WO2025143870A1 - Pharmaceutical composition comprising enavogliflozin as active ingredient for preventing or treating alopecia areata - Google Patents

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

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

Alopecia areata is a chronic recurrent hair loss disease that occurs in about 2% of the world's population and is a relatively common disease that can occur in both men and women of all ages. It is known that genetic predisposition and autoimmune response are the most important causes of alopecia areata, and it can be affected by various environmental factors. However, the exact pathogenesis has not been identified yet, and its treatment method is also unclear.

The most important cause of alopecia areata is immunological factors, which occur when T cells are activated due to certain stimuli, mistakenly recognize the hair follicle as a foreign substance, attack it, and induce an immune response, resulting in alopecia areata. A recent nonclinical study suggested a new mechanism in which alopecia areata occurs through an immune response induced by the activation of NLRP3 inflammasome, and an increase in the expression of NLRP3, ASC, Caspase-1, and IL-1β, which form NLRP3 inflammasome in the scalp hair follicles of alopecia areata patients, was reported (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]).

Currently, various treatments such as steroids and janus kinase (JAK) inhibitors are being used to treat alopecia areata. However, the time it takes for the effects to appear, the high cost, the side effects that can occur with long-term use, and the relapse rate that occurs when the medication is discontinued are raising the need for new treatments.

Sodium-glucose co-transporter (SGLT) is a glucose transport protein, SGLT-1 is expressed in the small intestine, liver, kidney, and heart, and SGLT-2 is mainly expressed in the kidney. SGLT-2 inhibitors are a new type of antidiabetic drug that decreases glucose reabsorption in the proximal nephron and increases 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 such effects is enavogliflozin, which is disclosed in Korean Patent Publication No. 2014-0022086 (Patent Document 1).

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

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]).

The relationship between increased BHB and alopecia areata has not been reported yet. However, based on previously reported literature, the present inventors focused on the possibility that administration of SGLT-2 inhibitors can prevent or treat alopecia areata by increasing BHB and thereby suppressing NLRP3 inflammasome activation, thereby regulating inflammation, independently of glycemic control by SGLT-2 inhibitors.

[Prior art literature]

[Patent Document]

Republic of Korea Patent Publication No. 2014-0022086

[Non-patent literature]

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

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

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

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.

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.

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.

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

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.

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

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

In the following examples, it was confirmed that administration of inavogliflozin tended to increase plasma BHB concentrations and decreased IL-1β secreted when NLRP3 inflammasome was activated. In addition, it was confirmed that inavogliflozin had the potential to improve inflammation that occurs when alopecia areata develops by dose-dependently reducing the expression of inflammatory factors NLRP3, Caspase-1, ASC, and IL-1β proteins. In addition, it was confirmed that inavogliflozin could improve alopecia areata by reducing the expression of NKG2D, which is expressed in pathogenic cells in alopecia areata and causes hair follicle destruction.

The term "inavogliflozin" in this specification 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 through urine.

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

[Chemical Formula 1]

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.

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.

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.

Although inavogliflozin 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 may exhibit complementary or synergistic effects when administered in combination, so a pharmaceutical composition containing inavogliflozin can be administered in combination with other alopecia areata treatments.

In one embodiment of the present invention, the pharmaceutical composition may be co-administered with one or more drugs selected from corticosteroids, minoxidil, and JAK inhibitors.

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

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

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 show that the JAK inhibitor tofacitinib (brand name Xeljanz®) could successfully treat alopecia areata, and since then, many studies have shown that JAK inhibitors can be effective in promoting hair regrowth in alopecia areata. Tofacitinib is an old 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, ritlecitinib, duruxolitinib, etc.

In another embodiment, the pharmaceutical composition of the present invention can be used in combination with immunotherapy for the treatment of alopecia areata. Here, immunotherapy for the treatment of alopecia areata refers to topical immunotherapy, also called contact immunotherapy. Immunotherapy agents cause an allergic rash (allergic contact dermatitis) at the site of application, and although the exact cause is not known, it is known to change the body's immune response around the hair follicle and promote hair regrowth. The most commonly used drug, i.e. immunotherapy agent, applied topically to the area of alopecia areata in immunotherapy is squaric acid dibutyl ester (SADBE) or diphencyprone (DPCP).

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

The pharmaceutical composition according to the present invention may additionally contain 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.

As used herein, 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 one or more of these components can be mixed and used. Additionally, antioxidants, buffers, bacteriostatic agents, and other conventional additives can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

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.

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.

To formulate it as 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 for unit dose in an ampoule or vial.

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

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 according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the 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 in a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.

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

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.

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.

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 the presence or absence of other complications.

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.

Inavogliflozin used as an effective 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 form, 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.

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°

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°

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°

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°

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°

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.

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.

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.

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

When the average particle size of inabogliflozin exceeds 15 um, the 5-minute dissolution rate was very low, less than 40% of the total content of inabogliflozin, and the 30-minute dissolution rate was also less than 80%, indicating that the final dissolution rate was inadequate.

When fine particle size differentiation of the drug is required, the drug can be pulverized using a common mill capable of fine particle differentiation, such as a Z-mill, a hammer mill, a ball mill, or a fluid energy mill. In addition, a size classification method, such as a sieving method using a sieve or an air current classification, can be used to refine the particle size of the drug. 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.: H.A.Lieberman, L.Lachman, J.B.Schwartz (Chapter 3: SIZE REDUCTION)].

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 are accumulated in order of smallness and becomes 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 drugs are accumulated in order of smallness and becomes 10%, D(50) represents the particle diameter at the point where the particle sizes of the drugs are accumulated in order of smallness and becomes 50%, and D(90) represents the particle diameter at the point where the particle sizes of the drugs are accumulated in order of smallness and becomes 90%.

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, by routine experimentation. For example, laser diffraction is sensitive to particle volume and provides a volume average particle size, which, at constant density, corresponds to a weight average particle size.

In the present invention, the particle size distribution of drug particles can be measured using a commercially available device based on laser diffraction/scattering method 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 to appear on a detector, and the particle diameter distribution is obtained by interpreting this light scattering pattern according to Mie theory. 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.

In a specific embodiment of the present invention, the compound of Chemical Formula 1 may be included in less than 1 part by weight relative to 100 parts by weight of the total pharmaceutical composition.

The appropriate daily dose 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.

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

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

Examples of excipients include lactose (including hydrated), 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.

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.

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

Examples of other additives include lubricants and colorants.

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 oils, white lead, titanium oxide, microcrystalline cellulose, macrogol 4000 and 6000, isopropyl myristate, calcium hydrogen phosphate and mixtures thereof.

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.

In a specific example 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 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 it exceeds 8 parts by weight relative to 100 parts by weight of the total pharmaceutical composition, the amount of disintegrant in the post-mixing portion may increase, which may deteriorate the overall flowability of the granules.

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 granule flowability due to the occurrence of fine powder. 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 a 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.

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

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.

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.

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.

Since the dissolution rate of the active ingredient in the pharmaceutical composition affects the maximum blood concentration (Cmax) and the area under the blood concentration-time curve (AUC) when administering the drug, conversely, it is important to adjust the dissolution rate of the pharmaceutical composition to implement the appropriate Cmax and AUC. Since inabogliflozin 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 by the 2nd dissolution test method (paddle method) of the Korean Pharmacopoeia. For specific conditions, refer to the following experimental example.

The present invention also comprises

A pharmaceutical composition comprising a granule mixture comprising a pre-mixed granule and a post-mixed portion comprising a compound of formula 1 or a pharmaceutically acceptable salt thereof is provided.

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 uniformity of drug content and uniformity of formulation.

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

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

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

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

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.

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.

In a specific example of the present invention, the particle size of the microcrystalline cellulose in the pre-mixed granules may be 130 um or less, preferably 60 um to 130 um. 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 under the above conditions, a formulation having 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 um 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 found to be poor.

Meanwhile, the particle size of the microcrystalline cellulose in the post-mixing unit may be 130 um or more, preferably 130 um to 250 um. 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 um, it was confirmed that the Carr's index value indicating the physical properties of the final granules was not appropriate, and the granule flowability became weak.

When the microcrystalline cellulose in the pre-mixed granules is relatively compared with the microcrystalline cellulose in the post-mixed portion, it was found that, while it is desirable for the particle size of the microcrystalline cellulose included in the pre-mixed granules to be small, 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 included in the pre-mixed granules.

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.

In a specific example of the present invention, the weight ratio of the excipient in the pre-mixed granule and the excipient in the post-mixed portion may be from 4:1 to 1:1. As the ratio of microcrystalline cellulose in the post-mixed portion increases, the flowability of the granule becomes better, but the content deviation increases, so it was found that it is desirable to match the weight ratio within the above appropriate range.

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.

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 not preferable in terms of bioavailability.

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.

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.

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.

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

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

The inabogliflozin of the present invention improves inflammation that occurs when alopecia areata occurs by increasing BHB (β-hydroxybutyrate) to inhibit NLRP3 inflammasome, decreasing IL-1β, and decreasing the expression of inflammatory factors NLRP3, Caspase-1, ASC, and IL-1β proteins, while decreasing the expression of NKG2D, which causes hair follicle destruction, and thus can be usefully used for the prevention or treatment of alopecia areata.

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

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.

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

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

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.

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.

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.

The advantages and features of the present invention, and the method for achieving them, will become clear 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, and these embodiments are provided only to make the disclosure of the present invention complete and to fully inform a person having ordinary skill in the art to which the present invention belongs of the scope of the invention, and the present invention is defined only by the scope of the claims.

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

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

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. Then, ^1.5x107 cells/mouse were injected intra-derma into C3H/Hej mice (recipients) that were 10 weeks old or older to create an alopecia areata model.

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

Test group Methods for inducing alopecia areata Test substance Dosage
(mg/kg) Route of administration Number of doses Number of heads G1 Lymph node cell injection Vehicle (NC) N/A PO QD 6 G2 Enavogliflozin (ENA) 0.1 6 G3 1 6

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℃ for use.

After the injection of lymph node cells into mice, the test substances were divided into groups and administered to the mice after selecting the mice at 4 weeks when alopecia areata was visually confirmed. Individual selection was performed at a time when alopecia areata was induced in about 50% of all mice after the injection of lymph node cells. Mice with alopecia areata in more than 50% of the mice, mice with alopecia areata 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.

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

(1) Measurement of disease-free ratio

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.

For statistical analysis of data, Prism 10.0 software (GraphPad Software Inc., San Diego, CA, USA) was used. The experimental results were expressed as the mean ± standard error of the mean (SEM). For statistical methods, one-way ANOVA was performed to verify the difference between each group, and for post hoc tests for each type, the Tukey function verification method (Tukey's test) or Dunn's multiple comparison test was used. The significance level was set at p<0.05 .

Figure 1 and Table 2 show the percentage of mice in which alopecia areata did not occur according to one embodiment of the present invention.

Disease-free ratio Week Vehicle ENA 0.1 mg/kg ENA 1 mg/kg 0 4/6 (66.7%) 3/6 (50%) 3/6 (50%) 2 2/6 (33.3%) 2/6 (33.3%) 2/6 (33.3%) 3 2/6 (33.3%) 2/6 (33.3%) 3/6 (50%) 4 2/6 (33.3%) 1/6 (16.7%) 3/6 (50%) 5 1/6 (16.7%) 1/6 (16.7%) 3/6 (50%) 8 0/5 (0%) 1/5 (20%) 2/6 (33.3%) 10 0/5 (0%) 1/5 (20%) 2/6 (33.3%) 12 0/5 (0%) 1/5 (20%) 1/6 (16.7%)

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

(2) Measurement of the size of the alopecia areata (AA lesion size)

The size of alopecia areata was measured by taking pictures once every two weeks using a digital single-lens reflex camera (DSLR) and expressing the symptom area as a percentage of the total area of the mouse using Image J (National Institutes of Health). The occurrence of alopecia areata was measured over the entire back and belly of the mouse.

For statistical analysis of data, Prism 10.0 software (GraphPad Software Inc., San Diego, CA, USA) was used. The experimental results were expressed as the mean ± standard error of the mean (SEM). For statistical methods, one-way ANOVA was performed to verify the difference between each group, and the post hoc test for each type was performed using the Tukey function or Dunn's multiple comparison test. The significance level was set at p<0.05 .

As can be seen in Figures 2 and 3, in the last 12 weeks of administration, the size of alopecia areata was smaller in the 1 mg/kg inavogliflozin group and the 0.1 mg/kg inavogliflozin group 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 alopecia areata.

(3) Measurement of plasma BHB concentration

Fasting was induced by removing food for 24 hours before blood collection after the end of the test. The collected blood was placed in an anticoagulant-treated tube and centrifuged at 13,000 rpm for 10 minutes to obtain the supernatant. Afterwards, the protein in the blood was 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).

For statistical analysis of data, Prism 10.0 software (GraphPad Software Inc., San Diego, CA, USA) was used. The experimental results were expressed as the mean ± standard error of the mean (SEM). For statistical methods, one-way ANOVA was performed to verify the difference between each group, and the post hoc test for each type was performed using the Tukey function or Dunn's multiple comparison test. The significance level was set at p<0.05 .

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

(4) Histological analysis

At the 12th week, when the test substance administration was terminated, 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), caspase1 (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.

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, which 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 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, which indicates that inflammation related to NLRP3 inflammasome can be improved in the skin of the alopecia areata mouse model.

(5) Analysis of changes in inflammatory factor protein expression

At the 12th week, the end point of test substance administration, the skin was removed, lysed, and the protein concentration was measured using a BCA protein assay kit (Bio-Rad, Hercules, CA, USA). The protein (20 μg) was separated by SDS-PAGE (polyacrylamide gel electrophoresis) and absorbed onto a PVDF (polyvinylidene fluoride) membrane (Millipore Corp, Bedford, MA). The membrane was reacted in PBS containing 0.1% Tween-20 and 5% skim milk (non-fat milk), and reacted using a primary antibody diluted with 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).

For statistical analysis of data, Prism 10.0 software (GraphPad Software Inc., San Diego, CA, USA) was used. The experimental results were expressed as the mean ± standard error of the mean (SEM). For statistical methods, one-way ANOVA was performed to verify the difference between each group, and the post hoc test for each type was performed using the Tukey function or Dunn's multiple comparison test. The significance level was set at p<0.05 .

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 inabogliflozin treatment groups compared to the vehicle group. Based on Figs. 5 and 6, it can be confirmed that the inflammation that occurs when alopecia areata develops can be improved as the expression of inflammatory factors is reduced in the skin tissue of the alopecia areata mouse model when inabogliflozin is treated.

(6) Flow cytometry analysis of lymph nodes

At the end of the experiment, skin-draining lymph node cells from mice were removed from the nitrogen tank and used for flow cytometry. The cells were stained with antibodies to L/D, CD3, CD4, CD8, CD44, CD49d, CD62L, and NKG2D without additional stimulation and analyzed. The stained cells were subjected to quantitative analysis of cell surface proteins using 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).

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

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)].

As shown in Fig. 7, the ratio of pathogenic cells (CD44 ^s-hi ) in lymph node cells 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 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 pathogenic cells in alopecia areata and causes hair follicle destruction.

Claims (6)

A pharmaceutical composition for preventing or treating alopecia areata, comprising inabogliflozin as an active ingredient. In claim 1, the pharmaceutical composition is a pharmaceutical composition for preventing or treating alopecia areata for oral or parenteral administration. A pharmaceutical composition for preventing or treating alopecia areata in claim 1, wherein a single administration dose of the inabogliflozin is 0.1 mg to 0.5 mg. A pharmaceutical composition for preventing or treating alopecia areata in claim 1, wherein the pharmaceutical composition is administered once a day. A pharmaceutical composition for preventing or treating alopecia areata in claim 1, wherein the pharmaceutical composition is administered in combination with one or more drugs selected from corticosteroids, minoxidil, and JAK inhibitors. A pharmaceutical composition for preventing or treating alopecia areata in claim 1, wherein the pharmaceutical composition is used in combination with immunotherapy for treating alopecia areata.

Images