WO2020009522A1 - Pharmaceutical composition for prevention or treatment of prion disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of a prion disease which is one of the neurodegenerative diseases, the composition comprising, as an active ingredient, radotinib or a pharmaceutically acceptable salt thereof.

Description

Pharmaceutical compositions for the prevention or treatment of prion diseases

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of prion disease, which is one of degenerative neurological diseases, comprising radotinib or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention was made by the task number HI16C0965 under the support of the Ministry of Health and Welfare, the research and management institution of the task is Korea Health Industry Development Institute, the research project name is infectious disease risk technology development project, the research project name is the development of treatment for prion disease, research period 2016.05.17 ~ 2020.12.31.

Prions accumulate in the central nervous system and form plaques known as amyloids, which disrupt normal tissue structure, leading to neurodegenerative disease. This phenomenon is characterized by the appearance of "holes" in the tissue due to the sponge-like structure produced by the vacuole formation of neurons. Other histological changes include abnormal astrogliosis and no inflammatory response. The incubation period of prion disease is quite long, but once symptoms develop, the disease progresses rapidly, leading to brain damage and death.

Prion, Stanly B. Prusiner, who studied Creutzfeldt-Jakob Disease (CJD), isolated infectious proteins in 1982 and lacked nucleic acids, unlike viroids or other viruses, small RNA pathogens. The term is used to refer to the distinction "proteinaceous infectious particles" (Proteinaceous + Infectious + viriON = PRION).

The discovery of this prion protein (PrP) originates from the first protein (PrP ^Sc ) found in sheep's scrapie disease, which is resistant to proteinase K, a protease. It is a highly contagious protein with a size of 27-30 kDa. On the other hand, there is a prion protein also found in normal people, the size of about 33kDa is required for normal cell function is called cellular prion protein (PrP ^C ). Unlike PrP ^Sc , PrP ^C is completely lost when treated with proteinase K. That is, the prion disease occurs when the normal prion protein form PrP ^C is transformed into a disease-causing abnormality PrP ^Sc , which is basically a basis for revealing that the prion disease is a disease of protein structure.

Prion disease is caused by a prion conversion, a conformational change in which protein structure is changed to abnormal infectious prion protein (PrP ^Sc ) by post-translational processing, in which normal prion protein (PrP ^C ) has not yet been identified. It is known that these infectious prion proteins accumulate in the cerebral tissues of infected individuals, leading to prion disease specific sponge changes.

This process involves a period of months or years of natural infection, depending on the species of infection or peripheral infection, and microfold cells (M cells) and enterocytes (enterocytes) in the small intestine wall at the beginning of infection. Pathogenic prion proteins are absorbed by macrophages and follicular dendritic cells (FDCs), which are immune cells, and are deposited in the spleen along the peripheral nerves in the intestinal wall. Finally, infection is transmitted to the cerebral neurons through the spinal cord nerves, and at the end, infectious prions accumulate throughout the cerebrum. In this process, the conversion of normal prion protein (PrP ^C ) to pathogenic prion protein (PrP ^Sc ) occurs continuously in various organs and tissues of infected individuals and in certain organ tissues (spleen, appendix, tonsil and spinal cord tissue) Although pathogenic prion proteins are known to deposit, lesions are characterized only by the degeneration of the cerebral nervous system and the formation of vacuoles in the form of sponges in the cerebrum.

The amino acid sequence of the normal and infectious prion proteins is the same, except that in the tertiary structure of the protein, the infectious prion protein has a high content of β-sheet structure and is resistant to proteinase K (PK), a protease. Known as To date, many features on the structure of prion proteins have been reported, but the ways in which normal prion proteins are converted to infectious prion proteins and the associated post-translational processes are unknown.

Currently, prion diseases have been identified as kuru in humans, sporadic Creutzfeldt-Jakob disease (sCJD), familial Creutzfeldt-Jakob disease (familial CJD), variant Creutzfeldt-Jakob disease (vCJD), and Gerstmann- Gertmann-Straussler-Scheinker Syndrome (GSS), fatal familial insomnia (FFI), and in animals, scrapie in sheep, transmissible mink encephalopathy, deer and elk Chronic wasting disease, and bovine spongiform encephalopathy (BSE, commonly referred to as "mad cow disease").

BSE occurred collectively in the UK in the late 1980s, which is believed to be due to industrial feed. However, it is not yet clear whether BSE appears to be the primary sporadic in cattle or in sheep scrapie. The characteristics of these prion diseases are mainly invading the brain, have a period of incubation for many years, and when they develop, they rapidly deteriorate, leading to death, and pathologically, spongi-form changes, infectious, inflammatory and immune responses. It doesn't happen.

Creutzfeldt-Jakob disease (CJD) is a representative prion disease that occurs in humans. Unlike kururu, which occurs only in certain regions, it is a spongiform encephalopathy that occurs around the world. It is characteristic of 1 ~ 1.5hz EEG observed in the test. Sporadic CJD (sporadic CJD), which occurs sporadically, is the most common prion disease in humans, accounting for about 85% of human CJD disease. 10-15% of CJDs are hereditary; familial CJD (fCJD), Gerstmann-Strauss-Shanker Disease (GSS), and fatal familial insomnia (FFI) are all dominant caused by modification of the PrP gene. It is an inherited prion disease. Although infectious prion disease is within 1% of all patients, and infection is not a major cause of overall CJD, infectivity of prion is an important biological feature.

CJD occurs all over the world. The incidence of sCJD is about one in one million people. Although CJD has been reported to have a large number of locally clustered populations, mutations of the specific PrP genes have been observed in each cluster. Finding out what common causal factors work for sporadic and familial CJD disease has not been successful. Although research has been conducted on the path of infection, in addition to the strain CJD (vCJD) caused by eating cows infected with BSE, it has not been demonstrated by epidemiologic studies that eating Scrapie-infected sheep or goats is responsible for CJD in humans. Did. In addition, studies with Syrian hamsters have shown that eating prion can cause infection, but does not produce infection more effectively than inoculating prion directly into the cerebrum.

About 25% of patients with sporadic CJD (sCJD) suffer from nonspecific symptoms weeks or months before the onset of characteristic clinical symptoms, including helplessness and weakness, changes in appetite, changes in sleep habits, weight loss, loss of concentration, and transient time. And place confusion, hallucinations, and emotional disorders. Physical symptoms such as visual impairment, dizziness, balance disorder and sensory impairment of the limbs are also common symptoms.

However, there are some clinical differences between sporadic CJD (sCJD) and variant CJD (vCJD). Sporadic CJD shows dementia in the early stages and the onset age is 55-70 years high, but variant CJD shows dementia symptoms in the early stages of mental disorders, sensory central abnormalities, and ataxia. Also, the incidence age is generally low, between 19 and 39 years, and EEG findings are not characteristic. Most patients survive for six to twelve months after the onset of clinical manifestations, and some survive for more than five years.

There is no way to prevent or treat CJD properly. Many attempts have been made to treat prion disease, but once clinical symptoms have appeared, no treatment has been effective.

Recent studies have shown that amphotericin B, a fungal infection treatment, inhibits the proliferation of abnormal prion proteins, and a substance called photalocyanine tetrasulfate is responsible for the formation of abnormal prion proteins. Although strongly inhibited, both drugs had no therapeutic effect after the onset of clinical symptoms.

In addition, the antiviral agent amantidine, the rare drug pentosan polysulphate, and the antimalarial quinacrine have been tried, but the effect is insignificant. Peptides and antibody vaccines that modulate immunity show anti-prion effects, but remain in the experimental phase of cells and animals, and they are not achieving satisfactory results.

Prion disease has no special treatment or treatment to date, and the purpose of the ongoing treatment is to alleviate the symptoms of patients. Therefore, there is a need to find a new way to treat prion disease, an advanced neurodegenerative disease that goes beyond control.

As we enter the aging society, the number of degenerative neuropathies is expected to increase exponentially with the rapid increase of the elderly population, and the number of patients with prion is expected to increase even more than now.

In addition, concerns about the pandemic of mad cow disease (BSE), a representative animal prion disease that has occurred in animals and has been issued around the world, have not disappeared.

As a potential therapeutic agent for such neurodegenerative diseases, effects associated with the treatment of degenerative neurological diseases have been reported in selective Bcr-Abl kinase inhibitors used as therapeutic agents for leukemia (Chinese Patent CN 102406648 A). From this, if the Bcr-Abl kinase inhibitors can inhibit the degeneration of normal prions to pathogenic prions and inhibit the deposition formation of the pathogenic prions, they may be sufficiently applicable to the treatment of prion diseases, which are degenerative neurological diseases.

Therefore, the present inventors have tried to develop a novel therapeutic agent for prion disease that inhibits the deposition formation of the prion protein, and as a result, doratinib or a pharmaceutically acceptable salt thereof can be usefully used as a prophylactic and therapeutic agent for prion disease, a neurodegenerative disease. By confirming that the present invention was completed, the present invention was completed.

Therefore, the present invention is to provide a novel approach for the treatment of prion disease has not yet found a cure method as its technical problem.

In order to solve the above problems, the present invention is a selective Bcr-Abl kinase inhibitor and a neurodegenerative disease comprising Radotinib, a drug used for treating Philadelphia chromosome positive (Ph +) chronic myelogenous leukemia (CML) as an active ingredient. It provides a pharmaceutical composition for the prevention or treatment of one prion disease.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for the prophylaxis or treatment of prion disease, which is one of neurodegenerative diseases, which comprises doratinib or a pharmaceutically acceptable salt thereof represented by the following formula (I) as an active ingredient.

The present invention also provides a method of preventing or treating a prion disease in a therapeutic subject, comprising administering to the therapeutic subject a therapeutically effective amount of doratinib or a pharmaceutically acceptable salt thereof represented by Formula (I) above. do.

The present invention also provides the use of doratinib or a pharmaceutically acceptable salt thereof represented by the formula (I) for use in the prevention or treatment of prion diseases.

Radotinib is a type of receptor tyrosine kinase inhibitor, 4-methyl-N- (3- (4-methyl-1H-imidazol-1-yl) -5- (trifluoromethyl) phenyl) -3- (4- It may also be referred to as pyrazin-2-yl-pyrimidin-2-ylamino) benzamide.

Radotinib is known to inhibit one or more tyrosine kinases such as c-Abl, Bcr-Abl, receptor tyrosine kinases PDGFR, Flt3, VEGF-R, EGF-R and c-Kit, and are known to be used in lung cancer, stomach cancer, It is known to have excellent anticancer effects against cancers of various areas such as colon cancer, pancreatic cancer, liver cancer, prostate cancer, breast cancer, chronic or acute leukemia, blood cancer, brain tumor, bladder cancer, rectal cancer, cervical cancer and lymphoma. However, no literature and patents have been reported to date that doratinib is effective against prion disease, one of neurodegenerative diseases.

Radotinib may be used in the form of the free base itself or a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable" refers to a physiologically acceptable and, when administered to humans, typically does not cause an allergic or similar reaction, the salt is a pharmaceutically acceptable free acid (free acid) Acid addition salts formed by this may be exemplified, but may not be limited thereto. Acid addition salts include doratinib, 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 or tartaric acid. It may be formed from a pharmaceutically acceptable inorganic or organic acid selected from, but may not be limited thereto. These salts can be prepared by known methods, for example by treating the doratinib with the appropriate acid in the presence of a suitable solvent.

Radotinib may be in crystalline form or in the form of a solvent compound (eg, a hydrate) and both forms are within the scope of the present invention. The term "solvent compound" is a complex of various stoichiometry formed by a solute (in the present invention, a compound such as doratinib) and a solvent. Such solvents should not interfere with the biological activity of the solute. For example, the solvent may be water, ethanol, methanol, ethyl acetate or acetone, but may not be limited thereto. Solvation methods are generally known in the art.

Pharmaceutically acceptable salts of ladotinib may include both unsolvated and solvated forms.

The pharmaceutical composition of the present invention may comprise a therapeutically effective amount of doratinib. As used herein, "therapeutically effective amount" refers to an amount that exhibits a higher response than a negative control, and preferably an amount sufficient to treat or prevent prion disease, which is a neurodegenerative disease of mammals including humans. Treatment doses when the patient is a human generally range from 10 mg to 2000 mg / day, more specifically 30 mg to 1000 mg / day, depending on the severity of the condition and whether doratinib is administered alone or in combination with other drugs. It may be, but may not be limited thereto. For example, the pharmaceutical compositions of the present invention may be administered via oral or parenteral routes once or divided daily. However, the therapeutically effective amount may be appropriately changed depending on various factors such as the disease and its severity, the patient's age, weight, health condition, sex, route of administration and duration of treatment.

According to one embodiment of the invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. For example, the carrier may be inert, and may include sugars, corn starch, wheat starch, rice starch and potato starch, including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and the like. Celluloses including starch, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, and the like, and fillers such as gelatin, polyvinylpyrrolidone, and the like. . In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, but may not be limited thereto.

For example, the pharmaceutical composition may further include, but may not be limited to, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives.

The pharmaceutical compositions according to the invention can be used for the prevention or treatment of prion diseases which are neurodegenerative diseases of animals, for example mammals, in particular humans.

Specific examples of prion diseases include Kuru, Creutzfeldt-Jacob Disease (CJD), Gerstmann-Straussler-Scheinker Syndrome (GSS), and fatal family in humans. Sex insomnia (FFI), fatal sporadic insomnia, and in animals, bovine spongiform encephalopathy (BSE), mad cow disease, sheep scrapie, and transmissible minking encephalopathy (transmissible) mink encephalopathy, chronic wasting disease of deer and elk, feline spongiform encephalopathies, and the like.

Specific examples of the Creutzfeldt-Jakob disease (CJD), sporadic Creutzfeldt-Jakob disease (sCJD), familial Creutzfeldt-Jakob disease (familial CJD), variant Creutzfeldt-Jakob disease (vCJD), clinic Creutzfeldt-Jakob disease (iatrogenic) CJD) and the like, but is not limited thereto.

The route of administration of the pharmaceutical composition of the present invention may be administered parenterally or parenterally. Parenteral routes of administration may include, but are not limited to, for example, various routes of transdermal, nasal, abdominal, muscle, subcutaneous, intravenous, and the like.

For example, when administered parenterally, the pharmaceutical compositions of the present invention may be formulated according to methods known in the art in the form of injections, transdermal administration, suppositories, aerosols and nasal inhalants together with suitable parenteral carriers. have. Such injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, solvents or dispersion media comprising water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycols, etc.), mixtures thereof and / or vegetable oils Can be. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be further included. In addition, the injection may in most cases further comprise an isotonic agent such as sugar or sodium chloride. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, PA, a prescription generally known in pharmaceutical chemistry.

According to one embodiment of the invention, the pharmaceutical composition may be for oral administration to a subject.

According to one embodiment of the present invention, the oral dosage form is powder, powder, granule, tablet, capsule, oral dispersible tablet, dragee, aerosol, gel, pill, soft capsule, suspension, emulsion, aqueous medicine, Syrups, elixirs, wafers and sachets.

For inhaled dosages, ladotinib is in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be delivered conveniently. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, capsules and cartridges for use in an inhaler or blower may be formulated to contain a powder mixture of suitable powder bases.

Other pharmaceutically acceptable carriers may be referred to those described in the following references (Remington's Pharmaceutical Sciences, 19th Edition, 1955, Mack Publishing Company, Easton, PA).

The pharmaceutical composition of the present invention can effectively prevent or treat prion disease, which is one of neurodegenerative diseases, by including doratinib or a pharmaceutically acceptable salt thereof as an active ingredient. In particular, doratinib or a pharmaceutically acceptable salt thereof, which is an active ingredient of the pharmaceutical composition of the present invention, has an effect of inhibiting degeneration of normal prions to pathogenic prions and inhibiting the formation of pathogenic prions.

1 is a comparative survival graph results of a hamster control group treated with a vehicle after infection in an intraperitoneal 263K scrapie prion inoculated hamster model, and a hamster group administered orally intragastrically with doratinib after infection.

FIG. 2 shows: (i) an infected group who died 146 days after infection in a vehicle treated group after intraperitoneal infection with 263K scrapie prion, (ii) a normal control (negative control) at 146 days post infection and (iii) 263K- Brain tissue was recovered from the doratinib treated group (day 146 days post-infection with no clinical symptoms and healthy appearance; right brain extraction), and (iv) hamsters who died at 154 days in the 263K-doratinib treated group. The result was obtained by measuring the cerebral deposition level of pathogenic infectious prion protein by Western blot test.

FIG. 3 shows (i) an infected group who died 146 days after infection in a vehicle treated group after intraperitoneal infection with 263K scrapie prion, (ii) a normal control (negative control) at 146 days post infection and (iii) 263K- Each of the radotinib treated groups (the 146 days post-infection) showed no clinical symptoms and seemed healthy. Left brain extraction was performed, followed by perfusion of the brain tissues and immunohistochemical staining. Results of measurement of cerebral deposition levels and patterns.

Figure 4 is a section of the cerebellum tissue of the hamster infected with 263K scrapie prion, treated with doratinib and cultured for 5 weeks after treatment with a new doratinib drug at the corresponding concentration when replacing the culture medium every three days, the cerebellum of each experimental group The homogenates were prepared from tissue culture sections and the levels of pathogenic infectious prion protein were measured by Western blot test.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily practice the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

Example 1

In this example, the survival curves and the levels of infectious prion protein deposition in hamsters intraperitoneally infected with 263K scrapie prion were tested for the drug effect of doratinib through Western blot test.

1) Experimental Animal

6 weeks old. 90-100 g of golden Syrian hamster

2) Test Method:

A) 100 μl of 1% homogenate of 263K scrapie prion cerebral infected hamster brain tissue was inoculated intraperitoneally into golden Syrian hamster (6 weeks old, 90-100 g) and infected with scrapie prion (1 mg). / kg, daily except Sunday) is dissolved in 0.5% carboxymethylcellulose (CMC, vehicle) orally administered intragastricly. Negative control (Neg CTL) was administered orally intragastrically to doratinib (100 mg / kg, daily except Sunday) in the normal group, and vehicle (10) in the 263K intraperitoneal injection infected group with the positive control (Pos CTL). ml / kg) was administered orally intragastrically, using Survival curves of each hamster group was confirmed using GraphPad Prism 4.0.

B) Western blot test

After protein quantification, 20 μg of total protein in 10% brain homogenate of each hamster group was treated with some samples with protease (proteinase K, PK, 5-20 μg / ml, 1 hr, 37 ℃). After electrophoresis, proteins were separated by mass, followed by electrophoresis on nitrocellulose (NC) membranes, and blocking the NC membranes with 5% skimmed milk, followed by 3F4 or 3F10 anti-i-PrP antibodies (1: 3000 ~). 1: 10000) and incubated overnight at 4 ° C. and incubated with goat anti-mouse antibody-HRP for 1 hour at room temperature, and chemiluminescence was measured using SuperSignal West Pico (Thermo Scientific).

The results are as shown in Figures 1 and 2, the graph of Figure 1 obtained a statistically significant survival curve in the pharmacological test of oral administration of doratinib in the stomach to hamsters intraperitoneally infected with 263K scrapie prion. 2 was confirmed through the Western blot test that the effect of inhibiting and delaying the cerebral deposition of infectious prion with pathogenicity.

The control group administered intragastrically with a vehicle with 0.5% CMC after peripheral intraperitoneal infection with 263K scrapie prion died at 118, 119, 134 and 146 days post-infection, whereas the intragastric oral group of doratinib (100 mg / kg) was infected after infection. Died at 124, 135, 136, 140, 145, 150, 154, 164, 166, 177, 198 and 223 days. These results indicate that the orally administered group of doratinib shows a statistically significant survival curve compared to the control group (FIG. 1).

One normal control-ladotinib treated group (CTL + Radotinib) at 146 days post-infection, in contrast to the infected group (263K + CMC) who died 146 days after infection in the vehicle treated group after intraperitoneal infection with 263K scrapie prion. , healthy) and one 263K-ladotinib treatment group (263K + Radotinib, observed 146 days post-infection, with no clinical signs and healthy appearance. Right brain extraction) was performed to kill brain tissue and 263K abdominal infection. The brain tissue of one hamster that died at 154 days in the post-ladotinib treatment group was also extracted and examined for cerebral deposition levels of pathogenic infectious prion protein via Western blot (FIG. 2).

In the normal control group, no prion protein was deposited after the proteolytic enzyme PK treatment, so that no infectious prion was detected in the normal control group (lane 2 of FIG. 2), and the CMC (vehicle) treatment group (negative control group) after 263K intraperitoneal infection. ) Confirmed significant deposits of PK resistant pathogenic prion protein after 146 days (lane 4 in FIG. 2).

Very small amounts of pathogenic prion protein were found to deposit in the brain tissues of hamster individuals who appeared healthy in the intragastric oral group of doratinib after 263K intraperitoneal infection at 146 dpi (146 days post infection) (lane 8 in FIG. 2). This deposition amount was very small compared to the amount of pathogenic prion protein deposited in the brain tissue of the deceased hamster 154 days after 263K abdominal cavity infection (lane 6 in Figure 2).

These findings indicate that doratinib, administered orally intragastrically, inhibits 263K scrapie prion infections from the periphery, or that doratinib passes through the blood-brain barrier (BBB) and directly inhibits cerebral tissue. It is judged to suppress. This result is thought to be a difference between hamster individuals infected with the 263K scrapie prion peritoneal abdominal cavity.

That is, in Figure 2, the hamster who died at 154 dpi in the doratinib treatment group showed a 263K scrapie prion clinical manifestation and died, and a significant amount of pathogenic prion protein was deposited in cerebral tissue. Very few pathogenic prions were deposited in the 146 dpi hamster's cerebral tissue after 263K scrapie prion infection, which showed no clinical symptoms and appeared healthy. The results demonstrate that hamster cerebral tissue from each 263K-ladotinib treated group that died after 154 dpi had deposited much less pathogenic prion at the time of 146 dpi survival than at the end of clinical symptoms.

These results confirmed that doratinib orally administered intragastricly during infection of peripheral abdominal scrapie prion has the effect of inhibiting and delaying cerebral deposition of infectious prion with pathogenicity.

Example 2

In the present embodiment, the control group, the 263K scrapie prion infected group and the doratinib treated group were perfused, the cerebral tissues were fixed, the brain sections were prepared, and immunohistochemical staining was performed to confirm the deposition level of protease-resistant prion protein. The test was conducted using Immunohistochemistry.

1) Test Method:

After treatment with protease (PK) (20 μg / ml, 5 min, room temperature), the brain tissue sections of each group were blocked with goat serum, and the 3F10 antibody was used as the primary antibody. Anti-mouse IgG was used as an antibody to measure the level of deposition of prion protein and protease resistant infectious prion in each group.

As a result, as shown in FIG. 3, it was confirmed that doratinib orally administered intragastricly during infection of the peripheral abdominal scrapie prion of hamster could inhibit cerebral deposition of infectious prion with pathogenicity.

One normal control-ladotinib treated group (CTL + Radotinib, at 146 days post-infection) compared to the infected group (263K + CMC) who died 146 days after infection in the vehicle treated group after intraperitoneal infection with 263K scrapie prion. 3F10 anti-PrP antibody after perfusion of healthy) and one 263K-Radotinib-treated group (263K + Radotinib, which showed no clinical symptoms at day 146 post-infection. Immunohistochemical staining was performed to determine cerebral deposition levels and aspects of infectious prion protein with pathogenicity (FIG. 3).

Similar to the Western blot observation, it was observed that the prion protein was not deposited after the proteolytic enzyme PK treatment (CTL-PK-Radotinib of FIG. 3). However, a significant amount of PK resistant pathogenic prion protein was observed after 146 days in the CMC (vehicle) group after intraperitoneal infection with 263K scrapie prion (263K-PK-CMC in FIG. 3). In addition, deposition of very faint pathogenic prion proteins was observed in the left brain tissues of hamsters administered orally in the stomach of doratinib after 263K intraperitoneal infection, which had no clinical symptoms and healthy at 146 dpi (263K-Radotinib and 263K-PK-Radotinib in FIG. ).

Similar to Western blot results, these test results confirmed that rabitinib orally administered intragastricly could inhibit cerebral deposition of pathogenic infectious prions upon infection with peripheral abdominal scrapie prion.

Example 3

In this example, a test was conducted to confirm the effect of inhibiting the pathogenic prion production of doratinib using cerebellum tissue ex vivo.

1) Test Method:

Sections of the cerebellum of the golden Syrian hamster at postnatal 12, p12 were sectioned. Cerebellar sections were exposed to 1% homogenate of 263K scrapie prion cerebral infected hamster brain tissue at 4 ° C. for 1 hour and incubated at 37 ° C. by treating doratinib at a concentration of 10, 20, or 40 μM. When the tissue culture solution was exchanged at 3 days intervals, dolatinib was dissolved in the culture solution at each concentration, and the control group was exposed only to the culture medium instead of the infected brain homogenate and all cerebellar tissues were cultured for 5 weeks.

As a result, as shown in Figure 4, it was confirmed that doratinib treated with ex vivo cerebellar tissue culture medium inhibited the generation of pathogenic prion from 263K scrapie prion infection.

Twelve days old golden Syrian hamster cerebellar tissue was sectioned and infected with 263K scrapie prion, followed by treatment with 10, 20, or 40 μM of radotinib in the culture medium and fresh doratinib at the appropriate concentrations for 3 weeks. After incubation, homogenates were prepared from cerebellar tissue culture sections of each experimental group to measure the deposition level of infectious prion protein with pathogenicity (FIG. 4).

As shown in FIG. 4, no pathogenic prion was deposited in cultured cerebellar tissue of the normal control group (lane 2 in FIG. 4), but PK resistant prion was detected in the 263K scrapie prion cerebral tissue inoculation group (lane 4 in FIG. 4). ).

However, it was confirmed that in the treatment group of ladotinib, the deposition of PK resistant pathogenic prion was suppressed depending on the treatment concentration (lane 5-7 in FIG. 4).

These results indicate that doratinib treated in ex vivo cerebellar tissue culture medium inhibited the prion conversion or prion amplification reaction from the beginning of 263K scrapie prion infection or inhibited the formation of pathogenic prion by inhibiting pathogenic prion deposition.

Example 4

In this example, the blood brain barrier barrier test of doratinib was performed in mice.

Experimental Animals: 6-week-old male ICR mouse

2) Test substance: Radotinib

3) Test Method:

The following tests were conducted to determine the distribution of doratinib administered in vivo and its distribution in the brain, which is the point of action.

ICR mice, 6 weeks old, and females were orally administered with a dose of 50 mg / kg of the test substance doratinib, respectively. Before dosing, blood was collected at 1, 2, 3, 4, 6, 8, and 12 hours after dosing to separate plasma, and brains were extracted and analyzed for doratinib concentration by LC-MS / MS.

As a result, as shown in Table 1, radotinib has been confirmed pharmacokinetic values in plasma and brain tissue, showing that the absorption rate of doratinib in plasma and brain tissue is excellent, in particular, doratinib absorption ratio in plasma Permeability in the brain tissue was 0.74%, indicating that the blood-brain barrier was excellent.

That is, from the above experimental results, it was confirmed that doratinib has excellent blood brain barrier permeability. Blood-brain barrier permeability is one of the very important factors in the development of drugs for the treatment of brain diseases, it can be interpreted that doradinib is more effective in the treatment of neurodegenerative prion disease.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is shown by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. do.

Claims (12)

1. A pharmaceutical composition for the prophylaxis or treatment of prion disease, a neurodegenerative disease, which comprises as an active ingredient Radotinib or a pharmaceutically acceptable salt thereof represented by Formula (I):

   
2. The pharmaceutical composition according to claim 1, wherein doratinib or a pharmaceutically acceptable salt thereof inhibits or eliminates the production and deposition of modified prions.
3. The method of claim 1, wherein the prion disease is Kuru, Creutzfeldt-Jacob Disease (CJD), Gerstmann-Straussler-Scheinker Syndrome (GSS), fatal familial insomnia (FFI), fatal sporadic insomnia, bovine spongiform encephalopathy (BSE), mad cow disease, scrapie, transmissible mink encephalopathy, and chronic chronic pharmaceutical composition, characterized in that it is selected from the group consisting of wasting disease) and feline spongiform encephalopathies.
4. 4. The Creutzfeldt-Jakob disease according to claim 3, wherein Creutzfeldt-Jakob disease is sporadic Creutzfeldt-Jakob disease (sCJD), familial Creutzfeldt-Jakob disease (vCJD), variant Creutzfeldt-Jakob disease (vCJD), and immature Creutzfeldt-Jakob disease (iatrogenic CJD). Pharmaceutical composition, characterized in that selected from the group consisting of.
5. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier.
6. A pharmaceutical composition according to claim 1 for administration to an animal.
7. The pharmaceutical composition of claim 6, wherein the animal is a mammal.
8. 8. The pharmaceutical composition of claim 7, wherein the mammal is a human.
9. The pharmaceutical composition according to any one of claims 1 to 8, which is for oral administration.
10. The method of claim 9, wherein the oral dosage form is powder, powder, granule, tablet, capsule, oral dispersible tablet, dragee, aerosol, gel, pill, soft capsule, suspension, emulsion, aqueous medicine, syrup, elixir Pharmaceutical composition, characterized in that it is selected from the group consisting of elixir, wafer and sachet.
11. The pharmaceutical composition according to any one of claims 1 to 8, which is for parenteral administration.
12. The pharmaceutical composition of claim 11, wherein the form of parenteral administration is selected from the group consisting of injections, transdermal administrations, suppositories, aerosols and nasal inhalants.

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