seeded in poly-L-lysine coated flasks. After 14 days in culture, microglia was separated by shaking for 45 min in an orbital shaker at 250 rpm/min, and oligodendrocytes were separated from the astrocytes by continuous shaking overnight at 300 rpm/min.
• FCS fetal calf serum
• the cell suspension obtained was filtered and then centrifuged at 1000 rpm during 5 min.
• the pellet containing the oligodendrocytes was resuspended in N medium containing a 1 :1 mixture of DMEM:F12, glucose 0.6%, insuline 25 ⁇ g/ml, N2 mix 1%, B27 0.5% and HEPES 0.5%, and plated in poly-L-lysine 24-well plates (5x10 4 cells/well)
• the cell cultures were kept for 2 days in N medium in the absence or the presence of 5x10 "7 M TFA-12. Medium was changed every each day. Cells were fixed in PFA 4% and used for morphological and immunocytochemical analysis.
• Frontal slices of postnatal 2 (P2) mice brains were cut at 500 ⁇ m using a vibratome as described in Lavdas et al. (2006).
• the slices were placed in a collagen- coated Millicell-CM culture inserts in 1 ml of medium consisting of a mixture of Neurobasal medium 50%, Hanks' buffered salt solution 25%, glutamine 1 mM, horse serum 25% and glucose 5 mg/ml.
• Brain slices were gradually transferred to N serum- free medium and cultured in the absence or the presence of 5x10 "7 M TFA-12 during 6 days.
• slices were fixed with 4 % paraformaldehyde and washed with PBS. Fixed tissue was cryopreserved, sectioned at 12 ⁇ m with a freezing microtome and used for immunostaining.
• Embryonic human brains are obtained from 7-8 week-old fetuses. Aborted fetuses are collected in hibernation medium following a protocol that is in line with the recommendations of the French bioethic committee (CCNE) and the European legislation.
• CCNE French bioethic committee
• the Student's t-test was used for statistical analysis. Data are expressed as mean ⁇ SEM or percentage of MBP+ cells/mm 2 .
• TFA-12 promotes oligodendrocyte differentiation
• GaIC+ cells a terminal marker of differentiated oligodendrocytes
• N1 medium alone or N1 supplemented with 5.10 "7 M of TFA-12 the number of GaIC+ differentiated oligodendrocytes was significantly increased by TFA- 12 treatment (19% + 0.17) compared to controls cells in N1 medium alone (14% +_ 0.14) ( Figure 6).
• Oligodendrocyte precursors were plated and maintained in culture in the presence of 30% N1 B104-conditioned medium to induce proliferation of the cells. After that, the medium was changed and the cultures were treated with or without 5x10 "7 M TFA-12 during 2 days to assess the effect of TFA-12 on oligodendroglial lineage, lmmunostaining shows the expression of different markers in each experimental condition. As can be seen in Figure 7, TFA-12 treatment induced a significant increase of GaIC+ cells compared to control group.
• TFA-12 treatment promotes myelination in vitro
• Mouse brain slices were used as a more complex model to evaluate the effect of TFA-12 on oligodendrocyte differentiation during development.
• P2 brain slices were transferred to a serum-free medium and cultured for 6 days in the presence or the absence of 5x10 "7 M TFA-12.
• TFA12-treated slices show an increase in the number of double-labelled CC1+/MBP+ cells compared to controls ( Figure 9). Ongoing experiments are being performed to further evaluate the extent of this effect.
• TFA-12 enhances the generation of oligodendrocytes from human neural precursors
• Cells were isolated from ganglionic eminences of a 7.5 week-old human embryo and maintained in culture in the presence of EGF and bFGF. Once the spheres were formed, they were cultured as adherent cells for 5 days with N medium in the presence of EGF and bFGF to allow cells to migrate outside of the spheres. After this, growth factors were removed from the culture and 2x10 "7 M TFA-12 was added during 5 days, lmmunocytochemical staining was performed to evaluate the cell population present around the spheres with and without the treatment.
• FIG. 10a Data show that TFA-12 induced changes in the composition of the population of the spheres compared to control.
• the number of A2B5+ cells after TFA-12 treatment increased significantly respect to the control group ( Figure 10a).
• FIGs 10a-10c show that after 5 days of TFA-12 treatment, there was an increase in the number of A2B5+ cells, Olig2+ cells and double-labelled A2B5+/Olig2+ cells. Since the data for A2B5 and Olig2 suggested a possible increase in neuronal or oligodendroglial population, ⁇ 3-tubulin staining was evaluated, as an indicator of neuronal lineage.
• Figure 10d shows that TFA-12 induced a significant decrease in the number of ⁇ 3-tubulin+ cells, suggesting that this factor is able to induce a pro- oligodendroglial commitment of human neural precursor cells.
• TFA-12 acts as an antagonist the Notch-signaling pathway
• CG4 differentiation assay were carried out in presence of the soluble form of the Notch receptors agonist Jaggedi .
• Activation of the Notch signaling pathway, via the standard Notch ligands Jaggedi or Deltal was previously reported to prevent the differentiation of oligodendrocyte precursor cells into myelinating oligodendrocytes and therefore to maintain these cells in an undifferentiated proliferating stage (Wang et 1998; Genoud et al., 2002).
• CG4 differentiation assays were carry out during 3 days in N1 alone, N1 +Jagged1/Fc (4 ⁇ g/ml), and N1 +Jagged1/Fc (4 ⁇ g/ml) supplemented with 5.10 '7 M of TFA-12. Quantification of the number of 04+ cells, after 3 days of differentiation reveals a significant 2 fold decrease of the number of 04+ oligodendrocytes in N1 +Jagged/Fc, compared to N1 alone, indicating that activation of the Notch signaling in OPCs prevents their differentiation, as previously described.
• Hu, Q. D. et al. F3/contactin acts as a functional ligand for Notch during oligodendrocyte maturation. Ce// 115, 163-75 (2003). John, G. R. et al. Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation. Nat Med 8, 1115-21 (2002).

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