EP2370075A1 - Verwendung von geranyl-geranyl-transferase bei der behandlung von rückenmarksläsionen - Google Patents

Verwendung von geranyl-geranyl-transferase bei der behandlung von rückenmarksläsionen

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Publication number
EP2370075A1
EP2370075A1 EP09799369A EP09799369A EP2370075A1 EP 2370075 A1 EP2370075 A1 EP 2370075A1 EP 09799369 A EP09799369 A EP 09799369A EP 09799369 A EP09799369 A EP 09799369A EP 2370075 A1 EP2370075 A1 EP 2370075A1
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EP
European Patent Office
Prior art keywords
compound
spinal cord
ggti
formula
geranyl
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EP09799369A
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English (en)
French (fr)
Inventor
Isabelle Boquet
Dorothée BUTTIGIEG
Jean-Chrétien NORREEL
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Pharmaxon
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Pharmaxon
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    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4174—Arylalkylimidazoles, e.g. oxymetazolin, naphazoline, miconazole
    • 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

Definitions

  • the present invention relates to compounds of general formula (I).
  • the present invention relates to the use of these compounds as a medicament.
  • the present invention finds particular application in the fields of human and veterinary medicine.
  • references in brackets ([]) refer to the list of references at the end of the text.
  • the medullary lesion causes dysfunction of the voluntary motility (paralysis) or reflex (spasticity), chronic pain, sensory disorders of the superficial type (cutaneous sensations) or deep (position of the body in space), neuro-vegetative disorders (sudden change in temperature or blood pressure), genito- sexual, bladder and intestinal disorders (spasmodic or flaccid sphincters), and respiratory disorders in case of high cervical lesion,
  • the biological response following a spinal cord injury can be divided into 3 stages: an acute, secondary and then chronic phase.
  • the acute phase which begins in the seconds following the trauma, there is a necrosis of the cells in the lesion zone, with the cell contents (ions, glutamate) being spilled into the extracellular medium, which is likely to lead to the death of the cells. other cells.
  • the appearance of small edema due to the rupture of the blood-brain barrier is also observed.
  • the secondary phase that begins within minutes of injury is dominated by lesion extension, immune response, and apoptotic cell death.
  • glial scar mainly composed of astrocytes but also of oligodendrocytes (Fawcett, JW, and Asher, RA (1999), The Brain Research Bulletin 49, 377-391 [1].
  • Astrocytes contribute to the maintenance of the blood-brain barrier that limits the central nervous system. They form a three-dimensional network between neurons and contribute to their proper development and survival. In adults and under normal conditions, astrocytes are immobile, star-shaped, with no preferential orientation in the gray matter where they predominate while they are extended and organized rostro-caudally in the white matter.
  • the astrocytes of the glial scar are characterized by hyperplasia and hypertrophy. There is a reorientation of these to the site of inflammation. Division, elongation and migration of astrocytes to the lesion lead to the formation of the glial scar.
  • Astrocytes will express on their surface highly glycosilated and sulphated proteins, proteoglycans, inhibitory molecules of neuronal regrowth. These hyperactivated cells will form a tight mesh constituting a physical and chemical barrier inhibiting axonal regrowth.
  • Oligodendrocytes, including myelin that surrounds neuronal extensions of a protective sheath, are also found in the glial scar.
  • MAG Myelin Assocoiated Glycoprotein
  • Omgp Oligodendrocyte Myelin Glycoprotein
  • Prenylation is an irreversible post-translational modification consisting of a covalent grafting by thioether bond of an isoprene lipid, famesylpyrophosphate (15 carbon atoms) or geranylgeranylpyrophosphate (20 carbon atoms), on a consensus sequence of the end COOH-terminal protein, usually a GTAPase.
  • the role of prenylation is major because it intervenes in the subcellular localization of proteins and thus in their mode of operation.
  • FTase farnesyl transferase
  • GTTase II geranylgeranyl transferase type II
  • XCC or CXC motifs Zhang, FL, and Casey, PJ (1996) Protein prenylation: molecular mechanisms and functional consequences. Biochemistry 65, 241-269. [2]).
  • the nature of the transferred isoprene depends on the nature of the amino acid X.
  • the FTase reacts with the CAAX sequence when X is for example a serine or a methionine.
  • GGTase I When X is leucine, isoleucine or phenylalanine, the protein is recognized by GGTase I. There are inhibitors of these highly selective enzymes, and FTase inhibitors (lonafarnib, tipifarnib) are involved in clinical trials of treatment of certain cancers in which there is overactivation of the Ras protein.
  • FTase inhibitors lonafarnib, tipifarnib
  • GGtase I the small GTPases of the Rho family (RhoA, RhoC, Rac1, Rac2, cdc42) known for their involvement in survival and cell motility.
  • RhoA protein in particular is a target for the treatment of spinal cord injury (Dergham, P., Ellezam, B., Essagian, C., Avedissian, H., Lubell, WD, and McKerracher, L. (2002). J Neurosci 22, 6570-6577 [3]).
  • Rac1 may be implicated in the persistence of neuropathic pain after spinal cord injury (Tan, AM, Stamboulian, S., Chang, YW, Zhao, P., Hains, AB, Waxman, SG, and Hains, BC (2008) Neuropathic pain memory is maintained by radially-regulated dendritic spine remodeling after spinal cord injury.J Neurosci 28, 13173-13183. [4])
  • chondroitinase ABC partially restores neurological functions (Bradbury, EJ, Moon, LD, Popat , RJ, King, VR, Bennett, GS, Patel, PN, Fawcett, JW, and McMahon, SB (2002), Chondroitinase ABC promoted functional recovery after spinal cord injury, Nature 416, 636-640 [5]).
  • thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury (Proceedings of the National Academy of Sciences of the United States of America [8]).
  • Cell transplants are also contemplated, so oligodendrocyte precursors derived from human embryonic stem cells (hESCs) were grafted into adult rats at different times after spinal cord injury at 7 days or 10 months.
  • the transplanted cells survived and differentiated into oligodendrocytes, migrating a few millimeters and allowed for individuals treated at 7 days remyelination of axons and partial recovery of locomotion (Sharp, J., Frame, J., Siegenthaler, M., Nistor, G., and Keirstead, HS (2009) Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor CeII Transplants Improve Recovery after Cervical Spinal Cord Injury.Stem cells (Dayton, Ohio). [9]).
  • the present invention precisely makes it possible to solve the aforementioned problems and disadvantages of the state of the art by providing compounds of formula (I) and their use as a medicament.
  • the present invention also relates to the use of geranyl-geranyl transferase inhibitors for the treatment of spinal cord lesions.
  • R 1 is hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 6 -C 10 aryl or an alkali metal cation (Na +, K +) :
  • R 2 is a hydrogen atom, an alkyl to C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6, alkoxy Cl to C 6, an aryl C 6 -C 1 0, a halogen atom (F, Cl, Br, I), -OH, -NO 2 , -CN, -CO 2 H, -SO 3 H, -NHNH 2 , -SH, -NH 2 , HO -NH-, isobutyl, secbutyl or benzyl;
  • R 3 and R 4 independently of one another a hydrogen atom, an alkyl to C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6;
  • R5 is five-membered heterocycle or heteroaryl Ci-C 6, for the manufacture of a medicament.
  • the compounds of the invention make it possible to promote axonal regrowth and thus restore nerve connections.
  • the compounds of the invention also make it possible to inhibit the migration of astrocytes at the level of the lesion of the spinal cord.
  • alkyl refers to, for example, a linear or branched, cyclic or acyclic saturated hydrocarbon radical.
  • the alkyl radical may comprise from 1 to 20 carbon atoms, preferably from 1 to 10 carbon atoms, more particularly from 1 to 8 carbon atoms. carbon, even more particularly from 1 to 6 carbon atoms.
  • an alkyl radical may be a methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl or tert-pentyl radical.
  • alkenyl refers to, for example, a linear or branched, cyclic or acyclic unsaturated hydrocarbon radical comprising at least one carbon-carbon double bond.
  • the alkenyl radical may comprise from 2 to 20 carbon atoms, preferably from 2 to 10 carbon atoms, more particularly from 1 to 8 carbon atoms, even more particularly from 2 to 6 carbon atoms.
  • an alkenyl radical may be an allyl, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl or similar radicals.
  • alkynyl refers to, for example, a linear or branched, cyclic or acyclic unsaturated hydrocarbon radical comprising at least one carbon-carbon triple bond.
  • the alkynyl radical may comprise from 2 to 20 carbon atoms, preferably from 2 to 10 carbon atoms, more particularly from 1 to 8 carbon atoms, even more particularly from 2 to 6 carbon atoms.
  • an alkynyl radical may be an ethynyl, 2-propynyl (propargyl), 1-propynyl or similar radicals.
  • alkoxy refers to an alkyl radical, as defined above, attached to the remainder of the molecule via an oxygen atom.
  • the alkyl radical may comprise from 1 to 20 carbon atoms, preferably from 1 to 10 carbon atoms, more particularly from 1 to 8 carbon atoms, even more particularly from 1 to 6 carbon atoms.
  • an alkoxy radical may be methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, n-hexoxy, or a similar radical.
  • aryl refers to, for example, a mono-, bi- or tricyclic hydrocarbon system comprising one, two or three cycles satisfying Hekel's aromaticity rule.
  • an aryl radical may be a phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl or similar group.
  • the aryl radicals may comprise from 6 to 20 carbon atoms, especially from 6 to 14 carbon atoms, more particularly from 6 to 10 carbon atoms.
  • alkylenyl refers to, for example, a divalent, linear or branched, cyclic or acyclic saturated hydrocarbon system.
  • the alkylenyl radical may comprise from 1 to 20 carbon atoms, preferably from 1 to 10 carbon atoms, more particularly from 1 to 8 carbon atoms, even more particularly from 1 to 6 carbon atoms.
  • an alkylenyl radical may be a methylenyl, ethylenyl, n-propylenyl, isopropylenyl, n-butylenyl, n-pentylenyl, n-hexylenyl, or similar radicals.
  • halogen atom refers to, for example, an atom selected from fluorine, chlorine, bromine and iodine.
  • heteroaryl refers to alkyl as defined above comprising a hetero atom selected from sulfur, nitrogen and oxygen.
  • the arylalkyl and alkylaryl radicals may comprise, for example, from 7 to 25 carbon atoms, especially from 7 to 20 carbon atoms and in particular from 7 to 15 carbon atoms.
  • R 5 may be selected from the group consisting of the following heterocycles:
  • n 0, 1 or 2;
  • R 1A is H, C 1-4 alkyl;
  • R 1D represents H or C1-alkyl or a C1-C4 heteroalkyl with at least one heteroatom selected from nitrogen and / or sulfur.
  • R 2 may be selected from the group consisting of isobutyl, secbutyl or benzyl.
  • R 5 may be of formula (XIV), in which R 1A is H, and R 1D is H, or 2-aminoethylthio
  • the compound used can be the compound of formula (I) in which
  • R1 represents H
  • R2 represents isobutyl
  • R3 represents H
  • R4 represents H
  • R5 is of formula VR 1D (XIV) in which R 1A represents H, and
  • R 1D represents H, or the compound used may be the compound of formula (I) in which R 1 represents CH 3 ,
  • R2 represents an isobutyl
  • R3 represents H
  • R4 represents H
  • R5 represents -CH-NH 2 -CH 2 -SH for the manufacture of a medicament.
  • the compound used can be the compound of formula (XXIII)
  • the compound used may be the compound of formula (XXIII).
  • the term "geranyl geranyl transferase inhibitor” is intended to mean all the compounds known to those skilled in the art and / or commercially available; it may be, for example, the compounds described in US Pat. No. 6,693. 123, US 6,627,610, US 6,221,865, US 6,204,293, US 5,965,539 and US 5,789,558.
  • the present invention also relates to the different enantiomers of the abovementioned compounds of the invention.
  • the subject of the present invention is also the various polymorphs of the compounds of the invention.
  • polymorph it is understood crystals of molecules having different physical properties due to the arrangement of molecules in the crystal lattice.
  • drug is understood to mean, for example, any substance or compound having curative and / or preventive properties with regard to pathologies, injuries, trauma, human or animal diseases. It may be for example a pharmaceutical product for human and / or veterinary use.
  • the medicament may be in any form known to those skilled in the art, for example a tablet, capsule, patch, in liquid form, for example a solution suitable for oral administration, intraperitoneal, intravenous, intramuscular, transcutaneous, or intrathecal.
  • the drug may also be in the form of a prodrug.
  • pro-drug is meant a derivative of a compound that comprises an additional moiety that is capable of being detached in vivo from the parent molecule to release the active compound.
  • a prodrug may be an ester that is cleaved in vivo to release the compound.
  • the medicament is a medicament for the treatment of lesions of the central nervous system, in particular for the treatment of spinal cord lesions.
  • cerebral lesion or spinal cord In the present, by lesion of the central nervous system, it is understood cerebral lesion or spinal cord.
  • cerebral lesion it is understood, for example, a traumatic brain injury (traumatic brain injury) or medical (stroke, cerebral compression).
  • traumatic brain injury traumatic brain injury
  • stroke cerebral compression
  • spinal cord injury means, for example, traumatic or medical injury to the spinal cord, ischaemia, degeneration of the spinal cord, e.g. neurodegenerative pathology or cancer. as intraspinal tumors; the lesion may be for example a total section of the spinal cord separating it into two parts, a partial section of the spinal cord or a medullary compression of traumatic or medical origin.
  • pharmaceutically acceptable salts, esters or ester salts means, for example, salts, esters and ester salts of the compound of the invention or any derivative thereof which are effective for example for the treatment of spinal cord injuries.
  • they may be salts, for example, basic or acidic salts.
  • the compounds of the invention may be for example hydrochloride salts, hydrobromide.
  • Pharmaceutically acceptable salts are known to those skilled in the art, for example, they may be pharmaceutically acceptable salts described in SM Berge et al., Describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1 -19 [11].
  • Pharmaceutically acceptable salts of the compounds of the present invention include those derived from bases and organic and / or inorganic acids.
  • Examples of pharmaceutically acceptable, non-toxic salts are, for example, amine group salts formed with an inorganic acid such as hydrochloric acid, bromic acid, phosphoric acid, sulfuric and perchloric acid or with an organic acid.
  • an inorganic acid such as hydrochloric acid, bromic acid, phosphoric acid, sulfuric and perchloric acid or with an organic acid.
  • Other pharmaceutically acceptable salts further include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pect
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to the present invention or a salt thereof and a pharmaceutically acceptable carrier.
  • pharmaceutical composition is understood to mean a liquid composition, an emulsion, an ointment, a mousse, a paste, a scored tablet, a capsule, an effervescent tablet, a dermal patch or an injectable device, without the shape being limiting.
  • pharmaceutical composition is a liquid composition, for example a composition adapted for injection by a syringe, or pump.
  • the term "pharmaceutically acceptable carrier” means an inert diluent agent, such as sorbitol, sugar, mannitol, microcrystalline cellulose, starch, sodium chloride, sodium phosphate, calcium, calcium phosphate, calcium sulphate, lactose, for example lactose monohydrate.
  • the compound of the invention or salt thereof may be used in a concentration of between 1 nM and 10 mM, preferably between 1 .mu.M and 1 mM and even more preferably between 10 .mu.M and 1 mM.
  • the compounds of the present invention in the pharmaceutical composition, may be in racemic form or in enantiomerically enriched or even pure form.
  • the compounds of the present invention or salts thereof can also be used for the treatment of spinal cord injuries.
  • the compounds of the invention may be administered to a subject to be treated, that is to say for example a subject having a spinal cord injury.
  • treatment it is generally understood that the compounds of the invention may be used independently in humans or animals.
  • subject to be treated is meant, for example, a human being or a mammal.
  • it may be a subject who has a traumatic or pathological lesion of the spinal cord.
  • an animal preferably a mammal and even more preferably a human being.
  • animal is understood to mean humans as well as non-human beings, at any stage of their development, including, for example, mammals, birds, reptiles and fish.
  • the non-human being may be a mammal, for example a rodent, a mouse, a rat, a monkey, a dog, a cat, a sheep, cattle, a primate or a pig.
  • the animal may also be, for example, a transgenic animal or a human clone.
  • the compounds of the present invention may be administered, for example one to three times a day, once every two days, once every three days, four days, five days, six days or once a week.
  • the compounds of the present invention are administered orally, intraperitoneally, intravenously, intramuscularly, transdermally, or intrathecally.
  • the compound of the present invention can be obtained by any chemical synthesis method known to those skilled in the art.
  • FIG. 1 shows schematically the technique of the test injury ("scratch assay"). It represents a diagram of a monolayer of astrocytes in culture (A). A wound (z) is mechanically performed in the monolayer (B), which will close in time by recolonization of the area by the cells (scratch assay) (C).
  • FIG. 1 Photograph of a well (p) of 96-well culture plate (pc) not shown, representing the labeling of cells with calcein (fluorescent marker of living cells). The wound (z) is visualized by a black surface devoid of cells.
  • 3 photographs of 3 wells of a 96-well plate showing 3 treatment conditions: a negative control well treated with the carrier solution or with an inactive compound in which no residual streak is detected (A), a well treated by a cell migration blocking compound (C), a well treated with a toxic compound, which generates a surface greater than the starting surface, due to cell death.
  • FIG. 4 shows curves showing the concentration corresponding to 50% inhibition of migration (IC50) of simvastatin (A) and lovastatin (B) on the astrocyte migration test.
  • the abscissa represents the logarithm of the molar concentration and ordered the percentage of molar inhibition.
  • FIG. 5 shows the route of synthesis of cholesterol.
  • Statins act upstream by inhibiting the synthesis of L-mevalonate by HMG-CoA reductase. From the mevalonate are produced the prenatal residues geranylgeranyl-pyrophosphate and farnesyl-pyrophosphate which will be anchored on target proteins via geranylgeranyl transferases or farnesyl transferase.
  • - Figure 6 is a bar graph showing the inhibition of astrocyte migration in culture in the test "scratch assay" by the compound of formula (XXIV) (GGTI-298) at different concentrations. The effect is dependent on the concentration of the GGTI-298 compound. The positive control is lovastatin. The ordinate represents the inhibition of migration and the abscissa the culture conditions that is to say the test compound and its concentration.
  • FIG. 7 is a bar graph showing the inhibition of astrocyte migration in culture in the "scratch assay" test by the compound of formula (XXIII) (GGTI-2133). The effect is dependent on the concentration of the compound.
  • the positive control is lovastatin.
  • the ordinate represents the inhibition of migration and the abscissa the culture conditions that is to say the test compound and its concentration.
  • FIG. 8 is a curve for determining the IC50 of compound GGTI-2133 in the scratch assay model.
  • the abscissa represents the decimal logarithm of the molar concentration and the ordered the percent inhibition.
  • FIG. 9 is a bar graph showing the percentage of neural shoot as a function of the concentration of GGTI-2133. This compound stimulates the neuritic growth of cortical neurons cultured on an inhibitory substrate composed of myelin. Stimulation of neuritic growth is 20% at the concentration of 15 ⁇ M of GGTI-2133 in the culture medium.
  • FIG. 10 is a bar graph representing the percentage of neuritic (ordered) growth as a function of the concentration of GGTI-2133
  • GGTI-2133 stimulates neuritic growth of cortical neurons cultured on a permissive substrate composed of poly-D-Lysine (PDL). However, this effect of GGTI-2133 is weaker than the effect of the observed compound on neurons cultured on an inhibitory substrate.
  • 11 is a bar graph showing the effect of GGTI-2133 on cell survival. Cortical neurons grown on a permissive substrate (PDL) are treated with different concentrations of GGTI-2133 (abscissa). A slight positive effect is observed on the survival of GGTI-2133 cells. In culture conditions on inhibitory substrate (myelin) no effect of the compound on the survival of neurons is observed.
  • the abscissa represents the culture conditions that is to say the test compound and its concentration and ordered the percentage of the number of cells.
  • the purpose of this example was to identify novel compounds that can decrease the density of the glial scar following spinal cord injury.
  • an in vitro model of astrocyte migration was used: the colonization of an injury performed in a mat of confluent astrocytic cells (scratch assay). Small commercial molecules (sigma-aldrich) were tested to identify candidates with an inhibitory effect on astrocyte migration.
  • Tissues are enzymatically dissociated in 9 mL of Trypsin-EDTA (Gibco Trade Mark) 0.5% diluted
  • DMEM Dulbecco's Modified Eagle's Medium
  • Complete DMEM 1 mM sodium / pyruvate (Gibco brand filed), 3mM penicillin (Sigma trademark) and 0.8mM streptomycin (Sigma trademark) (hereinafter referred to as "Complete DMEM").
  • the mixture was incubated at 37 ° C for 10 min and then an additional 10 min at 37 ° C in the presence of DNAse (1 mg / mL, Sigma trademark) to remove cell DNA released during trypsin treatment.
  • the cell suspension was then centrifuged for 1 min at 800 rpm.
  • the pellet was taken up in 5 mL of complete DMEM.
  • the suspension is sieved to remove cell clumps (70 ⁇ m, BD Falcon) and washed with about 20 ml of complete DMEM.
  • After centrifugation for 5 minutes at 800 rpm the pellet was taken up in 24 ml of complete DMEM and then placed in 3 dishes of 75 cm 2 (Techno Plastic AG Switzerland registered trademark) due to 8 ml per dish.
  • the medium was renewed after 24 hours of incubation to remove the dead cells and then performed every 48 hours until reaching confluence (after about a week).
  • the cultures were then stirred for 12 hours at 250 rpm (Heidolph Titramax 100 shaker) at room temperature and then re-seeded in 8 dishes and incubated at 37 ° C., 5% CO 2 'at confluence.
  • the cell purity was evaluated at 95% (immunocytochemical labeling with an antibody directed against the GFAP protein).
  • the wells of a 96-well plate were previously incubated for 1 h at 37 ° C. with 50 ⁇ l of poly-D-lysine (10 ⁇ g / ml, Sigma registered trademark). ). The wells were then rinsed three times with 100 ml of filtered distilled water and dried. Each well was inoculated with 20,000 cells from a confluent primary culture of astrocytes. For this, the cells were resuspended with trypsin, rinsed and counted in the Malassez cell (Microgravure Preciss registered trademark) and 100 ⁇ L a cell suspension at 200,000 cells / mL, were deposited in each well.
  • Malassez cell Microgravure Preciss registered trademark
  • the culture medium used was Minimum Essential Medium (MEM, Invitrogen trademark, ref 11090-081) supplemented with 10% fetal calf serum (Gibco trademark), 1mM sodium / pyruvate (Gibco registered trademark), 2mM of glutamine (Sigma trademark), 3mM penicillin (Sigma trademark) and 0.8mM streptomycin (Sigma trademark).
  • MEM Minimum Essential Medium
  • the use of MEM rather than DMEM is justified by a lower concentration of fluorescent elements, which reduces the background noise during acquisition with the flash cytometer.
  • the culture was automated using a TECAN Miniprep 75/2 robot.
  • the cells thus deposited in the plates were incubated at 37 ° C., 5% CO 2 for 24 hours before depositing the compounds to be tested.
  • the molecules were tested at different concentrations. Each final concentration is obtained by depositing in the wells 2 .mu.l of a stock solution of the compound prepared in 25% DMSO. The compounds are distributed in an automated manner by a TECAN Miniprep 75/2 robot in the wells containing 100 ⁇ l of culture medium. The final concentration of DMSO is thus identical for each condition and equal to 0.5%. In each plate columns 1 and 12 are systematically reserved for the negative control (DMSO 0.5%).
  • a "scratch" or injury
  • a brush provided with 96 metal peaks (VP scientific; ref VP408FH).
  • 3 passes with the brush were performed ( Figure 1).
  • Figure 2 shows the appearance of the well just after the injury was created and the calcein staining. The scratch appears as a black surface devoid of cells.
  • the effect of test compounds on astrocyte migration is measured by calculating the area that was not recolonized by the cells after 3 days of incubation (J3), (black surface). This parameter is measured by the program in an automated way for all the wells of each plate. Thus, the larger the area of the lesion on day 3, the more the compound inhibits migration. This program also makes it possible to discriminate between toxic compounds (the surface at J3 is greater than the area at OJ) of those blocking migration or having no effect ( Figure 3).
  • statins (simvastatin, fluvastatin, lovasatin) were selected for their inhibitory effect on astrocyte migration.
  • the IC50 inhibition of astrocyte migration of simvastatin and lovastatin is shown in Figure 4.
  • Statins inhibit cholesterol synthesis, by acting on I 1 HMG-CoA reductase, an enzyme that transforms 3- hydroxy-3-methylglutaryl-CoA at L-mevalonate.
  • RhoA protein in particular is a therapeutic target known in the context of the medullary lesion (cethrin / Ba-210, Alseres therapeutics).
  • the inventors of the present invention sought to determine whether the inhibitory effect of statins on astrocyte migration was due to indirect inhibition of GTPase prenylation.
  • inhibitors of the prenylation of Rho proteins, Rac1, cdc42, GGTase type I inhibitors, on the astrocyte migration test (scratch assay) were tested.
  • Example 2 Effect of inhibitors of geranylgeranyl transferase type I on astrocyte migration in vitro.
  • the purpose of this example was to determine the action of a type of prenylation inhibitors on astrocyte migration in vitro.
  • the targeted enzyme here is geranyl geranyl transferase type I (GGTase I), inhibited by GGTI-298 compound, C 27 H 33 N 3 O 3 SC 2 HF 3 O 2 and GGTI-2133, C 27 H 2B N 4 O 3 -C 2 HF 3 O 2 , (Sigma trademark).
  • the compounds were tested in primary astrocyte cultures at several concentrations ranging from 10 ⁇ M to 0.5 nM, in particular 10 ⁇ M, 5 ⁇ M, 50 ⁇ M, 5 nm and 0.5 nM (FIGS. 6, 7) on the model.
  • astrocyte migration according to the protocol described in Example 1.
  • Lovastatin 10 .mu.M and 1 .mu.M (Sigma) was used as a positive control.
  • Astrocyte culture in 96-well plates The protocol used is identical to that described in Part A-2 of Example I.
  • FIGS. 6 and 7 show the percentage inhibition of astrocyte migration relative to the negative control well after three days of incubation as a function of the treatment administered, that is to say, respectively test of the compound of formula XXIV (GGTI- 298) and the compound of formula XXIII (GGTI-2133) at the following different concentrations: 10 ⁇ M, 5 ⁇ M, 50 ⁇ M, 5 ⁇ M and 5 nM for compound GGTI-298 and 100 ⁇ M, 50 ⁇ M, 10 ⁇ M, 5 ⁇ M, 50 ⁇ M , 5OnM and 5 nM for the compound GGTI-2133.
  • Figure 6 shows the results obtained with GGTI-298.
  • this figure represents the percentage inhibition as a function of the concentration of GGTI-298 in the culture medium.
  • the different concentrations tested were 0 ⁇ M, 5 ⁇ M, 50 ⁇ M, 5 nM and 0.5 nM.
  • the GGtase type I inhibitor used showed a classical dose effect with a significant inhibition efficiency of cell migration compared with the negative control, lovastatin at 10 ⁇ M. It is equivalent to that of lovastatin at a concentration of 1 ⁇ M ( Figure 6).
  • the inventors have thus shown here that the compound GGTI-298 inhibitor of GGTase I is capable of mimicking the effect of lovastatin on the astrocyte migration test. Therefore, it appears that the effect of lovastatin on astrocyte migration is at least partially due to an inhibition of the prenylation of GGTase type I target GTPases.
  • Mevalonate sensitizes the nociceptive transmission in the mouse spinal cord. Bread 134, 285-292. [13]). It has been shown in this paper that a local application on the spinal cord of GGTI-2133 was only able to inhibit mevalonate-induced hyperalgesia.
  • Figure 7 shows the results obtained with GGTI-2133.
  • this figure represents the percentage inhibition as a function of the concentration of GGTI-2133 in the culture medium.
  • the different concentrations tested were 100 ⁇ M, 50 ⁇ M, 25 ⁇ M, 10 ⁇ M, 5 ⁇ M, 50 ⁇ M and 5 nm.
  • Lovastatin corresponds to the negative control and was used in a concentration of 10 .mu.M and 1 .mu.M.
  • GGTI-2133 at the various concentrations tested inhibited astrocyte cell migration in vitro (FIG. 7).
  • the inventors have also surprisingly demonstrated that GGTI-2133 has a similar action to the compound GGTI-298 and that it is not toxic on astrocytes even at high concentration (100 ⁇ M and 50 ⁇ M).
  • Figure 8 shows the inhibition curve of the concentration of astrocyte migration as a function of the decimal logarithm of the concentration of the test compound. From this curve, the 50% inhibition concentration of the astrocyte migration (IC50) of the GGTI-2133 compound was evaluated at approximately 30 ⁇ M (FIG. 8), according to the protocol described in the Yung-Chi Cheng document. , William H prussof: Relationship between the constant inhibition and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction - Biocher. Pharmacol (Basso, D.M., Beattie, M.S., and Bresnahan, J.C. (1995), A sensitive and reliable locomotor rating scale for open field testing in rats, Journal of Neurotrauma 12, 1-21 [14]).
  • Example 3 Analysis of the ability of the GGTI-2133 compound to promote neuritic growth of neurons cultured on an inhibitory substrate (myelin) and cellular toxicity.
  • the homogenization buffer is composed of 1 mM Tris-HCl, 1 1.5 mM CaCl 2, 1 mM spermidine (Sigma), aprotinin 25 ⁇ g / ml (Sigma trademark), leupeptin 25 ⁇ g / ml (Sigma registered trademark), pepstatin At 5 ⁇ g / ml (Sigma trademark), 2.3 dehydro 2-desoxy-N-acetylneuraminic acid 15 ⁇ g / ml (Sigma trademark), sucrose pH 7.4.
  • the proteins contained in the homogenization buffer are protease inhibitors, with the exception of the 2,3 dehydro-2-desoxy-N-acetylneuraminic which protects the sugar groups of the proteins.
  • the homogenate was centrifuged at 500g for 10 min to remove the waste and the cores, denser than the other constituents. The supernatant was recovered and then separated on a three-phase sucrose gradient (19%, 25.5% and 35.5%) in an ultracentrifuge for 3h, 54000g at 4 ° C to separate plasma membranes from myelin. The fraction containing myelin corresponding to interphase 19 / 25.5% sucrose was washed several times and then removed. For each preparation, the amount of myelin to be used to have the desired inhibitory properties on the neurite shoot has been defined.
  • the 96-well plates were coated with Poly-D-Lysine for 2 hours at 37 ° C. and then 40 ⁇ l of the myelin preparation were deposited per well. The plates were allowed to dry under host for 12 hours.
  • a pregnant mouse (January) was euthanized by elongation of the cervical vertebrae and the 15.5 day embryos were removed from the uterus and placed in cold HBSS. After extracting the embryos from their amniotic pocket, the brains were recovered and dissected under a binocular loupe. The anterior part of the brain was cut off and the two hemispheres were separated, and the meninges removed. The cortex was dissected and placed in a 15 mL falcon (Trade Mark) tube containing HBSS and kept cold; At the end of the dissection, HBSS was replaced with 1 ml of 5 mg / mL trypsin (sigma trademark) to dissociate the tissues.
  • falcon Trade Mark
  • FIG. 9 represents the percentage of neuritic growth obtained in the presence of myelin and in the presence of myelin and of GGTI-2133 compound at the following concentrations: 15 ⁇ M, 10 ⁇ M and 5 ⁇ M.
  • the negative control corresponds to myelin alone and the positive control to the presence of a permissive subtrate Poly-D-lysine (PDL).
  • FIG. 10 represents the percentage of neuritic growth obtained in the absence of myelin and in the presence of myelin and of the compound GGTI-2133 at the following concentrations: 15 ⁇ M, 10 ⁇ M and 5 ⁇ M.
  • the results obtained by the inventors and represented in FIGS. 9 and 10 clearly demonstrate that GGTI-2133 makes it possible to stimulate neurite growth in the presence of myelin (FIG. 9) more significantly than on a permissive substrate (Poly-D-lysine). , Figure 9). This clearly demonstrates that at least one aspect of this stimulation is a response of neurons specific to inhibitory signals.
  • the compound GGTI-2133 exhibits no cellular toxicity and this surprisingly compared with the statins which they are at the concentrations used, on cortical neurons. Indeed, the inventors have demonstrated that regardless of the concentration of compound GGTI-2133, namely 15 ⁇ M, 10 ⁇ M or 5 ⁇ M, no toxicity has been observed on the cells as shown in FIG. 11 represents the percentage of the number of cells present in the culture as a function of the presence in the culture medium of GGTI-2133 at a concentration of 15 ⁇ M, 10 ⁇ M and 5 ⁇ M alone or in the presence of myelin.
  • GGTI-2133 is a compound that can be used according to the present invention for the treatment of the spinal cord because of its safety on this tissue, its inhibitory action on the model of formation of the glial scar (astrocyte migration) its stimulating effect on a model of neuronal regeneration in an inhibitory environment (neuronal growth on myelin).
  • geranylgeranyl transferase type I inhibitors could be used in the treatment of lesions of the central nervous system, in particular the lesions of the spinal cord.
  • the compounds of the present invention may be formulated in a liquid composition adapted for intramedullary administration by means of a syringe, for example in a vehicle solution composed of 0.9% NaCl, 0.5% to 20% DMSO, or 0.9% NaCl, 5% DMSO, 5% Cremophore or any other animal or vegetable oil of the compound of formula XXIII.
  • Example 5 Treatment of rats having suffered a spinal cord injury.
  • the lesion causes a loss of rat motility in the hind limbs.
  • the study of post-lesional motor recovery is performed by the BBB test at 1, 7, 14, 21, 28 and 35 days postoperatively. This motor test was developed by Drs. Basso, Beattie and Breshnahan at Ohio State University (Basso, DM, Beattie, MS, and Bresnahan, JC (1995).) A sensitive and reliable locomotor rating scale for open field testing in rats, Journal of Neurotrauma 12, 1-21 [14]).
  • This scoring system makes it possible to precisely analyze the voluntary movements of the hind legs of the animal.
  • the rat is placed in a standardized open field of
  • This score takes into account the mobility of the hip, knee and ankle joints, the weight support, the position of the steps during walking and their coordination, and the position of the tail.
  • Rho signaling pathway targeted to promote spinal cord repair. J Neurosci 22, 6570-6577.
  • Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416, 636-640
  • thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury. Proceedings of the National Academy of Sciences of the United States of America.

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EP09799369A 2008-12-05 2009-12-04 Verwendung von geranyl-geranyl-transferase bei der behandlung von rückenmarksläsionen Withdrawn EP2370075A1 (de)

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US6627610B1 (en) 1992-05-29 2003-09-30 Jeffrey Glenn Method for inhibition of viral morphogenesis
US5965539A (en) 1993-05-18 1999-10-12 Univeristy Of Pittsburgh Inhibitors of prenyl transferases
US5789558A (en) 1994-01-31 1998-08-04 Merck & Co., Inc. Protein prenyltransferase
US6693123B2 (en) 1995-11-06 2004-02-17 University Of Pittsburgh Inhibitors of protein isoprenyl transferases
US6221865B1 (en) 1995-11-06 2001-04-24 University Of Pittsburgh Inhibitors of protein isoprenyl transferases
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