Pharmaceutical composition comprising enavogliflozin as active ingredient for preventing or treating alopecia areata

Abstract

The present disclosure relates to a pharmaceutical composition comprising enavogliflozin as an active ingredient for preventing or treating alopecia areata. Enavogliflozin of the present disclosure increases β-hydroxybutyrate (BHB) to inhibit NLRP3 inflammasome and reduce IL-1β, and reduces the expression of inflammatory proteins such as NLRP3, Caspase-1, ASC, and IL-1β to alleviate inflammation associated with the onset of alopecia areata while decreasing the expression of NKG2D, which contributes to follicular destruction, thereby being able to be advantageously used to prevent or treat alopecia areata.

Description

Pharmaceutical composition for preventing or treating alopecia areata containing inabogliflozin as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating alopecia areata, comprising inabogliflozin as an active ingredient.

[0002] Alopecia areata (Alopecia areata) is a chronic, recurrent hair loss disorder that affects approximately 2% of the global population. It is a relatively common condition and can occur in both men and women of all ages. Genetic predisposition and autoimmune responses are known to be the most important causes of Alopecia areata, although various environmental factors can also play a role. However, the exact pathogenesis remains unknown, and its treatment remains unclear.

[0003] The most important cause of alopecia areata is immunological factors. T cells are activated due to certain stimuli, etc., and mistakenly recognize hair follicles as foreign substances and attack them, triggering an immune response, which leads to alopecia areata. A recent non-clinical study suggested a new mechanism in which alopecia areata occurs through an immune response due to NLRP3 inflammasome activation. It was reported that increased expression of NLRP3, ASC, Caspase-1, and IL-1β, which form the NLRP3 inflammasome, was observed in the scalp hair follicles of patients with alopecia areata (see Non-Patent Document 1, [JM Shin et al., Scientific Reports (2017) 7:44127]). In particular, a study was conducted to confirm that administering MCC950, a substance known as an NLRP3 inhibitor, to a mouse model of alopecia areata inhibits NLRP3 inflammasome activity, thereby alleviating the inflammatory response and improving alopecia areata (see Non-patent Document 2, [K Hashimoto et al., Exp Dermatol. 2022 Feb;31(2):133-142]).

[0004] Currently, various treatments for alopecia areata, including steroids and Janus kinase (JAK) inhibitors, are being used. However, the time it takes for the drugs to take effect, their high cost, the potential side effects of long-term use, and the high relapse rate after discontinuation of the medications are raising the need for new treatments.

[0005] Sodium-glucose co-transporter (SGLT) is a glucose transport protein. SGLT-1 is expressed in the small intestine, liver, kidney, and heart, while SGLT-2 is primarily expressed in the kidney. SGLT-2 inhibitors are a new class of antidiabetic drugs that reduce glucose reabsorption in the proximal nephron, thereby increasing glucose excretion through an insulin-independent mechanism. One of the drugs developed as an SGLT-2 inhibitor that can be useful as a treatment for type 2 diabetes due to this effect is enavogliflozin, which is disclosed in Korean Patent Publication No. 2014-0022086 (Patent Document 1).

[0006] During the research on another medicinal use of inavogliflozin, the present inventors noted that β-hydroxybutyrate (hereinafter, BHB) increased when fasting for 24 hours after taking inavogliflozin in normal subjects and type 2 diabetic clinical trials.

[0007] Recently, non-patent literature 3 reported that SGLT2 inhibitors attenuate NLRP3 inflammasome activation and IL-1β secretion, which affect both type 2 diabetes and cardiovascular disease, by increasing serum BHB and decreasing serum insulin (see non-patent literature 3, [SR Kim et al., Nature Communications (2020) 11:2127]).

[0008] The relationship between increased BHB and alopecia areata has not yet been reported. However, based on previously reported literature, the present inventors focused on the possibility that SGLT-2 inhibitors, independently of their glycemic control, increase BHB, thereby suppressing NLRP3 inflammasome activation and thus regulating inflammation, thereby preventing or treating alopecia areata.

[0009] [Prior Art Literature]

[0010] [Patent Document]

[0011] Republic of Korea Patent Publication No. 2014-0022086

[0012] [Non-patent literature]

[0013] JM Shin et al., Scientific Reports (2017) 7:44127

[0014] K Hashimoto et al., Exp Dermatol. 2022 Feb;31(2):133-142

[0015] SR Kim et al., Nature Communications (2020) 11:2127

[0016] As a result of the above research, the inventors of the present invention confirmed that inabogliflozin has an excellent effect in preventing or treating alopecia areata by improving inflammation through inhibition of NLRP3 inflammasome activation, thereby completing the present invention.

[0017] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating alopecia areata, which contains inabogliflozin as an active ingredient.

[0018] Another object of the present invention is to provide a method for preventing or treating alopecia areata, which comprises administering a pharmaceutical composition for preventing or treating alopecia areata containing inabogliflozin as an active ingredient to a subject in need thereof.

[0019] Another object of the present invention is to provide a use of inabogliflozin for preparing a pharmaceutical composition for preventing or treating alopecia areata.

[0020] The present invention relates to a pharmaceutical composition for preventing or treating alopecia areata, comprising Enavogliflozin as an active ingredient, and to the prevention or treatment of alopecia areata using the same. It has been confirmed that the pharmaceutical composition according to the present invention is significantly superior in preventing or treating alopecia areata.

[0021] Hereinafter, the present invention will be described in more detail.

[0022] One specific example of the present invention provides a pharmaceutical composition for preventing or treating alopecia areata, comprising inabogliflozin as an active ingredient.

[0023] In the following examples, it was confirmed that administration of inavogliflozin tends to increase plasma BHB concentrations and decreases IL-1β secreted when NLRP3 inflammasome is activated. In addition, inavogliflozin dose-dependently decreased the expression of inflammatory factors NLRP3, Caspase-1, ASC, and IL-1β proteins, confirming the possibility of improving inflammation that occurs in the onset of alopecia areata. In addition, it was confirmed that inavogliflozin can improve alopecia areata by reducing the expression of NKG2D, which is expressed in pathogenic cells in alopecia areata and causes hair follicle destruction.

[0024] The term "inavogliflozin" used herein is an SGLT-2 (Sodium-Glucose Cotransporter 2) inhibitor, a drug that selectively inhibits SGLT-2, which is involved in the reabsorption of glucose in the kidneys, thereby preventing glucose from being absorbed in the body and excreting it in the urine.

[0025] In the present invention, “inabogliflozin” may have a structure represented by the following chemical formula 1.

[0026] [Chemical Formula 1]

[0027]

[0028] The term “prevention” as used herein means any act of inhibiting or delaying the onset of alopecia areata by administering a pharmaceutical composition according to the present invention.

[0029] The term “treatment” as used herein means any action by which alopecia areata is improved or beneficially altered by administration of the pharmaceutical composition according to the present invention.

[0030] Another specific embodiment of the present invention is a method for preventing or treating alopecia areata, which comprises administering to a subject in need thereof a pharmaceutical composition for preventing or treating alopecia areata, which comprises inabogliflozin as an active ingredient. In one specific embodiment of the present invention, the pharmaceutical composition for preventing or treating alopecia areata, which comprises inabogliflozin as an active ingredient, can be used in the form of single administration or combination administration.

[0031] Although inabogliflozin can be used as a single therapy for the prevention or treatment of alopecia areata, alopecia areata treatments are administered in combination for various purposes, and since they can exhibit complementary or synergistic effects when administered in combination, a pharmaceutical composition containing inabogliflozin can be administered in combination with other alopecia areata treatments.

[0032] In one embodiment of the present invention, the pharmaceutical composition may be administered in combination with one or more drugs selected from corticosteroids, minoxidil, and JAK inhibitors.

[0033] Corticosteroids are frequently used to treat mild to moderate alopecia areata. The route of administration of corticosteroids administered in combination with inavogliflozin is not particularly restricted and can be oral or parenteral.

[0034] Minoxidil, which is commonly prescribed to alleviate hair loss symptoms, can also be administered orally or in combination with inavogliflozin.

[0035] JAK inhibitors, a type of immunomodulatory agent, are known to help suppress inflammation by reducing cytokines. A clinical report published in 2014 was the first to demonstrate that the JAK inhibitor tofacitinib (brand name Xeljanz®) could successfully treat alopecia areata. Since then, numerous studies have shown that JAK inhibitors may be effective in promoting hair regrowth in alopecia areata. Tofacitinib is an older drug that is still prescribed off-label for alopecia areata. As of 2022, the FDA has approved two JAK inhibitors, baricitinib and ritlecitinib, for the treatment of severe alopecia areata. Another JAK inhibitor, deuruxolitinib, is also in clinical trials for the treatment of alopecia areata. Therefore, JAK inhibitors that can be co-administered with inavogliflozin include, but are not limited to, tofacitinib, baricitinib, ritrecitinib, duruxolitinib, etc.

[0036] In another embodiment, the pharmaceutical composition of the present invention may be used in combination with immunotherapy for the treatment of alopecia areata. Immunotherapy for alopecia areata here refers to topical immunotherapy, also known as contact immunotherapy. Immunotherapy agents cause an allergic rash (allergic contact dermatitis) at the site of application. While the exact cause is unknown, this is known to alter the body's immune response around hair follicles and promote hair regrowth. The most commonly used drugs, i.e., immunotherapy agents, applied topically to areas of alopecia areata in immunotherapy are squaric acid dibutyl ester (SADBE) or diphencyprone (DPCP).

[0037] In one embodiment of the present invention, the pharmaceutical composition may be for oral or parenteral use.

[0038] The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier in addition to the active ingredient, inabogliflozin of chemical formula 1, or a pharmaceutically acceptable salt thereof, and may be formulated together with the carrier.

[0039] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0040] In the present invention, the pharmaceutical composition may have a dosage form for oral administration, such as a tablet or capsule. In one specific embodiment of the present invention, the pharmaceutical composition may have a dosage form of a tablet.

[0041] In the present invention, the pharmaceutical composition may have a formulation for parenteral administration. For example, but not limited to, a formulation for parenteral administration containing the composition of the present invention as an active ingredient may be formulated in an injectable form, such as subcutaneous injection, intravenous injection, or intramuscular injection.

[0042] To formulate it into an injectable formulation, the composition of the present invention can be prepared as a solution or suspension by mixing it in water with a stabilizer or buffer, and this can be formulated into a unit dose form in an ampoule or vial.

[0043] Alternatively, the composition of the present invention may be formulated into various forms for parenteral administration, such as eye drops, microneedles, patches, and depots.

[0044] The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease, and the effective dosage level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. That is, the total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered through a fractionated treatment protocol in which multiple doses are administered over a long period of time. Taking all of the above factors into consideration, it is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0045] The appropriate daily dose of inabogliflozin identified during clinical trials is 0.1 mg to 0.5 mg.

[0046] When the above pharmaceutical composition is formulated as a unit dosage form, the content of the active ingredient in the pharmaceutical composition may be 0.1 mg to 0.5 mg.

[0047] The pharmaceutical composition according to the present invention may be administered once to three times a day, for example, once a day, but is not limited thereto.

[0048] In the present invention, the dosage of inabogliflozin that can be used for the prevention or treatment of alopecia areata is not particularly limited, and can be appropriately adjusted depending on the severity of the disease of the subject of administration, weight, age, gender, and presence or absence of other complications.

[0049] Since the above method for preventing or treating alopecia areata uses a pharmaceutical composition for preventing or treating alopecia areata, any overlapping content between the two is omitted to avoid excessive description in the specification.

[0050] Inavogliflozin used as an active ingredient in the present invention can be synthesized through known prior literature. In the present invention, inavogliflozin can be crystalline or amorphous. For example, inavogliflozin can be inavogliflozin crystalline form A, crystalline form B, crystalline form C, crystalline form D, crystalline form E or inavogliflozin amorphous, which are reported to have the following X-ray diffraction spectra through Korean Patent Publication No. 2017-0142904 or Korean Patent Application No. 2022-0123673.

[0051] Crystalline Form A: A crystalline form having an X-ray diffraction (XRD) spectrum comprising peaks at 2[θ] values ​​selected from 6.2°±0.2°, 7.2°±0.2°, 8.8°±0.2°, 17.6°±0.2°, 19.0°±0.2°, 22.5°±0.2°, and 25.1°±0.2°.

[0052] Crystalline Form B: A crystalline form having an X-ray diffraction (XRD) spectrum comprising peaks at 2[θ] values ​​selected from 7.0°±0.2°, 14.9°±0.2°, 17.7°±0.2°, 18.8°±0.2°, 20.6°±0.2°, 21.8°±0.2°, and 23.5°±0.2°.

[0053] Crystalline Form C: A crystalline form having an X-ray diffraction (XRD) spectrum comprising peaks at 2[θ] values ​​selected from 5.6°±0.2°, 7.3°±0.2°, 15.7°±0.2°, 17.2°±0.2°, 18.9°±0.2°, 21.2°±0.2°, and 21.9°±0.2°.

[0054] Crystalline Form D: A crystalline form having an X-ray diffraction (XRD) spectrum comprising peaks at 2[θ] values ​​selected from 5.5°±0.2°, 7.2°±0.2°, 15.3°±0.2°, 17.2°±0.2°, 17.6°±0.2°, 18.9°±0.2°, and 21.1°±0.2°.

[0055] Crystalline Form E: A crystalline form having an X-ray diffraction (XRD) spectrum comprising peaks at 2[θ] values ​​selected from 4.93°±0.2°, 6.12°±0.2°, 7.43°±0.2°, 8.89°±0.2°, 9.74°±0.2°, 14.79°±0.2°, 15.79°±0.2°, 16.11°±0.2°, 19.79°±0.2°, and 22.83°±0.2°

[0056] The above crystal forms A, B, C, D and E can be characterized by X-ray diffraction spectra each having four or more peaks, for example, four, five, six, seven, eight or more, at the above-mentioned 2[θ] values.

[0057] Although not limited thereto, the pharmaceutical composition comprising inabogliflozin according to the present invention may have the composition of the pharmaceutical composition of PCT / KR2022 / 014640.

[0058] For example, the pharmaceutical composition of the present invention may be a pharmaceutical composition comprising a compound of formula 1 or a pharmaceutically acceptable salt thereof, an excipient, a disintegrant, and a binder as an active ingredient, and wherein the average particle size of the compound of formula 1 is 15 μm or less.

[0059] In a specific example of the present invention, the average particle size of inabogliflozin may be 15 um or less, preferably 10 um or less.

[0060] When the average particle size of inabogliflozin exceeds 15 um, the 5-minute dissolution rate is very low, less than 40% of the total content of inabogliflozin, and the 30-minute dissolution rate is also less than 80%, so the final dissolution rate is found to be inadequate.

[0061] If micronization of drug particle size is required, the drug can be milled using a conventional mill capable of micronizing particles, such as a Z-mill, hammer mill, ball mill, or fluid energy mill. In addition, a size classification method, such as a sieving method using a sieve or air current classification, can be used to refine the drug particle size. Methods for controlling the desired particle size are well known in the art. For example, see the following literature: [Pharmaceutical dosage forms: volume 2, 2nd edition, Ed.: HALieberman, L. Lachman, J. B. Schwartz (Chapter 3: SIZE REDUCTION)].

[0062] In this specification, the particle size of a drug is expressed based on a particle size distribution such as D(X) = Y (where X and Y are positive numbers). D(X) = Y means that when the particle size distribution of a drug obtained by measuring the particle diameter of a certain drug in a formulation is expressed by a cumulative curve, the particle diameter at the point where the particle sizes accumulate from the smallest to the largest is X% (% is calculated based on number, volume, or weight), is Y. For example, D(10) represents the particle diameter at the point where the particle sizes of the drug accumulate from the smallest to the smallest, D(50) represents the particle diameter at the point where the particle sizes of the drug accumulate from the smallest to the smallest, and D(90) represents the particle diameter at the point where the particle sizes of the drug accumulate from the smallest to the smallest.

[0063] Whether the particle size distribution D(X) represents a percentage of the total cumulative particles by number, volume, or weight depends on the method used to measure the particle size distribution. Methods for measuring particle size distributions and the type of percentages associated with them are well known in the art. For example, when measuring particle size distributions by the well-known laser diffraction method, the X value in D(X) represents the percentage calculated by volume average. It is well known to those skilled in the art that particle size distribution measurement results obtained by a particular method can be correlated with those obtained by other techniques based on experience through routine experiments. For example, laser diffraction is sensitive to particle volume and provides a volume average particle size, which corresponds to a weight average particle size at constant density.

[0064] In the present invention, the particle size distribution of drug particles can be measured using a commercially available device based on laser diffraction and scattering based on Mie theory. For example, measurement is performed using a commercially available device such as the Mastersizer laser diffraction device from Malvern Instruments. This device irradiates particles with a helium-neon laser beam and a blue light-emitting diode, causing scattering to occur, and a light scattering pattern appears on a detector. By interpreting this light scattering pattern according to Mie theory, the particle diameter distribution is obtained. Either a dry or wet method can be used for the measurement. In one embodiment of the present invention, the average particle size of inavogliflozin may be a volume average particle size obtained by laser diffraction.

[0065] In a specific example of the present invention, the compound of Chemical Formula 1 may be included in an amount of less than 1 part by weight based on 100 parts by weight of the total pharmaceutical composition.

[0066] The appropriate daily dosage of inabogliflozin identified during clinical trials is 0.1 mg to 0.5 mg. When the pharmaceutical composition is formulated as a unit dosage form, the content of the active ingredient in the pharmaceutical composition may be 0.1 mg to 0.5 mg.

[0067] The pharmaceutical composition according to the present invention comprises pharmaceutically acceptable additives in addition to the compound of chemical formula 1, which is an active ingredient.

[0068] The pharmaceutical composition of the present invention includes excipients, disintegrants, and binders as additives.

[0069] Examples of excipients include lactose (including hydrate), dextrin, mannitol, sorbitol, starch, microcrystalline cellulose (e.g., Celphere™), silicified microcrystalline cellulose (e.g., Prosolv™), calcium phosphate hydrate, anhydrous calcium phosphate, calcium carbonate, sugars, or mixtures thereof. In embodiments of the present invention, a preferred excipient is microcrystalline cellulose.

[0070] Examples of disintegrants include crospovidone, croscarmellose sodium, sodium starch glycolate, and low-substituted hydroxypropyl cellulose. In a specific embodiment of the present invention, a preferred excipient is croscarmellose sodium.

[0071] Examples of binders include polyvinylpyrrolidone, povidone, gelatin, starch, sucrose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylalkylcellulose (e.g., hydroxypropylmethylcellulose), and mixtures thereof. In an embodiment of the invention, a preferred binder is hydroxypropylcellulose.

[0072] Examples of other additives include lubricants and colorants.

[0073] The above-mentioned lubricants include stearic acid, stearic acid salts (e.g., magnesium stearate), light anhydrous silicic acid, talc, corn starch, carnauba wax, magnesium silicate, synthetic aluminum silicate, hydrogenated oil, white lead, titanium dioxide, microcrystalline cellulose, macrogol 4000 and 6000, isopropyl myristate, calcium hydrogen phosphate, and mixtures thereof.

[0074] In a specific embodiment of the present invention, the excipient may be included in an amount of 80 to 95 parts by weight based on 100 parts by weight of the total pharmaceutical composition.

[0075] In a specific embodiment of the present invention, the disintegrant may be included in an amount of 2 to 8 parts by weight relative to 100 parts by weight of the total pharmaceutical composition. If the disintegrant is included in an amount of less than 2 parts by weight relative to 100 parts by weight of the total pharmaceutical composition, the initial disintegration power may be low, which may delay the dissolution rate, and this may affect the Cmax in the body. In addition, if the amount of the disintegrant is included in an amount of more than 8 parts by weight relative to 100 parts by weight of the total pharmaceutical composition, the amount of the disintegrant in the post-mixing portion may increase, which may reduce the overall flowability of the granules.

[0076] In a specific embodiment of the present invention, the binder may be included in an amount of 3 to 10 parts by weight relative to 100 parts by weight of the total pharmaceutical composition. If the binder is included in an amount of less than 3 parts by weight relative to 100 parts by weight of the total pharmaceutical composition, it may be difficult to form and maintain suitable dry granules, which may affect the maintenance of homogeneous dispersion of the main ingredient and the flowability of the granules due to the generation of fine particles. In addition, if the binder is included in an amount of more than 10 parts by weight relative to 100 parts by weight of the total pharmaceutical composition, granules having strong binding force are formed, which may affect the solubility of the granule particles that are initially disintegrated during dissolution, which may also affect the Cmax in the body.

[0077] The pharmaceutical composition according to the present invention may be an immediate-release formulation.

[0078] In one specific embodiment of the present invention, the pharmaceutical composition may have a dissolution rate of 50% or more, preferably 60% or more, of the total content of the active ingredient after 5 minutes.

[0079] In one specific embodiment of the present invention, the pharmaceutical composition may have a dissolution rate of 80% or more, preferably 80% or more, of the total content of the active ingredient after 15 minutes.

[0080] In one specific embodiment of the present invention, the pharmaceutical composition may have a dissolution rate of 85% or more, preferably 90% or more, of the total content of the active ingredient after 30 minutes.

[0081] The dissolution rate of the active ingredient in the pharmaceutical composition affects the peak blood concentration (Cmax) and the area under the blood concentration-time curve (AUC) when the drug is administered. Therefore, conversely, it is important to adjust the dissolution rate of the pharmaceutical composition to achieve appropriate Cmax and AUC. Since inavogliflozin has a Tmax of 1 to 2 hours, the drug absorption rate from above is considered important. The above dissolution rate was measured under the condition of a dissolution volume of 1.2 according to the dissolution test method 2 (paddle method) of the Korean Pharmacopoeia. For specific conditions, please refer to the experimental example below.

[0082] The present invention also

[0083] A pharmaceutical composition comprising a granule mixture comprising a premixed granule and a postmixed granule comprising a compound of formula 1 or a pharmaceutically acceptable salt thereof is provided.

[0084] During the study on formulation of the compound of chemical formula 1, the inventors of the present invention confirmed that preparing granules and formulating them into tablets, etc. is advantageous in terms of drug content uniformity and formulation uniformity.

[0085] In the above pharmaceutical composition, the granules are prepared by mixing pre-mixed granules and a post-mixed portion.

[0086] The above premixed granules may include a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof, an excipient, a binder, and a lubricant.

[0087] Additionally, the post-mixing portion may include excipients, disintegrants, and lubricants.

[0088] Descriptions of excipients, binders, disintegrants, lubricants, etc. are the same as those described above, so they are omitted to avoid duplicate description.

[0089] In a specific embodiment of the present invention, the pre-mixed granules and the post-mixed portion may each include an excipient. More specifically, the pre-mixed granules and the post-mixed portion may each include microcrystalline cellulose as an excipient.

[0090] It was found that the microcrystalline cellulose included in the above pre-mixed granules and post-mixed portion affects the uniformity of drug content depending on its particle size and bulk density.

[0091] In a specific example of the present invention, the particle size of the microcrystalline cellulose in the pre-mixed granules may be 130 μm or less, preferably 60 μm to 130 μm. The bulk density of the microcrystalline cellulose in the pre-mixed granules may be 0.26 to 0.33. When the particle size and bulk density of the microcrystalline cellulose in the pre-mixed granules are as above, a formulation with a low deviation (SD) of content uniformity can be secured. When the particle size of the microcrystalline cellulose in the pre-mixed granules is 130 μm or less, the content uniformity of the pre-mixed granules, the content uniformity of the final granules, and the formulation all exhibited good levels, and the Carr's index value indicating the physical properties of the final granules was also good, so it was confirmed that the flowability of the formulation was also excellent. On the other hand, when the particle size of microcrystalline cellulose in the premixed granules exceeded 130 μm, both the content uniformity of the premixed granules and the content uniformity of the final granules showed a large deviation and were not suitable, and the formulation uniformity was also not good.

[0092] Meanwhile, the particle size of the microcrystalline cellulose in the post-mixing unit may be 130 μm or more, preferably 130 μm to 250 μm. The bulk density of the excipient in the post-mixing unit may be 0.28 to 0.37. When the particle size of the microcrystalline cellulose in the post-mixing unit is less than 130 μm, it was confirmed that the Carr's index value, which indicates the physical properties of the final granules, was not appropriate and the granule flowability became poor.

[0093] When the microcrystalline cellulose in the pre-mixed granules is relatively compared to the microcrystalline cellulose in the post-mixed portion, it is found that, unlike the microcrystalline cellulose contained in the pre-mixed granules, it is desirable for the particle size of the microcrystalline cellulose in the post-mixed portion to be relatively large compared to the particle size of the microcrystalline cellulose contained in the pre-mixed granules.

[0094] It was confirmed that not only the particle size of microcrystalline cellulose in the pre-mixed granules and the microcrystalline cellulose in the post-mixed portion, but also the weight ratio of the excipients in the pre-mixed granules and the excipients in the post-mixed portion affected the drug content uniformity.

[0095] In a specific example of the present invention, the weight ratio of the excipients in the pre-mixed granules and the excipients in the post-mixed portion may be 4:1 to 1:1. As the proportion of microcrystalline cellulose in the post-mixed portion increases, the flowability of the granules improves, but the content deviation increases, so it was determined that it is desirable to maintain the weight ratio within the above-described appropriate range.

[0096] Meanwhile, in the pharmaceutical composition according to the present invention, the binder may be at least one selected from the group consisting of hydroxypropylcellulose, povidone, copovidone, and hypromellose.

[0097] In one specific example of the present invention, the binder is hydroxypropyl cellulose and may have a weight average molecular weight of less than 200,000. If hydroxypropyl cellulose having a weight average molecular weight of 200,000 or more is used, both the 5-minute dissolution rate and the 30-minute dissolution rate are low, which is undesirable in terms of bioavailability.

[0098] Meanwhile, with respect to Carr's index, which is used as a measure of flowability in formulation, it is preferable that the Carr's index of the granules be 21 to 25.

[0099] Although not limited thereto, the granules in the pharmaceutical composition of the present invention may be dry granules. In another specific embodiment, the granules may be wet granules.

[0100] In the present invention, the pharmaceutical composition may have a dosage form for oral administration, such as a tablet or capsule. In one specific embodiment of the present invention, the pharmaceutical composition may have a dosage form of a tablet.

[0101] In a preferred embodiment, the pharmaceutical composition may comprise the compound of formula 1 in a dosage of 0.3 mg.

[0102] The pharmaceutical composition according to the present invention may be administered orally once a day, but is not limited thereto.

[0103] The inabogliflozin of the present invention increases BHB (β-hydroxybutyrate) to suppress NLRP3 inflammasome, reduce IL-1β, and reduce the expression of inflammatory factors NLRP3, Caspase-1, ASC, and IL-1β proteins, thereby improving inflammation that occurs when alopecia areata occurs, while reducing the expression of NKG2D, which causes hair follicle destruction, and thus can be usefully used for the prevention or treatment of alopecia areata.

[0104] Figure 1 is a graph showing the ratio of mice that did not develop alopecia areata according to one embodiment of the present invention.

[0105] Figure 2 is a graph showing the ratio of the size of alopecia areata that occurred in mice with alopecia areata according to one embodiment of the present invention.

[0106] Figure 3 is a diagram showing representative comparative photographs of each material treatment group of mice with circular alopecia according to one embodiment of the present invention.

[0107] FIG. 4 is a graph showing BHB production confirmed in the plasma of a mouse with alopecia areata according to one embodiment of the present invention.

[0108] FIG. 5 is a diagram showing changes in protein expression of inflammatory factors through staining in skin tissue of a mouse with circular alopecia according to one embodiment of the present invention.

[0109] Figure 6 is a diagram showing changes in protein expression of inflammatory factors in lymph node cells of a mouse with circular alopecia according to one embodiment of the present invention.

[0110] FIG. 7 is a diagram showing changes in the expression of NKG2D confirmed when alopecia areata develops in pathogenic cells isolated from lymph node cells of a mouse in which alopecia areata develops according to one embodiment of the present invention.

[0111] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention. The present invention is defined solely by the scope of the claims.

[0112]

[0113] Example 1. Evaluation of the efficacy of inabogliflozin on alopecia areata in a mouse model of alopecia areata.

[0114] Eight-week-old C3H / Hej female mice were obtained and provided with sufficient solid food (antibiotic-free) and water until the day of the experiment. They were acclimated to an environment with a temperature of 23±2℃, a humidity of 55±10%, and a 12-hour light-dark cycle before being used in the experiment.

[0115] A model of alopecia areata was created using the lymph node cell injection method. Lymph node cells were isolated from mice (donors) with alopecia areata and expanded using Dynabead Mouse T-activator CD3 / CD28, Human Recombinant IL-2, Mouse Recombinant IL-7, and Mouse Recombinant IL-15. 1.5 x 10 cells were injected into C3H / Hej mice (recipients) aged 10 weeks or older. 7 A circular hair loss model was created by injecting cells / mouse intra-dermal injection.

[0116] To evaluate the efficacy of inabogliflozin on alopecia areata in a mouse model of alopecia areata produced by lymph node cell injection, test groups were set up and evaluated as shown in Table 1.

[0117] Test group Alopecia areata induction method Test substance Administration dose (mg / kg) Administration route Number of administrations Number of animals G1 Lymph node cell injection method Vehicle (NC) N / APOQD6 G2 Enavogliflozin (ENA) 0.16 G3 16

[0118] The test substances used were 0.1 mg / kg and 1 mg / kg of inabogliflozin. The vehicle group was prepared with 1% Tween 80 in 0.5% Carboxymethyl cellulose sodium salt and stored at 4℃ before use.

[0119] After lymph node cell injection into mice, test substances were administered to the mice by selecting them into groups at the 4th week when alopecia areata was visually confirmed. Individual selection was performed at a time when alopecia areata was induced in approximately 50% of all mice after lymph node cell injection. Mice with alopecia areata occurring in more than 50% of the mice, mice with alopecia areata occurring in less than 50% of the mice, and mice with no alopecia areata were selected, so that a total of 6 mice were in each group.

[0120] The administration period of the test substance was 12 weeks, and the vehicle group and the inabogliflozin group were administered orally (PO injection) once daily (QD).

[0121]

[0122] (1) Measurement of the disease-free ratio

[0123] The incidence of alopecia areata was expressed as the ratio of the number of mice that did not develop alopecia areata to the total number of individuals.

[0124] Statistical analysis of the data was performed using Prism 10.0 software (GraphPad Software Inc., San Diego, CA, USA). The experimental results are expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was performed to verify differences between groups. Post hoc tests for each type were performed using Tukey's test or Dunn's multiple comparison test. The significance level was set at p<0.05.

[0125] Figure 1 and Table 2 show the percentage of mice that did not develop alopecia areata according to one embodiment of the present invention.

[0126] Disease-free ratioWeekVehicleENA 0.1 mg / kgENA 1 mg / kg04 / 6 (66.7%)3 / 6 (50%)3 / 6 (50%)22 / 6 (33.3%)2 / 6 (33.3%)2 / 6 (33.3%)32 / 6 (33.3%)2 / 6 (33.3%)3 / 6 (50%)42 / 6 (33.3%)1 / 6 (16.7%)3 / 6 (50%)51 / 6 (16.7%)1 / 6 (16.7%)3 / 6 (50%)80 / 5 (0%)1 / 5 (20%)2 / 6 (33.3%)100 / 5 (0%)1 / 5 (20%)2 / 6 (33.3%)120 / 5 (0%)1 / 5 (20%)1 / 6 (16.7%)

[0127] As shown in Figure 1 and Table 2, in the vehicle group, alopecia areata was observed in 100% of all mice 12 weeks after lymph node cell injection (8 weeks after substance administration). In the inavogliflozin-administered group, alopecia areata was observed in 80-83.3% by 16 weeks after lymph node cell injection (12 weeks after substance administration). These results indicate that inavogliflozin protects against or improves the occurrence of alopecia areata.

[0128]

[0129] (2) Measurement of the size of the alopecia areata (AA lesion size)

[0130] The size of alopecia areata was measured by taking photographs every two weeks using a digital single-lens reflex camera (DSLR) and expressing the area affected as a percentage of the total area of ​​the mouse using ImageJ (National Institutes of Health). The occurrence of alopecia areata was measured over the entire back and belly of the mouse.

[0131] Statistical analysis of the data was performed using Prism 10.0 software (GraphPad Software Inc., San Diego, CA, USA). Experimental results are expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was performed to examine differences between groups. Post-hoc tests for each type were performed using the Tukey function or Dunn's multiple comparison test. The significance level was set at p<0.05.

[0132] As shown in Figures 2 and 3, in the last 12 weeks of administration, the size of alopecia areata was smaller in the 1 mg / kg and 0.1 mg / kg inavogliflozin groups compared to the vehicle group. The 1 mg / kg inavogliflozin group showed a significant difference compared to the vehicle group. These results indicate that inavogliflozin protects against or improves the onset of alopecia areata.

[0133]

[0134] (3) Measurement of plasma BHB concentration

[0135] After the test, fasting was induced by removing food for 24 hours before blood collection. The collected blood was placed in anticoagulant-treated tubes and centrifuged at 13,000 rpm for 10 minutes to obtain the supernatant. Blood proteins were then concentrated using a 10 kDa molecular weight cut-off (MWCO) spin filter, and the BHB concentration was confirmed using a BHB assay kit (MAK041, Sigma, MO, USA).

[0136] Statistical analysis of the data was performed using Prism 10.0 software (GraphPad Software Inc., San Diego, CA, USA). Experimental results are expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was performed to examine differences between groups. Post-hoc tests for each type were performed using the Tukey function or Dunn's multiple comparison test. The significance level was set at p<0.05.

[0137] As can be seen in Figure 4, although there was no statistical significance compared to the vehicle group, a tendency was observed for the plasma BHB concentration to increase with administration of 1 mg / kg of inabogliflozin.

[0138]

[0139] (4) Histological analysis

[0140] At the 12th week, the end point of test substance administration, the skin was removed and fixed in 10% formalin solution. After making a paraffin block, the tissue was cut, slides were made, and stained using ImmPRESS Anti-Rabbit Ig Kit (Catalog No. MP-7401) or ImmPRESS Anti-Rat Ig Kit (Catalog No. MP-7404). NLRP3 (768319, Invitrogen, CA, USA), caspase-1 (Ab138483, Abcam, Cambridge, UK), ASC (NBP1-78977, Novusbio, CO, USA), and IL-1β (AF5103, Affinity biosciences, OH, USA) were used as primary antibodies. The tissues were analyzed using an optical microscope (PANORAMIC MIDI II) and the tissue slides were photographed at 100x magnification, and the expression level of each slide was read using Case Viewer software.

[0141] As can be seen in Fig. 5, in the vehicle group, brown staining was observed in the epidermis, dermis, and hair follicles of the skin. This means that NLRP3, Caspase-1, ASC, which are components of the NLRP3 inflammasome, which is an inflammation target, and IL-1β, which is secreted when the NLRP3 inflammasome is activated, were highly expressed in the skin of the alopecia areata mouse model. Compared to the vehicle group, it was confirmed that the expression of inflammatory factors was reduced in the group administered 1 mg / kg of inavogliflozin, indicating that inflammation related to the NLRP3 inflammasome in the skin of the alopecia areata mouse model can be improved.

[0142]

[0143] (5) Analysis of changes in inflammatory factor protein expression

[0144] At week 12, the end point of test substance administration, skin was removed and lysed, and protein concentration was measured using a BCA protein assay kit (Bio-Rad, Hercules, CA, USA). Protein (20 μg) was separated by SDS-PAGE (polyacrylamide gel electrophoresis) and adsorbed to a PVDF (polyvinylidene fluoride) membrane (Millipore Corp, Bedford, MA). The membrane was reacted in phosphate-buffered saline (PBS) containing 0.1% Tween-20 and 5% skim milk (non-fat milk), and the reaction was performed using a primary antibody diluted in 0.2% BSA (in PBS). After washing three times, the membrane was reacted with HRP-conjugated secondary antibody (anti-rabbit IgG antibody (PI-1000) or anti-mouse IgG antibody (PI-2000), Vector Labs Inc., Burlingame, CA, USA), and the bands were visualized using an ECL advance kit (ATTO, Tokyo, Japan).

[0145] Statistical analysis of the data was performed using Prism 10.0 software (GraphPad Software Inc., San Diego, CA, USA). Experimental results are expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was performed to examine differences between groups. Post-hoc tests for each type were performed using the Tukey function or Dunn's multiple comparison test. The significance level was set at p<0.05.

[0146] As shown in Fig. 6, it was confirmed that the inflammatory factors NLRP3, Caspase-1, ASC, and IL-1β proteins were significantly reduced in a dose-dependent manner in the 0.1 and 1 mg / kg inavogliflozin treatment groups compared to the vehicle group. Based on Figs. 5 and 6, it can be confirmed that the inflammation that appears at the onset of alopecia areata can be improved as the expression of inflammatory factors is reduced in the skin tissue of the alopecia areata mouse model when inavogliflozin is treated.

[0147]

[0148] (6) Flow cytometry analysis of lymph nodes

[0149] At the end of the experiment, skin-draining lymph node cells from mice were removed from the nitrogen tank and used for flow cytometry. Cells were analyzed by staining with antibodies against L / D, CD3, CD4, CD8, CD44, CD49d, CD62L, and NKG2D without additional stimulation. Stained cells were subjected to quantitative analysis of cell surface proteins using a FACSymphony A1 (Becton, Dickinson and Company, BD). Afterwards, MFI and positive cell population were confirmed using FlowJo (Becton, Dickinson and Company, BD, v. 10.0).

[0150] Experimental results are expressed as the mean ± standard error of the mean (SEM). GraphPad Prism version 7 software was used to determine statistical significance. The Mann-Whitney test was used to verify the significance of flow cytometry data, and a p value of 0.05 or less was considered statistically significant.

[0151] It is known that when alopecia areata develops, virtual memory T cells (TVM cells) become pathogenic cells due to inflammatory cytokines, and these cytokines upregulate the expression of NKG2D, destroying hair follicles and contributing to the worsening of alopecia areata symptoms (see Non-patent Document 5, [Seok J et al., Nature Immunology 24, 1308-1317 (2023)].

[0152] As can be seen in Figure 7, the pathogenic cells (CD44) in the lymph node cells s-hi ) The ratio did not show a significant decrease in the inavogliflozin administration group compared to the vehicle group, but the MFI value of NKG2D, which is expressed in the pathogenic cells when alopecia areata develops, showed a significant decrease at 1 mg / kg of inavogliflozin. This indicates that inavogliflozin can improve alopecia areata by reducing the expression of NKG2D, which is expressed in the pathogenic cells in alopecia areata and causes hair follicle destruction.

Claims

1. A pharmaceutical composition for preventing or treating alopecia areata, containing inabogliflozin as an active ingredient.

2. In the first paragraph, the pharmaceutical composition is a pharmaceutical composition for preventing or treating alopecia areata for oral or parenteral administration.

3. A pharmaceutical composition for preventing or treating alopecia areata in the first paragraph, wherein a single administration dose of the inabogliflozin is 0.1 mg to 0.5 mg.

4. A pharmaceutical composition for preventing or treating alopecia areata in the first paragraph, wherein the pharmaceutical composition is administered once a day.

5. A pharmaceutical composition for preventing or treating alopecia areata in the first paragraph, wherein the pharmaceutical composition is administered in combination with one or more drugs selected from corticosteroids, minoxidil, and JAK inhibitors.

6. A pharmaceutical composition for preventing or treating alopecia areata in accordance with paragraph 1, wherein the pharmaceutical composition is used in combination with immunotherapy for treating alopecia areata.

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