阅读说明：本技术 使用曲美替尼和标志物治疗神经退化性疾病的给药方法和剂量方案 (Methods of administration and dosage regimens for treating neurodegenerative diseases using trametinib and markers ) 是由 韩成镐 金美娟 千允宣 于 2020-05-22 设计创作，主要内容包括：本发明涉及一种使用曲美替尼和标志物治疗神经退化性疾病的给药方法和剂量方案。所述给药方法和剂量方案诱导神经再生和基因表达的变化。(The present invention relates to a method and dosage regimen for the treatment of neurodegenerative diseases using trametinib and a marker. The methods and dosage regimens induce nerve regeneration and changes in gene expression.)

1. A pharmaceutical composition comprising trametinib for use in treating a patient diagnosed with a neurodegenerative disease, at a daily dose effective to induce a change in the level of one or more markers in a biological sample obtained from the patient after at least four weeks of daily administration as compared to before administration.

2. The pharmaceutical composition of claim 1, wherein the daily dose is effective to induce at least a 1.3-fold, at least a 1.5-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, at least a 6-fold, at least a 7-fold, at least an 8-fold, at least a 9-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, or at least a 100-fold change in the level of one or more markers in a biological sample obtained from a patient.

3. The pharmaceutical composition of claim 1, wherein the daily dose is effective to reduce the level of one or more markers in a biological sample obtained from the patient by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% after administration for at least four weeks as compared to before administration.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein each of the one or more markers is encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctsk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Admp, Vpps, Admp, Vpmin, Pck, and Pck 13.

5. The pharmaceutical composition of claim 4, wherein the human homolog is selected from the group consisting of: GABRB, GABRR, GLRA, NR3C, CDKL, GRIN2, PLCXD, CHRM, CHRNA, CHRNB, NEFL, PLD, ADRA1, CHRNB, SLC6A, SLC18A, CDH, NEUROD, NKX-1, CXCL, REST, SYT, DISC, IRX, MDM, SOX, GRIP, PAX, BMP, CPNE, NUMB, ADRA 8A, TRIM, OTP, IL1RAPL, CPEB, TNFRSF12, HSPB, OPRM, LMX1, CLCF, ASPM, MECP, NTF, VEGFA, LRP, FEZ, ATP6V0, RNASE, CTSK, ACR, PRSS, LAMP, PRDX, UNC13, BAG, TIAL, ADRB, HPS, KAMAP, CCR, GIGIGIGIGIGIG, SEASE, HMPSSN, CPSN, VPSN, VPGB 13, and HMGB.

6. The pharmaceutical composition of any one of claims 1-3, wherein the one or more markers is a protein associated with lysosomal activity.

7. The pharmaceutical composition of claim 6, wherein the protein associated with lysosomal activity is a cathepsin.

8. The pharmaceutical composition of claim 7, wherein the cathepsin is selected from the group consisting of: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein said trametinib is administered at oral doses of 0.5 and 2 mg/day.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the neurodegenerative disease is selected from: alzheimer's Disease (AD), Mild Cognitive Impairment (MCI), dementia, vascular dementia, senile dementia, frontotemporal dementia (FTD), dementia with Lewy Bodies (LBD), Parkinson's disease, Multiple System Atrophy (MSA), corticobasal degeneration (CBD), corticobasal degeneration (PD), Progressive Supranuclear Palsy (PSP), progressive subanalysis disease), Huntington HD (Huntington's disease), amyotrophic lateral sclerosis (ALS, Logru-Gehrig's disease), primary lateral sclerosis (PLS; primary cerebral palsy), progressive regional paralysis (PBP), progressive regional paralysis (HSP), systemic paralysis (HSP), systemic paralysis (P, systemic paralysis), systemic paralysis (P, systemic paralysis (HSP), systemic paralysis (HSP), systemic paralysis (HSP, systemic paralysis, Creutzfeldt-Jakob disease (CJD), Multiple Sclerosis (MS), and Guillain-Barre syndrome (GBS).

11. The pharmaceutical composition of claim 10, wherein the neurodegenerative disease is Alzheimer's Disease (AD).

12. A pharmaceutical composition comprising trametinib for use in treating a patient diagnosed with a disease associated with lysosomal dysfunction or autophagic tide.

13. The pharmaceutical composition of claim 12, wherein the disease is selected from: lysosomal storage diseases, spinocerebellar atrophy, oculopharyngeal muscular dystrophy, prion diseases, fatal familial insomnia, alpha-1 antitrypsin deficiency, dentatorubral pallidoluysian atrophy, X-linked spinobulbar muscular atrophy, neuronal intranuclear hyaline inclusion body disease, multiple sclerosis, glaucoma and age-related macular degeneration.

14. The pharmaceutical composition of claim 13, wherein the lysosomal storage disease is selected from the group consisting of: mannosidosis, aspartylaminoglucosuria, juvenile neuronal ceroid lipofuscinosis (JNCCL, juvenile Barton's or CLN3 disease), cystinosis, Fabry's disease, gaucher's disease types I, II and III, glycogen storage disease type II (Pompe disease), Gm 2-gangliosidosis type II (sandhoff disease), metachromatic brain protein dystrophy, mucolipidosis types I, II/III and IV, mucopolysaccharidosis (Hewler's disease and variants thereof, Hunter's disease, Sanfilippo A, B, C and D syndrome, Morkola A and B syndrome, Maroteeaux-Lamy and Sly syndrome), Niemann-pick disease types A/B, C1 and C2, and Sindler disease types I and II.

15. A pharmaceutical composition comprising trametinib for use in treating a patient diagnosed with a disease associated with neuronal damage.

16. The pharmaceutical composition of claim 15, wherein the disease is selected from: glaucoma stroke, head trauma, spinal cord injury, optic nerve injury, ischemia, hypoxia, multiple sclerosis, multiple system atrophy, virus-related neuropathy, including acquired immunodeficiency syndrome (AIDS) -related neuropathy, infectious mononucleosis with polyneuritis, viral hepatitis with polyneuritis, Guillain-Barre syndrome, botulism-related neuropathy, toxic polyneuropathy including lead and alcohol-related neuropathy, nutritional neuropathy including subacute joint degeneration, vasculopathy including systemic lupus erythematosus-related neuropathy, sarcoidosis-related neuropathy, cancerous neuropathy, pressure neuropathy, carpal tunnel syndrome, hereditary neuropathy such as peroneal muscle atrophy, and peripheral nerve injury associated with spinal cord injury.

17. The pharmaceutical composition of claim 16, wherein the disease is an ocular injury, an ocular disease, or an optic neuropathy selected from the group consisting of: toxic amblyopia, optic atrophy, elevated retinopathy, eye movement disorder, third cerebral nerve paralysis, fourth cerebral nerve paralysis, sixth cerebral nerve paralysis, internuclear ophthalmoplegia, gaze paralysis, free radical damage to the eye, ischemic optic neuropathy, toxic optic neuropathy, ocular ischemic syndrome, optic neuritis, optic nerve infection, optic neuritis, optic neuropathy, optic papillary edema, optic neuritis, retrobulbar optic neuritis, retinal concussion, glaucoma, macular degeneration, retinal pigment degeneration, retinal detachment, tractive tear, diabetic retinopathy, iatrogenic trommel's disease, and optic nerve drusen.

18. A pharmaceutical composition comprising trametinib for use in treating a patient diagnosed with a disease associated with myelin sheath damage or demyelination of nerve fibers.

19. The pharmaceutical composition of claim 18, wherein the disease is selected from: multiple sclerosis, acute disseminated encephalomyelitis, transverse myelitis, Sheer's disease, Crohn's disease, clinically isolated syndrome, Alexander disease, Kanawan disease, Kekahn's syndrome, Palmer's disease, optic neuritis, optic neuromyelitis, HTLV-I related myelopathy, hereditary leukoencephalopathy, Guillain-Barre syndrome, Central pontine myelinolysis, deep leukocythemia, progressive multifocal leukoencephalopathy, demyelinating HIV encephalitis, demyelinating radiation injury, acquired toxic metabolic disorders, reversible posterior encephalopathy syndromes, Central pontine myelinolysis, leukodystrophy, adrenoleukodystrophy, Krabbe's globulopathy and/or metachromatic brain protein dystrophy, myelogenous cervical spondylosis due to cervical stenosis, traumatic injury to the brain or spinal cord, and stroke and neonatal hypoxic injury.

20. A composition for determining the therapeutic efficacy of a MEK1/2 inhibitor on a neurodegenerative disease, a disease associated with lysosomal dysfunction or autophagic tide, a disease associated with neuronal injury, or a disease associated with myelin sheath injury or demyelination of nerve fibers comprising a probe or antibody that specifically binds to a marker encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctsk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Admp, Vpps, Admp, Vpmin, Pck, and Pck 13.

21. The composition of claim 20, wherein the human homolog is selected from the group consisting of: GABRB, GABRR, GLRA, NR3C, CDKL, GRIN2, PLCXD, CHRM, CHRNA, CHRNB, NEFL, PLD, ADRA1, CHRNB, SLC6A, SLC18A, CDH, NEUROD, NKX-1, CXCL, REST, SYT, DISC, IRX, MDM, SOX, GRIP, PAX, BMP, CPNE, NUMB, ADRA 8A, TRIM, OTP, IL1RAPL, CPEB, TNFRSF12, HSPB, OPRM, LMX1, CLCF, ASPM, MECP, NTF, VEGFA, LRP, FEZ, ATP6V0, RNASE, CTSK, ACR, PRSS, LAMP, PRDX, UNC13, BAG, TIAL, ADRB, HPS, KAMAP, CCR, GIGIGIGIGIGIG, SEASE, HMPSSN, CPSN, VPSN, VPGB 13, and HMGB.

22. A composition for determining the therapeutic efficacy of a MEK1/2 inhibitor on a neurodegenerative disease, a disease associated with lysosomal dysfunction or autophagy tide, a disease associated with neuronal injury, or a disease associated with myelin sheath injury or demyelination of nerve fibers comprising an antibody that specifically binds to a marker protein associated with lysosomal activity.

23. The composition of claim 22, wherein the marker protein associated with lysosomal activity is a cathepsin.

24. The composition of claim 23, wherein the cathepsin is selected from the group consisting of: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L.

25. The composition of any one of claims 20 to 24, wherein the MEK1/2 inhibitor is trametinib.

26. The composition of any one of claims 20 to 25, wherein the therapeutic efficacy of the MEK1/2 inhibitor is achieved by comparing the level of one or more markers in a biological sample obtained from a patient diagnosed with the disease or condition following administration of trametinib with (a) the level of one or more markers in a biological sample obtained from the patient prior to the start of administration of trametinib, or (b) the level of one or more markers in a biological sample obtained from a healthy subject without the disease or condition.

27. A method of detecting the level of a marker in a biological sample obtained from a patient diagnosed with a disease selected from a neurodegenerative disease, a disease associated with lysosomal dysfunction or autophagy tide, a disease associated with neuronal injury, or a disease associated with myelin damage or demyelination of nerve fibers using a probe or antibody that specifically binds to the marker to provide information about the efficacy of a MEK1/2 inhibitor for the treatment of the disease, wherein the marker is encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctsk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Admp, Vpps, Admp, Vpmin, Pck, and Pck 13.

28. The method of claim 27, wherein the human homolog is selected from the group consisting of: GABRB, GABRR, GLRA, NR3C, CDKL, GRIN2, PLCXD, CHRM, CHRNA, CHRNB, NEFL, PLD, ADRA1, CHRNB, SLC6A, SLC18A, CDH, NEUROD, NKX-1, CXCL, REST, SYT, DISC, IRX, MDM, SOX, GRIP, PAX, BMP, CPNE, NUMB, ADRA 8A, TRIM, OTP, IL1RAPL, CPEB, TNFRSF12, HSPB, OPRM, LMX1, CLCF, ASPM, MECP, NTF, VEGFA, LRP, FEZ, ATP6V0, RNASE, CTSK, ACR, PRSS, LAMP, PRDX, UNC13, BAG, TIAL, ADRB, HPS, KAMAP, CCR, GIGIGIGIGIGIG, SEASE, HMPSSN, CPSN, VPSN, VPGB 13, and HMGB.

29. A method of detecting the level of a marker in a biological sample obtained from a patient diagnosed with a disease selected from the group consisting of a neurodegenerative disease, a disease associated with lysosomal dysfunction or autophagy tide, a disease associated with neuronal damage, or a disease associated with myelin damage or demyelination of nerve fibers using a probe or antibody that specifically binds to the marker to provide information about the efficacy of a MEK1/2 inhibitor in the treatment of the disease, wherein the marker is a protein associated with lysosomal activity.

30. The method of claim 29, wherein the protein associated with lysosomal activity is a cathepsin.

31. The method of claim 30, wherein the cathepsin is selected from: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L.

32. The method of any one of claims 27 to 31, wherein the MEK1/2 inhibitor is trametinib.

Technical Field

The present invention relates to a method and dosage regimen for the treatment of neurodegenerative diseases using trametinib. The invention also includes a method of treating a neurodegenerative disease or other disease associated with lysosomal dysfunction, autophagic tide, nerve injury, myelin sheath injury, or demyelination of nerve fibers using trametinib and one or more markers at levels that are altered by administration of trametinib.

Background

Neurodegenerative diseases such as Alzheimer's Disease (AD) and Parkinson's Disease (PD) are prevalent in the elderly population, and the number of patients is rapidly increasing as society ages. Furthermore, reports on early onset neurodegenerative diseases are not uncommon. Therefore, there is great interest in developing therapies that help to arrest disease progression and to restore damaged brain tissue.

The exact cause of such neurodegenerative diseases has not been determined. As is currently known, neuronal cells at specific locations in the brain (e.g., hippocampus or substantia nigra) are damaged, leading to a defect in the neural network between a reduced number of neurons, resulting in various symptoms of neurodegenerative diseases.

Research has been conducted in various fields to find a treatment method. Although some drugs have been approved for relief of symptoms associated with alzheimer's disease, parkinson's disease and other neurodegenerative diseases, these drugs are limited to short-term effects and the undamaged brain tissue associated with side effects is repaired or restored. More recently, trametinib (SNR1611,) Proved to be effective in inducing neuron differentiation and promoting neuron and Neural Stem Cell (NSCs) survival even if A beta exists in vitro_1-42As described in U.S. pre-grant publication No. 2018/0169102, and is incorporated by reference herein in its entirety. Thus, administration of trametinib and other MEK1/2 inhibitors has been proposed as a method of protecting neurons from neuronal loss or damage and inducing neurogenesis, thereby treating the symptoms of neurodegenerative diseases and restoring brain tissue damaged thereby.

MEK1/2 inhibitors are designed to be useful as anti-cancer agents, and a great deal of research on these drugs has been used to treat cancer. For the therapeutic use of MEK1/2 inhibitors in neurodegenerative diseases, particularly for administration to elderly patients, there is a need to develop suitable methods of administration and dosage regimens that result in effective treatment of neurodegenerative diseases and acceptable side effects.

Disclosure of Invention

Technical problem

The present disclosure relies on the following findings: administration of trametinib in an effective amount for more than four weeks induces genetic, structural and functional changes associated with nerve regeneration and enables survival of neuronal-like cells differentiated in the brain of an animal model of Alzheimer's Disease (AD). Since trametinib targets multiple pathways that promote recovery of neuronal function in the degenerated brain, these data predict that daily administration of an effective amount of trametinib can reverse at least four weeks the functional deficits associated with neurodegenerative diseases, and can be used to treat AD as well as other neurodegenerative diseases.

Technical scheme

Accordingly, in a first aspect, a method is provided for treating a neurodegenerative disease (e.g., AD) by administering trametinib daily for at least four weeks.

In some embodiments, the method comprises the step of administering trametinib daily for at least four weeks to a patient diagnosed with a neurodegenerative disease.

In some embodiments, trametinib is administered for at least five weeks. In some embodiments, trametinib is administered for at least six weeks. In some embodiments, trametinib is administered for at least seven weeks. In some embodiments, trametinib is administered for at least eight weeks. In some embodiments, trametinib is administered for at least nine weeks. In some embodiments, trametinib is administered for at least three months.

In some embodiments, the trametinib is administered at a daily oral dose effective to induce at least a 1.3-fold change in the level of one or more markers in the brain of the patient, or in a biological sample obtained from the patient, after at least four weeks of administration as compared to before the administration of trametinib. In some embodiments, the daily oral dose is effective to induce at least a 1.5-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, at least a 6-fold, at least a 7-fold, at least an 8-fold, at least a 9-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, or at least a 100-fold change in the level of one or more markers in the brain of the patient or in a biological sample obtained from the patient.

In some embodiments, the trametinib is administered at a daily oral dose effective to reduce the level of one or more markers in the brain of the patient or a biological sample obtained from the patient by at least 20% after at least four weeks of administration as compared to before administration of trametinib. In some embodiments, the daily oral dose is effective to reduce the level of one or more markers in the brain of the patient or a biological sample obtained from the patient by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%.

In one embodiment, each of the one or more markers is encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcx d, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Admp, Vpps, Admp, Vpmin, Pck, and Pck 13. The human homolog of the mouse gene can be GABRB, GABRR, GLRA, NR3C, CDKL, GRIN2, PLCXD, CHRM, CHRNA, CHRNB, NEFL, PLD, ADRA1, CHRNB, SLC6A, SLC18A, CDH, neuod, NKX-1, CXCL, REST, SYT, DISC, IRX, MDM, SOX, GRIP, PAX, BMP, CPNE, NUMB, ATP8A, TRIM, OTP, IL1RAPL, CPEB, TNFRSF12, HSPB, OPRM, LMX1, CLCF, ASPM, MECP, NTF, VEGFA, LRP, FEZ, ATP6V0, RNASE, CTSK, ACR, PRSS, PRDX, UNC13, rgn, TIAL, adbanb, ASSs, CCKAR, cgar, pcgun 152, pcgun, hmgnk, pcsmox, vpgimb, and vpgimb.

In some embodiments, the one or more markers are proteins associated with lysosomal activity. In some embodiments, the protein associated with lysosomal activity is a glycosyl hydrolase or protease. In some embodiments, the glycosyl hydrolase is selected from: beta-hexosaminidase, beta-galactosidase, beta-galactocerebrosidase, and beta-glucuronidase. In some embodiments, the protease is a cathepsin. In some embodiments, the cathepsin is selected from: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L.

In some embodiments, trametinib is administered in an amount to provide an average peak trametinib concentration (C) in the brain of at least 0.25ng/g_max) The dosage of (a). In some casesIn an example, trametinib is administered to provide a mean peak brain trametinib concentration in the brain of at least 0.5ng/g (C)_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide a mean peak brain trametinib concentration (C) in the brain of at least 0.75ng/g_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide a mean peak brain trametinib concentration (C) of at least 1ng/g in the brain_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide a mean peak brain trametinib concentration (C) in the brain of at least 1.5ng/g_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide a mean peak brain trametinib concentration (C) in the brain of at least 2ng/g_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide a mean peak brain trametinib concentration (C) in the brain of at least 5ng/g_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide a mean peak brain trametinib concentration (C) in the brain of at least 10ng/g_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide a mean peak brain trametinib concentration (C) in the brain of at least 15ng/g_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide an average peak brain trametinib concentration (C) in the brain of between 0.25 and 20ng/g_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide an average peak brain trametinib concentration (C) in the brain of between 0.25 and 5ng/g_max) The dosage of (a).

In some embodiments, trametinib is administered at an oral dose of between 0.5 and 2 mg/day. In some embodiments, trametinib is administered at an oral dose greater than 0.5 and less than 2 mg/day. In some embodiments, trametinib is administered at an oral dose greater than 0.75 and less than 2 mg/day. In some embodiments, trametinib is administered at an oral dose greater than 1 and less than 2 mg/day. In some embodiments, trametinib is administered at an oral dose greater than 0.75 and less than 1.25 mg/day. In some embodiments, trametinib is administered at an oral dose of 0.5 mg/day. In some embodiments, trametinib is administered at an oral dose of 1 mg/day. In some embodiments, trametinib is administered at an oral dose of 1.5 mg/day. In some embodiments, trametinib is administered at an oral dose of 2 mg/day. In some embodiments, the trametinib is administered as a tablet.

In some embodiments, the patient does not have the BRAF V600E or V600K mutation. In some embodiments, the patient is free of cancer.

In some embodiments, the neurodegenerative disease is selected from: alzheimer's Disease (AD), Mild Cognitive Impairment (MCI), dementia, vascular dementia, senile dementia, frontotemporal dementia (FTD), dementia with Lewy Bodies (LBD), Parkinson's disease, Multiple System Atrophy (MSA), corticobasal degeneration (CBD), corticobasal degeneration (PD), Progressive Supranuclear Palsy (PSP), progressive subanalysis disease), Huntington HD (Huntington's disease), amyotrophic lateral sclerosis (ALS, Logrub-Gehrig's disease), primary lateral sclerosis (PLS; primary cerebral amy), Progressive Bulbar Paralysis (PBP), progressive bulbar paralysis (HSP), chronic myelogenous paralysis (PSP), chronic myelogenous paralysis (P), chronic myelogenous paralysis (HSP) and chronic myelogenous paralysis (HSP) in a, Creutzfeldt-Jakob disease (CJD), Multiple Sclerosis (MS), and Guiland-Barre syndrome (GBS). In some embodiments, the neurodegenerative disease is Alzheimer's Disease (AD).

In some embodiments, the method further comprises the step of detecting the level of one or more markers in a sample obtained from the patient. In one embodiment, each of the one or more markers is encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctsk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Admp, Vpps, Admp, Vpmin, Pck, and Pck 13.

In some embodiments, each of the one or more markers is a protein associated with lysosomal activity. In some embodiments, the protein associated with lysosomal activity is a glycosyl hydrolase or protease. In some embodiments, the glycosyl hydrolase is selected from: beta-hexosaminidase, beta-galactosidase, beta-galactocerebrosidase, and beta-glucuronidase. In some embodiments, the protease is a cathepsin. In some embodiments, the cathepsin is selected from: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L.

In some embodiments, the sample is obtained after the step of administering trametinib. In some embodiments, the samples are obtained at a plurality of time points after the step of administering trametinib. In some embodiments, the method further comprises the step of obtaining the sample. In some embodiments, the method further comprises the step of detecting the level of one or more markers in a control sample obtained from the patient prior to the step of administering trametinib. In some embodiments, the method further comprises the step of obtaining the control sample. In some embodiments, the sample is obtained by brain biopsy. In some implementations, the sample is any biological sample obtained from an individual including a bodily fluid, a bodily tissue, a cell, a secretion, or other source. In some implementations, the bodily fluid or secretion includes blood, urine, saliva, stool, pleural fluid, lymph, sputum, ascites, prostatic fluid, cerebrospinal fluid (CSF), or any other bodily secretion or derivative thereof. In some embodiments, the blood is selected from whole blood, plasma, serum, Peripheral Blood Mononuclear Cells (PBMC), or any component of blood.

In some implementations, the method further includes the step of determining the therapeutic efficacy of trametinib administered to the patient based on changes in the levels of the one or more markers. In some implementations, the method further includes the step of determining the duration or dose of the subsequent administration of trametinib. In some implementations, the method further includes the step of discontinuing the administration of trametinib based on the determined efficacy of the treatment. In some implementations, the method further includes the step of continuing to administer trametinib based on the determined efficacy of the treatment. In some implementations, the method further includes the step of adjusting the administration of trametinib based on the determined treatment efficacy. In some implementations, the method further comprises: (a) detecting the level of one or more markers in a biological sample obtained from the patient after administration of trametinib, (b) comparing the level detected in (a) to the level of one or more markers in a biological sample obtained from the patient prior to administration of trametinib, or (c) comparing the level detected in (a) to the level of one or more markers in a biological sample obtained from a healthy subject without a disease of interest.

In another aspect, the invention discloses a method of enhancing lysosomal activity in a target tissue, comprising the step of administering metrinib to a subject, wherein the subject is diagnosed with a disease associated with lysosomal dysfunction or autophagic tide.

In some embodiments, the disease is selected from the group consisting of lysosomal storage diseases, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar atrophy, oculopharyngeal muscular dystrophy, prion diseases, fatal familial insomnia, alpha-1 antitrypsin deficiency, dentatorubral-pallidoluysian atrophy, frontotemporal dementia, progressive supranuclear palsy, X-linked spinobulbar muscular atrophy, intranuclear hyaline inclusion body disease, multiple sclerosis, glaucoma, and age-related macular degeneration.

In some embodiments, the lysosomal storage disease is selected from: mannosidosis, aspartyl glucosaminouria, juvenile neuronal ceroid lipofuscinosis (JNCCL, juvenile Barton's syndrome) or CLN3 disease), cystinosis, Fabry's disease, gaucher's disease types I, II and III, glycogen storage disease type II (Pompe's disease), GM 2-gangliosidosis type II (sandhoff's disease), metachromatic brain protein dystrophy, mucolipidosis type I, II/III and IV, mucopolysaccharidosis (Hewler's disease and variants thereof, Hunter's disease, Filippo A, B, C and D syndrome, Morkola A and B syndrome, Maroteeaux-Lamy and Sly syndrome), Niemann-pick disease types A/B, C1 and C2, and Sindleler disease types I and II.

In one aspect, the present disclosure provides a method of inducing axonal neogenesis in a target tissue comprising the step of administering trametinib to a subject, wherein the subject is diagnosed with a disease associated with neuronal damage.

In some embodiments, the disease is selected from: glaucoma, stroke, head trauma, spinal cord injury, optic nerve injury, ischemia, hypoxia, neurodegenerative diseases, multiple sclerosis and multiple system atrophy. In some embodiments, the disease is selected from: diabetic neuropathy; viral-associated neuropathy; acquired Immune Deficiency Syndrome (AIDS) -related neuropathy; infectious mononucleosis with polyneuritis; viral hepatitis with polyneuritis; Guillain-Barre syndrome; neuropathy associated with botulism; toxic polyneuropathy including lead and alcohol related neuropathy; vegetative neuropathy including subacute joint degeneration; vasculopathy including neuropathy associated with systemic lupus erythematosus; neuropathy associated with sarcoidosis; cancerous neuropathy; pressure neuropathy (e.g., carpal tunnel syndrome); hereditary neuropathy such as peroneal muscular atrophy; and peripheral nerve damage associated with spinal cord injury.

In some embodiments, the disease is an ocular trauma, an ocular disease, or an optic neuropathy selected from the group consisting of: toxic amblyopia, optic atrophy, elevated retinopathy, eye movement disorder, third cerebral nerve paralysis, fourth cerebral nerve paralysis, sixth cerebral nerve paralysis, internuclear ophthalmoplegia, gaze paralysis, free radical damage to the eye, ischemic optic neuropathy, toxic optic neuropathy, ocular ischemic syndrome, optic neuritis, optic nerve infection, optic neuritis, optic neuropathy, optic papillary edema, optic neuritis, retrobulbar optic neuritis, retinal concussion, glaucoma, macular degeneration, retinal pigment degeneration, retinal detachment, tractive tear, diabetic retinopathy, iatrogenic trommel's disease, and optic nerve drusen.

In one aspect, the present disclosure provides a method of treating a disease associated with myelin sheath damage or nerve fiber demyelination, comprising the step of administering trametinib to a subject, wherein the subject is diagnosed with the disease associated with myelin sheath damage or nerve fiber demyelination.

In some embodiments, the disease is selected from: multiple sclerosis, acute disseminated encephalomyelitis, transverse myelitis, Sheer's disease, Crohn's disease, clinically isolated syndrome, Alexander's disease, Kanawan's disease, Kekan syndrome, Palmer's disease, optic neuritis, neuromyelitis optica, HTLV-I related myelopathy, hereditary leukoencephalopathy, Guillain-Barre syndrome, Central pontine myelinolysis, deep leukoischemia, progressive multifocal leukoencephalopathy, demyelinating HIV encephalitis, demyelinating radiation injury, acquired toxic metabolic disorder, reversible posterior encephalopathy syndrome, Central pontine myelinolysis, leukodystrophy, adrenoleukodystrophy, Krabbe's globulopathy, and/or metachromatic encephalopathy. Other diseases in which demyelination occurs include myelogenous cervical spondylosis caused by cervical stenosis, traumatic injury to the brain or spinal cord, and hypoxic injury to the central nervous system including stroke and neonatal hypoxic injury.

In some embodiments of the above aspects, trametinib is administered for at least four weeks. In some embodiments, trametinib is administered for at least five weeks. In some embodiments, trametinib is administered for at least six weeks. In some embodiments, trametinib is administered for at least seven weeks. In some embodiments, trametinib is administered for at least eight weeks. In some embodiments, trametinib is administered for at least nine weeks. In some embodiments, trametinib is administered for at least three months.

In some embodiments, the trametinib is administered at a daily oral dose effective to induce a change in the level of one or more markers in a target tissue of the patient or a biological sample obtained from the patient of at least 1.3-fold after administration compared to before administration of trametinib for at least four weeks. In some embodiments, the daily oral dose is effective to induce at least a 1.5-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, at least a 6-fold, at least a 7-fold, at least an 8-fold, at least a 9-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, or at least a 100-fold change in the level of one or more markers in a target tissue of the patient or a biological sample obtained from the patient.

In some embodiments, the trametinib is administered at a daily oral dose effective to reduce the level of one or more markers in a target tissue of a patient or a biological sample obtained from the patient by at least 20% after administration of trametinib compared to before administration. In some embodiments, the daily oral dose is effective to reduce the level of one or more markers in a target tissue of a patient or a biological sample obtained from the patient by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%.

In some embodiments, the one or more markers are encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctsk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Admp, Vpps, Admp, Vpmin, Pck, and Pck 13.

In some embodiments, each of the one or more markers is selected from the group consisting of: beta-hexosaminidase, beta-galactosidase, beta-galactocerebrosidase, and beta-glucuronidase. In some embodiments, the protease is a cathepsin. In some embodiments, the cathepsin is selected from: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L. Preferably, the protease is cathepsin B.

In some embodiments, administration of trametinib provides a mean peak concentration of trametinib (C) in the target tissue of at least 0.25ng/g_max). In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Is at least 0.5 ng/g. In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Is at least 0.75 ng/g. In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Is at least 1 ng/g. In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Is at least 1.5 ng/g. In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Is at least 2 ng/g. In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Is at least 5 ng/g. In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Is at least 10 ng/g. In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Is at least 15 ng/g. In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Between 0.25 and 20 ng/g. In some embodiments, the mean peak concentration of trametinib (C) in the target tissue_max) Between 0.25 and 5 ng/g.

In some embodiments, the target tissue is brain.

In some embodiments, trametinib is administered at an oral dose of between 0.5 and 2 mg/day. In some embodiments, trametinib is administered at an oral dose greater than 0.5 and less than 2 mg/day. In some embodiments, trametinib is administered at an oral dose greater than 0.75 and less than 2 mg/day. In some embodiments, trametinib is administered at an oral dose greater than 1 and less than 2 mg/day. In some embodiments, trametinib is administered at an oral dose greater than 0.75 and less than 1.25 mg/day. In some embodiments, trametinib is administered at an oral dose of 0.5 mg/day. In some embodiments, trametinib is administered at an oral dose of 1 mg/day. In some embodiments, trametinib is administered at an oral dose of 1.5 mg/day. In some embodiments, trametinib is administered at an oral dose of 2 mg/day.

In another aspect, a pharmaceutical composition comprising trametinib is provided for use in the above methods.

Another aspect of the invention provides a composition for determining the therapeutic efficacy of a MEK1/2 inhibitor, such as trametinib, for a neurodegenerative disease, a disease associated with lysosomal dysfunction or autophagic tide, a disease associated with neuronal injury, or a disease associated with myelin sheath injury or demyelination of nerve fibers, comprising a probe or antibody that specifically binds to one or more of the markers described above.

In some embodiments, the therapeutic efficacy of the MEK inhibitor is determined by comparing the level of one or more markers in a biological sample obtained from a patient diagnosed with the disease or disorder after administration of trametinib to (a) the level of one or more markers in a biological sample obtained from the patient prior to the start of administration of trametinib, or (b) the level of one or more markers in a biological sample obtained from a healthy subject without the disease or disorder.

Another aspect includes methods of detecting marker levels using probes or antibodies that specifically bind to one or more of the above-described markers to provide information about the therapeutic efficacy of a MEK1/2 inhibitor, such as trametinib, on neurodegenerative diseases, diseases associated with lysosomal dysfunction or autophagic tide, diseases associated with neuronal injury, or diseases associated with myelin damage or demyelination of nerve fibers.

Advantageous effects

The present disclosure relies on the following findings: administration of trametinib in an effective amount for more than four weeks induces genetic, structural and functional changes associated with nerve regeneration and enables survival of neuronal-like cells differentiated in the brain of an animal model of Alzheimer's Disease (AD). Since trametinib targets multiple pathways that promote recovery of neuronal function in the degenerated brain, these data predict that daily administration of an effective amount of trametinib can reverse at least four weeks the functional deficits associated with neurodegenerative diseases, and can be used to treat AD as well as other neurodegenerative diseases.

Drawings

Figure 1 shows concentration-time curves of trametinib in brain (top) and plasma (bottom) after a single oral administration of trametinib in mice.

FIG. 2 provides representative images of Western blot analysis of pERKs and ERKs in mouse whole brain lysates. ERKs were included as loading internal controls.

Figures 3A and B show data obtained from brain lysate analysis of normal mice administered trametinib in a time-dependent manner. Figures 3A and B track, in gene ontology terms, up-regulated genes (figure 3A) and down-regulated genes (figure 3B) associated with biological processes at each time point in the brain.

FIGS. 4A to C and 4D to G show genes involved in synaptic, neurogenesis, lysosomal and autophagosome activities that exhibited significant changes in mRNA expression levels by administration of trametinib compared to the vehicle treated group. The values in fig. 4D to G represent fold-change (FC) in mRNA expression levels of the trametinib-treated group compared to the vehicle-treated group.

Fig. 5A shows the normalized EPSC slope in LTP recordings from CA1 recording electrodes, where the baseline balance at 3 minutes prior to TBS induction (red arrow) was measured and the subsequent 20 minute recordings in WT-support (black circle; n ═ 8 slices of 6 mice), 5 XFAD-support (blue squares; n ═ 4 slices of 3 mice) and 5 XFAD-trametinib (red triangle; n ═ 6 slices of 3 mice) were measured. Representative EPSCs of each type are shown as baseline (light color) and response at 20 minutes (vivid color). Scale bar: 20ms, 100 pA. Fig. 5B is a graph comparing the average of the normalized EPSC slopes from 15.5 minutes to 20 minutes.

FIG. 6A shows the average ratio of alternation over 3 minutes as measured in the Y-maze test. Figure 6B provides the average ratio of the number of surveys over 3 minutes measured and calculated in the novelty identification test. P values were obtained by analysis of variance (ANOVA; analysis of variance) tests. P <0.05, between the WT-vector group (n ═ 5), the 5 XFAD-vector group (n ═ 4) and the 5 XFAD-trametinib group (n ═ 5).

Figure 7 provides an immunofluorescence stain image and quantification of neurite/axon length and swollen axon area in the cortex V of eight month-old 5XFAD mice. Five-month-old mice were administered vehicle and 0.1 mg/kg/day trametinib for 3 months. N-3 sagittal sections from each mouse, and n-3 mice per group. Normalized to WT-vector group. Scale bar, 50 μm. Scale bar, 50 μm. P values were obtained by T-test. P <0.05, p <0.005 and p <0.001 between the WT-vector group and the 5 XFAD-vector group. # p <0.05, # # p <0.005 and # # # p <0.001 are between the 5 XFAD-vector group and the 5 XFAD-trametinib group.

Figure 8 provides an immunofluorescence stain image and quantification of neurite/axon length and swollen axon regions in cortical layer V from thirteen month old 5XFAD mice. Twelve months old 5XFAD mice were dosed with vehicle and trametinib for 1 month. N-3 serial sagittal sections from each mouse, with each group of n-3 mice. Standardized to a scale bar of 5 XFAD-support set, 50 μm. P values were obtained by T-test. P <0.05, p <0.005 and p <0.001 between the 5 XFAD-vector and 5 XFAD-trametinib groups.

Figure 9 is an image of a representative western blot analysis of brain cortex lysate from the mouse of figure 7 for a given protein.

Figure 10 is an image of a representative western blot analysis of brain cortex lysate from the mouse of figure 8 for a given protein.

Fig. 11A and B provide immunofluorescence staining images and quantification showing changes in dendritic spines caused by trametinib (SNR1611) in primary cortical neurons. P values were obtained by T-test. And pair ofIn comparison with groups,. about.p<0.05，***p<0.001. And Abeta₄₂Treatment group (n ═ 17) comparison, ### p<0.001。

Figure 12 is an image of representative western blot analysis of mouse brain cortex lysates for the indicated proteins.

FIG. 13A is an image of a representative Western blot analysis of SH-SY5Y cell lysate against a given protein. FIG. 13B shows the use of trametinib (Tra) and/or A β as compared to untreated controls_1-42Quantification of LC3II/LC3I (left) and mature cathepsin B (right) in treated cells. P values were obtained by T-test. P compared to control group<0.05，**p<0.005. and-Abeta₄₂Comparison of treatment groups, # # # p<0.001(LC3II/LC 3I: n-5, cathepsin B: n-6).

Figure 14A is an image of a representative western blot analysis of primary cortical neurons for a given protein. FIG. 14B shows treatment with trametinib (SNR1611) and/or A β as compared to untreated controls_1-42Quantification of mature cathepsin B in treated neurons. P values were obtained by T-test. P compared to untreated control group<0.05. And Abeta₄₂Treatment group (n ═ 5) comparison, # p<0.05。

Fig. 15A and B provide immunofluorescence images of LC3, LAMP1, and lysosomal probe (lysotracker) and quantification of co-staining rate and number of lysosomal probe stain (punta) of cells in SH-SY5Y cells. Scale bar, 10 μm. P values were obtained by T-test. P<0.05、**p<0.005 and x p<0.001 in control group with trametinib or control with Abeta_1-42In the meantime. # p<0.005 and # # # p<0.001 at A.beta._1-42And Abeta_1-42Between/trametinib.

Fig. 16A and B are immunofluorescence images of LC3, LAMP1, and lysosomal probe (fig. 16A) and quantification of the cell rate of LC3 and LAMP1 antibody staining (top of fig. 16B) and the number of lysosomal probe stain per cell in primary cortical neurons (bottom of fig. 16B). Scale bar, 20 μm. P values were obtained by T-test. P<0.05、**p<0.005 and x p<0.001 in control group and trametinib or control group and Abeta_1-42In the meantime. # p<0.05、##p<0.005 and # # # p<0.001 at Aβ_1-42And Abeta_1-42Between/trametinib.

FIGS. 17A, B and C are representative Western blot analyses of SH-SY5Y cell lysates for the indicated proteins.

Fig. 18A is an image of representative western blot analysis of primary cortical neurons for a given protein. FIGS. 18B and C show treatment with trametinib (SNR1611) and/or A β as compared to untreated controls_1-42Quantification of p-mTOR/mTOR and p-ULK1(s757)/ULK1 in oligomer-treated neurons. P values were obtained by T-test. P compared to untreated control group<0.05. And Abeta₄₂Treatment group comparison, # p<0.05 (fig. 18B; n-5, fig. 18C; n-4).

Figure 19 provides immunofluorescence images (left) and quantification (right) of apoptotic cells, LC3 and LAMP1 in cortex V. The arrows in the LC3/LAMP1 map indicate the co-stained regions. N-3 sagittal sections from each mouse, and n-3 mice per group. Scale bar, 10 or 50 μm. P values were obtained by T-test. # p <0.05 and # p <0.005 between the 5 XFAD-vector group and the 5 XFAD-trametinib group, and # p <0.001 between the WT-vector group and the 5 XFAD-vector group.

FIGS. 20A and B are cathepsin B (CTSB) levels in plasma of eight month old 5XFAD mice (FIG. 20A) and thirteen month old 5XFAD mice (FIG. 20B) following administration of trametinib (SNR 0.05: trametinib 0.05 mg/kg/day, SNR 0.1: trametinib 0.1 mg/kg/day) and donepezil. P values were obtained by T-test. P <0.05 compared to the 5 XFAD-vector group.

FIG. 21 is an immunofluorescent stained image of A β, active caspase 3 and Tau protein in Neural Stem Cells (NSCs) from adult Tg2576 mice. NSCs were treated with 100nM trametinib for 48 hours under undifferentiated or differentiated conditions. The medium conditions for undifferentiated cells contained 10ng/ml EGF and 10ng/ml bFGF. Growth factors were excluded from differentiation conditions. Scale bar, 20 μm.

Fig. 22A and B provide immunofluorescence images of LAMP1 and LC3 and the pooling of the two signals in NSCs from adult Tg2576 mice (fig. 22A) and magnification of the pooled images (fig. 22B). NSCs were treated with 100nM trametinib for 48 hours under either undifferentiated ("UD") or differentiated ("D") conditions. The medium conditions for undifferentiated cells contained 10ng/ml EGF and 10ng/ml bFGF. Growth factors were excluded from differentiation conditions. Yellow arrows indicate the combined signal of LAMP1 and LC 3. White arrows only indicate LAMP1 signal.

FIG. 23 shows Myelin Basic Protein (MBP) staining in the cortex of eight months old 5XFAD mice after dosing with trametinib (SNR 0.05: trametinib 0.05 mg/kg/day, SNR 0.1: trametinib 0.1 mg/kg/day) and donepezil.

The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.

Detailed Description

1. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the following meanings assigned to them.

As used herein, the term "MEK 1/2 inhibitor" refers to a compound that inhibits the function of both MEK1 and MEK 2.

An exemplary MEK1/2 inhibitor is trametinib (GSK-1120212, GSK1120212, JTP74057 or JTP-74057). The chemical name of trametinib is acetamide, N- [3- [ 3-cyclopropyl-5- [ (2-fluoro-4-iodophenyl) amino]-3,4,6, 7-tetrahydro-6, 8-dimethyl-2, 4, 7-trioxopyridino [4,3-d]Pyrimidin-1 (2H) -yl]Phenyl radical]. Its molecular formula is C₂₆H₂₃FIN₅O₄And has a molecular weight of 615.39. Trametinib has the chemical structure of formula 1.

Formula 1

In the commercial productsIn (b), trametinib is in the form of a dimethylsulfoxide solvate. In the invention described herein, trametinib may be used in the form of the free base or a pharmaceutically acceptable salt or solvate, including dimethylsulfoxide solvate. Examples of possible solvates are hydrates, dimethyl sulfoxide, acetic acid, ethanol, nitromethane, chlorobenzene, 1-pentanol, isopropanol, ethylene glycol, 3-methyl-1-butanol, etc.

As used herein, the term "therapeutically effective dose" or "effective amount" refers to an administered dose or amount that produces a desired effect. In the context of the present methods, a therapeutically effective amount is an amount effective to treat a symptom or improve a disease state in a subject having a neurodegenerative disease. As used herein, the term "sufficient amount" refers to an amount sufficient to produce a desired effect.

2. Other explanation conventions

Ranges recited herein are to be understood as shorthand for all values falling within the range, including the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from 1, 2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

Unless otherwise indicated, reference to a compound having one or more stereocenters refers to each stereoisomer thereof and all combinations of stereoisomers thereof.

3. Methods of treating neurodegenerative diseases

In a first aspect, a method for treating a patient suffering from a neurodegenerative disease is presented. The method comprises administering trametinib daily to a patient diagnosed with a neurodegenerative disease for at least four weeks. In some embodiments, trametinib is administered to provide a mean peak concentration of trametinib (C) in the brain of at least 0.25ng/g_max)。

A variety of delivery methods can be used to administer trametinib in the methods described herein. In a currently preferred embodiment, trametinib is delivered by oral administration.

3.1. Subjects receiving trametinib therapy

3.1.1. Patients with neurodegenerative diseases

In the examples below, we demonstrate that trametinib has multifaceted therapeutic effects that promote the recovery of degenerated brain neuronal function. Thus, the methods described herein are useful for treating neurodegenerative diseases characterized by cortical degeneration, such as alzheimer's disease. In the examples below, we also show that trametinib promotes lysosomal activity; accordingly, trametinib is useful for treating diseases characterized by lysosomal dysfunction or autophagic tide dysfunction. In the examples below, we also show that trametinib induces axonogenesis (axogenesis) in the nervous system; accordingly, trametinib is useful for treating diseases that can be controlled or cured by inducing axonogenesis, such as diseases characterized by neuronal damage, including neuronal death, neurodegeneration, physically damaged nerves and/or neurite damage, axonal lesions, and reduced potential for axonal growth. In the examples below, we also show that trametinib protects or repairs myelin sheaths around nerve cell axons; accordingly, trametinib is useful for treating diseases associated with myelin sheath damage or demyelination of nerve fibers.

Neurodegenerative diseases that can be treated by the methods provided herein include, but are not limited to, dementia, vascular dementia, senile dementia, frontotemporal dementia (FTD), Lewy Body Dementia (LBD), Parkinson's disease (PD; Parkinson's disease), multiple system atrophy (MSA; multiple system atrophy), corticobasal degeneration (CBD; corticobasal degeneration), progressive supranuclear palsy (PSP; progressive subapertular palsy), Huntington's disease (HD; huntton's disease), Amyotrophic Lateral Sclerosis (ALS), Lou-Gehrig's disease), primary lateral sclerosis (CJ; primary systemic sclerosis), progressive bulbar palsy (PBP; PLSP; hypertrophic bulbar paralysis), progressive bulbar paralysis (HSP; Parkinson's disease), progressive bulbar paralysis (HSP), progressive focal paralysis (HSP; Parkinson's disease; HSP; Parkinson's; HSP; Parkinson's; HSP; Parkinson's; Parkinson's; HSP, Parkinson's; P; Parkinson's; HSP, Parkinson's; P; HSP, Parkinson's; P; Parkinson's; P; Parkinson's; P; Parkinson's; P, P; P, P, Multiple Sclerosis (MS), Guillain-Barre syndrome (GBS), and Mild Cognitive Impairment (MCI).

In some embodiments, the patient selected for treatment has Alzheimer's Disease (AD). AD patients may have mild AD, moderate AD, or severe AD. In some embodiments, the patient has early-onset AD. In some embodiments, the patient has late-onset AD. In some embodiments, AD patients exhibit high serum albumin to globulin ratios and high levels of C-reactive protein, which indicates inflammation. In some embodiments, AD patients do not exhibit elevated inflammation biomarkers such as CRP or elevated serum albumin to globulin ratios. In some embodiments, the patient exhibits zinc deficiency in various regions throughout the brain. In some embodiments, AD patients exhibit high plasma protease levels. In some embodiments, the protease is a cathepsin. In some embodiments, the cathepsin is selected from: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L. In some embodiments, the protease is cathepsin B.

In some embodiments, the patient has one or more symptoms, such as memory loss, language problems, unpredictable behavior, and personality and behavioral changes. In some embodiments, the patient does not have any behavioral symptoms. In some embodiments, the patient has a change in one or more biomarkers associated with AD.

In some embodiments, the patient has Mild Cognitive Impairment (MCI). In some embodiments, the patient has memory impairment and memory difficulties. In some embodiments, the patient has abnormal memory function with a record having an educational adjustment threshold (top score of 25 points) with a score below the logical memory ii sub-scale (delayed paragraph recall) from the wecker memory scale-revision: a) less than or equal to 8 for 16 years and more, b) less than or equal to 4 for 8 to 15 years of education, c) less than or equal to 2 for 0 to 7 years of education. In some embodiments, the patient's Mini-mental state exam score is between 24 and 30 (inclusive). In some embodiments, the patient has a clinical dementia rating scale score of 0.5 and a memory box score of at least 0.5. In some embodiments, the patient's general cognitive and functional performance is sufficiently preserved so that it cannot be diagnosed with AD.

In some embodiments, the neurodegenerative disease involves abnormal activation of MAPK. In some embodiments, the neurodegenerative disease involves abnormal activation of the MAPK/ERK pathway. In some embodiments, the neurodegenerative disease involves abnormal endosomal-lysosomal function.

In preferred embodiments, the patient does not have BRAF V600E or V600K mutations and the patient does not have cancer.

3.1.2. Patients with diseases associated with lysosomal dysfunction or autophagy tide

In the examples, we demonstrate that trametinib promotes lysosomal activity by inducing autophagosome-lysosomal fusion through modulation of the rapamycin target protein (mTOR; mammalin target of rapamycin) and Transcription factor EB (TFEB; Transcription factor EB) pathways. Accordingly, trametinib is useful for treating diseases characterized by lysosomal dysfunction or autophagic tide dysfunction. Such diseases include, but are not limited to, lysosomal storage diseases, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, huntington's disease, spinocerebellar atrophy, oculopharyngeal muscular dystrophy, prion diseases, fatal familial insomnia, alpha-1 antitrypsin deficiency, dentatorubral pallidoluysian atrophy, frontotemporal dementia, progressive supranuclear palsy, X-linked spinobulbar muscular atrophy, intranuclear clear inclusion body disease, multiple sclerosis, glaucoma, and age-related macular degeneration. Lysosomal storage diseases include, but are not limited to, mannosidosis, aspartyl glucosaminuria, juvenile neuronal ceroid lipofuscinosis (JNCL, juvenile Barton's or CLN3 disease), cystinosis, Fabry's disease, gaucher's disease types I, II and III, glycogen storage disease type II (Pompe's disease), Gm 2-gangliosidosis type II (sandhoff's disease), metachromatic brain protein dystrophy, mucolipidosis types I, II/III and IV, mucopolysaccharidosis (Hewler's disease and its variants, Hunter's disease, Sanfilippo A, B, C and D syndrome, Morkolo A and B syndrome, Maroteaux-Lamy and Sly syndrome), Niemann-gram's disease types A/B, C1 and C2, and Sindler's disease types I and II.

3.1.3. Patients with diseases associated with neuronal damage

In the examples, we demonstrate that trametinib induces axonogenesis in the nervous system (axogenesis). Accordingly, trametinib is useful for treating diseases that can be controlled or cured by inducing axonogenesis, such as diseases characterized by neuronal damage, including, but not limited to, neuronal death, neurodegeneration, physically damaged nerves and/or axonal damage, axonal pathology, or diminished axonal growth potential. Such diseases include, but are not limited to, glaucoma stroke, head trauma, spinal cord injury, optic nerve injury, ischemia, hypoxia, neurodegenerative diseases, multiple sclerosis, and multiple system atrophy. The disease also includes diabetic neuropathy; viral-associated neuropathy; including acquired immunodeficiency syndrome (AIDS) -associated neuropathy; infectious mononucleosis with polyneuritis; viral hepatitis with polyneuritis; Guillain-Barre syndrome; neuropathy associated with botulism; toxic polyneuropathy including lead and alcohol related neuropathy; vegetative neuropathy including subacute joint degeneration; vasculopathy including neuropathy associated with systemic lupus erythematosus; neuropathy associated with sarcoidosis; cancerous neuropathy; pressure neuropathy (e.g., carpal tunnel syndrome); hereditary neuropathy such as peroneal muscular atrophy; and peripheral nerve damage associated with spinal cord injury. The disease also includes an eye injury or disease (e.g., toxic amblyopia, optic atrophy, high vision disorder, eye movement disorder, third cerebral nerve paralysis, fourth cerebral nerve paralysis, sixth cerebral nerve paralysis, internuclear ophthalmoplegia, gaze paralysis, free radical damage to the eye, etc.) or an optic neuropathy (e.g., ischemic optic neuropathy, toxic optic neuropathy, ocular ischemic syndrome, optic neuritis, optic nerve infection, optic neuritis, optic neuropathy, optic papillary edema, optic neuritis, retrobulbar optic neuritis, retinal concussion, glaucoma, macular degeneration, retinitis pigmentosa, retinal detachment, tear due to birth, diabetic retinopathy, iatrogenic stretch-dependent keratopathy, and optic nerve drusen, etc.)

3.1.4. Patients with diseases associated with damaged myelin sheath

In the examples, we demonstrate that trametinib protects or repairs the myelin sheath surrounding nerve cell axons. Accordingly, trametinib is useful for treating diseases characterized by myelin damage or demyelination of nerve fibers, such as multiple sclerosis, acute disseminated encephalomyelitis, transverse myelitis, Sheer's disease, Crohn's disease, clinically isolated syndrome, Alexander's disease, Kanawan's disease, Kekan syndrome, Palmer's disease, optic neuritis, neuromyelitis optica, HTLV-I related myelopathy, hereditary leukoencephalopathy, Guillain-Barre syndrome, Central pontine myelinolysis, deep leukoischemia, progressive multifocal leukoencephalopathy, demyelinating HIV encephalitis, demyelinating radiation injury, acquired toxic metabolic disorder, reversible posterior encephalopathy syndrome, Central pontine myelinolysis, leukodystrophy, adrenoleukodystrophy, Krabe's globulopathy, and/or metachromatic encephaloprotein dystrophy. Other diseases in which demyelination occurs include myelogenous cervical spondylosis caused by cervical stenosis, traumatic injury to the brain or spinal cord, and hypoxic injury to the central nervous system including stroke and neonatal hypoxic injury.

3.2. Administration of trametinib

3.2.1. Period of time

Administering a therapeutically effective amount of trametinib to the selected patient daily for at least four weeks. In some embodiments, trametinib is administered for a period of time sufficient to induce neural differentiation. In some embodiments, trametinib is administered for a period of time sufficient to induce nerve regeneration. In some embodiments, trametinib is administered for a period of time sufficient to induce lysosomal activity. In some embodiments, trametinib is administered for a period of time sufficient to enhance autophagosome-lysosomal fusion. In some embodiments, trametinib is administered for a period of time sufficient to induce neuritogenesis. In some embodiments, trametinib is administered for a period of time sufficient to protect newly formed axons in the nervous system. In some embodiments, trametinib is administered for a period of time sufficient to induce repair or protection of myelin.

In certain embodiments, trametinib is administered for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, or at least ten weeks. In certain embodiments, trametinib is administered for at least one month, at least two months, at least three months, or four months. In some embodiments, trametinib is administered for about six weeks, seven weeks, eight weeks, nine weeks, ten weeks, or more. In some embodiments, trametinib is administered for about one month, two months, three months, four months, five months, six months, twelve months, or longer.

In some embodiments, trametinib is administered for a period of time sufficient to induce expression of genes involved in the formation of synapses in the brain. In some embodiments, trametinib is administered for a period of time sufficient to induce expression of genes involved in proliferation of neuroblasts in the brain. In some embodiments, trametinib is administered for a period of time sufficient to induce expression of genes involved in axonal growth in the brain. In some embodiments, trametinib is administered for a period of time sufficient to induce expression of genes involved in an immune response in the brain. In some embodiments, trametinib is administered for a period of time sufficient to induce expression of genes involved in lysosomal and/or autophagosome activity. In some embodiments, trametinib is administered for a period of time sufficient to induce expression of genes involved in synapse formation, neuroblast proliferation, axon growth, lysosomal activity, and autophagosome activity in the brain.

In some embodiments, trametinib is administered until a change in the level of one or more markers is detected. In one embodiment, each of the one or more markers is encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctsk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Admp, Vpps, Admp, Vpmin, Pck, and Pck 13. In some embodiments, each of the one or more markers is a protein associated with lysosomal activity. In some embodiments, the protein associated with lysosomal activity is a glycosyl hydrolase or protease. In some embodiments, the glycosyl hydrolase is selected from: beta-hexosaminidase, beta-galactosidase, beta-galactocerebrosidase, and beta-glucuronidase. In some embodiments, the protease is a cathepsin. In some embodiments, the cathepsins are selected from: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L. These proteins can be used as markers for measuring the efficacy of MEK1/2 inhibitors (such as trametinib).

In some embodiments, trametinib is administered until the level of one or more markers reaches at least 1.3x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 100x, 200x, or 1000x of the level prior to or without trametinib administration. In some embodiments, trametinib is administered until the level of one or more markers reaches at most 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1x, 0.05x, or 0.01x of the level prior to or without trametinib administration.

In some embodiments, trametinib is administered until the level of one or more markers reaches a fixed or predetermined level of at least 1.3x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 100x, 200x, or 1000 x. In some embodiments, trametinib is administered until the level of one or more markers reaches at most 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1x, 0.05x, or 0.01x of a fixed or predetermined level.

In some embodiments, trametinib is administered until a desired therapeutic outcome is detected. In some embodiments, the desired therapeutic result is a change in a behavioral symptom of the patient. In some embodiments, trametinib is administered until unacceptable toxicity occurs.

3.2.2. Dosage form

Trametinib is administered in a therapeutically effective dose. In the methods described herein, a therapeutically effective dose is a dose effective to treat a neurodegenerative disease in a subject. In a particular embodiment, the therapeutically effective dose is a dose effective to treat AD in the subject.

In some embodiments, a therapeutically effective dose is a dose sufficient to induce neural differentiation. In some embodiments, a therapeutically effective dose is a dose sufficient to induce nerve regeneration. In some embodiments, a therapeutically effective dose is a dose sufficient to induce lysosomal activity. In some embodiments, a therapeutically effective dose is a dose sufficient to induce neuritogenesis. In some embodiments, the therapeutically effective dose is a dose sufficient to enhance autophagosome-lysosomal fusion in the subject. In some embodiments, a therapeutically effective dose is a dose sufficient to protect newly formed axons in the nervous system. In some embodiments, a therapeutically effective dose is a dose sufficient to induce myelin repair or protection.

In some embodiments, a therapeutically effective dose is a dose sufficient to induce expression of genes involved in synaptic formation in the brain. In some embodiments, a therapeutically effective dose is a dose sufficient to induce expression of genes involved in proliferation of neuroblasts in the brain. In some embodiments, the therapeutically effective dose is a dose sufficient to induce expression of genes involved in axonal growth in the brain. In some embodiments, a therapeutically effective dose is a dose sufficient to induce expression of genes involved in axonogenesis. In some embodiments, a therapeutically effective dose is a dose sufficient to induce expression of a gene involved in enhancing lysosomal activity. In some embodiments, a therapeutically effective dose is a dose sufficient to induce expression of genes involved in an immune response in the brain. In some embodiments, a therapeutically effective dose is sufficient to induce expression of a gene involved in lysosomal and/or autophagosome activity. In some embodiments, the therapeutically effective dose is a dose sufficient to induce expression of genes involved in synapse formation, neuroblast proliferation, axon growth, lysosomal activity, and autophagosome activity in the brain.

In some embodiments, trametinib is administered at a dose sufficient to induce a change in the level of one or more markers. In some embodiments, trametinib is administered at a dose sufficient to cause a change in the level of one or more markers in a target tissue of a patient or a biological sample obtained from the patient. In one embodiment, each of the one or more markers is encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctsk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Admp, Vpps, Admp, Vpmin, Pck, and Pck 13. The human homolog of the mouse gene can be GABRB, GABRR, GLRA, NR3C, CDKL, GRIN2, PLCXD, CHRM, CHRNA, CHRNB, NEFL, PLD, ADRA1, CHRNB, SLC6A, SLC18A, CDH, neuod, NKX-1, CXCL, REST, SYT, DISC, IRX, MDM, SOX, GRIP, PAX, BMP, CPNE, NUMB, ATP8A, TRIM, OTP, IL1RAPL, CPEB, TNFRSF12, HSPB, OPRM, LMX1, CLCF, ASPM, MECP, NTF, VEGFA, LRP, FEZ, ATP6V0, RNASE, CTSK, ACR, PRSS, PRDX, UNC13, rgn, TIAL, adbanb, ASSs, CCKAR, cgar, pcgun 152, pcgun, hmgnk, pcsmox, vpgimb, and vpgimb. In some embodiments, each of the one or more markers is a protein associated with lysosomal activity. In some embodiments, the protein associated with lysosomal activity is a glycosyl hydrolase or protease. In some embodiments, the glycosyl hydrolase is selected from: beta-hexosaminidase, beta-galactosidase, beta-galactocerebrosidase, and beta-glucuronidase. In some embodiments, the protease is a cathepsin. In some embodiments, the cathepsin is selected from: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L. These proteins can be used as marker proteins for measuring the effect of MEK1/2 inhibitors (e.g. trametinib).

In some embodiments, trametinib is administered at a mean peak concentration (C) that provides at least 0.25ng/g of trametinib in the brain_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide a mean peak concentration (C) of trametinib of at least 0.5, 0.75, 1, 1.25, 1.50, 1.75, or 2ng/g in the brain_max) The dosage of (a). In some embodiments, trametinib is formulated to provide an average peak concentration of trametinib (C) in the brain of at least 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3ng/g_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide an average peak concentration of trametinib (C) in the brain of about 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20ng/g_max) The dosage of (a). In some embodiments, trametinib is formulated to provide an average peak concentration of trametinib (C) in the brain of between 0.25 to 20, 0.25 to 10, 0.25 to 5, 0.5 to 5, 2.5 to 10, and 1 to 5ng/g_max) The dosage of (a).

In some embodiments, trametinib is formulated to provide a mean peak concentration of trametinib (C) in CSF of at least 0.25ng/ml_max) The dosage of (a). In some embodiments, trametinib is formulated to provide a mean peak concentration (C) of trametinib of at least 0.5, 0.75, 1, 1.25, 1.50, 1.75, or 2ng/ml in CSF_max) The dosage of (a). In some embodiments, trametinib is formulated to provide a mean peak concentration of trametinib (C) in CSF of at least 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3ng/ml_max) The dosage of (a). In some embodimentsWherein trametinib is present in the CSF to provide an average peak concentration (C) of trametinib of about 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20ng/ml in CSF_max) The dosage of (a). In some embodiments, trametinib is administered at a dose that provides a mean peak concentration (Cmax) of trametinib in CSF of between 0.25 to 20, 0.25 to 10, 0.25 to 5, 0.5 to 5, 2.5 to 10, and 1 to 5 ng/ml.

In some embodiments, trametinib is administered in an amount to provide an average peak concentration of trametinib (C) in the brain of no more than 4.4ng/g_max) The dosage of (a). In some embodiments, trametinib is administered in an amount to provide an average peak concentration of trametinib (C) in the brain of no more than 2ng/g_max) The dosage of (a). In some embodiments, trametinib is formulated to provide an average peak concentration of trametinib (C) in the brain of no more than 1.8ng/g, no more than 1.6ng/g, no more than 1.4ng/g, no more than 1.2ng/g, no more than 1ng/g, no more than 0.8ng/g, no more than 0.6ng/g, or no more than 0.4ng/g_max) The dosage of (a).

In some embodiments, trametinib is formulated to provide a mean peak concentration of trametinib (C) in CSF of no more than 4.4ng/ml_max) The dosage of (a). In some embodiments, trametinib is formulated to provide a mean peak concentration of trametinib (C) in CSF of no more than 2ng/ml_max) The dosage of (a). In some embodiments, trametinib is formulated to provide an average peak concentration of trametinib (C) in CSF of no more than 1.8ng/ml, no more than 1.6ng/ml, no more than 1.4ng/ml, no more than 1.2ng/ml, no more than 1ng/ml, no more than 0.8ng/ml, no more than 0.6ng/ml, or no more than 0.4ng/ml in CSF_max) The dosage of (a).

In some embodiments, trametinib is formulated to provide a mean peak concentration (C) of trametinib in plasma that does not exceed 22.2ng/ml_max) The dosage of (a). In some embodiments, trametinib is formulated to provide a mean peak concentration (C) of trametinib in plasma of no more than 20ng/ml, no more than 18ng/ml, no more than 16ng/ml, no more than 14ng/ml, no more than 12ng/ml, no more than 10ng/ml, no more than 8ng/ml, no more than 6ng/ml, or no more than 4ng/ml_max) The dosage of (a).

In some embodiments, trametinib is administered at a dose that provides an area under the concentration curve (AUC) of trametinib in the brain of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 ng-h/g. In some embodiments, trametinib is administered at a dose that provides an area under the brain concentration curve for trametinib of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, or 300ng · h/g. In some embodiments, trametinib is administered at a dose that provides an area under the brain concentration curve for trametinib of about 20 to about 700 ng-h/g, about 20 to about 600 ng-h/g, about 30 to about 500 ng-h/g, about 50 to about 400 ng-h/g, about 50 to about 300 ng-h/g, about 50 to about 200 ng-h/g, about 50 to about 100 ng-h/g, about 60 to about 300 ng-h/g, about 30 to about 200 ng-h/g.

In some embodiments, trametinib is administered at a dose that provides an area under the concentration curve (AUC) of trametinib in CSF of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 ng-h/ml. In some embodiments, trametinib is administered at a dose that provides an area under the concentration curve of trametinib in CSF of about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, or 300ng · h/ml. In some embodiments, trametinib is administered at a dose that provides an area under the CSF concentration curve for trametinib of about 20 to about 700 ng-h/ml, about 20 to about 600 ng-h/ml, about 30 to about 500 ng-h/ml, about 50 to about 400 ng-h/ml, about 50 to about 300 ng-h/ml, about 50 to about 200 ng-h/ml, about 50 to about 100 ng-h/ml, about 60 to about 300 ng-h/ml, about 30 to about 200 ng-h/ml.

In some embodiments, trametinib is administered at a mean peak concentration (C) that provides at least 0.25ng/ml of trametinib in plasma_max) The dosage of (a). In some embodiments, trametinib provides an average of trametinib of at least 0.5, 0.75, 1, 1.25, 1.50, 1.75, 2, 2.25, 2.50, 2.75, 3, 3.25, 3.50, 3.75, 4, 4.25, 4.50, 4.75, or 5ng/ml in plasmaPeak concentration (C)_max) The dosage of (a). In some embodiments, trametinib is present in the plasma at a peak concentration that provides a mean concentration of at least 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.6 ng, 5.6, 9, or/ml of trametinib (C/ml)_max) The dosage of (a). In some embodiments, trametinib is formulated to provide a mean peak concentration (C) of trametinib of about 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30ng/ml in plasma_max) The dosage of (a). In some embodiments, trametinib is formulated to provide an average peak concentration (C) of trametinib in plasma of between 1 to 200, 1 to 150, 1 to 100, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 10 to 100, 15 to 90, 20 to 80, 2.5 to 50, 2.5 to 25, 2.5 to 10, and 3 to 50ng/ml_max) The dosage of (a).

In some embodiments, trametinib is administered at a dose that provides an area under the plasma concentration curve of trametinib of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng-h/mL. In some embodiments, trametinib is administered at a dose that provides an area under the plasma concentration curve of trametinib of about 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 400, or 500 ng-h/mL. In some embodiments, trametinib is administered at a dose that provides an area under the plasma concentration curve of trametinib of about 20 to about 700 ng-h/mL, about 20 to about 600 ng-h/mL, about 30 to about 500 ng-h/mL, about 50 to about 400 ng-h/mL, about 50 to about 300 ng-h/mL, about 50 to about 200 ng-h/mL, about 50 to about 100 ng-h/mL, about 100 to about 500 ng-h/mL.

In some embodiments, trametinib is administered at a dose of between 0.5 and 2 mg/day. In some embodiments, trametinib is administered at a dose of between 0.75 and 2 mg/day. In some embodiments, trametinib is administered at a dose of between 1 and 2 mg/day. In some embodiments, trametinib is administered at a dose of between 0.75 and 1.25 mg/day. In some embodiments, trametinib is administered at a dose of between 0.5 and 1 mg/day. In some embodiments, trametinib is administered at a dose of 0.5 mg/day. In some embodiments, trametinib is administered at a dose of 1 mg/day. In some embodiments, trametinib is administered at a dose of 1.5 mg/day. In some embodiments, trametinib is administered at a dose of 2 mg/day.

In some embodiments, trametinib is administered at a dose greater than 0.5 mg/day and less than 2 mg/day. In some embodiments, trametinib is administered at a dose greater than 0.75 mg/day and less than 2 mg/day. In some embodiments, trametinib is administered at a dose greater than 1 mg/day and less than 2 mg/day. In some embodiments, trametinib is administered at a dose greater than 0.75 mg/day and less than 1.25 mg/day. In some embodiments, trametinib is administered at a dose greater than 0.5 mg/day and less than 1 mg/day.

In a preferred embodiment, each dose is a daily dose delivered as a single oral intake. In some embodiments, each dose is divided into several oral intakes. In some embodiments, each dose is divided into equal ingested doses. In some embodiments, each dose is divided into unequal ingested doses. In a preferred embodiment, each dose is administered at regular intervals.

4. Detection of markers

In another aspect, a method of testing the therapeutic outcome of a drug (e.g., a MEK1/2 inhibitor, such as trametinib) in a neurodegenerative subject is provided. The method comprises the step of measuring the level of one or more markers in a sample obtained from the subject.

In some embodiments, the methods provided herein further comprise the step of testing a sample obtained from the subject for expression of one or more markers. Expression of one or more markers can be tested by measuring protein or by measuring mRNA using methods known in the art, such as Western blotting, enzyme-linked immunosorbent assay (ELISA), RT-PCR, qPCR, immunoelectrophoresis, protein immunoprecipitation, and protein immunostaining. The method may employ various methods of measuring the amount of mRNA or protein.

In some embodiments, the methods provided herein further comprise the step of measuring the level of one or more marker proteins in a sample obtained from the subject. The level of one or more marker proteins can be measured using various protein assays known in the art. For example, the sample may be contacted with an antibody specific for the marker under conditions sufficient to form an antibody-marker complex, and the complex then detected. The presence of protein biomarkers can be detected by a variety of means, such as western blotting, ELISA, immunoelectrophoresis, protein immunoprecipitation, protein immunostaining, two-dimensional polyacrylamide gel electrophoresis (SDS-PAGE), Fluorescence Activated Cell Sorting (FACS), and flow cytometry.

The levels of one or more markers may be measured at multiple time points, and the amounts measured at different time points may be compared. Changes in the levels of one or more markers over time can be used to determine the therapeutic effect of a MEK1/2 inhibitor (e.g. trametinib) on a patient.

In one embodiment, each of the one or more markers is encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctsk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Admp, Vpps, Admp, Vpmin, Pck, and Pck 13.

In some embodiments, each of the one or more markers is a protein associated with lysosomal activity. The protein associated with lysosomal activity can be a glycosyl hydrolase or a protease. Said glycosyl hydrolase is selected from the group consisting of: beta-hexosaminidase, beta-galactosidase, beta-galactocerebrosidase, and beta-glucuronidase. In some embodiments, the protease may be a cathepsin. In some embodiments, the cathepsin may be selected from: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L.

In some embodiments, the level of one or more markers previously known to be associated with a neurodegenerative disease is measured.

In some embodiments, the one or more markers are selected from: (1) markers previously known to be associated with neurodegenerative diseases (e.g., AD); (2) a protein or mRNA encoded by a human homolog of a mouse gene selected from the group consisting of: gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Plud, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrfsf 12, pb, Oprm, Lmx1, Clcf, Aspm, Mecp, Ntf, Vegfa, Lrp, Hrp, 6v0, RNase, Ctk, Acr, Prss, Lamp, Prdx, Uncp, Bancr 13, Bancr, Adrn, Admp, Vpx, Vpmin, Pck 13, and Pck 13; and (3) proteins associated with lysosomal activity, such as glycosyl hydrolases or proteases. The glycosyl hydrolase is selected from the following groups: beta-hexosaminidase, beta-galactosidase, beta-galactocerebrosidase, and beta-glucuronidase. In some embodiments, the protease may be a cathepsin. In some embodiments, the cathepsin may be selected from: cathepsin S, cathepsin D, cathepsin B, cathepsin K and cathepsin L.

In some embodiments, the level of one or more markers is measured in a sample obtained after the step of starting the administration of trametinib. Samples may be obtained at one or more time points after the step of starting administration of trametinib. For example, samples are obtained one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen weeks after the start of dosing of trametinib. In some embodiments, the sample is obtained one, two, three, four, five or six months after the start of dosing trametinib. In some embodiments, the sample is obtained immediately after the step of starting administration of trametinib. In some embodiments, the samples are obtained at two different time points after the step of starting the administration of trametinib. In some embodiments, samples are obtained at three different time points after starting dosing trametinib. In some embodiments, the samples are obtained at four, five, six, seven, or eight different time points after the step of starting dosing trametinib.

In some embodiments, the level of one or more markers is measured in a control sample obtained prior to the start of dosing trametinib. In some embodiments, the level of one or more markers is measured in a biological sample obtained from a healthy subject without the disease of interest. In some embodiments, the method further comprises the step of comparing the level of one or more markers in a control sample obtained before starting the administration of trametinib to a sample obtained after the administration of trametinib. In some embodiments, the method further comprises the step of comparing the level of one or more markers in a healthy subject without the disease of interest to the level in a sample obtained from the patient before or after the start of the administration of trametinib. Comparison of levels of one or more markers can be used to determine the therapeutic effect of trametinib. In some embodiments, the level of one or more markers may be used to determine the appropriate trametinib administration duration or dose to achieve a desired therapeutic result. In some embodiments, a time course analysis of one or more markers is performed. In some embodiments, the level of one or more markers may be used to determine a method of subsequently administering trametinib, e.g., the duration and dose of trametinib. In some embodiments, the levels of one or more markers may be used to identify individuals more likely to experience the beneficial effects of exposure to trametinib as compared to individuals similar without the biomarker.

Samples for testing for markers can be obtained by any method known in the art. For example, the sample may be obtained by brain biopsy. In some embodiments, the sample is obtained by stereotactic brain biopsy. In some embodiments, the sample is obtained from a body fluid or secretion of the patient, such as blood, cerebrospinal fluid (CSF), urine, body fluid, saliva, stool, pleural fluid, lymph, sputum, ascites, prostatic fluid, or any other bodily secretion or derivative thereof. Blood samples include whole blood, plasma, serum, Peripheral Blood Mononuclear Cells (PBMC), or any blood component.

In another aspect, compositions for determining the therapeutic effect of a MEK1/2 inhibitor, e.g., trametinib, are disclosed that include probes for specifically detecting the markers. In another aspect, kits for such purposes are also provided. Such kits may include a carrier, e.g., a vial, a tube, etc., separated to receive one or more containers in a closed space, and each container includes one of the individual components used in the method. For example, one of the containers may include a probe that is detectably labeled or potentially detectably labeled. Such probes may be antibodies or polynucleotides specific for proteins or mrnas, respectively. Such kits typically include the above-described container and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is for a particular application, and may also indicate the direction of use in vivo or in vitro, such as described above.

A typical example is a kit comprising a container, a label on the container, and a composition contained within the container, wherein the composition comprises a primary antibody that binds to a protein or autoantibody biomarker, and the label on the container indicates that the composition can be used to assess the presence of such protein or antibody in a sample, wherein the kit comprises instructions for using the antibody to assess the presence of the biomarker protein in a particular sample type. The kit may also include a set of instructions and materials for preparing a sample and applying antibodies to the sample. The kit may include a primary antibody and a secondary antibody, wherein the secondary antibody is conjugated to a label.

5. Pharmaceutical compositions and unit dosage forms

In yet another aspect, the present disclosure provides pharmaceutical compositions and unit dosage forms comprising trametinib for use in the treatment of neurodegenerative diseases (e.g., AD).

In typical embodiments, trametinib is formulated for oral administration. In some embodiments, trametinib is a formulation with an inert diluent or with an edible carrier. In various embodiments, trametinib is encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the diet food. For oral therapeutic administration, the active compound may be combined with excipients and used in the form of ingestible tablets, oral tablets, coated tablets, buccal tablets, capsules, elixirs, dispersions, suspensions, solutions, syrups, wafers, patches, powders for oral administration, and the like.

Tablets, troches, pills, capsules and the like may also include one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid, and the like; lubricants such as magnesium stearate and the like; sweeteners such as sucrose, lactose or saccharin; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings, for example tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. It may be desirable that the materials in the dosage form or pharmaceutical composition be pharmaceutically pure and substantially non-toxic in the amounts used.

Some compositions or dosage forms may be liquid, or may include a solid phase dispersed in a liquid.

In some embodiments, the oral dosage form may include silicified microcrystalline cellulose, e.g.For example, about 20% (wt/wt) to about 70% (wt/wt), about 10% (wt/wt) to about 20% (wt/wt), about 20% (wt/wt) to about 40% (wt/wt), about 25% (wt/wt) to about 30% (wt/wt), about 40% (wt/wt) to about 50% (wt/wt), or about 45% (wt/wt) to about 50% (wt/wt) of silicified microcrystalline cellulose may be present in an oral dosage form or unit of an oral dosage form.

In some embodiments, the oral dosage form may include a cross-linked polyvinylpyrrolidone, such as crospovidone. For example, about 1% (wt/wt) to 10% (wt/wt), about 1% (wt/wt) to 5% (wt/wt), or about 1% (wt/wt) to about 3% (wt/wt) of the crosslinked polyvinylpyrrolidone may be present in the oral dosage form or units of the oral dosage form.

In some embodiments, oral dosage forms may include, for exampleFumed silica of (2). For example, from about 0.1% (wt/wt) to about 10% (wt/wt), from about 0.1% (wt/wt) to about 1% (wt/wt), or from about 0.4% (wt/wt) to about 0.6% (wt/wt) of the fumed silica can be present in the oral dosage form or unit of the oral dosage form. In some embodiments, the oral dosage form can include magnesium stearate. For example, about 0.1% (wt/wt) to 10% (wt/wt), about 0.1% (wt/wt) to 1% (wt/wt), or about 0.4% (wt/wt) to about 0.6% (wt/wt) magnesium stearate can be present in an oral dosage form or unit of an oral dosage form. Oral dosage forms comprising zoledronic acid or another diphosphate salt may includeIncluded in pharmaceutical products are oral dosage forms comprising more than one unit.

Trametinib can be formulated for other methods of administration, such as sublingual, rectal, intranasal, parenteral, transdermal or topical administration, or injection. Solutions of the active compound as a free acid or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. The dispersant may also have an oil dispersed therein or in glycerin, liquid polyethylene glycol, and mixtures thereof. Under normal conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

In a preferred embodiment, each unit of the oral dosage form contains an effective amount for daily administration. In some embodiments, each unit of the oral dosage form contains 0.1 to 3mg trametinib. In some embodiments, each unit of the oral dosage form contains 0.2 to 3mg trametinib. In some embodiments, each unit of the oral dosage form contains 0.3 to 3mg trametinib. In some embodiments, each unit of the oral dosage form contains 0.4 to 3mg trametinib. In some embodiments, each unit of the oral dosage form contains 0.5 to 3mg trametinib. In some embodiments, the oral dosage form contains 0.5 to 2.5mg trametinib per unit. In some embodiments, the oral dosage form contains 0.5 to 2mg trametinib per unit. In some embodiments, the oral dosage form contains 0.75 to 2.5mg trametinib per unit. In some embodiments, each unit of the oral dosage form contains 1 to 2mg trametinib. In some embodiments, the oral dosage form contains 0.75 to 1.25mg trametinib per unit. In some embodiments, each unit of the oral dosage form contains 0.2mg trametinib. In some embodiments, each unit of the oral dosage form contains 0.25mg trametinib. In some embodiments, each unit of the oral dosage form contains 0.5mg trametinib. In some embodiments, each unit of the oral dosage form contains 1mg trametinib. In some embodiments, each unit of the oral dosage form contains 1.5mg trametinib. In some embodiments, each unit of the oral dosage form contains 2mg trametinib. In some embodiments, each unit of the oral dosage form contains 2.5mg trametinib. In some embodiments, each unit of the oral dosage form contains 3mg trametinib.

In some embodiments, each unit of the oral dosage form is a MEKINIST tablet containing 0.5mg, 1mg, or 2mg trametinib. In some embodiments, each 0.5mg tablet contains 0.5635mg trametinib dimethyl sulfoxide, corresponding to 0.5mg of trametinib unsolvated precursor. In some embodiments, 1.127mg trametinib dimethyl sulfoxide per 1mg tablet is equivalent to 1mg of trametinib unsolvated parent. In some embodiments, 2.254mg trametinib dimethyl sulfoxide per 2mg tablet is equivalent to 1mg of trametinib unsolvated parent.

In some embodiments, the tablet contains about 25% to about 89% by weight of one or more excipients. In some embodiments, the excipient is substantially free of water. The one or more excipients may be selected from microcrystalline cellulose, powdered cellulose, pregelatinized starch, lactose, dibasic calcium phosphate, lactitol, mannitol, sorbitol, and maltodextrin. In some embodiments, the amount of unsolvated trametinib is no more than about 20%. Pharmaceutical compositions described in U.S. patent nos. 8,580,304 and 9,271,941, which are incorporated by reference in their entirety, may be used in various embodiments of the present disclosure.

The tablet may also include a core comprising colloidal silicon dioxide, croscarmellose sodium, hydroxypropylmethyl cellulose, magnesium stearate (vegetable derived), mannitol, microcrystalline cellulose and sodium lauryl sulphate. The tablet may also include a coating comprising hydroxypropyl methylcellulose, red iron oxide, yellow iron oxide, polyethylene glycol, polysorbate 80, and/or titanium dioxide.

6. Examples of the embodiments

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.). But some experimental error and deviation should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp, base pair; kb, kilobases; pl, picoliter; s or sec, seconds; min, min; h or hr, hours; aa, an amino acid; nt, nucleotide, and the like.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology, within the skill of the art

6.1. Example 1: crossing the blood brain barrier

Trametinib was confirmed to cross the blood brain barrier (BBB; blood-brain-bridge barrier) after a single oral administration to normal mice. The brain/plasma exposure ratio (AUC) of the highest dose group was 47.7% (fig. 1). Trametinib was also found to exert its MEK1/2 inhibition in the brain of normal mice after oral administration by causing a significant decrease in pERK expression (fig. 2). The p-ERKs/ERKs ratio decreased by 22.5%, 33.7%, 50% and 45.6% after one, two, three and four weeks of administration, respectively, compared to the vehicle treated group. These results indicate that trametinib penetrates the BBB.

6.2. Example 2: time course of gene expression changes in brain following administration of trametinib

To evaluate the transcriptional profile in the brain, a number of RNA-Seq were performed using the whole brain of normal mice after oral administration of trametinib. Gene ontology terms were enriched at each time point of the week, indicating correlations in cell function, such as synaptic potentials, nervous system development, immune responses, and incorrect protein folding in temporal patterns (fig. 3A). Reduction of MEK-ERK signaling by administration of trametinib between the first and second weeks of administration can be indirectly demonstrated by a reduction in expression of genes involved in FGF receptor signaling and GPCR signaling. The reduced telomere-associated gene expression observed at the fourth week of trametinib administration (fig. 3B) indicates that it is closely associated with neuronal maturation or terminally differentiated neurons activated by neurogenesis.

Our results indicate that the first week of trametinib administration appears to be a critical period for the establishment of neuronal communication, evidenced by transcriptional changes in synapse formation. In the second week, we observed the expression of the genes related to the proliferation of neuroblasts and to the axon growth, and then in the third and fourth weeks, the expression of the genes of the immune response and the wrong protein modification response, respectively. It is important to note that both the increase in the gene set associated with neurogenesis and the induction of lysosomal activity occurred during the four week period as shown by the gene expression heatmap (fig. 4A to G). The genes listed in figures 4A-G are genes that showed a Fold Change (FC) absolute value of at least 1.3 between the vector treated group and the trametinib treated group in at least one of the one week, two weeks, three weeks, or four weeks dosing. Its FC is between-2.26 and 3.71.

Proteins or mrnas encoded by human homologues of the genes shown in figures 4A to G or neurotransmitters associated with protein receptors (GABA, glutamate, acetylcholine, monoamines such as dopamine, etc.) may be used as biomarkers to determine whether a beneficial effect has occurred in individuals receiving trametinib for the treatment of neurodegenerative diseases. The genes are Gabrb, Gabrr, Gla, Nr3c, Cdkl, Grin2, Plcxd, Chrm, Chrna, Chrnb, Nefl, Pld, Adra1, Chrnb, Slc6a, Slc18a, Cdh, Neurod, Nkx-1, Cxcl, Rest, Syt, Disc, Irx, Mdm, Sox, Grip, Pax, Bmp, Cpne, Numb, Atp8a, Trim, Otp, Il1rapl, Cpeb, Tnfrsf12, Hspb, Oprm, Clcf, Aspm, Membf, Ntff, Vegfa, HLlmx, 6v0, ase, Cnsk, Acr, Prss, Lancmp, Prdmp, Uncp, Bancp 13, Adcp, Adpsc, Vpx, Vppsn, Vpkapn, Vpx, Vpmin, Cmpp 13, Vpmin, Cmsk 13 and Pcn 13. The genes are associated with synapse formation, neurogenesis, lysosomal function, and/or autophagosomes.

6.3. Example 3: time course of brain function changes following administration of trametinib

To verify functional recovery of the neural network, we tested cortical neural activity in 5XFAD mice. Cognitive dysfunction in AD is closely related to cortical atrophy and memory impairment caused by neuronal loss and neural network collapse. The fine interaction between Hippocampal Structures (HPFs) and corresponding cortical regions is responsible for information transfer and incorporation. To examine the ability of memory formation in the hippocampus, excitatory postsynaptic currents (EPSCs; excitatory postsynaptic currents) were recorded in the hippocampal CA1 region to compare the long-term potentiation (LTP; long-term potentiation) between eight-month-old wild-type and 5XFAD mice. LTP in the orally administered trametinib animals in 5XFAD mice was significantly reduced and restored to levels nearly comparable to wild-type controls (FIGS. 5A and B).

This is consistent with the results of behavioral studies using the Y-maze and novelty recognition assays, which confirmed the recovery of memory formation in mice administered with trametinib (fig. 6A and B).

6.4. Example 4: structural changes in the brain following administration of trametinib

To determine whether the functional recovery of trametinib to the brain is due to structural recovery of neurons, we examined morphological recovery of axons and dendrites, which are key components constituting neuronal networks. Staining of the dendritic and axonal markers Map2 and Tau indicated that trametinib treatment resulted in recovery of dendritic and axonal length in the cortex of eight and thirteen month-old 5XFAD mice, whereas 5XFAD mice dosed with vehicle showed reduced length malformations. In addition, spherical swollen axons, which is an index of deterioration of axons due to accumulation of A β plaques in the brains of AD patients and aged monkeys, were also significantly reduced in the trametinib-administered group as compared with the vehicle-administered group (FIGS. 7 and 8). Increased expression of the pre-synaptic and post-synaptic markers synaptophysin-95 also revealed a contribution of trametinib in synaptic connection recovery (fig. 9 and 10). These results indicate that trametinib, in addition to conferring neuroprotective effects by enhancing axons and dendrites against amyloid plaque toxicity, also induces the formation of neuronal synapses.

We also examined the effect of trametinib on synapse formation in primary cortical neurons from ICR mouse embryos by examining changes in dendritic spine formation. Dendritic spines are small protrusions that are present in large numbers on the surface of the dendrites. They are the postsynaptic component of most excitatory synapses. The number, size and shape of the dendritic spinesDefines neuronal function and provides a structural basis for synaptic plasticity. Trametinib (100nM) increased the number of dendritic spines by 24% compared to vehicle treated groups. In A beta_1-42Under oligomer-induced neurotoxicity conditions, the number of dendritic spines was reduced by 24% compared to vehicle-treated groups (CTL), and compared to Abeta_1-42The number of trametinib treatments increased by 80% compared to the treatment group alone (fig. 11A and 11B). The increase in the number of dendritic spines by trametinib indicates that a β is responsible for the increase in the number of dendritic spines_1-42Induced synaptic dysfunction is restored by trametinib.

6.5. Example 5: trametinib enhances lysosomal activity through autophagosome-lysosomal fusion

We examined the possibility that trametinib reduced apoptosis and enhanced lysosomal activity in cerebral cortex V in 5XFAD mice. The enhancement of autophagosomal activity was demonstrated by several markers in the brain of eight months old 5XFAD mice (fig. 12). Autophagosome marker LC 3-ii was increased in the vector-administered group, and further increased in the trametinib-administered group. The level of mature cathepsin B, one of the lysosomal proteases, was reduced in vehicle-administered 5XFAD mice. In contrast, increased levels of mature cathepsin B were observed in the group administered trametinib, suggesting that lysosomal degradation of neurotoxic proteins can be induced by trametinib (fig. 12).

The ability of trametinib to induce autophagosomal activity was also demonstrated in a neuronal cell line (SH-SY5Y) (fig. 13A and B). Similar to the results obtained with 5XFAD mice, with A.beta._1-42The level of pERK and LC 3-II was increased in oligomer-treated SH-SY5Y cells. When treating A beta with trametinib_1-42LC 3-II/LC-I levels at cell level vs. Abeta_1-42Cells treated alone increased 65% and levels of mature cathepsin B were greater than A β_1-42The cells treated alone increased 44% (fig. 13B). Even in the presence of A beta_1-42In the case of oligomers, degradation of p62 was also observed as a marker of autophagy flux upon treatment with trametinib (fig. 13A). Similar results were observed in primary cortical neurons (fig. 14A). In particular, the level of mature cathepsin B as compared to vehicle-treated Controls (CTL)The treatment increased by 54%. When using A beta_1-42When oligomers treated primary cortical neurons, levels of mature cathepsin B were reduced by 26% compared to untreated neurons (CTL), but compared to A β_1-42The level of treatment with trametinib was increased by 73% compared to the treatment group alone.

We then performed immunocytochemical analysis to confirm the induction of lysosomal activity by autophagosome-lysosomal fusion in SH-SY5Y cells. We have found that even at A.beta._1-42Cells treated with trametinib also showed increased LC3-LAMP1 co-staining in the presence of oligomers (fig. 15A and B). To assess lysosomal acidification, we measured lysosomal probe-positive acidic spots. In the presence of A beta_1-42In the case of oligomers, trametinib treatment resulted in an increase in lysosomal probe-positive acidic spots (fig. 15A and B).

We also investigated the effects of these on primary cortical neurons. Even at A beta_1-42Neurons treated with trametinib also showed increased co-staining of LC3-LAMP1 and lysosomal probe-positive acidic spots in the presence of oligomers (fig. 16A and B). These results suggest that trametinib activates lysosomal degradation of the neurotoxin by inducing autophagy-lysosomal fusion. Further confirmation of the involvement of trametinib in autophagosome-lysosomal fusion was provided by the use of bafilomycin a1, which disrupts autophagosome and lysosomal fusion by inhibiting V-ATPase. In A beta_1-42Treatment with trametinib and bafilomycin a1 in the presence of oligomers abolished the effect of trametinib on increasing mature cathepsin B and decreasing p62 (fig. 17A).

To examine the mechanism by which trametinib triggered autophagy flux, changes in the downstream mediators of autophagy were measured in SH-SY5Y cells using western blot. Inactivation of mTOR resulted in dephosphorylation of Ser758 by ULK1 (Ser 757 in humans and mice) and subsequent induction of autophagy. In fact, we observed trametinib at a β_1-42mTOR and ULK1 phosphorylation on Ser758 in SH-SY5Y cells was inhibited in the presence of the oligomers (fig. 17B). In A beta_1-42Similar results were observed in oligomer-treated primary cortical neurons (fig. 18A to C). And do notTreatment group comparison by Abeta_1-42Treatment, the p-mTOR/mTOR ratio increased 2-fold. In the presence of trametinib (100nM) and Abeta_1-42In the co-treated group, with Abeta_1-42This ratio was reduced by 67% compared to the treatment group alone (fig. 18B). In addition, the neurons were defined by A β as compared to untreated neurons_1-42The ratio of acidified ULK1 to total ULK1 on Ser757 (poulk 1(S757)) in treated neurons increased by 51% compared to Α β_1-42This ratio was found with trametinib and A β compared to neurons treated alone_1-42There was a 29% reduction in co-treated neurons. We also tested whether trametinib affects the translocation of TFEB, a transcription factor EB that regulates lysosomal and autophagy-related genes. ERK2 and mTORC1 phosphorylated TFEB on Ser142, thereby inhibiting translocation from the cytoplasm to the nucleus and preventing transcription of lysosome and autophagy-related genes. We demonstrate trametinib in the presence of A β_1-42Nuclear translocation of TFEB was induced in the case of oligomers (fig. 17C) and demonstrated that trametinib dephosphorylates TFEB and localizes it to the nucleus.

In addition to the increase of autophagosome-lysosomal fusion, a decrease in apoptosis was also observed in the trametinib-administered group. To determine if the increase in autophagic tides induced a decrease in toxic proteins and resulted in a decrease in apoptosis, we examined co-staining of LC3-LAMP1 and expression of the apoptosis marker active caspase 3 in the cortex of 5XFAD mice. An increase in LC3-LAMP1 co-stained cells and a decrease in apoptosis were observed in the trametinib-administered group (fig. 19).

Taken together, these findings indicate that trametinib inhibits a β by increasing autophagosome-lysosomal fusion and downregulating mTOR pathway promoting lysosomal activity_1-42The induced cell death, together with the induction of NSC differentiation into neuronal lineage.

In the presence of toxic Abeta_1-42Under conditions of oligomeric or amyloid plaques, autophagic tide and lysosomal activity are known to be reduced, leading to eventual cell death. In this study, we demonstrated that trametinib restores autophagic tide and lysosomal activity in toxic environments by inducing autophagosome-lysosomal fusion. As to its mechanism of action, trametinib inhibits mTOR acidification and reduces Ser7Ulk1 at 58 is phosphorylated, which in turn may increase the interaction of ULK1 with AMPK to induce autophagy. Maintenance of protein homeostasis by lysosomal activation not only induces protection by removing neurotoxins, but also reverses age-related phenotypes by intracellular metabolic activation (rejuvenation effect). Therefore, lysosomal activation and induction of neurogenesis may act synergistically to produce a restorative effect in the brain of AD patients.

We also examined whether changes in endogenous molecular levels associated with lysosomal activity could be detected in the plasma of 5XFAD mice treated with trametinib. Plasma cathepsin B levels tended to decrease in eight month-old 5XFAD mice dosed with trametinib at 0.05 and 0.1 mg/kg/day (SNR0.05, SNR0.1) and reached statistical significance in the 0.1 mg/kg/day treated group (56.16% decrease in the 0.05 mg/kg/day group and 99.2% decrease in the 0.1 mg/kg/day group compared to the vehicle-treated 5XFAD mouse group) (fig. 20A). Donepezil-administered group also showed a statistically significant decrease in cathepsin B levels compared to 5 XFAD-vehicle (97.13% decrease compared to vehicle-treated 5XFAD mouse group) (fig. 20A). Plasma cathepsin B levels in thirteen month old 5XFAD mice treated with trametinib (SNR 0.1) at 0.1 mg/kg/day tended to be reduced, although not statistically significant, compared to the 5 XFAD-vector group (Veh) (fig. 20B).

There are reports of elevated plasma cathepsin B levels in Alzheimer's disease patients compared to healthy controls (comparison of the peripheral blood lysosomal enzyme expression levels of Morena, F. et al. light and severe Alzheimer's disease and MCI patients: Effect on regenerative medicine procedures. Int J Mol Sci 18, doi:10.3390/ijms18081806 (2017); Sundoff, J. et al. higher cathepsin B levels in the plasma of Alzheimer's disease patients compared to healthy controls. J. Alzheimer's disease J22, 1223-1011230, doi:10.3233/JAD-2010-101023(2010)), Alzheimer's disease patients.

Our results indicate that endogenous molecules associated with lysosomal activity can be used as biomarkers to determine whether trametinib produces a beneficial effect in individuals with neurodegenerative diseases. Such molecules include glycosyl hydrolases, such as beta-hexosaminidase, beta-galactosidase, beta-galactocerebrosidase, and beta-glucuronidase, as well as proteases, such as cathepsins, including cathepsin S, cathepsin D, cathepsin B, cathepsin K, and cathepsin L.

6.6. Example 6: induction of axonogenesis and protection of newly formed axons

In NSCs isolated from Tg2576 AD model mice, trametinib reduced active caspase 3 and strongly induced the differentiation of NSCs into neuron-like cells (fig. 21). The Tg 2576-derived NSC expresses the human transgenic protein A β and is considered to be an in vitro AD model similar to some of the cellular changes observed in vivo (world J.Stem cells 2013; 5 (4): 229-.

We observed a significant increase in cells with bipolar morphology in trametinib-treated Tg2576 NSCs, with prolonged neurites (FIG. 21). Expression of autophagosomes and lysosomes was significantly increased in trametinib-treated NSCs as indicated by increased staining with LC3 and LAMP1 (fig. 22A). Autophagosome-lysosomal fusion increased significantly in axon-like elongated elements as well as in the soma of trametinib-treated NSCs (yellow arrows in fig. 22B). These results imply that trametinib induces axonogenesis and activates lysosomal degradation in newly formed axons, which shows its potential as a therapeutic for diseases associated with axonopathy.

6.7. Example 7: restoration of myelin

Myelin is an insulating layer around the axons of nerve cells that allows electrical impulses to be rapidly and efficiently transmitted along the nerve cells. We examined the effect of trametinib on myelin using antibodies against myelin basic protein (MBP; myelin basic protein) as the major component of myelin (FIG. 23). Eight months of age 5XFAD mice treated with vehicle showed significant impairment of myelin sheath compared to wild type mice of the same age. However, the MBP level of eight months old 5XFAD mice treated with trametinib (SNR 0.1) at 0.05 mg/kg/day (SNR 0.05) or 0.1 mg/kg/day returned to levels comparable to wild-type mice. In contrast, the MBP levels of the 5XFAD mice treated with donepezil were as low as the MBP levels of the 5XFAD mice treated with vehicle. These results indicate that damaged myelin in 5XFAD mice can be restored by treatment with trametinib. Restoration of myelin can enhance the activation of neuronal cell communication in the cerebral cortex.

6.8. Example 8: therapeutic effect of trametinib on humans

Trametinib to provide a mean peak concentration of trametinib (C) in the brain of at least 0.25ng/g_max) Is administered to a patient suffering from alzheimer's disease. Dosing was performed daily for at least four weeks. Administering trametinib in such an amount and reducing behavioral and/or physiological symptoms associated with Alzheimer's disease during such period

6.9. Test method

Animals:b6SJL-Tg (APPSwFlLon, PSEN 1M 146L L286V) (5XFAD) mice were purchased from Jackson Laboratory (The Jackson Laboratory) (MMRRC stock number: 34840-JAX) and The experimental procedures were performed according to protocols approved by The Institutional Animal Care and Use Committee (IACUC) of KPCLab (approval number: P171011) or mediflon DBT Inc (approval number: mediflon 2017-1). The C57BL/6 mice were obtained from OrientBio Inc. and were reviewed and approved by the Seal university Hospital IACUC (approval No.: 16-0043-C1a0) or the International biomedical research institute IACUC (approval No.: 2017-0107) in compliance with relevant ethical regulations and animal procedures. ICR mice for pharmacokinetic analysis were obtained from OrientBio inc, and the experimental procedure was approved by IACUC from KPCLab (approval No.: P171011).

Trametinib treatment:trametinib (medchexpress, octocrylene, n.j.) was micronized and suspended in a vehicle containing 5% mannitol, 1.5% hydroxypropyl methylcellulose, and 0.2% sodium lauryl sulfate. For pharmacokinetic analysis, 0.05, 0.2 and 0.8mg/kg trametinib was orally administered to seven week old normal mice (ICR, n ═ 5 per group) as a single administration. Mice were sacrificed at each identical time point. For studies of pERK expression changes and whole cell RNA sequencing studies in normal mice, 0.1 mg/kg/day trametinib (in 4% dimethyl sulfoxide (DMSO) + 96% corn oil) was orally administered to six week old C57BL/6 mice (n ═ 3 per group). Mice were sacrificed one, two, three or four weeks after dosing.5XFAD mice (male, n-7 to 10 per group) were divided into vehicle and trametinib treatment groups. Twelve month old mice received vehicle or 0.1mg/kg trametinib by oral gavage once a day for 1 month (these mice are referred to as "13 month old 5XFAD mice" in the examples). Five month old mice were orally gavaged once daily with vehicle, 0.05mg/kg or 0.1mg/kg trametinib or 2mg/kg donepezil for 3 months (these mice were referred to as "eight month old 5XFAD mice" in the examples). Blood was collected after the treatment was completed. Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes and centrifuged to obtain plasma. All mice were sacrificed by perfusion and brain samples were subjected to biochemical and immunohistochemical analysis.

Whole cell RNA sequencing:RNA was isolated from the whole mouse brain and a cDNA library for RNA sequencing was prepared using the TruSeq strunded mRNA Prep kit (Illumina, san diego, ca) according to the manufacturer's instructions (1). The library was sequenced on the Illumina Nextseq500 platform, and reads were mapped to the reference mouse mm10 genome using Tophat v2.0.13. The total number of reads mapped to the transcriptome was 24,532 genes, and genes counted as 0 were removed in at least one sample prior to differential expression analysis. After removing the genes counted as 0, there were 18,727 genes in total. To define Differentially Expressed Genes (DEG), we set a strict statistical cut-off and False Discovery Rate (FDR) for Fold Changes (FC) of ≧ 1.3<0.05. A total of 500 DEGs were identified between the vector-treated group and the trametinib-treated group in the first week, 498 DEGs in the second week, 446 genes in the third week, and 538 genes in the fourth week. Gene ontology is performed by the gene ontology program of the gene ontology federation. Thermographic analysis was performed by R studio using the DEG list associated with synapses, neurogenesis and lysosomes.

Electrophysiology:

preparing brain slices: artificial cerebrospinal fluid (ASCF; artifical cerebropine fluids) was prepared as described in the following components: high sucrose ACSF (mM): 0.5CaCl₂、2.5KCl、1.25NaH₂PO₄、5MgSO₄Sucrose 205, 5-hydroxyethyl piperazine ethanethiosulfonic acid (HEPES), 10-glucose, 26-NaHCO₃(pH 7.3-7.4, mOsm 300-: 126NaCl, 3.5KCl, 1.25NaH₂PO₄、1.6CaCl₂、1.2MgSO₄10 glucose, 26NaHCO₃5HEPES (pH 7.3-7.4, mOsm 300-310). ACSF was prepared fresh daily as needed.

All experiments were performed using eight month old 5XFAD mice. High sucrose ACSF was maintained on ice and passed through 95% O₂/5％CO₂Is injected saturated for at least 20 minutes. Animals were euthanized with carbon dioxide, brains were harvested rapidly in less than four minutes, and stored in pre-oxygenated high sucrose ASCF for two minutes. To make the slices, the cerebral hemisphere is divided sagittally. Cortex and hippocampal segments were coronal sectioned to 300 μm for each hemisphere by a VF-200 vibratory microtome (precision Instrument, USA). For the cultivation of the sections, they were immersed in a nylon mesh at 95% O₂/5％CO₂Was incubated at 32 to 34 ℃ for 30 minutes and at room temperature for another 30 minutes before first recording.

Whole cell patch clamp LTP recording: sections were recorded perfused in 2ml/min oxygenated ACSF for 30 minutes before starting the experiment in a patch clamp chamber at 28 to 30 ℃. For whole-cell patch clamp, we used 4 to 8M Ω borosilicate glass electrodes (A-M Systems, USA) pulled from Micropipette pump P-1000(Sutter instrument, USA). The intracellular solution comprises (in mM): 140K-gluconate at 290mOsm, 10KCl, 1EGTA, 10HEPES, 4Na₂ATP、0.3Na₂GTP and pH 7.3 is adjusted with KOH. A HEKA EPC-10 dual amplifier (HEKA Elektronik, Germany) was used. Slice images were monitored under an upright Eclipse FN1 microscope (nikon, japan) by infrared differential interference contrast (IR-DIC) optics at 400X magnification.

Excitatory postsynaptic currents (EPSCs) were recorded in a voltage clamp pattern with-70 mV hold potential in CA1 pyramidal neurons. The access resistance in the recording unit is below 40M omega with a tolerance of 20%. For stimulating neurons, the bipolar electrode (FHC, usa) in the external stimulator Iso-flexa.m. p.i., israel) was located in the lateral Schaffer collateral (Schaffer collilator) at a distance of 200 to 400 μm from the recording electrode. Test stimulation pulses were applied at the same site with 30 to 40% intensity every 30 seconds, starting from the maximum EPSC amplitude, for long-term potentiation (LTP) induction 3 minutes before the next Theta pulse stimulation (TBS). The TBS consisted of four 10 second spaced sequences, each consisting of 10 pulses at 5Hz and 4 pulses at 100 Hz. EPSC was recorded 20 minutes after TBS application. Data were filtered at 1KHz and analyzed using Clampfit software (Molecular devices, USA).

Immunohistochemical analysis:mice were perfused with ice-cold Phosphate Buffered Saline (PBS) and then perfused with 4% Paraformaldehyde (PFA). Brains were dissected and analyzed by immunohistochemistry. For paraffin sections, the brain hemispheres were sagittal embedded in paraffin and prepared as 5 μm sections. Sections were deparaffinized and antigen retrieval was performed in citrate buffer (pH 6.0). For immunostaining, sections were stained with anti-Map 2 (Millipore), burlington, massachusetts), anti-Tau (Cell signaling, denfrost, massachusetts), anti-pNFh (Biolegend, san diego, ca), anti-active caspase 3(Cell signaling), anti-MBP (R)&D systems, Minneapolis, Minnesota), anti-LAMP 1(Abcam, Cambridge, UK), anti-LC 3(Cell signaling), or anti-Dcx (Abcam) antibodies. This step was followed by incubation with either Alexa Fluor 488-conjugated anti-goat IgG (seemer fly, waltham, massachusetts) or Alexa Fluor 555-conjugated anti-rabbit IgG secondary antibody (Thermo). Sections were counterstained with DAPI. LSM700 laser scanning confocal microscope (Carl Zeiss, Heildenheim, germany) was used. For Diaminobenzidine (DAB) staining, immunohistochemistry was performed using peroxidase substrate in DAB kit (Vector Laboratories inc., clingamm, ca). NeuN-positive and active caspase 3-positive cells from the cortical layer V were counted using the Icy micromanager program (nstitut Pasteur, Paris, France). The length of Map2 and Tau-positive dendrites and axons was measured using the Icy program.

Cell cultureCulturing:SH-SY5Y neuroblastoma cells at 37 ℃ with 5% CO₂DMEM (Dulbecco's Modified Eagle Medium) Medium/Ham's F-12 nutrient mixture (Invitrogen, Calsbarded, Calif.) supplemented with 10% heat-inactivated fetal bovine serum containing 100 units/ml penicillin and 100. mu.g/ml streptomycin. Primary cortical neuron cultures were derived from day eighteenth embryos of ICR mice (E18). Dissociated cells were plated on poly-D-lysine coated glass coverslips in neural stem cell culture medium and supplemented with 2% B27(Invitrogen), 100 units/ml penicillin, 100 μ g/ml streptomycin, and 2 ml-glutamine. Primary NSCs from normal mice were isolated from the subventricular zone (SVZ) of the brains of eight-week-old C57BL/6 mice. NSCs from the brain of adult Tg2576 mice were obtained from seoul national university hospital. Neurospheres were cultured as described previously (m.y.kim, b.s.moon, k.y.choi, in vitro isolation and maintenance of cortical neural progenitor cells. molecular biology methods 1018,3-10 (2013)). For neurosphere cultures from normal mice, cells were isolated from brain tissue and at 25cm²The cells were grown in suspension in N2 medium in flasks (SPL, Kyogi tract, Korea). bFGF (20ng/ml, peptaik (Peprotech), prinston, new jersey) and human EGF (20ng/ml, peptaik) were added to the medium to allow the cells to form neurospheres. For analysis, NSCs are cultured as monolayers. For NSC differentiation, neurospheres were isolated from TrypLE (Invitrogen) and plated on 15. mu.g/ml poly-L-ornithine- (Merck, St. Louis, Mo.) and 10. mu.g/ml fibronectin (Gibco) coated dishes and cultured for 2 days in bFGF and EGF depleted N2 medium. For neurosphere cultures from Tg2576 mice, cells were at 75cm²Flasks (SPL) were grown in suspension in DMEM/F12 supplemented with B27 supplement. bFGF (10ng/ml) and human EGF (10ng/ml) were added to the medium to allow the cells to form neurospheres. To induce differentiation and A β expression, neurospheres were pipetted off and plated on 15 μ g/ml poly-L-ornithine and 10 μ g/ml fibronectin (Gibco) -coated dishes and cultured for 2 days in bFGF and EGF depleted medium.

Protein extraction and western blotting:ice-cold phosphorous for cells and tissuesAcid salt buffered saline (PBS) was washed twice and then Ripa buffer (10mM HEPES, 1.5mM MgCl)₂10mM KCl, 0.01M DTT, protease inhibitor, pH 7.9). The lysate was centrifuged at 13,000rpm for 20 minutes at 4 ℃ and the protein content in the supernatant was determined using the Bradford assay (Bio-rad), Heracles, Calif.). For subcellular separation, cells were lysed with lysis buffer (250mM sucrose, 20mM HEPES, pH 7.4, 10mM KCl, 1.5mM MgCl) containing protease inhibitors₂1mM EGTA, 1mM EDTA and 1mM DTT) were lysed on ice for 30 minutes and then centrifuged at 720g for 5 minutes at 4 ℃. The supernatant was centrifuged at 15,000rpm for 10 minutes at 4 ℃ and the resulting supernatant was used as a cytoplasmic fraction. After centrifugation at 720g for 5 minutes, the pellet was washed with lysis buffer and dissolved in nuclear lysis buffer (50mM Tris HCl, pH 8.0, 150mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol) as nuclear fraction. Proteins from each sample were subjected to 8% to 15% SDS-PAGE and resolved proteins were transferred to nitrocellulose or polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk powder in Tris buffered saline/tween 20(TBST) for 1 hour at room temperature and then incubated with anti-phospho-erk (Cell signaling), anti-LAMP 1(Abcam), anti-LC 3(Cell signaling), anti-cathepsin b (Cell signaling), anti-p 62(Cell signaling), anti-p 62(5114, Cell signaling), anti-phospho-mTOR (5536, Cell signaling), anti-mTOR (2983, Cell signaling), anti-phospho-ULK 1(8054, Cell signaling), anti-TFEB (852501, Biolegend), and anti-gapdh (signaling) overnight at 4 ℃. After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Thermo) or goat anti-rat IgG antibody (Thermo) for 2 hours at room temperature. Peroxidase activity was visualized by enhanced chemiluminescence. The detected signals were quantified using the LAS-4000 system (fuji film, tokyo, japan).

Quantitative PCR (qRT-PCR):quantitative PCR analysis was performed on NSCs as previously described (j. konirova et al, modulated DISP3/PTCHD2 expression affects neural stem cell fate decision Sci Rep 7, 41597 (2017)). Using TRIzol (Invit)rogen) extracted total RNA from the cells. Reverse transcription was performed using M-MLV reverse transcriptase (Invitrogen). According to manufacturer's guidelines, SYBR is used^TMGreen PCR master mix (Thermo) was subjected to qRT-PCR. The results are shown relative to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

Immunocytochemistry:SH-SY5Y cells or adult NSCs were placed on glass coverslips coated with poly-L-ornithine/laminin or poly-D-lysine, respectively. After washing 3 times with PBS for 5 minutes, the cells were fixed in 10% formalin for 15 minutes at room temperature. Cells were then washed with PBS and permeabilized in 0.1% Triton X-100 for 2 minutes. Cells were placed in PBS blocking solution containing 5% BSA for 1 hour at room temperature and incubated with anti-active caspase 3(Cell signaling), anti- Α β (thermo), anti-tau (Cell signaling), anti-LC 3(Cell signaling) and anti-LAMP 1(Abcam) in blocking buffer for 2 hours at room temperature. After washing, cells were incubated overnight with goat anti-rabbit antibody conjugated to Alexa Fluor 488(Thermo) and/or goat anti-mouse antibody conjugated to Alexa Fluor 555 (Thermo). After washing, the cells were incubated with 4', 6-diamidino-2-phenylindole (DAPI) for 5 minutes. The coverslips were mounted using mounting medium (Biomeda, foster city, ca) and viewed by confocal microscopy using LSM700 microscopy (Carl Zeiss). The percentage of coefficients is calculated using pixels above the fluorescence intensity threshold. The in vivo-lysozyme pH was estimated using the lysosomal Red fluorescent probe (LysoTracker Red) DND-99(L7528, Invitrogen) according to the manufacturer's instructions. Cells were incubated with 500nM for 30 min at 37 ℃. The fluorescence intensity was observed under a confocal microscope using an LSM700 microscope (Carl Zeiss). The amount of lysosomal probe staining was analyzed with software Icy.

Behavioral testing

Y-maze test: animals were placed in the center of the Y-maze and their activity was recorded for 3 minutes. The Y-maze is a three-arm maze with an angle of 120 between all arms (40cm long by 15cm high). Video tracking was performed using intelligent video tracking software (Panlab, usa) and the order and number of entries into each arm were recorded. Spontaneous alternation was calculated when a mouse entered three arms in succession without accessing the previous arm.

Novelty identification test: to test novelty recognition, mice were habituated to an open field (40cm x 40 cm). In the training trial, mice were placed in an open field with two identical objects, each object for 10 minutes. The next day, the test trial was run for 3 minutes, replacing one of the two familiar objects with a new one. Using intelligent video tracking software (Panlab, usa) for video tracking, the recognition of familiarity and novelty was calculated as the percentage of time spent on new objects compared to the time spent exploring all objects.

Plasma cathepsin B levels:plasma was collected from eight and thirteen month old 5XFAD mice using EDTA tubes and stored at-80 ℃ until use. Cathepsin B ELISA kits (Novus Biologicals, centuries, colorado) were used to analyze cathepsin B levels in plasma. To a 96-well plate, 100. mu.l of a standard solution (0 to 10ng/ml) and 100. mu.l of plasma were added, and incubated at 37 ℃ for 90 minutes. Mu.l of biotinylated detection antibody working solution was added to each well immediately and incubated at 37 ℃ for 1 hour. The solution in each well was decanted and washed 3 times with 350. mu.l of wash buffer. Then 100. mu.l of HRP-conjugated working solution was added to each well and incubated at 37 ℃ for 30 minutes. The solution was decanted from each well and the washing process was repeated five times. 90 μ l of substrate reagent was added to each well and the dish was protected from light and incubated at 37 ℃ for 15 minutes. 50 μ l of stop solution was added to each well and the plate was read with a microplate reader with a reading set at 450 nm.

Incorporation of references

All publications, patents, patent applications, and other documents cited in this application are incorporated by reference in their entirety for all purposes, as if each individual publication, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

Equivalents (I)

While various specific embodiments have been illustrated and described, the above description is not intended to be limiting. It will be understood that various changes may be made without departing from the spirit and scope of the invention. Many variations will become apparent to those of ordinary skill in the art upon reading the present specification.