Treatment of Neurological Disorders

Abstract

The present invention is directed to 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for the treatment of diseases mediated by protein misfolding, the heat shock factor 1 (HSF1) pathway, or the nuclear factor erythroid 2-related factor 2 (NRF2) pathway.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 747,961, filed October 19, 2018, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to therapeutic agents and methods for the treatment of diseases mediated by protein misfolding, the heat shock factor 1 (HSF1) pathway, or the nuclear factor erythroid 2-related factor 2 (NRF2) pathway. [Background technology]

[0003] background In normal cells, proteostasis is maintained by regulating protein expression, folding, modification, translocation, and ultimately degradation. To achieve this, cells utilize sophisticated mechanisms to ensure the proper performance of these processes in response to cellular stress. A critical aspect of cellular proteostasis is the utilization of chaperone proteins, which stabilize protein structure, assist in the correct folding and unfolding of proteins, and facilitate the assembly of multimeric protein complexes. Chaperone proteins, including αB crystallin, heat shock protein 27 (HSP27), HSP40, HSP70, and HSP90, as well as class I and class II chaperonins, function individually or as part of larger heterocomplexes to prevent protein misfolding, accumulation of misfolded proteins, and protein aggregation. Chaperone proteins can promote cell survival by stabilizing and refolding misfolded proteins, as well as by inhibiting apoptosis.

[0004] Heat shock transcription factor 1 (HSF1, HGNC:5224 https: / / www.ncbi.nlm.nih.gov / gene / 3297) is a master activator of chaperone protein gene expression. HSF1 promotes the expression of genes encoding chaperone proteins in response to cellular stress. HSF1 also regulates the expression of genes involved in other aspects of cell survival, including protein degradation, ion transport, signal transduction, energy generation, carbohydrate metabolism, endoplasmic reticulum trafficking, and cytoskeleton organization.

[0005] Stress-dependent regulation of HSF1 is a multistep process controlled by intricate regulatory mechanisms. Under basal conditions, HSF1 exists primarily as an inactive monomer in the cytoplasm and is inhibited in part through the activity of the chaperone proteins HSP90, HSP70, HSP40, the chaperonin-containing t-complex polypeptide 1 (TCP1) ring complex (TRiC), and other co-chaperones that form inhibitory complexes with HSF1. In response to proteotoxic stress, HSF1 appears to dissociate from HSP90, HSP70, HSP40, and TRiC. This allows HSF1 to homotrimerize, accumulate in the nucleus, and bind to heat shock elements in the promoter regions of target stress-responsive genes, such as those encoding chaperone proteins, after activating post-translational modifications (including phosphorylation).

[0006] Protein chaperones play a critical role in protein synthesis, de novo folding, refolding, disaggregation, oligomer assembly, trafficking, modification, maturation and degradation, either via the ubiquitin-proteasome system and / or autophagy (including chaperone-mediated autophagy) of cellular client proteins. The HSF1 pathway has been implicated in a diverse range of diseases, including cancer, neurodegenerative, metabolic, inflammatory and cardiovascular diseases, involving a variety of cells and tissues, including neurons, heart, muscle, spleen and liver.

[0007] Protein misfolding, accumulation of misfolded proteins, and protein aggregation are hallmarks of neurodegenerative diseases. In patients with Parkinson's disease, Parkinson's dementia, or dementia with Lewy bodies, Lewy bodies are observed in the cytoplasm of neurons in the substantia nigra of the brain. The main components of these aggregates are α-synuclein, phosphorylated α-synuclein, hyperphosphorylated tau, leucine-rich repeat kinase (LRRK2), and a protein fragment named trans-active DNA-binding protein 43 (TDP-43) aggregates. In Alzheimer's disease, there are two main types of protein deposits. Amyloid plaques are deposited extracellularly within the brain parenchyma and around the walls of cerebral blood vessels, and their main component is a 40-42 residue peptide named β-amyloid protein. Neurofibrillary tangles are present in the cytoplasm of degenerated neurons and are composed of aggregates of hyperphosphorylated tau protein. Up to 50% of Alzheimer's disease patients are also known to have TDP-43 aggregates in the central nervous system (CNS).In patients with Huntington's disease, intranuclear deposits of polyglutamine-expanded forms of the mutated huntingtin protein are a typical feature of the brain. Patients with amyotrophic lateral sclerosis (ALS) have misfolded and / or aggregated proteins, primarily TDP-43 (either mutant or dysregulated), and / or misfolded ubiquitinated aggregates of TDP43, a protein encoded by TAR DNA-binding protein 43, as well as misfolded superoxide dismutase (SOD1), dipeptide repeat proteins from the expanded hexanucleotide repeat C9orf72, hyperphosphorylated tau, fused in sarcoma (FUS), ataxin-2 (ATXN2), and heterogeneous nuclear ribonucleoproteins (hnRNPs) in the somas and axons of motor neurons and / or interneurons. Finally, the brains of humans and animals with diverse forms of transmissible spongiform encephalopathies are characterized by the accumulation of protease-resistant aggregates of prion proteins.

[0008] Pharmacological activation of HSF1 and the HSF1 pathway is a promising means for therapeutic intervention in diseases involving the HSF1 pathway, particularly diseases involving protein misfolding, accumulation of misfolded proteins, and protein aggregation. HSF1 activators represent a novel therapeutic approach that may slow, halt, or reverse the underlying disease process of diseases involving the HSF1 pathway.

[0009] One group of diseases that can be treated by therapeutic intervention involving HSF1 pathway is mitochondrial disease.Mitochondrial disease is a genetic disorder driven by genetic mutations in mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) of genes that are transcribed and translated into mitochondrial proteins responsible for mitochondrial function.These mutations cause misfolding and aggregation of mutated mitochondrial proteins / enzymes, which impair mitochondrial functions including oxidative phosphorylation, fatty acid oxidation, Krebs cycle, urea cycle, gluconeogenesis and ketogenesis (Gorman et al. Nat Rev Dis Primers. 2016, 2, 1-22).

[0010] HSF1 activation has been shown to restore damaged mitochondrial proteostasis and improve mitochondrial function, remove terminally dysfunctional mitochondria via mitophagy, and regenerate new mitochondria via mitochondrial biogenesis. These improvements in mitochondrial function and biogenesis lead to increased oxidative phosphorylation, thermogenesis, and energy expenditure (Gomez-Pastor et al. Nat Rev Mol Cell Biol. 2018, 19, 4-19).

[0011] Diseases / disorders due to mitochondrial dysfunction as a result of genetic mutations include childhood-onset mitochondrial disease-Leigh syndrome; Alpers-Huttenlocher syndrome; Childhood myocerebrohepatopathy spectrum (MCHS); Ataxic neuropathy spectrum (ANS); Myoclonic epilepsy-myopathy-sensory ataxia (MEMSA); Saengers syndrome; MEGDEL syndrome; Pearson syndrome; and Congenital Lactic Acidosis (CLA); Adult-onset mitochondrial disease-Leber hereditary optic neuropathy (LHON); Kearns-Sayre syndrome (KSS); Mitochondrial myopathy-lactic acidosis-strokelike episodes (MELAS) syndrome; Myoclonic epilepsy with ragged-red fibers (MERRF); Neuropathic ataxia retinitis pigmentosa (NARP); Chronic progressive external ophthalmoplegia syndrome (CPEO); and Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome.

[0012] Another group of diseases that can be treated by therapeutic intervention involving the HSF1 pathway are lysosomal storage diseases (LSDs). LSDs are disorders driven by genetic mutations that result in impaired function of lysosomal and membrane proteins, mainly lysosomal hydrolases. Lysosomes are essential for maintaining cellular homeostasis by recycling cellular components. The severity of lysosomal storage diseases is also an indicator of the vitality of lysosomal function. Clinical phenotypes associated with LSDs include severe neurodegeneration, systemic disease, and premature death driven by protein misfielding and aggregation, impaired lysosomal trafficking and autophagy, oxidative stress, endoplasmic reticulum stress response, impaired calcium homeostasis, and loss of lysosomal stability (Platt. Nat Rev Drug Discov. 2018, 17, 133-150; Ingemann, Kirkegaard. J Lipid Res. 2014, 55, 2198-2210).

[0013] Activation of HSF1 inhibits protein misfolding and aggregation caused by genetic mutations, and upregulation of heat shock chaperones has been shown to improve the enzymatic function of misfolded proteins by refolding them. Another benefit of activating HSF1 is that it enhances cell viability by inhibiting lysosomal membrane permeabilization and increasing lysosomal catabolism (Ingemann, Kirkegaard. J Lipid Res. 2014, 55, 2198-2210).

[0014] Disease groups that can be treated by therapeutic intervention involving the HSF1 pathway include GM1 gangliosidosis, GM2-gangliosidosis, alpha-mannosidosis, beta-mannosidosis, aspartylglucosaminuria, lysosomal acid lipase deficiency, Wolman disease, cystinosis, Chanarin-Dorfman syndrome, Danon disease, Fabry disease (types I and II), Farber disease, fucosidosis, galactosialidosis, Gaucher disease (types I, II, III, IIIC, saponin C deficiency), Krabbe disease, metachromatic leukodystrophy, Hurler syndrome, Hurler- Scheie syndrome, Hunter syndrome, Sanfilich syndrome type A, Sanfilich syndrome type B, Sanfilich syndrome type C, Sanfilich syndrome type D, Morquio syndrome type A, Morquio syndrome type B, hyaluronidase deficiency, Maroteaux-Lamy syndrome, Sly syndrome, Sialidosis, Leroy disease, Pseudo-Hurler polydystrophy, Mucolipidosis type IIIC, Mucolipidosis type IV, Multiple sulfatase deficiency, Niemann-Pick disease (types A, B, C1, C2 and D), CLN6 disease - atypical late-infantile type, Late-onset mutations early juvenile; Batten-Spielmeier-Voght disease / juvenile NCL / CLN3 disease; Finnish variant late infantile CLN5; Jansky-Bielschowski disease / late infantile CLN2 / TPP1 disease; Kuhus / adult-onset NCL / CLN4 disease; Northern epilepsy / variant late infantile CLN8 These include: CLN8), Santaburi-Hartia / infantile CLN1 / PPT disease, Pompe disease (glycogen storage disease type II), pyknodysostosis, Sandhoff disease (infantile, juvenile and adult onset), Schindler disease (types I and III), Schindler disease type II / Kanzaki disease, Salla disease, infantile sialic acid storage disease, spinal muscular atrophy with progressive myoclonic epilepsy (SMAPME), Tay-Sachs disease, Christianson syndrome, Lowe syndrome, Charcot-Marie-Tooth disease, Eunice-Baron syndrome, bilateral temporal-occipital polymicrogyria (BTOP), and X-linked hypercalciuric nephrolithiasis.

[0015] Another group of diseases that can be treated by therapeutic intervention involving the HSF1 pathway are tauopathies. Tauopathies are neurodegenerative disorders driven by intracellular tau protein misfolding and aggregation caused by genetic mutations in the MAPT gene and / or post-translational modifications of tau protein. Tau protein aggregates have been demonstrated to correlate with cognitive decline in multiple neurodegenerative diseases. And both hyperphosphorylated soluble tau and insoluble tau protein have been shown to be neurotoxic. In fact, soluble hyperphosphorylated tau is taken up by neurons and acts as a template for cytoplasmic tau misfolding. Further studies have also shown that tau protein aggregation is driven by abnormal liquid-liquid phase separation / stress granules that maintain and enhance aggregation.

[0016] Activation of HSF1 has been shown to upregulate molecular chaperones that inhibit tau protein misfolding and aggregation, refold misfolded tau protein, disaggregate tau oligomers and aggregates (Patterson et al. Biochemistry. 2011, 50, 10300-10310; Baughman et al. J. Biol. Chem. 2018, 293, 2687-2700), and degrade terminally aggregated tau protein via the ubiquitin proteasome system and autophagy, including chaperone-mediated autophagy (Boland et al. Nat Rev Drug Discov. 2018, 17, 660-688). HSF1 activation also improves synaptic plasticity, neuronal viability and neurotransmitter release at synapses (Gomez-Pastor et al. Nat Rev Mol Cell Biol. 2018, 19, 4-19). Upregulation of molecular chaperones has been demonstrated to inhibit protein misfolding and aggregation, refold misfolded proteins, disaggregate aggregated proteins, and degrade terminally misfolded and aggregated proteins via autophagy. These proteins include α-synuclein, TDP-43, FUS, ATXN2, amyloid beta, polyglutamine (polyQ)-expanded proteins, polytrinucleotide repeat expansions, polyhexanucleotide repeat expansions, misfolded SOD1, and multiple other misfolded proteins resulting from genetic mutations.

[0017] Diseases primarily driven by MAPT gene mutations and / or post-translational modifications of tau include progressive supranuclear palsy, corticobasal degeneration, Pick's disease, frontotemporal lobar degeneration-tau, argyrophilic grain disease, subacute sclerosing panencephalitis, Christianson syndrome, postencephalitic parkinsonism, Guadeloupe parkinsonism, spinocerebellar ataxia type 11, chronic traumatic encephalopathy, age-related tau astrogliopathy (ARTAG), globular glial tauopathy, and primary age-related tauopathy (PART).

[0018] Diseases / disorders in which tau plays a major role in the disease but which are primarily driven by beta-amyloid include Alzheimer's disease, cerebral amyloid angiopathy, vascular dementia, and Down's syndrome.

[0019] Diseases / disorders in which tau plays a major role in the disease but which are primarily driven by alpha-synuclein include Parkinson's disease, dementia with Lewy bodies, Parkinson's disease dementia, brain iron accumulation neurodegeneration, diffuse neurofibrillary tangles with calcification, multiple system atrophy, and Alzheimer's disease.

[0020] Diseases / disorders in which tau plays a major role in the disease but are primarily prion-driven include Creutzfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Straussler-Scheinker syndrome, and kuru.

[0021] Diseases / disorders in which tau plays a major role in disease but is primarily driven by other factors include Huntington's disease, Familial British Dementia, Familial Danish Dementia, Parkinson's Disease Dementia of Guam, Frontotemporal Lobar Degeneration-C9ORF72, Myotonic Dystrophy, Niemann-Pick Disease Type C, Neuronal Ceroid Lipofuscinosis, and Inclusion Body Myositis.

[0022] Other genetic diseases can also be treated by therapeutic intervention involving the HSF1 pathway. Genetic diseases are diseases / disorders driven by genetic mutations that produce dysfunctional proteins and cause loss of function of translated proteins. These diseases tend to produce heterogeneous clinical phenotypes. Activation of the heat shock response, primarily through HSF1 activation, generates multi-molecular chaperones that attenuate protein misfolding and refold dysfunctional proteins to restore the function of some, but not all, proteins / enzymes, thus slowing and / or inhibiting disease progression.

[0023] Disorders that are typically genetic include Alexander disease, aortic medial amyloidosis, apo AI amyloidosis, apo AII amyloidosis, apo AIV amyloidosis, autosomal dominant hyper-IgE syndrome, Bloom syndrome, Brown-Vialetto-Van Laere syndrome, Cockayne syndrome, Cushing's disease, cystic fibrosis, dentatorubral-pallidoluysian atrophy (DRPLA), Duchenne palsy, Eales disease, familial amyloidosis of the Finnish type, familial amyloidotic neuropathy, familial dementia, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Friedreich's ataxia, glycogen storage disease type IV (Andersen's disease), hereditary lattice corneal degeneration, Leber's hereditary optic neuropathy, hereditary spastic paraplegia (HS). P), Hutchinson-Gilford disease, Kugelberg-Welander syndrome, Lou Gehrig's disease, light- or heavy-chain amyloidosis, Mallory bodies, Paget's disease of bone (PDB), Pelizaeus-Merzbacher disease, primary lateral sclerosis (PLS), Saengers syndrome, sickle cell disease, spinal-bulbar muscular atrophy (SBMA) (also known as Kennedy disease), variant Creutzfeldt-Jakob disease, Werdnig-Hoffmann disease, and Werner syndrome.

[0024] Other protein misfolding and age-related diseases may also be treated by therapeutic intervention involving the HSF1 pathway. The heat shock response is a cytoprotective response mechanism in cells, including neurons, that are under cellular stress. In several degenerative diseases, including neurodegenerative diseases, the heat shock response is suboptimal. Furthermore, the heat shock response in cells in response to stress has been shown to decrease with age and to be the cause of several degenerative diseases (Klaips et al. J Cell Biol. 2018, 217, 51-63; Chiti, Dobson. Annu. Rev. Biochem. 2017, 86, 27-68; Labbadia, Morimoto. Annu. Rev. Biochem. 2015, 84, 435-464; Morimoto. Cold Spring Harb Symp Quant Biol. 2011, 76, 91-99).

[0025] Activation of the heat shock response, primarily via HSF1 activation, generates multi-molecular chaperones that inhibit protein misfolding and aggregation, refold misfolded proteins, and disaggregate aggregated proteins. HSF1 activation also reduces oxidative stress, improves mitochondrial function, initiates mitochondrial biogenesis, and improves synaptic plasticity and neuronal survival (Gomez-Pastor et al. Nat Rev Mol Cell Biol. 2018, 19, 4-19).

[0026] Protein misfolding and age-related diseases include amyotrophic lateral sclerosis, ataxia retinitis pigmentosa, ataxic neuropathy spectrum, ataxia telangiectasia, atherosclerosis, atrial fibrillation, autism spectrum disorder, benign focal atrophy, atrial amyloidosis, cardiovascular disease (including coronary artery disease, myocardial infarction, stroke, restenosis and arteriosclerosis), cataracts, intracerebral hemorrhage, cerebrovascular disease, lactate-like disease, and cerebrovascular disease. Corneal amyloidosis due to ferrin, Critical Illness Myopathy (CIM), Crohn's disease, Lichen amyloidosis, Demyelinating disorders, Dentatorubral-Pallidoluysian Atrophy (DRPLA), Depressive disorders, Type II diabetes mellitus, Dialysis amyloidosis, Endotoxin shock, Fibrinogen amyloidosis, Glaucoma, Ischemia, Ischemic conditions (including ischemia / reperfusion injury, myocardial ischemia, stable angina, unstable angina, stroke, ischemic heart disease and cerebral ischemia) , lactic acidosis-strokelike episodes (MELAS) syndrome, lysozyme amyloidosis, macular degeneration, medullary thyroid carcinoma, meningoencephalitis, multiple sclerosis, necrotizing enterocolitis, neurofibromatosis, amyloid-producing odontogenic (Pymborg) tumor, pituitary prolactinoma, post-traumatic stress disorder, presenile dementia, prion diseases (including Creutzfeldt-Jakob disease (CJD), also known as transmissible spongiform encephalopathies or TSEs), progressive glomerulonephritis, These include progressive muscular atrophy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, pulmonary alveolar proteinosis, retinal ganglion cell degeneration in glaucoma, retinal ischemia, retinal vasculitis, retinitis pigmentosa with rhodopsin mutations, schizophrenia, seminal vesicle amyloidosis, senile cataract, senile systemic amyloidosis, serpinopathies, subarachnoid hemorrhage, temporal lobe epilepsy, transient ischemic attacks, ulcerative colitis, and valosin-containing protein (VCP)-related disorders.

[0027] Nuclear factor erythroid transcription factor 2-related factor 2 (NRF2, HGNC:7782, https: / / www.ncbi.nlm.nih.gov / gene / 4780) regulates the expression of genes involved in cellular defense against damage by oxidants, electrophiles, and inflammatory agents, as well as in the maintenance of mitochondrial function, cellular redox, and protein homeostasis. The NRF2 protein contains seven functional domains, named the NRF2-ECH homology (Neh) 1-7 domains. Through its Neh2 domain, NRF2 binds one of the major negative regulators, Kelch-like ECH-associated protein 1 (Keap1). In addition, Neh1 is responsible for the formation of heterodimers with small musculoaponeurotic fibrosarcoma (sMaf) proteins and mediates binding to antioxidant / electrophile response element (ARE / EpRE) sequences in the promoter regions of Nrf2 target genes. The C-terminal Neh3 is another transactivation domain that recruits chromoATPase / helicase DNA-binding protein 6 (CHD6). Neh4 and Neh5 are transactivation domains that recruit cAMP response element-binding protein (CREB)-binding protein (CBP) and / or receptor-associated coupling factor 3 (RAC3). The Neh6 domain mediates interaction with a third negative regulator, β-transducin repeat-containing protein (β-TrCP). Furthermore, the Neh7 domain mediates binding to retinoid X receptor alpha (RXRα), another negative regulator of NRF2.

[0028] NRF2 levels are primarily regulated by ubiquitination and proteasomal degradation. Keap1 mediates Cul3 / Rbx1-dependent ubiquitination of NRF2 after binding to the Neh2 domain. In addition, the Neh6 domain contains a phosphodegron for β-TrCP / Cullin1-mediated ubiquitination. Synoviolin (Hrd1) and WDR23-DDB1-Cul4 are two other ubiquitin ligases that have been shown to be involved in the proteasomal degradation of NRF2.

[0029] NRF2 is a short-lived protein under homeostatic conditions. Under stress conditions, NRF2 is stabilized and translocated to the nucleus, where it binds to ARE / EpRE sequences in the promoters of target genes and activates their transcription. Targets of NRF2 include genes encoding detoxifying, antioxidant, and anti-inflammatory proteins, as well as proteins involved in regulating autophagy and clearance of damaged proteins, such as proteasome subunits. Activation of NRF2 leads to upregulation of proteins involved in the synthesis of glutathione, the main intracellular small molecule antioxidant, and NADPH, which provides the reduced equivalent for regenerating reduced glutathione (GSH) from its oxidized form GSSG. NRF2 also participates in the maintenance and regulation of mitochondrial function through activation of mitophagy. NRF2 inhibits the transcription of genes encoding proinflammatory cytokines and suppresses proinflammatory responses after exposure to ultraviolet light or lipopolysaccharide. Such comprehensive cytoprotective functions suggest the potential benefit of therapeutic targeting of NRF2 against neurodegeneration.

[0030] NRF2 activators have pleiotropic effects on multiple neurodegenerative disease pathways and show great promise for neuroprotection in these disorders. (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, as an NRF2 activator, has beneficial effects on critical drivers of neurodegeneration including redox imbalance, inflammation, mitochondrial dysfunction and altered proteostasis / autophagy in pathogenesis.

[0031] Pharmacological activation of NRF2 and the NRF2 pathway is another promising avenue for therapeutic intervention in diseases involving the NRF2 pathway. NRF2 activators are novel therapeutic strategies that may slow, halt, or reverse the underlying disease process of diseases involving the NRF2 pathway.

[0032] Combined pharmacological activation of HSF1 and NRF2, as well as combined HSF1 and NRF2 pathways, is another promising avenue for therapeutic intervention in diseases involving both the NRF1 and NRF2 pathways. Combined HSF1 / NRF2 activators are a novel therapeutic strategy that may slow, halt, or reverse the underlying disease process of diseases involving the HSF1 and NRF2 pathways. Summary of the Invention [Problem to be solved by the invention]

[0033] Thus, there is a need for compositions that have utility in activating HSF1 and / or NRF2. [Means for solving the problem]

[0034] Summary of the Invention 6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is a type of aporphine that has activity against dopamine receptors. It may also have effects on serotonergic and adrenergic receptors. 6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol does not actually contain morphine or its skeleton, and does not bind to opioid receptors. The prefix apo refers to the fact that it is a morphine derivative. 6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol has two enantiomers, namely (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. (6aR)-6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is a central nervous system penetrant catecholamine compound that is a dual activator of HSF1 and NRF2 transcription factors.

[0035] (6aR)-6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is a potent dopamine agonist currently approved for the treatment of Parkinson's disease. (6aR)-6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is also known as R-(-)-10,11-dihydroxyaporphine and is represented by the following chemical structure: [ka]

[0036] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, the enantiomer of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, is a weak dopamine antagonist and does not exhibit side effects related to dopamine agonism after administration. (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, also known as S-(+)-10,11-dihydroxyaporphine, is represented by the following chemical structure: [ka]

[0037] The present invention is predicted from the surprising finding, demonstrated through screening and testing, that 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is a potent HSF1 activator and can significantly affect protein misfolding, accumulation of misfolded proteins, and protein aggregation.

[0038] Thus, 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be used in methods for activating HSF1, increasing the transcription levels of genes positively regulated by HSF1, i.e. activating the HSF1 pathway to increase the cellular levels of protein chaperones and / or co-chaperones, reduce the frequency of protein misfolding, reduce the accumulation of misfolded proteins, and reduce protein aggregation in cells. 6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may further be used in methods for treating diseases mediated by protein misfolding, accumulation of misfolded proteins, protein aggregation, or by reduced HSF1 activity. HSF1 activation by 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is determined by upregulation of HSF1 target genes.

[0039] Specifically, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol are converted to orthoquinone moieties under oxidative stress, which is a pathological mechanism of neurodegenerative diseases. (6aS)-6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is classified as a pro-electrophilic and pathologically activated drug, and is an electrophile 3 compound (Satoh et al. Free Radic Biol Med. 2013, 65, 645-657; Satoh et al. J Neurochem. 2011, 119, 569-578; Satoh et al. ASN Neuro 2015, 1-13).

[0040] The orthoquinone moiety of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol acts as a Michael acceptor for Michael addition of specific cysteine ​​residues of regulators of transcription factors. Upon formation of Hsp90-(6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol orthoquinone adduct under cellular stress, Hsf1 monomers are released from the complex of Hsp90, Hsp70, Hsp40, Hsf1, and TRiC, homotrimerize, and translocate to the nucleus where they bind to heat shock elements (HSEs) and activate the transcription and translation of downstream genes that enhance neuronal survival and function (Gomez-Pastor et al. Nat Rev Mol Cell Biol. 2018, 19, 4-19; Naidu, Dinkova-Kostova. FEBS J. 2017, 284 1606-1627). The genes activated are estimated to be between 50 and 200 genes and encode protein chaperones (including heat shock protein 70 (Hsp70)), synapsin, postsynaptic density protein 95 (PSD95), brain-derived neurotrophic factor (BDNF), and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α).

[0041] Protein chaperones, including Hsp70 and Hsp40, as well as heat shock cognate 70 / heat shock protein A8 (Hsc70 / HSPA8), are responsible for inhibiting protein misfolding and aggregation, refolding misfolded proteins, disaggregating aggregated proteins, and removing terminally folded and / or aggregated proteins via the ubiquitin proteasome system (UPS) and autophagy, including chaperone-mediated autophagy (CMA). These chaperones are also involved in the inhibition of pathological stress granule formation, disaggregation of pathological stress granules, and removal of terminally formed abnormal stress granules via autophagy. Disaggregation of pathological stress granules has been demonstrated to restore dysfunctional nucleocytoplasmic transport, a pathological mechanism of multiple neurodegenerative diseases, through the release of nucleocytoplasmic transport factors trapped in abnormal stress granules.

[0042] Hsp70 has been shown to attenuate the formation of pro-inflammatory cytokines during the symptomatic phase of neurodegenerative diseases through inhibition of IkBα phosphorylation, which is upstream of the NF-kB signaling pathway.

[0043] Neurodegenerative diseases driven by impaired protein homeostasis include all tauopathies, including amyotrophic lateral sclerosis, motor neuron disease, frontotemporal dementia, Alzheimer's disease, FTLD-tau, progressive supranuclear palsy, corticobasal degeneration, and chronic traumatic encephalopathy (Gomez-Pastor et al. Nat Rev Mol Cell Biol. 2018,19,4-19; Li, Gotz, Nat Rev Drug Discov. 2017,12,863-883), Parkinson's disease, dementia with Lewy bodies, pathological polyglutamine expansion disorders including Huntington's disease, hereditary spastic paraplegia, spastic ataxia, Marinesco-Sjögren's syndrome, Charcot-Marie-Tooth disease type 2L, juvenile parkinsonism, distal hereditary motor neuropathy, dominantly inherited myopathies, Angelman syndrome, Nakajo-Nishimura syndrome, DCMA syndrome, desmin-related myopathy, spinocerebellar degeneration type 3 / Machado-Joseph disease (SCA3 / MJD), and Paget's disease (Labbadia,Morimoto.Annu Rev Biochem.2015,84,435-464). Lysosomal storage disorders including Niemann-Pick disease type C, Gaucher disease, Fabry disease, Sandhoff disease, Tay-Sachs disease, Wolman disease, Pompe disease, mucolipidosis type II, mucolipidosis type IV, multiple sulfatase deficiency, galactosialidosis, neuronal ceroid lipofuscinosis, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type IV, and metachromatic leukodystrophy (Platt. Nat Rev Drug Discov. 2018, 17, 133-150; Ingemann, Kirkegaard. J Lipid Res. 2014, 55, 2198-2210), as well as sporadic inclusion body myositis (Ahmed et al. Sci Transl Med. 2016, 8, 331).

[0044] In one aspect, the present invention provides a method of activating HSF1 in a cell, comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. As used herein, the term "effective amount" refers to an amount that produces a desired effect or result, e.g., an amount that produces activation of HSF1.

[0045] In a related aspect, the invention provides a method for increasing transcription of genes transactivated by HSF1 in a cell, the method comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0046] In another aspect, the present invention provides a method for increasing cellular levels of a protein chaperone and / or co-chaperone in a cell, comprising contacting said cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0047] In another aspect, the invention provides a method of reducing the frequency of protein misfolding or accumulation of misfolded proteins including TDP-43, SOD1, hyperphosphorylated tau, dipeptide repeat proteins from hexanucleotide repeat expansions associated with C9orf72, beta amyloid, alpha synuclein, phosphorylated alpha synuclein, polyglutamine repeat expansions, FUS, ATXN2, heterogeneous nuclear ribonucleoproteins (hnRNPs), and prion proteins in a cell, comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0048] In another aspect, the present invention provides a method of increasing the lifespan of a cell, comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0049] In one embodiment, the cell in one of the above aspects or other aspects herein is a cell type or derived from a tissue selected from any one or more of adrenal gland, bone marrow, brain, breast, bronchus, caudate nucleus, cerebellum, cerebral cortex, cervix, uterus, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, myocardium, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid, placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including the duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid, tonsils, bladder and vagina. In further embodiments, the brain cells are from brain tissue selected from the cerebrum (including the cerebral cortex, basal ganglia (often called the striatum), and olfactory bulb), cerebellum (including the dentate nucleus, interpositus nucleus, fastigial nucleus, and vestibular nuclei), diencephalon (including the thalamus, hypothalamus, etc., and the posterior part of the pituitary gland), and brainstem (including the pons, substantia nigra, and medulla oblongata). In further embodiments, the brain cells are selected from neurons or glial cells (e.g., astrocytes, oligodendrocytes, or microglia). In further embodiments, the neurons are sensory neurons, motor neurons, interneurons, or brain neurons.

[0050] In one embodiment, the cell is an animal cell, such as a mammalian cell. In a further embodiment, the cell is a human cell or a non-human cell. In a further embodiment, the cell is in vitro, in vivo, or ex vivo.

[0051] In another embodiment, the cell is a pathological cell. In another embodiment, the cell is a pathological cell from a patient suffering from the following diseases or disorders:

[0052] In another aspect, the invention provides a method for treating an animal having a disease or disorder that would benefit from increased HSF1 activation, or for preventing or reducing the risk of acquiring a disease or disorder in an animal, comprising administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. In one embodiment, the animal is a mammal. In another embodiment, the mammal is a human or non-human mammal. In a further embodiment, the mammal is a human. In another embodiment, the disease or disorder is caused by protein misfolding, accumulation of misfolded proteins, or protein aggregation. In another embodiment, the disease is age-related tau astrogliopathy (ARTA), Alexander disease, Alpers-Huttenlocher syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxic neuropathy spectrum, ataxia retinitis pigmentosa (NARP), critical illness myopathy (CIM), primary age-related tauopathy (PART), aortic medial amyloidosis, apo AI amyloidosis, apo AII amyloidosis, apo AIV amyloidosis, argyrophilic grain disease, ataxia telangiectasia, atrial fibrillation, autosomal dominant hyper IgE syndrome, atrial amyloidosis, Bloom's syndrome, cardiovascular disease (including coronary artery disease, myocardial infarction, stroke, restenosis and arteriosclerosis), cataracts, cerebral amyloidosis, Angiopathy, Christianson syndrome, chronic traumatic encephalopathy, chronic progressive external ophthalmoplegia syndrome (CPEO), Cockayne syndrome, congenital lactic acidosis (CLA), corneal amyloidosis due to lactoferrin, corticobasal degeneration, Crohn's disease, Cushing's disease, lichen amyloidosis, cystic fibrosis, dentatorubral-pallidoluysian atrophy (DRPLA), dialysis amyloidosis, diffuse neurofibrillary tangles with calcification, Down's syndrome, endotoxic shock, familial amyloidosis of the Finnish type, familial amyloid neuropathy, familial British dementia (FBD), familial Danish dementia (FDD), familial dementia, fibrinogen amyloidosis, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS),Friedreich's ataxia, frontotemporal lobar degeneration, glaucoma, glycogen storage disease type IV (Andersen's disease), Guadeloupe parkinsonism, hereditary lattice corneal degeneration, Huntington's disease, inclusion body myositis / myopathy, inflammation, inflammatory bowel disease, ischemic conditions (including ischemia / reperfusion injury, myocardial ischemia, stable angina, unstable angina, stroke, ischemic heart disease and cerebral ischemia), light or heavy chain amyloidosis, lysosomal storage diseases (including aspartylglucosaminuria), Fabry disease, Batten disease, cystinosis, Farber disease, fucosidosis, galactosidosis, Gossypii disease, Gerhardt disease (including types 1, 2 and 3), Gm1 gangliosidosis, Hunter disease, Hurler-Scheie disease, Krabbe disease, α-mannosidosis, Kearns-Sayre syndrome (KSS), lactic acidosis-strokelike episodes (MELAS) syndrome, Leber hereditary optic neuropathy (LHON), β-mannosidosis, Maroteaux-Lamy disease, MEGDEL syndrome (also known as 3-methylglutaconic aciduria with hearing loss, encephalopathy and Leigh-like syndrome), metachromatic leukodystrophy, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome, Morquio syndrome type A , Morquio syndrome type B, mucolipidosis type II, mucolipidosis type III, myoclonic epilepsy-myopathy-sensory ataxia, mitochondrial myopathy, myoclonic epilepsy with ragged red fibers (MERRF), Niemann-Pick disease (including types A, B, and C), neurogenic muscle weakness, Pearson syndrome, Pompe disease, Sandhoff disease, Sanfilich syndrome (including types A, B, C, and D), Schindler disease, Schindler-Kanzaki disease, Saengers syndrome, sialidosis, Sly syndrome, Tay-Sachs disease, Wolman disease, lysozyme amyloidosis thyroid cancer, mitochondrial myopathy, multiple sclerosis, multiple system atrophy, myotonic dystrophy, cerebral iron-storing neurodegeneration, neurofibromatosis, neuronal ceroid lipofuscinosis, amyloidogenic odontogenic (Pymborg) tumor, Guam Parkinson's dementia, Parkinson's disease, peptic ulcer, Pick's disease, pituitary prolactinoma, postencephalitic parkinsonism, prion diseases (also known as transmissible spongiform encephalopathies or TSEs, Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease,Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, and kuru), progressive supranuclear palsy, pulmonary alveolar proteinosis, retinal ganglion cell degeneration in glaucoma, retinitis pigmentosa with rhodopsin mutations, seminal vesicle amyloidosis, senile systemic amyloidosis, serpinopathies, sickle cell disease, spinal and bulbar muscular atrophy (SBMA) (also known as Kennedy disease), spinocerebellar degeneration (spinal Cerebellar degeneration type 1, spinocerebellar degeneration type 2, spinocerebellar degeneration type 3 (including Machado-Joseph disease), spinocerebellar degeneration type 6, spinocerebellar degeneration type 7, spinocerebellar degeneration type 8 and spinocerebellar degeneration type 17), subacute sclerosing panencephalitis, tauopathy, type II diabetes mellitus, vascular dementia, Werner syndrome, atherosclerosis, autism spectrum disorder (ASD), benign focal atrophy, Duchenne palsy, hereditary spastic paraplegia (HSP), Kugelberg-Welander syndrome, Lou Gehrig's disease, necrotizing enterocolitis, Paget's disease of bone (PDB), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, valosin-containing protein (VCP)-related disorder, or Werdnig-Hoffmann disease, transient ischemic attack, ischemia, intracerebral hemorrhage, senile cataract, retina ischemia, retinal vasculitis, Brown-Vialette-Van Lare syndrome, Eales disease, meningoencephalitis, post-traumatic stress disorder, Charcot-Marie-Tooth disease, macular degeneration, X-linked spinal and bulbar atrophy, presenile dementia, depressive disorder, temporal lobe epilepsy, Leber's hereditary optic atrophy, cerebrovascular disease, subarachnoid hemorrhage, schizophrenia, demyelinating disorders, and Pelicheus-Merzbacher disease.

[0053] In one embodiment, the disease is selected from any one or more of lysosomal storage diseases (e.g., Niemann-Pick disease type C, Gaucher disease), inclusion body myositis, spinocerebellar degeneration, spinal-bulbar muscular atrophy, or a disease related thereto.

[0054] In another embodiment, the disease is a neurological disease.

[0055] In another embodiment, the disease is selected from any one or more of amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, Parkinson's dementia, cerebral iron accumulation neurodegeneration, diffuse neurofibrillary tangles with calcification, multiple system atrophy, cerebral amyloid angiopathy, vascular dementia, Down's syndrome, Creutzfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Straussler-Scheinker syndrome, kuru, familial British dementia, familial Danish dementia, Parkinson's dementia of Guam, myotonic dystrophy, neuronal ceroid lipofuscinosis, or related diseases.

[0056] In another embodiment, the disease is selected from any one or more of frontotemporal dementia, cerebral iron accumulation neurodegeneration, diffuse neurofibrillary tangles with calcification, multiple system atrophy, cerebral amyloid angiopathy, vascular dementia, Down's syndrome, Creutzfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Straussler-Scheinker syndrome, kuru, familial British dementia, familial Danish dementia, myotonic dystrophy, neuronal ceroid lipofuscinosis, or a disease related thereto.

[0057] In another embodiment, the disease is selected from Friedreich's ataxia, multiple sclerosis, mitochondrial myopathy, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, argyrophilic grain disease, subacute sclerosing panencephalitis, Christianson's syndrome, postencephalitic parkinsonism, Guadeloupe parkinsonism, age-associated tau astrogliopathy (ARTA), and primary age-associated tauopathy (PART), Pick's disease, or a disease related thereto.

[0058] In another aspect, the present invention provides a method of increasing life span in an animal or treating a disease or disorder that results in accelerated aging or other abnormal aging processes, comprising administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. In one embodiment, the animal is a mammal. In another embodiment, the mammal is a human or non-human mammal. In one embodiment, the disease or disorder is selected from Werner syndrome, Hutchinson-Gilford disease, Bloom's syndrome, Cockayne's syndrome, ataxia-telangiectasia, and Down's syndrome.

[0059] In a related aspect, the invention provides a method for treating premature aging due to chemical or radiation exposure. In one embodiment, the premature aging is due to exposure to chemotherapeutic agents, radiotherapeutic agents, or UV radiation. In a further embodiment, the UV radiation is artificial, e.g., from a tanning bed, or solar UV radiation, i.e., exposure to sunlight.

[0060] In another aspect, the present invention provides an in vitro method of screening a candidate therapeutic agent(s) for its ability to activate the HSF1 pathway, comprising: (1) exposing induced astrocytes derived from fibroblast stem cells to a candidate therapeutic agent; (2) comparing the amount of misfolded SOD1 between the induced astrocytes exposed to the candidate therapeutic agent and a control cell, e.g., an induced astrocyte that has not been exposed to the candidate therapeutic agent (an unexposed induced astrocyte); The present invention provides an in vitro method comprising:

[0061] 6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may also be used in a method for activating NRF2 and / or reducing oxidative stress in a cell. (6aS)-6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may further be used in a method for treating a disease or disorder mediated by increased oxidative stress or reduced NRF2 activity. 6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may further be used in a method for reducing inflammation or treating a disease or disorder mediated by inflammation.

[0062] Activation of the NRF2 transcription factor through binding of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to cysteine ​​residues on Keap1 leads to the liberation of NRF2, which translocates to the nucleus and binds to antioxidant response elements (AREs), driving the transcription and translocation of over 250 downstream genes encoding proteins that reduce oxidative stress, provide an anti-inflammatory response, improve mitochondrial function and biogenesis, and autophagic removal of terminally misfolded and aggregated neurotoxic proteins (Dinkova-Kostova et al. FEBS J. 2018, doi:10.1111 / febs.14379).

[0063] To overcome some of the technical barriers to directly measuring NRF2 (including the lack of sensitive antibodies to detect low-abundance NRF2 protein and the relative stability of NRF2 mRNA during pathway activation), researchers have developed novel strategies to monitor NRF2 pathway activity, including (a) the use of stable reporter cell lines in which luciferase expression is controlled by one or more ARE sequences, (b) automated high-content imaging of cell lines expressing fluorescently labeled Nrf2 or target gene products, and (c) transcriptomic analysis of dynamic fluctuations in gene signatures (e.g., from ChIP data) shown to be representative of a large number of Nrf2-regulated genes (Mutter et al., Biochem Soc Trans. 2015, 43, 657-662).

[0064] Activation of the NRF2 transcription factor has been shown to modulate pathological mechanisms and provide neuroprotection in neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal lobar degeneration / frontotemporal dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Friedreich's ataxia, and multiple sclerosis (Dinkova-Kostova et al. FEBS J. 2018, doi:10.1111 / febs.14379; Cuadrado et al. Pharmacol Rev. 2018, 70, 348-383; Dinkova-Kostova, Kazantsev. Neurodegener. Dis. Manag. 2017, 7, 97-100; Johnson, Johnson. Free Radic Biol Med. 2015, 88, 253-267).

[0065] In one aspect, the invention provides a method of activating NRF2 in a cell, the method comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0066] In a related aspect, the invention provides a method of increasing transcription of genes transactivated by NRF2 in a cell, the method comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0067] In one embodiment, the cell in one of the above aspects or other aspects herein is a cell type or derived from a tissue selected from any one or more of adrenal gland, bone marrow, brain, breast, bronchus, caudate nucleus, cerebellum, cerebral cortex, cervix, uterus, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, myocardium, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid, placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including the duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid, tonsils, bladder and vagina. In further embodiments, the brain cells are from brain tissue selected from the cerebrum (including the cerebral cortex, basal ganglia (often called the striatum), and olfactory bulb), cerebellum (including the dentate nucleus, interpositus nucleus, fastigial nucleus, and vestibular nuclei), diencephalon (including the thalamus, hypothalamus, etc., and the posterior part of the pituitary gland), and brainstem (including the pons, substantia nigra, and medulla oblongata). In further embodiments, the brain cells are selected from neurons or glial cells (e.g., astrocytes, oligodendrocytes, or microglia). In further embodiments, the neurons are sensory neurons, motor neurons, interneurons, or brain neurons.

[0068] In one embodiment, the cell is an animal cell, such as a mammalian cell. In a further embodiment, the cell is a human cell or a non-human cell. In a further embodiment, the cell is in vitro, in vivo, or ex vivo.

[0069] In another embodiment, the cell is a pathological cell. In another embodiment, the cell is a pathological cell from a patient suffering from the following diseases or disorders:

[0070] In another aspect, the present invention provides a method for treating an animal having a disease or disorder that would benefit from increased NRF2 activation, or for preventing or reducing the risk of acquiring a disease or disorder in an animal, comprising administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. In one embodiment, the animal is a mammal. In another embodiment, the mammal is a human or non-human mammal. In a further embodiment, the mammal is a human. In another embodiment, the disease or disorder is selected from the group consisting of age-related tau astrogliopathy (ARTA), ALS, Alzheimer's disease, argyrophilic grain disease, asthma, cerebral amyloid angiopathy, cerebral ischemia, Christianson syndrome, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, diffuse neurofibrillary tangles with calcification, Down's syndrome, emphysema, familial British dementia, familial Danish dementia, fatal familial insomnia, Friedreich's ataxia, frontotemporal dementia, Gerstmann-Straussler-Human dementia, and others. The disease is selected from any one or more of Scheinker's syndrome, Guadeloupe Parkinsonism, Huntington's disease, kuru, mitochondrial myopathy, multiple sclerosis, multiple system atrophy, myotonic dystrophy, cerebral iron accumulation neurodegeneration, neuronal ceroid lipofuscinosis, Parkinson's disease dementia, Parkinson's disease, Guam Parkinson's disease dementia, Pick's disease, postencephalitic parkinsonism, primary age-related tauopathy (PART), progressive supranuclear palsy, pulmonary fibrosis, sepsis, septic shock, subacute sclerosing panencephalitis, vascular dementia, or a disease related thereto.

[0071] The above and other features and advantages of the present invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. Such description is meant to be illustrative and not limiting of the present invention. Obvious variations of the disclosed (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol crystalline complex in this document, such as those described by the drawings and examples, will be readily apparent to those skilled in the art having this disclosure, and such variations are considered to be part of the present invention. [Brief description of the drawings]

[0072] [Figure 1] Results of HSF1 and NRF2 gene expression compared to Gapdh following (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administration in a preclinical in vivo model. [Diagram 2] Results of HSF1 and NRF2 gene expression compared to Actb following (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administration in a preclinical in vivo model. [Diagram 3] Results of HSF1 and NRF2 gene expression relative to Gapdh in mouse cortical tissue 6 hours after the final administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in a 7-day repeated administration study in mice. [Figure 4] Results of HSF1 and NRF2 gene expression relative to Gapdh in mouse cortical tissue 24 hours after the final administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in a 7-day repeated administration study in mice. [Diagram 5]Hspa1a gene expression in mouse brain tissue after the final dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in a 4-day repeated dose study in mice. Two-way ANOVA with repeated measures with Dunnett's post-hoc test. *p<0.5. [Figure 6] Hspa8 gene expression in mouse brain tissue after the last dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in a 4-day repeated dose study in mice. Two-way ANOVA with repeated measures with Dunnett's post-hoc test. **p<0.01. [Figure 7A] Mouse weight over time. Mouse weights for the 3 month cohort plotted as mean + / - SD (n=6). There is no significant difference between animals in the two (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment groups and vehicle-treated animals. [Figure 7B] Mouse weight over time. Mouse weights for the 6 month cohort plotted as mean + / - SD (n=14). There is a significant decrease in body weight for 2.5 mg / kg treated animals compared to vehicle treated animals between 121 days and 6 months of age. Two-way ANOVA with repeated measures with Dunnett's post-hoc test. *p<0.5, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 8] Rotarod performance over time. Rotarod performance over the course of the test plotted as mean fall latency + / - SD (n=14). There is a significant increase in rotarod performance in the 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treated group compared to the vehicle treated group. Two-way ANOVA with Dunnett's post-hoc test. *p<0.5. [Figure 9A] Catwalk gait analysis at 3 and 6 months showing forelimb base of support (BOS). [Figure 9B] Catwalk gait analysis at 3 and 6 months showing hindlimb base of support (BOS). [Figure 9C] Catwalk gait analysis at 3 and 6 months showing % of time on diagonal limbs. [Figure 10A] Catwalk gait analysis at 3 and 6 months showing % time on all three legs. [Figure 10B] Catwalk gait analysis at 3 and 6 months showing % of time on all four legs. [Figure 11A] Catwalk gait analysis showing changes in forelimb BOS between 3 and 6 months of age. [Figure 11B] Catwalk gait analysis showing changes in hindlimb BOS between 3 and 6 months of age. [Figure 12A] Catwalk gait analysis showing the change in % time spent on the diagonal paw between 3 and 6 months of age. There is a significant difference in the change in % time spent on the diagonal paw between the 2.5 mg / kg twice daily and vehicle groups, with the vehicle having a decrease in time spent on the diagonal paw between 3 and 6 months and the 2.5 mg / kg twice daily group having a slight increase. There is a significant decrease in % time spent on the 3 paws in the 2.5 mg / kg twice daily group compared to the vehicle group. One-way ANOVA with Dunnett's post-hoc test. *p<0.5. n=8 / group. [Figure 12B] Catwalk gait analysis showing the change in % time spent on three paws between 3 and 6 months of age. There is a significant difference in the change in % time spent on the diagonal paw between the 2.5 mg / kg twice daily and vehicle groups, with the vehicle having a decrease in time spent on the diagonal paw between 3 and 6 months and the 2.5 mg / kg twice daily group having a slight increase. There is a significant decrease in % time spent on three paws in the 2.5 mg / kg twice daily group compared to the vehicle group. One-way ANOVA with Dunnett's post-hoc test. *p<0.5. n=8 / group. [Figure 13] Catwalk gait analysis showing the change in % time spent on all four legs between 3 and 6 months of age. [Figure 14]Compound muscle action potential (CMAP) amplitudes and repetitive stimulation. CMAP plotted as individual values ​​and mean + / - SD (n=14). Two-way ANOVA with Sidac post-hoc test. [Figure 15] Relative CMAP at 6 months calculated based on individual CMAP at 3 months. [Figure 16] Repeated stimulation at 6 weeks and 3 months of age plotted as % of first stimulation, mean + / - SD for each stimulation (n=14). [Figure 17] Repeated stimulation at 6 months of age plotted as % of the first stimulation, mean + / - SD for each stimulation (n=14). [Figure 18] qPCR results for 3 month cortical tissue. Mean relative mRNA levels from 3 month cohort cortical tissue normalized to Gapdh and vehicle + / - SD (n=6). Two-way ANOVA with Dunnett's post-hoc test. *p<0.5, **p<0.01, ****p<0.0001. [Figure 19] qPCR results for 6 month cortical tissue. Mean relative mRNA levels from 6 month cohort cortical tissue normalized to Gapdh and vehicle + / - SD (n=7). Two-way ANOVA with Dunnett's post-hoc test. ****p<0.0001. [Figure 20] Protein quantification data demonstrated that (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol induced a significant increase in NQO1 after 48 h treatment at 10 μM in induced astrocytes from healthy individuals (CTR, n=3); patients with C9orf72 mutations (C9orf72, n=3); sporadic ALS patients (sALS, n=3) and patients with SOD1 mutations (SOD1, n=3). Statistical test: One-way ANOVA with multiple comparison test. Statistically, 10 μM (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (shown as drug) is compared with DMSO treatment. *p<0.1, **p<0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Detailed Description of the Preferred Embodiments definition The term "(6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol" means R-(-)-10,11-dihydroxyaporphine, as well as prodrugs, salts, solvates, hydrates, and co-crystals thereof.

[0074] The term "(6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol" means S-(+)-10,11-dihydroxyaporphine, as well as prodrugs, salts, solvates, hydrates, and co-crystals thereof.

[0075] The term "6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol" refers to (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol or (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,1 1-diol, or the racemic forms of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, as well as prodrugs, salts, solvates, hydrates, and co-crystals thereof.

[0076] As used herein, the terms "treat", "treating" or "treatment" refer to alleviating, reducing or eliminating one or more symptoms or characteristics of a disease, which may be curing, palliating, preventing or slowing the progression of the disease.

[0077] The term "effective amount" refers to an amount that induces activation of HSF1 and / or NRF2, as well as the desired effect or outcome, as applicable or specified. The term "therapeutically effective amount" refers to an amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, alone or in combination with other active ingredients, that induces a desired biological or pharmacological response, such as an amount that is effective to prevent, alleviate, or ameliorate the symptoms of a disease or disorder; effective to slow, stop, or reverse the process or progression of an underlying disease; effective to partially or completely restore the function of a cell; or effective to prolong the survival of the subject being treated.

[0078] As used herein, the term "HSF1 activation" or "activation of HSF1" refers to the dissociation of HSF1 from inhibitory complexes (including Hsp40, Hsp70, TRiC and Hsp90) in the cytoplasm and the accumulation of homotrimeric HSF1 in the nucleus.

[0079] As used herein, the term "NRF2 activation" or "activation of NRF2" refers to the dissociation of NRF2 from the NRF2 regulator Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and accumulation of NRF2 in the nucleus.

[0080] The term "patient" or "subject" includes mammals, including non-human animals, particularly humans. In one embodiment, the patient or subject is a human. In another embodiment, the patient or subject is a human male. In another embodiment, the patient or subject is a human female.

[0081] The terms "significant" or "significantly" are determined by t-test at a significance level of 0.05.

[0082] The present invention relates to methods of use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for activating HSF1, activating the HSF1 pathway, increasing the transcription levels of genes positively regulated by HSF1, increasing the levels of protein chaperones and / or co-chaperones, reducing the amount of protein misfolding, or reducing the accumulation of misfolded proteins in cells, tissues, or animals.

[0083] The present invention further relates to a method of using 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for the treatment of a disease or disorder mediated by protein misfolding or accumulation of misfolded proteins, or to prevent, alleviate, ameliorate or reduce the risk of acquiring a disease or disorder, by activating the HSF1 pathway.The present invention further relates to a method of using 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for extending / increasing the life span of a cell, tissue, organ or animal.

[0084] Thus, in one aspect, the present invention provides a method for activating HSF1 in a cell, the method comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0085] In a related aspect, the invention provides a method of increasing transcription of a gene transactivated by HSF1 in a cell, comprising contacting said cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. In one embodiment, the gene encodes a protein selected from any one or more of PPARGC1A (PGC1 alpha), DLG4 (PSD95), SYN1 (synapsin), BDNF, HSP70, HSP40 (cysteine ​​string protein alpha, including auxilin), HSPA8 (HSC70), HSPB8, or BAG3.

[0086] In another aspect, the present invention provides a method of increasing protein chaperone and / or co-chaperone levels in a cell comprising contacting said cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. In one embodiment, the protein chaperone and / or co-chaperone is selected from any one or more of HSP70, HSP40 (including cysteine ​​string protein alpha, auxilin), HSPA8 (HSC70), HSPB8, or BAG3.

[0087] In another aspect, the present invention provides a method for (a) reducing protein misfolding in a cell in relation to the frequency or rate at which protein misfolding occurs, (b) reducing the accumulation of misfolded proteins in a cell, or (c) reducing protein aggregation, particularly the aggregation of misfolded proteins, in a cell, comprising contacting said cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0088] In another aspect, the present invention provides a method of increasing the lifespan of a cell, comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0089] In one embodiment, the cell in one of the above aspects or other aspects or embodiments herein is a cell type or derived from a tissue selected from any one or more of adrenal gland, bone marrow, brain, breast, bronchus, caudate nucleus, cerebellum, cerebral cortex, cervix, uterus, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, myocardium, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid, placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including the duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid, tonsils, bladder and vagina. In further embodiments, the brain cells are from brain tissue selected from the cerebrum (including the cerebral cortex, basal ganglia (often called the striatum), and olfactory bulb), cerebellum (including the dentate nucleus, interpositus nucleus, fastigial nucleus, and vestibular nuclei), diencephalon (including the thalamus, hypothalamus, etc., and the posterior part of the pituitary gland), and brainstem (including the pons, substantia nigra, and medulla oblongata). In further embodiments, the brain cells are selected from neurons or glial cells (e.g., astrocytes, oligodendrocytes, or microglia). In further embodiments, the neurons are sensory neurons, motor neurons, interneurons, or brain neurons.

[0090] In one embodiment, the cell is an animal cell, such as a mammalian cell. In a further embodiment, the cell is a human cell or a non-human cell. In a further embodiment, the cell is in vitro, in vivo, or ex vivo.

[0091] In another embodiment, the cell is a pathological cell. In another embodiment, the cell is a pathological cell from a patient suffering from a disease or disorder disclosed herein.

[0092] In another aspect, the invention provides a method of treating an animal having a disease or disorder (a) whose symptoms are prevented, alleviated or ameliorated by HSF1 activation, or (b) whose disease process or progression is slowed, stopped or reversed by HSF1 metabolization, comprising administering to said animal a therapeutically effective amount of a pharmaceutical composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. In one embodiment, the animal is a mammal. In a further embodiment, the mammal is a human. In another embodiment, the mammal is a non-human.

[0093] In another aspect, the present invention provides a method of (a) treating an animal having a disease or disorder that would benefit from HSF1 activation, or (b) preventing or reducing the risk of acquiring said disease or disorder, comprising administering to said animal a therapeutically effective amount of a pharmaceutical composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. In one embodiment, the animal is a mammal. In another embodiment, the mammal is a human or non-human mammal. In another embodiment, the disease or disorder is caused by protein misfolding, accumulation of misfolded proteins, or protein aggregation. In another embodiment, the disease is Age-related Tau Astrogliopathy (ARTA), Alexander's Disease, Alpers-Huttenlocher Syndrome, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Ataxic Neuropathy Spectrum, Ataxia Retinitis Pigmentosa (NARP), Critical Illness Myopathy (CIM), Primary Age-related Tauopathy (PART), Aortic Medial Amyloidosis, Apo AI Amyloidosis, Apo AII Amyloidosis, Apo AIV Amyloidosis, Argyrophilic Grain Disease, Ataxia-Telangiectasia, Atrial Fibrillation, Autosomal Dominant Hyper IgE Syndrome, Atrial Amyloidosis, Bloom's Syndrome, Cardiovascular Disease (including coronary artery disease, myocardial infarction, stroke, restenosis and arteriosclerosis), Cataract, Cerebral Amyloid Angiopathy, Christian's Syndrome, Chronic Obstructive Tauopathy (CHTA), ... Son syndrome, chronic traumatic encephalopathy, chronic progressive external ophthalmoplegia syndrome (CPEO), Cockayne syndrome, congenital lactic acidosis (CLA), corneal amyloidosis due to lactoferrin, corticobasal degeneration, Crohn's disease, Cushing's disease, lichen amyloidosis, cystic fibrosis, dentatorubral-pallidoluysian atrophy (DRPLA), dialysis amyloidosis, diffuse neurofibrillary tangles with calcification, Down's syndrome, endotoxic shock, familial amyloidosis of the Finnish type, familial amyloid neuropathy, familial British dementia (FBD), familial Danish dementia (FDD), familial dementia, fibrinogen amyloidosis, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Friedreich's ataxia, frontotemporal lobar degeneration, glaucoma,Glycogen storage disease type IV (Andersen's disease), Guadeloupe parkinsonism, hereditary lattice corneal degeneration, Huntington's disease, inclusion body myositis / myopathy, inflammation, inflammatory bowel disease, ischemic conditions (including ischemia / reperfusion injury, myocardial ischemia, stable angina, unstable angina, stroke, ischemic heart disease and cerebral ischemia), light or heavy chain amyloidosis, lysosomal storage diseases (aspartylglucosaminuria, Fabry disease, Batten disease, cystinosis, Farber disease, fucosidosis, galactosidosis, Gaucher disease (including types 1, 2 and 3), Gm1 gangliosidosis, Hansen's disease, α-mannosidosis, Kearns-Sayre syndrome (KSS), lactic acidosis-strokelike episodes (MELAS) syndrome, Leber's hereditary optic neuropathy (LHON), β-mannosidosis, Maroteaux-Lamy disease, MEGDEL syndrome (also known as 3-methylglutaconic aciduria with hearing loss, encephalopathy and Leigh-like syndrome), metachromatic leukodystrophy, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome, Morquio syndrome type A, Morquio syndrome type B, mucolipidosis type II, mucolipidosis type III, type III, myoclonic epilepsy-myopathy-sensory ataxia, mitochondrial myopathy, myoclonic epilepsy with ragged red fibers (MERRF), Niemann-Pick disease (including types A, B, and C), neurogenic muscle weakness, Pearson syndrome, Pompe disease, Sandhoff disease, Sanfilich syndrome (including types A, B, C, and D), Schindler disease, Schindler-Kanzaki disease, Saengers syndrome, sialidosis, Sly syndrome, Tay-Sachs disease, Wolman disease, lysozyme amyloidosis, Mallory bodies, medullary thyroid carcinoma, mitochondrial myopathy, Multiple sclerosis, Multiple system atrophy, Myotonic dystrophy, Myotonic dystrophy, Cerebral iron storage neurodegeneration, Neurofibromatosis, Neuronal ceroid lipofuscinosis, Amyloidogenic odontogenic (Pymborg) tumor, Guam Parkinson's dementia, Parkinson's disease, Peptic ulcer, Pick's disease, Pituitary prolactinoma, Postencephalitic parkinsonism, Prion diseases (also known as transmissible spongiform encephalopathies or TSEs, Creutzfeldt-Jakob disease (CJD), Variant Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, Fatal familial insomnia,and kuru), progressive supranuclear palsy, pulmonary alveolar proteinosis, retinal ganglion cell degeneration in glaucoma, retinitis pigmentosa with rhodopsin mutations, seminal vesicle amyloidosis, senile systemic amyloidosis, serpinopathies, sickle cell disease, spinal and bulbar muscular atrophy (SBMA) (also known as Kennedy disease), spinocerebellar degeneration (including spinocerebellar degeneration type 1, spinocerebellar degeneration type 2, spinocerebellar degeneration type 3 (Machado-Joseph disease), spinocerebellar degeneration type 6, spinocerebellar degeneration type 7, spinocerebellar degeneration type 8 and spinocerebellar degeneration type 17), subacute sclerosing panencephalitis, tauopathies, type II diabetes mellitus, vascular dementia, Werner syndrome, atherosclerosis, autism spectrum disorder (ASD), benign focal atrophy, Duchenne palsy, hereditary spastic paraplegia (HSP), Kugelberg-Welander syndrome group, Lou Gehrig's disease, necrotizing enterocolitis, Paget's disease of bone (PDB), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, valosin-containing protein (VCP)-related disorder, or Werdnig-Hoffmann disease, transient ischemic attack, ischemia, intracerebral hemorrhage, senile cataract, retinal ischemia, retinal vasculitis, Brown-William's disease, and other conditions. selected from any one or more of: Alette-Van Lare syndrome, Eales' disease, meningoencephalitis, post-traumatic stress disorder, Charcot-Marie-Tooth disease, macular degeneration, X-linked spinal-bulbar muscular atrophy (Kennedy disease), presenile dementia, depressive disorder, temporal lobe epilepsy, Leber's hereditary optic atrophy, cerebrovascular disease, subarachnoid hemorrhage, schizophrenia, demyelinating disorders, and Pelicheus-Merzbacher disease;

[0094] In another embodiment, the disease is a neurological disease.

[0095] In one embodiment, the disease is selected from any one or more of lysosomal storage diseases (e.g., Niemann-Pick disease type C, Gaucher disease), inclusion body myositis, spinocerebellar degeneration, spinal-bulbar muscular atrophy, or a disease related thereto.

[0096] In another embodiment, the disease is selected from one or more of ALS, frontotemporal dementia, Huntington's disease, Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, Parkinson's dementia, brain iron accumulation neurodegeneration, diffuse neurofibrillary tangles with calcification, multiple system atrophy, cerebral amyloid angiopathy, vascular dementia, Down's syndrome, Creutzfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Straussler-Scheinker syndrome, kuru, familial British dementia, familial Danish dementia, Parkinson's dementia of Guam, myotonic dystrophy, neuronal ceroid lipofuscinosis, or a disease related thereto.

[0097] In another embodiment, the neurological disease is selected from any one or more of Friedreich's ataxia, multiple sclerosis, mitochondrial myopathy, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, argyrophilic grain disease, subacute sclerosing panencephalitis, Christianson's syndrome, postencephalitic parkinsonism, Guadeloupe parkinsonism, age-associated tau astrogliopathy (ARTA), and primary age-associated tauopathy (PART), Pick's disease, or a disease related thereto.

[0098] In another aspect, the present invention provides a method of increasing life span in an animal, and treating a disease or disorder that results in accelerated aging or other abnormal aging processes, comprising administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. In one embodiment, the animal is a mammal. In another embodiment, the mammal is a human or non-human mammal. In one embodiment, the disease or disorder is selected from Werner syndrome, Hutchinson-Gilford disease, Bloom's syndrome, Cockayne's syndrome, ataxia-telangiectasia, and Down's syndrome.

[0099] In a related aspect, the invention provides a method for treating premature aging due to chemical or radiation exposure. In one embodiment, the premature aging is due to exposure to chemotherapeutic agents, radiotherapeutic agents, or UV radiation. In a further embodiment, the UV radiation is artificial, e.g., from a tanning bed, or solar UV radiation, i.e., exposure to sunlight.

[0100] Physical exercise leads to muscle adaptations, including muscle atrophy caused by muscle protein catabolism or muscle hypertrophy caused by muscle protein accretion. In muscle hypertrophy, new proteins are formed. The increased presence of molecular chaperones would act to enhance the stability of these rapidly forming new proteins by preventing misfolding and catabolism. 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may therefore be used to stabilize new proteins by reducing their misfolding and catabolism in situations of enhanced protein turnover, for example after physical exercise. Thus, in another aspect, the present invention relates to a method of increasing muscle hypertrophy or reducing muscle atrophy in an animal after physical exercise, comprising administering to said animal a therapeutically effective amount of a pharmaceutical composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0101] The invention further provides the use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for the preparation of a medicament for treating a human having any one of the diseases or disorders disclosed herein, or for use in any of the methods of the invention, comprising the administration of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to the human.

[0102] In another aspect, the present invention provides an in vitro method of screening a candidate therapeutic agent(s) for its ability to activate the HSF1 pathway, comprising: (a) exposing induced astrocytes derived from fibroblast stem cells to said candidate therapeutic agent; (b) comparing the amount of misfolded SOD1 between the induced astrocytes exposed to the candidate therapeutic agent and a control cell, such as an induced astrocyte that has not been exposed to the candidate therapeutic agent (i.e., an unexposed induced astrocyte); The present invention provides an in vitro method comprising:

[0103] The present invention relates to a method of use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to activate NRF2 or to activate the NRF2 pathway.

[0104] The present invention further relates to a method of reducing oxidative stress in a cell, comprising administering to said cell an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0105] In one aspect, the invention provides a method of activating NRF2 in a cell, the method comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0106] In a related aspect, the invention provides a method of increasing transcription of genes transactivated by NRF2 in a cell, the method comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0107] In one embodiment, the cell in one of the above aspects or other aspects herein is a cell type or derived from a tissue selected from any one or more of adrenal gland, bone marrow, brain, breast, bronchus, caudate nucleus, cerebellum, cerebral cortex, cervix, uterus, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, myocardium, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid, placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including the duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid, tonsils, bladder and vagina. In further embodiments, the brain cells are from brain tissue selected from the cerebrum (including the cerebral cortex, basal ganglia (often called the striatum), and olfactory bulb), cerebellum (including the dentate nucleus, interpositus nucleus, fastigial nucleus, and vestibular nuclei), diencephalon (including the thalamus, hypothalamus, etc., and the posterior part of the pituitary gland), and brainstem (including the pons, substantia nigra, and medulla oblongata). In further embodiments, the brain cells are selected from neurons or glial cells (e.g., astrocytes, oligodendrocytes, or microglia). In further embodiments, the neurons are sensory neurons, motor neurons, interneurons, or brain neurons.

[0108] In one embodiment, the cell is an animal cell, such as a mammalian cell. In a further embodiment, the cell is a human cell or a non-human cell. In a further embodiment, the cell is in vitro, in vivo, or ex vivo.

[0109] In another embodiment, the cell is a pathological cell. In another embodiment, the cell is a pathological cell from a patient suffering from the following diseases or disorders:

[0110] In another aspect, the present invention provides a method for treating an animal having a disease or disorder that would benefit from increased activation of NRF2 or combined activation of HSF1 and NRF2, or for preventing or reducing the risk of acquiring a disease or disorder in an animal, comprising administering to said animal a therapeutically effective amount of a pharmaceutical composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. 6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may further be used in a method for treating a disease or disorder in an animal mediated by increased oxidative stress or by reduced NRF2 activity, comprising administering to said animal an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. 6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may further be used in a method for reducing inflammation in an animal or for treating a disease or disorder mediated by inflammation comprising administering to the animal an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0111] In one embodiment, the animal is a mammal. In another embodiment, the mammal is a human or non-human mammal. In a further embodiment, the mammal is a human. In another embodiment, the disease or disorder is age-related tau astrogliopathy (ARTA), ALS, Alzheimer's disease, argyrophilic grain disease, asthma, cerebral amyloid angiopathy, cerebral ischemia, Christianson syndrome, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, diffuse neurofibrillary tangles with calcification, Down's syndrome, emphysema, familial British dementia, familial Danish dementia, fatal familial insomnia, Friedreich's ataxia, frontotemporal dementia, Gerstmann-Straussler-Lewis syndrome, cerebrovascular disease, cerebrovascular accident ... The disease is selected from any one or more of Scheinker's syndrome, Guadeloupe Parkinsonism, Huntington's disease, kuru, mitochondrial myopathy, multiple sclerosis, multiple system atrophy, myotonic dystrophy, cerebral iron accumulation neurodegeneration, neuronal ceroid lipofuscinosis, Parkinson's disease dementia, Parkinson's disease, Guam Parkinson's disease dementia, Pick's disease, postencephalitic parkinsonism, primary age-related tauopathy (PART), progressive supranuclear palsy, pulmonary fibrosis, sepsis, septic shock, subacute sclerosing panencephalitis, vascular dementia, or a disease related thereto.

[0112] The pharmaceutical composition of the present invention comprises a therapeutically effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharma- ceutically acceptable excipient. The term "excipient" refers to a pharma- ceutically acceptable inert substance used as a carrier for the therapeutically active ingredient (6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol), and includes anti-adhesives, binders, coatings, disintegrants, fillers, diluents, solvents, flavorings, bulkants, colorants, glidants, dispersants, wetting agents, lubricants, preservatives, adsorbents and sweeteners. The choice of excipient(s) will depend on factors such as the specific mode of administration and the nature of the dosage form. Solutions or suspensions used for injection or infusion may include sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes including autoinjectors, or multiple dose vials made of glass or plastic.

[0113] The pharmaceutical formulation of the present invention may be any pharmaceutical dosage form. The pharmaceutical formulation may be, for example, a tablet, a capsule, a nanoparticulate material, such as a granulated particulate material or a powder, a lyophilized material for reconstitution, a liquid solution, a suspension, an emulsion or other liquid form, an injectable suspension, a solution, an emulsion, etc., a suppository, or a topical or transdermal preparation or patch. The pharmaceutical formulation generally contains about 1 to about 99% by weight of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and 99 to 1% by weight of suitable pharmaceutical excipients. In one embodiment, the dosage form is an oral dosage form. In another embodiment, the dosage form is a parenteral dosage form. In another embodiment, the dosage form is an enteral dosage form. In another embodiment, the dosage form is a topical dosage form. In one embodiment, the pharmaceutical dosage form is a unit dose. The term "unit dose" refers to the amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered to a patient in one dose.

[0114] In some embodiments, a pharmaceutical composition of the invention is delivered to a subject via a parenteral, enteral, or topical route.

[0115] Examples of parenteral routes of the present invention include, but are not limited to, any one or more of the following: intraabdominal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intravesical, intracardiac, intracartilaginous, intrasacral, intracavity, intracavity, intracerebral, intracisternal, intracorneal, intracoronary, intracavernous, intracranial, intradermal, intraspinal, intraductal, intraduodenal, intradural, epidermal. intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intrabone marrow, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intraocular, intranasal, intraspinal, intrasynovial, intratendon, intratesticular, intrathecal, intrathoracic, intrarenal tubule, intratumor, intratympanic, intrauterine, intravascular, intravenous (bolus or infusion), intraventricular, intravesical, and / or subcutaneous.

[0116] Enteral administration routes of the present invention include administration to the gastrointestinal tract via the mouth (oral), stomach (intragastric), and rectum (rectal). Intragastric administration typically involves the use of a tube through the nostrils (NG tube) or a tube in the esophagus (PEG tube) that leads directly to the stomach. Rectal administration typically involves a rectal suppository. Oral administration includes sublingual and buccal administration.

[0117] Topical administration includes administration to a body surface such as the skin or mucous membranes, including intranasal and pulmonary administration. Transdermal forms include creams, foams, gels, lotions, or ointments. Intranasal and pulmonary forms include liquids and powders, such as liquid sprays.

[0118] Dosage may vary according to the dosage form used, the sensitivity of the patient, and the route of administration.Dosage and administration are adjusted to provide sufficient level of active agent(s) or to maintain desired effect.Factors that may be considered include: severity of disease state, overall health of the subject, age, weight and sex of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivity, and tolerability / response to treatment.

[0119] In one embodiment, the daily dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered to the patient is selected from up to 200 mg, 175 mg, 150 mg, 125 mg, 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 30 mg, 25 mg, 20 mg, 15 mg, 14 mg, 13 mg, 12 mg, 11 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, or up to 2 mg. In another embodiment, the daily dose is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 2,000 mg, 3,000 mg, 4,000 mg, or at least 5,000 mg. In another embodiment, the daily dose is 1-2 mg, 2-4 mg, 1-5 mg, 5-7.5 mg, 7.5-10 mg, 10-15 mg, 10-12.5 mg, 12.5-15 mg, 15-17.7 mg, 17.5-20 mg, 20-25 mg, 20-22.5 mg, 22.5-25 mg, 25-30 mg, 25-27.5 mg, 27.5-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, or 45-50 mg, 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 5-200 mg, 5-300 mg, 5-400 mg, 5-500 mg, 5-600 mg, 5-700 mg, 5-800 mg, 5-900 mg, 5-1,000 mg, 5-2,000 mg, 5-5,000 mg, or more than 5,000 mg.

[0120] In another embodiment, the dose of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered to a patient is 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 350 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 400 mg, 45 mg, 50 mg, 50 mg, 60 mg, 70 mg, 70 mg, 80 mg, 80 mg, 90 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 2 In one embodiment, the dose is selected from 0 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 2,000 mg, 3,000 mg, 4,000 mg, or 5,000 mg. In one embodiment, the dose is administered by a route selected from any one of oral, buccal, or sublingual administration.In another embodiment, the dose is administered by injection, such as subcutaneous, intramuscular, or intravenous.In another embodiment, the dose is administered by inhalation or intranasal administration.

[0121] As a non-limiting example, the dose of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered by subcutaneous injection may be about 3-50 mg / day administered in divided doses. A dose of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered by subcutaneous injection may be about 1-6 mg, preferably about 1-4 mg, 1-3 mg, or 2 mg. Other embodiments include doses in the range of about 5-5,000 mg, preferably about 100-1,000 mg, 100-500 mg, 200-400 mg, 250-350 mg, or 300 mg. Subcutaneous infusion would be preferred in those patients who require more than 10 divided injections per day. The continuous subcutaneous infusion dose can be 1 mg / hour / day, generally increased to 4 mg / hour depending on response.

[0122] The fine particle dose of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered by pulmonary administration, e.g., by inhalation using a pressurized metered dose inhaler (pMDI), dry powder inhaler (DPI), soft mist inhaler, nebulizer, or other device, may range from about 0.5 to 15 mg, preferably from about 0.5 to 8 mg or from 2 to 6 mg. Other embodiments include doses in the range of about 5 to 5,000 mg, preferably from about 100 to 1,000 mg, 100 to 500 mg, 200 to 400 mg, 250 to 350 mg, or 300 mg. The nominal dose (ND), i.e., the amount of drug metered into a receptacle (also known as the metered dose), of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered by pulmonary administration may be in the range of, for example, 0.5-15 mg, 3-10 mg, 10-15 mg, 10-12.5 mg, 12.5-15 mg, 15-17.7 mg, 17.5-20 mg, 20-25 mg, 20-22.5 mg, 22.5-25 mg, 25-30 mg, 25-27.5 mg, 27.5-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, or 45-50 mg. Other embodiments include ranges of about 5-5,000 mg, preferably about 100-1,000 mg, 100-500 mg, 200-400 mg, 250-350 mg, or 300 mg.

[0123] Long-acting pharmaceutical compositions may be administered daily (preferably 10 times or less per day), every other day, every 3-4 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times per week, or once every two weeks depending on the half-life and clearance rate of the particular compound.

[0124] In embodiments of any of the above methods and compositions, the 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, a prodrug of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, or salts, solvates, hydrates, and cocrystals thereof are a racemic mixture of the R and S enantiomers, or are enriched in the R enantiomer (i.e., the 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in the composition). the ratio of R enantiomer to S enantiomer for all of the tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol or for all of the 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered is 5:1 to 1,000:1, 10:1 to 10,000:1, or 100:1 to 100,000:1, or the ratio of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in the composition as a whole the 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol enantiomer is at least 98% R enantiomer, 99% R enantiomer, 99.5% R enantiomer, 99.9% R enantiomer, or does not contain an observable amount of the S enantiomer) or is enriched in the S enantiomer (i.e., all of the 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in the composition or all of the 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol administered the ratio of S-enantiomer to R-enantiomer of the 10,11-diol is between 5:1 and 1,000:1, 10:1 and 10,000:1, or 100:1 and 100,000:1, or the 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol enantiomers in the composition as a whole are at least 98% S-enantiomer, 99% S-enantiomer, 99.5% S-enantiomer, 99.9% S-enantiomer, or contain no observable amount of R-enantiomer).

[0125] The invention further provides an in vitro or ex vivo method of activating HSF1 in a cell; increasing transcription of genes transactivated by HSF1 in a cell; increasing protein chaperone and / or co-chaperone levels (such as one or more of HSP70, HSP40 (including cysteine ​​string protein alpha, auxilin), HSPA8 (HSC70), HSPB8, or BAG3) in a cell; or reducing protein misfolding, accumulation of misfolded proteins, or aggregated proteins in a cell, comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (such as (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol).

[0126] Suitably, the misfolded or aggregated protein may be selected from any one of TDP-43, SOD1, hyperphosphorylated tau, hexanucleotide repeat expansion C9orf72, beta amyloid, alpha synuclein, polyglutamine repeat expansion, FUS, hnRNP, ATXN2, or prion proteins.

[0127] Suitably, in the method of the present invention, the cell may be of a cell type or be derived from tissue selected from any one or more of the following: adrenal gland, bone marrow, brain, breast, bronchus, caudate nucleus, cerebellum, cerebral cortex, cervix, uterus, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, cardiac muscle, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid gland, placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid gland, tonsil, bladder or vagina.Suitably, the brain cell may be derived from brain tissue selected from cerebrum, cerebellum, diencephalon and brain stem.Suitably, the brain cell may be selected from neurons (such as sensory neurons, motor neurons, interneurons or brain neurons), astrocytes, oligodendrocytes or microglia.

[0128] Suitably the cell may be an animal cell (such as a human cell).

[0129] Suitably, the cells are associated with a variety of conditions, including age-associated tau astrogliopathy (ARTA), Alexander disease, Alpers-Huttenlocher syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxic neuropathy spectrum, ataxia retinitis pigmentosa (NARP), critical illness myopathy (CIM), primary age-associated tauopathy (PART), aortic medial amyloidosis, apo AI amyloidosis, apo AII amyloidosis, apo AIV amyloidosis, argyrophilic grain disease, ataxia telangiectasia, atrial fibrillation, autosomal dominant hyper IgE syndrome. group, atrial amyloidosis, Bloom's syndrome, cardiovascular disease, coronary artery disease, myocardial infarction, stroke, restenosis, arteriosclerosis, cataract, cerebral amyloid angiopathy, Christianson syndrome, chronic traumatic encephalopathy, chronic progressive external ophthalmoplegia syndrome (CPEO), Cockayne's syndrome, congenital lactic acidosis (CLA), lactoferrin-induced corneal amyloidosis, corticobasal degeneration, Crohn's disease, Cushing's disease, lichen amyloidosis, cystic fibrosis, dentatorubral-pallidoluysian atrophy (DRPLA), dialysis amyloidosis, diffuse neurogenic fibrillary tangles with calcification, Dow Streptococcus aureus syndrome, endotoxic shock, familial amyloidosis of the Finnish type, familial amyloid neuropathy, familial British dementia (FBD), familial Danish dementia (FDD), familial dementia, fibrinogen amyloidosis, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Friedreich's ataxia, frontotemporal lobar degeneration, glaucoma, glycogen storage disease type IV (Andersen's disease), Guadeloupe parkinsonism, hereditary lattice corneal degeneration, Huntington's disease, inclusion body myositis / myopathy, inflammation, inflammatory bowel disease, ischemic state, ischemia / reperfusion injury , myocardial ischemia, stable angina, unstable angina, stroke, ischemic heart disease and cerebral ischemia, light or heavy chain amyloidosis, lysosomal storage diseases, aspartylglucosaminuria, Fabry disease, Batten disease, cystinosis, Farber disease, fucosidosis, galactoside sialidosis, Gaucher disease types 1, 2 or 3, Gm1 gangliosidosis, Hunter disease, Hurler-Scheie disease, Krabbe disease, alpha-mannosidosis, Kearns-Sayre syndrome (KSS), lactic acidosis-strokelike episodes (MELAS) syndrome, Leber's hereditary optic neuropathy (LHON),β-mannosidosis, Maroteaux-Lamy disease, MEGDEL syndrome (also known as 3-methylglutaconic aciduria with deafness, encephalopathy and Leigh-like syndrome), metachromatic leukodystrophy, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome, Morquio syndrome type A, Morquio syndrome type B, mucolipidosis type II, mucolipidosis type III, myoclonic epilepsy-myopathy-sensory ataxia, mitochondrial myopathy, myoclonic epilepsy with ragged red fibers (MERRF), Niemann-Pick disease types A, B or C, neurogenic muscle weakness, Pi Asson syndrome, Pompe disease, Sandhoff disease, Sanfilich syndrome types A, B, C, or D, Schindler disease, Schindler-Kanzaki disease, Saengers syndrome, Sialidosis, Sly syndrome, Tay-Sachs disease, Wolman disease, Lysozyme amyloidosis, Mallory bodies, Medullary thyroid carcinoma, Mitochondrial myopathy, Multiple sclerosis, Multiple system atrophy, Myotonic dystrophy, Myotonic dystrophy, Cerebral iron storage neurodegeneration, Neurofibromatosis, Neuronal ceroid lipofuscinosis, Amyloidogenic odontogenic (Pinborg) tumor, Guam Parkinson's disease dementia, ... Son's disease, peptic ulcer disease, Pick's disease, pituitary prolactinoma, postencephalitic parkinsonism, prion diseases (transmissible spongiform encephalopathies), variant Creutzfeldt-Jakob disease including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, kuru, progressive supranuclear palsy, pulmonary alveolar proteinosis, retinal ganglion cell degeneration in glaucoma, retinitis pigmentosa with rhodopsin mutations, seminal vesicle amyloidosis, senile systemic amyloidosis, serpinopathies, sickle cell disease, spinal and bulbar muscular atrophy (SBMA), spinocerebellar degeneration , spinocerebellar degeneration type 1, spinocerebellar degeneration type 2, spinocerebellar degeneration type 3 (including Machado-Joseph disease), spinocerebellar degeneration type 6, spinocerebellar degeneration type 7, spinocerebellar degeneration type 8, and spinocerebellar degeneration type 17), subacute sclerosing panencephalitis, tauopathy, type II diabetes mellitus, vascular dementia, Werner syndrome, atherosclerosis, autism spectrum disorder (ASD), benign focal atrophy, Duchenne palsy, hereditary spastic paraplegia (HSP), Kugelberg-Welander syndrome, Lou Gehrig's disease, necrotizing enterocolitis, Paget's disease of bone (PDB), primary lateral sclerosis (PLS),have, or are at risk of acquiring, a disease or disorder selected from any one or more of: progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, valosin-containing protein (VCP)-related disorder, or Werdnig-Hoffmann disease, transient ischemic attack, ischemia, intracerebral hemorrhage, senile cataract, retinal ischemia, retinal vasculitis, Brown-Viarette-Van Lare syndrome, Eales disease, meningoencephalitis, post-traumatic stress disorder, Charcot-Marie-Tooth disease, macular degeneration, X-linked spinal-bulbar muscular atrophy (Kennedy disease), presenile dementia, depressive disorder, temporal lobe epilepsy, Leber's hereditary optic atrophy, cerebrovascular disease, subarachnoid hemorrhage, schizophrenia, demyelinating disorder, and Pelicheus-Merzbacher disease;

[0130] The present invention also relates to a therapeutically effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (a composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol) for use in: 1) treating an animal having a disease or disorder that would benefit from increased HSF1 activation in the subject; 2) preventing or reducing the risk of acquiring a disease or disorder in a subject by increasing HSF1 activation; and / or 3) increasing muscle hypertrophy or reducing muscle atrophy in an animal following physical exercise. Suitably the disease or disorder is selected from the group consisting of age-associated tau astrogliopathy (ARTA), Alpers-Huttenlocher syndrome, Alexander disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxic neuropathy spectrum, ataxia retinitis pigmentosa (NARP), critical illness myopathy (CIM), primary age-associated tauopathy (PART), aortic medial amyloidosis, apoAI. Amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, Argyrophilic grain disease, Ataxia telangiectasia, Atrial fibrillation, Autosomal dominant hyper-IgE syndrome, Atrial amyloidosis, Bloom's syndrome, Cardiovascular disease, Coronary artery disease, Myocardial infarction, Stroke, Restenosis, Atherosclerosis, Cataract, Cerebral amyloid angiopathy, Christianson syndrome, Chronic traumatic encephalopathy, Chronic progressive external ophthalmoplegia syndrome (CP) EO), Cockayne syndrome, congenital lactic acidosis (CLA), corneal amyloidosis due to lactoferrin, corticobasal degeneration, Crohn's disease, Cushing's disease, amyloidosis, cystic fibrosis, dentatorubral-pallidoluysian atrophy (DRPLA), dialysis amyloidosis, diffuse neurofibrillary tangles with calcification, Down's syndrome, endotoxic shock, familial amyloidosis of the Finnish type, familial amyloid neuropathy, familial British dementia (FBD), familial Danish dementia (FDD), familial dementia, fibrinogen amyloidosis, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Friedreich's ataxia, frontotemporal lobar degeneration, glaucoma, glycogen storage disease type IV (Andersen's disease), Guadeloupe parkinsonism, hereditary lattice corneal degeneration, Huntington's disease,Inclusion body myositis / myopathy, inflammation, inflammatory bowel disease, ischemic conditions, ischemia / reperfusion injury, myocardial ischemia, stable angina, unstable angina, stroke, ischemic heart disease and cerebral ischemia, light or heavy chain amyloidosis, lysosomal storage diseases, aspartylglucosaminuria, Fabry disease, Batten disease, cystinosis, Farber disease, fucosidosis, galactosidioside sialidosis, Gaucher disease types 1, 2 or 3, Gm1 gangliosidosis, Hunter disease, Hurler-Scheie disease, Krabbe disease, alpha-mannosidosis, Kearns-Sayre syndrome (KSS), lactic acidosis and stroke Myoclonus-like seizures (MELAS) syndrome, Leber's hereditary optic neuropathy (LHON), beta-mannosidosis, Maroteaux-Lamy disease, MEGDEL syndrome (also known as 3-methylglutaconic aciduria with hearing loss, encephalopathy and Leigh-like syndrome), metachromatic leukodystrophy, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome, Morquio syndrome type A, Morquio syndrome type B, mucolipidosis type II, mucolipidosis type III, myoclonus-like epilepsy-myopathy-sensory ataxia, mitochondrial myopathy, myoclonus-like epilepsy with ragged red fibers (MER RF), Niemann-Pick disease types A, B or C, neurogenic muscle weakness, Pearson syndrome, Pompe disease, Sandhoff disease, Sanfilich syndrome types A, B, C or D, Schindler disease, Schindler-Kanzaki disease, Saengers syndrome, sialidosis, Sly syndrome, Tay-Sachs disease, Wolman disease, lysozyme amyloidosis, Mallory bodies, medullary thyroid carcinoma, mitochondrial myopathy, multiple sclerosis, multiple system atrophy, myotonic dystrophy, myotonic dystrophy, cerebral iron storage neurodegeneration, neurofibromatosis, neuronal ceroid lipofuscinosis, amyloidogenic odontopathy Pathogenic (Pinborg) tumors, Guam Parkinson's dementia, Parkinson's disease, peptic ulcers, Pick's disease, pituitary prolactinoma, postencephalitic parkinsonism, prion diseases (transmissible spongiform encephalopathies), variant Creutzfeldt-Jakob disease (including CJD), Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, kuru, progressive supranuclear palsy, pulmonary alveolar proteinosis, retinal ganglion cell degeneration in glaucoma, retinitis pigmentosa with rhodopsin mutations, seminal vesicle amyloidosis, senile systemic amyloidosis, serpinopathies,Sickle cell disease, Spinal-bulbar muscular atrophy (SBMA), Spinocerebellar degeneration, Spinocerebellar degeneration type 1, Spinocerebellar degeneration type 2, Spinocerebellar degeneration type 3 (including Machado-Joseph disease), Spinocerebellar degeneration type 6, Spinocerebellar degeneration type 7, Spinocerebellar degeneration type 8, Spinocerebellar degeneration type 17), Subacute sclerosing panencephalitis, Tauopathy, Type II diabetes, Vascular dementia, Werner syndrome, Atherosclerosis, Autism spectrum disorder (ASD), Benign focal atrophy, Duchenne palsy, Hereditary spastic paraplegia (HSP), Kugelberg-Welander syndrome, Lou Gehrig's disease, Necrotizing enterocolitis, Paget's disease of bone (PDB), Primary lateral sclerosis (PLS), Progressive bulbar palsy (PBP), Progressive The present invention may be selected from any one or more of: percutaneous muscular atrophy (PMA), pseudobulbar palsy, spinal muscular atrophy (SMA), ulcerative colitis, valosin-containing protein (VCP)-related disorder, or Werdnig-Hoffmann disease, transient ischemic attack, ischemia, intracerebral hemorrhage, senile cataract, retinal ischemia, retinal vasculitis, Brown-Viarette-Van Lare syndrome, Eales disease, meningoencephalitis, post-traumatic stress disorder, Charcot-Marie-Tooth disease, macular degeneration, X-linked spinal-bulbar muscular atrophy (Kennedy disease), presenile dementia, depressive disorder, temporal lobe epilepsy, Leber's hereditary optic neuropathy, cerebrovascular disorder, subarachnoid hemorrhage, schizophrenia, demyelinating disorder, and Pericheis-Merzbacher disease.

[0131] Suitably the disease or disorder may be selected from any one or more of lysosomal storage diseases, inclusion body myositis, spinocerebellar degeneration, or spinal-bulbar muscular atrophy.

[0132] Suitably the lysosomal storage disease may be selected from Niemann-Pick disease type C or Gaucher disease.

[0133] Suitably the disease or disorder may be selected from any one or more of ALS, frontotemporal dementia, Huntington's disease, Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, Parkinson's dementia, cerebral iron accumulation neurodegeneration, diffuse neurofibrillary tangles with calcification, multiple system atrophy, cerebral amyloid angiopathy, vascular dementia, Down's syndrome, Creutzfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Straussler-Scheinker syndrome, kuru, familial British dementia, familial Danish dementia, Parkinson's dementia of Guam, myotonic dystrophy, neuronal ceroid lipofuscinosis, or a disease related thereto.

[0134] Suitably the disease or disorder may be selected from any one or more of Friedreich's ataxia, multiple sclerosis, mitochondrial myopathy, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, argyrophilic grain disease, subacute sclerosing panencephalitis, Christianson syndrome, age-associated tau astrogliopathy (ARTA), primary age-associated tauopathy (PART), or Pick's disease.

[0135] Suitably, the subject or animal may be a mammal, such as a non-human animal or a human.

[0136] The invention further provides in vitro or ex vivo uses of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (e.g., (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol) to activate NRF2 in a cell, or to activate both HSF1 and NRF2 in a cell, or to reduce oxidative stress in a cell. The invention further provides an in vitro or ex vivo method of activating NRF2 in a cell, or activating both HSF1 and NRF2 in a cell, or reducing oxidative stress in a cell, comprising contacting the cell with an effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (e.g., (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol). Suitably, activation of NRF2 may include dissociation of NRF2 from Kelch-like ECH-associated protein.

[0137] Suitably, in the uses and / or methods of the invention the cell may be a cell type or derived from a tissue selected from any one or more of the adrenal gland, bone marrow, brain, breast, bronchus, caudate nucleus, cerebellum, cerebral cortex, cervix, uterus, colon, endometrium, epididymis, esophagus, fallopian tube, gallbladder, myocardium, hippocampus, kidney, liver, lung, lymph node, nasopharynx, oral mucosa, ovary, pancreas, parathyroid, placenta, prostate, rectum, salivary gland, seminal vesicle, skeletal muscle, skin, small intestine (including the duodenum, jejunum and ileum), smooth muscle, spleen, stomach, testis, thyroid, tonsils, bladder or vagina.

[0138] Suitably, the cell is a member of a group of disorders associated with age-related tau astrogliopathy (ARTA), ALS, Alzheimer's disease, argyrophilic grain disease, asthma, cerebral amyloid angiopathy, cerebral ischemia, Christianson syndrome, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, diffuse neurofibrillary tangles with calcification, Down's syndrome, emphysema, familial British dementia, familial Danish dementia, fatal familial insomnia, Friedreich's ataxia, frontotemporal dementia, Gerstmann-Sträussler-Scheinker syndrome, Guadeloupe parkinsonism, Huntington's disease, and the like. The mouse may be derived from an animal having, or at risk of acquiring, a disease or disorder selected from any one or more of: myotonic dystrophy, encephalomyelitis, kuru, mitochondrial myopathy, multiple sclerosis, multiple system atrophy, myotonic dystrophy, cerebral iron accumulation neurodegeneration, neuronal ceroid lipofuscinosis, Parkinson's disease dementia, Parkinson's disease, Parkinson's dementia of Guam, Pick's disease, postencephalitic parkinsonism, primary age-related tauopathy (PART), progressive supranuclear palsy, pulmonary fibrosis, sepsis, septic shock, subacute sclerosing panencephalitis, vascular dementia, or a condition related thereto.

[0139] The present invention also relates to a therapeutically effective amount of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (or a composition comprising 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol) for use in: 1) treating an animal having a disease or disorder that would benefit from increased NRF2 activation, or would benefit from a combination of increased HSF1 and increased NRF2; or 2) preventing or reducing the risk of acquiring a disease or disorder in an animal by increasing NRF2 activation, or by increasing the activation of both HSF1 and NRF2. Suitably, the disease or disorder is selected from the group consisting of age-related tau astrogliopathy (ARTA), ALS, Alzheimer's disease, argyrophilic grain disease, asthma, cerebral amyloid angiopathy, cerebral ischemia, Christianson syndrome, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, diffuse neurofibrillary tangles with calcification, Down's syndrome, emphysema, familial British dementia, familial Danish dementia, fatal familial insomnia, Friedreich's ataxia, frontotemporal dementia, Gerstmann-Straussler-Schein syndrome, and / or cerebrovascular disease. The disease may be selected from any one or more of Carr's syndrome, Guadeloupe Parkinsonism, Huntington's disease, Kuru, mitochondrial myopathy, multiple sclerosis, multiple system atrophy, myotonic dystrophy, cerebral iron accumulation neurodegeneration, neuronal ceroid lipofuscinosis, Parkinson's disease dementia, Parkinson's disease, Guam Parkinson's disease dementia, Pick's disease, postencephalitic parkinsonism, primary age-related tauopathy (PART), progressive supranuclear palsy, pulmonary fibrosis, sepsis, septic shock, subacute sclerosing panencephalitis, vascular dementia, or a disease related thereto.

[0140] Suitably the animal may be a mammal, such as a non-human mammal or a human.

[0141] Suitably, in the methods, compositions and / or uses of the second agent of the invention, 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (such as (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol) may be administered or formulated for administration at a dose of 0.12 mg / kg or more. Suitably, 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be administered at a dose between 10 and 5000 mg / day.

[0142] Suitably, in the methods, compositions and / or uses of the second agent of the invention, 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (such as (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol) may be administered or formulated for administration in any suitable manner, for example parenterally, enterally or topically.

[0143] Suitably, in the methods, compositions and / or uses of the second agent of the invention, 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol (such as (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol) may be administered or formulated for administration by oral, sublingual, buccal, pulmonary, intranasal, intravenous, intramuscular or subcutaneous administration.

[0144] Another embodiment of the present invention includes the use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for slowing the fall of CMAP or improving CMAP. Alternatively, 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be for use in slowing the fall of CMAP or improving CMAP, optionally for use in treating a disease or disorder by slowing the fall of CMAP or improving CMAP.

[0145] Another embodiment of the invention encompasses the use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to improve muscle strength. Alternatively, 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be for use in improving muscle strength, optionally in the treatment of a disease or disorder by improving muscle strength.

[0146] Another embodiment of the present invention includes the use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for controlling body weight during the treatment of frontotemporal dementia. Alternatively, 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be for use in controlling body weight, optionally during the treatment of frontotemporal dementia or in patients with frontotemporal dementia.

[0147] One embodiment encompasses the use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for increasing the expression of the heat shock protein Hspa8. Alternatively, the 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be for use in increasing the expression of the heat shock protein Hspa8, optionally for use in the treatment of a disease or disorder by increasing the expression of the heat shock protein Hspa8.

[0148] Another embodiment encompasses the use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for increasing the expression of the heat shock protein Hspa1a. Alternatively, 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol may be for use in increasing the expression of the heat shock protein Hspa1a, optionally for use in the treatment of a disease or disorder by increasing the expression of the heat shock protein Hspa1a.

[0149] Yet another embodiment encompasses the use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for the preparation of a medicament for increasing heat shock protein Hspa8 or Hspa1a.

[0150] Another embodiment encompasses the use of 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for the preparation of an orally administered medicament for increasing heat shock proteins Hspa8 or Hspa1a.

[0151] Working Example The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0152] Further, it should be understood that all values ​​are approximate and are provided for illustrative purposes. All references cited and discussed herein are incorporated by reference in their entirety to the same extent as if each reference was individually incorporated by reference.

[0153] The ability of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to induce HSF1-regulated genes in the central nervous system was evaluated in a preclinical in vivo model.

[0154] Example 1: In vivo pharmacokinetic study via subcutaneous administration methodology Wild-type mice (3 mice / group) were administered 0, 0.5, 1.5, 5, and 10 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride subcutaneously once daily for 7 days. One mouse in the 10 mg / kg group was actually administered 15 mg / kg on day 1. To prepare the dosing solutions, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol was pre-measured into a bijou and stored in foil. Each dose was made up fresh daily in filter-sterilized vehicle (0.9% (w / v) saline, 0.05% (w / v) sodium metabisulfite, and 1% (w / v) ascorbic acid, pH 3.5). Tissues were collected at the expected peak mRNA expression (6 hours after the last dose) and expected trough expression (24 hours after the last dose). During tissue collection, spinal cord and cortex were collected from each animal. RNA was immediately extracted using the Rneasy® Lipid Tissue Mini Kit. RNA was treated with DNase and converted to cDNA. Gene expression measurements included Pgc1a, Dnajb1, Hspa1a, which are upregulated by HSF1 activation. Gene expression measurements included Gclm and Nqo1, which are upregulated by NRF2 activation.

[0155] result The effect of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol on gene expression induction was evaluated in a wild-type mouse model. Six hours after the last dose, 10 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed 2.2-fold Hspa1a gene induction and 1.3-fold Dnajb1 gene induction compared to Gapdh. Repeated doses of 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol also gave 1.8-fold Pgc1a gene induction. At 24 hours after the last dose, 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed 2.3-fold higher Hspa1a gene induction and 1.7-fold higher Pgc1a gene induction compared to Gapdh.

[0156] At 6 hours after the final dose, 10 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed 2.0-fold higher Hspa1a gene induction and 1.5-fold higher Pgc1a gene induction compared to Actb. At 24 hours after the final dose, 1.5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed 2.3-fold higher Hspa1a gene induction and 1.7-fold higher Pgc1a gene induction compared to Actb.

[0157] The results of Hspa1a and Dnajb1 gene induction by (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed that (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 0.5 mg / kg or more could activate HSF1-regulated genes in mice (Figures 1 and 2).

[0158] Gene expression including Gclm and Nqo1 regulated by NRF2 activation was also measured as shown in Figures 1 and 2. At 6 hours after the last administration, 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed 1.5-fold Gclm gene induction compared to Gapdh. At 24 hours after the last administration, 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed 1.5-fold Gclm gene induction and 1.3-fold Nqo1 gene induction compared to Gapdh.

[0159] At 6 hours after the final dose, 10 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed 1.4-fold Gclm gene induction compared to Actb. At 24 hours after the final dose, 10 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed 1.2-fold Gclm gene induction and 1.4-fold Gclm gene induction compared to Actb.

[0160] Example 2: In vivo pharmacokinetic study via subcutaneous administration methodology Wild-type mice (3 mice / group) were administered 0, 0.5, 1.5, 5, and 10 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride subcutaneously once daily for 7 days. One mouse in the 10 mg / kg group was actually administered 15 mg / kg on day 1. To prepare the dosing solutions, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol was pre-weighed into bijoux and stored in foil. Each dose was made up fresh daily in filter-sterilized vehicle (0.9% (w / v) saline, 0.05% (w / v) sodium metabisulfite, and 1% (w / v) ascorbic acid, pH 3.5). Tissues were collected at the expected peak mRNA expression (6 hours after the last dose) and expected trough expression (24 hours after the last dose). During tissue collection, spinal cord and cortex were collected from each animal. RNA was immediately extracted using the Rneasy® Lipid Tissue Mini Kit. RNA was treated with DNase and converted to cDNA. Gene expression measurements included Hspa1a, Hspa8, Dlg4, Syn1, and Dnajb1, which are upregulated by HSF1 activation. Gene expression measurements included Gclm, Hmox1, Nqo1, and Pgc1a, which are upregulated by NRF2 activation.

[0161] result The consequences of gene expression induction by (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol were evaluated in a wild-type mouse model.

[0162] At 6 hours after the final administration, compared to Gapdh, 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol exhibited 1.84-fold Hspa1a gene induction, 1.56-fold Hspa8 gene induction, 1.46-fold Dlg4 gene induction, 1.68-fold Syn1 gene induction, and 1.31-fold Dnajba gene induction, as shown in Figure 3.

[0163] At 24 hours after the final administration, compared to Gapdh, 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol exhibited 2.25-fold Hspa1a gene induction, 2.62-fold Hspa8 gene induction, 0.90-fold Dlg4 gene induction, 1.47-fold Syn1 gene induction, and 1.17-fold Dnajba gene induction, as shown in Figure 4.

[0164] Expression of genes regulated by NRF2 activation, including Gclm and Nqo1, was also measured as shown in Figs. 3 and 4 .

[0165] At 6 hours after the final administration, compared to Gapdh, 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol exhibited 1.51-fold Gclm gene induction, 2.02-fold Hmox1 gene induction, 1.04-fold Nqo1 gene induction, 1.71-fold Pgc1a gene induction, and 2.39-fold Nrf1 gene induction, as shown in Figure 3.

[0166] At 24 hours after the final administration, compared to Gapdh, 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol exhibited 1.45-fold Gclm gene induction, 2.11-fold Hmox1 gene induction, 1.32-fold Nqo1 gene induction, 1.70-fold Pgc1a gene induction, and 1.58-fold Nrf1 gene induction, as shown in Figure 4.

[0167] The gene induction results showed that (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 0.5 mg / kg or more could activate HSF1-regulated genes in mice.

[0168] Example 3: In vivo pharmacokinetic study via oral administration In vivo testing and tissue processing Wild-type mice were orally administered 25 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol once daily for 4 days.

[0169] A total of 36 mice were randomly divided into 12 groups. Each group had 3 mice. After oral administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol once a day for 4 consecutive days, the brains of mice from each group were harvested individually at the following time points: 0, 5, 15, 30 min, 1, 2, 3, 4, 8, 12, 24, and 48 h after the last oral administration. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed on one quarter of the whole brain from each mouse in each time group. Before RNA extraction, the brain tissue was immediately frozen in liquid nitrogen and transferred to -80 °C until further use.

[0170] RT-qPCR Total RNA from 36 frozen brain samples was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA, 15596018) according to the manufacturer's instructions. After extraction, the integrity of the total RNA was checked by 1% agarose gel stained with ethidium bromide and Nanodrop (Thermo Fisher, USA). 2 μg of total RNA was then used for reverse transcription with M-MLV reverse transcriptase (Invitrogen, 28025-013) after gDNA removal (NEB, M0303S). qRT-PCR analysis was performed using a LightCycler® 480 Probe Master (4887301001, Roche, Basel, Switzerland) and a Roche LightCycler480 (Roche). Data from triplicate experiments were subjected to statistical analysis and target genes were normalized to the level of actin beta (Actb) mRNA. Primers are listed below. [Table 1]

[0171] result Test compounds increase gene expression of the heat shock proteins Hspa8 and Hspa1a.

[0172] To evaluate the ability of test compounds to activate heat shock genes, RT-PCR was performed to detect the mRNA expression of Hspa8 and Hspa1a in brain samples. Our results show that the mRNA expression level of Hspa8 exhibited a slight increase in groups 10 and 11 compared to group 1 (Table 1). Similarly, the mRNA level of Hspa1a peaked in group 9 and exhibited a significant increase in groups 9 and 10 compared to group 1 (Table 1, Figures 5 and 6). [Table 2]

[0173] Example 4: TDP-43 Q331K In vivo pharmacological testing in mouse models Tg(Prnp-TARDBP * Q331K)103Dwc(TDP-43 Q331K Transgenic mice expressing the ALS-associated Q331K mutation (huTDP-43), which is targeted to the brain and spinal cord by the mouse prion protein promoter, * Expression of myc-tagged human TAR DNA-binding protein carrying the nucleotide sequence Q331K. TDP-43 Q331K Transgenic mice may be useful in studying motor dysfunction in amyotrophic lateral sclerosis, a neurodegenerative disorder. Q331KThe mouse model was initially characterized at the Sheffield Institute for Translational Neuroscience, brought from Jackson laboratories (USA) (Control Number: 017933). All experiments involving mice were performed in accordance with the Animal (Scientific Procedures) Act 1986 and approved by the Sheffield University Ethical Review Committee Project Applications and Amendments Sub-Committee and by the UK Animal Procedures Committee (London, UK).

[0174] The mouse population was kept in a specific pathogen free (SPF) environment in accordance with the Home Office code of practice for the housing and care of animals used in scientific procedures before being transferred to conventional animal facilities for experiments.

[0175] Test Plan This in vivo pharmacological study was performed using TDP-43 Q331K This study was designed to test the efficacy of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol on the progression of motor and cognitive decline in a mouse model. In addition, tissues were collected at the end of the experiment for target engagement (gene expression) analysis of Nrf2 and HSF1 target genes.

[0176] Transgenic female animals were randomized after genotyping into three different treatment groups: vehicle once daily, 2.5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol twice daily, and 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol once daily. Doses were selected based on the results of previous internal studies. Animals were weighed once daily prior to dosing and dosed subcutaneously at 10 ml / kg with a solution of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 0.5 mg / ml for the 5 mg / kg dose and 0.25 mg / ml for the 2.5 mg / kg dose. When animals were dosed twice daily, doses were separated by at least 6 hours and dosing solutions were made fresh for the second dose on the same day.

[0177] The study was designed with two main cohorts of mice. One cohort was administered from 25 days to 6 months of age, and the mice underwent behavioral testing throughout the study, constituting 14 mice / group. The other satellite cohort was administered from 25 days to 3 months of age, did not undergo behavioral testing, and comprised 6 mice / group for target engagement and histological evaluation. Behavioral tests performed during the experiment were the accelerating rotarod test, gait analysis, and continuous physiological tests.

[0178] Weighing Animals were weighed each day prior to dosing to calculate dose volume. For animals dosed twice daily, morning body weights were used for the second dose.

[0179] In the 3-month cohort, there were no significant differences in body weight between either of the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol-treated groups compared to the vehicle-treated group (Figure 7).

[0180] In the 6-month cohort, there was a significant decrease in body weight in the 2.5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol-treated group compared to the vehicle-treated group from 121 days of age to the end of the study (Figure 7).

[0181] Accelerated rotarod test Mice were tested on an accelerating rotarod (Jones & Roberts for mice, model 7650) once a week from 40 days of age until the end of the study. The rotarod accelerates from 4 rpm to 40 rpm over a period of 300 seconds. Mice are placed on the rotarod and the time it takes to fall off the rotarod (fall latency) is recorded. On each test day, each mouse is tested twice on the rotarod with a short break between trials, and the best result from the two trials is recorded. The rotarod test was performed at the same time (afternoon) each week.

[0182] Before the first rotarod test, mice were trained on the rotarod for three consecutive days with two trials each. These results were recorded but not used in the data analysis.

[0183] Rotarod results: TDP-43 Q331K Consistently declines over time in mouse models. At one time point (19 weeks of age), rotarod performance was significantly increased in mice treated with 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol compared to vehicle. Increased rotarod performance indicates improved coordination and motor function (Figure 8).

[0184] Catwalk Gait Analysis Gait analysis was performed at 3 and 6 months of age using the Catwalk gait analysis system 7.1 (Noldus Information Technology BV, The Netherlands) and analyzed using Catwalk software 7.1, which calculated a number of different gait parameters, such as step length, base of support (BOS) and swing time, as well as step pattern and the % of time spent on 2, 3 or 4 legs.

[0185] On the day of testing, mice were placed on a glass runway and allowed to run back and forth freely. Approximately six consistent straight runs were recorded by the camera for each mouse. The three best runs with the closest total running time were selected for analysis for each mouse. For these three runs, footprints were displayed in consecutive frames using software, and then multiple gait parameters were analyzed for each run. Using Excel, the average of all three runs was calculated for each mouse, followed by the average per group for each gait parameter.

[0186] Gait analysis was performed using the Catwalk system (Noldus) at both 3 and 6 months of age with 8 animals per group and time point. Traditionally in this model, the base of support increases with age, as shown in Figures 9-13, representing the wobbly or "swimming" gait described in this strain. (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol did not restrict this swimming gait. Vehicle-treated mice showed a decrease in the amount of time spent on the diagonal paw and an increase in the percentage % of time spent on 3 or 4 paws, indicating an unsteady gait. These gait parameters are relatively stable at 3-6 months, although the opposite is true for both the 2.5 and 5 mg / kg dose groups.

[0187] Electrophysiological testing (CMAP and repetitive stimulation) Compound muscle action potential (CMAP) and repetitive stimulation electrophysiological assessments were performed at 6 weeks, 3 months, and 6 months of age.

[0188] Mice were anesthetized with isoflurane gas and then kept under anesthesia gas with a nose cone for the duration of the experiment. Body temperature was maintained with a heating pad. To allow skin contact of the ring electrode, hair on the left lower leg was removed with an electric razor followed by a hair removal cream. The ring electrode was covered with conductive paste and placed around the heel and thigh of the shaved limb. The ring was tightened so that there was no gap between the skin and the electrode, but not so tight that blood flow was altered. The ground electrode was placed at the base of the tail and the stimulating electrode was placed higher up the leg as close as possible to the sciatic nerve.

[0189] CMAP was obtained by pulsing the ischial tuberosity with a 0 ms duration electric current. The electrode position was tested before the final pulse to ensure correct electrode placement by visualizing the results of the pulse. The stimulation current was then increased until no further increase in CMAP was observed.

[0190] Repetitive stimulation was performed after calculating the CMAP. The electrode was held stable while delivering 10 pulses at 10 Hz through the stimulating electrode. Each of the 10 stimuli produced a stimulation amplitude and area. Data was normalized so that the first stimulation was 100%.

[0191] At 6 weeks of age, there are no significant differences between the different treatment groups when compared to CMAP. The means are similar to previous experiments in this disease model at this age. At 3 months of age, there is a significant decrease in the 5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment group compared to the vehicle treatment group. At 6 months of age, there is a significant increase in both (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treatment groups compared to vehicle treatment animals (Figure 14).

[0192] Considering the variability between different subjects, the relative CMAP values ​​at 6 months were also calculated based on the CMAP values ​​of individual animals (Figure 15). Both the 2.5 and 5 mg / kg treatment groups showed significant improvement in relative CMAP compared to the vehicle group, indicating improvement in electrophysiological parameters by (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

[0193] Repeated stimulation is plotted as a % of the first stimulation to show the reduced response to multiple stimulations. Q331K In the model, a decreased response to 10 stimuli at 3 and 6 months of age was observed. At 6 weeks of age, there is a significant difference between the groups treated with 2.5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol twice daily compared to vehicle-treated mice at the time of the last stimuli, with the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol-treated mice having a greater decrease in response from the first stimuli. This may be the main reason for the difference, as repeated stimuli are difficult at 6 weeks due to the small size of the mice and their muscles at this age, and therefore we do not usually see differences so early in this disease model. There is no significant difference in repeated stimuli between the treatment groups at 3 months, with a greater decrease in response to stimuli at 3 months compared to 6 weeks. At 6 months of age, at stimulation times 5 and 7, there was a significant increase in responding in the 2.5 mg / kg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol treated group compared to the vehicle treated group (Figures 16 and 17).

[0194] Tissue collection Tissues were collected 24 hours after the last morning dose. At the time of tissue collection, all animals were overdosed with an intraperitoneal injection of pentobarbital (JML, M042) (2.5 mg / kg). In the 6-month cohort (n=14), 7 animals / group were perfused and tissues were fixed for histology. In these animals, once the animals lost foot reflexes, the chest cavity was opened and perfused transcardially with 10 ml PBS followed by 10 ml 4% PFA. The brain and spinal cord were removed and stored overnight in 4% PFA before being replaced with PBS. The lumbar portion of the spinal cord was dissected and embedded. For the remaining 7 animals / group, once the animals lost foot reflexes, blood was extracted by cardiac puncture. Blood was collected and stored in 1 ml RNALater (ThermoFisher). The spinal cord was removed and the upper part was flash frozen in liquid nitrogen for protein analysis, and the lower part was stored in RNALater and stored at −20° C. The cortex was removed and dissected into four parts, the anterior left part was stored in RNALater, and the other three parts were flash frozen in liquid nitrogen.

[0195] For the 3 month cohort, tissue was collected in a similar manner to the flash frozen tissue for the 6 month cohort, except that tissue was stored in RNALater for the 6 month cohort and immediately processed for RNA extraction for the 3 month cohort.

[0196] RNA extraction RNA was extracted from the lower spinal cord and cortex using the RNeasy Lipid Tissue Mini Kit (Qiagen, 74804) according to the manufacturer's protocol. Briefly, tissues were homogenized in a small volume (150 μl) of QIAzol using a handheld homogenizer in a fume hood. Once homogenized, the total volume of QIAzol was increased to 1 ml. Samples were incubated at room temperature for 5 minutes, after which 200 μl of chloroform (Honeywell) was added and samples were shaken vigorously for 15 seconds. The mixed samples were then incubated at room temperature for 3 minutes, before being centrifuged at 4° C. and 12,000 g for 15 minutes in a benchtop microcentrifuge.

[0197] The upper aqueous phase of the sample was transferred to a new labelled tube and one volume of 70% ethanol was added. The sample was vortexed and 700 μl of sample was transferred to an RNeasy mini spin column attached to a 2 ml collection tube. The sample was centrifuged at room temperature and 13,000 rpm for 15 seconds and the flow-through was discarded. The remaining sample was added to the spin column, the sample was spun again and the flow-through was discarded. A volume of 700 μl of RW1 buffer was added to the spin column and these were spun at 13,000 rpm for 15 seconds. The flow-through was discarded. 500 μl of RPE buffer was added to the column, which was spun and the flow-through was discarded. An additional 500 μl of RPE buffer was added to the column and the sample was centrifuged at 13,000 rpm for 2 minutes. To further dry the membrane, the spin column was placed in a new 2 ml tube and centrifuged at full speed for 1 minute. Finally, the column was placed into a new 1.5 ml tube and 30 μl of RNeasy-free water was added. These were centrifuged at 13,000 rpm for 1 minute and the flow-through was pooled.

[0198] RNA quantification Directly after extraction, RNA was quantified and checked for purity by spectrophotometry using a Nanodrop ND-1000 (Thermo Scientific). Total RNA concentration, as well as A260 / 280 and A260 / 230 ratios were measured to check the purity of the samples.

[0199] cDNA synthesis All water used throughout the cDNA synthesis and qPCR protocols was DEPC-treated water, which was made by adding 1 ml of DEPC (BioChemica) to 1 L of MQ water and autoclaving.

[0200] cDNA was synthesized from RNA using the following method: First, any potential DNA was digested from the sample using RNase-free DNase and DNase buffer (Roch Diagnostics, 04716728001). A volume of 1 μl of DNase and 1 μl of 10× DNase buffer was added to 2000 ng of RNA sample (total volume 10 μl). This was then incubated at 37° C. for 10 minutes. DNase was inactivated with 1 μl of 25 mM sterile DEPC-treated EDTA (Ameresco) and incubated at 75° C. for 10 minutes.

[0201] A volume of 1 μl of DN6 (random hexamer primer, Sigma Aldrich) and 1 μl of deoxyribonucleotide triphosphates (dNTPs, Bioline, Bio-39053) were added to each reaction and these were incubated at 75° C. for 5 minutes to denature the RNA. Samples were immediately placed on ice to prevent refolding of the RNA, and 2 μl of DTT, 4 μl of 5× buffer, and 1 μl of reverse transcriptase (RT) enzyme were added to all tubes (all Invitrogen, 28025-13). These were placed in a PCR machine (G-storm) and run with the following protocol: 25° C. for 10 minutes, 42° C. for 1 hour, 85° C. for 5 minutes, then hold at 10° C. Once the protocol was completed, 40 μl of DEPC H2O was added, samples were vortexed briefly, and cDNA was stored at −20° C.

[0202] qPCR Primers (Sigma Aldrich) were diluted to 100 μM with DEPC H2O. Primers were further diluted to create mixtures containing both forward and reverse primers at optimized concentrations for each target.

[0203] All qPCR experiments were performed using 96-well qPCR plates (Bio-Rad, MLL9651) with optical strip lids (Bio-Ras, TCS0803) or 384-well plates (Bio-Rad, HSP3865) with clear plate seals.

[0204] Cycle threshold (cT) values, amplification curves and melt peaks were analyzed and extracted using CFX Maestro software (Bio-Rad) and further analyzed using Excel (Microsoft) and GraphPad Prism 7. Relative mRNA levels were determined by normalization to internal controls using the ΔΔCT method and normalization to vehicle samples.

[0205] At 3 months, cortical samples from mice (normalized to Gapdh, n=6) showed significant upregulation of Hspa1a, Nqo1, Sqstm1, and GSR in the 5 mg / kg group compared to the vehicle-treated group. Upregulation of Nqo1, Osgin1, and GSR was also observed in the 2.5 mg / kg group (Figure 18).

[0206] At 6 months, cortical samples from mice (normalized to Gapdh, n=7) showed significant upregulation of Hspa1a at dose levels of 2.5 mg / kg twice daily and 5 mg / kg once daily (FIG. 19).

[0207] Example 5: Protein analysis in in vitro pharmacological testing In vitro modeling of neurodegeneration has made impressive advances in the past decade, mainly due to the reprogramming of adult human fibroblasts into induced pluripotent stem cells (iPSCs) and induced neural progenitor cells (iNPCs), which has provided the ALS research field with the opportunity to model familial and sporadic forms of the disease in vitro.

[0208] NPCs taken from autopsy spinal cords of ALS patients have already been successfully differentiated into motor neurons, astrocytes and oligodendrocytes. Deriving astrocytes using this method avoids the induction of major epigenetic changes. However, the availability of autopsy samples is limited. In addition, the drawbacks of reprogramming astrocytes from human-derived iPSCs include the time-consuming protocol and the complex and highly variable maturation time of astrocytes.

[0209] Therefore, a promising alternative to iPSC sources is the direct reprogramming of fibroblasts into astrocytes from an immunologically matched host. Instead of generating iPSCs, direct reprogramming involves the use of lineage transcription factors to convert adult somatic cells into another cell type. This technique has been used to generate subpopulations of neural cell lineages such as cholinergic, dopaminergic, and motor neurons. Using direct reprogramming techniques, astrocytes were derived from fibroblasts of ALS patients, tripotent iNPCs were derived from ALS patients, and controls were generated within a month. When these cells were differentiated into astrocytes, they showed similar toxicity to autopsy-derived astrocytes towards motor neurons in co-culture, making them a useful tool in the development of drug screens.

[0210] methodology Induced NPCs were generated from adult fibroblasts from patients diagnosed with ALS and age-matched healthy controls utilizing previously reported approaches (Kim et al PNAS,2001.108(19),7838-7843; Meyer et al.,PNAS,2014.111(2),829-832). Induced NPCs are differentiated into iAstroctes by culturing the precursors in induced astrocytes (iAstrocytes) medium for a total of 7 days with a medium change on day 3.

[0211] Induced astrocytes from human donors were treated with 0.1% DMSO, 10 μM (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol or 10 μM riluzole for 48 h and then harvested. Cells were detached from 10 cm dishes and cell pellets were lysed in ice-cold IP lysis buffer (150 mM NaCl, 50 mM HEPES, 1 mM EDTA, 1 mM DTT, 0.5% (v / v) Triton X-100, protease inhibitor cocktail, pH 8.0) for 15 min and further homogenized using a 25-gauge needle and syringe. Protein samples were separated by SDS-polyacrylamide gel electrophoresis and then semi-dried transferred onto nitrocellulose membranes. Membranes were blotted with anti-NQO1-1:1000 (5% milk / TBST); rabbit; abcam; ab34173 overnight at 4°C and anti-beta-actin-1:5,000 (5% milk / TBST); mouse; abcam; ab6276 clone AC-15 overnight at 4°C.

[0212] Western blot analysis Protein quantification data from Western blot analysis demonstrated that (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol induced a significant increase in NQO1 after 48 h treatment at 10 μM in human iAstrocytes. As shown in Figure 20, human iAstrocytes were derived from healthy individuals (CTR, n=3), patients with C9orf72 mutations (C9orf72, n=3); sporadic ALS patients (sALS, n=3) and patients with SOD1 mutations (SOD1, n=3).

Claims

1. A pharmaceutical composition for the treatment of amyotrophic lateral sclerosis (ALS) not caused by a superoxide dismutase 1 (SOD1) gene mutation, comprising a therapeutically effective amount of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.

2. 2. The pharmaceutical composition of claim 1, wherein the therapeutically effective amount is at least 0.12 mg / kg.

3. 2. The pharmaceutical composition of claim 1, wherein the therapeutically effective amount is from 5 mg / day to 5000 mg / day.

4. The pharmaceutical composition according to any one of claims 1 to 3, which is formulated for oral administration.

5. The pharmaceutical composition according to any one of claims 1 to 3, which is formulated for subcutaneous administration.

Images