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WO2022192944A1 - Polythérapie pour le traitement de la sla - Google Patents

Polythérapie pour le traitement de la sla Download PDF

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Publication number
WO2022192944A1
WO2022192944A1 PCT/AU2022/050222 AU2022050222W WO2022192944A1 WO 2022192944 A1 WO2022192944 A1 WO 2022192944A1 AU 2022050222 W AU2022050222 W AU 2022050222W WO 2022192944 A1 WO2022192944 A1 WO 2022192944A1
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Prior art keywords
inhibitor
tyrosine kinase
kit
fyn
pdgfr
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Inventor
Samuel BRENNAN
Zheng SU
Phillip WEST
Matthew KEON
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Genieus Genomics Pty Ltd
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Genieus Genomics Pty Ltd
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Priority claimed from AU2021900742A external-priority patent/AU2021900742A0/en
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Publication of WO2022192944A1 publication Critical patent/WO2022192944A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present disclosure relates to the field of therapeutic methods suitable for treating neurodegenerative diseases.
  • Neurodegenerative diseases are a set of devastating, relentlessly progressive conditions that result in the dysfunction and death of specific groups of neurons. Many NDs are associated with increasing age and the public health burden caused by these diseases is predicted to get even more significant as populations around the world include a greater proportion of elderly people (1). There are currently very few treatments available for any neurodegenerative disease, and many are aimed at controlling symptoms rather than dealing with underlying molecular causes. Accordingly, there remains an unmet clinical need for effective drug therapies in the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the present disclosure is based on the surprising finding that inhibiting a particular combination of intracellular signalling pathways is effective in normalising caspase- dependent apoptosis in motor neurones derived from ALS patients and can lead to improvements in motor function together with reductions in gliosis and neurodegeneration in a mouse model of ALS.
  • the present disclosure provides a method of treating a neurodegenerative disease, disorder or condition in a subject, said method including the step of administering a therapeutically effective amount of two or more of:
  • the method of this aspect includes administering the therapeutically effective amount of:
  • the mTOR pathway inhibitor the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR and the unfolded protein response pathway inhibitor; or
  • c the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR and the inflammasome inhibitor; to the subject.
  • the present disclosure relates to use of two or more of:
  • an inflammasome inhibitor in the manufacture of a medicament for the treatment of a neurodegenerative disease, disorder or condition in a subject.
  • this aspect includes use of:
  • the mTOR pathway inhibitor the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR and the unfolded protein response pathway inhibitor; or
  • the present disclosure provides a composition comprising two or more of:
  • an inflammasome inhibitor for use in the treatment of a neurodegenerative disease, disorder or condition in a subject.
  • composition of this aspect comprises:
  • the mTOR pathway inhibitor the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR and the unfolded protein response pathway inhibitor; or
  • the mTOR pathway inhibitor suitably is or comprises one or more of an mTOR inhibitor, a PI3K inhibitor, an AKT inhibitor, an EGFR inhibitor and a BTK inhibitor. More particularly, the mTOR inhibitor may be or comprise rapamycin or an analogue or derivative thereof.
  • the inhibitor of at least one tyrosine kinase suitably is or comprises one or more of masitinib, imatinib, nilotinib, dasatinib and bosutinib. More particularly, the inhibitor of at least one tyrosine kinase can be or comprise masitinib or a pharmaceutically acceptable salt or solvate thereof.
  • the unfolded protein response pathway inhibitor is or comprises a PKR-like endoplasmic reticulum kinase (PERK) inhibitor.
  • the PERK inhibitor can be selected from the group consisting of GSK2656157, GSK2606414, ISRIB and any combination thereof.
  • the inflammasome inhibitor and/or the unfolded protein response pathway inhibitor suitably is or comprises an inhibitor of eIF2a phosphorylation, such as ISRIB.
  • the neurodegenerative disease, disorder or condition is or comprises a motor neurone disease (MND), such as amyotrophic lateral sclerosis (ALS).
  • MND motor neurone disease
  • the ALS may be familial or sporadic ALS. In one example, the ALS is sporadic ALS.
  • FIG. 1 Caspase activity at day 10 post-differentiation in ALS-derived and healthy control motor neurons.
  • Figure 3 Fold change in caspase activity at day 10 post-differentiation in ALS-derived and healthy control motor neurons.
  • Figure 4 Fold change in caspase activity at day 14 post-differentiation in ALS-derived and healthy control motor neurons.
  • Drag combination reduces inflammatory behaviour of LPS induced astrocytes.
  • Inflammatory Astrocytes produce high levels of IL-8 at 24 and 48 hours after exposure to LPS+IL1B.
  • MCC950 a positive control inflammasome inhibitor
  • the drag combination (rapamycin, masitinib & MCC950) reduced IL-8 levels across the board with the greatest reduction occurring in the 24 hour IL1B + LPS exposure condition.
  • Drag combination reduces inflammatory behaviour of LPS induced astrocytes. Inflammatory Astrocytes produce high levels of IL-6 at 24 and 48 hours after exposure to LPS+IL1B.
  • MCC950 an inflammasome inhibitor significantly reduced IL-6 secretion from these cells.
  • the drug combination rapamycin, masatinib & MCC950
  • Rapamycin reduces cell death in in vitro models of ALS.
  • ALS affected motor neurons have increased caspase activity compared to healthy motor neurons in both no treatment and solvent controls.
  • Staurosporine (SSP) a potent inducer of caspase activity, was used as a positive control and raised Caspase activity in both healthy and ALS patient derived MNs.
  • SSP Staurosporine
  • Both rapamycin as a single agent and in a triple combination that includes Masitinib reduces caspase activity in ALS patient derived MNs compared to the solvent treated cells.
  • Figure 8 Drag intervention timeline for treatment of animals and summary of behavioral analyses. The dosing schedule begun when mice were 4 weeks of age and concluded at week 12 when the animals underwent a final functional assessment followed by sacrificing the animals and harvesting tissues of interest.
  • Figure 9 Tissue collected from mouse brain and spinal cord for immunohistochemistry, fluorimetry and microscopy.
  • FIG. 10 Timeline for the experiments. Each group consisted of 7 animals for a total of 35 animals in the experiment, 28 of which are iTDP-43 mice. Functional tests occurred fortnightly after week 4.
  • FIG. 12 The data shows some examples of the drag combination offering improved performance over either drag alone.
  • the combination treatment prevented the clasping phenotype for longer than either treatment by itself. Gait abnormalities were also reduced at the conclusion of the study indicating some potential protection or recovery from this phenotype.
  • FIG. 15 Motor function assessment by rotarod test in iTDP-43 A315T mice.
  • the rotarod results are represented as a line graph with mean ⁇ SEM (a) or a column graph with individual values per mouse and mean ⁇ SEM (b).
  • n 7-8 mice/group.
  • FIG. 16 Motor function assessment by wire hang test in iTDP-43 A3l3 T mice.
  • the wire hang results are represented as a line graph with mean ⁇ SEM (a) or a column graph with individual values per mouse and mean ⁇ SEM (b).
  • n 7-8 mice/group.
  • Two-way repeated measures ANOVA with Tukey’s post-test *, p ⁇ 0.05.
  • FIG. 17 Motor function assessment by grip strength test in iTDP-43 A3151 mice.
  • the grip strength results are represented as a line graph with mean ⁇ SEM (a) or a column graph with individual values per mouse and mean ⁇ SEM (b).
  • n 7-8 mice/group.
  • Two-way repeated measures ANOVA with Tukey’s post-test *, p ⁇ 0.05.
  • FIG. 1 Motor function assessment by rotarod (RR), wire hang and grip strength tests in iTDP-43 A315T mice.
  • FIG. 19 Staining for CD68-positive microglia.
  • Figure 20 Staining for GFAP-positive astroglia.
  • FIG. 21 Body weight changes in treated mice. Rapamycin reduces body weight, but co-administration of Masitinib reduces the impact somewhat. Detailed description
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • a bacterium includes a plurality of such bacteria
  • a reference to “an allergen” is a reference to one or more allergens.
  • the inventors have surprisingly shown for the first time that a combination therapy of masitinib and rapamycin together with GSK2606414 or MCC950 synergistically and positively modulates cellular dysfunction, such as cell apoptosis and cytokine secretion, in ALS patient-derived motor neurons and human astrocytes and therefore offers promise as a treatment for neurodegenerative diseases, disorders and conditions broadly. Additionally, the present inventors have demonstrated that the combination therapy of masitinib and rapamycin demonstrates improvements in motor function and associated reductions in gliosis and neurodegeneration in a well-established mouse model of ALS.
  • a method of treating a neurodegenerative disease, disorder or condition in a subject including the steps of: administering a therapeutically effective amount of two or more of: (a) an mTOR pathway inhibitor; (b) an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR; (c) an unfolded protein response pathway inhibitor; and (d) an inflammasome inhibitor; to the subject to thereby treat the neurodegenerative disease, disorder or condition in the subject.
  • compositions comprising two or more of: (a) an mTOR pathway inhibitor; (b) an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR; (c) an unfolded protein response pathway inhibitor; and (d) an inflammasome inhibitor; and optionally a pharmaceutically-acceptable carrier, diluent or excipient.
  • the composition is suitable for use in the treatment of a neurodegenerative disease, disorder or condition in a subject.
  • an inflammasome inhibitor in the manufacture of a medicament for the treatment of a neurodegenerative disease, disorder or condition in a subject.
  • the term “ subject ” includes but is not limited to mammals inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs).
  • the subject is a human.
  • inhibitor refers to a molecule having the ability to inhibit a biological function of a target polypeptide or protein.
  • selective inhibition or “selectively inhibit” refers to the agent's ability to preferentially reduce the target signalling activity, such as a kinase activity, as compared to off-target signalling activity, via direct or indirect interaction with the target.
  • the inhibitors described for the present disclosure refer to a compound or a substance that acts against or blocks, at least in part, the expression, physiological function or activity, such as the kinase activity, of a target polypeptide or protein.
  • the inhibitor is an antibody or antigen-binding fragment thereof, an inhibitory nucleic acid molecule, such as a shRNA, a siRNA, including divalent siRNAs, or a miRNA, against the expression of the target protein, an inhibitory polypeptide, such as dominant-negative polypeptides or aptamers, or a small molecule inhibitor.
  • inhibition by the various inhibitors described herein need not be absolute to elicit a biological effect, such as treatment of a neurodegenerative disease, disorder or condition, in the subject. Accordingly, inhibition of a target protein by an inhibitor provided herein can be partial (e.g., an expression, a function and/or an activity of the target protein is reduced by about 20%, 30%, 40%, 50%, 60% or 70%, 80%, 90%, 95%, 96%, 97%, 98% and 99%, including any intermediate value therebetween) in the presence of a compound or agent as described herein.
  • the present methods, uses and compositions disclosed herein can encompass any particular combination of two or more (e.g., 2, 3 or 4) of the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, the unfolded protein response pathway inhibitor and the inflammasome inhibitor.
  • the methods described herein include administration of a therapeutically effective amount of the mTOR pathway inhibitor, such as rapamycin, and the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, such as masitinib.
  • the methods described herein include administration of a therapeutically effective amount of rapamycin and masitinib.
  • the methods described herein include administration of a therapeutically effective amount of the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, and optionally the unfolded protein response pathway inhibitor, such as GSK2606414.
  • the methods described herein include administration of a therapeutically effective amount of the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, and the unfolded protein response pathway inhibitor.
  • the methods described herein include administration of a therapeutically effective amount of the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, and optionally the inflammasome inhibitor, such as MCC950 or ISRIB.
  • the methods described herein include administration of a therapeutically effective amount of the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, and the inflammasome inhibitor.
  • the composition described herein comprises the mTOR pathway inhibitor, such as rapamycin, and the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, such as masitinib.
  • the composition described herein comprises rapamycin and masitinib.
  • the composition described herein comprises the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, and optionally the unfolded protein response pathway inhibitor, such as GSK2606414 or ISRIB.
  • the composition described herein comprises the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, and the unfolded protein response pathway inhibitor.
  • the composition described herein comprises the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, and optionally the inflammasome inhibitor, such as MCC950 or ISRIB.
  • the compositions described herein comprise the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, and the inflammasome inhibitor.
  • a neurodegenerative disease, disorder or condition any disease, disorder and/or condition that comprises a progressive decline and/or deterioration in the structure, function, signalling and/or population of the neurons or neural tissue in an animal.
  • the neurodegenerative disease, disorder or condition can be or comprise a neuromuscular disease, disorder or condition.
  • a neuromuscular disease, disorder or condition refers to any disease, disorder and/or condition that comprises a progressive decline and/or deterioration in the structure, function, signalling and/or population of the neurons or neural tissue that innervate and/or communicate, whether directly or indirectly, with the muscles of an animal.
  • the aetiology of a neurodegenerative disease, disorder or condition may involve, but is not limited to, inflammation, genetic mutations, protein misfolding and/or aggregation, autoimmune disorders, mitochondrial dysfunction, defective axonal transport, aberrant apoptosis and/or autophagy and elevated oxidative stress and/or reactive oxygen species (ROS) production.
  • ROS reactive oxygen species
  • neurodegenerative diseases, disorders or conditions can include Parkinson’s disease and related disorders, Huntington’ s disease, Alzheimer’s disease and other forms of dementia, Spinocerebellar ataxia, Friedreich ataxia, Tay-Sachs disease, Lewy body disease, Parkinson’s disease and related disorders, Prion diseases (e.g.
  • Creutzfeldt-Jakob disease Multiple sclerosis (MS), Pick disease, Shy-Drager syndrome, pontocerebellar hypoplasia, neuronal ceroid lipofuscinoses, Gaucher disease, neurodegeneration with brain iron accumulation, spastic ataxia/paraplegia, supranuclear palsy, mesolimbocortical dementia, thalamic degeneration, cortical- striatal- spinal degeneration, cortical -basal ganglionic degeneration, cerebrocerebellar degeneration, Leigh syndrome, post-polio syndrome, hereditary muscular atrophy, encephalitis, neuritis, hydrocephalus and the motor neurone diseases, such as ALS.
  • the subject with a neurodegenerative disease, disorder or condition may be undergoing a treatment regimen (preventative and/or therapeutic).
  • the subject may have been determined to either (i) have an existing neurodegenerative disease, disorder or condition; or (ii) be predisposed to such a disease, disorder or condition.
  • the neurodegenerative disease, disorder or condition described herein is a motor neurone disease (MND).
  • MNDs are a form of neurodegenerative diseases that typically involve the motor neurons of an affected subject.
  • motor neurons are nerve cells that control the voluntary muscles of the trunk, limbs and phalanges, as well as those muscles that influence speech, swallowing and respiration.
  • the clinical symptoms of a MND may include muscle weakness and/or wasting, muscle cramps, dysphagia, slurred speech, muscle tremors/fasciculations, reduced cognition, dyspnoea, respiratory failure, fatigue and weight loss without limitation thereto.
  • MNDs include, but are not limited to, amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig’s disease), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy and spinal muscular atrophy (SMA).
  • ALS amyotrophic lateral sclerosis
  • PLS primary lateral sclerosis
  • PMA progressive muscular atrophy
  • PBP progressive bulbar palsy
  • SMA spinal muscular atrophy
  • the MND may be ALS, PLS, PMA, PBP, pseudobulbar palsy or SMA.
  • the MND is ALS.
  • the ALS may be familial or sporadic ALS. In one example, the ALS is sporadic ALS. In another example, the ALS is familial ALS.
  • the disease, disorder or condition of this aspect may be at least in part mediated or associated with TDP-43, such as aberrant or abnormal TDP-43 aggregation.
  • TDP-43 is a highly conserved nuclear protein, and one of the major components of protein inclusions that typify the neurodegenerative diseases Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Dementia with ubiquitin inclusions.
  • ALS Amyotrophic Lateral Sclerosis
  • mRNA stabilization adenotriglyceride
  • cleavage, hyperphosphorylation and ubiquitination of TDP-43 can occur.
  • TDP-43 cytoplasmic accumulation and aggregation of TDP-43.
  • Abnormal accumulation of TDP-43 is observed at the site of lesions of many neurodegenerative diseases, which appears to imply involvement in the cause of nerve degeneration in these diseases.
  • Increased cytoplasmic localization of TDP-43 in brains and spinal cords of patients termed as “pre-inclusions”. These pre-inclusions of TDP-43 can induce eIF2a phosphorylation and the ISR.
  • the subject has ALS, which is mediated or caused at least in part by: (a) cleavage, hyperphosphorylation and ubiquitination of TDP-43; and/or (b) TDP- 43 aggregation.
  • the subject may have an ALS-linked mutation in TDP-43, as are well known in the art.
  • the ALS is at least in part caused by the phosphorylation of TDP-43 at serine 403/404 and 409/410.
  • mTOR pathway inhibitor also referred to herein interchangeably as “mTOR pathway specific drug”, refers to an inhibitor of the expression or activation, or both expression and activation, of a member of the mTOR pathway.
  • an mTOR pathway inhibitor can inhibit the expression or activation, or both, of AKT, mTOR, pTSC2, HIFla, pS6, p4EBPl, PI3K, STAT3, Bruton’ s tyrosine kinase (BTK), epidermal growth factor receptor (EGFR) and GSiGa/b as well as any receptor or receptor ligand that activates any component of the mTOR pathway.
  • BTK tyrosine kinase
  • EGFR epidermal growth factor receptor
  • GSiGa/b GSiGa/b
  • the mTOR pathway inhibitor can be or comprise one or more of an AKT inhibitor, an mTOR inhibitor, an EGFR inhibitor, a BTK inhibitor, a TSC2 inhibitor, a HIFla inhibitor, a S6 inhibitor, a 4EBP1 inhibitor, a PI3K inhibitor, a STAT3 inhibitor and a GSK3 ⁇ / ⁇ inhibitor. More particularly, the mTOR pathway inhibitor is or comprises one or more of an mTOR inhibitor, a PI3K inhibitor, an AKT inhibitor, an EGFR inhibitor and a BTK inhibitor. Even more particularly, the mTOR pathway inhibitor can be or comprise an mTOR inhibitor.
  • mTOR inhibitor refers to a compound or a ligand that inhibits at least one activity of an mTOR protein, such as, for example, the serine/threonine protein kinase activity on at least one of its substrates (e.g ., p70S6 kinase 1, 4E-BP1, AKT/PKB and eEF2).
  • the mTOR inhibitors of the present disclosure are suitably able to bind directly to and inhibit mTORCl, mTORC2 or both mTORCl and mTORC2 by binding to mTORCl and/or mTORC2.
  • mTOR inhibitors for use in the present disclosure can be active site inhibitors. These are mTOR inhibitors that bind to the ATP binding site (also referred to as ATP binding pocket) of mTOR and inhibit the catalytic activity of both mTORCl and mTORC2. Accordingly, in one aspect, an mTOR inhibitor for use in the present disclosure competes with ATP for binding to the ATP-binding site on mTORCl and/or mTORC2.
  • Exemplary ATP-competitive mTOR kinase inhibitors include dactolisib, voxtalisib, BGT226, SF1126, PKI-587 and NVPBE235.
  • the mTOR inhibitor can be a limus drug, such as rapamycin or a derivative or analogue thereof (e.g., a rapalog).
  • rapalogs refers to compounds that specifically bind to the mTOR FRB domain (FKBP rapamycin binding domain), are structurally related to rapamycin, and retain mTOR inhibiting properties. Rapalogs include esters, ethers, oximes, hydrazones, and hydro xylamines of rapamycin, as well as compounds in which functional groups on the rapamycin core structure have been modified, for example, by reduction or oxidation. Pharmaceutically acceptable salts of such compounds are also considered to be rapamycin derivatives. It will be appreciated that rapalogs, like rapamycin, typically selectively inhibit mTORCl relative to mTORC2.
  • Exemplary limus drugs include, but are not limited to, rapamycin, temsirolimus (CCT 779), everolimus (RAD001), ridaforolimus (AP -23573), deforolimus (MK-8669), zotarolimus (ABT-578), pimecrolimus, tacrolimus (FK-506), CC-115 and CC-223.
  • the mTOR inhibitor is or comprises rapamycin or an analogue or derivative thereof.
  • tyrosine kinases are enzymes that transfer the terminal phosphate of ATP to tyrosine residues of proteins thereby activating or inactivating signal transduction pathways associated with said proteins.
  • Tyrosine kinases can be categorized as receptor type and non-receptor type.
  • Signal transduction mediated by receptor tyrosine kinases is typically initiated by the extracellular interaction with a specific growth factor or ligand, typically followed by receptor dimerization, stimulation of the intrinsic protein tyrosine kinase activity and receptor transphosphorylation.
  • tyrosine kinase inhibitor therefore refers to an agent or molecule that at least partly inhibits one or more (e.g., 1, 2, 3, 4, 5 etc) tyrosine kinases, such as c-Kit, Lyn, Fyn, CSF1R and/or PDGFR, thereby interfering with the aforementioned associated cell signalling processes.
  • C-Kit also known as cluster of differentiation 117 (CD117) or mast/stem cell growth factor receptor (SCFR)
  • CD117 cluster of differentiation 117
  • SCFR mast/stem cell growth factor receptor
  • C-kit is a type of receptor tyrosine kinase found on the surface of many different types of cells. It binds to a substance called stem cell factor (SCF) and typically functions to promote growth in certain types of blood cells.
  • SCF stem cell factor
  • C-kit may also be found in higher than normal amounts, or in a changed form, on some types of cancer cells.
  • Lyn is a member of the Src family of protein tyrosine kinases, which is mainly expressed in hematopoietic cells, neural tissues, the liver and adipose tissue. As such, this non receptor tyrosine-protein kinase plays an important role in the regulation of innate and adaptive immune responses, hematopoiesis, responses to growth factors and cytokines, integrin signalling and cellular responses to DNA damage and genotoxic agents.
  • Fyn belongs to the Src family of nonreceptor tyrosine kinases.
  • the Fyn protein associates with the p85 subunit of phosphatidylinositol 3 -kinase (PI3K) and interacts with the fyn-binding protein and plays a role in many biological processes including regulation of cell growth and survival, cell adhesion, integrin-mediated signalling, cytoskeletal remodelling, cell motility, immune response and axon guidance.
  • PI3K phosphatidylinositol 3 -kinase
  • Platelet-derived growth factor receptors are cell surface tyrosine kinase receptors for the platelet-derived growth factors (PDGFs). It is envisaged that the term “PDGFR” as provided herein includes any of the recombinant or naturally-occurring forms of the platelet-derived growth factor receptor (PDGFR) protein, such as PDGFRa and PDGFR , or variants or homologs thereof that retain PDGFR protein activity.
  • Colony stimulating factor- 1 receptor (CSF-1R or CSF1R) is a tyrosine-protein kinase that acts as cell-surface receptor for CSF1 and interleukin 34 (IL34) and plays an role in the regulation of survival, proliferation and differentiation of hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes. It promotes the release of proinflammatory chemokines in response to IL34 and CSF1, and thereby plays an important role in innate immunity and inflammatory processes.
  • CSF-1R or CSF1R is a tyrosine-protein kinase that acts as cell-surface receptor for CSF1 and interleukin 34 (IL34) and plays an role in the regulation of survival, proliferation and differentiation of hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes. It promotes the release of proinflammatory chemokines in response to IL34 and CSF1,
  • the inhibitor of at least one tyrosine kinase can be considered to be or comprise an inhibitor or modulator of a mast cell function or activity (such as mast cell degranulation), a macrophage function or activity and/or a microglial cell function or activity. Consequently, the inhibitor of at least one tyrosine kinase may inhibit activation of an inflammatory process, such as proinflammatory cytokine release, in the central nervous system.
  • inhibitors of at least one tyrosine kinase include masitinib, imatinib, cromolyn sodium, midostaurin, BLU-285, bosutinib, ibrutinib, LAS189386, DP-2618, fostamatinib, nilotinib, dasatinib, sunitinib, axitinib, pazopanib, toceranib, GW2580, pexidartinib, BLZ945, linifanib, OSI-930, sunitinib, emactuzumab, FPA008, quizartinib, axitinib, motesanib, cediranib, JNJ-28312141, Ki-20227, MLN-518, sorafenib, and SU- 14813.
  • the inhibitor of at least one tyrosine kinase is or comprises one or more
  • the inhibitor of at least one tyrosine kinase is or comprises masitinib or a pharmaceutically acceptable salt or solvate thereof (e.g., masitinib mesylate).
  • Masitinib (AB1010) is a small molecule agent that selectively inhibits specific tyrosine kinases such as c-Kit, PDGFR, Lyn, Fyn, CSF1R, lymphocyte-specific protein tyrosine kinase (Lck), focal adhesion kinase (FAK) and fibroblast growth factor receptor 3 (FGFR3).
  • masitinib The chemical name for masitinib is 4-(4-methylpiperazin-l-ylmethyl)-N- [4-methyl-3- (4-pyridin-3ylthiazol-2-ylamino) phenyl ]benzamide (CAS number 790299-79-5).
  • Masitinib was first described in U.S. Pat. No. 7,423,055 and EP1525200B1 and a detailed procedure for the synthesis of masitinib mesylate is provided in WO 2008/098949. Because of its selective activity against CSF1R, masitinib is able to inhibit the CSF1/CSF1R signalling pathway thereby regulating CSFIR-dependent cells, such as microglia. Similarly, and by virtue of its activity against c-Kit, Fyn and Fyn, masitinib is also able to inhibit the function of mast cells.
  • An unfolded protein response pathway inhibitor generally refers to an active agent that suppresses expression, activity and/or function of at least one UPR-related gene, protein, or signalling pathway.
  • the term “Unfolded Protein Response ” (UPR) or the “ Unfolded Protein Response pathway ” refers to an adaptive response to the accumulation of unfolded proteins in the ER and includes the transcriptional activation of genes encoding chaperones and folding catalysts and protein degrading complexes as well as translational attenuation to limit further accumulation of unfolded proteins. Both surface and secreted proteins are synthesized in the endoplasmic reticulum (ER) where they need to fold and assemble prior to being transported.
  • the UPR is organized into three specific branches, each controlled by one of three respective transducers of the canonical pathway, that is, IRE1, PERK and ATF6.
  • the UPR pathway inhibitor can be an inhibitor of the expression, a function and/or an activity of IRE1, PERK and/or ATF6.
  • UPR pathway inhibitors include GSK2656157; GSK2606414; ISRIB (integrated stress response inhibitor); 4-(2- aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF); 5-aminoimidazole-4- carboxamide ribonucleotide (AICAR); 4-phenylbutyrate (4-PBA); bile acids (e.g., UDCA and TUDCA); Binding immunoglobulin protein (BiP); ceapins; extendin-4; IC87144; IRE1 inhibitors; metformin; rapamycin; salubrinal; SRT1720; STF-083010; toyocamycin; and vatic anol B.
  • the unfolded protein response pathway inhibitor is or comprises a PKR-like endoplasmic reticulum kinase (PERK) inhibitor.
  • PERK refers to a protein also known as “ PKR-like endoplasmic reticulum kinase” and “ eIF2aK3 ” (eIF2a kinase 3).
  • An exemplary activity of PERK (which may be inhibited by the agent described herein) is phosphorylation of eIF2 ⁇ .
  • the PERK inhibitor may be any as are known in the art, such as those disclosed in in International Publication Nos. W02015/056180, W02014/161808 and WO2017/216792.
  • the PERK inhibitor can include one or more of GSK2656157, GSK2606414 and ISRIB. In one specific example, the PERK inhibitor is ISRIB.
  • inflammasomes are cytosolic multiprotein oligomers of the innate immune system responsible for the activation and mediation of various inflammatory responses.
  • Inflammasomes can include the NLR-class of inflammasomes, such as NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, and NLRC4 (IPAF), as well as interferon-inducible protein AIM2 (AIM2).
  • NLR-class of inflammasomes such as NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, and NLRC4 (IPAF)
  • AIM2 interferon-inducible protein AIM2
  • the NLR-class of inflammasomes each have a nucleotide-binding oligomerization domain (NOD), which is bound by ribonucleotide-phosphates (rNTP) and can facilitate self-oligomerization as well as a C-terminal leucine-rich repeat (LRR), which serves as a ligand-recognition domain for other receptors (e.g., TLR) or microbial ligands.
  • NOD nucleotide-binding oligomerization domain
  • LRR C-terminal leucine-rich repeat
  • the result of any inflammasome activation is the activation of the protease caspase-1. Caspase-1 cleaves pro-IL-Ib and pro-IL-18 into their active forms, which then precipitate a wider inflammatory reaction.
  • Multiple inflammasome types can be present in the brain and spinal cord, including but not limited to, the NLRP1 inflammasome, the NLRP3 inflamma
  • inflammasome inhibitor refers to any compound capable of inhibiting the expression, formation, activity and/or function of inflammasomes (e.g., an NLRP3 inflammasome), in a cell, such as a CNS cell, including inhibiting the expression and/or function of the proteins in the NLRP3/IL- 1 b pathway.
  • Inflammasome inhibitors can include, but are not limited to, an NLRP1 inflammasome inhibitor, an NLRP3 inflammasome inhibitor, an NLRP6 inflammasomes inhibitor, an NLRP7 inflammasomes inhibitor, an NLRP12 inflammasome inhibitor, an NLRC4 inflammasome inhibitor, and/or an AIM2 inflammasome inhibitor.
  • Such inflammasome inhibitors can include compounds or a combination of compounds that inhibit the expression, formation, activity and/or function of one or more proteins in the NLRP3/IL-i pathway.
  • Inhibitors of proteins in the NLRP3/IL-I b pathway include, but are not limited to, NLRP3 inflammasome inhibitors, TXNIP inhibitors, ASC inhibitors, NEK7 inhibitors, Gasdermin D inhibitors, capspase-11 inhibitors, capsase-1 inhibitors, IL-Ib inhibitors, IL-18 inhibitors and combinations thereof and pharmaceutical compositions thereof.
  • the inflammasome inhibitor is or comprises an NLRP3 inflammasome inhibitor.
  • NLRP3 refers to NOD-like receptor family, pyrin domain containing 3 inflammasome or NACHT, LRR and PYD domains-containing protein 3 (NALP3 ; also known as cryopyrin, cold induced autoinflammatory syndrome 1 (CIAS1), caterpiller- like receptor 1.1 (CLR1.1) or Pyrin Domain-Containing Apafl-Like Protein 1 (PYPAF1)).
  • NLRP3 is a component of a multiprotein oligomer consisting of the NLRP3 protein, a structural co-factor protein called thioredoxin-interacting protein (TXNIP), ASC (apoptosis-associated speck- like protein containing a CARD) and pro-caspase 1.
  • NLRP3 is involved in inflammation and the immune response. In the presence of activating stimuli, this complex forms, recruits, and activates caspase-1, resulting in the cleavage and maturation of the pro-inflammatory cytokines IL-Ib and IL-18. These cytokines are released from the cell via a form of necrotic cell death called pyroptosis, where they go on to promote inflammation.
  • Exemplary NLRP3 inflammasome inhibitors include ISRIB, glyburide, 2- mercaptoethane sulfonate sodium (Mesna), CY-09, MCC950, 3,4-Methylenedioxy ⁇ - nitrostyrene (MNS), Tranilast (N-[3',4'-dimethoxycinnamoyl]-anthranilic acid, TR), OLT1177, Qridonin, 16673-34-0, JC124, FCl lA-2, parthenolide, VX-740, VX-765, beta-hydroxybutyrate (BHB), Z-VAD-FMK, Bay 11-7082, aloe vera, curcumin, artesunate, dapansutrile, glybenclamide, Epigallocatechin-3-gallate (EGCG), Genipin, red ginseng extract (RGE), isoliquiritigenin (ILG), NBC 6, NBC 19 INF 39, OX
  • the NLRP3 inflammasome inhibitor is or comprises ISRIB.
  • ISRIB is an inhibitor of the NLRP3 inflammasome, by way of preventing transcription of LONP1, which is necessary for mitophagy leading to oxidative stress and NLRP3 inflammasome activation (Onat et al., J Am Coll Cardiol. 2019 Mar, 73 (10) 1149-1169).
  • ISRIB may also be considered to be an unfolded protein response pathway inhibitor.
  • the unfolded protein response pathway inhibitor and/or the inflammasome inhibitor are or comprise an inhibitor of eIF2a phosphorylation, such as ISRIB.
  • the inhibitor of eIF2a phosphorylation can refer to an agent or compound that inhibits phosphorylation of the 51 st serine residue of eIF2a.
  • the inhibitor of eIF2a phosphorylation can include, for example, low molecular weight compounds or small molecules that specifically inhibit the binding or interaction between a protein kinase (e.g., a protein kinase, such as PERK, PKR and GCN2, that is activated in the event of stress) and eIF2a.
  • Methods of treating a neurodegenerative disease, disorder or condition may be prophylactic, preventative or therapeutic and suitable for treatment of the neurodegenerative disease, disorder or condition in mammals, particularly humans.
  • “treating”, “treat' or “treatment” refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of a neurodegenerative disease, disorder or condition after the neurodegenerative disease, disorder or condition and/or its symptoms have at least started to develop.
  • preventing refers to therapeutic intervention, course of action or protocol initiated prior to the onset of a neurodegenerative disease, disorder or condition and/or a symptom of the neurodegenerative disease, disorder or condition so as to prevent, inhibit or delay or development or progression of the neurodegenerative disease, disorder or condition or the symptom thereof.
  • therapeutically effective amount describes a quantity of a specified agent, such as the composition or combination therapy described herein, sufficient to achieve a desired effect in a subject being treated with that agent or combination of agents.
  • this can be the amount of a composition comprising two or more of an mTOR pathway inhibitor, an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, an inflammasome inhibitor and an unfolded protein response pathway inhibitor, necessary to reduce, alleviate and/or prevent a neurodegenerative disease, disorder or condition.
  • a “ therapeutically effective amount ” is sufficient to reduce or eliminate a symptom of a neurodegenerative disease, disorder or condition.
  • a “therapeutally effective amount ’ may be an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent disease progression.
  • the therapeutically effective amount of two or more of an mTOR pathway inhibitor, an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, an inflammasome inhibitor and an unfolded protein response pathway inhibitor is sufficient to prevent or inhibit further neurodegeneration, gliosis and/or astrogliosis in the subject.
  • the aforementioned agents prevent or inhibit apoptosis of neurons, such as motor neurons, in the subject.
  • the therapeutically effective amount of two or more of an mTOR pathway inhibitor, an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, an inflammasome inhibitor and an unfolded protein response pathway inhibitor is sufficient to prevent or inhibit a decline or decrease in motor function of the subject.
  • administering may improve or prevent or inhibit a decline in one or more indices or indicators of neuromuscular function of the subject.
  • administration of two or more of an mTOR pathway inhibitor, an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, an inflamma some inhibitor and an unfolded protein response pathway inhibitor prevents or ameliorates stiffness, tremors, muscle spasms, poor muscle control, and/or pain sensations in the brain of a subject.
  • the methods described herein further include the step of administering a further therapeutic agent to the subject (i.e., in addition to two or more of an mTOR pathway inhibitor, an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, an inflammasome inhibitor and an unfolded protein response pathway inhibitor).
  • a further therapeutic agent i.e., in addition to two or more of an mTOR pathway inhibitor, an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, an inflammasome inhibitor and an unfolded protein response pathway inhibitor.
  • two or more of an mTOR pathway inhibitor, an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, an inflammasome inhibitor and an unfolded protein response pathway inhibitor may be administered in combination with the further therapeutic agent which aims to treat or prevent a disease, disorder or condition described herein (e.g., a neurodegenerative disease).
  • two or more of an mTOR pathway inhibitor, an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, an inflammasome inhibitor and an unfolded protein response pathway inhibitor described herein may be co-administered with (simultaneously or sequentially) a therapeutic agent for the treatment of a neurodegenerative disease, disorder or condition.
  • Non-limiting examples of such therapeutic agents include a gene therapy (e.g., delivery of genes encoding neurotrophic or neuroprotective factors), a cell therapy (e.g., a stem cell therapy), and/or a molecularly targeted agent (e.g., edaravone, riluzole, thickened riluzole, riluzole oral film and dextromethorphan HBr and quinidine sulfate).
  • the present methods further include the step of administering a therapeutically effective amount of a molecularly targeted agent for the subject’s neurodegenerative disease, disorder or condition, such as ALS, to the subject.
  • the current methods can also improve the prognosis of the subject being treated.
  • administration of two or more of an mTOR pathway inhibitor, an inhibitor of at least one tyrosine kinase selected from c-Kit, Lyn, Fyn, CSF1R and PDGFR, an inflammasome inhibitor and an unfolded protein response pathway inhibitor to the subject with a neurodegenerative disease, disorder or condition may reduce the probability of a clinical worsening event (e.g., hospitalization for the neurodegenerative disease, disorder or condition, initiation of additional therapy or a combination thereof) during the treatment period.
  • a clinical worsening event e.g., hospitalization for the neurodegenerative disease, disorder or condition, initiation of additional therapy or a combination thereof
  • the methods described herein provide a reduction of at least about 25%, at least about 50%, at least about 75% or at least about 80%, in probability of a clinical worsening event during the treatment period.
  • a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject.
  • the effective amount of an agent useful for reducing, alleviating and/or preventing a neurodegenerative disease, disorder or condition will be dependent on the subject being treated, the type and severity of any associated disease, disorder and/or condition (e.g., disease progression), and the manner of administration of the therapeutic composition.
  • composition described herein is administered to a subject as a pharmaceutical composition comprising a pharmaceutically-acceptable carrier, diluent or excipient.
  • a pharmaceutical composition comprising a pharmaceutically-acceptable carrier, diluent or excipient.
  • any dosage form and route of administration such as those provided herein, may be employed for providing a subject with the composition provided herein.
  • pharmaceutically-acceptable carrier diluent or excipient
  • a solid or liquid filler diluent or encapsulating substance that may be safely used in systemic administration.
  • a variety of carriers well known in the art may be used.
  • These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen- free water.
  • Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.
  • compositions of the present disclosure suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre determined amount of one or more therapeutic agents of the present disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing the agents of the present disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective.
  • the dose administered to a patient should be sufficient to effect a beneficial response in a patient over an appropriate period of time.
  • the quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.
  • the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase, the inflammasome inhibitor and the UPR pathway inhibitor may be administered simultaneously, concurrently, sequentially, successively, alternately or separately in any particular combination and/or order.
  • the mTOR pathway inhibitor can be administered (i) prior to; (ii) after; or (iii) simultaneously with, the administration of the inhibitor of at least one tyrosine kinase, the inflammasome inhibitor and/or the UPR pathway inhibitor.
  • the inhibitor of at least one tyrosine kinase can be administered (i) prior to; (ii) after; or (iii) simultaneously with, the administration of the mTOR pathway inhibitor, the inflammasome inhibitor and/or the UPR pathway inhibitor.
  • the UPR pathway inhibitor can be administered (i) prior to; (ii) after; or (iii) simultaneously with, the administration of the mTOR pathway inhibitor, the inflammasome inhibitor and/or the inhibitor of at least one tyrosine kinase.
  • the inflammasome inhibitor can be administered (i) prior to; (ii) after; or (iii) simultaneously with, the administration of the mTOR pathway inhibitor, the UPR pathway inhibitor and/or the inhibitor of at least one tyrosine kinase.
  • the mTOR pathway inhibitor can be administered (i) prior to; (ii) after; or (iii) simultaneously with, the administration of the inhibitor of at least one tyrosine kinase.
  • administration of two or more of the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase, the inflammasome inhibitor and the UPR pathway inhibitor results in treatment or prevention of neurodegenerative disease, disorder or condition that is greater than such treatment or prevention from administration of one, two or three of the said inhibitors in the absence of one, two or three of the remaining recited inhibitors.
  • Simultaneous administration typically includes administration at substantially the same time. This form of administration may also be referred to as “concomitant” administration.
  • Concurrent administration includes administering the active agents within the same general time period, for example on the same day(s) but not necessarily at the same time.
  • Alternate administration includes administration of one agent during a time period, for example over the course of a few days or a week, followed by administration of another agent during a subsequent period of time, for example over the course of a few days or a week, and then repeating the pattern for one or more cycles.
  • Sequential or successive administration includes administration of one agent during a first time period (for example over the course of a few days or a week) using one or more doses, followed by administration of another agent during a second and/or additional time period (for example over the course of a few days or a week) using one or more doses.
  • An overlapping schedule may also be employed, which includes administration of the active agents on different days over the treatment period, not necessarily according to a regular sequence. Variations on these general guidelines may also be employed, such as according to the agents used and the condition of the subject.
  • kits may further comprise a package insert comprising printed instructions for simultaneous, concurrent, sequential, successive, alternate or separate use of the inhibitors in the treatment and/or prevention of a neurodegenerative disease, disorder or condition, as described herein, in a patient in need thereof.
  • the aforementioned kits are suitably for use in a method of treating and/or preventing a neurodegenerative disease, disorder or condition, as described herein.
  • two or more of the mTOR pathway inhibitor, the inhibitor of at least one tyrosine kinase, the inflammasome inhibitor and the UPR pathway inhibitor can be formulated together in a composition that optionally includes a pharmaceutically acceptable carrier, excipient or diluent.
  • Human iPSC-derived motor neurons were purchased from iXCells Biotechnologies (San Diego, USA); these included Diseased ALS motor neurons (#40HU-006) and healthy motor neurons (#40HU-005). These were plated in Matrigel-coated (80 pg/mL) plates and grown in motor neuron maintenance medium (#MD-0022) as per instructions from iXCells Biotechnologies. Matrigel was purchased from Corning (#354277 ; Lot number 9322016) and diluted in DMEM-F12 medium (Thermo Fischer Scientific, #31330-038) for coating. Cells were incubated in a normoxia incubator, at 37°C with 5% CO2.
  • Caspase-Glo 3/7 assay reader mode Spectroscopic mode: Luminescence Intensity Presets: Ultra-Glo Emission (l): 545-50 nm Intergration time: 1 s Well scan: Matrix scan - 5x5 Focal height: Automatic focus Optic: bottom Temperature: 37°C
  • results show that the drug combination is more effective at reducing caspase expression in ALS-derived motor neurons when compared to Rapamycin alone (Ligures 1-4 and 7). It is possible that we are observing an incomplete effect of both Rapamycin and the drug combination. Glial cells are driving factors in ALS patients and animal models (Phatnani, Guarnieri et al. 2013, Juliani, Vassileff et al. 2021), and the cross talk between these cells is not assessed in pure cell culture. By way of example, both Rapamycin and Masitinib may be of use in glial cells as well. It is known that aggregates are spread to glial cells in ALS (Ishii, Kawakami et al.
  • Human astrocytes were purchased from iXCells Biotechnologies (San Diego, USA - #10HU-035). These were plated in plates coated with Poly-L-Lysine (Sigma- Aldrich, #P4832) and cultured in Astrocytes medium (#MD-0039) fully supplemented with fetal bovine serum, growth factors and antibiotics as per instructions from iXCells Biotechnologies upon purchase of the Astrocyte culture medium kit. Astrocytes were sub-cultured as per detailed protocol provided by iXCell Biotechnologies. For stimulation, human IL-Ib (Sigma- Aldrich, # SRP3083) and Lipopolysaccharide (Sigma- Aldrich, # L4391). Cells were incubated in a normoxia incubator, at 37°C with 5% CO2.
  • the growth medium was collected, and particulates were removed by centrifugation at 500 x g for 15 mins. The supernatant was then transferred into clean Eppendorf tubes and stored at
  • ELISA kits were purchased from Sigma- Aldrich and the assay was mostly run as per manufacturer’s protocol, except for adjustments in the dilution range for protein standards so that there were up to 12 data points. The following kits were used: IL-6 ELISA kit (#RAB0306, Lot Number 0115L0140); IL-8 ELISA kit (#RAB0319, Lot Number 0121L0143); TNL-a ELISA kit (#RAB0476, Lot Number 1125L0193).
  • IL-8 secretion from IL1B + LPS-treated astrocytes increased by >1 order of magnitude at 48 hours compared to 24 hours.
  • MCC950 reduced IL-8 secretion from IL1B + LPS stimulated astrocytes at 24 and 48 hours. It was most effective at 48 hours and only marginally effective at 24 hours.
  • the drug combination of rapamycin, masitinib and MCC950 reduced IL-8 secretion levels across the board with the greatest reduction occurring in the 24 hour IL1B + LPS exposure condition (Figure 5).
  • IL-6 secretion from IL1B + LPS-treated astrocytes increased by >1 order of magnitude at 48 hours compared to 24 hours.
  • MCC950 reduced IL-6 secretion from IL1B + LPS stimulated astrocytes at 48 hour only. It was most effective at 48 hours and only marginally effective at 24 hours.
  • the drug combination reduced IL-6 levels across the board with the greatest reduction occurring in the 24 hour IL1B + LPS exposure condition (Ligure 6).
  • the data in Examples 1 and 2 supports a polypharmacy approach to the treatment of ALS which includes the backbone of a tyrosine kinase inhibitor, like masitinib, and an mTOR inhibitor, like rapamycin, and optionally further including a PERK inhibitor and/or an NLRP3 inflammasome inhibitor.
  • a tyrosine kinase inhibitor like masitinib
  • an mTOR inhibitor like rapamycin
  • a PERK inhibitor and/or an NLRP3 inflammasome inhibitor optionally further including a PERK inhibitor and/or an NLRP3 inflammasome inhibitor.
  • Both rapamycin and masitinib have undergone safety and efficacy trials in sALS patients.
  • This combination therapy approach is aimed at tyrosine kinase inhibition to decrease neuroinflammation (masitinib) and mTOR inhibition to enhance autophagy (rapamycin).
  • This drug combination is aimed primarily at glial cells specifically which also suffer from aggregates and perpetuate inflammatory phenotypes that cause further disease progression .
  • the same activity of the combined therapy of masitinib and rapamycin is expected to assist motor neurons clear aggregates and decrease inflammatory signalling.
  • the results herein testing combinations of rapamycin and masitinib with MCC950 or GSK2606414 on motor neurons and astrocytes stimulated with pro-inflammatory cytokines suggest that this combination may be effective at decreasing motor neuron apoptosis ( Figures 1-4 and 7) and decreasing secretion of inflammatory cytokines from stimulated astrocytes ( Figures 5 & 6).
  • Example 3 The present Example tested the drug combination of Rapamycin and Masitinib in the iTDP43 mouse model as this mouse model recapitulates ALS with high fidelity.
  • Rapamycin dosage 10 mg/kg intraperitoneally, three times a week (Wang et ah, 2012), but rapamycin has been safely administered at 8 mg/kg/day (Bitto et al., 2016).
  • Masitinib has been used in human ALS patients alongside Riluzole successfully at 4.5 mg/kg/day (Mora et al., 2020).
  • a formulation consisting of 8 mg/kg Rapamycin and 4.5 mg/kg Masitinib was administered to ALS mice once a day intraperitoneally.
  • Rapamycin and masitinib dosing was scheduled at 4 weeks of age using a single dose.
  • Motor functional assessment including rotarod performance test, grip strength test and hanging wire test, also began at 4 weeks of age and continued at 2-week intervals.
  • cortex and spinal cord were extracted and histological analysis was performed. This was then followed by permeabilising and staining sections of the motor cortex and spinal cord with GFAP for detection of Astrocytes and CD68 for microglia ( Figures 8 and 9). Each tissue section was assessed for microgliosis, astrogliosis and the potential loss of motor neurons.
  • Mouse Protocol There were 5 experimental groups in this experiment, each group consisting of 7 animals each for a total of 35 animals ( Figures 8-10):
  • mice Four-week-old ALS (iTDP-43 A315T ) mice were intraperitoneally injected daily with saline, 8 mg/kg rapamycin, 4.5 mg/kg masitinib or 8 mg/kg rapamycin and 4.5 mg/kg masitinib for 8 weeks. Rotarod, wire hang and grip strength testing were performed at 4, 6, 8, 10 and 12 weeks ( Figure 14). Healthy control animals were also intraperitoneally injected daily with saline and motor performance was also assessed.
  • each group at each timepoint was normalized to the saline iTDP-43 A315T group from that timepoint.
  • rapamycin-treated iTDP-43 A315T mice and rapamycin + masitinib -treated iTDP-43 A315T mice spent more time on the rotarod than saline-treated iTDP-43 A315T mice and appeared to be mostly comparable to healthy control animals (Figure 15a).
  • rapamycin + masitinih- treated animals spent significantly more time on the rotarod than saline-treated iTDP-43 A315T mice ( Figure 15b).
  • Masitinib-treated iTDP-43 A315T mice also showed a non-significant trend towards increased time on the rotarod compared with saline-treated iTDP-43 A315T mice, particularly at 12 weeks ( Figure 15a, b).
  • iTDP-43 A315T mice from each treatment group had comparable grip strength (Figure 17a, b).
  • healthy control animals showed a non-significant trend towards increased grip strength compared with iTDP-43 A315T mice.
  • the grip strength of rapamycin-treated iTDP-43 A315T mice was significantly reduced compared with healthy mice.
  • mice The histopathology from these mice demonstrates evidence of reduced microgliosis and astrogliosis in iTDP-43 A315T treated with the rapamycin + masitinib combination therapy ( Figures 19 and 20)
  • mice may exhibit variable phenotypes, with a subgroup that does not respond to rapamycin and masitinib treatment, while another subgroup of animals that does respond to the drug combination treatment, with reduced gliosis and neurodegeneration as evidenced from the histopathology of these mice.
  • Zhao, J., et al., mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy. Proceedings of the National Academy of Sciences, 2015. 112(52): p. 15790.

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Abstract

La présente invention concerne des méthodes thérapeutiques, et une composition associée, comprenant un inhibiteur de la voie mTOR, et un inhibiteur d'au moins une tyrosine kinase choisie parmi c-Kit, Lyn, Fyn, CSF1R et PDGFR ; et comprenant en outre au moins l'un d'un inhibiteur de la voie de réponse aux protéines non repliées, ou d'un inhibiteur de l'inflammasome, pour le traitement de maladies, troubles ou états neurodégénératifs, tels que la sclérose latérale amyotrophique (SLA). Spécifiquement, la présente invention concerne l'administration de Rapamycine et de Mastinib, en combinaison avec soit MCC950, soit GSK2606414 pour le traitement de la SLA.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12091413B2 (en) 2020-05-08 2024-09-17 Halia Therapeutics, Inc. Inhibitors of NEK7 kinase
WO2023171806A1 (fr) * 2022-03-11 2023-09-14 株式会社幹細胞＆デバイス研究所 Agent thérapeutique ou prophylactique pour des maladies associées à des dysfonctionnements mitochondriaux

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