WO2018026150A1 - Pharmaceutical composition for prevention or treatment of neurodegenerative diseases - Google Patents

Pharmaceutical composition for prevention or treatment of neurodegenerative diseases Download PDF

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WO2018026150A1
WO2018026150A1 PCT/KR2017/008229 KR2017008229W WO2018026150A1 WO 2018026150 A1 WO2018026150 A1 WO 2018026150A1 KR 2017008229 W KR2017008229 W KR 2017008229W WO 2018026150 A1 WO2018026150 A1 WO 2018026150A1
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radotinib
synuclein
pharmaceutical composition
disease
nilotinib
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PCT/KR2017/008229
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French (fr)
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Dong Yeon Kim
Jae Soo Shin
Dae Jin Cho
Gong Yeal Lee
Hong Youb Kim
Hea Un Lee
Choong Am Ahn
Song Ei SONG
Jong Seong Park
Soung Young KANG
Hye Lin Park
Hyun Soo Ju
Hyo Jung Yun
Han Seok Ko
Sae Bom Lee
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Il-Yang Pharm. Co., Ltd.
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Publication of WO2018026150A1 publication Critical patent/WO2018026150A1/en

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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
    • A61K31/495—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
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • 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
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/008—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy comprising drug dissolved or suspended in liquid propellant for inhalation via a pressurized metered dose inhaler [MDI]

Definitions

  • the present invention relates to a pharmaceutical composition for prevention or treatment of neurodegenerative diseases comprising an effective amount of radotinib or its pharmaceutically acceptable salts.
  • Neurodegenerative disease refers to a disease in which neuronal cells are destroyed for some reason and thereby causing abnormality of brain function.
  • Typical neurodegenerative diseases are, for example, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.
  • Parkinson's disease is one of the most common neurodegenerative diseases. It is a progressive disease among the degenerative nervous diseases. The average onset age is around 60 ⁇ that is, Parkinson's disease is found mainly in older people. It is known that more than 1% of people older than 60 suffer from Parkinson's disease. In Korea, an aging society, patients with Parkinson's disease have been increasing steadily. The male-to-female ratio is 3:2; it is slightly higher in men. About 60,000-100,000 people in Korea, about 1.5 million people in the United States, and about 10 million people worldwide suffer from Parkinson's disease. In the case of the United States, the prevalence of Parkinson's disease is expected to more than double by 2040. According to epidemiological studies, in China, about 5 million patients with Parkinson's disease are expected in 2030.
  • Parkinson's disease is a progressive neurodegenerative disease characterized by Parkinsonian symptoms such as slow motion, static tremor, muscle rigidity, walking with dragging feet, and bending posture. Such symptoms are caused due to imperfect dopamine production and action in the substantia nigra and subsequent decrease of motor cortex stimulation. Chronic and progressive, serious hypermetamorphosis and mild language impairment often appear. The incidence rate is about 1 out of 1,000, but it increases with age.
  • Parkinson's disease occurs in about one out of 100 in people older than 50. In the United States, incidence rate in higher in white people than black people.
  • the immediate cause of Parkinson's disease is the slow death of neurons in the substantia nigra and corpus amygdaloideum.
  • Most studies have focused on neurons in the substantia nigra which send dopaminergic axons to the caudate nucleotoxin and the cornu cutaneum. These axons excite D1 receptors and inhibit D2 receptors.
  • inhibitory output from the globus pallidus to the thalamus increases due to loss of dopamine in both receptors.
  • Parkinson's disease and dopamine are closely related, but dopamine is not only associated with Parkinson's disease.
  • Dopamine one of the neurotransmitters that transmit intercellular signals, is produced from neuronal cells in the brain. If the mutual transmission of the neurotransmitters is abnormally suppressed or overly increased, it will lead to abnormalities in the whole function of nervous system. There are neuronal cells in the brain that broadly use dopamine in the frontal lobe which relates to thinking, in the limbic system which relates to emotion, and in the pituitary gland which relates to hormones.
  • Parkinson's Since James Parkinson's first description of Parkinson's disease, there was no effectual treatment until the 1960s. In the 1950s, it was observed that injection of reserpine, which causes depletion of dopamine, into a white rat leads to symptoms of Parkinson's disease. Autopsies of the patients revealed that the nervous cells in the substantia nigra had disappeared.
  • the dopamine described above is used as a neurotransmitter that transmits intercellular signals. When dopamine is injected directly into the body, it cannot reach the neuronal cells in the brain. This is because of the blood-brain barrier between blood and brain tissue, which allows the passage of only the substances necessary for the brain. For this reason, dopamine itself cannot pass through the barrier.
  • Parkinson's disease occurs due to depletion of dopamine in the corpus striatum. Therefore, supplement of dopamine is the main way of treating Parkinson's disease, and the most powerful drug is levodopa.
  • Levodopa has chemical similarity with dopamine, and is actually a precursor of dopamine. Levodopa can pass through the blood-brain barrier, and once it has passed, it is metabolized to dopamine in the brain and eventually becomes available in neuronal cells.
  • dopamine agonist medicines such as ropinirole and pramipexole that act as levodopa which is converted into dopamine in the brain.
  • These drugs are not dopamine, but are designed to respond to neurotransmission like dopamine. They are used either alone or as an adjuvant to levodopa for patients with early Parkinson's disease.
  • the anti-Parkinson effect of dopamine is caused by acting on the D2 receptor.
  • the effect of the D1 or D3 receptor on the symptoms of Parkinson's disease is not yet known.
  • Dopamine agonists may cause confusion or hallucination in elderly patients, and leg edema in some patients.
  • MAO-B monoamine oxidase inhibitor
  • MAO-B inhibitor has a neuroprotective effect that the other drugs do not have.
  • MAO-B inhibitors can be used for patients with early-stage to late-stage Parkinson's disease.
  • Trihexyphenidyl, benztropine mesylate, biperiden and diphenhydramine as anticholinergics help control tremors or contractures, but they are less effective than dopaminergic drugs.
  • Amantadine has several mechanisms of action. It promotes dopamine secretion at the nerve endings, and also blocks the reuptake of dopamine, the antimuscarinic effect and the glutamate receptor at the nerve endings. Although amantadine can help control symptoms by dopaminergic action, it has side effects such as livedo reticularis, ankle edema, hallucinations, confusion, and do on. Antiglutamatergic action is helpful in the control of dyskinesia caused by levodopa.
  • Parkinson's disease is performed on patients who are not adequately treated with medication and their life is affected thereby.
  • "Parkinson's disease is not adequately treated” does not mean that there is no response to levodopa or dopamine agonist but it does mean that the drug cannot be administered in a sufficient dose because of disruption of normal activity due to changes in movement and dyskinesia, or other kinds of side effects.
  • Parkinson's disease is still impossible to cure.
  • the mortality rate from Parkinson's disease is increasing every year not only in Korea but throughout the world. There is much interest in the development of drugs for treatment of Parkinson's disease.
  • the present invention is intended to provide a novel approach for treatment of neurodegenerative diseases including Parkinson's disease for which a full recovery method has yet to be found.
  • the present invention provides a pharmaceutical composition for prevention or treatment of neurodegenerative diseases, comprising radotinib as an active ingredient which is a selective Bcr-Abl kinase inhibitor and is used as a medicament for treatment of Philadelphia chromosome-positive (Ph + ) chronic myelogenous leukemia (CML).
  • radotinib as an active ingredient which is a selective Bcr-Abl kinase inhibitor and is used as a medicament for treatment of Philadelphia chromosome-positive (Ph + ) chronic myelogenous leukemia (CML).
  • the present invention can provide a pharmaceutical composition for prevention or treatment of neurodegenerative diseases comprising radotinib represented by Chemical Formula (I) below or pharmaceutically acceptable salt thereof as an active ingredient.
  • Radotinib is one of the receptor tyrosine kinase inhibitors, and is also represented by 4-methyl-N-[3-(4-methylimidazol-1-yl)-5-trifluoromethyl-phenyl]-3-[4-(3-methylpyrazin-2-yl)-pyrimidin-2-ylamino)benzamide.
  • radotinib inhibits one or more tyrosine kinases ⁇ for example, c-Abl, Bcr-Abl and the receptor tyrosine kinases such as PDGFR, Flt3, VEGF-R, EGF-R and c-Kit.
  • radotinib has excellent anticancer effects on various cancers such as lung cancer, gastric cancer, colon cancer, pancreatic cancer, hepatoma, prostatic cancer, breast cancer, chronic or acute leukemia, hematologic malignancy, brain tumor, bladder cancer, rectal cancer, cervical cancer and lymphoma.
  • radotinib has no report about the effect of radotinib on neurodegenerative diseases comprising Parkinson's disease.
  • Radotinib may be used as itself or in the form of a pharmaceutically acceptable salt.
  • pharmaceutically acceptable means that the salt is physiologically acceptable and normally causes no allergic or other similar adverse reactions when administered to a human.
  • the salt may be, but is not limited thereto, an acid addition salt formed from a pharmaceutically acceptable free acid.
  • the acid addition salt may be formed from radotinib and a pharmaceutically acceptable inorganic or organic acid which is selected from, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, acetic acid, propionic acid, ascorbic acid, citric acid, malonic acid, fumaric acid, maleic acid, lactic acid, salicylic acid, sulfamic acid and tartaric acid.
  • These salts may be prepared by a method known in the art ⁇ for example, by treating radotinib with an appropriate acid in the presence of an appropriate solvent.
  • Radotinib may be in the form of a crystalline or a solvate (e.g., hydrate), and both forms are within the scope of the present invention.
  • solvate refers to complexes in various stoichiometry formed by a solute (in the present invention, a compound such as radotinib) and a solvent. Such solvents should not impede the biological activity of the solute.
  • the solvent may be, but is not limited thereto, water, ethanol, methanol, ethyl acetate or acetone.
  • the method for solvation is generally known in the art.
  • a pharmaceutically acceptable salt of radotinib may include both solvated form and non-solvated form.
  • the pharmaceutical composition according to the present invention may comprise a pharmaceutically effective amount of radotinib.
  • pharmaceutically effective amount refers to an amount required to show the desired effect as compared with a negative control group, and preferably it refers to an amount sufficient for the prevention or treatment of neurodegenerative diseases including Parkinson's disease in mammals, including humans.
  • the therapeutic dose may be 50 mg to 2,000 mg/day, and more preferably 100 mg to 1,000 mg/day, depending on the seriousness of the status and whether radotinib is administered alone or in combination with another drug(s).
  • the pharmaceutical composition may be administered via oral or parenteral route once a day or dividedly.
  • the pharmaceutically effective amount may be properly changed depending on the particular disease and severity thereof, age, body weight, physical conditions and sex of patients, administration route, treatment period, or other various factors.
  • the neurodegenerative disease may be Parkinson's disease.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.
  • the carrier may be inert, and may be selected from, but is not limited thereto, fillers such as sugar including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, etc., starch including corn starch, wheat starch, rice starch, potato starch, etc., celluloses including cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, etc., gelatin, polyvinylpyrrolidone, or the like.
  • a disintegrating agent such as crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate, etc. may be added, but is not limited thereto.
  • the pharmaceutical composition may additionally include an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, an antiseptic, or the like, but is not limited thereto.
  • the pharmaceutical composition of the present invention may be used for prevention or treatment of neurodegenerative diseases in animals such as mammals, especially in humans.
  • the administration route of the pharmaceutical composition of the present invention may include oral and parenteral routes, but is not limited thereto.
  • Examples of the parenteral administration route may include transdermal, intranasal, intraabdominal, intramuscular, subcutaneous and intravenous routes, etc., but is not limited thereto.
  • the pharmaceutical composition of the present invention may be formulated into an injection, a transdermal system, a suppository, an aerosol or a nasal inhaler together with an adequate carrier for parenteral administration according to a method known in the art.
  • the injection should be sterilized and protected from contamination by microorganisms such as bacteria and fungi.
  • examples of an adequate carrier may include, but are not limited to, a solvent or a suspension medium such as water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), a mixture thereof and/or vegetable oil.
  • the carrier may be Hank's balanced salt solution, Ringer's solution, phosphate buffered saline (PBS) or sterile water for injection containing triethanolamine, or isotonic solution such as 10% ethanol, 40% propylene glycol and 5% dextrose.
  • the injection may further include various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. for protection from contamination by microorganisms.
  • the injection may include an isotonic agent such as sugar or sodium chloride in most cases.
  • the pharmaceutical composition may be orally administered to a subject.
  • the form of oral administration may be selected from powder, pulvis, granule, tablet, capsule, mouth-dispersible tablet, sugar-coated tablet, aerosol, gel, pill, soft capsule, suspension, emulsion, aqueous medicine, syrup, elixir, wafer and sachet.
  • radotinib may be conveniently delivered in the form of an aerosol spray delivered from a pressurized pack or a nebulizer by the use of an adequate propellant compound such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other appropriate gases.
  • an adequate propellant compound such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other appropriate gases.
  • a unit dosage may be determined with a valve which delivers a weighed amount.
  • a capsule or a cartridge used in the inhaler or insufflator may be formulated to include a powder mixture of proper powder matrix.
  • the pharmaceutical composition of the present invention comprises radotinib or a pharmaceutically acceptable salt thereof as an active ingredient, and thus it shows an effect of prevention or treatment of neurodegenerative diseases such as Parkinson's disease.
  • radotinib has a superior characteristic of blood-brain barrier penetration, which is a very important factor of a medicament for neurodegenerative disease.
  • Figure 1A shows a schematic diagram of the schedule of the induction of a Parkinson's disease model by injecting ⁇ -synuclein preformed fibrils (PFFs) and the treatment of radotinib.
  • Figure 1B is a graph showing the effect of radotinib for enhancing the cell survival rate of neuronal cells analyzed by using Alamar Blue assay.
  • Figure 2 represents a graph showing an effect of radotinib for reducing neuronal cell death rate, which is determined by using lactate dehydrogenase (LDH) assay in ⁇ -synuclein PFFs-induced neuronal cell model.
  • LDH lactate dehydrogenase
  • Figure 3 represents the staining images and a graph showing the effect of radotinib for reducing neuronal cell death rate analyzed by using TUNEL staining in ⁇ -synuclein PFFs-induced neuronal cell model.
  • Figure 4 represents the staining images and a graph showing the effect of radotinib for reducing Lewy Body (LB)/Lewy Neurites (LN)-like pathology observed in a neuronal cell model by ⁇ -synuclein PFFs induction.
  • Figure 5 represents the results of western blot assay showing the effect of radotinib for inhibiting c-Abl activity in ⁇ -synuclein PFFs-induced neuronal cell model.
  • Figure 6 represents the staining images and a graph showing that radotinib treatment reduced accumulation of A53T ⁇ -synuclein in HEK293T having A53T ⁇ -synuclein point mutation.
  • Figure 7 represents graphs showing the inhibitory effect of radotinib on ⁇ -synuclein formation in blood and brain in A53T transgenic mice.
  • Figure 8 represents graphs showing the effect of radotinib for inhibition of ⁇ -synuclein formation, increase of dopamine production and recovery of motor ability in brain ⁇ -synuclein-administered disease model.
  • Figure 9 represents a graph showing that after radotinib administration, ⁇ -synuclein migrated to the autophagic compartment containing ⁇ -synuclein degrading enzyme, in brain ⁇ -synuclein-administered disease model.
  • Figure 10 represents graphs showing that after radotinib administration, ⁇ -synuclein and Parkin which relates to autophagic clearance migrated to the autophagic compartment containing ⁇ -synuclein degrading enzyme, in A53T transgenic mice.
  • Figure 11 represents graphs showing the effect of radotinib for increasing Parkin activity in human M17 neuroblastoma cells and brain ⁇ -synuclein-administered disease model.
  • Figure 12 represents graphs showing the effect of radotinib for inhibiting caspase-3 activity which relates to apoptosis, in A53T transgenic mice and brain ⁇ -synuclein-administered disease model.
  • Example 1 the effect of radotinib for enhancing survival rate of neuronal cells in ⁇ -synuclein PFFs-induced neuronal cell model was examined.
  • Example 2 the effect of radotinib for reducing neuronal cell death in ⁇ -synuclein PFFs-induced neuronal cell model was examined by using lactate dehydrogenase (LDH) assay.
  • LDH lactate dehydrogenase
  • the cells were treated with ⁇ -synuclein PFFs to induce Parkinson's disease model.
  • cells were treated with 1 ⁇ M of radotinib and DMSO as a control, and the radotinib treatment group was further treated with radotinib every other day while culturing for 14 days.
  • LDH assay was performed for determining cell death rate by measuring LDH level secreted from damaged cells.
  • neuronal cell death rate was increased (27%) by treatment of ⁇ -synuclein PFFs, but it was decreased (17%) by treatment of radotinib ( Figure 2).
  • Example 3 the effect of radotinib for reducing neuronal cell death in ⁇ -synuclein PFFs-induced neuronal cell model was examined by using TUNEL staining.
  • the cells were treated with ⁇ -synuclein PFFs to induce Parkinson's disease model.
  • cells were treated with 1 ⁇ M of radotinib and DMSO as a control, and the radotinib treatment group was further treated with radotinib every other day while culturing for 14 days.
  • FIG. 3A shows TUNEL-positive neuronal cells.
  • Figure 3B is a quantitative result of the number of TUNEL-positive neuronal cells.
  • the number of TUNEL-positive neuronal cells was increased (52%) by treatment of ⁇ -synuclein PFFs, but it was decreased (36%) in radotinib treatment group. That is, radotinib suppressed neuronal cell death induced by ⁇ -synuclein PFFs.
  • Example 4 the effect of radotinib for reducing LB/LN-like pathology in ⁇ -synuclein PFFs-induced neuronal cell model was examined.
  • ⁇ -synuclein a major Parkinson's disease-inducing factor
  • LB Lewy Body
  • LN Lewy Neurites
  • the cells were treated with ⁇ -synuclein PFFs and cultured for 10 days.
  • the cells were immunostained by pSer129, an anti- ⁇ -synuclein antibody, to detect LB/LN-like pathology.
  • Figure 4A is the result of pSer129 immunostaining of accumulated ⁇ -synuclein.
  • Figure 4B is the result of quantifying ⁇ -synuclein signal.
  • ⁇ -synuclein was accumulated to form LB/LN-like pathology in the control group.
  • LB/LN-like pathology was reduced by about 50%. That is, ⁇ -synuclein PFFs-induced LB/LN-like pathology was reduced by radotinib.
  • Example 5 the effect of radotinib for inhibiting c-Abl activity in ⁇ -synuclein PFFs-induced neuronal cell model was examined.
  • This experiment was performed to confirm whether radotinib inhibits the increase of c-Abl kinase activity, one of the features found in patients with Parkinson's disease.
  • the cells were treated with ⁇ -synuclein PFFs at 7 DIV.
  • c-Abl activity was measured at 14 DIV by western blot with cell lysate.
  • Figure 5A is the result of western blot showing the amount of p-Tyr245 c-Abl protein.
  • Figure 5B is the quantitative graph thereof.
  • c-Abl activity was increased in ⁇ -synuclein PFFs-induced models (1.4), and radotinib significantly inhibited the increased c-Abl activity (0.8).
  • Example 6 the effect of radotinib for reducing A53T ⁇ -synuclein accumulation in HEK293T cell model was examined.
  • the cells were transfected with myc-tagged A53T ⁇ -synuclein plasmid.
  • the transfected cells were treated with 1 ⁇ M of radotinib, and myc-A53T ⁇ -synuclein accumulation was observed by ⁇ -synuclein immunostaining in comparison with the control group which was treated with PBS.
  • Figure 6A represents images showing immunostaining of HEK293T cells overexpressing myc-tagged A53T ⁇ -synuclein. Arrows indicate accumulated A53T ⁇ -synuclein.
  • Figure 6B is a quantitative result of accumulated A53T ⁇ -synuclein by using image J software (NIH, Bethesda). In myc-A53T ⁇ -synuclein overexpressing model, accumulation of myc-A53T ⁇ -synuclein was observed (36). Radotinib reduced the accumulation of myc-A53T ⁇ -synuclein (7). That is, accumulation of ⁇ -synuclein was reduced by radotinib in a disease model induced by A53T point mutation. Values are represented as mean ⁇ SEM (P*** ⁇ 0.001).
  • Example 7 an in vivo test was conducted to compare the inhibitory effect of radotinib and nilotinib on the formation of ⁇ -synuclein.
  • Nilotinib (4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]benzamide) is a selective Bcr-Abl kinase inhibitor of the tyrosine kinase inhibitors family, similar to radotinib. Nilotinib has been used as a therapeutic agent for treatment of Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia. It has been reported that nilotinib showed neuroprotective effects in genetically modified or toxin-induced Parkinson's disease animal models.
  • radotinib of the present invention is a selective Bcr-Abl kinase inhibitor similar to nilotinib, radotinib has safer and better pharmacokinetic properties than nilotinib and thus it has improved effect of prevention and treatment of neurodegenerative disease.
  • A53T mutation is characterized by an increase of ⁇ -synuclein polymers. Based on this character, 7- to 8-month-old male A53T transgenic mice were orally administered 3 mg/kg or 10 mg/kg of radotinib or nilotinib, respectively, for 6 weeks, once a day. Blood and brain were then isolated therefrom, and the amount of ⁇ -synuclein therein was measured by ELISA (Enzyme-Linked ImmunoSorbent Assay).
  • Example 8 comparative tests of inhibition of ⁇ -synuclein formation, dopamine production and motor ability were conducted using radotinib and nilotinib in a brain ⁇ -synuclein-administered disease model.
  • Lentiviral ⁇ -synuclein was stereotactically administered bilaterally into the substantia nigra in the brain of male C58BL/6 mice for 3 weeks. Subsequently, 10 mg/kg of radotinib or nilotinib was orally administered thereto once a day for 3 weeks, and the amounts of ⁇ -synuclein and dopamine in the brain and blood were determined by ELISA.
  • the change of motor ability was tested by rotarod test and pole test. The rotarod test measures how long the mouse stays on a rotating rod, and the pole test measures how long it takes the mouse to move from the top of a fixed rod to the bottom. Therefore, if the value of the rotarod test is higher and the value of the pole test is lower, it can be determined that the motor ability is better.
  • radotinib's effect is superior to that of nilotinib on the inhibition of ⁇ -synuclein formation, increase of dopamine production and improvement of motor ability in the brain ⁇ -synuclein-administered disease model.
  • Example 9 a comparative test of the degree of promoting autophagy clearance of ⁇ -synuclein according to administration of radotinib and nilotinib was conducted.
  • Lentiviral ⁇ -synuclein was stereotactically administered bilaterally into the substantia nigra in the brain of male C58BL/6 mice for 3 weeks. Subsequently, 10 mg/kg of radotinib or nilotinib was orally administered thereto once a day for 3 weeks, and the substantia nigra was isolated from the mice and AV (Autophagic Vacuole) was separated therefrom. The amounts of ⁇ -synuclein in AV-10 (Phagophores+Autophagosomes), AV-20 (Autophagosomes) and lysosome in AV was determined by ELISA.
  • Example 10 a comparative test of the degree of promoting autophagy clearance of accumulated ⁇ -synuclein and Parkin according to administration of radotinib and nilotinib was conducted.
  • A53T mice which are characterized by increase of ⁇ -synuclein oligomerization, were orally administered with 10 mg/kg of radotinib or nilotinib once a day for 3 weeks.
  • the substantia nigra was separated and Autophagic Vacuole (AV) was isolated therefrom.
  • the levels of ⁇ -synuclein and Parkin in AV-10 (Phagophores+Autophagosomes), AV-20 (Autophagosomes) and lysosome were determined by ELISA.
  • Example 11 the increase of Parkin activity (a major factor for ⁇ -synuclein removal) was tested using radotinib and nilotinib, and the result was compared.
  • Parkin promotes protein degradation through ubiquitination. Therefore, the activity of Parkin plays a major role in the removal of ⁇ -synuclein. To evaluate the effect of radotinib administration on the activity of Parkin, the following test was conducted.
  • human M17 neuroblastoma cells were treated with 10 ⁇ M of radotinib or nilotinib, and E3 activity of Parkin was determined by ELISA.
  • lentiviral ⁇ -synuclein was stereotactically administered bilaterally into the substantia nigra in the brain of male C58BL/6 mice for 3 weeks.
  • 10 mg/kg of radotinib or nilotinib was orally administered once a day for 3 weeks, and the brains were isolated from the mice to evaluate the brain Parkin level by ELISA.
  • the activity of E3 (indicating the activity of ubiquitin) was significantly increased in M17 cells treated with radotinib or nilotinib, respectively.
  • the radotinib treatment group showed 2.7 times higher activity than the nilotinib treatment group.
  • the Parkin level was improved in ⁇ -synuclein-treated mice according to the administration of radotinib or nilotinib when compared to the control group.
  • the radotinib treatment group showed 2.6 times higher Parkin level than the nilotinib treatment group.
  • Example 12 the effect of radotinib for inhibiting caspase-3 (a protein involved in apoptosis) activity was tested compared to nilotinib.
  • caspase-3 a protein involved in apoptosis, tends to increase in Parkinson's disease model.
  • radotinib administration inhibits caspase-3 activity, the following test was conducted.
  • a disease model was produced by stereotactical bilateral administration of lentiviral ⁇ -synuclein into the substantia nigra in the brain of male C58BL/6 mice for 3 weeks. 10 mg/kg of radotinib or nilotinib was orally administered once a day for 3 weeks into the disease model and A53T transgenic mice (7- to 8-months old) characterized by an increase of ⁇ -synuclein polymers, and the changes of caspase-3 level were measured.
  • Example 13 penetration of radotinib through the blood-brain barrier was tested in mice compared to nilotinib.
  • Radotinib and nilotinib were orally administered at a dose of 50 mg/kg into 6-week-old male ICR mice. Before and 1, 2, 3, 4, 5, 7 and 12 hours after administration, plasma was isolated from blood samples and the brain was extracted, and the concentrations of radotinib and nilotinib were analyzed by LC-MS/MS.
  • radotinib has a blood-brain barrier penetrability that is three times higher than that of nilotinib.
  • the blood-brain barrier penetrability is one of the most important factors in the development of a medicament for treatment of brain diseases, and it is expected that radotinib is more effective for treatment of neurodegenerative diseases.
  • radotinib has effects of reducing ⁇ -synuclein PFFs-induced neurotoxicity in neuronal cells, reducing ⁇ -synuclein PFFs-induced LB/LN-like pathology, reducing accumulation of A53T ⁇ -synuclein and inhibiting ⁇ -synuclein PFFs-induced c-Abl activity.
  • radotinib inhibits ⁇ -synuclein formation in blood and the brain.
  • ⁇ -synuclein level was reduced by 65-73% in the radotinib treatment group compared to the nilotinib treatment group, and the radotinib treatment group showed about 2.4 times higher effect than the nilotinib treatment group in restoration of dopamine secretion and motor ability.
  • Radotinib showed an almost 2-fold higher ethological effect than nilotinib which has a similar effect in the treatment of CML. This is possible because, when the two drugs were administered at the same dose (50 mg/kg), the distribution of radotinib in the brain tissue was about 3 times higher than nilotinib due to high blood-brain barrier penetration rate although the plasma absorption of radotinib was slightly lower than nilotinib.

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Abstract

The present invention relates to a pharmaceutical composition for prevention or treatment of neurodegenerative diseases comprising an effective amount of radotinib or its pharmaceutically acceptable salts.

Description

PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF NEURODEGENERATIVE DISEASES
The present invention relates to a pharmaceutical composition for prevention or treatment of neurodegenerative diseases comprising an effective amount of radotinib or its pharmaceutically acceptable salts.
Neurodegenerative disease refers to a disease in which neuronal cells are destroyed for some reason and thereby causing abnormality of brain function. Typical neurodegenerative diseases are, for example, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.
Parkinson's disease is one of the most common neurodegenerative diseases. It is a progressive disease among the degenerative nervous diseases. The average onset age is around 60―that is, Parkinson's disease is found mainly in older people. It is known that more than 1% of people older than 60 suffer from Parkinson's disease. In Korea, an aging society, patients with Parkinson's disease have been increasing steadily. The male-to-female ratio is 3:2; it is slightly higher in men. About 60,000-100,000 people in Korea, about 1.5 million people in the United States, and about 10 million people worldwide suffer from Parkinson's disease. In the case of the United States, the prevalence of Parkinson's disease is expected to more than double by 2040. According to epidemiological studies, in China, about 5 million patients with Parkinson's disease are expected in 2030.
Parkinson's disease is a progressive neurodegenerative disease characterized by Parkinsonian symptoms such as slow motion, static tremor, muscle rigidity, walking with dragging feet, and bending posture. Such symptoms are caused due to imperfect dopamine production and action in the substantia nigra and subsequent decrease of motor cortex stimulation. Chronic and progressive, serious hypermetamorphosis and mild language impairment often appear. The incidence rate is about 1 out of 1,000, but it increases with age.
Parkinson's disease occurs in about one out of 100 in people older than 50. In the United States, incidence rate in higher in white people than black people. The immediate cause of Parkinson's disease is the slow death of neurons in the substantia nigra and corpus amygdaloideum. Most studies have focused on neurons in the substantia nigra which send dopaminergic axons to the caudate nucleotoxin and the cornu cutaneum. These axons excite D1 receptors and inhibit D2 receptors. In the case of Parkinson's disease, inhibitory output from the globus pallidus to the thalamus increases due to loss of dopamine in both receptors. Researchers presume that normal people lose a little over 1% of neurons in the substantia nigra every year, starting from about age 45. While most human beings have more neurons than they need, some start with fewer neurons and some lose neurons at a faster rate. When the number of viable neurons in the substantia nigra decreases to 20-30% of that of normal people, symptoms of Parkinson's disease begin to appear. The more cell loss, the more severe the symptoms.
Parkinson's disease and dopamine are closely related, but dopamine is not only associated with Parkinson's disease. Dopamine, one of the neurotransmitters that transmit intercellular signals, is produced from neuronal cells in the brain. If the mutual transmission of the neurotransmitters is abnormally suppressed or overly increased, it will lead to abnormalities in the whole function of nervous system. There are neuronal cells in the brain that broadly use dopamine in the frontal lobe which relates to thinking, in the limbic system which relates to emotion, and in the pituitary gland which relates to hormones.
Since James Parkinson's first description of Parkinson's disease, there was no effectual treatment until the 1960s. In the 1950s, it was observed that injection of reserpine, which causes depletion of dopamine, into a white rat leads to symptoms of Parkinson's disease. Autopsies of the patients revealed that the nervous cells in the substantia nigra had disappeared. The dopamine described above is used as a neurotransmitter that transmits intercellular signals. When dopamine is injected directly into the body, it cannot reach the neuronal cells in the brain. This is because of the blood-brain barrier between blood and brain tissue, which allows the passage of only the substances necessary for the brain. For this reason, dopamine itself cannot pass through the barrier.
The main symptoms of most cases of Parkinson's disease occur due to depletion of dopamine in the corpus striatum. Therefore, supplement of dopamine is the main way of treating Parkinson's disease, and the most powerful drug is levodopa.
Levodopa has chemical similarity with dopamine, and is actually a precursor of dopamine. Levodopa can pass through the blood-brain barrier, and once it has passed, it is metabolized to dopamine in the brain and eventually becomes available in neuronal cells.
There are dopamine agonist medicines such as ropinirole and pramipexole that act as levodopa which is converted into dopamine in the brain. These drugs are not dopamine, but are designed to respond to neurotransmission like dopamine. They are used either alone or as an adjuvant to levodopa for patients with early Parkinson's disease. The anti-Parkinson effect of dopamine is caused by acting on the D2 receptor. The effect of the D1 or D3 receptor on the symptoms of Parkinson's disease is not yet known. Dopamine agonists may cause confusion or hallucination in elderly patients, and leg edema in some patients.
Differently from levodopa and dopamine agonists which supersede the role of dopamine, some drugs are used to prevent gradual dying of dopaminergic neuronal cells. Type B monoamine oxidase inhibitor (MAO-B) is the typical drug. MAO-B inhibitor has a neuroprotective effect that the other drugs do not have. Unlike the other drugs, MAO-B inhibitors can be used for patients with early-stage to late-stage Parkinson's disease.
Trihexyphenidyl, benztropine mesylate, biperiden and diphenhydramine as anticholinergics help control tremors or contractures, but they are less effective than dopaminergic drugs.
Amantadine has several mechanisms of action. It promotes dopamine secretion at the nerve endings, and also blocks the reuptake of dopamine, the antimuscarinic effect and the glutamate receptor at the nerve endings. Although amantadine can help control symptoms by dopaminergic action, it has side effects such as livedo reticularis, ankle edema, hallucinations, confusion, and do on. Antiglutamatergic action is helpful in the control of dyskinesia caused by levodopa.
Surgical treatment of Parkinson's disease is performed on patients who are not adequately treated with medication and their life is affected thereby. "Parkinson's disease is not adequately treated" does not mean that there is no response to levodopa or dopamine agonist but it does mean that the drug cannot be administered in a sufficient dose because of disruption of normal activity due to changes in movement and dyskinesia, or other kinds of side effects.
However, most of all, the appropriate drugs should be applied in consideration of patients' motile and nonmotile symptoms rather than uniform treatment, because patients with Parkinson's disease are not all the same. However, despite many achievements and therapeutic advances, Parkinson's disease is still impossible to cure. The mortality rate from Parkinson's disease is increasing every year not only in Korea but throughout the world. There is much interest in the development of drugs for treatment of Parkinson's disease.
As a potential therapeutic agent for Parkinson's disease, treatment effect on Parkinson's disease has been reported in selective Bcr-Abl kinase inhibitors which are used as medicine for leukemia. For example, Chinese Patent Publication No. CN 102406648 A discloses the use of imatinib mesylate for degenerative nervous diseases. It has been further reported that medicines for leukemia such as ponatinib have the effect associated with the treatment of Parkinson's disease.
However, these drugs have failed to be developed as a therapeutic agent for Parkinson's disease because they could not pass through the blood-brain barrier in in vivo experiments. Therefore, there is a need for the development of a novel therapeutic agent capable of efficiently treating Parkinson's disease.
The present invention is intended to provide a novel approach for treatment of neurodegenerative diseases including Parkinson's disease for which a full recovery method has yet to be found.
In order to resolve the above technical problem, the present invention provides a pharmaceutical composition for prevention or treatment of neurodegenerative diseases, comprising radotinib as an active ingredient which is a selective Bcr-Abl kinase inhibitor and is used as a medicament for treatment of Philadelphia　chromosome-positive (Ph+)　chronic　myelogenous　leukemia　(CML).
Hereinafter, the present invention is explained in more detail.
According to an aspect of the present invention, the present invention can provide a pharmaceutical composition for prevention or treatment of neurodegenerative diseases comprising radotinib represented by Chemical Formula (I) below or pharmaceutically acceptable salt thereof as an active ingredient.
Figure PCTKR2017008229-appb-I000001
Radotinib is one of the receptor tyrosine kinase inhibitors, and is also represented by 4-methyl-N-[3-(4-methylimidazol-1-yl)-5-trifluoromethyl-phenyl]-3-[4-(3-methylpyrazin-2-yl)-pyrimidin-2-ylamino)benzamide.
It has been known that radotinib inhibits one or more tyrosine kinases―for example, c-Abl, Bcr-Abl and the receptor tyrosine kinases such as PDGFR, Flt3, VEGF-R, EGF-R and c-Kit. In addition, it has been known that radotinib has excellent anticancer effects on various cancers such as lung cancer, gastric cancer, colon cancer, pancreatic cancer, hepatoma, prostatic cancer, breast cancer, chronic or acute leukemia, hematologic malignancy, brain tumor, bladder cancer, rectal cancer, cervical cancer and lymphoma. However, there is no report about the effect of radotinib on neurodegenerative diseases comprising Parkinson's disease.
Radotinib may be used as itself or in the form of a pharmaceutically acceptable salt. Herein, the term "pharmaceutically acceptable" means that the salt is physiologically acceptable and normally causes no allergic or other similar adverse reactions when administered to a human. The salt may be, but is not limited thereto, an acid addition salt formed from a pharmaceutically acceptable free acid. The acid addition salt may be formed from radotinib and a pharmaceutically acceptable inorganic or organic acid which is selected from, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, acetic acid, propionic acid, ascorbic acid, citric acid, malonic acid, fumaric acid, maleic acid, lactic acid, salicylic acid, sulfamic acid and tartaric acid. These salts may be prepared by a method known in the art―for example, by treating radotinib with an appropriate acid in the presence of an appropriate solvent.
Radotinib may be in the form of a crystalline or a solvate (e.g., hydrate), and both forms are within the scope of the present invention. The term "solvate" refers to complexes in various stoichiometry formed by a solute (in the present invention, a compound such as radotinib) and a solvent. Such solvents should not impede the biological activity of the solute. For example, the solvent may be, but is not limited thereto, water, ethanol, methanol, ethyl acetate or acetone. The method for solvation is generally known in the art.
A pharmaceutically acceptable salt of radotinib may include both solvated form and non-solvated form.
The pharmaceutical composition according to the present invention may comprise a pharmaceutically effective amount of radotinib. Herein, the term "pharmaceutically effective amount" refers to an amount required to show the desired effect as compared with a negative control group, and preferably it refers to an amount sufficient for the prevention or treatment of neurodegenerative diseases including Parkinson's disease in mammals, including humans. In the case that the patient is a human, the therapeutic dose may be 50 mg to 2,000 mg/day, and more preferably 100 mg to 1,000 mg/day, depending on the seriousness of the status and whether radotinib is administered alone or in combination with another drug(s). For example, the pharmaceutical composition may be administered via oral or parenteral route once a day or dividedly. However, the pharmaceutically effective amount may be properly changed depending on the particular disease and severity thereof, age, body weight, physical conditions and sex of patients, administration route, treatment period, or other various factors.
For example, the neurodegenerative disease may be Parkinson's disease.
In an embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. For example, the carrier may be inert, and may be selected from, but is not limited thereto, fillers such as sugar including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, etc., starch including corn starch, wheat starch, rice starch, potato starch, etc., celluloses including cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, etc., gelatin, polyvinylpyrrolidone, or the like. In addition, a disintegrating agent such as crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate, etc. may be added, but is not limited thereto.
For example, the pharmaceutical composition may additionally include an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, an antiseptic, or the like, but is not limited thereto.
The pharmaceutical composition of the present invention may be used for prevention or treatment of neurodegenerative diseases in animals such as mammals, especially in humans.
The administration route of the pharmaceutical composition of the present invention may include oral and parenteral routes, but is not limited thereto. Examples of the parenteral administration route may include transdermal, intranasal, intraabdominal, intramuscular, subcutaneous and intravenous routes, etc., but is not limited thereto.
For example, in case of parenteral administration, the pharmaceutical composition of the present invention may be formulated into an injection, a transdermal system, a suppository, an aerosol or a nasal inhaler together with an adequate carrier for parenteral administration according to a method known in the art. The injection should be sterilized and protected from contamination by microorganisms such as bacteria and fungi. In the case of injection, examples of an adequate carrier may include, but are not limited to, a solvent or a suspension medium such as water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), a mixture thereof and/or vegetable oil. More preferably, the carrier may be Hank's balanced salt solution, Ringer's solution, phosphate buffered saline (PBS) or sterile water for injection containing triethanolamine, or isotonic solution such as 10% ethanol, 40% propylene glycol and 5% dextrose. The injection may further include various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. for protection from contamination by microorganisms. In addition, the injection may include an isotonic agent such as sugar or sodium chloride in most cases. These formulations are described in the publication (Remington's Pharmaceutical Sciences, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania), which is well known in the pharmaceutical chemistry field.
In an embodiment of the present invention, the pharmaceutical composition may be orally administered to a subject.
In an embodiment of the present invention, the form of oral administration may be selected from powder, pulvis, granule, tablet, capsule, mouth-dispersible tablet, sugar-coated tablet, aerosol, gel, pill, soft capsule, suspension, emulsion, aqueous medicine, syrup, elixir, wafer and sachet.
In case of an inhaler, radotinib may be conveniently delivered in the form of an aerosol spray delivered from a pressurized pack or a nebulizer by the use of an adequate propellant compound such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other appropriate gases. In case of a pressurized aerosol, a unit dosage may be determined with a valve which delivers a weighed amount. For example, a capsule or a cartridge used in the inhaler or insufflator may be formulated to include a powder mixture of proper powder matrix.
Other pharmaceutically acceptable carriers may refer to the following publication (Remington's Pharmaceutical Sciences, 19th Edition, 1995, Mack Publishing Company, Easton, Pennsylvania).
The pharmaceutical composition of the present invention comprises radotinib or a pharmaceutically acceptable salt thereof as an active ingredient, and thus it shows an effect of prevention or treatment of neurodegenerative diseases such as Parkinson's disease. Especially, radotinib has a superior characteristic of blood-brain barrier penetration, which is a very important factor of a medicament for neurodegenerative disease.
Figure 1A shows a schematic diagram of the schedule of the induction of a Parkinson's disease model by injecting α-synuclein preformed fibrils (PFFs) and the treatment of radotinib. Figure 1B is a graph showing the effect of radotinib for enhancing the cell survival rate of neuronal cells analyzed by using Alamar Blue assay.
Figure 2 represents a graph showing an effect of radotinib for reducing neuronal cell death rate, which is determined by using lactate dehydrogenase　(LDH)　assay in α-synuclein PFFs-induced neuronal cell model.
Figure 3 represents the staining images and a graph showing the effect of radotinib for reducing neuronal cell death rate analyzed by using TUNEL staining in α-synuclein PFFs-induced neuronal cell model.
Figure 4 represents the staining images and a graph showing the effect of radotinib for reducing Lewy Body (LB)/Lewy Neurites (LN)-like pathology observed in a neuronal cell model by α-synuclein PFFs induction.
Figure 5 represents the results of western blot assay showing the effect of radotinib for inhibiting c-Abl activity in α-synuclein PFFs-induced neuronal cell model.
Figure 6 represents the staining images and a graph showing that radotinib treatment reduced accumulation of A53T α-synuclein in HEK293T having A53T α-synuclein point mutation.
Figure 7 represents graphs showing the inhibitory effect of radotinib on α-synuclein formation in blood and brain in A53T transgenic mice.
Figure 8 represents graphs showing the effect of radotinib for inhibition of α-synuclein formation, increase of dopamine production and recovery of motor ability in brain α-synuclein-administered disease model.
Figure 9 represents a graph showing that after radotinib administration, α-synuclein migrated to the autophagic compartment containing α-synuclein degrading enzyme, in brain α-synuclein-administered disease model.
Figure 10 represents graphs showing that after radotinib administration, α-synuclein and Parkin which relates to autophagic clearance migrated to the autophagic compartment containing α-synuclein degrading enzyme, in A53T transgenic mice.
Figure 11 represents graphs showing the effect of radotinib for increasing Parkin activity in human M17 neuroblastoma cells and brain α-synuclein-administered disease model.
Figure 12 represents graphs showing the effect of radotinib for inhibiting caspase-3 activity which relates to apoptosis, in A53T transgenic mice and brain α-synuclein-administered disease model.
Hereinafter, the present invention is explained in more detail with the following examples. However, the following examples are only intended to facilitate understanding of the present invention, and the protection scope of the present invention is not limited thereto.
Example 1
In Example 1, the effect of radotinib for enhancing survival rate of neuronal cells in α-synuclein PFFs-induced neuronal cell model was examined.
1) Cell line
Primary cortical neurons separated from 15.5 dpc (day post coitum) C57BL/6 mice
2) Methods
A) After a 7-day culture of primary cortical neurons separated from 15.5 dpc C57BL/6 mice, the cells were treated with α-synuclein PFFs at 7 DIV (days in vitro) to induce Parkinson's disease model. At this time, cells were treated with 1 μM of radotinib and DMSO as a control, and the radotinib treatment group was further treated with radotinib every other day while culturing for 14 days (Figure 1A).
B) On the 21st day of culture (21 DIV), Alamar Blue assay was performed to determine cell viability. As a result, cell viability was reduced by about 43% due to α-synuclein PFFs treatment, but radotinib was able to restore 31% of cell viability (Figure 1B). Values are represented as mean ± SEM (one-way ANOVA, Tukey's multiple comparison test, P* < 0.05, P** < 0.01, n = 7-10).
That is, this experiment confirmed that radotinib restores the decrease of cell viability of neuronal cells induced by α-synuclein PFFs.
Example 2
In Example 2, the effect of radotinib for reducing neuronal cell death in α-synuclein PFFs-induced neuronal cell model was examined by using lactate dehydrogenase　(LDH)　assay.
1) Cell line
Primary cortical neurons separated from 15.5 dpc C57BL/6 mice
2) Methods
After a 7-day culture (7 DIV) of primary cortical neurons, the cells were treated with α-synuclein PFFs to induce Parkinson's disease model. At this time, cells were treated with 1 μM of radotinib and DMSO as a control, and the radotinib treatment group was further treated with radotinib every other day while culturing for 14 days.
At 21 DIV, LDH assay was performed for determining cell death rate by measuring LDH level secreted from damaged cells. As a result, neuronal cell death rate was increased (27%) by treatment of α-synuclein PFFs, but it was decreased (17%) by treatment of radotinib (Figure 2). Values are represented as mean ± SEM (one-way ANOVA, Tukey's multiple comparison test, P** < 0.01, P*** < 0.001, n = 4).
That is, through this experiment using LDH assay, it was confirmed that radotinib suppresses neuronal cell death induced by α-synuclein PFFs.
Example 3
In Example 3, the effect of radotinib for reducing neuronal cell death in α-synuclein PFFs-induced neuronal cell model was examined by using TUNEL staining.
1) Cell line
Primary cortical neurons separated from 15.5 dpc C57BL/6 mice
2) Methods
After a 7-day culture (7 DIV) of primary cortical neurons, the cells were treated with α-synuclein PFFs to induce Parkinson's disease model. At this time, cells were treated with 1 μM of radotinib and DMSO as a control, and the radotinib treatment group was further treated with radotinib every other day while culturing for 14 days.
At 21 DIV, neuronal cell death was observed by TUNEL staining. A) Figure 3A shows TUNEL-positive neuronal cells. B) Figure 3B is a quantitative result of the number of TUNEL-positive neuronal cells. As a result, the number of TUNEL-positive neuronal cells was increased (52%) by treatment of α-synuclein PFFs, but it was decreased (36%) in radotinib treatment group. That is, radotinib suppressed neuronal cell death induced by α-synuclein PFFs. Values are represented as mean ± SEM (one-way ANOVA, Tukey's multiple comparison test, P* < 0.05, P** < 0.01, n = 3-5).
That is, through this experiment using TUNEL staining, it was confirmed that radotinib suppresses neuronal cell death induced by α-synuclein PFFs.
Example 4
In Example 4, the effect of radotinib for reducing LB/LN-like pathology in α-synuclein PFFs-induced neuronal cell model was examined.
1) Cell line
Primary cortical neurons separated from 15.5 dpc C57BL/6 mice
2) Methods
When primary cortical neurons are cultured for a certain time after α-synuclein PFFs treatment, α-synuclein, a major Parkinson's disease-inducing factor, accumulates in neurons and a disease model is prepared. This accumulation of α-synuclein is also known as Lewy Body (LB)/Lewy Neurites (LN)-like pathology, and is one of the main features of Parkinson's disease.
At 7 DIV, the cells were treated with α-synuclein PFFs and cultured for 10 days. At 17 DIV, the cells were immunostained by pSer129, an anti-α-synuclein antibody, to detect LB/LN-like pathology.
A) Figure 4A is the result of pSer129 immunostaining of accumulated α-synuclein. B) Figure 4B is the result of quantifying α-synuclein signal. After treatment of α-synuclein PFFs, α-synuclein was accumulated to form LB/LN-like pathology in the control group. On the other hand, in the radotinib treatment group, LB/LN-like pathology was reduced by about 50%. That is, α-synuclein PFFs-induced LB/LN-like pathology was reduced by radotinib. Values are represented as mean ± SEM one-way ANOVA, Tukey's multiple comparison test, P*** < 0.001, P**** < 0.0001, n = 4).
That is, through this experiment, it was confirmed that α-synuclein PFFs-induced LB/LN-like pathology is reduced by radotinib.
Example 5
In Example 5, the effect of radotinib for inhibiting c-Abl activity in α-synuclein PFFs-induced neuronal cell model was examined.
1) Cell line
Primary cortical neurons separated from 15.5 dpc C57BL/6 mice
2) Methods
This experiment was performed to confirm whether radotinib inhibits the increase of c-Abl kinase activity, one of the features found in patients with Parkinson's disease. After culturing primary cortical neurons, the cells were treated with α-synuclein PFFs at 7 DIV. After culturing for 7 days, c-Abl activity was measured at 14 DIV by western blot with cell lysate.
A) Figure 5A is the result of western blot showing the amount of p-Tyr245 c-Abl protein. B) Figure 5B is the quantitative graph thereof. As a result, c-Abl activity was increased in α-synuclein PFFs-induced models (1.4), and radotinib significantly inhibited the increased c-Abl activity (0.8). Values are represented as mean ± SEM (one-way ANOVA, Tukey's multiple comparison test, P*** < 0.001, P**** < 0.0001, n = 3).
That is, through this experiment, it was confirmed that c-Abl kinase activity which was increased by PFF is decreased by radotinib.
Example 6
In Example 6, the effect of radotinib for reducing A53T α-synuclein accumulation in HEK293T cell model was examined.
1) Cell line
Wild-type HEK293T cells
2) Methods
For inducing accumulation of A53Y α-synuclein in wild-type HEK293T cells, the cells were transfected with myc-tagged A53T α-synuclein plasmid. The transfected cells were treated with 1 μM of radotinib, and myc-A53T α-synuclein accumulation was observed by α-synuclein immunostaining in comparison with the control group which was treated with PBS.
A) Figure 6A represents images showing immunostaining of HEK293T cells overexpressing myc-tagged A53T α-synuclein. Arrows indicate accumulated A53T α-synuclein. B) Figure 6B is a quantitative result of accumulated A53T α-synuclein by using image J software (NIH, Bethesda). In myc-A53T α-synuclein overexpressing model, accumulation of myc-A53T α-synuclein was observed (36). Radotinib reduced the accumulation of myc-A53T α-synuclein (7). That is, accumulation of α-synuclein was reduced by radotinib in a disease model induced by A53T point mutation. Values are represented as mean ± SEM (P*** < 0.001).
That is, through this experiment, it was confirmed that accumulation of α-synuclein induced by A53T point mutation is reduced by radotinib.
Example 7
In Example 7, an in vivo test was conducted to compare the inhibitory effect of radotinib and nilotinib on the formation of α-synuclein.
Nilotinib (4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]benzamide) is a selective Bcr-Abl kinase inhibitor of the tyrosine kinase inhibitors family, similar to radotinib. Nilotinib has been used as a therapeutic agent for treatment of Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia. It has been reported that nilotinib showed neuroprotective effects in genetically modified or toxin-induced Parkinson's disease animal models.
Through the experimentations in this and the following working examples, it has been found that although radotinib of the present invention is a selective Bcr-Abl kinase inhibitor similar to nilotinib, radotinib has safer and better pharmacokinetic properties than nilotinib and thus it has improved effect of prevention and treatment of neurodegenerative disease.
1) Experimental animals
7- to 8-month-old male A53T transgenic mice
2) Drugs
Radotinib and nilotinib
3) Methods
A53T mutation is characterized by an increase of α-synuclein polymers. Based on this character, 7- to 8-month-old male A53T transgenic mice were orally administered 3 mg/kg or 10 mg/kg of radotinib or nilotinib, respectively, for 6 weeks, once a day. Blood and brain were then isolated therefrom, and the amount of α-synuclein therein was measured by ELISA (Enzyme-Linked ImmunoSorbent Assay).
As a result, as shown in Figure 7 and Table 1 below, expression of α-synuclein was significantly increased in both the brain and blood of A53T transgenic mice, and the α-synuclein level was reduced in a dose-dependent manner according to the administration of radotinib and nilotinib. In addition, the same dose of radotinib reduced α-synuclein by about 58-69% more than that of nilotinib.
Figure PCTKR2017008229-appb-T000001
That is, through this experiment, it was confirmed that radotinib's inhibitory effect is superior to that of nilotinib on the formation of α-synuclein.
Example 8
In Example 8, comparative tests of inhibition of α-synuclein formation, dopamine production and motor ability were conducted using radotinib and nilotinib in a brain α-synuclein-administered disease model.
1) Experimental animals
Male C58BL/6 mice
2) Drugs
Radotinib and nilotinib
3) Methods
To confirm the change of blood α-synuclein level according to administration of α-synuclein into the brain, the inhibition of formation of α-synuclein by radotinib, and the relationship between said processes and Parkin, the following experimentation was performed.
Lentiviral α-synuclein was stereotactically administered bilaterally into the substantia nigra in the brain of male C58BL/6 mice for 3 weeks. Subsequently, 10 mg/kg of radotinib or nilotinib was orally administered thereto once a day for 3 weeks, and the amounts of α-synuclein and dopamine in the brain and blood were determined by ELISA. The change of motor ability was tested by rotarod test and pole test. The rotarod test measures how long the mouse stays on a rotating rod, and the pole test measures how long it takes the mouse to move from the top of a fixed rod to the bottom. Therefore, if the value of the rotarod test is higher and the value of the pole test is lower, it can be determined that the motor ability is better.
As a result, as shown in Figure 8 and Table 2 below, it was confirmed that the expressions of α-synuclein in the brain and blood were significantly reduced by administration of radotinib or nilotinib. In addition, the level of dopamine and motor ability were almost restored. These changes were more pronounced in the radotinib group. In comparison to the nilotinib group, α-synuclein was reduced by 65-73% in the radotinib group. Furthermore, dopamine secretion and motor ability recovery of the radotinib group were 2.4-fold superior to those of the nilotinib group.
Figure PCTKR2017008229-appb-T000002
That is, through this experiment, it was confirmed that radotinib's effect is superior to that of nilotinib on the inhibition of α-synuclein formation, increase of dopamine production and improvement of motor ability in the brain α-synuclein-administered disease model.
Example 9
In Example 9, a comparative test of the degree of promoting autophagy clearance of α-synuclein according to administration of radotinib and nilotinib was conducted.
1) Experimental animals
Male C57BL/6 mice
2) Drugs
Radotinib and nilotinib
3) Methods
In order to confirm that autophagy clearance of accumulated α-synuclein is promoted by radotinib administration, the following test was conducted.
Lentiviral α-synuclein was stereotactically administered bilaterally into the substantia nigra in the brain of male C58BL/6 mice for 3 weeks. Subsequently, 10 mg/kg of radotinib or nilotinib was orally administered thereto once a day for 3 weeks, and the substantia nigra was isolated from the mice and AV (Autophagic Vacuole) was separated therefrom. The amounts of α-synuclein in AV-10 (Phagophores+Autophagosomes), AV-20 (Autophagosomes) and lysosome in AV was determined by ELISA.
As a result, as shown in Figure 9 and Table 3 below, the level of α-synuclein in AV-20 and lysosome was increased after administration of radotinib and nilotinib when compared to the control group. It seemed that α-synuclein migrated to AV-20 and lysosome (which contain various enzymes) for degradation. These changes were more pronounced in the radotinib group. About 2.5 times more α-synuclein was observed in AV-20 and lysosome in the radotinib group compared to the nilotinib group.
Figure PCTKR2017008229-appb-T000003
That is, through this experiment, it was confirmed that the amount of α-synuclein in AV-20 and lysosome is increased by radotinib administration to promote autophagy clearance, and such an effect is even better than nilotinib.
Example 10
In Example 10, a comparative test of the degree of promoting autophagy clearance of accumulated α-synuclein and Parkin according to administration of radotinib and nilotinib was conducted.
1) Experimental animals
A53T transgenic mice
2) Drugs
Radotinib and nilotinib
3) Methods
To confirm that autophagy clearance of the accumulated α-synuclein and Parkin are promoted by radotinib administration, the following test was conducted.
A53T mice, which are characterized by increase of α-synuclein oligomerization, were orally administered with 10 mg/kg of radotinib or nilotinib once a day for 3 weeks. The substantia nigra was separated and Autophagic Vacuole (AV) was isolated therefrom. The levels of α-synuclein and Parkin in AV-10 (Phagophores+Autophagosomes), AV-20 (Autophagosomes) and lysosome were determined by ELISA.
As a result, as shown in Figure 10 and Table 4 below, it was confirmed that the accumulation of α-synuclein and Parkin in the substantia nigra was increased generally as the age of A53T mice increased. In addition, high amounts of α-synuclein and Parkin were found in AV-20 and lysosome in case of administration of radotinib and nilotinib compared to the control group. These results suggest that α-synuclein and Parkin migrated to AV-20 and lysosome (which contain various enzymes) for degradation. These changes were more pronounced in the radotinib group. About 2.5 times more α-synuclein and Parkin were observed in AV-20 and lysosome in the radotinib group compared to the nilotinib group in 5-month-old mice.
Figure PCTKR2017008229-appb-T000004
That is, through this experiment, it was confirmed that autophagy clearance of accumulated α-synuclein and Parkin is promoted by administration of radotinib, and such an effect is superior to nilotinib.
Example 11
In Example 11, the increase of Parkin activity (a major factor for α-synuclein removal) was tested using radotinib and nilotinib, and the result was compared.
1) Experimental animals
C57BL/6 male mice
2) Cell line
Human M17 neuroblastoma cell
3) Drugs
Radotinib and nilotinib
4) Methods
Parkin promotes protein degradation through ubiquitination. Therefore, the activity of Parkin plays a major role in the removal of α-synuclein. To evaluate the effect of radotinib administration on the activity of Parkin, the following test was conducted.
First, human M17 neuroblastoma cells were treated with 10 μM of radotinib or nilotinib, and E3 activity of Parkin was determined by ELISA. In addition, lentiviral α-synuclein was stereotactically administered bilaterally into the substantia nigra in the brain of male C58BL/6 mice for 3 weeks. And then, 10 mg/kg of radotinib or nilotinib was orally administered once a day for 3 weeks, and the brains were isolated from the mice to evaluate the brain Parkin level by ELISA.
As shown in Figure 11 and Table 5 below, the activity of E3 (indicating the activity of ubiquitin) was significantly increased in M17 cells treated with radotinib or nilotinib, respectively. Especially, the radotinib treatment group showed 2.7 times higher activity than the nilotinib treatment group. In addition, the Parkin level was improved in α-synuclein-treated mice according to the administration of radotinib or nilotinib when compared to the control group. The radotinib treatment group showed 2.6 times higher Parkin level than the nilotinib treatment group.
Figure PCTKR2017008229-appb-T000005
That is, through this experiment, it was confirmed that Parkin (a major factor for α-synuclein removal) is activated by radotinib, and such an effect of radotinib is superior to that of nilotinib.
Example 12
In Example 12, the effect of radotinib for inhibiting caspase-3 (a protein involved in apoptosis) activity was tested compared to nilotinib.
1) Experimental animals
C57BL/6 mice and A53T transgenic mice
2) Drugs
Radotinib and nilotinib
3) Methods
The activity of caspase-3, a protein involved in apoptosis, tends to increase in Parkinson's disease model. To confirm that radotinib administration inhibits caspase-3 activity, the following test was conducted.
A disease model was produced by stereotactical bilateral administration of lentiviral α-synuclein into the substantia nigra in the brain of male C58BL/6 mice for 3 weeks. 10 mg/kg of radotinib or nilotinib was orally administered once a day for 3 weeks into the disease model and A53T transgenic mice (7- to 8-months old) characterized by an increase of α-synuclein polymers, and the changes of caspase-3 level were measured.
As shown in Figure 12 and Table 6 below, activity of caspase-3 was found to be highly increased in both disease models. After administration of radotinib or nilotinib, the activity of caspase-3 was decreased to a level similar to that of the control group. These effects were higher in the radotinib treatment group than the nilotinib treatment group, especially 58% higher in α-synuclein model and 82% higher in A53T transgenic mice.
Figure PCTKR2017008229-appb-T000006
That is, it was confirmed that excessive apoptosis in the midbrain substantia nigra was more efficiently reduced by radotinib administration than nilotinib administration.
Example 13
In Example 13, penetration of radotinib through the blood-brain barrier was tested in mice compared to nilotinib.
1) Experimental animals
6-week-old male ICR mice
2) Drugs
Radotinib and nilotinib
3) Method
To confirm the in vivo absorption of administered radotinib and its distribution in the brain as a site of action, the following test was conducted using nilotinib as a control.
Radotinib and nilotinib were orally administered at a dose of 50 mg/kg into 6-week-old male ICR mice. Before and 1, 2, 3, 4, 5, 7 and 12 hours after administration, plasma was isolated from blood samples and the brain was extracted, and the concentrations of radotinib and nilotinib were analyzed by LC-MS/MS.
As shown in Table 7 below, A) pharmacokinetic values of radotinib and nilotinib in plasma and brain tissue were determined, and B) from the result above, it was possible to compare the absorption rate of radotinib and nilotinib by plasma and brain tissue. Radotinib showed a slightly lower absorption rate than nilotinib in plasma, but it showed 3.28 times higher AUC0-24 than nilotinib in the brain. These results indicate that radotinib's effect on penetration of blood-brain barrier is superior to that of nilotinib.
Figure PCTKR2017008229-appb-T000007
From the above results, it was confirmed that radotinib has a blood-brain barrier penetrability that is three times higher than that of nilotinib. The blood-brain barrier penetrability is one of the most important factors in the development of a medicament for treatment of brain diseases, and it is expected that radotinib is more effective for treatment of neurodegenerative diseases.
From the experiments for confirming the excellent therapeutic effect of radotinib on neurodegenerative diseases including Parkinson's disease, it was proved that radotinib has effects of reducing α-synuclein PFFs-induced neurotoxicity in neuronal cells, reducing α-synuclein PFFs-induced LB/LN-like pathology, reducing accumulation of A53T α-synuclein and inhibiting α-synuclein PFFs-induced c-Abl activity.
In animal studies, it was confirmed that radotinib inhibits α-synuclein formation in blood and the brain. In the brain α-synuclein-administered disease model, α-synuclein level was reduced by 65-73% in the radotinib treatment group compared to the nilotinib treatment group, and the radotinib treatment group showed about 2.4 times higher effect than the nilotinib treatment group in restoration of dopamine secretion and motor ability. These are the results of activation of Parkin by radotinib, and subsequent acceleration of α-synuclein migration to autophage for degradation to promote autophage clearance. These results are also related to inhibition of caspase-3 protein which is involved in apoptosis.
Radotinib showed an almost 2-fold higher ethological effect than nilotinib which has a similar effect in the treatment of CML. This is possible because, when the two drugs were administered at the same dose (50 mg/kg), the distribution of radotinib in the brain tissue was about 3 times higher than nilotinib due to high blood-brain barrier penetration rate although the plasma absorption of radotinib was slightly lower than nilotinib.
The above description of the present disclosure is provided for the purpose of illustration, and it would be understood by those skilled in the art that various changes and modifications may be made without changing the technical conception and essential features of the present disclosure. Thus, it is clear that the above-described examples are illustrative in all aspects and do not limit the present disclosure. For example, each component described as being of a single type can be implemented in a distributed manner. Likewise, components described as distributed can be implemented in a combined manner.
The scope of the present disclosure is defined by the following claims rather than by the detailed description of the embodiment. It should be understood that all modifications and embodiments conceived from the meaning and scope of the claims and their equivalents are included in the scope of the present disclosure.

Claims (10)

  1. A pharmaceutical composition for prevention or treatment of a neurodegenerative disease, comprising radotinib represented by Chemical Formula (I) below or a pharmaceutically acceptable salt thereof as an active ingredient:
    Figure PCTKR2017008229-appb-I000002
    (I)
  2. The pharmaceutical composition according to Claim 1, wherein the neurodegenerative disease is Parkinson's disease.
  3. The pharmaceutical composition according to Claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  4. The pharmaceutical composition according to Claim 1 for administration to an animal.
  5. The pharmaceutical composition according to Claim 4, wherein the animal is a mammal.
  6. The pharmaceutical composition according to Claim 5, wherein the mammal is a human.
  7. The pharmaceutical composition according to Claim 1 for oral administration to a subject.
  8. The pharmaceutical composition according to Claim 7, wherein the form of oral administration is selected from powder, pulvis, granule, tablet, capsule, mouth dispersible tablet, sugar-coated tablet, aerosol, gel, pill, soft capsule, suspension, emulsion, aqueous medicine, syrup, elixir, wafer and sachet.
  9. The pharmaceutical composition according to Claim 1 for parenteral administration to a subject.
  10. The pharmaceutical composition according to Claim 9, wherein the form of parenteral administration is selected from injection, transdermal system, suppository, aerosol and nasal inhaler.
PCT/KR2017/008229 2016-08-01 2017-07-31 Pharmaceutical composition for prevention or treatment of neurodegenerative diseases WO2018026150A1 (en)

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