KR102106032B1 - Pharmaceutical composition for treating multiple sclerosis comprising pracinostat - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment of multiple sclerosis, including pracinostat. Pracinostat according to the present invention can be useful as a new treatment for multiple sclerosis because it can control inflammatory cells by selectively inducing the expression of the immunomodulatory enzyme IDO only in inflammatory lesions in the central nervous system.

Description

Pharmaceutical composition for treating multiple sclerosis comprising pracinostat}

The present invention induces the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) only in inflammatory lesions in the central nervous system, thereby treating prascinostat that can treat multiple sclerosis. It is about use.

Multiple sclerosis is an autoimmune disease that occurs in the central nervous system and has pathological features of demyelination and axon injury of nerve cells due to inflammation. The most common symptom is sensory and motor paralysis, and the symptoms vary depending on the area of inflammation. During the course of the disease, relapses and remissions can be repeated and progressively worsened. It can occur in all age groups, but is more common in adults 20-40 years of age, especially in women. The incidence of whites in Europeans is high, and prevalence is high in Northern Europe, the northern United States, southern Canada, and southern Australia. Through this, it is assumed that multiple sclerosis is caused by a combination of genetic and environmental factors.

Symptoms repeat relapse and remission according to changes in the autoimmune state, and over time, recurrence repeats, and damage accumulates, so symptoms do not improve completely and the disorder remains. Therefore, early treatment to reduce the frequency of recurrence and active anti-inflammatory treatment to minimize acute phase damage in case of relapse are key. In acute relapses, high-dose corticosteroids are used as standard treatment, and relapsing remitting multiple sclerosis is beta interferon, glatiramer acetate, and fingolimod disease. It is used as a palliative treatment. In spite of primary disease alleviation treatment, mitosantrone or natalizumab is used in cases of frequent recurrence or secondary progressive multiple sclerosis, but the therapeutic effect is negligible.

Autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis and is useful for the development of disease causes and therapeutics. The EAE model revealed that myelin antigen-specific Th1 and Th17 cells are the major etiological cells. In particular, it was recently confirmed that the unbalance between Th17 cells and Treg cells is one of the major causes. In addition, a new fact has been revealed that inflammatory reactions are caused by various immune mechanisms, such as deposition of inflammatory macrophages and promoting inflammation of glial cells. This suggests that not only the inflammatory cells migrated from the periphery but also the inflammatory activity of cells residing in the nerve tissue should be included in the treatment strategy.

Pracinostat (Pracinostat) is 3- [2-butyl-1- (2-diethylaminoethyl) -1 H -benzoimidazol-5-yl] -N -hythoxyacrylami-hydrochloride structure It is a histone deacetylase inhibitor (HDAC inhibitor). It strongly inhibits Class I, II and IV HDACs and has excellent pharmacokinetic properties. It has excellent cancer cell death inducing effect, and is currently in phase 3 clinical trials for acute myeloid leukemia.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan metabolic enzyme whose expression is induced by an initial inflammatory reaction, thereby suppressing excessive inflammatory reactions and inducing immune tolerance. Have IDO inhibits the proliferation of inflammatory T cells and induces the production of Treg by generating kynurenine (KYN), a metabolite that functions as an immunosuppressive function simultaneously with induction of tryptophan depletion. Recently, KYN was found to be an endogenous ligand of the aryl hydrocarbon receptor (ARH), an immunomodulatory transcription factor, and it was clear that IDO inhibits the function of overactivated immune cells by the mechanism of IDO-KYN-AHR. . It has been confirmed through animal experiments and clinical studies that lowering of IDO expression and lowering activity are associated with various inflammatory diseases. In addition, it has been reported that inducing the expression of IDO by external administration of a substance can treat intractable inflammatory diseases.

existing IDO inducers (TLR9 agonists, anti-4-1BB, CTLA-4Ig) promote in vivo interferon-gamma (IFN-γ) production to induce IDO expression. Therefore, it also carries the risk of promoting an inflammatory reaction. In addition, existing immunosuppressive drugs such as calcineurin inhibitors and steroids block interferon-gamma production, and thus cannot be combined with these drugs. Therefore, a novel substance capable of selectively inducing IDO expression to a lesion by an interferon-gamma-independent mechanism may be an innovative treatment for multiple sclerosis.

An object of the present invention is to provide a pharmaceutical composition for the treatment of multiple sclerosis containing a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

1. A pharmaceutical composition for treating an inflammatory disease or preventing recurrence of an inflammatory disease in a subject, comprising a compound represented by the following formula (I): or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier:

[Formula I]

R ¹ is of the formula — (CR ³ R ⁴ ) _m — (CR ⁵ R ⁶ ) _n — (CR ⁷ R ⁸ ) _o —NR ⁹ R ¹⁰ ;

R ² is selected from the group consisting of F, cyano, C ₂ -C ₆ alkenyl or C ₂ -C ₆ alkynyl substituted or unsubstituted alkyl, and = O substituted or unsubstituted heteroalkyl;

R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently selected from the group consisting of H and methyl;

R ⁹ and R ¹⁰ are each independently selected from the group consisting of H, hydroxyalkyl and alkyl;

m, n and o are integers independently selected from the group consisting of 0, 1, 2, 3 and 4, respectively.

2. Formula I is a compound represented by the following Formula II, a pharmaceutical composition:

[Formula II]

.

.

3. The inflammatory disease is multiple sclerosis, pharmaceutical composition.

4. The pharmaceutical composition is administered orally, pharmaceutical composition.

5. The subject is indolamine 2,3-dioxygenase (indoleamine 2,3-dioxygenase; IDO) is not knocked out, pharmaceutical composition.

6. The compound is a pharmaceutical composition that increases the expression of IDO in the subject.

7. The pharmaceutical composition, the expression of IDO is increased in the neurons of the subject.

8. Health functional food for alleviating inflammatory diseases or preventing recurrence of inflammatory diseases, comprising a compound represented by the formula (I) or a food-acceptable salt thereof:

[Formula I]

R ¹ is of the formula — (CR ³ R ⁴ ) _m — (CR ⁵ R ⁶ ) _n — (CR ⁷ R ⁸ ) _o —NR ⁹ R ¹⁰ ;

R ² is selected from the group consisting of F, cyano, C ₂ -C ₆ alkenyl or C ₂ -C ₆ alkynyl substituted or unsubstituted alkyl, and = O substituted or unsubstituted heteroalkyl;

R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently selected from the group consisting of H and methyl;

R ⁹ and R ¹⁰ are each independently selected from the group consisting of H, hydroxyalkyl and alkyl;

m, n and o are integers independently selected from the group consisting of 0, 1, 2, 3 and 4, respectively.

9. Formula I is a compound represented by the following Formula II, health functional food:

[Formula II]

.

.

10. The inflammatory disease is multiple sclerosis, health functional food.

According to the present invention, the expression of IDO in the inflammatory site of the spinal cord is induced by oral administration of pracinostat in an animal model of multiple sclerosis of autoimmune encephalomyelitis (EAE), thereby reducing the severity of the disease, reducing histopathological damage, and etiological cytokines. Since it exhibits a reduction and removal effect of tissue-infiltrating inflammatory cells, prasinostat can effectively treat multiple sclerosis.

1 shows the effect of treating prascinostat for multiple sclerosis.
Figure 2 shows the effect of prasinostat IDO-dependent disease treatment.
Figure 3 shows the results of induction of IDO expression in neurons in inflammatory lesions of prasinostat.
Figure 4 shows the effect of treatment of inflammation by the mechanism of IDO-AhR Prasinostat.

Hereinafter, the present invention will be described in more detail through the following examples. However, the present invention is not limited by these examples.

The term "compound of the present invention" as used herein means both the compound represented by the formula (I) and pharmaceutically acceptable salts thereof:

[Formula I]

R ¹ is of the formula — (CR ³ R ⁴ ) _m — (CR ⁵ R ⁶ ) _n — (CR ⁷ R ⁸ ) _o —NR ⁹ R ¹⁰ ;

R ² is selected from the group consisting of F, cyano, C ₂ -C ₆ alkenyl or C ₂ -C ₆ alkynyl substituted or unsubstituted alkyl, and = O substituted or unsubstituted heteroalkyl;

R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently selected from the group consisting of H and methyl;

R ⁹ and R ¹⁰ are each independently selected from the group consisting of H, hydroxyalkyl and alkyl;

m, n and o are integers independently selected from the group consisting of 0, 1, 2, 3 and 4, respectively.

In one embodiment of the present invention, the compound represented by the formula (I) is a compound represented by the formula (II), prasinostat, 3- [2-butyl-1- (2-diethylaminoethyl) -1 H -benzoimidazole -5-yl] -N -hythoxyacrylamide or 3- [2-butyl-1- (2-diethylaminoethyl) -1 H -benzoimidazol-5-yl] -N -hythoxyacrylami- It may be hydrochloride:

[Formula II]

.

.

The prasinostat can inhibit the activity of myelin-specific inflammatory T cells.

The prasinostat can inhibit the activity of innate immune cells.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease, an animal model of multiple sclerosis in humans with inflammatory cell infiltration and myelin loss in the central nervous system.

The present inventors confirmed that multiple sclerosis is treated when ingested with pracinostat and shows superior efficacy than the existing treatment drug, pincolimod. An important finding is that the expression of IDO is induced only in neurons in the inflamed region by prasinostat, and the major etiological cells, Th17 cells and inflammatory macrophages, are effectively removed by the IDO-KYN-AHR mechanism, resulting in a therapeutic effect. Was confirmed.

The term “pharmaceutically acceptable salt” as used herein is a salt that retains the desired biological activity of the target compound and exhibits minimal undesirable toxic effects, which is a salt with a pharmaceutically acceptable acid or base. It is a salt. Pharmaceutically acceptable acids include mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid, double phosphoric acid, hydrobromic acid or nitric acid, citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesul Organic acids such as phosphonic acid, salicylic acid, stearic acid, benzenesulfonic acid or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metals containing sodium or potassium and alkaline earth metal hydroxides containing calcium or magnesium and organic bases such as alkyl amines, aryl amines or heterocyclic amines.

Those skilled in the art will further appreciate that certain compounds of the present invention, present in crystalline form, or various solvates thereof, may exhibit polymorphism, the ability to occur with various crystalline structures. These various crystalline forms are typically known as "polymorphs". The present invention includes all such polymorphs. Polymorphs have the same chemical composition, but differ in packing, geometrical arrangement and other technical properties of the crystalline solid state. Thus, polymorphs can have different physical properties, such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns that can be used for identification. Those skilled in the art will appreciate that different polymorphs can be prepared, for example, by changing or adjusting the reaction conditions or reagents used in the preparation of the compound. For example, changes in temperature, pressure, or solvent can produce polymorphs.

In addition, one polymorph can spontaneously convert to another polymorph under certain conditions.

The present invention also includes various deuterated forms of the compound represented by formula (I). Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom. The skilled person will know how to synthesize the deuterated form of the compound represented by formula (I). Commercially available deuterated starting materials can be used for the preparation of deuterated forms of compounds of formula (I), or they can be synthesized using conventional techniques using deuteration reagents.

The compounds of the present invention and compositions comprising them can be administered in a variety of dosage forms. In one embodiment of the present invention, the pharmaceutical composition comprising the compound of the present invention may be formulated in a form suitable for oral, rectal, parenteral, nasal or transdermal administration, or administration by inhalation or suppository.

The method of administration is not particularly limited, but any method of oral or parenteral administration may be used, and systemic or topical administration is possible, but systemic administration is more preferred and oral administration is most preferred. The administration to the patient's body is preferably oral administration, and specifically, 5-10 administrations once a day are basic, but further administration may be used. Moreover, the administration time may be a short time or a long-time administration. Liquid dispersions for oral administration can be syrups, emulsions and suspensions.

The pharmaceutical composition of the present invention can be formulated by a method known by those skilled in the art. For example, it can be used parenterally in the form of an injectable solution of a sterile solution or suspension with water or other pharmaceutically acceptable liquid, if necessary. For example, in a suitable combination with a pharmaceutically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, excipient, vehicle, preservative, binder, etc. It can be formulated by admixing in the form of a unit dose required for pharmaceutical practice recognized as. The active ingredient amount in the formulation is to obtain a suitable dose in the indicated range.

In addition, the sterile composition for injection may be prescribed according to the usual formulation practice using an excipient such as distilled water for injection. As an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, suitable dissolution aids, for example For example, alcohols, specifically ethanol, poly alcohols, for example, propylene glycol, polyethylene glycol, nonionic surfactants, for example, polysorbate 80 (TM), can be used in combination with HCO-50. Sesame oil and soybean oil are mentioned as an oily liquid, and can be used together with benzyl benzoate and benzyl alcohol as a dissolution aid.

Examples of injection formulations can be administered by intravenous injection, intraarterial injection, selective intraarterial injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intraventricular injection, intracranial injection, intramedullary injection, etc. Preferably intravenous injection.

In addition, buffers such as phosphate buffers, sodium acetate buffers, analgesic agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, antioxidants can be combined. The prepared injection solution is usually filled into a suitable ampoule.

Suspensions and emulsions can contain, for example, natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol as carriers. Suspensions or solutions for intramuscular injection, together with the active compound, in a pharmaceutically acceptable carrier, such as sterile water, olive oil, ethyl oleate, glycols, such as propylene glycol, and, if necessary, suitable amounts Lidocaine hydrochloride.

The compounds of the present invention may exist in solid or liquid form. In the solid state, the compounds of the present invention may exist in crystalline or amorphous form, or mixtures thereof. For compounds of the present invention in crystalline form, the skilled artisan will recognize that pharmaceutically acceptable solvates in which solvent molecules are incorporated into the crystalline lattice during crystallization can be formed. Solvates can include non-aqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine and ethyl acetate, or they can include water as a solvent incorporated into the crystalline lattice. Solvates, which are solvents in which water is incorporated into a crystalline lattice, are typically referred to as “hydrates”. Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. The present invention includes all such solvates.

The present invention also includes prodrugs that react in vivo to provide the compounds of the present invention.

The compounds of the present invention can be prepared based on synthetic routes apparent to those skilled in the art.

When further providing administration of one or more drugs to the compositions or foods of the invention, they may be administered simultaneously, sequentially or separately. These can be packaged together or separately. Also, they need not be administered simultaneously or they need not be in the same dosage form. Individual administration is the drug is administered as part of the same overall dosing regimen, and can be administered on the same day or on different days. Simultaneous means that the drugs must be taken together or formulated as a single composition. Sequential means that the drugs are administered at approximately the same time, preferably within about 1 hour of each other.

As used herein, the term "pharmaceutically acceptable carrier" is used when administering a compound of the present invention as a diluent, adjuvant, excipient or vehicle, and is approved by a national regulatory agency, or an animal, more specifically a human being. It is listed in the commonly known pharmacopoeia for use in the. The pharmaceutical carrier may be liquid such as water or oil, and the oil includes oil derived from petroleum, animal, plant or synthetic materials, and oils such as peanut oil, soybean oil, mineral oil and sesame oil. Pharmaceutically acceptable carriers include saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica and urea. When administered to a patient, the compounds of the present invention and pharmaceutically acceptable carriers are preferably sterile. Salt solutions, liquid dextrose and glycerol solutions can also be used as liquid carriers, particularly injectable solutions. Suitable pharmaceutical carriers may also include additives such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water and ethanol. The composition of the present invention may contain a small amount of wetting agent, emulsifying agent, or pH buffering agent as necessary. The compositions of the present invention may take solutions, emulsions, sustained release formulations, or other formulations suitable for use.

 The term “inflammatory disease” as used herein includes, but is not limited to, rheumatoid arthritis, Crohn's disease, asthma, Alzheimer's disease, multiple sclerosis, dry, ulcerative colitis, osteoarthritis or ankylosing spondylitis.

The therapeutically effective amount of the pharmaceutical composition is not particularly limited, preferably 5 mg / kg to 50 mg / kg cell / kg, more preferably 10 mg / kg to 40 mg / kg, and 20 mg / kg It is most preferable that it is 30 mg / kg.

<Example 1> Prascinostat treatment effect of multiple sclerosis

In order to investigate the therapeutic effect of prascinostat according to the present invention on multiple sclerosis, induce autoimmune encephalomyelitis in C57BL / 6 mice as follows and evaluate the efficacy by administering pracinostat (Selleckchem, Cat No. S1515) Did.

Test Day 0 Myelin oligodendrocyte glycoprotein _35-55 (myelin oligodendrocyte glycoprotein _35-55 ; MOG _35-55 , Peptron) (200 ㎍), heat-treated Mycobacterium tuberculosis, heat killed mycobacterium tuberculosis, Difco, Cat No. 231141) (500 μg), adjuvant (Complete freund's adjuvant, Sigma aldrich, Cat No. F5506) was mixed and then immersed for 7 minutes. 100 μl subcutaneous injection of the submerged peptide into both flanks of C57BL / 6 mice (7 weeks old, female, 17 ± 2 g), followed by pertussis toxin, Sigma aldrich, Cat No. P2980 ) (200 ng) 100 μl was administered to the tail vein. On the second day of the experiment, the same amount of pertussis toxin was administered intravenously. The mice were checked to see if immersion was leaking from the injected site, and visually observed from the 7th day of the experiment to determine whether multiple sclerosis was onset.

In the prasinostat administration group according to the present invention, a cremophor EL-ethanol mixture (1: 1, v / v) corresponding to 15% (v / v) of the administration dose of 30 mg / kg prasinostat per mouse. ), Then diluted in phosphate buffered saline, and administered 200 µl orally every day 10 times from Experiment 13 to Experiment 22. In addition, treatment of multiple sclerosis by administering 200 μl of Fingolimod, a treatment for multiple sclerosis, which is currently used in clinical practice as a drug control, at 1 mg / kg per mouse, 30 times daily from Experiment 13 to Experiment 42 Efficacy was compared, and a solvent (Vehicle) control group was administered with a Cremophor EL-ethanol-phosphate buffered saline mixture (7.5: 7.5: 85, v / v / v) in the same manner as the pracinostart administration group. The multiple sclerosis index was visually observed with a severe index system classified into 0-5 steps from the 7th day of the experiment and recorded at intervals of 2 days.

The symptoms of autoimmune encephalomyelitis were indexed according to the following items.

0 No symptoms

1 Power is lost at the tail

2 The tail is weak and the hind legs are weakened

3 hind leg paralysis

4 Paralysis of the hind legs and weakness of the forelimbs

5 Mortality or death

As a result of the analysis, it was confirmed that in the experimental group, multiple sclerosis developed from the 7th day of the experiment, and an acute reaction with a severity index of 3.0 or higher was induced by 100% on the 18th day of the experiment. The mice in the solvent control group had a severity index of 3.08 ± 0.41 on the 18th day of the experiment with acute reaction period and 2.91 ± 0.41 on the 34th day of the chronic reaction period with the experiment. The solvent control group showed a relapse-remitting pattern and a high severity index throughout the experiment. The pingolimod treatment group was 1.67 ± 0.17 on the 20th day of the experiment, and 1.0 ± 0 on the 34th day of the experiment, showing a relieving acute and chronic response effect compared to the solvent control group. However, in the group administered with pingolimod, the severity index began to increase from day 42 of the experiment immediately after the end of dosing, and showed a high severity index of 3.67 ± 0.33 on the 46th day of the experiment. The compound prasinostat 30 mg / kg administration group of the present invention was able to confirm a statistically significant therapeutic effect compared to the solvent control group from the 22nd day of the experiment. Even after discontinuation of administration (1.25 ± 0.25 on the 34th day of the experiment and 1.42 ± 0.42 on the 54th day of the experiment), a statistically significant treatment effect was maintained compared to the solvent control group (*, p <0.05 compared to the solvent control group; **, p <0.01; ** *, p <0.001, see FIG. 1). This treatment maintenance effect showed a statistically significant difference compared to the group administered with pingolimod from day 44 of the experiment (+, p <0.05; ++, p <0.01; +++, p <compared to the group administered with pingolimod). 0.001, see FIG. 1). Prasinostat of the present invention showed excellent initial treatment and continuous recurrence prevention effect when administered 30 mg / kg.

A tissue sample was prepared by extracting the spinal cord of the mouse on the 18th day of the experiment, in which the 5 times oral administration of prasinostat of the present invention was completed. After the spinal cord of the extracted mouse is fixed with 4% paraformaldehyde (sigma aldrich), paraffin infiltration is performed using a tissue processor (Leica), and a paraffin block is fabricated using a tissue embedding (Embedding center, Leica). Did. The prepared paraffin block was cut to a thickness of 5 μm with a microtome (Leica) to prepare a tissue section.

Hematoxylin (100%) & eosin (0.15%) staining was performed on the completed tissue sections to perform histopathological analysis of the spinal cord. Histological analysis was performed using Nanozoomer 2.0 RS (Hamammatsu).

As a result of histopathological analysis, inflammatory cell infiltration of the spinal cord lesions was significantly reduced in the tissue treated with pracinostat compared to the solvent control group (see FIG. 1).

The mRNA sample was prepared by extracting the spinal cord of mice on the 18th day of the experiment, in which 5 times the oral administration of prasinostat of the present invention was completed. To extract the mRNA, spinal cord tissue was ground with a homogenizer to obtain a homogeneous suspension. MRNA from the homogeneous suspension was extracted by phenol-chloroform sedimentation using Trizol reagent (Invitrozen, Cat No. 15596018). Synthesis of cDNA by reverse transcription from the isolated RNA and confirmation of inflammatory cytokine expression by real-time polymerase chain reaction (real-time PCR) using iQ SYBR-Green Supermix (Bio-rad) in CFX96 (Bio-rad) detection system Did. The relative value of the amount of enzyme expression was compared in the ΔΔct method using GAPDH as a control enzyme. One fold was set using the WT mouse spinal cord as a control.

The annealing temperature of the real-time polymerase chain reaction was set to 58 ° C, and was performed under the conditions of 45 cycles, and the following primer sequences were used. Mouse IL-17A forward, 5'-TTT AAC TCC CTT GGC GCA AAA-3 'and reverse, 5'-CTT TCC CTC CGC ATT GAC AC-3'; Mouse IL-6 forward, 5'-CAT GTT CTC TGC GAA ATC GTG G-3 'and reverse, 5'-AAC GCA CTA GGT TTG CCG AGT A-3'; Mouse TNF-α forward, 5′-CCA CAC CGT CAG CCG ATT TG-3 ′ and reverse, 5′-CAC CCA TTC CCT TCA CAG AGC-3 ′; Mouse IFN-γ forward, 5′-ATG AAC GCT ACA CAC TGC ATC-3 ′ and reverse, 5′-CCA TCC TTT TGC CAG TTC CTC-3 ′; Mouse IL-23 forward, 5'-ATG CTG GAT TGC AGA GCA GTA-3 'and reverse, 5'-ACG GGG CAC ATT ATT TTT AGT CT-3'; Mouse CSF2 forward, 5'-GGC CTT GGA AGC ATG TAG AGG-3 'and reverse, 5'-GGA GAA CTC GTT AGA GAC GAC TT-3'; Mouse IL-1β forward, 5'-CTC GTG CTG TCG GAC CCA TAT-3 'and reverse, 5'-TTG AAG ACA AAC CGC TTT TCC A-3'; Mouse GAPDH forward, 5'-TTC ACC ACC ATG GAG AAG GC-3 'and reverse, 5'-GGC ATG GAC TGT GGT CAT GA-3'.

The expression levels of inflammatory cytokines IL-17A, IL-6, TNF-α, IFN-γ, IL-23, CSF2 (GM-CSF), and IL-1β in spinal cord lesions are significantly higher than those of the solvent control group due to the administration of prasinostat. Decrease (*, p <0.05; **, p <0.01; ***, p <0.001 compared to the solvent control, see FIG. 1).

Therefore, prasinostat of the present invention has a therapeutic effect in a mouse model of multiple sclerosis, and it is possible to present a useful treatment strategy as a novel multiple sclerosis oral treatment agent because the efficacy of preventing recurrence persists even after administration is discontinued.

<Example 2> Pracinostat IDO-dependent disease treatment effect

Autoimmune encephalomyelitis in wild type (WT, normal) mice and IDO-knockout (IDO-KO, gene deficiency) mice as follows to determine whether the therapeutic effect of autoimmune encephalomyelitis of prasinostat according to the present invention is IDO dependent And induction of prasinostat was administered to evaluate its efficacy.

The disease was induced in WT C57BL / 6 mice and IDO-KO C57BL / 6 mice (7 weeks old, female, 17 ± 2 g) in the same manner as in FIG. 1A, and prasinostat (30 mg / kg, 1 day) Once a total of 10 times from 13th to 22nd) and a solvent control group were administered orally. The autoimmune encephalomyelitis symptoms were evaluated according to the same criteria as in FIG. 1.

The 30 mg / kg administration group of prasinostat in WT mice showed a statistically significant treatment effect compared to the solvent control group from the 16th day of the experiment, and the effect continued until the end of the experiment. On the other hand, the IDO-KO mouse's prasinostat administration group showed a high severity similar to that of the IDO-KO mouse solvent control group, and no disease alleviation effect was observed. And compared to the pracinostat administration group of the WT mouse, the disease severity of the pracinostat administration group of the IDO-KO mouse was significantly higher than that of the administration group of the IDO-KO mouse. , p <0.05; ++, p <0.01; +++, p <0.001, see FIG. 2).

The spinal cord of the mouse was extracted on the 18th day of the experiment on which the 5th oral administration of the prasinostat of the present invention was completed, and the tissue was stained in the same manner as in “Fig. 1B”.

As a result of histopathological analysis, IDO-KO mice showed no effect of reducing inflammatory cell infiltration by administration of prasinostat (see FIG. 2).

On the 18th day of the experiment in which the oral administration of prasinostat 5 times of the present invention was completed, the spinal cord of the mouse was extracted to confirm the expression of inflammatory cytokines in the same manner as in FIG. 1.

In the case of WT mice, the expression levels of inflammatory cytokines IL-17A, IL-6, TNF-α, IFN-γ, and IL-1β in the spinal cord lesion were significantly reduced compared to the solvent control group due to the administration of prasinostat, but IDO-KO Mice were not affected by prasinostat administration (see FIG. 2).

Therefore, it was confirmed that the prasinostat of the present invention has an effect of treating multiple sclerosis depending on IDO.

<Example 3> Induction of IDO in neurons in inflammatory lesions of prasinostat

It was confirmed that the disease treatment effect of prasinostat according to the present invention is IDO dependent. Thus, in order to identify IDO expressing and expressing cells of prasinostat, induction of autoimmune encephalomyelitis in C57BL / 6 mice as shown in FIG. 1A, administration of pracinostat 5 times, extraction of spinal cord lesions was conducted. . Protein samples, mRNA samples, and tissue samples were prepared from the extracted spine.

First, in order to investigate IDO protein expression in the spine, a homogenous suspension was obtained by grinding with a homogenizer. The homogeneous suspension is 5 mM DL-Dithiothreitol (DTT, Fluka, Cat No. 2224687), 5 mM EDTA (pH 8.0), 1X protease inhibitor cocktail (SIGMA FAST) in mammalian cell PE LB buffer (G-Biosciences, Cat No. 2224687) Protease inhibitor Cocktail Tablets, EDTA-free, Sigma-Aldrich, Cat No. S8830) was dissolved in the added solution. The homogeneous suspension was used at a concentration of 20 μg.

Protein was separated on 15% SDS-PAGE and transferred to a nitrocellulose membrane (Whatman). The membrane of the transferred protein was blocked with 5% skim milk (skim-milk, sigma aldrich). Anti-IDO (Adipogen, Cat No. AG-25A-0032-C100, 1: 2,000 dilution), ß-Actin (Santa cruze, Cat No. sc-1615-R, 1: 1,000 dilution) overnight on the blocked membrane Treatment. Expression of each protein was confirmed through LAS-3000 (Fujifilm, Tokyo, Japan), and the changes of proteins were compared using Multi Gauge v.2.2 software (Fujifilm).

IDO expression was significantly increased in the spinal cord lesions of autoimmune encephalomyelitis by 5 times orally administering prasinostat of the present invention (see FIG. 3).

Next, to quantify IDO expression, spinal cord tissue was ground with a homogenizer to obtain a homogeneous suspension. MRNA from the homogeneous suspension was extracted by phenol-chloroform sedimentation using Trizol reagent (Invitrozen, Cat No. 15596018). Synthesis of cDNA by reverse transcription from the separated RNA and IDO expression was confirmed by real-time polymerase chain reaction (real-time PCR) using iQ SYBR-Green Supermix (Bio-rad) in a CFX96 (Bio-rad) detection system. The relative value of the amount of enzyme expression was compared in the ΔΔct method using GAPDH as a control enzyme. One fold was set using the WT mouse spinal cord as a control.

The annealing temperature of the real-time polymerase chain reaction was set to 58 ° C, and was performed under the conditions of 45 cycles, and the following primer sequences were used. Mouse IDO forward, 5'-CAC CAT GGC GTA TGT GTG GAA-3 'and reverse, 5'-TGC CAG GAC ACA GTC TGC ATA AG-3'; Mouse GAPDH forward, 5'-TTC ACC ACC ATG GAG AAG GC-3 'and reverse, 5'-GGC ATG GAC TGT GGT CAT GA-3'.

The increase in IDO mRNA expression by prasinostat administration showed a statistically significant difference compared to the solvent control group (*, p <0.05; **, p <0.01; ***, as compared to the solvent control group).

Next, in order to identify the cells expressing IDO by prasinostat, tissue samples were prepared in the same manner as in FIG. 1A.

Subsequently, for the analysis of immunohistochemistry, tissue sections are dehydrated with xylene and ethanol (EtOH), and then an antigen unmasking solution (Vector, Cat No. H-3301) Was restored. The anti-IDO (Adipogen, Cat No. AG-25A-0032-C100, 1: 100) was treated overnight with a tissue treated with a CAS block (CAS block, Thermo Fisher, Cat No. 00-8120) and blocked. After treating the secondary antibody against IDO biotinylated anti-rabbit antibody (Biotinylated anti-rabbit Ab, Vector, Cat No. BA-1100, 1: 100 dilution) for 2 hours, Vectastain ABC kit (Vector, Cat No. .PK-4000) for 1 hour. Counter-staining with DAB (3,3-diaminobenzidine) and observed with Nanozoomer 2.0 RS (Hamammatsu).

It was confirmed by histochemical method that IDO is selectively expressed in cells morphologically seen as neurons in the spinal cord lesion of autoimmune encephalomyelitis due to oral administration of prasinostat of the present invention (see FIG. 3).

In order to more clearly confirm the expression of IDO in neurons, dual immunofluorescence was performed. The tissue sections were dehydrated with xylene and ethanol (EtOH) and then recovered with an antigen unmasking solution (Vector, Cat No. H-3301). Anti-NEUN (Millipore, Cat No. MAB377, 1: 100 dilution) and anti-IDO (Adipogen, Cat No. AG-25A-) in tissues treated and blocked with a solution of CAS block (Thermo Fisher, Cat No. 00-8120) 0032-C100, 1: 100) overnight. Secondary antibody Alexa 488 bound anti-mouse antibody against NEUN (Molecular probe, Cat No. A-11001, 1: 100 dilution) and secondary antibody Alexa 594 bound anti-rabbit antibody against IDO (Molecular probe, Cat No. A-11037, 1: 100 dilution) was treated for 2 hours, followed by control staining with DAPI and observed by fluorescence in situ hybridization.

Through this, it was confirmed through fluorescence microscopic observation that IDO expressing cells were NEUN ⁺ neurons (see FIG. 3).

 Therefore, it was found that the prasinostat of the present invention induces IDO expression in neurons in spinal cord lesions of an experimental multiple sclerosis mouse model and has therapeutic efficacy.

<Example 4> Prasinostat's inflammation treatment effect by IDO-AhR mechanism

It was confirmed that the treatment effect of multiple sclerosis of prasinostat according to the present invention is achieved by neuronal IDO expression. In order to investigate whether the induction of AhR activation after the increase in IDO expression is a mechanism of sub-action, C57BL / 6 mice were induced with autoimmune encephalomyelitis as follows, and the efficacy was evaluated by administering prasinostat and the AhR inhibitor CH-223191.

After inducing the disease in the same manner as in “Fig. 1A”, prasinostar was administered orally. At this time, in the group administered with the AhR inhibitor CH-223191, a cremophor EL-ethanol mixture (1: 1, v / v) corresponding to 15% (v / v) of the dose of 10 mg / kg CH-223191 per mouse was used. After completely dissolving in, diluted in phosphate buffered saline, 200 μl was administered intraperitoneally 5 times daily from the 13th to the 17th experiment.

As a result, the group treated with prasinostat 30 mg / kg showed statistically significant treatment effect compared to the solvent control group. However, it was confirmed that the treatment effect of prasinostat was not observed at all by administration of the AhR inhibitor CH-223191 (see FIG. 4).

To further analyze the effect of the AhR inhibitor CH-223191 on the treatment effect of prasinostat histopathologically, tissue staining was performed in the same manner as in “Fig. 1B”.

As a result of histopathological analysis, there was no effect of reducing the infiltration of inflammatory cells by prasinostat by administration of the AhR inhibitor CH-223191 (see FIG. 4).

In order to further confirm the effect of the AhR inhibitor CH-223191 on the treatment effect of prasinostat by cytokine analysis, the expression of inflammatory cytokines was confirmed in the same manner as in FIG.

As a result of the analysis, there was no effect of reducing the cytokine inflammation caused by prasinostat by administration of the AhR inhibitor CH-223191 (see FIG. 4).

Therefore, it was confirmed that the prasinostat of the present invention has the efficacy of treating multiple sclerosis by inducing the sub-mechanism of AhR activation after increasing IDO expression.

Claims (10)

A pharmaceutical composition for the treatment or prevention of multiple sclerosis comprising a compound represented by Formula II or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier:
[Formula II]

.
delete delete The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered orally.
According to claim 1, Indoleamine 2,3-dioxygenase (indoleamine 2,3-dioxygenase; IDO) is intended to be administered to a subject not knocked out, pharmaceutical composition.
delete The pharmaceutical composition of claim 5, which acts on an inflammatory lesion in the central nervous system of the subject.
Health functional food for alleviating or preventing multiple sclerosis comprising a compound represented by the following formula (II) or a food-acceptable salt thereof:
[Formula II]

.
delete delete

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