MX2007006796A - Use of selected compounds for protection of neurones and oligodendrocytes in the treatment of multiple sclerosis - Google Patents

Abstract

Certain compounds, including 5,6-dihydro-3,9-dihydroxyindolo[2,1-a-isoquinolin12yl)[4-[2­-(1-piperidinyl) ethoxy]phenyl]- methanone and arzoxifen, are useful for providing protection to oligodendrocytes and neurons of multiple sclerosis patients.

Description

USE OF SELECTED COMPOUNDS FOR THE PROTECTION OF NEURONS AND OLIGODENDOCYTES IN THE TREATMENT OF THE MULTIPLE SCLEROSIS FIELD OF THE INVENTION The present invention relates to methods for treating multiple sclerosis. In particular, the present invention relates to the protection of neurons and / or oligodendrocytes in patients with multiple sclerosis with certain compounds described herein, their isomers, racemates, enantiomers, their salts, and with drugs that contain them.

BACKGROUND OF THE INVENTION Multiple sclerosis (MS) is an autoimmune disease that leads to the loss of CNS myelin (central nervous system), the death of oligodendrocyte-like cells and axonal destruction, causing serious functional defects. MS occurs 2-3 times higher in women than in men (Duquette, et al., 1992. Can. J. Neurol, Sci. 19: 466-71) and estrogens reduce the severity of the disease during the second and third trimesters of pregnancy (Confavreux et al., 1998. N Eng J Med 339: 285-291), while the clinical symptoms of MS have been reported to worsen after administration (Evron et al., 1984 Am. J. Reprod Immunol., 5: 109-1 13; Mertin and Rumjanek 1985. J. Neurol, Sci. 68: 15-24, Grossman, 1989. J. Steroid Biochem. 34: 241-245, Singer et al. al., 1998. N. Engl. J. Med. 339: 285-291). Treatment with estriol reduces gadolinium-enhanced lesions and volume on MRI (Voskuhl and Palaszynski, 2001. Neuroscientist, 7 (3): 258-270, Sicotte et al., 2002. Ann Neurol, 52: 421-428). . In addition, estrogen causes changes in the immune response, alleviation of clinical symptoms and increased myelin formation in rodents with EAE (experimental allergic encephalomyelitis) (Curry and Heim 1966. Nature 81: 1263-1272, Kim et al., 1999 Neurology 52: 1230-1238; Ito et al., 2002. Clin Immunol. 102 (3): 275-282). Estrogens have been reported to protect oligodendrocytes from cell death induced by cytotoxicity (Takao et al., 2004. J Neurochem 89: 660-673) and it has been reported that 17p-estradiol (E2) accelerates the generation of multiple interconnected processes on oligodendrocytes (Zhang et al., 2004. J Neurochem 89: 674-684).

There is growing evidence that estrogens play a direct protective role in response to degenerative disease and injury by increasing cell survival, favoring axonal regrowth, regenerative responses, synaptic transmission, and neurogenesis. In the CNS, there is a greater synthesis of estrogens and a greater expression of estrogen receptors in the sites of injury (García-Segura et al., 2001. Prog. In Neurobiol., 63: 29-60.) And demonstrated estrogen-mediated cellular protection in a series of in vitro models of neurodegeneration, including cite-toxicity, excitotoxicity and ß-amyloid-induced oxidative stress (Behl et al., 1995. Biochem. Biophys. Res. Commun. 216,473-482; Goodman et al., 1996. J. Neurochem 66: 1836-1844, Green et al., 1997. J. Neurosci.17: 51-1 -515; Behl et al., 1999. Trends Pharmacol. Sci. 20: 441 -444). Recent clinical studies suggest that a replacement therapy with estrogens can also decrease the risk and delay the onset and progression of Alzheimer's disease and schizophrenia. (A review can be found in García-Segura et al., 2001. Prog. In Neurobiol 63: 29-60.) E2, a lipophilic hormone that can cross the blood-brain barrier, maintains brain systems that serve the function of awakening, attention, mood and cognitive activity (Lee and McEwan, 2001. Annu. Rev. Pharmacol. &Toxicol 41: 569-591.). In addition, both natural estrogens and synthetic selective estrogen receptor modulators (SERM), such as tamoxifen, reduce neuronal injury caused by ischemic stroke, while either E2 or raloxifene protect neurons against 1-methyl-induced toxicity. 4-phenyl-1, 2,3,6-tetrahydropyridine (Callier, et al., 2001. Synapse 41: 131-138; Dhandapani and Brann, 2003. Endocrine 21: 59-66).

The neuroprotective effects of estrogens are mediated through the modulation of bcl-2 expression, the activation of the c-AMP signaling pathways and of mitogen-activated quinases, the modulation of intracellular calcium homeostasis, the increase of antioxidant activity and / or the activation of estrogen receptors (ER) that can act as hormone-regulated transcription factors (Mangelsdorf, et al., 1995. Cell 83: 835-839; Katzenellenbogen, et al. ., 1996. Mol Endocrinol 10: 1 9-131, Singer et al., 1996. Neurosci Lett 212: 13-16, Singer et al., 1998. Neuroreport 9: 2565-2568, Singer et al. , 1999. Neurosci, Lett, 212: 13-16, Weaver et al., 1997. Brain Res 761: 338-341, Watters and Dorsa, 1998. J. Neurosci 18: 6672-6680, Singh et al. , 1999. J. Neurosci 19: 1 79-188, Alkayed et al., 2001. J. Neurosci 21: 7543-7550, García-Segura et al., 2001. Prog. In Neurobiol 63: 29-60 ). Two characterized estrogen receptors, ERa and ER, belong to the family of hormone receptors of class I that function as nuclear transcription factors. ERa and ERp (in the form of mRNA or protein) are expressed in types of neural cells that include Schwann cells, myelin-forming cells of the peripheral nervous system and neurons of the CNS, astrocytes and oligodendrocytes (Miranda and Toran-Allerand , 1992; Santagati, et al., 1994; Kuiper, et al., 1996; Mosselman, et al., 1996; Thi et al., 1998; Platania, et al., 2003). In oligodendrocytes, CNS myelin-forming cells that are lost in MS, ERa are reported to be nuclear, whereas ERp is cytoplasmic, with in vivo immunoreactivity easily detectable in the cytoplasm and myelin sheaths (Zhang et al. ., 2004. J Neurochem 89: 674- 684). Recently Arvanitis et al., 2004 (J Neurosci Res. 75: 603-613) have reported an ER with similarities with ER in myelin isolated from the CNS, the myelin sheath of the spinal cord and brain sections and in the membrane Plasma of oligodendrocytes.

Imitate and / or enhance the beneficial effects of estrogens in MS by means of small molecules that are ligands in the ERp, or compounds that preferentially mimic the effects of estrogens in sites other than the classical ERa may have advantages for the treatment of MS because the small molecules would be exempt from the unwanted hormonal effects of estrogens which are mediated by ERa. These other ER sites may include newly identified ER-Xs, which have been identified in neurons and is experimentally regulated (Toran-Allerand 2004. Endocrinology 145: 1069-1074), or GPR30, which allows estrogens to trigger different pathways that they integrate the signaling of the cell surface with gene transcription (Kanda and Watanabe 2003. J Invest Derm 121: 771 -780).

These compounds can also be used to treat or prevent the development of other demyelinating diseases, including Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, Guillian-Barre syndrome. , and disorders in which the glial cells that form myelin (oligodendrocytes or Schwann cells) are damaged, including spinal cord injuries, neuropathies, and nerve injuries.

SUMMARY OF THE INVENTION Certain compounds, including 5,6-dihydro-3,9-dihydroxyindolo [2,1-α-isoquinolinyl-2-yl] [4- [2- (1-piperidinyl) ethoxy-phenyl] -methanone and arzoxifen, are useful in providing protection to oligodendrocytes and the neurons of patients with multiple sclerosis.

DETAILED DESCRIPTION OF THE INVENTION The invention also relates to the acid addition salts of the preceding compounds with organic or inorganic acids. Compounds containing one or more asymmetric centers have isomeric forms; these isomers and mixtures form part of the invention. The racemates and enantiomers of these compounds also form part of the invention.

The terms used have the meanings defined in this specification. a) Pharmaceutically acceptable salts "refers to an acid addition salt or base addition salt, it being possible to achieve any of them with the compounds of the present invention.

A "pharmaceutically acceptable acid addition salt" is any non-toxic organic or inorganic acid addition salt of the base compounds represented by Formula I. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric, and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include mono, di and tricarboxylic acids. Illustrative of said acids are, for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymelic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2- phenoxybenzoic acid, p-toluenesulfonic acid and sulphonic acids such as methanesulfonic acid and 2-hydroxyethanesulfonic acid. Salts of mono- or di-acids can be formed, and such salts can exist in hydrated or substantially amorphous form. In general, the acid addition salts of these compounds are more soluble in water and in different hydrophilic organic solvents, and in comparison with their free base forms, they generally show higher melting points.

"Pharmaceutically acceptable base addition salts" refers to non-toxic organic or inorganic base addition salts of the compounds of Formula I. Examples are alkali metal or alkaline earth metal hydroxides such as sodium, potassium, calcium, magnesium or magnesium hydroxides. barium; ammonia, and aliphatic, alicyclic or aromatic amines such as methylamine, trimethylamine and picoline. The selection of the appropriate salt may be important so that the ester is not hydrolyzed. The person skilled in the art will know the selection criteria for the suitable salt.

"Patient" means a warm-blooded animal, such as for example rats, mice, dogs, cats, guinea pigs and primates such as humans.

"Treat" or "treatment" means any treatment including, but not limited to, relief of symptoms, elimination of cessation of symptoms on a temporary and permanent basis, or prevention or slowing of the onset of symptoms and progression of the disorder or condition indicated.

"Therapeutically effective amount" means an amount of the compound that is effective to treat the disorder or condition mentioned.

"Pharmaceutically acceptable carrier" is a solvent, dispersant, excipient, adjuvant or other non-toxic material that is mixed with the compound of the present invention to allow the formation of a pharmaceutical composition, ie, a dosage form that can be administered to the patient . An example of this vehicle is a pharmaceutically acceptable oil which is typically used for parenteral administration. f) "Stereoisomers" is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. This includes isomers that are mirror image (enantiomers), geometric isomers (cis / trans) and isomers of compounds with more than one chiral center that are not mirror images of each other.

To treat a patient suffering from a condition described above, a selected compound may be administered in any form or mode that makes the compound bioavailable in therapeutically effective amounts, including orally, sublingually, buccally, subcutaneously, intramuscularly, intravenously, transdermally , Intranasal, rectal, topical and the like. A person skilled in the art of preparing formulations can determine the proper form and mode of administration, depending on the particular characteristics of the compound selected for the condition or disease to be treated, the stage of the disease, the condition of the patient and others. important circumstances. For example, see Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (1990), incorporated herein by reference.

The compositions of the present invention may be administered orally, for example, in the form of tablets, troches, capsules, elixirs, suspensions, solutions, syrups, wafers, chewing gums and the like, and may contain one or more of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; slip agents such as colloidal silicon dioxide; and sweetening agents such as sucrose or saccharin can be added or a flavoring agent such as peppermint, methyl salicylate or orange flavor. When the dosage unit form is a capsule, it may contain, in addition to the materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil. Other forms of dosage unit may contain various other materials that modify the physical form of the dosage unit, for example, as coatings. Thus, the tablets or pills can be coated with sugar, lacquer or other enteric coating agents. A syrup may contain, in addition to the present compounds, sucrose as a sweetening agent and some preservatives, dyes and flavoring agents.

The compounds of this invention can also be administered topically, and when this is done the vehicle can suitably comprise a solution, ointment or gel base. The base may comprise, for example, one or more petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.

The solutions or suspensions may also include one or more of the following adjuvants: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and tonicity adjusting agents such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials.

The dosage range in which the compounds of the invention exhibit their ability to act therapeutically may vary depending on the particular compound, the severity of the condition, the patient, the formulation, other underlying disease states that the patient suffers from, and other medications that they can be administered concurrently to the patient. Generally, the compound of Formula I will present its therapeutic activities in doses of between about 0.001 mg / kg of body weight of the patient / day to approximately 100 mg / kg of body weight of the patient / day.

The content of all publications and patents described in this document are incorporated by reference.

NEUROPROTECTION TEST Cells from a human neuroblastoma cell line, SK-N-SH, are cultured at 50,000 cells / well in 96-well Costar Biocoat plates coated with poly-D-lysine in EMEM (Eagle's Minimum Essential Medium with Earle salts ) containing penicillin / streptomycin, L-glutamine, sodium pyruvate, non-essential amino acids and sodium bicarbonate. The cells were reproduced overnight in an incubator at 37 ° C in 5% C02. The next day, the medium was removed and replaced by new medium. The cells were previously treated with SERM for 1 hour and SIN-1 (3-morpholino-sidnonimine, which produces peroxynitrite) was added to give a final concentration of 2 or 10mM. After 24 hours, the medium was removed and assayed for its LDH activity using the Promega cytotox 96 assay kit (catalog no. G1780). The results were calculated as the percentage of protection against the SIN-1 toxicity.

WESTERN TRANSFERS OF ERK1 / 2 SK-N-SH cells were cultured at 2 x 106 cells / well in 6-well polystyrene culture plates, in 2 ml of EMEM containing penicillin / streptomycin, L-glutamine, sodium pyruvate, non-essential amino acids and sodium bicarbonate. The cells were reproduced overnight at 37 ° C in 5% C02.

Next, 200μ? of medium and the cells were dosed with 200μ? of compound prepared up to 10 times the final concentration in the medium. After incubating for the appropriate time, the medium was removed by aspiration and the cells were washed twice with cold PBS. These were then lysed with 10 μm? of RIPA buffer containing protease and phosphatase inhibitors.

For Western blots, 20μg of protein was denatured at 95 ° C in Laemmli sample buffer containing beta-mercaptoethanol, then loaded on SDS gels with Tris Glycine and a gradient of 4-20% and electrophoresed at 70 ° C. volts until its completion. The proteins were transferred to nitrocellulose membranes and used as probes for phospho-ERK1 / 2 and total ERK1 / 2 using the appropriate antibodies. The bands were detected using chemiluminescent substrate for Western ECL transfer. For the phospho-ERK ELISA, the Assay Designs ELISA kit was used.

Bcl-2 LUCIFERASA SK-N-MC Bcl-2 (neo) clone 218 was seeded at 25,000 cells per well in Packard View plates in EMEM medium free of phenol red containing penicillin / streptomycin, L-glutamine, sodium pyruvate, non-essential amino acids, sodium bicarbonate and G4 8 200ug / ml. The cells were reproduced overnight in an incubator at 37 ° C in 5% C02.

On day 2, the medium was removed and replaced by serum-free EMEM containing ITS supplement (BD Biosciences No. 35 4352). The medium was loaded again on days 3 and 4; On day 4, the cells were dosed with compounds, in a final volume of 10 μm. Twenty-four hours after dosing, 10 μm were added. of SteadyGlo (Promega # E2510) and the luciferase was measured in a Packard Topcount liquid scintillation counter.

Toxicity test of OLIGODENDROCITOS Primary oligodendrocyte progenitor cells were obtained from rat rats of postnatal rats (Sprague Dawley) 2-3 days old. The meninges were separated and the tissue mechanically dissociated. The cells were spread on sheets on T75 flasks and fed with DMEM + 10% FBS.

Enriched PLOs were collected by mechanical separation of the astrocytic monolayer and expanded in serum-free medium (SFM) supplemented with the mitogens, PDGF-AA (10 ng / mL) and FGF-2 (10 ng / mL).

To generate mature oligodendrocytes, the progenitor cells were changed to SFM supplemented with IGF-1 (10 ng / mL) 24 hours after spreading them on sheets and the cells were grown under these conditions for 7 days before the experimental tests.

The cells were spread on plates in 96-well plates, 10,000 per well. The medium was changed by fresh medium and the cells were pretreated with the compounds for 1 hour. Toxins were added to give the following final concentrations: Sin-1 10mM Pirogalol 500μ? Ceramide C2 100μ? Camptothecin 10μ? After 24 hours, the medium was separated and tested for its LDH activity using the Promega cytotox 96 assay kit (catalog no. G1780). The results were calculated as the percentage of protection against toxicity induced by toxins.

These compounds have been tested for their efficacy in neuroprotection against cell death caused by toxic agents such as SIN-1 (3-morpholino-sidnonimine, which produces peroxynitrite), ceramide C2, camptothecin, staurosporine, SNAP (S-nitroso-N) -acetylpenicillamine, which produces nitric oxide) and pyrogallol (which produces superoxide anion). The target cells tested in vitro are: human neuroblastoma cell lines [SK-N-SH, SH-SY5Y] and primary cultures of rodent oligodendrocyte progenitor cells and their mature counterparts. The protection by these compounds of the SERM type has been compared with that of 17-p-estradiol and tamoxifen. (See Table I below). The mechanism of action of this neuroprotection has been investigated with respect to the use of a classic nuclear ERa or ß (genomic) and an assessment of the function for phosphorylation of MAPK p40 / p42 (ERK1 / 2).

Results PHARMACOLOGICAL COMPOSITION It seems that both compounds tested protect the neurons and oligodendrocytes. It seems that this is mediated by the upregulation of ERK1 / 2 phosphorylation, which is confirmed by the inhibition of neuroprotection by U-0126, a MEK inhibitor, specific for the ERK pathway.

Claims (2)

CLAIMS:

1 .- A method for treating patients with multiple sclerosis by protecting their neurons or oligodendrocytes, which comprises administering to a patient having multiple sclerosis a therapeutically effective amount of a compound selected from the group consisting of 5,6-dihydro-3,9- dihydroxyindolo [2,1-α-isoquinolin-12] -1- [4- [2- (1-piperidinyl) ethoxy-phenyl] -methanone and arzoxifen, its isomers, racemates and enantiomers and the pharmaceutically acceptable salts of said compounds.

2 - . 2 - The method according to claim 1, wherein said effective amount is administered daily and is in the range of about 0.001 to about 100 mg / kg of body weight of the patient / day.