HK1111343A - Use of selected compounds for protection of neurones and oligodendrocytes in the treatment of multiple sclerosis - Google Patents

Description

Use of selected compounds for protecting neurons and oligodendrocytes in the treatment of multiple sclerosis

Technical Field

The present invention relates to methods of treating multiple sclerosis. In particular, the present invention relates to the use of certain compounds disclosed herein, and their isomers, racemates, enantiomers, salts thereof and medicaments containing them, for protecting neurons and/or oligodendrocytes in patients with multiple sclerosis.

Background

Multiple Sclerosis (MS) is an autoimmune disease that results in Central Nervous System (CNS) myelin loss, oligodendrocyte death, and axonal destruction, leading to serious functional deficits. MS is more frequently found in women than in men 2 to 3 times (Duquette et al, 1992.Can. J. Neurol. Sci.19: 466-71.), estrogen reduces the severity of the disease in the third and last trimester of pregnancy (Confavreux et al, 1998.N Eng J Med 339: 285-291), but exacerbation of clinical symptoms of MS after delivery has been reported (Evron et al, 1984.am. J. reprod. Immunol.5: 109-113; Mertin and Rumjanek, 1985, J. Neurol Sci.68: 15-24; Grossman, 1989.J. Steroid. Bio34: 241 Med 245; Confavreux et al, 1998, N. Engl. J. 339: 285: 291). Treatment with estriol reduced gadolinium-enhanced lesion lesions and Magnetic Resonance Imaging (MRI) volume (Voskuhl and Palaszynski, 2001, neuroscientist.7 (3): 258-. Furthermore, estrogens cause shifts in immune response, improved clinical symptoms and increased myelin formation in rodent EAE (Experimental allergic encephalomyelitis) models (Curry and Heim, 1966, Nature 81: 1263-. It has been reported that estrogens protect oligodendrocytes from cytotoxicity induced cell death (Takao et al, 2004.J. neurochem.89: 660-673); 17 β -estradiol (E2) was reported to promote multiple, interrelated processes on oligodendrocytes (Zhang et al, 2004, J.neurohem.89: 674-684).

There is increasing evidence that estrogens play a direct protective role in response to degenerative diseases and damage caused by enhancing cell survival, axon growth, regenerative responses, synaptic transmission, and neurogenesis. Within the central nervous system, estrogen synthesis is increased and estrogen receptor expression is enhanced at the site of injury (Garcia-Segura et al, 2001, prog.in Neurobiol.63: 29-60); estrogen-mediated cytoprotective effects have been identified in a number of in vitro models of neurodegeneration, including β -amyloid-induced cytotoxicity, excitotoxicity, and oxidative stress (Behl et al, 1995, biochem. Biophys. Res. Commun.216, 473-482; Goodman et al, 1996. Neurochem.66: 1836-1844; Green et al, 1997, J.Neurosci.17: 511-515; Behl et al, 1999.Trends Pharmacol.Sci.20: 441-444). Recent clinical studies have shown that estrogen replacement therapy may also reduce risk and delay the onset and progression of alzheimer's disease and schizophrenia. (for review see Garcia-Segura et al, 2001, prog. in Neurobiol.63: 29-60). The blood brain barrier crossing lipophilic hormone E2 maintains the arousal, attention, mood and cognitive abilities of the brain system (sub-serving) (Lee and McEwan, 2001.annu. rev. pharmacol. & toxicol.41: 569-. In addition, both natural estrogens and synthetic Selective Estrogen Receptor Modulators (SERMs), such as tamoxifen, reduce neuronal damage caused by ischemic stroke, while E2 or raloxifene protect neurons against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced toxicity (Callier et al, 2001, Synapse 41: 131-.

The neuroprotective effect of estrogen is mediated by the regulation of bcl-2 expression, activation of the cAMP and mitogen activated kinase signaling pathways, regulation of intracellular calcium homeostasis, enhancement of antioxidant activity and/or activation of Estrogen Receptors (ER) as hormone-regulated transcription factors (Mangelsdorf et al, 1995.Cell 83: 835-839; Katzenllenbogen et al, 1996.mol. Endocrinol.10: 119-131; Singer et al, 1996.Neurosci. Lett.212: 13-16; Singer et al, 1998.Neuroreport 9: 2565-2568; Singer et al, 1999. Neurosci.667212: 13-16; Weaver et al, 1997.Brain Res.761: 338-341; ttters and Dorsa, 1998.J. Neurosci.667.2001: 2001-16; Weiber et al, 1997. Brachy. J. Ser. 19: 7563; Neurospora. J. 10: 759; Neurospora. J. 10-19; Neurospora. 10. Alurospora. K.1999). Both identified estrogen receptors, ER α and ER β, belong to the class I hormone receptor family, which functions as nuclear transcription factors. ER α and ER β (in mRNA or protein form) are expressed in a variety of neural cells, including Schwann cells (i.e., myelin-forming cells of the peripheral nervous system), central nervous system neurons, 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 (i.e., myelin-forming cells in the central nervous system that are lost in MS), ER α is reported to be nuclear and ER β is cytoplasmic, and in vivo immunoreactivity within the cytoplasm and myelin sheath can be readily detected (Zhang et al, 2004.J neurohem 89: 674-. Recently, Arvanitis et al, 2004(J Neurosci Res.75: 603-613) reported ER-like ER β in the cell membranes of isolated CNS myelin, myelin sheath of the spinal cord and brain, and oligodendrocytes.

Mimicking and/or enhancing the beneficial effects of estrogen in MS by means of small molecules that are ligands of ER β or compounds that preferentially mimic the effects of estrogen at sites other than the classical ER α may be beneficial in the treatment of MS, as small molecules will avoid the undesirable "hormonal" effects of estrogen mediated by ER α. These other ER sites can include the recently identified ER-X, which is identified in neurons and is developmentally regulated (Toran-Allerand 2004, Endocrinology 145: 1069-1074); or GPR30, which allows estrogens to trigger different pathways for integration of cell surface signaling with gene transcription (Kanda and Watanabe 2003, J.invest.Derm.121: 771-780).

These compounds are also useful in the treatment or prevention of other demyelinating diseases, including sand-horse-picture disease, peyer's disease, encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, guillain-barre syndrome, and diseases in which myelin forming glial cells (oligodendrocytes or schwann cells) are destroyed, including the development of spinal cord injury, neuropathy, and nerve injury.

Summary of The Invention

Certain compounds, including 5, 6-dihydro-3, 9-dihydroxyindolo [2, 1-a ] isoquinolin-12-yl) [4- [2- (1-piperidinyl) ethoxy ] phenyl ] -methanone and azoxifene, are useful in providing protection to oligodendrocytes and neurons in multiple sclerosis patients.

Detailed Description

The invention also relates to an addition salt formed by the compound and inorganic acid or organic acid.

Compounds containing one or more asymmetric centers have isomeric forms; these isomers and mixtures form part of the present invention. Racemates and enantiomers of these compounds also form part of the present invention.

The terms used herein have the meanings defined in the specification.

a) "pharmaceutically acceptable salt" refers to any acid addition salt or base addition salt that may be prepared with the compounds of the present invention.

"pharmaceutically acceptable acid addition salts" refers to any non-toxic organic or inorganic acid addition salts of basic compounds of formula I. Illustrative inorganic acids which can form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids and acid metal salts such as disodium hydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which can form suitable salts include mono-, di-and tri-carboxylic acids. Illustrative of such acids are acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic, p-toluenesulfonic acid, and sulfonic acids such as methanesulfonic and 2-hydroxyethanesulfonic acid. Salts of mono-or dibasic acids may be formed, and such salts may exist in either hydrated or substantially anhydrous form. In general, the acid addition salts of these compounds are relatively soluble in water and various hydrophilic organic solvents and generally exhibit higher melting points than their free base forms.

"pharmaceutically acceptable base addition salts" means non-toxic organic or inorganic base addition salts of the compounds of formula (I). Examples thereof are alkali metal or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide or barium hydroxide; ammonia and aliphatic, alicyclic or aromatic organic amines, such as methylamine, trimethylamine and picoline. It may be important to select the appropriate salt so that the ester does not hydrolyze. Selection criteria for appropriate salts are known to those skilled in the art.

b) "patient" refers to warm-blooded animals such as rats, mice, dogs, cats, guinea pigs, and primates such as humans.

c) "treatment" refers to any form of treatment, including but not limited to, temporary or permanent alleviation of symptoms, elimination of causes of symptoms, or prevention or slowing of the appearance of symptoms and progression of the disease or disorder.

d) "therapeutically effective amount" refers to an amount of a compound effective to treat the disease or condition.

e) A "pharmaceutically acceptable carrier" is a non-toxic solvent, dispersant, excipient, adjuvant, or other material that is mixed with the compounds of the present invention to form a pharmaceutical composition, i.e., a dosage form capable of administration to a patient. An example of such a carrier is a pharmaceutically acceptable oil commonly used for parenteral administration.

f) "stereoisomers" is a generic term for all isomers of individual molecules that differ only in the spatial orientation of the atoms. It includes mirror image isomers (enantiomers), geometric isomers (cis or trans), and isomers of compounds having more than one chiral center that are not mirror images of each other (diastereomers).

In treating a patient suffering from one of the conditions described above, the selected compound may be administered in any form or mode that allows a therapeutically effective amount of the compound to be bioavailable, including orally, sublingually, buccally, subcutaneously, intramuscularly, intravenously, transdermally, intranasally, rectally, topically, etc. One skilled in the art of pharmacy will be able to determine the appropriate form and mode of administration depending on the particular nature of the compound selected for the condition or disease being treated, the stage of the disease, the condition of the patient, and other relevant circumstances. See, for example, Remington's Pharmaceutical Sciences, 18 th edition, Mack publishing company (1990), which is incorporated herein by reference.

The pharmaceutical compositions of the present invention may be administered orally, e.g., 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: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose; disintegrating agents, such as alginic acid, Primogel or corn starch, and the like; lubricants, such as magnesium stearate or Sterotex; glidants, such as colloidal silicon dioxide; and sweeteners, such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil. Other unit dosage forms may also contain other various materials which modify the physical form of the dosage unit, for example, coatings. Thus, tablets or pills may be coated with sugar, shellac, or other enteric coating material. Syrups may contain, in addition to the compounds of the invention, sucrose as a sweetening agent and certain preservatives, dyes, colors and flavors.

The compounds of the invention may also be administered topically, in which case the carrier may suitably comprise a solution, ointment or gel base. The base may, for example, comprise one or more of petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.

The solution or suspension may also contain one or more of the following adjuvants: sterile diluents such as water for injection, physiological saline, non-volatile 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 acetate, citrate or phosphate; and substances for regulating the osmotic pressure, such as sodium chloride or glucose. The parenteral preparation may be enclosed in ampoules, disposable syringes or multi-dose vials.

The dosage range at which a compound of the invention will exhibit its therapeutic ability will vary depending on the particular compound, the severity of the condition, the patient, the formulation, other underlying disease from which the patient is suffering, and other drugs which may be co-administered to the patient. In general, the compounds of formula (I) will exhibit their therapeutic activity at a dosage range of from about 0.001mg/kg patient body weight/day to about 100mg/kg patient body weight/day.

The contents of all publications and patents discussed herein are hereby incorporated by reference.

Neuroprotective assay

SK-N-SH cells from a human neuroblastoma cell line were plated at 50,000 cells/well on poly-D-lysine coated plates in Costar Biocoat 96-well medium containing penicillin/streptomycin, L-glutamine, sodium pyruvate, non-essential amino acidsAnd sodium bicarbonate EMEM (Eagle minimal essential medium with Earle salts). Cells in 5% CO₂Was cultured overnight in a 37 ℃ incubator. The next day, the medium was removed and replaced with fresh medium. Cells were pretreated with SERM for 1 hour and SIN-1 (3-morpholino sydnimine (sydnonimine) was added to generate peroxynitrite ions) to give a final concentration of 2 or 10 mM. After 24 hours, the medium was removed and assayed for LDH activity using the Promega cytox 96 kit (catalog No. G1780). The results were calculated as% protection against SIN-1 toxicity.

ERK1/2 WESTERNS blotting

SK-N-SH cells are arranged according to a 2X 10⁶The cells/well were plated on 6-well polystyrene plates in 2ml EMEM containing penicillin/streptomycin, L-glutamine, sodium pyruvate, non-essential amino acids and sodium bicarbonate. Cells were in 5% CO₂And cultured overnight at 37 ℃.

The following day, 200. mu.l of the medium was removed and 200. mu.l of a compound was added to the cells at a concentration 10 times as high as the final concentration in the medium. After incubation for a suitable time, the medium was aspirated off and the cells were washed twice with cold PBS. The cells were then lysed with 100 μ l RIPA buffer containing protease and phosphatase inhibitors.

For western blotting, 20 μ g of protein was denatured at 95 ℃ in Laemmli sample buffer containing β -mercaptoethanol, then loaded onto a gradient of 4-20% Tris glycine SDS gel and electrophoresed to completion at 70 volts. The proteins were transferred to nitrocellulose membranes and phospho-ERK 1/2 and total ERK1/2 were probed with appropriate antibodies. Color bands were detected using ECLwestern blotting chemiluminescent substrate. The phospho-ERK ELISA used was an ELISA kit from Assay Designs.

Bcl-2 luciferase

SK-N-MC Bcl-2(neo) clone 218 was plated at 25,000 cells/well on PackardView plates in a medium containing penicillin/streptomycin, L-glutamineAmide, sodium pyruvate, non-essential amino acids, sodium bicarbonate and 200ug/ml G418 phenol red free EMEM. Cells in 5% CO₂Was cultured overnight in a 37 ℃ incubator.

The following day, the medium was removed and replaced with serum-free EMEM with ITS supplement (BDBiosciences # 354352). Changing the culture medium again on the third and fourth days; on the fourth day, compound was added to the cells to a final volume of 100. mu.l. 24 hours after compound addition, 100. mu.l SteadyGlo (Promega # E2510) was added and luciferase was measured in a Packard Topcount liquid scintillation counter.

Oligodendrocyte cytotoxicity assay

Primary rat oligodendrocyte progenitor cells were obtained from the brains of rats (Sprague Dawley) 2-3 days after birth. The meningeal membrane is removed and the tissue is mechanically stripped. Cells were plated in T75 flasks and fed with DMEM + 10% FBS.

Concentrated OLPs were collected from astrocytic monolayers by mechanical separation and expanded in serum-free medium (SFM) supplemented with mitogens PDGF-AA (10ng/ml) and FGF-2(10 ng/ml).

To generate mature oligodendrocytes, the progenitor cells were transferred to SFM supplemented with IGF-1(10ng/ml) after 24 hours in culture, the cells were grown under these conditions for 7 days, and then subjected to experimental analysis.

Cells were plated in 96-well plates with 10,000 cells per well. The medium was changed to fresh medium and the cells were pre-treated with the compound for 1 hour. Toxins were added to give the following final concentrations:

Sin-1 10mM

phthalic triphenol 500 mu M

C2 ceramide 100. mu.M

Camptothecin 10 mu M

After 24 hours, the medium was removed and assayed for LDH activity using the Promega cytox 96 kit (catalog No. G1780). The results were calculated as% protection against toxin-induced toxicity.

The neuroprotective efficacy of these compounds against cell death caused by toxic substances such as SIN-1 (3-morpholino-sydnonimine, which produces peroxynitrite ion), C2 ceramide, camptothecin, staurosporine, SNAP (S-nitroso-N-acetylpenicillamine, which produces nitric oxide), and pyrogallol (which produces superoxide anion) was evaluated. The target cells evaluated in vitro were: primary cultures of human neuroblastoma cell lines [ SK-N-SH, SH-SY5Y ] and rodent oligodendrocyte progenitor cells and their mature copies. The protective effect of these SERM-like compounds was compared with 17- β -estradiol and tamoxifen (see table 1 below). The mechanism of action of this neuroprotective effect was investigated by using classical nuclear (genomic) ER α or β and evaluation of the effect on MAPK p40/p42(ERK1/2) phosphorylation.

Results

Pharmacological composition

Both compounds tested showed protective effects on neurons and oligodendrocytes. This appears to be mediated by upregulation of ERK1/2 phosphorylation as evidenced by inhibition of the neuroprotective effects of U-O126, a MEK inhibitor specific for the ERK pathway.

Claims (2)

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

2. The method of claim 1, wherein the effective amount is administered daily at from about 0.001 to about 100mg/kg of patient body weight/day.