KR20220152073A - Pharmaceutical composition for treatment or prevention of Crohn's disease comprising pyrimethamine as an active ingredient - Google Patents

Abstract

The present disclosure relates to a pharmaceutical composition for treating or preventing Crohn's disease, and a method for treating or preventing Crohn's disease. Accordingly, the problem to be solved by the present disclosure is to provide a medicinal use of a compound useful for the treatment or prevention of Crohn's disease. That is, the problem to be solved by the present disclosure is to provide the pharmaceutical composition effective for the treatment or prevention of Crohn's disease.

Description

Pharmaceutical composition for treatment or prevention of Crohn's disease comprising pyrimethamine as an active ingredient

The present disclosure relates to pharmaceutical compositions for treating or preventing Crohn's disease. That is, the present disclosure relates to pharmaceutical uses and/or methods of treatment related to Crohn's disease.

Crohn's disease is a type of inflammatory bowel disease that can affect any part of the gastrointestinal tract from the mouth to the anus. Symptoms of Crohn's disease include abdominal pain, diarrhea, fever, bloating, and weight loss. The exact cause of Crohn's disease is unknown, but it is believed to be caused by a combination of an individual's genetics, immune and bacterial factors. Currently, there is no drug or surgical procedure that can fundamentally treat Crohn's disease, and the treatment of Crohn's disease patients is only about relieving symptoms, maintaining remission, or preventing recurrence.

Acute treatment for Crohn's disease is usually first with antibiotics to treat bacterial infections, then anti-inflammatory drugs such as aminosalicylates or corticosteroids to reduce inflammation may be used. However, continuous use of antibiotics can change the intestinal flora, and there is a risk of overgrowth of pathogens such as Clostridium difficile in the intestine. In addition, anti-inflammatory agents such as aminosalicylates can only be used to the extent of maintaining treatment for a small number of Crohn's disease patients, and many Crohn's disease patients require additional immunosuppressive agents. In addition, corticosteroid drugs can cause serious side effects such as gastrointestinal diseases such as pancreatitis and peptic ulcer, neuropsychiatric diseases such as hypertension and depression, or cataracts when used for a long time, and are not generally used for long-term treatment.

Drugs used to treat the symptoms of Crohn's disease include anti-inflammatory drugs such as 5-aminosalicylic acid (5-ASA) or prednisone, as well as immunosuppressive drugs such as azathioprine (available as a prodrug of 6-mercaptopurine) or methotrexate. modulator; Antibody medicines such as infliximab, adalimumab, certolizumab, vedolizumab, ustekinumab, or natalizumab may be used. have. When the symptoms of Crohn's disease are alleviated, maintenance management through the above-mentioned drug therapy can be entered in order to prevent the recurrence of symptoms.

However, since the antibody drugs are expensive and require repeated injections, patients are highly resistant to them. In the case of these antibody drugs, there is a possibility that patients who responded to the drug may develop resistance within several years due to immunogenic side effects. In addition, antibody drugs have disadvantages such as difficult storage because low temperature storage is essential. Therefore, it is necessary to develop a new drug having excellent treatment effect of Crohn's disease while solving the above disadvantages of existing drugs for the treatment of Crohn's disease.

Pyrimethamine, on the other hand, was discovered in the early 1950s and is used as a treatment for malaria, and is now used as a preventive agent against toxoplasmosis, cystoisosporiasis, or pneumonia in AIDS patients. . However, it is unknown what pharmacological effects pyrimethamine exhibits in immune-related diseases.

Accordingly, the problem to be solved by the present disclosure is to provide a medicinal use of a compound useful for the treatment or prevention of Crohn's disease.

That is, the problem to be solved by the present disclosure is to provide a pharmaceutical composition effective for the treatment or prevention of Crohn's disease.

Another problem to be solved by the present disclosure is to provide an effective method for preventing or treating Crohn's disease.

As a result of intensive research efforts to develop an effective therapeutic agent for Crohn's disease, the present inventors have surprisingly found that pyrimethamine is useful for treating or preventing Crohn's disease. In addition, the present inventors confirmed this effect through an in vivo experiment using a Crohn's disease animal model. Antibody medicines that have been conventionally used for the treatment of Crohn's disease are highly resistant to patients because they are expensive and require repeated injections. Since the therapeutic use of pyrimethamine newly discovered through the present invention can replace conventional medicines used for the treatment of Crohn's disease, it is possible to solve the problems of the existing antibody medicines.

The present disclosure provides a pharmaceutical composition for treating or preventing Crohn's disease comprising pyrimethamine or a pharmaceutically acceptable salt thereof as an active ingredient. Such a therapeutic or preventive effect can be confirmed from preclinical test results using Crohn's disease animal models, as based on the experimental results confirmed by the present inventors described below.

In another aspect of the present disclosure, the present disclosure provides Crohn's disease comprising administering a therapeutically effective amount of pyrimethamine or a pharmaceutically acceptable salt thereof to a subject (preferably a human) in need of treatment or prevention of Crohn's disease. A treatment or prevention method is provided. That is, the present disclosure provides a pharmaceutical use for treating or preventing Crohn's disease of pyrimethamine or a pharmaceutically acceptable salt thereof.

In one aspect of the present disclosure, the pharmaceutically acceptable salt of pyrimethamine may be a salt with an organic acid, an inorganic acid, or an acidic amino acid. Salts with organic acids are acetic acid, acetic acid, propionic acid, isobutyric acid, oxalic acid, maleic acid, malonic acid, succinic acid, glutaric acid, salicylic acid, suberic acid, fumaric acid, manderic acid, phthalic acid, benzenesulfonic acid, p -Salts with torylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid or the like. Salts with inorganic acids may include salts with hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonate, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydrogen iodide or phosphorous acid and the like. Salts with acidic amino acids may include salts with aspartic acid, glutamic acid, and the like. Preferably, the pharmaceutically acceptable salt of pyrimethamine may be a salt with any one selected from the group consisting of acetic acid, acetic acid, maleic acid, fumaric acid, succinic acid, glutaric acid and salicylic acid.

In one aspect of the present disclosure, a pharmaceutical composition for the treatment or prevention of Crohn's disease containing pyrimethamine or a pharmaceutically acceptable salt thereof as an active ingredient, when administered to a patient with Crohn's disease, has TNF-α, IL-1β, and IL Levels of -6 can be suppressed. The TNF-α, IL-1β, and IL-6 are known as cytokines involved in the mechanism of inducing inflammation in Crohn's disease. In the present disclosure, the therapeutic or preventive effect of pyrimethamine on Crohn's disease is not only the result of Crohn's disease index measurement using an animal model (Experimental Example 2) and histopathological experiment (Experimental Example 3), but also TNF-α through ELISA analysis , IL-1β, and inhibition of IL-6 levels (Experimental Example 4) can also be confirmed indirectly.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of pyrimethamine or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. As used herein, "effective amount" refers to an amount of pyrimethamine sufficient to slow or minimize the expansion of the inflammatory response in Crohn's disease or to provide therapeutic benefit in the treatment or management of Crohn's disease.

In one preferred aspect of the present disclosure, the effective dosage of pyrimethamine or a pharmaceutically acceptable salt thereof is about 0.1 to about 5 mg/kg (human body weight)/day, preferably about 0.2 mg/day in terms of pyrimethamine. to about 3 mg/kg (human body weight)/day, more preferably from about 0.3 to about 2.5 mg/kg (human body weight)/day, even more preferably from about 0.4 to about 1.25 mg/kg (human body weight)/day. .

These human doses can be calculated according to FDA guidelines. The effective dose in the mouse model of the present disclosure is converted to the human effective dose according to the following formula (see J Basic Clin Pharm . 2016 Mar;7(2):27-31, FDA guideline, Table 1).

[human effective dose mg/kg/day] = [mouse effective dose mg/kg/day]*0.081

The term "about" when used in the context of any number or range in the disclosure herein generally means that it may represent a deviation of ±20%, preferably ±10%, from the center value.

In another aspect of the present disclosure, the dosage of the above-mentioned pyrimethamine or a pharmaceutically acceptable salt thereof is about 6 to 300 mg/day (in terms of weight converted to pyrimethamine) based on an adult whose body weight is 60 kg. day, preferably about 12 to about 180 mg/day, more preferably about 18 to about 150 mg/day, even more preferably about 24 to about 75 mg/day. The adult body weight of 60 kg is based on the guidelines of "Guidance for Industry - Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" Guidance for Industry (fda.gov).

The dosage range set based on the body weight of an adult of 60 kg may be administered at one time or divided (eg, twice a day).

The present inventors performed an experiment using an animal (mouse) model to confirm the therapeutic effect of pyrimethamine on Crohn's disease, and when using a dose of about 5 or 15 mg/kg/day based on the weight of the mouse, the mouse model found that the treatment effect of Crohn's disease was remarkably excellent. Dosage ranges for the mouse model can be derived from the experimental examples disclosed herein below.

In Experimental Examples 2 and 3 disclosed herein, in the mouse group administered with pyrimethamine at a dose of 5 mg/kg/day or 15 mg/kg/day, the Crohn's disease index was significantly improved and histopathologically It was confirmed that the treatment effect appeared. In addition, when looking at the ELISA test results of Experimental Example 4 disclosed herein, TNF-α, IL-1β, TNF-α, IL-1β, And it was confirmed that the level of IL-6 was significantly lowered.

For the treatment of Crohn's disease, the pyrimethamine or pharmaceutically acceptable salts thereof described herein may be administered in the following manner.

Oral administration

In one aspect of the present disclosure, the pyrimethamine or pharmaceutically acceptable salts thereof described herein may be administered orally as follows.

The oral cavity is a concept including swallowing. Oral administration allows a pharmaceutical composition according to the present disclosure to enter the gastrointestinal tract or be directly absorbed from the mouth into the bloodstream, for example, by buccal or sublingual administration.

Compositions suitable for oral administration may be in solid, liquid, gel, or powder form, and may have formulations such as tablets, lozenges, capsules, granules, powders, and the like. .

Compositions for oral administration may optionally be enteric coated and exhibit delayed or sustained release through the enteric coating. That is, the composition for oral administration according to the present disclosure may be a dosage form with an immediate or modified release pattern.

Liquid formulations may include solutions, syrups and suspensions, and such liquid compositions may be contained in soft or hard capsules. Such formulations may include a pharmaceutically acceptable carrier such as water, ethanol, polyethylene glycol, cellulose, or oil. The formulation may also contain one or more emulsifying and/or suspending agents.

In a tablet formulation, the amount of drug active ingredient may be present from about 0.05% to about 95%, more typically from about 2% to about 70% by weight of the total weight of the tablet. Tablets may also contain a disintegrant comprising from about 0.5% to about 35% by weight, more usually from about 2% to about 25% by weight of the dosage form. Examples of disintegrants include, but are not limited to, lactose, starch, sodium starch glycolate, crospovidone, croscarmellose sodium, maltodextrin, or mixtures thereof.

Suitable glidants included for making into tablets may be present in amounts from about 0.1% to about 10% by weight, and include talc, silicon dioxide, stearic acid, calcium, zinc or magnesium stearate, sodium stearyl fumarate, and the like. Although it can be used as a lubricant, the lubricant according to the present disclosure is not limited to the types of these additives.

Gelatin, polyethylene glycol, sugar, gum, starch, polyvinylpyrrolidone, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc. may be used as a binder for preparing tablets. Mannitol, xylitol, lactose, dextrose, sucrose, sorbitol, starch, microcrystalline cellulose, etc. may be used as suitable diluents for preparing tablets, but the binder according to the present disclosure is limited to the types of these additives. it is not going to be The binder may be used in an amount of about 0.1% to about 20% by weight based on the total weight of the tablet, and the diluent may be used in an amount of about 0.1% to about 20% by weight based on the total weight of the tablet.

Optionally, the solubilizing agent that may be included in the tablet may be used in an amount of about 0.1% to about 3% by weight based on the total weight of the tablet, for example, polysorbate, sodium lauryl sulfate, sodium dodecyl sulfate, propylene carbonate, Diethylene glycol monoethyl ether, dimethylisosorbide, polyoxyethylene glycolated natural or hydrogenated castor oil, HCOR ^™ (Nikkol), oleyl ester, Gelucire ^™ , caprylic/caprylic mono/ Diglyceride, sorbitan fatty acid ester, Solutol HS ^TM and the like may be used in the pharmaceutical composition according to the present disclosure, but the solubilizing agent according to the present disclosure is not limited to the specific type of such solubilizing agent.

Parenteral Administration

In another aspect of the present disclosure, the pyrimethamine or pharmaceutically acceptable salts thereof described herein may be administered parenterally as follows.

The parenteral administration includes administration directly into the blood stream, muscle, or intestine. Suitable methods for parenteral administration include intravenous, intramuscular, subcutaneous intraarterial, intraperitoneal, intrathecal, intracranial injection, and the like. include Suitable devices for parenteral administration include injectors (including needle and needleless syringes) and infusion methods.

Compositions for parenteral administration may be formulations with an immediate or modified release pattern, and the modified release pattern may be a delayed or sustained release pattern.

Most parenteral formulations are liquid compositions, and these liquid compositions are aqueous solutions containing active ingredients, salts, buffers, tonicity agents and the like according to the present disclosure.

Parenteral formulations may also be prepared in dried form (eg lyophilized) or as sterile non-aqueous solutions. These formulations may be used with a suitable vehicle such as sterile water. Solubility-enhancing agents may also be used in the preparation of parenteral solutions.

The pharmaceutical composition according to the present disclosure includes pyrimethamine or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition of the present disclosure exerts a significant effect on the treatment or prevention of Crohn's disease.

1 is a result of evaluating the Crohn's disease index on the 14th day after oral administration of Example (pyrimethamine) or Comparative Example (mesalazine) in a Crohn's disease animal model (G1: normal group, G2: disease-induced group, G3: disease induction and mesalazine 200 mg/kg/day administration group, G4: disease induction and pyrimethamine 5 mg/kg/day administration group, G5: disease induction and pyrimethamine 15 mg/kg day administration group).
Figure 2 quantifies the histopathological results on inflammation, crypt cell damage, ulceration, and edema when each drug is orally administered in a Crohn's disease animal model.
FIG. 3 shows the levels of TNF-α, IL-1β, and IL-6 measured and quantified through ELISA analysis using colon tissue lysate from a Crohn's disease animal model.

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, embodiments according to the present disclosure may be modified in many different forms, and the scope of the present disclosure should not be construed as being limited to the following examples. Embodiments of the present disclosure are provided to more completely explain the present invention to those skilled in the art.

Experimental Example 1: Preclinical test preparation and implementation

C57BL/6 Mice were allowed to freely ingest a 2% dextrin sodium sulfate (DSS) solution from the onset of Crohn's disease (Day 0) to Day 6 (replacement of normal negative water in the morning of Day 6) to induce intestinal disease. The 2% DSS solution is freshly made and replaced every 3 days. To mice with intestinal disorders, mesalazine (MSZ) 200 mg/kg/day twice/day, pyrimethamine (PYR) 5 mg/kg/day once/day, or pyrimethamine (PYR) 15 mg /kg/day once/day was orally administered for 14 days, and symptoms were recorded. The composition of each experimental group is shown in Table 1 (N=6/group, ***/**/* A significant difference at p<0.001/p<0.01/p<0.05 level compared to the G2: disease-induced group) . During the observation period after administration, the type, date of onset, and severity of general symptoms, including death, were observed once a day, recorded for each individual, and scored according to the criteria (disease activity index, DAI) presented below (Cooper, HS , Murthy, SN, Shah, RS, and Sedergran, DJ (1993). Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab. Invest . 69, 238-249.).

Body weight loss (%): 0 (< 2%), 1 (≥2-<5%), 2 (≥5-<10%), 3 (≥10-<15%), 4 (≥15%) . Stool type: 0 (Normal), 1 (softer stool), 2 (moderate diarrhea), 3 (diarrhea). Bleeding: 0 (no rectal bleeding), 1 (weak hemoccult), 2 (blood in stool), 3 (fresh rectal bleeding)

army gender number of animals
(number of animals) animal number Whether Crohn's disease is caused administration substance Administration method dose
(mg/kg) dose amount
(mL/kg) G1 M 6 1-6 N 0.5% MC oral- - 10 G2 M 6 7-12 Y 0.5% MC oral- - 10 G3 M 6 13-18 Y MSZ oral- 200 10 G4 M 6 19-24 Y PYR oral- 5 10 G5 M 6 25-30 Y PYR oral- 15 10

Experimental Example 2: Crohn's disease index measurement

The Crohn's disease index of each group from G1 to G5 is shown in Table 2 and FIG. 1. Looking at the results shown in Table 2 and FIG. 1, no statistically significant improvement was observed in the mesalazine 200 mg/kg/day QD oral administration group (G3). On the other hand, in the case of pyrimethamine 5 or 15 mg/kg/day administration groups (G4, G5), the Crohn's disease index tended to improve in a concentration-dependent manner.

army animal number Day 0 Day 14 Bleeding Stool type BW loss Total Bleeding Stool type BW loss Total G1
0.5% MC
PO One 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 MEAN 0 0 SD 0 0 G2
0.5% MC
PO 7 0 0 0 0 0 One 2 3 8 0 0 0 0 - - - - 9 0 0 0 0 One 2 4 7 10 0 0 0 0 0 2 4 6 11 0 0 0 0 0 One One 2 12 0 0 0 0 One 3 3 7 MEAN 0 5 SD 0 2.3 G3
MSZ
200 mpk
PO 13 0 0 0 0 0 2 4 6 14 0 0 0 0 - - - - 15 0 0 0 0 0 2 2 4 16 0 0 0 0 0 One 2 3 17 0 0 0 0 0 2 0 2 18 0 0 0 0 0 2 3 5 MEAN 0 4 SD 0 1.6 G4
PYR
5 mpk
PO 19 0 0 0 0 0 2 4 6 20 0 0 0 0 - - - - 21 0 0 0 0 0 2 One 3 22 0 0 0 0 0 One 0 One 23 0 0 0 0 0 2 4 6 24 0 0 0 0 0 2 3 5 MEAN 0 4.2 SD 0 2.2 G5
PYR
15 mpk
PO 25 0 0 0 0 0 2 2 4 26 0 0 0 0 - - - - 27 0 0 0 0 0 One 0 One 28 0 0 0 0 0 2 One 3 29 0 0 0 0 One 2 2 5 30 0 0 0 0 0 2 3 5 MEAN 0 3.6 SD 0 1.7

(Mice objects 8, 14, 20, and 26 died before the 14th day, so monitoring was stopped)

Experimental Example 3: Histopathological observation

Colon tissues were collected from the animal models of the G1 to G5 groups, and specimens for histopathological examination were prepared through general tissue processing procedures such as trimming, dehydration, paraffin embedding, and cutting. After fixing the specimen, hematoxylin & eosin (H&E) was performed, and histopathological changes were observed using an optical microscope (Olympus BX53, Japan). For histopathological microscopy, the most severe lesions were selected for the large intestine, and inflammation (0-5 points), crypt cell damage (0-4 points), ulcers (0-3 points), and edema (0-1 points) were evaluated. ) After evaluation by scoring for each, it is shown in Table 3 and FIG. As a result, the pyrimethamine 15 mg/kg/day PO administration group (G5) showed better efficacy than the existing Crohn's disease treatment drug mesalazine administration group on inflammatory reactions and ulcers caused by immune cell infiltration. In addition, the anti-inflammatory and anti-ulcer effects of pyrimethamine showed concentration dependence.

Histopathological test (unit: score) Experiment group G1 G2 G3 Inflammation 0.0±0.0 4.6±0.9 ^** 4.6±0.5 ^** crypt cell damage 0.0±0.0 3.8±1.1 ^** 3.4±0.9 ^** ulcer 0.0±0.0 2.6±0.9 ^** 2.4±0.9 ^** edema 0.0±0.0 1.0±0.0 ^** 1.0±0.0 ^** N 6 5 5 Experiment group G4 G5 Inflammation 4.0±1.0 ^** 3.8±0.8 ^** crypt cell damage 3.6±1.7 ^** 3.0±1.0 ^** ulcer 2.2±1.1 ^** 1.8±0.8 ^** edema 1.0±0.0 ^** 0.8±0.4 ^* N 5 5

(G1: Normal control, G2: Vehicle control, G3: MSZ, G4: PYR 5 mg/kg, G5: PYR 15 mg/kg. **/* A significant difference at p<0.01/p<0.05 level compared to the G1)

Experimental Example 4: ELISA analysis

IL-1β, IL-6, and TNF-α (Abcam, ab100704; Abcam, ab222503; Abcam, ab208348) were measured using colon tissue lysates. The measurement of the cytokines was performed according to the protocol provided by the manufacturer using a commercially available ELISA kit, and the results are shown in Table 4 and FIG. 3. As a result of ELISA analysis, the levels of TNF-α and IL-6 in the pyrimethamine 5 mg/kg group (G4) and the pyrimethamine 15 mg/kg group (G5) were significantly lower than those in the control group (G2) to which the excipient was administered. . In addition, the IL-1β level of the pyrimethamine 5 mg/kg group (G4) and the pyrimethamine 15 mg/kg group (G5) tended to be lower than that of the control group (G2) to which the vehicle was administered. It is considered to be consistent with the clinical symptom evaluation and histopathological examination results.

ELISA assay (unit: ng/mg) Experiment group G1 G2 G3 TNF-α 0.016±0.005 0.045±0.006 ^*** 0.038±0.012 ^*** IL-1β 0.022±0.005 0.052±0.023 ^** 0.044±0.005 ^* IL-6 0.026±0.005 0.050±0.015 ^** 0.030±0.007 ^# N 6 5 5 Experiment group G4 G5 TNF-α 0.037±0.003 ^***,# 0.034±0.002 ^***,## IL-1β 0.046±0.012 ^* 0.035±0.006 IL-6 0.032±0.008 ^# 0.030±0.010 ^# N 5 5

(G1: Normal control, G2: Vehicle control, G3: MSZ, G4: PYR 5 mg/kg, G5: PYR 15 mg/kg. ***/**/* A significant difference at p<0.001/p<0.01 /p<0.05 level compared to the G1. ##/# A significant difference at p<0.01/p<0.05 level compared to the G2.)

Under the present experimental conditions, the body weight levels of the pyrimethamine 5 mg/kg group (G4) and the pyrimethamine 15 mg/kg group (G5) tended to be higher than those of the excipient control group (G2). In addition, in view of the clinical symptoms, histopathological examination, and ELISA test results, the Crohn's disease index or related factors in the pyrimethamine-administered groups (G4, G5) were significantly lower than in the control group (G2) administered with the excipient, or or showed a low tendency, it was confirmed that Crohn's disease was improved in the pyrimethamine-administered group. That is, in the dextran sodium sulphate (DSS)-induced CD (Crohn's disease) model using C57BL/6 mice, it is thought that pyrimethamine administration can help improve Crohn's disease.

Claims (3)

A pharmaceutical composition for treating or preventing Crohn's disease comprising pyrimethamine or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered at a dose of 0.1 to 5 mg/(human body weight)kg/day in terms of pyrimethamine or a pharmaceutically acceptable salt thereof, in terms of pyrimethamine. composition. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits the levels of TNF-α, IL-1β, and IL-6 when administered to a patient with Crohn's disease.

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