WO2021096270A1 - Ampk 억제기능과 아연항상성 조절기능에 기반한 다발성 경화증 치료용 약학적 조성물 - Google Patents

Ampk 억제기능과 아연항상성 조절기능에 기반한 다발성 경화증 치료용 약학적 조성물 Download PDF

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WO2021096270A1
WO2021096270A1 PCT/KR2020/015932 KR2020015932W WO2021096270A1 WO 2021096270 A1 WO2021096270 A1 WO 2021096270A1 KR 2020015932 W KR2020015932 W KR 2020015932W WO 2021096270 A1 WO2021096270 A1 WO 2021096270A1
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indol
amino
thiazol
group
methylene
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English (en)
French (fr)
Korean (ko)
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김양희
엄재원
서상원
최보영
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Zincure Corp
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Zincure Corp
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Priority to US17/776,693 priority Critical patent/US12343335B2/en
Priority to EP20887409.9A priority patent/EP4066832A4/en
Priority to KR1020227017186A priority patent/KR102770734B1/ko
Priority to JP2022528544A priority patent/JP7398840B2/ja
Priority to CN202080093043.2A priority patent/CN114929217A/zh
Publication of WO2021096270A1 publication Critical patent/WO2021096270A1/ko
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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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to a pharmaceutical composition for the treatment of multiple sclerosis, and more particularly, to a pharmaceutical composition for the treatment of multiple sclerosis based on an AMPK inhibitory function and a zinc homeostasis control function.
  • MS Multiple sclerosis
  • the mechanism is involved.
  • the treatment of multiple sclerosis has been mainly steroids and immunosuppressants. This is based on the fact that the main mechanism of multiple sclerosis is an autoimmune mechanism, and it is an attempt to control the disease by weakening the body's immune function.
  • the above treatments have a distinct effect in the acute phase of the disease, they do not play a role in suppressing or reducing the recurrence of the disease in the long term.
  • beta-globulin is injected through the spinal fluid or skin.
  • the downside is that you have to keep receiving it.
  • Korean Patent No. 1324647 discloses a composition for treating or preventing multiple neurosclerosis and a screening method thereof.
  • LeuSH L-Leucinethiol
  • the present invention provides a pharmaceutical composition for the treatment of multiple sclerosis based on an AMPK inhibitory function and a zinc homeostasis modulating function that effectively treats multiple sclerosis due to a neuroprotective effect without side effects. It aims to do.
  • these problems are exemplary, and the scope of the present invention is not limited thereby.
  • a pharmaceutical composition for the treatment of multiple sclerosis or encephalomyelitis containing a compound having the structure of the following formula 1 as an active ingredient:
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms, or an unsubstituted alkoxy group, an amine group.
  • a carboxyl group or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3)
  • R 6 is hydrogen or a methyl group
  • R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • a method for treating multiple sclerosis in a subject comprising administering to a subject suffering from multiple sclerosis a therapeutically effective amount of a compound having the structure of Formula 1 below:
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms or an unsubstituted alkoxy group, amine A group, a carboxyl group, or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3), and R 6 is a hydrogen or methyl group And R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • a method for treating encephalomyelitis in a subject comprising administering to a subject suffering from encephalomyelitis a therapeutically effective amount of a compound having the structure of Formula 1 below:
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms or an unsubstituted alkoxy group, amine A group, a carboxyl group, or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3), and R 6 is a hydrogen or methyl group And R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms or an unsubstituted alkoxy group, amine A group, a carboxyl group, or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3), and R 6 is a hydrogen or methyl group And R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms or an unsubstituted alkoxy group, amine A group, a carboxyl group, or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3), and R 6 is a hydrogen or methyl group And R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • the pharmaceutical composition for the treatment of multiple sclerosis based on the AMPK inhibitory function and zinc homeostasis control function of the present invention made as described above is treated with a novel compound having AMPK activity inhibitory function and zinc homeostasis control function to prevent spinal cord injury due to multiple sclerosis. It can be used to develop new treatments that can overcome behavioral disorders. Of course, the scope of the present invention is not limited by these effects.
  • 1A is a diagram showing a timeline for an experimental design according to an embodiment of the present invention.
  • 1H10 was administered intraperitoneally once a day, and mice were sacrificed 21 days after immunization.
  • 1B is a graph analyzing EAE clinical scores for the vehicle administration group.
  • 1C is a graph analyzing EAE clinical scores for the 1H10 administration group.
  • FIG. 2A is a microscopic photograph observing representative microglia/macrophage activation in the spinal cord of sham-operated and MOG35-55 immunized mice (vehicle or 1H10). F4/80 (red), DAPI-stained nuclei (blue), and demyelinated regions were confirmed by reduced MBP staining (green). Scale bar, 50 ⁇ m.
  • 2D is a representative image of Iba-1- (green) and CD68- (red) immune positive cells as images merged into vehicle- and 1H10-treated EAE mice. Nuclei stained with DAPI (blue). Scale bar, 50 ⁇ m.
  • Figure 2g is a graph analyzing the colocalization scatterplots of Iba-1 with CD68.
  • 3A is a micrograph showing a spinal cord section stained for cresyl violet to detect infiltration of SMS mononuclear cells. Scale bar, 100 ⁇ m.
  • 3C is a micrograph showing the expression of T and B cells stained with antibodies to cell surface molecules such as CD4, CD8 and CD20.
  • 3D is a graph analyzing the intensity of CD4, CD8 and CD20 immune responses in the thoracic spinal cord treated with or without 1H10 in sham-operated or EAE mice.
  • 3E is a graph analyzing the percent area of CD4, CD8 and CD20 immune responses in the thoracic spinal cord treated with or without 1H10 in sham-operated or EAE mice.
  • 3F is a representative immunofluorescence image showing CD8+ T cells co-labeled with phospho-AMPK ⁇ 1/2 in the spinal cord from vehicle- and 1H10-treated EAE mice.
  • 3G is a graph showing the analysis of colocalization scatterplots of phospho-AMPK ⁇ 1/2 and CD8.
  • 4A is a photomicrograph showing a portion of the spinal cord stained with TSQ to detect zinc accumulation. Scale bar, 100 ⁇ m.
  • 4B is a micrograph showing a portion of the spinal cord stained for anti-mouse immunoglobulin G (IgG) to detect endogenous IgG. Scale bar, 100 ⁇ m.
  • IgG immunoglobulin G
  • 4E is a double-labeled confocal micrograph of CD31 + endothelial cells (red) and endogenous mouse IgG molecules (green) in the white matter of the spinal cord from vehicle- and 1H10-treated EAE mice. Scale bar, 20 ⁇ m.
  • Figure 4f is an immunofluorescence image showing the expression of MMP-9 in the white matter of the spinal cord. Scale bar, 50 ⁇ m.
  • Figure 5a is a graph showing the expression of the fluorescent substance FluoZin-3 as verified by the 1H10 treatment of the present invention to control the homeostasis of zinc.
  • FIG. 5B is a graph showing the expression of free zinc fluorescently labeled drug (FluoZin-3) to verify the regulation of zinc homeostasis according to the 1H10 treatment of the present invention.
  • 6A is a timeline showing the experimental design by analyzing the long-term protection effect of 1H10 after EAE induction. After 1H10 was administered intraperitoneally once a day for the entire period, mice were sacrificed 45 days after the initial immunization.
  • 6B is a graph analyzing the long-term protective effect of 1H10 after induction of EAE, and analyzing the EAE clinical score for the vehicle.
  • Figure 6c is a graph analyzing the long-term protective effect of 1H10 after EAE induction, and analyzing the EAE clinical score for the 1H10 administration group.
  • FIG. 7 is a diagram of the results of research showing that loss of homeostasis of zinc may cause multiple sclerosis.
  • MS multiple sclerosis
  • 9 is a graph analyzing the neuroprotective effect of 25 similar compounds having a similar structure based on the structural similarity of 1H10 of the present invention.
  • 10 is a graph analyzing the inhibitory effect of oxidative stress according to 25 novel compounds and 1H10 treatment.
  • 11 is a graph analyzing the inhibitory effect of oxidative stress according to 8 novel compounds and 1H10 treatment.
  • 12 is a graph analyzing the effect of inhibiting excitotoxicity according to 8 novel compounds and 1H10 treatment.
  • 13 is a graph analyzing the effect of inhibiting apoptosis according to 8 novel compounds and 1H10 treatment.
  • 14 is a graph showing the results of a zinc binding assay according to 25 novel compounds and 1H10 treatment.
  • 15 is a graph analyzing AMPK ⁇ 2 inhibitory activity according to 25 novel compounds and 1H10 treatment.
  • 16 is a graph showing the results of self-toxicity analysis according to 4 novel compounds and 1H10 treatment.
  • FIG. 17 is an immunofluorescence photograph of GFAP (green) as an analysis of the activity of astrocytes in spinal cord tissue according to the 1H10 treatment of the present invention.
  • GFAP green
  • an abnormal increase astrogliosis
  • MOG35-55 immunized mice vehicle or 1H10 was observed.
  • Scale bar 50 ⁇
  • FIG. 19 shows expression of IFN gamma and TNF alpha among cytokines in the spinal cord of the mouse on the 21st day after induction of EAE mice.
  • IFN- ⁇ , green C, H
  • TNF- ⁇ , red tumor necrosis factor alpha
  • D I
  • double label confocal micrographs AE
  • Nuclei stained with DAPI blue
  • B G
  • Scale bar 20 ⁇
  • AMP-activated protein kinase as used herein is a heterologous trimer protein composed of a catalytic ⁇ subunit ( ⁇ 1 or ⁇ 2), and two regulatory subunits ( ⁇ and ⁇ ). AMPK is phosphorylated and activated when the cellular energy level is low, and again regulates cellular metabolism to regulate gene expression over a long period of time to restore the level of ATP. It is known that an increase in the AMP/ATP ratio, a change in cellular pH and redox state, and an increase in the creatine/phosphocreatine ratio activate AMPK.
  • Zinc used in this document is a substance abundantly present in the entire body, including the central nervous system, and plays a very important role in synaptic plasticity and learning of nerve cells. However, excessive zinc accumulation or severe deficiency is toxic to neurons. Intracellular accumulation of zinc is a major cause of nerve damage after acute neurological diseases such as stroke, epilepsy, traumatic brain injury, and hypoglycemia, and occurs in Alzheimer's disease, a chronic disease. It is well known that it causes the generation of plaques.
  • a pharmaceutical composition for the treatment of multiple sclerosis or encephalomyelitis containing a compound having the structure of the following formula 1 as an active ingredient:
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms, or an unsubstituted alkoxy group, an amine group.
  • a carboxyl group or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3)
  • R 6 is hydrogen or a methyl group
  • R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • the unsubstituted alkyl group may be methyl, ethyl, propyl or butyl
  • the substituted alkyl group is fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, or It may be iodomethyl, diiodomethyl, or triraiodomethyl
  • the halogen may be fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).
  • the compound is (5Z)-5-(1H-Indol-3-ylmethylene)-2-[2-(trifluoromethyl) phenyl]amino-1,3-thiazol-4(5H)-one,
  • the compound may have an effect of preventing demyelination, a neuroprotective effect, reducing tissue damage, preventing apoptosis, or inhibiting inflammatory infiltration.
  • a method for treating multiple sclerosis in a subject comprising administering to a subject suffering from multiple sclerosis a therapeutically effective amount of a compound having the structure of Formula 1 below:
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms or an unsubstituted alkoxy group, amine A group, a carboxyl group, or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3), and R 6 is a hydrogen or methyl group And R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • a method for treating encephalomyelitis in a subject comprising administering to a subject suffering from encephalomyelitis a therapeutically effective amount of a compound having the structure of Formula 1 below:
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms or an unsubstituted alkoxy group, amine A group, a carboxyl group, or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3), and R 6 is a hydrogen or methyl group And R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms or an unsubstituted alkoxy group, amine A group, a carboxyl group, or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3), and R 6 is a hydrogen or methyl group And R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • R 1 to R 5 are each independently hydrogen, a hydroxy group, a halogen, a substituted or unsubstituted alkyl group having 1 to 7 carbon atoms, a substituted alkoxy group having 1 to 7 carbon atoms or an unsubstituted alkoxy group, amine A group, a carboxyl group, or R 2 and R 3 together form a -O-(CH 2 ) n -O- ring or a substituted or unsubstituted benzene ring (n is an integer of 1 to 3), and R 6 is a hydrogen or methyl group And R 7 is hydrogen or halogen, in the above formula Is a single bond or a double bond).
  • the effective amount of the compound may vary depending on the type of the affected part of the patient, the application site, the number of treatments, the treatment time, the formulation, the condition of the patient, the type of adjuvant, and the like.
  • the amount used is not particularly limited, but may be 0.01 ⁇ g/kg/day to 10 mg/kg/day.
  • the daily dose may be administered once a day, divided into 2-3 times a day at appropriate intervals, or intermittently administered at intervals of several days.
  • the compound may be contained in an amount of 0.1-100% by weight based on the total weight of the composition.
  • the pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions.
  • suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions may be used in the preparation of pharmaceutical compositions.
  • solid or liquid additives for preparation may be used in the preparation of pharmaceutical compositions.
  • the additive for formulation may be either organic or inorganic.
  • excipients include lactose, sucrose, sucrose, glucose, cornstarch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide.
  • a binder for example, polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, arabic rubber, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, And dextrin and pectin.
  • the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any colorant that is permitted to be added to pharmaceuticals can be used. These tablets and granules can be appropriately coated with a sugar coat, gelatin coating, or other necessary. In addition, preservatives, antioxidants, etc. can be added as needed.
  • the pharmaceutical composition of the present invention may be prepared in any formulation conventionally prepared in the art (for example, Remington's Pharmaceutical Science, the latest edition; Mack Publishing Company, Easton PA), and the form of the formulation is not particularly limited. . These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a formula generally known for all pharmaceutical chemistry.
  • the compound in the pharmaceutical composition of the present invention, can be administered orally or parenterally, preferably parenteral administration by intravenous injection, subcutaneous injection, intracerebroventricular injection, and intracerebrospinal fluid injection. , It can be administered by intramuscular injection and intraperitoneal injection.
  • Zinc Transporter 3 which is located at the nerve endings and regulates the movement of zinc transporter 3 (ZnT3), which is located at the nerve endings, suppresses the symptoms of multiple sclerosis and tissue damage of the spinal cord white matter. As the results are released one after another, interest in zinc and multiple sclerosis is increasing.
  • nerve cell damage processes such as zinc neurotoxicity and excitatory toxicity are accompanied by a decrease in ATP and an increase in AMP, and the AMPK activity, which is activated by AMP, is evident, and when the increased AMPK activity is lowered, nerve cell damage is reduced.
  • AMPK activity which is activated by AMP
  • AMPK activity is associated with damage and pathological involvement of the central nervous system in neurodegenerative diseases such as dementia, Parkinson's disease, Huntington's chorea, and multiple sclerosis. Accordingly, the present inventors have conducted a study on the neurotoxic mechanism centered on AMPK, which is the most important in metabolic regulation, and based on the preceding research, the present inventors conducted a study on the development of a new AMPK inhibitor to screen for a new compound, and the AMPK inhibitor ((Z)-5 -((1H-indol-3-yl)methylene)-2-((3-hydroxyphenyl)amino)thiazol- 4(5H)-one, named "1H10", formula 2) was discovered.
  • the present invention has shown that the 1H10 can suppress the onset of disease at various levels after induction of autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis. Specifically, 1H10 treatment showed excellent symptom relief effect in the behavioral experiment and the incidence rate was also suppressed. In addition, it was confirmed that demyelination and immune cell activity of the spinal cord were reduced, and immune cells were prevented from invading into the demyelinating site. This suggests that the 1H10 drug is related to the mechanism of inhibition of AMPK activity.
  • EAE autoimmune encephalomyelitis
  • the pharmaceutical composition for the treatment of multiple sclerosis based on the AMPK inhibitory function and zinc homeostasis control function of the present invention can overcome spinal cord damage and behavioral disorders caused by multiple sclerosis by treating a novel compound with AMPK activity inhibitory function and zinc homeostasis control function. It provides a new therapeutic agent that can be used.
  • the novel drug can act as a zinc chelator in the process of nerve cell damage. In particular, in the case of 1H10, it can act as a zinc ionophore as well as chelation, so it can be developed as an active zinc homeostasis modulator.
  • the experimental animal used in the present invention is an 8-week-old C57BL/6 female mouse supplied from Daehan Biolink. These mice were raised in an environment where temperature and humidity were controlled, and food and water were supplied freely.
  • the mouse cortical neurons used in the present invention were extracted and cultured from the brain of a mouse embryo, and Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY) to which 5% fetal bovine serum (FBS) and 5% horse serum (HS) were added. , US) was incubated at 95% humidity, 5% CO 2 and 37°C temperature conditions. For activation and differentiation of the cells, the cells were proliferated at a density of 2 x 10 4 cells in a 24-well tissue culture plate and cultured in MEM medium without FBS and HS before treatment with zinc and compounds.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • HS horse serum
  • the present inventors induce an autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, by subcutaneous injection of Myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide antigen into C57BL/6 female mice (Fig. 1a).
  • EAE autoimmune encephalomyelitis
  • MOG35-55 Myelin oligodendrocyte glycoprotein 35-55
  • CFA Complete freund's adjuvant
  • mice were injected with the same amount of CFA containing MOG35-55 and Mycobacterium tuberculosis H37Ra in both sides.
  • Pertussis toxin (4 ⁇ g/ml, List Biological Laboratories, USA) was injected intraperitoneally on the day of immunization and on the second day after immunization. After the immunization, the weight and clinical symptoms of the mice were measured every day, and the degree of progression of EAE was evaluated based on specific clinical symptoms. Details on this are described below.
  • EAE clinical symptom evaluation used in the present invention was evaluated based on previous studies (Jones et al., J Neuroimmunol . Aug 13;199(1-2):83-93. 2008). Specifically, for the evaluation of clinical symptoms of EAE, the motion of the mice was evaluated daily according to the following criteria.
  • score 0 no symptoms; score 0.5, partial paralysis of the tail or slight abnormal gait; score 1.0, complete or partial tail palsy and mild hindlimb palsy; score 1.5, complete tail palsy and mild hindlimb palsy; score 2.0, tail palsy and moderate hind limb weakness (proven that feet often fall out while walking in a cage); score 2.5, no weight in hind legs, but slight movement; score 3.0, complete hind limb paralysis; score 3.5, paralysis of the hind limbs and mild weakness in the forelimbs; score 4.0, limbs are completely paralyzed, but the head is moving; score 4.5, moribund state; score 5.0, death.
  • the present inventors anesthetized mice with urethane on the 21st day after EAE induction, and then perfused with 4% paraformaldehyde to the heart to fix the spinal cord, and after extracting the spinal cord, mononuclear cells from the spinal cord The infiltrate was observed through cresyl violet staining.
  • the spinal cord was cut to a thickness of 30 um in a cryostat, and the slices were obtained, and then placed on a gelatin-coated slide and dried at room temperature for 30 minutes. It was immersed in 100% and 70% ethanol solutions for 3 minutes each, followed by staining in 0.1% cresyl violet solution for 15 minutes.
  • the present inventors On the 21st day after EAE induction, the present inventors anesthetized the mouse with urethane, and then perfused with 4% paraformaldehyde to the heart to fix the spinal cord, and after extracting the spinal cord, the infiltration of immune cells in the spinal cord was observed. To this end, immunohistochemistry was performed using CD4 and CD8 used as markers for T cells, and CD20 antibodies used as markers for B cells. The frozen sections were reacted with 3% hydrogen peroxide for 15 minutes at room temperature to remove endogenous peroxidase, and then the primary antibodies rat anti-CD4 (1:50, BD Bioscience, CA, USA), CD8 (1:50).
  • IgG Immunoglobulin G
  • the IgG is the most abundant immunoglobulin in plasma, and it is easy to identify it, so the presence of IgG in the spinal cord is used to monitor damage to the blood-brain barrier and extravasation of proteins (Ruth and Feinerman, Acta Neuropathol. 76(4): 380-7.1988).
  • the frozen sections were reacted with biotinylated horse anti-mouse IgG (1:250, Vector Laboratories, USA) at room temperature for 2 hours and then reacted with ABC regent in the same manner as above, and then colored with DAB solution.
  • the present inventors anesthetized mice with urethane on the 21st day after EAE induction, and then perfused with 4% paraformaldehyde to the heart to fix the spinal cord.After the spinal cord was excised, the tissue demyelination and microglia Immunofluorescence staining was performed with myelin basic protein (MBP) and F4/80 (Microglia/Macrophage) antibodies to test the activity of cells/macrophages.
  • MBP myelin basic protein
  • F4/80 Microglia/Macrophage
  • Iba-1 Ionized calcium binding adapter molecule-1
  • CD68 which are used as markers for microglia/macrophages, are used as markers for microglia/macrophages to analyze the phenotype of strongly active microglia/macrophages in the white matter part of the damaged spinal cord.
  • Cluster of Differentiation 68 Biimmunofluorescence staining was performed using an antibody.
  • AMPK AMP-activated protein kinase
  • Matrix metallopeptidase 9 In order to compare and analyze the activity of (MMP-9), immunofluorescence staining was performed using an MMP-9 antibody. To test the activity of astrocytes in the spinal cord, immunofluorescence staining was performed using an antibody of glial fibrillary acidic protein (GFAP), and the expression of cytokines such as tumor necrosis factor (TNF) alpha and interferon (IFN) gamma were compared and analyzed. For this, double immunofluorescence staining was performed using TNF-alpha and IFN gamma antibodies.
  • GFAP glial fibrillary acidic protein
  • TNF tumor necrosis factor
  • IFN interferon
  • the frozen sections were reacted with 3% hydrogen peroxide for 15 minutes at room temperature to remove endogenous peroxidase, and then the primary antibody, rat anti-MBP (1:200, Abcam, UK).
  • the present inventors examined the spinal cord tissue after the evaluation of clinical symptoms of EAE-induced mice was completed by the histochemical method of N-(6 methoxy 8 quinolyl) para toluenesulfonamide (TSQ), which is a zinc staining method.
  • TSQ N-(6 methoxy 8 quinolyl) para toluenesulfonamide
  • zinc is present at the end of the nerve axons in gray matter, and only very small amounts are observed in the white matter.
  • the mice were anesthetized with 5% isoflurane, and the spinal cord was removed without perfusion and rapidly cooled with dry ice. The unfixed frozen tissue was cut to a thickness of 20 ⁇ m in a -15°C cryostat to obtain a section.
  • TSQ fluorescence was observed using an Olympus IX70 fluorescence microscope with a wavelength of 360 nm/490 nm and evaluated using an INFINITY3-1 CCD cooled digital color camera (Lumenera Co., Canada) and INFINITY analysis software.
  • FluoZin-3 a substance that shows fluorescence when bound to free zinc, was treated on neurons of cultured mice, exposed to zinc (300 ⁇ M) for 15 minutes, and then the zinc was removed. The zinc concentration in nerve cells was measured for 60 minutes.
  • the present inventors observed the demyelination of spinal cord tissue and the activity of microglia/macrophages through immunofluorescence staining.
  • the white matter of the spinal cord to be stained in green color
  • the green staining was lowered in the tissues of EAE mice, and the red color indicating the activity of microglia/macrophages appeared strongly (FIG. 2A).
  • the decrease in MBP means that myelin covering the axon of the nerve is damaged (FIG. 2B).
  • the EAE-induced demyelination and microglia/macrophage activity were significantly reduced in the spinal cord of animals to which 1H10 was continuously administered (FIG. 2C).
  • the present inventors observed the activity of astrocytes in spinal cord tissue through immunofluorescence staining. As a result, it was confirmed that the green color indicating the activity of astrocytes was strongly distributed in the spinal cord white matter (white matte) of the experimental group in which EAE was induced, whereas the activity of astrocytes was significantly suppressed in the experimental group administered with 1H10 (Fig. 17 and 18).
  • the present inventors observed the invasion of monocytes in the spinal cord of the mouse on the 21st day after induction of EAE mice by cresyl violet staining.
  • AMPK activity is known to enhance the survival of T cells.
  • phosphorylation of AMPK in CD8(+) T cells infiltrated into the spinal cord after EAE induction was increased, but was observed to be significantly decreased in the experimental group to which 1H10 was administered (FIGS. 3f to 3h).
  • the above results suggest that inhibition of AMPK by 1H10 of the present invention can alleviate symptoms of EAE by reducing the survival of autoreactive T cells infiltrated into the spinal cord.
  • the present inventors observed expression of IFN gamma and TNF alpha among cytokines in the spinal cord of the mouse on the 21st day after induction of EAE mice through immunofluorescence staining. As a result, it was observed that the expression of IFN gamma in the spinal cord white matter was increased in the experimental group to which 1H10 was administered after EAE induction, whereas the expression of TNF alpha was decreased (FIGS. 19 and 20).
  • the present inventors observed abnormal accumulation of zinc in the EAE-induced spinal cord white matter, damage to the blood brain barrier (BBB), and the activity of MMP-9 by the administration of 1H10.
  • mice administered with vehicle or mice administered with only 1H10 mice administered with only 1H10 (Sham group)
  • IgG staining due to BBB damage was not found in white matter or gray matter.
  • mice 3 weeks after injection of MOG IgG infiltration was significantly increased in both white matter and gray matter, and this phenomenon was found to be significantly reduced by the administration of 1H10 (FIGS. 4c to 4e).
  • the present inventors administered 1H10 intraperitoneally once a day for the entire period after induction of mouse EAE, and then sacrificed the mouse 45 days after the initial immunization to observe clinical symptoms and incidence (FIG. 6A), and 1H10 was administered. It was observed that the clinical symptoms and incidence rate significantly decreased in the experimental group (FIGS. 6b to 6d). This proves that the administration of 1H10 is effective in relieving the symptoms of EAE for a long time in the acute phase (21 days) as well as the chronic phase (45 days).
  • 1H10 of the present invention based on the structural similarity of 1H10 of the present invention, 25 similar compounds having similar structures were purchased from a compound library manufacturer (InterBioScreen, Russia; Akos, Germany). Thereafter, ZnCl 2 (400 ⁇ M) was treated for 10 minutes to induce zinc toxicity in the cerebral cortical neurons of the cultured mice, and after 12.5 hours, the selected 25 compounds and the previously selected drug 1H10 were used. Treatment (20 ⁇ M) was performed and the inhibition of cell death was observed through cell viability assay (Cell Counting Kit-8, Dojindo).
  • the present inventors observed the effects of inhibiting oxidative stress in addition to zinc toxicity for the eight drugs and 1H10 of the above examples. Specifically, oxidative damage induced neurotoxicity by treating the cortical neurons of mice with H 2 O 2 (100 ⁇ M) and FeCl 2 (100 ⁇ M) for about 4 hours and 20 hours, respectively, and the selected 8 drugs and 1H10 was treated (20 ⁇ M). After that, cytotoxicity was observed through LDH (Lactate Dehydrogenase) analysis.
  • LDH Lacate Dehydrogenase
  • the present inventors observed the effect of inhibiting excitotoxicity on the 8 drugs of the above example and 1H10. Specifically, excitotoxicity was induced by treatment with NMDA (N-methyl-D-aspartate, 50 ⁇ M) on the cortical neurons of mice for 3 hours, and treatment with the selected 8 drugs and 1H10 (20 ⁇ M) and LDH cells As a result of observing toxicity, 6 drugs (4B01, 4B08, 4C01, 4C03, 4C04, 4C08) except for 4B04 and 4C06 were found to significantly inhibit excitatory toxicity by NMDA (FIG. 12).
  • the present inventors observed the effect of inhibiting apoptosis on the 8 drugs of the above example and 1H10. Specifically, neurotoxicity caused by apoptosis was induced by treatment with Etoposide (ETPS, 10 ⁇ M) on cortical neurons of mice for 20 hours. Thereafter, the selected 8 drugs and 1H10 were treated (20 ⁇ M) and LDH cytotoxicity was observed. As a result, 5 drugs (4B01, 4B08, 4C01, 4C06, 4C08) except 4B04, 4C03, 4C04 showed ETPS toxicity. It was found to significantly inhibit (Figure 13).
  • Etoposide Etoposide
  • the present inventors measured the recombinant AMPK ⁇ 2 enzyme activity by treatment with 1H10 and 25 compounds (10 ⁇ M each) and the previously well-known AMPK inhibitor compound C (CC, 10 ⁇ M) through KinaseProfilerTM Service (Eurofins, UK). As a result, 4A06, 4B02, 4C04, 4C05, 4C06, 4C08, 4D01 showed similar AMPK inhibitory effects to 1H10 (FIG. 15).
  • the present inventors determined the self-toxicity of 4 drugs (4B01, 4B08, 4C01, 4C08) that showed a protective effect in common against all cytotoxicity (zinc toxicity, oxidative damage, excitotoxicity, apoptosis) with 1H10. Compared. Specifically, in the cerebral cortical neuron culture of mice, each 40 ⁇ M of the drug was treated and LDH cytotoxicity was observed after 24 or 48 hours.
  • the compounds according to an embodiment of the present invention have protective effects on various toxic mechanisms such as excitotoxicity, oxidative stress, apoptosis, and zinc neurotoxicity related to conventional multiple sclerosis.
  • MMP-9 activity was reduced through zinc chelation to prevent the permeation and accumulation of immune cells in the white matter of the spinal cord, thereby reducing the autoimmune response, thereby suppressing the occurrence of multiple sclerosis disease. Therefore, 1H10 of the present invention can be used in the development of drugs capable of replacing steroids and immunosuppressants having serious side effects due to long-term administration and controlling fundamental problems.

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PCT/KR2020/015932 2019-11-14 2020-11-12 Ampk 억제기능과 아연항상성 조절기능에 기반한 다발성 경화증 치료용 약학적 조성물 Ceased WO2021096270A1 (ko)

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US17/776,693 US12343335B2 (en) 2019-11-14 2020-11-12 Pharmaceutical composition for treating multiple sclerosis on basis of AMPK inhibitory function and zinc homeostasis control function
EP20887409.9A EP4066832A4 (en) 2019-11-14 2020-11-12 Pharmaceutical composition for treating multiple sclerosis on basis of ampk inhibitory function and zinc homeostasis control function
KR1020227017186A KR102770734B1 (ko) 2019-11-14 2020-11-12 Ampk 억제기능과 아연항상성 조절기능에 기반한 다발성 경화증 치료용 약학적 조성물
JP2022528544A JP7398840B2 (ja) 2019-11-14 2020-11-12 Ａｍｐｋ抑制機能と亜鉛恒常性調節機能に基づく多発性硬化症治療用の薬学的組成物
CN202080093043.2A CN114929217A (zh) 2019-11-14 2020-11-12 基于ampk抑制功能和锌稳态控制功能用于治疗多发性硬化的药物组合物

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