WO2024242530A1 - Composé destiné à la neuroprotection ou au traitement de maladies associées à l'alpha-synucléine et/ou à une neuro-inflammation - Google Patents

Composé destiné à la neuroprotection ou au traitement de maladies associées à l'alpha-synucléine et/ou à une neuro-inflammation Download PDF

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WO2024242530A1
WO2024242530A1 PCT/KR2024/095790 KR2024095790W WO2024242530A1 WO 2024242530 A1 WO2024242530 A1 WO 2024242530A1 KR 2024095790 W KR2024095790 W KR 2024095790W WO 2024242530 A1 WO2024242530 A1 WO 2024242530A1
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compound
disease
synuclein
alpha
cells
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Yoonsuk LEE
Juyoung JUNG
Yunhee Kim
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Yunovia Co Ltd
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Yunovia Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present disclosure relates to the compounds for neuroprotection, or treating diseases related to alpha-synuclein and/or neuro-inflammation, and their medical uses.
  • Neuroprotection refers to the relative preservation of neuronal structure and/or function. In the case of an ongoing insult (a neurodegenerative insult) the relative preservation of neuronal integrity implies a reduction in the rate of neuronal loss over time. It is a widely explored treatment option for many central nervous system (CNS) disorders including neurodegenerative diseases, stroke, traumatic brain injury, spinal cord injury, and acute management of neurotoxin consumption (e.g. methamphetamine overdoses). Neuroprotection aims to prevent or slow disease progression and secondary injuries by halting or at least slowing the loss of neurons.
  • CNS central nervous system
  • Neuroprotective treatments often can be useful in treating, ameliorating or preventing diseases or disorders related to such causes.
  • alpha-synuclein aggregates to form insoluble fibrils in pathological conditions characterized by Lewy bodies, such as Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, (pure) autonomic failure and both sporadic and familial cases with Alzheimer's disease. These disorders are known as synucleinopathies.
  • synucleinopathies In vitro models of synucleinopathies revealed that abnormal accumulation or aggregation of alpha-synuclein may lead to various cellular disorders including microtubule impairment, synaptic and mitochondrial dysfunctions, oxidative stress as well as dysregulation of calcium signaling, proteasomal and lysosomal pathway.
  • Alpha-synuclein is the primary structural component of Lewy body fibrils.
  • Neuro-inflammation is inflammation of the nervous tissue. It may be initiated in response to a variety of cues, including infection, traumatic brain injury, toxic metabolites, or autoimmunity.
  • CNS central nervous system
  • microglia are the resident innate immune cells that are activated in response to these cues.
  • Such neuro-inflammation plays an important role in diseases or disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, peripheral neuritis, neuropathic pain, complex region pain syndrome (CRPS), and it is known that these diseases or disorders can be improved if neuro-inflammation is ameliorated.
  • CRPS complex region pain syndrome
  • the objective of the present disclosure is to provide new medical or pharmaceutical uses of compounds for neuroprotection, or treating diseases related to alpha-synuclein and/or neuro-inflammation.
  • the present disclosure provides a pharmaceutical composition for neuroprotection; treating or preventing neuro-inflammation; or treating or preventing synucleinopathy, comprising 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient, and optionally an a pharmaceutically acceptable carrier.
  • the present disclosure also provides methods of using said compound and compositions comprising the compound of formula (I) or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for neuroprotection; treating or preventing neuro-inflammation; or treating or preventing synucleinopathy.
  • the neuroprotection is for treating or preventing neurodegenerative disease (including Parkinson's disease, Alzheimer's disease, and Huntington' disease), stroke, traumatic brain injury, or spinal cord injury; and for acute management of neurotoxin consumption (e.g. methamphetamine overdoses).
  • neurodegenerative disease including Parkinson's disease, Alzheimer's disease, and Huntington' disease
  • stroke traumatic brain injury, or spinal cord injury
  • neurotoxin consumption e.g. methamphetamine overdoses
  • the synucleinopathy is any one selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.
  • the present disclosure provides methods of protecting neurons; ameliorating neuro-inflammation; or modulating (e.g., inhibiting or antagonizing) toxic forms of ⁇ -synuclein, comprising contacting a sample or cell or biological system with an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • Also provided are methods of protecting neurons; treating or preventing neuro-inflammation; inhibiting or preventing alpha synuclein (expression); or treating or preventing synucleinopathy that includes administering a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject having or at risk from disease(s) or disorder(s) that can be treated or ameliorated by neuroprotection, or related to or caused by neuro-inflammation or abnormal alpha-synuclein.
  • Also provided are methods of treating neuro-inflammation, Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and/or Alzheimer's disease with Lewy bodies that include administering a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject having such disease or disorder.
  • 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same may achieve an excellent effect of preventing or treating neuro-inflammation and/or synucleinopathy; or inhibiting alpha synuclein expression.
  • FIG. 4 is the effect of reference compound (RI) MK801 on neuronal read outs.
  • A % TH positive neurons
  • B number of PSD95 positive synapses
  • C number of caspase 3 cells per TH neurons
  • D total number of caspase 3 positive cells after RI MK801 treatment.
  • Statistical analysis One-way ANOVA followed by Dunnett's multiple comparisons test versus LC. *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • FIG. 6 shows detergent free total alpha-synuclein in SH-SY5Y-a-syn cell extracts.
  • Total alpha-synuclein assessed with MSD assay after the treatment. Data are given as pg alpha-synuclein per ⁇ g total protein and are shown as bar graphs with group mean + SEM (n 6).
  • Statistical analysis One-way ANOVA followed by Dunnett's multiple comparisons test versus vehicle control (VC). *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • FIG. 8 shows autophagy marker LC3BII in SH-SY5Y-alpha-syn cell extracts.
  • Statistical analysis One-way ANOVA followed by Dunnett's multiple comparisons test versus vehicle control (VC). *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • FIG. 10 shows the number of synapses per image.
  • Statistical analysis One-way ANOVA followed by Dunnett's multiple comparisons test versus LC. *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • the compound of the present disclosure exhibits more desirable effects for the purpose of the present disclosure compared to compounds having similar structures.
  • a solvate, a hydrate, a prodrug, and/or a stereoisomer, or a pharmaceutically acceptable salt of the compound of formula (I) may be used as active ingredient in uses according to the present disclosure.
  • compositions comprising the compound of formula (I), or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof may be used for medical or pharmaceutical uses of the present disclosure.
  • the compound can protect neurons, and treat or ameliorate neuro-inflammation and/or any toxicities relate to alpha-synuclein in cells and biological systems of interest.
  • the compound find use in a variety of therapeutic applications, including neuroprotection, or diseases or disorders related to or caused by neuro-inflammation and/or abnormal alpha-synuclein.
  • the compound can inhibit or prevent alpha-synuclein (expression).
  • the compound also can protect neurons by inhibiting or preventing alpha-synuclein (expression).
  • the compound find use in neurodegenerative disease (including Parkinson's disease, Alzheimer's disease, and Huntington' disease), stroke, traumatic brain injury, spinal cord injury, and acute management of neurotoxin consumption (e.g.
  • the compound also find use in the synucleinopathy selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.
  • the synucleinopathy selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.
  • any of the compound described herein may also be referred to as a compound of formula (I) or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
  • compositions may be formulated in pharmaceutical compositions.
  • the pharmaceutical composition can include the compound of the present disclosure and at least one excipient (e.g., a pharmaceutically acceptable excipient).
  • the compounds described herein can find use in pharmaceutical compositions for administration to a subject in need thereof in a variety of therapeutic applications where neuroprotection is desirable. Specifically, the compounds can protect neurons by inhibiting alpha-synuclein (expression).
  • the compounds described herein also can find use in pharmaceutical compositions for administration to a subject in need thereof in a variety of therapeutic applications where treatment and/or amelioration of neuro-inflammation and/or alpha-synuclein toxicity is desirable.
  • the treatment and/or amelioration of alpha-synuclein toxicity may refer an inhibition or prevention of alpha-synuclein (expression).
  • the present disclosure provides pharmaceutical compositions comprising the compound described herein, a pharmaceutically acceptable salt thereof, or a prodrug thereof, and at least one pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient refers any ingredient other than the inventive compound described herein (for example, a vehicle capable of suspending or dissolving the active compound, or any other convenient pharmaceutically acceptable carriers, excipients or additives) and having the properties of being substantially nontoxic and non-inflammatory in a patient.
  • Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, dispensing, or dispersing agents, sweeteners, and waters of hydration.
  • the pharmaceutical composition comprises a compound as described herein, a pharmaceutically acceptable salt thereof, or a prodrug thereof in a therapeutically effective amount.
  • the pharmaceutical composition may be formulated according to any convenient methods, and may be prepared in various forms for oral administration such as tablets, pills, powders, nanoparticles, capsules, syrups, suspensions, emulsions and microemulsions, or in forms for non-oral administration such as preparations for intramuscular, intravenous or subcutaneous administration.
  • the pharmaceutical compositions provided herein can be administered by a variety of routes including, but not limited to, oral (enteral) administration, parenteral (by injection) administration, rectal administration, transdermal administration, intradermal administration, intrathecal administration, subcutaneous (SC) administration, intravenous (IV) administration, intramuscular (IM) administration, and intranasal administration.
  • the pharmaceutical composition could contain a pharmaceutically allowed carrier, excipient, or additive.
  • the pharmaceutical composition could be produced as medicine in the conventional method, and could be produced as various oral medicine such as tablet, pill, powder, capsule, syrup, emulsion, micro-emulsion, and so on, or could be produced as non-oral medicine such as muscular injection, vascular injection, or subcutaneous injection.
  • carrier is a solid carrier or a flowable carrier.
  • Suitable pharmaceutical carriers include those described in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005).
  • examples of the used additive or carrier could include cellulose, silicic calcium, corn starch, lactose, sucrose, dextrose, phosphoric acid calcium, stearic acid, stearic acid magnesium, stearic acid calcium, gelatin, talc, surfactant, suspension, emulsifying agent, diluting agent, and so on.
  • the additives or carrier could include water, saline water, glucose aqueous solution, similar sugar-soluble solution, alcohol, glycol, ether (e.g., polyethylene glycol 400), oil, fatty acid, fatty acid ester, glyceride, surfactant, suspension, emulsifying agent, and so on.
  • the pharmaceutical compositions are formulated for parenteral administration to a subject in need thereof. In some parenteral embodiments, the pharmaceutical compositions are formulated for intravenous administration to a subject in need thereof. In some parenteral embodiments, the pharmaceutical compositions are formulated for subcutaneous administration to a subject in need thereof.
  • aspects of the present disclosure include methods of protecting neurons; treating, preventing or ameliorating neuro-inflammation; and/or treating, preventing or ameliorating toxicity related to alpha-synuclein, particularly, abnormal forms of alpha-synuclein in a biological system or sample. Treating, prevention and/or ameliorating of alpha-synuclein toxicity may refer inhibiting or preventing alpha-synuclein (expression).
  • the method includes contacting a sample with the compound of this disclosure. In some embodiments, the method includes contacting a cell or biological system with the compound of this disclosure.
  • aspects of the present disclosure include methods of protecting neurons; treating, preventing or ameliorating neuro-inflammation; and/or treating, preventing or ameliorating toxicity related to alpha-synuclein using the compound described herein.
  • Such methods may include methods of protecting neurons; treating, preventing or ameliorating neuro-inflammation; and/or treating, preventing or ameliorating abnormal reactions related to alpha-synuclein in biological systems by contacting such systems with the compound of formula (I).
  • Biological systems may include, but are not limited to, cells, tissues, organs, bodily fluids, organisms, non-mammalian subjects, and mammalian subjects (e.g., humans, dogs, cats, mice, rats, rabbits, guinea pigs, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and, in particular, humans).
  • mammalian subjects e.g., humans, dogs, cats, mice, rats, rabbits, guinea pigs, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and, in particular, humans).
  • the method of protecting neurons; treating, preventing or ameliorating neuro-inflammation; and/or treating, preventing or ameliorating alpha-synuclein toxicity comprises contacting a biological system or sample comprising neuro-inflammation and/or abnormal alpha-synuclein with an effective amount of any of the compound or a pharmaceutically acceptable salt thereof as described herein, or a pharmaceutical composition as described herein.
  • the biological system or sample is in vitro .
  • the biological system or sample is in vivo .
  • the sample is a cell sample.
  • treating, preventing and/or ameliorating alpha-synuclein toxicity and/or abnormal alpha-synuclein may refer inhibiting or preventing alpha-synuclein (expression).
  • the present disclosure provides a method of protecting neurons in a subject using the subject compound as therapeutic agent, and compositions including the compound. Any diseases or disorders for which neuroprotection are thought to be useful by those of ordinary skill in the art are contemplated as diseases treatable using the compound of this disclosure.
  • the method includes administering to a subject having a disease or disorder requiring neuroprotection a therapeutically effective amount of the compound of formula (I).
  • the disease or disorder is neurodegenerative diseases (including Parkinson's disease, Alzheimer's disease, and Huntington' disease), stroke, traumatic brain injury, spinal cord injury, or neurotoxin consumption (e.g. methamphetamine overdoses).
  • the compound of formula (I) can protect neurons by reducing or inhibiting alpha-synuclein (expression).
  • neuroprotection of the present disclosure aims to prevent or slow disease progression and secondary injuries by halting or at least slowing the loss of neurons.
  • the present disclosure provides a method of protecting neurons in a subject having neurodegenerative disease(s) using the subject compound as therapeutic agent.
  • the method further comprises identifying a subject suffering from or at risk of a disease or disorder requiring neuroprotection.
  • the method further comprises identifying an underlying disease or condition associated with neuroprotection.
  • the present disclosure provides a method of treating, ameliorating or preventing diseases or disorders related to or caused by neuro-inflammation and/or alpha-synuclein toxicity in a subject using the subject compound as therapeutic agent, and/or compositions including the compound. Any diseases or disorders for which amelioration of neuro-inflammation and/or alpha-synuclein toxicity are thought to be useful by those of ordinary skill in the art are contemplated as diseases treatable using the compound of this disclosure.
  • the method includes administering to a subject having a disease or disorder related to or caused by neuro-inflammation and/or alpha-synuclein toxicity a therapeutically effective amount of the compound of formula (I).
  • the disease or disorder is synucleinopathy.
  • the synucleinopathy is Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, or Alzheimer's disease with Lewy bodies.
  • the disease or disorder is a disease or disorder which can be treated by ameliorating neuro-inflammation.
  • the disease or disorder which can be treated by ameliorating neuro-inflammation is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, peripheral neuritis, neuropathic pain, or complex region pain syndrome (CRPS).
  • Alzheimer's disease Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, peripheral neuritis, neuropathic pain, or complex region pain syndrome (CRPS).
  • CRPS complex region pain syndrome
  • the alpha-synuclein toxicity can be caused by overexpression of alpha-synuclein protein or alpha-synuclein aggregation.
  • treating, preventing and/or ameliorating of alpha-synuclein toxicity may refer inhibiting or preventing alpha-synuclein (expression).
  • the present disclosure provides a method of inhibiting or preventing alpha-synuclein (expression) in a subject using the subject compound as therapeutic agent, or compositions including the compound. Any diseases or disorders for which inhibition of alpha-synuclein (expression) are thought to be useful by those of ordinary skill in the art are contemplated as diseases treatable or preventable using the compound of this disclosure.
  • the method further comprises identifying a subject suffering from or at risk of a disease or disorder requiring amelioration of neuro-inflammation and/or alpha-synuclein toxicity.
  • the method further comprises identifying an underlying disease or condition associated with neuro-inflammation and/or alpha-synuclein toxicity.
  • the amount of the aforementioned pharmaceutical composition administered is the effective amount to treat or prevent illness for an entity or patient, and could be administered orally or non-orally according to the purpose.
  • the amount administered based on the active component is 0.01 to 1,000 mg per 1kg of body weight, more specifically 0.1 to 300 mg per 1kg.
  • 0.01 to 100 mg based on the active component is administered per 1kg of body weight per day, and more specifically 0.1 to 50 mg is administered once or several times.
  • the amount administered for a specific entity or patient can be determined based on many related factors including the patient's weight, age, sex, health status, diet, time of administration, method of administration, severity of the illness, and so on, must be understood to be able to be increased or decreased appropriately by the specialist, and the aforementioned amount of administration does not limit the scope of the present disclosure in any way.
  • a medical doctor or veterinarian with ordinary level of knowledge in the related technological field may determine and prescribe effective amount of the pharmaceutical composition. For example, a medical doctor or veterinarian may start with the amount of the compound under the present disclosure used in a pharmaceutical composition that is lower than the amount required to achieve the desired treatment effect, and may increase the amount administered gradually until the desired effect is achieved.
  • suitable routes of administration include oral, rectal, transmucosal (including sublingual and buccal), intranasal, topical, transdermal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intracerebroventricular injection, direct injections to the human brain, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • the compounds can also be administered in sustained or controlled release dosage forms, including nanoparticles, depot injections, osmotic pumps, electronic pumps, pills, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate. Sustained or controlled release dosage forms may be used to increase CNS exposure and minimize systemic exposure. It is also possible to combine at least two different routes of administration.
  • compositions of this disclosure may be administered alone, in combination with a compound according to another example of the present disclosure, or in simultaneous, separate or sequential concomitant administration with at least one other therapeutic agent, for example with other pharmaceutical active ingredients described herein.
  • a pharmaceutical composition of this disclosure includes within its scope at least one of the compounds in accordance with a specific example of the effective treatment amount as effective component, or a pharmaceutical composition that is contained in combination with a pharmaceutical carrier.
  • the compound in accordance with an embodiment of the present disclosure could be administered independently, in combination with a compound in accordance with another specific example, or simultaneously with one or more other treatment medications, for example simultaneously with an anticancer medicine (e.g., as described herein) or with an active pharmaceutical material, separately, or consecutively in conjunction.
  • alpha-synuclein (aSyn or ⁇ Syn)
  • Alpha-synuclein is a neuronal protein that regulates synaptic vesicle trafficking and subsequent neurotransmitter release. It is abundant in the brain, while smaller amounts are found in the heart, muscle and other tissues.
  • ⁇ -Synuclein is the major constituent of Lewy bodies as a common pathological signature of Lewy body diseases including Parkinson's disease, and Dementia with Lewy bodies.
  • ⁇ -Synuclein exists in various forms, such as oligomers, immature filaments, and filaments, and is a cause of disease.
  • filamentous structures are mainly found in Lewy bodies in nerve cells.
  • amyloid protein filaments are thermodynamically stable structures, and the process of converting toxic oligomers into amyloid structures is thought to be a product of efforts to reduce toxicity.
  • oligomers affect cell membrane permeability, resulting in abnormal calcium influx and cell death.
  • alpha-synuclein used with the terms “inhibit”, “prevent”, “ameliorate”, “treat” and so on may mean any forms of alpha-synuclein, including oligomers, aggregates and toxic forms.
  • oligomer oligomer
  • aggregate oligomeric form of alpha-synuclein
  • oligomeric forms of alpha-synuclein are known to be toxic, but the present disclosure is not limited to these specific forms of alpha-synuclein.
  • the present disclosure can be used for synucleinopathy based on excessive alpha-synuclein levels as well as these toxic forms of alpha-synuclein.
  • synucleinopathy also called ⁇ -synucleinopathy
  • ⁇ -synucleinopathy means a disease characterized by the (abnormal) accumulation of alpha-synuclein protein, particularly aggregates of alpha-synuclein protein in neurons, nerve fibres or glial cells.
  • synucleinopathy also means a disease characterized by specific forms, particularly, toxic forms, including oligomers or aggregates, of alpha-synuclein protein.
  • MSA multiple system atrophy
  • the term “about” refers to a ⁇ 10% variation from the nominal value unless otherwise indicated or inferred. Where a percentage is provided with respect to an amount of a component or material in a composition, the percentage should be understood to be a percentage based on weight, unless otherwise stated or understood from the context.
  • molecular weight is provided and not an absolute value, for example, of a polymer, then the molecular weight should be understood to be an average molecule weight, unless otherwise stated or understood from the context.
  • pharmaceutically acceptable excipient pharmaceutically acceptable diluent
  • pharmaceutically acceptable carrier pharmaceutically acceptable adjuvant
  • pharmaceutically acceptable adjuvant refers to an excipient, diluent, carrier, or adjuvant that is useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use.
  • pharmaceutically acceptable excipient includes both one and more than one such excipient, diluent, carrier, and/or adjuvant.
  • composition is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
  • a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject ( i.e. , the compound(s) in the pharmaceutical composition is pharmaceutical grade).
  • Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intranasal, intramuscular, subcutaneous, and the like.
  • subject to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs.
  • the subject is a human.
  • the subject is a non-human animal.
  • the terms "human,” “patient,” and “subject” are used interchangeably herein.
  • the terms “treat,” “treating” and “treatment” contemplate an action that occurs while a subject is suffering from the specified disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition.
  • Treatment includes: (i) prevention of the disease, disorder, or condition, i.e., reducing the incidence of and/or ameliorating the effect and/or duration of a disease, disorder, or condition from occurring in subjects that may get, be exposed to and/or be predisposed to the disease, disorder or condition, but may not yet have been diagnosed as having it; or are diagnosed as having the disease, disease, or condition; or are at risk of developing such disease, disorder, or condition; (ii) inhibition of the disease, disorder, or condition, i.e., preventing or delaying the onset of a disease, disorder, or condition; arresting further development or progression of a disease, disorder, or condition in a subject already suffering from or having one or more symptoms of the disease, disorder, or condition; or reducing the risk of a disease, disorder, or condition worsening; (iii) amelioration of the disease, disorder, or condition, i.e., attenuating, relieving, reversing or eliminating the disease, disorder, or
  • a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder or condition.
  • the term "therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.
  • a prophylactically effective amount of a compound is an amount sufficient to prevent a disease, disorder or condition, or one or more symptoms associated with the disease, disorder or condition, or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease, disorder or condition.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • the text refers to various embodiments of the present compounds, compositions, and methods.
  • the various embodiments described are meant to provide a variety of illustrative examples and should not be construed as descriptions of alternative species. Rather, it should be noted that the descriptions of various embodiments provided herein may be of overlapping scope.
  • the embodiments discussed herein are merely illustrative and are not meant to limit the scope of the present technology.
  • a pharmaceutical composition for neuroprotection; treating or preventing neuro-inflammation; or treating or preventing synucleinopathy comprising 3-(6-amino-1-(4-amino-3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-2-fluorobenzonitrile or a solvate, a hydrate, a prodrug, and/or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient.
  • neuroprotection is for treating or preventing neurodegenerative disease, stroke, traumatic brain injury, spinal cord injury; and for acute management of neurotoxin consumption.
  • synucleinopathy is any one selected from the group consisting of Parkinson's disease with Lewy bodies, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), (pure) autonomic failure, and Alzheimer's disease with Lewy bodies.
  • HPLC-MS analysis was carried out with gradient elution.
  • Medium pressure liquid chromatography (MPLC) was performed with silica gel columns in both the normal phase and reverse phase.
  • Reagents and condition (a) Pd(PPh 3 ) 4 , K 2 CO 3 , dioxane/H 2 O, 100 °C, 16h; (b) Fe, NH 4 Cl, EtOH/H 2 O, 60 °C, 1 h.
  • the residue was purified by prep-HPLC (column: Phenomenex Synergi C18 150*25mm* 10 ⁇ m; mobile phase: [water(0.225%FA)-ACN];B%: 16%-46%,11min), the 89% purity compound was obtained after first purification.
  • the residue was purified by prep-HPLC (column: Phenomenex Luna C18 150*25mm*10 ⁇ m; mobile phase: [water(0.225%FA)-ACN];B%: 18%-48%,10min).
  • the aim of the study was to test the compound of formula (I) at 7 concentrations (1, 3, 10, 30, 100, 300, 1000 [nM], HCl salt, DMSO) in MPP+ lesioned rat TH neurons and in SH-SY5Y cells.
  • SH-SY5Y cells human ⁇ Syn overexpressing cells
  • RA retinoic acid
  • Cells were treated with the compound at 7 concentrations or vehicle control.
  • the experiment was performed in either 48-well or 96-well plates for protein analysis or for mitochondrial profile and immunocytochemical staining, respectively.
  • Cells were harvested from 48-well plates after 48 h treatment phase. Total fractions were extracted and used for evaluation of human alpha-synuclein with MSD assay.
  • TH positive neurons and synapses The compound at 10-100 nM was able to rescue dopaminergic (TH positive) neurons and led to increased number of synapses compared to the lesion control.
  • Apoptotic cells The compound was able to significantly reduce the number of apoptotic cells in the MPP+ lesion assay at almost all tested concentrations, except the highest dose of 1000 nM.
  • Mitochondrial health The compound only showed trends towards rescued mitochondrial activity compared to the lesion control.
  • Alpha-synuclein Free total fraction was assessed with MSD assay. Due to absence of detergent this fraction represents mainly soluble monomers.
  • Mitochondrial health For the compound at medium concentrations the mitotracker showed trends towards increased signal, getting significant for the compound at 30 nM and 100 nM compared to the respective vehicle control.
  • Synapses A significant increase in the number of synapses was detected when comparing vehicle control with 10nM the compound-treated cells.
  • Rat line Sprague Dawley
  • CMF-HBSS Calcium and Magnesium free Hanks Balanced Salt Solution
  • the mesencephalon was chopped with a sterile razor blade in Chop solution (Hibernate-E without Calcium containing 2% B-27) and digested in 2 mg/ml papain (Worthington) dissolved in Hibernate-E without Calcium for 45 minutes at 30 °C.
  • the mesencephalon was triturated for 15 times with a fire-polished silanized Pasteur pipette in Hibernate-E without Calcium containing 2% B-27, 0.01% DNaseI, 1 mg/ml BSA, and 1 mg/ml Ovomucoid Inhibitor. Undispersed pieces were allowed to settle by gravity for 1 min and the supernatant was centrifuged for 5 min at 228 g.
  • the pellet was resuspended in Hibernate-E containing 2% B-27, 0.01% DNaseI, 1 mg/ml BSA, 1 mg/ml Ovomucoid Inhibitor and further diluted with Hibernate-E containing 2% B-27.
  • the pellet was resuspended in Neurobasal medium containing, 2% B-27, 0.5 mM glutamine, 1% Penicillin-Streptomycin. Cells were counted in a hemacytometer and were seeded in poly-D-lysine pre-coated 96 well plates at a density of 50.000 cells/well. 4 plates were seeded in total.
  • TH-plating medium [DMEM/F12, 10% serum (Horse serum), 5% Glucose stock (100 mg/ml) and 1% Penicillin-Streptomycin] for 24 h at 37 °C and 5% CO 2 .
  • TH treatment medium [DMEM/F12, 1% N2, 5% Glucose stock [100 mg/ml] and 1% Penicillin-Streptomycin].
  • DMEM/F12 10% serum (Horse serum), 5% Glucose stock (100 mg/ml) and 1% Penicillin-Streptomycin]
  • Plate1 seeded/treated twice, once for set1 and once for set2
  • the number of TH positive cells per MAP2 positive cells were measured using Gen05 software. PSD95 positive small spots were counted as synapses.
  • ACSF buffer 125 mM NaCl, 2.5 mM KCl, 26.2 mM NaHCO3, 1 mM NaH2PO4, 2.5 mM CaCl2, 1.25 mM MgCl2, 25 mM Hepes, 11 mM D-Glucose, pH 7.3
  • the overall number of caspase 3 positive cells as well as caspase 3 positive cells per TH positive cells were measured using Gen05 software. Additionally, the fluorescence area of TR channel was measured to quantify active mitochondria using Gen05 software (as % of VC).
  • SH-SY5Y- ⁇ -syn cells were kept in culture medium (DMEM medium, 10% FCS, 1% NEAA, 1% L-Glutamine, 100 ⁇ g/ml Gentamycin, 300 ⁇ g/ml Geneticin G-418) for ⁇ 2 days until 80-90% confluency.
  • DMEM medium 10% FCS
  • NEAA 1% NEAA
  • L-Glutamine 100 ⁇ g/ml Gentamycin, 300 ⁇ g/ml Geneticin G-418
  • RA ⁇ M retinoic acid
  • DIV1 differentiation start
  • samples were frozen at -80 °C at this step until further processing.
  • Human alpha-synuclein levels in the total fractions of all samples were determined by using a commercially available immunosorbent assay (Meso Scale Diagnostic, Cat. No. K151TGD) according to the manufacturer's protocol and plates were read on the MESO QuickPlex SQ 120 reader. Samples were applied at uniform total protein concentrations (1 ⁇ g/ml for soluble and total and 35 ⁇ g/ml for insoluble fraction) and evaluated in comparison to an adequate protein standard as pg human alpha-synuclein per ⁇ g total protein.
  • Samples were analysed for Total p62 (SQSTM1) with a commercially available immunosorbent assay kit from Mesoscale Diagnostics (MSD, Cat.No. K151MJD) at uniform total protein concentration of 25 ⁇ g/ml.
  • the immune assay was carried out according to the manual and plates were read on the MESO QuickPlex SQ 120 reader. Analyte levels were evaluated according to adequate calibration curves (recombinant human SQSTM1 produced in E.Coli from Prospec, Cat.No. PRO-806) as arbitrary units (AU).
  • ACSF buffer 125 mM NaCl, 2.5 mM KCl, 26.2 mM NaHCO 3 , 1 mM NaH 2 PO 4 , 2.5 mM CaCl 2 , 1.25 mM MgCl 2 , 25 mM Hepes, 11 mM D-Glucose, pH 7.3
  • the fluorescence area of TR channel was measured to quantify active mitochondria using Gen05 software.
  • MPP+ lesioned neurons were fixed and stained for MAP2, TH and PSD95 as neuronal marker.
  • the number of TH positive neurons in relation to total number of neurons was assessed and expressed as %TH neurons.
  • the compound of formula (I) treatment at 10-100 nM led to significant rescue of TH positive neurons compared to the vehicle treated lesion control (LC; FIG. 1). Interestingly, also the lower concentrations of the compound were able to significantly increase the number of PSD95 positive synapses compared to the lesion control (FIG. 2).
  • Mitochondrial health of neuronal cultures after MPP+ lesion and treatments was assessed using mitotracker dye.
  • the compound of formula (I) showed trends towards rescued mitochondrial activity compared to the lesion control (FIG. 5).
  • the amount of alpha-synuclein in total fractions of cell lysates was assessed by Mesoscale Discovery assay.
  • Mitochondrial health was again assessed using mitotracker dye.
  • the mitotracker showed trends towards increased signal, getting significant for the compound at 30 nM and 100 nM compared to the respective vehicle control (FIG. 9).
  • the number of synapses in SH-SY5Y-alpha-syn cells was assessed by quantification of synapsin positive spots.

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Abstract

De nouvelles utilisations d'un composé sont divulguées. Le composé de la présente divulgation est utile pour protéger des neurones ou traiter ou prévenir une neuro-inflammation et/ou une toxicité par l'alpha-synucléine. Ainsi, le composé de la présente divulgation peut être utilisé pour traiter ou prévenir des maladies ou des troubles qui nécessitent une neuroprotection ou sont provoqués par ou associés à une neuro-inflammation et/ou une toxicité par l'alpha-synucléine. La présente divulgation concerne également des méthodes d'utilisation dudit composé ou de ladite composition comprenant le composé destiné à traiter ou prévenir de telles maladies ou troubles.
PCT/KR2024/095790 2023-05-19 2024-05-17 Composé destiné à la neuroprotection ou au traitement de maladies associées à l'alpha-synucléine et/ou à une neuro-inflammation Ceased WO2024242530A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018091444A1 (fr) * 2016-11-15 2018-05-24 H. Lundbeck A/S Agents, utilisations et procédés pour le traitement d'une synucléinopathie
WO2018183192A1 (fr) * 2017-03-27 2018-10-04 Chase Therapeutics Corporation Compositions et méthodes de traitement des synucléinopathies
KR102180550B1 (ko) * 2020-05-20 2020-11-18 충남대학교산학협력단 FAF1 단백질의 발현을 억제하는 miRNA를 유효성분으로 함유하는 신경 퇴행성 질환 예방 또는 치료용 약학적 조성물
WO2022107044A1 (fr) * 2020-11-19 2022-05-27 Ildong Pharmaceutical Co., Ltd. Prévention et/ou traitement de troubles du système nerveux central
KR20220104760A (ko) * 2019-11-19 2022-07-26 일동제약(주) 아데노신 수용체 길항제 화합물

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018091444A1 (fr) * 2016-11-15 2018-05-24 H. Lundbeck A/S Agents, utilisations et procédés pour le traitement d'une synucléinopathie
WO2018183192A1 (fr) * 2017-03-27 2018-10-04 Chase Therapeutics Corporation Compositions et méthodes de traitement des synucléinopathies
KR20220104760A (ko) * 2019-11-19 2022-07-26 일동제약(주) 아데노신 수용체 길항제 화합물
KR102180550B1 (ko) * 2020-05-20 2020-11-18 충남대학교산학협력단 FAF1 단백질의 발현을 억제하는 miRNA를 유효성분으로 함유하는 신경 퇴행성 질환 예방 또는 치료용 약학적 조성물
WO2022107044A1 (fr) * 2020-11-19 2022-05-27 Ildong Pharmaceutical Co., Ltd. Prévention et/ou traitement de troubles du système nerveux central

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