KR102162744B1 - Pharmaceutical composition for treating inflammatory bowel disease comprising pracinostat - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating inflammatory bowel disease comprising pracinostat. The prascinostat according to the present invention can control inflammatory cells by selectively inducing the expression of immunomodulatory enzymes only in inflammatory lesions in intestinal tissues, and thus can be usefully used as a new therapeutic agent for inflammatory bowel disease.

Description

Pharmaceutical composition for treating inflammatory bowel disease comprising pracinostat {Pharmaceutical composition for treating inflammatory bowel disease comprising pracinostat}

The present invention relates to a medical use of prasinostat capable of treating inflammatory bowel disease by selectively inducing the expression of an immunomodulatory enzyme IDO only in inflammatory lesions in intestinal tissue.

Inflammatory bowel disease is a disease that causes chronic inflammation and ulcers in the intestine with repeated improvement and recurrence. Although the cause has not yet been identified, environmental and genetic factors affect bacteria normally present in the intestine. Our body's excessive immune response is considered to be an important factor. Ulcerative colitis and Crohn's disease are collectively referred to as inflammatory bowel disease. Crohn's disease can cause inflammation throughout the digestive tract from the mouth to the anus, and the inflammation spreads sporadically in several places, and is accompanied by deep ulcers. Abdominal pain and weight loss are the main symptoms. On the other hand, ulcerative colitis is caused by confined inflammation to the large intestine, mainly distributed continuously in shallow areas of the intestinal mucosa, and a typical symptom is bloody stool.

A method for curing inflammatory bowel disease is still unknown. The goal of treatment is to subside the inflammatory response, allow tissue damage to heal, and relieve symptoms such as diarrhea, bloody stools and abdominal pain. Existing drugs such as aminosalicylate, steroids, immunomodulators and antibiotics are used for the treatment of inflammatory bowel disease, but long-term administration is inevitable because it is difficult to cure, and side effects occur severely. Therefore, the drug effect can be selectively generated only in the inflamed area, so the development of a new therapeutic agent with low side effects is urgently required.

Induction of intestinal inflammation by DSS-induced colitis and CD4 ⁺ T cell adoptive transfer is a representative animal model of inflammatory bowel disease and is usefully used in the development of disease cause and treatment. Through a mouse model, it was found that the production of Th17 cells by IL-23 induction is a major pathogenic cell. In particular, it was recently confirmed that the unbalance of Th17 cells and Treg cells is one of the major causes of pathogenesis. In addition, a new fact has been revealed that an inflammatory reaction occurs by various immune mechanisms such as overactivation of innate immune cells and promotion of inflammation of intestinal epithelial cells. Therefore, it is necessary to develop a new therapeutic agent that ultimately restores tissue homeostasis by inducing immune tolerance with the removal of pathogenic cells.

Prasinostat is a 3-[2-butyl-1-(2-diethylaminoethyl)-1H-benzoimidazol-5-yl]-N-hydroxyacrylami-hydrochloride) structure that can be ingested. It is a histone deacetylase inhibitor (HDAC inhibitor). It strongly inhibits Class I, II and IV HDACs and has excellent pharmacokinetics. Due to its excellent cancer cell death induction effect, it is currently undergoing phase 3 clinical trials for acute myeloid leukemia.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan metabolizing enzyme. It is expressed by the initial inflammatory reaction, suppresses excessive inflammatory reactions, and induces immune tolerance. have. IDO inhibits the proliferation of inflammatory T cells and induces the generation of Tregs by inducing tryptophan depletion and simultaneously generating kynurenine (KYN), a metabolite that has an immunosuppressive function. As it was recently discovered that KYN is an endogenous ligand of the aryl hydrocarbon receptor (AHR), which is an immunomodulatory transcription factor, it has become clear that it suppresses the function of overactivated immune cells by the mechanism of IDO-KYN-AHR. It was confirmed through animal and clinical studies that decreased IDO expression and decreased activity were associated with various inflammatory diseases. In addition, it has been reported that inducing the expression of IDO by administration of substances from outside can treat intractable inflammatory diseases.

existing IDO inducers (TLR9 agonist, anti-4-1BB, CTLA-4Ig) induce IDO expression by promoting the production of interferon-gamma (IFN-γ) in vivo. So, it also carries the risk of promoting the inflammatory reaction. In addition, conventional immunosuppressive drugs such as calcineurin inhibitors and steroids block interferon-gamma production, making it impossible to co-administer them with these drugs. Therefore, the development of a novel substance that can selectively induce IDO expression in a lesion through an interferon-gamma-independent mechanism could be an innovative inflammatory bowel disease treatment.

An object of the present invention is to provide a pharmaceutical composition for treating enteroinflammatory diseases containing prasinostat or a pharmaceutically acceptable salt thereof as an active ingredient.

1. A pharmaceutical composition for treating inflammatory bowel 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 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 each an integer independently selected from the group consisting of 0, 1, 2, 3 and 4.

2. The pharmaceutical composition according to item 1, wherein the formula I is a compound represented by the following formula II:

[Formula II]

.

.

3. The pharmaceutical composition according to item 1 or 2, wherein the pharmaceutical composition is administered orally.

4. The pharmaceutical composition according to item 1 or 2, wherein the subject is not knocked out of indoleamine 2,3-dioxygenase (IDO).

5. The pharmaceutical composition according to item 1 or 2, wherein the compound increases the expression of IDO in the subject.

6. The pharmaceutical composition according to item 5, wherein the expression of IDO in the intestinal epithelial cells of the subject is increased.

7. Health functional food for alleviating inflammatory bowel disease, comprising a compound represented by the following formula (I) or a food acceptable salt thereof:

[Formula I]

R ¹ is 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 each an integer independently selected from the group consisting of 0, 1, 2, 3 and 4.

8. The health functional food according to item 7, wherein the formula I is a compound represented by the following formula II:

[Formula II]

.

.

According to the present invention, oral administration of prasinostat in an animal model of inflammatory bowel disease induces the expression of IDO in the intestinal inflammatory site, thereby reducing the severity of the disease, reducing histopathological damage, reducing pathogenic cytokines, and reducing tissue infiltration of inflammatory cells. Shows the effect of removal. Therefore, prascinostat can effectively treat inflammatory bowel disease.

1 shows the effect of prascinostat for treating inflammatory bowel disease.
Fig. 2 shows the effect of prasinostat on the treatment of IDO-dependent diseases.
3 shows the results of inducing IDO expression in intestinal epithelial cells in inflammatory lesions of prasinostat.
Figure 4 shows the therapeutic effect of pranisostat and anti-TNFα for inflammatory bowel disease.

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 refers to both the compound represented by formula (I) and its pharmaceutically acceptable salts:

[Formula I]

R ¹ is 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 each an integer independently selected from the group consisting of 0, 1, 2, 3 and 4.

In an embodiment of the present invention, the compound represented by Formula I is a compound represented by Formula II, prasinostat, 3-[2-butyl-1-(2-diethylaminoethyl)-1 H -benzoimidazole -5-yl] -N -Hyoxyacrylamide or 3-[2-butyl-1-(2-diethylaminoethyl)-1 H -benzoimidazol-5-yl] -N -Hyoxyacrylami- May be hydrochloride:

[Formula II]

.

.

It was confirmed that ingestion of prasinostat cured inflammatory bowel disease and showed superior efficacy than anti-TNF-alpha, an existing therapeutic drug. An important finding is that prascinostat induces the expression of IDO only in the intestinal epithelial cells at the inflamed area, and the IDO-KYN mechanism effectively inhibits the IL-23-Th17 response, which is a major etiology, resulting in a therapeutic effect. It was confirmed to appear.

The inflammatory bowel disease model can be made by a method known to a person skilled in the art. For example, DSS (Dextran Sulfate Sodium) may be mixed with drinking water and administered to mice, white papers, and hamsters for 5-7 days to prepare a DSS-inflammatory bowel disease model, but is not limited thereto. In the DSS inflammatory bowel disease model, acute colitis with edema, redness and ulcers, and bloody stool is induced throughout the large intestine, and neutrophil infiltration into the tissue is observed. Early lesions mainly occur in the left large intestine, and bloody stool, weight loss, and contraction of the large intestine appear, and as time passes, plasma cells and lymphocytes become infiltrated, resulting in chronic inflammation.

As used herein, the term “pharmaceutically acceptable salt” is a salt that retains the desired biological activity of the target compound and exhibits minimal undesirable toxic effects, and is a pharmaceutically acceptable salt with a pharmaceutically acceptable acid or base. It is a salt. Pharmaceutically acceptable acids include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric 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, ethanesol Both organic acids such as phonic acid, salicylic acid, stearic acid, benzenesulfonic acid or p-toluenesulfonic acid are included. Pharmaceutically acceptable bases include alkali metals including sodium or potassium and alkaline earth metal hydroxides including calcium or magnesium, and organic bases such as alkyl amines, aryl amines or heterocyclic amines.

One of ordinary skill in the art will further appreciate that certain compounds of the present invention, or various solvates thereof, that exist in crystalline form may exhibit polymorphism, which is the ability to occur in 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, geometry 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. One of ordinary skill in the art will recognize that different polymorphs can be prepared, for example, by varying or adjusting the reaction conditions or reagents used in the preparation of the compounds. For example, changes in temperature, pressure or solvent can produce polymorphs.

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

The invention also includes various deuterated forms of the compounds represented by formula (I). Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom. The skilled artisan 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 deuterated reagents.

The compounds of the present invention and compositions comprising the same 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 by suppositories.

The administration method is not particularly limited thereto, and any method of oral or parenteral administration may be used, and systemic administration or local administration may be possible, but systemic administration is more preferable, and oral administration is most preferable. Administration to the patient's body is preferably oral administration, specifically, 5-10 times a day administration is basic, but it may be administered more than that. In addition, the administration time may be short or sustained for a long time. Liquid dispersions for oral administration can be syrups, emulsions and suspensions.

The pharmaceutical composition of the present invention can be formulated by a known method by a person skilled in the art. For example, if necessary, it can be used parenterally in the form of an injection in a sterile solution or suspension of water or other pharmaceutically acceptable liquids. For example, a pharmaceutically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, excipients, vehicles, preservatives, binders, etc. are appropriately combined, and generally It can be formulated by blending in the unit dosage form required for the practice of pharmaceuticals recognized as. In the above formulation, the amount of the active ingredient is such that an appropriate dose within the indicated range can be obtained.

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

As an example of an injection formulation, for example, intravenous injection, intraarterial injection, selective intraarterial injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intraventricular injection, intracranial injection, intramedullary injection, etc. It is preferably intravenous injection.

In addition, buffers such as phosphate buffers, sodium acetate buffers, painless agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenols, and antioxidants may be combined. The prepared injection solution is usually filled in suitable ampoules.

Suspensions and emulsions may contain, as carriers, for example, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injection, together with the active compound, are pharmaceutically acceptable carriers such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and if necessary in suitable amounts It may contain 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. In the case of the compounds of the present invention in crystalline form, one of skill in the art will recognize that pharmaceutically acceptable solvates can be formed in which solvent molecules are incorporated into the crystalline lattice during crystallization. Solvates may include non-aqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine and ethyl acetate, or they may include water as a solvent incorporated into the crystalline lattice. Solvates, which are solvents in which water is incorporated into the crystal 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 on the basis of synthetic routes apparent to those skilled in the art.

When the composition or food of the present invention further provides for administration of one or more drugs, they may be administered simultaneously, sequentially or separately. They can be packaged together or separately. Also, they do not have to be administered simultaneously or they need to be in the same dosage form. Individual administrations can be administered on the same day or on different days, with the drug being administered as part of the same overall dosing treatment regimen. Concurrent means that drugs must be taken together or formulated as a single composition. Sequential means that the drugs are administered approximately simultaneously, preferably within about 1 hour of each other.

The term "pharmaceutically acceptable carrier" as used herein 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 in animals, more specifically in humans. It is listed in the generally known pharmacopoeia for use in The pharmaceutical carrier may be a liquid such as water or oil, and the oil includes oils derived from petroleum, animal, plant, or synthetic products, and oils such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutically acceptable carriers include saline, gum acacia, 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, especially 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 trace amounts of wetting agents, emulsifying agents, and pH buffering agents as necessary. The composition of the present invention may take a formulation suitable for solution, emulsion, sustained-release preparation or other use.

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

<Example 1> Effect of prasinostat on the treatment of inflammatory bowel disease

In order to investigate the therapeutic effect of prasinostat according to the present invention for inflammatory bowel disease, inflammatory bowel disease was induced in C57BL/6 mice as follows, and prascinostat (Selleckchem, Cat No. S1515) was administered as follows to evaluate its efficacy. Evaluated.

On day 0 of the experiment, a 1.5% DSS solution prepared by dissolving DSS (Detran sulfate sodium, MP biomedicals, Cat No. 160110) in 1.5% sterile distilled water was given to C57BL/6 mice (8 weeks old, female, 18±2 g) for 9 days. I let you drink. The 1.5% DSS solution was changed every 2 days. From the 0th day of the experiment, the body weight was measured every day to check whether inflammatory bowel disease occurred.

In the prascinostat administration group according to the present invention, a Cremophor EL-ethanol mixture (1:1, v/v) corresponding to 15% (v/v) of the administered dose of 30 mg/kg of prasinostat per mouse ), and then diluted in phosphate buffered saline, 200 μl each was orally administered 5 times daily from the 4th day to the 8th day of the experiment. In addition, a mixture of Cremophor EL-ethanol-phosphate buffered saline (7.5:7.5:85, v/v/v) was administered to the vehicle control group in the same manner as in the prascinostat administration group at 200 µl. Changes in weight were measured and recorded every day from the 0th day of the experiment.

As a result of the analysis, it was confirmed that in all experimental groups, body weight began to decrease from the 5th day of the experiment, and 100% of the intestinal inflammation, in which the body weight decreased by more than -10%, was induced on the 10th day of the experiment. The mice in the solvent control group showed a weight loss of -16.46% ± 5.23 on the 10th day of the experiment. The group administered with the compound prasinostat 30 mg/kg of the present invention was able to confirm a statistically significant treatment effect compared to the solvent control group from the 5th day of the experiment (compared to the solvent control group *, p<0.05; **, p<0.01; * **, p<0.001, see Fig. 1). Prasinostat of the present invention showed excellent anti-inflammatory effect when administered at 30 mg/kg.

On the 9th day of the experiment, on the 9th day of the experiment in which the oral administration of prascinostat of the present invention was completed, the large intestine was excised, and the length and weight of the large intestine were compared. Prasinostat increased the length of the colon and decreased the weight compared to the solvent control group. The weight: length ratio was also significantly reduced in the colon administered prasinostat compared to the solvent control group (*, p<0.05; **, p<0.01; ***, p<0.001, see FIG. 1B) compared to the solvent control group.

On the 9th day of the experiment where 5 oral administration of prascinostat of the present invention was completed, the colon of the mouse was excised to prepare a tissue sample. After fixing the intestine of the extracted mouse with 4% paraformaldehyde (sigma aldrich), paraffin infiltration was performed using a tissue processor (Leica), and a paraffin block was produced using a tissue embedding center (Leica). I 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 finished tissue section to perform histopathological analysis of the colon. Histological analysis was performed using Nanozoomer 2.0 RS (Hamammatsu).

The histopathological evaluation of the colon was measured by setting two major histological scales and then rating them by scale. The two main measures are epithelium damage and inflammatory cell infiltration, 1) epithelial damage is normal 0, goblet cell damage 1, goblet cell destruction 2, crypt damage and goblet cell damage 3, intestinal gland The standard value was set as destruction 4, and 2) inflammatory cell infiltration was normal 0, intestinal gland infiltration 1, infiltrate of the mucous membrane propria 2, mucosal ulcers due to excessive infiltration of the mucosa 3, and submucosa infiltration 4 After determining the reference value, it was measured for each tissue individual and evaluated for clinical severity by the sum of the reference values (0-8) for each of the two main scales.

As a result of histopathological analysis, inflammation of colon lesions was significantly reduced in the tissues administered with prasinostat compared to the solvent control group (*, p<0.05; **, p<0.01; ***, p<0.001, compared to the solvent control group). See Fig. 1C).

On the 9th day of the experiment, on the ninth day of the experiment, when the oral administration of prasinostat of the present invention was completed five times, the large intestine of the mouse was excised to prepare an mRNA sample. In order to extract mRNA, colon tissue was ground with a homogenizer to obtain a homogeneous suspension. The mRNA from the homogeneous suspension was extracted by phenol-chloroform precipitation method using Trizol reagent (Invitrozen, Cat No. 15596018). Synthesize cDNA from the isolated RNA by reverse transcription and check the expression of inflammatory cytokines by real-time PCR using iQ SYBR-Green Supermix (Bio-rad) in the CFX96 (Bio-rad) detection system. I did. The relative values of the enzyme expression levels were compared with the ΔΔct method using GAPDH as a control enzyme. A WT mouse colon was used as a control to establish a fold of 1.

The real-time polymerase chain reaction was performed under the conditions of 45 cycles with an annealing temperature of 58° C., and the following primer sequences were used. 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 TNF-α forward, 5'-CCA CAC CGT CAG CCG ATT TG-3' and reverse, 5'-CAC CCA TTC CCT TCA CAG AGC-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 IL-17A forward, 5'-TTT AAC TCC CTT GGC GCA AAA-3' and reverse, 5'-CTT TCC CTC CGC ATT GAC AC-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 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 the inflammatory cytokines IL-1β, TNF-α, IL-6, IL-17A, and IFN-γ in the colon lesion were significantly decreased compared to the solvent control group due to the administration of prasinostat (*, p< 0.05; **, p<0.01; ***, p<0.001, see Fig. 1).

Therefore, since the prascinostat of the present invention has an orally administered treatment effect in a mouse model of inflammatory bowel disease, it can propose a useful treatment strategy as a novel oral treatment for inflammatory bowel disease.

<Example 2> Effect of prasinostat on treatment of IDO-dependent diseases

To determine whether the therapeutic effect of prasinostat according to the present invention for inflammatory bowel disease is IDO-dependent, inflammatory bowel in wild-type (WT, normal) mice and IDO-knockout (IDO-KO, gene deficiency) mice as follows. Disease was induced and prascinostat was administered to evaluate its efficacy.

Disease was induced in WT C57BL/6 mice and IDO-KO C57BL/6 mice (8 weeks old, female, 18±2 g) in the same manner as in "Fig. 1A", and prasinostat (30 mg/kg, 1 day A total of 5 times from day 4 to day 8) and a solvent control group were administered orally. Symptoms of inflammatory bowel disease were evaluated based on the same criteria as in "Fig. 1".

The prascinostat 30 mg/kg administration group of WT mice showed statistically significant treatment effects compared to the solvent control group from the 5th day of the experiment, and the effect continued until the end of the experiment. On the other hand, the prascinostat-administered group of IDO-KO mice showed a high severity similar to that of the solvent control group of IDO-KO mice, and no disease alleviation effect was observed. In addition, compared to the prasinostat group of WT mice, the IDO-KO mouse prascinostat-treated group had a statistically significant disease severity (IDO-KO mouse prascinostat-treated group compared to the WT mouse prascinostat-treated group + , p<0.05; + +, p<0.01; + + +, p<0.001, see Fig. 2).

On the 9th day of the experiment, on the 9th day of the experiment in which the oral administration of prascinostat of the present invention was completed, the large intestine was excised, and the length and weight of the large intestine were compared. In IDO-KO mice, changes in colon morphology by administration of prasinostat could not be confirmed. The weight:length ratio was also significantly higher in the plasinostat-treated colon of IDO-KO mice compared to the prascinostat-treated colon of WT mice (IDO-KO mouse prasinostat-treated group compared to WT mouse prasinostat-treated group +, p <0.05; + +, p<0.01; + + +, p<0.001, see Fig. 2B).

On the 9th day of the experiment where the oral administration of prascinostat of the present invention was completed five times, the large intestine of the mouse was excised, and tissue stained in the same manner as in "Fig. 1B".

As a result of histopathological analysis, in IDO-KO mice, the effect of reducing inflammation of colon lesions by administration of prasinostat was not observed at all (see Fig. 2D).

On the 9th day of the experiment, on the 9th day of the experiment where the oral administration of prascinostat of the present invention was completed, the large intestine of the mouse was excised and the expression of inflammatory cytokines was confirmed in the same manner as in “Fig.

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

Therefore, it was confirmed that the prascinostat of the present invention has an IDO-dependent treatment effect for inflammatory bowel disease.

Example 3 Induction of IDO expression in intestinal epithelial cells in inflammatory lesions of prasinostat

It was confirmed that the disease treatment effect of prascinostat according to the present invention was IDO dependent. Accordingly, in order to identify the IDO-expressing and expressing cells of prasinostat, inflammatory bowel disease was induced in C57BL/6 mice as shown in "Fig. 1A", and after administration of prasinostat 5 times, colon lesions were extracted and the experiment was conducted. . An mRNA sample and a tissue sample were prepared from the extracted spine.

First, in order to investigate the expression of IDO in the large intestine, a homogenous suspension was obtained by grinding the large intestine tissue with a homogenizer. The mRNA from the homogeneous suspension was extracted by phenol-chloroform precipitation method using Trizol reagent (Invitrozen, Cat No. 15596018). From the isolated RNA, cDNA was synthesized by reverse transcription, and IDO expression was confirmed by real-time PCR using iQ SYBR-Green Supermix (Bio-rad) in a CFX96 (Bio-rad) detection system. The relative values of the enzyme expression levels were compared with the ΔΔct method using GAPDH as a control enzyme. A WT mouse colon was used as a control to establish a fold of 1.

The real-time polymerase chain reaction was performed under the conditions of 45 cycles with an annealing temperature of 58° C., 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; ***, see Fig. 3) compared to the solvent control group.

Next, in order to identify the cells expressing IDO by prascinostat, a tissue sample was prepared in the same manner as in "Fig. 1A".

Thereafter, for immunohistochemistry analysis, tissue sections are dehydrated with xylene and ethanol (EtOH), and then antigen unmasking solution (Vector, Cat No. H-3301). I restored it. Anti-IDO (Adipogen, Cat No. AG-25A-0032-C100, 1:100) was treated overnight on the blocked tissue by treating the CAS block (CAS block, Thermo Fisher, Cat No. 00-8120) solution. Secondary antibody against IDO Biotinylated anti-rabbit antibody (Biotinylated anti-rabbit Ab, Vector, Cat No. BA-1100, 1: 100 dilution) was treated for 2 hours, and then Vectastain ABC kit (Vector, Cat No. PK-4000) was reacted for 1 hour. Counter-staining with DAB (3,3-diaminobenzidine) and observation with Nanozoomer 2.0 RS (Hamammatsu).

It was confirmed by histochemical method that selective expression of IDO in cells morphologically seen as epithelial cells in colon lesions of inflammatory bowel disease due to oral administration of prascinostat of the present invention (see FIG. 3B).

Therefore, it was found that the prascinostat of the present invention has therapeutic efficacy by inducing IDO expression in epithelial cells in the colon lesion of the experimental inflammatory bowel disease mouse model.

<Example 4> Anti-TNFα (anti-Tumor Necrosis Factor alpha) compared to the treatment effect of prasinostat inflammatory bowel disease

In order to investigate the therapeutic effect of prascinostat according to the present invention and anti-TNFα for inflammatory bowel disease, as follows, induce inflammatory bowel disease in RAG-knockout (RAG-KO, gene deficiency) mice, and prascinostat (Selleckchem, Cat No. S1515) or anti-TNFα was administered to compare the efficacy.

The spleen extracted from IL-10-KO mice (8-10 weeks old, female, 18±4 g) on day 0 of the experiment was 1X penicillin/streptomycin (HyClone ^TM ), 5% FBS, 15 μg/ml DNase I (Sigma, Cat # 10104159001), and 1 mg/ml collagenase type II (sigma, Cat # 1148090) was added to the RPMI (HyClone ^TM ) dissociation solution (enzyme digestion) for 40 minutes at 37 ℃. CD4 ⁺ T cells were obtained from single cells obtained by passing through a 40-μM filter strainer through CD4 positive selection (Miltenyi, Cat # 130-117-043) of the autoMACS system. CD4 ⁺ cells of IL-10-KO mice were transferred to RAG1-KO mice (6 weeks old, female, 16±2 g) by tail vein at a number of 4×10 ⁶ . From the 0th day of the experiment, the severity of stool was measured twice a week to confirm the onset of inflammatory bowel disease.

In the prascinostat administration group according to the present invention, a Cremophor EL-ethanol mixture (1:1, v/v) corresponding to 15% (v/v) of the administered dose of 30 mg/kg of prasinostat per mouse ), and then diluted in phosphate buffered saline, 200 μl each was orally administered 10 times daily from the 33rd day to the 42nd day. In addition, as a drug control, anti-TNFα, an inflammatory bowel disease treatment currently used in clinical practice, was administered intraperitoneally at 25 mg/kg per mouse, twice a week for 2 weeks from the 27th to the 42nd of the experiment. In the solvent (Vehicle) control group, a mixture of Cremophor EL-ethanol-phosphate buffered saline (7.5:7.5:85, v/v/v) was administered in the same manner as in the prascinostat administration group at 200 μl. Fecal score was measured and recorded twice a week from the 27th day of the experiment.

Stool severity was indexed according to the following items.

0 no symptoms

1 somewhat soft stools

2 Shaped but soft stools

3 Shapeless and soft stools

4 diarrhea or bloody stool

As a result of the analysis, it was confirmed that in all experimental groups, enteritis developed from the 21st day of the experiment, and 100% of the enteritis with a severity index of 1.0 or higher was induced on the 27th day of the experiment. The mice in the solvent control group showed a severity index of 2.88 ± 0.38 on day 42 of the experiment. The group administered with the compound prasinostat 30 mg/kg of the present invention was able to confirm a statistically significant therapeutic effect compared to the solvent control group (*, p<0.05; **, p<0.01; ***, p compared to the solvent control group). <0.001, see Fig. 4).

These treatment effects showed a statistically significant difference compared to the anti-TNFα administration group (+, p<0.05; + +, p<0.01; + + +, p<0.001, see FIG. 4 compared to the anti-TNFα administration group. ). The prascinostat of the present invention showed excellent anti-inflammatory effect compared to the anti-TNFα administration group when administered with 30 mg/kg.

Claims (8)

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

.
The pharmaceutical composition of claim 1, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is administered orally. The pharmaceutical composition of claim 1 or 2, wherein indoleamine 2,3-dioxygenase (IDO) is administered to a subject that has not been knocked out.
The pharmaceutical composition according to claim 4, which increases the expression of IDO in the intestinal epithelial cells of the subject.
The pharmaceutical composition according to claim 1, which acts on inflammatory lesions of intestinal epithelial cells.
Health functional food for alleviation or prevention of inflammatory bowel disease, comprising a compound represented by the following Formula II or a food pharmaceutically acceptable salt thereof:
[Formula II]

.
The health functional food according to claim 7, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

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