Injectable preparations containing isoxazoline derivatives

Abstract

The present invention relates to an injectable formulation comprising, as an active pharmaceutical ingredient (API), an isoxazoline derivative useful as a caspase inhibitor, or a pharma- ceutical acceptable salt thereof. The injectable formulation according to the present invention comprises a first agent comprising a high-capacity API and a second agent which is a solvent capable of dissolving the first agent. The first agent and the second agent can be mixed together immediately before administration to prepare the formulation. Since the formulation contains the active API stably, it has an advantage that it can be expected to have an effective medicinal effect when administered to a patient.

Description

[Technical field]

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0010141 filed on January 24, 2022, and all contents disclosed in the documents of that Korean patent application are incorporated as part of this specification.

[0002] The present invention relates to an injectable preparation comprising, as an active pharmaceutical ingredient (API), an isoxazoline derivative useful as a caspase inhibitor, or a pharma- ceutically acceptable salt thereof. [Background technology]

[0003] Caspase is a cysteine ​​protease, and caspase inhibitors are compounds that can regulate inflammation and apoptosis caused by the action of caspases by inhibiting the action of caspases. Diseases that can be treated with these compounds to eliminate or alleviate symptoms include osteoarthritis, rheumatoid arthritis, degenerative arthritis, destructive bone disease, hepatitis virus-induced liver disease, acute hepatitis, hepatic sclerosis, hepatitis virus-induced brain damage, human sudden liver failure, sepsis, organ transplant rejection, ischemic heart disease, dementia, stroke, AIDS-induced brain damage, diabetes, and gastric ulcers.

[0004] As caspase inhibitors, various kinds of isoxazoline derivatives and prodrugs of isoxazoline derivatives are known. Among the isoxazoline derivatives, (R)-N-((2S,3S)-2-(fluoromethyl)-2-hydroxy-5-oxotetrahydrofuran-3-yl)-5-isopropyl-3-(isoquinolin-1-yl)-4,5-dihydroisoxazole-5-carboxamide (hereinafter referred to as "compound of formula 1") has already been proven to be effective in treating liver diseases caused by hepatitis viruses, liver fibrosis, and nonalcoholic steatohepatitis (NASH). [ka] Recently, there have been attempts to apply the compound of Formula 1 to diseases related to bones and joints, such as osteoarthritis (OA), rheumatoid arthritis, degenerative arthritis, and destructive bone disorders.

[0005] In the treatment of diseases related to bones and joints, the general method of drug administration is to administer the drug directly into the joint cavity, which is filled with synovial fluid, using a syringe. When administered orally, the drug has an effect on the whole body, but when administered directly into the joint cavity, the drug has a local effect, so it is possible to administer a high concentration of the drug and minimize side effects on the whole body.

[0006] The compound of Chemical Formula 1 has been shown to have therapeutic effects on conventional liver diseases when administered orally, but since the compound of Chemical Formula 1 is poorly soluble in water, it is difficult to formulate it into an injectable formulation.

[0007] Therefore, there is a need to develop a high-concentration injectable formulation that can use the compound of Chemical Formula 1 as an injection. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent Publication No. 0774999 (November 2, 2007) Isoxazoline derivatives and their manufacturing method [Patent Document 2] Korean Patent Application No. 2021-0102476 (August 4, 2021) Use of caspase inhibitors in alleviating or treating osteoarthritis [Non-patent literature]

[0009] [Non-Patent Document 1] Ratziu et al.,'A Phase2, Randomized, Double‐Blind, Placebo‐Controlled Study of GS‐9450 in Subjects with Nonalcoholic Steatohepatitis' HEPATOLOGY, Vol.55,No.2,2012. Summary of the Invention [Problem to be solved by the invention]

[0010] As a result of extensive research conducted by the present inventors to solve the above problems, the present invention aims to provide an injectable formulation containing a high content of the compound of Chemical Formula 1 as an active pharmaceutical ingredient (API). [Means for solving the problem]

[0011] The present invention relates to a first agent comprising a compound of Chemical Formula 1; and The second agent is an aqueous solution having a pH of 7 to 11 and containing a phosphate buffer for dissolving the compound of Formula 1, a surfactant, and NaOH as a pH adjuster. [ka]

[0012] The content of the compound of Chemical Formula 1 contained in the first agent of the injectable preparation of the present invention is 5 to 20 mg / ml based on the mixed solution of the first agent and the second agent.

[0013] The contents of the phosphate buffer, surfactant and NaOH contained in the second agent of the injectable preparation of the present invention are 100-130 mM, 0.2-5.0 (w / v)% and 12-42 mM, respectively, based on the second agent.

[0014] The injectable formulation of the present invention is used for the treatment or prevention of a caspase-related disease selected from osteoarthritis, rheumatoid arthritis, degenerative arthritis, destructive bone disorders, liver disease caused by hepatitis viruses, acute hepatitis, liver sclerosis, brain damage caused by hepatitis viruses, sudden human liver failure, sepsis, organ transplant rejection, ischemic heart disease, dementia, stroke, brain damage caused by AIDS, diabetes, and gastric ulcer.

[0015] The injectable preparation of the present invention is used within 30 minutes after mixing the first and second agents immediately before administration to a patient. Effect of the Invention

[0016] The injectable formulation comprising the compound of Chemical Formula 1 according to the present invention comprises a first agent in powder form comprising the compound of Chemical Formula 1; and a second agent which is a buffer solution for dissolving the first agent. The first agent in powder form is mixed with the second agent acting as an injection solvent just before administration to a patient to prepare a drug, and the drug is administered to a patient within 30 minutes, which is advantageous in that it has a high content of active API. [Brief description of the drawings]

[0017] [Figure 1] The content of active API is shown according to the pH of the solvent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention will now be described in more detail.

[0019] All technical terms used in the present invention are used in the same sense as commonly understood by those having ordinary skill in the art unless otherwise defined. In addition, although preferred methods and samples are described in this specification, similar or equivalent methods and samples are also included in the scope of the present invention. The contents of all publications cited as references in this specification are incorporated herein by reference in their entirety.

[0020] The present inventors have continued to conduct research in various ways to develop an injectable formulation containing, as an API, (R)-N-((2S,3S)-2-(fluoromethyl)-2-hydroxy-5-oxotetrahydrofuran-3-yl)-5-isopropyl-3-(isoquinolin-1-yl)-4,5-dihydroisoxazole-5-carboxamide of Chemical Formula 1, which is poorly soluble in water, and as a result have developed an injectable formulation comprising a first agent containing the API; and a second agent which is an aqueous solution containing a phosphate buffer, a surfactant, and a pH adjuster. [ka]

[0021] In one embodiment of the invention, the pH adjusting agent is NaOH, and the pH of the second agent is 7-11.

[0022] In one embodiment of the invention, the first agent in the injectable formulation is in powder form.

[0023] In one embodiment of the present invention, the content of Chemical Formula 1 is 5 to 20 mg / ml based on the mixed solution of the first agent and the second agent.

[0024] In one embodiment of the invention, the pH of the solution in which the first agent and the second agent are mixed is 6-8.

[0025] In one embodiment of the invention, the osmotic pressure of the solution in which the first agent and the second agent are mixed is 280 to 330 mOsm / kg.

[0026] In one embodiment of the invention, the phosphate buffer is a mixture of sodium phosphate dibasic heptahydrate and sodium phosphate monobasic monohydrate.

[0027] In one embodiment of the invention, the surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80.

[0028] In one embodiment of the invention, the surfactant is Polysorbate 80.

[0029] In one embodiment of the invention, the content of the surfactant is 0.2 to 5.0 (w / v)% based on the second agent.

[0030] In one embodiment of the invention, the content of NaOH is 12 to 42 mM based on the second agent.

[0031] In one embodiment of the invention, the injectable formulation is used to treat or prevent a disease selected from osteoarthritis, rheumatoid arthritis, degenerative arthritis, destructive bone disease, liver disease caused by hepatitis viruses, acute hepatitis, liver clots, brain damage caused by hepatitis viruses, sudden human liver failure, sepsis, organ transplant rejection, ischemic heart disease, dementia, stroke, brain damage caused by AIDS, diabetes, and gastric ulcers.

[0032] In one embodiment of the invention, the disease that the injectable preparation can treat or prevent is selected from osteoarthritis, rheumatoid arthritis, degenerative arthritis, and destructive bone disorders.

[0033] In one embodiment of the invention, the injectable formulation is used in a single dose in the form of a mixed solution of the first agent and the second agent.

[0034] In one embodiment of the invention, the injectable preparation is administered into the joint cavity after the first and second agents are mixed.

[0035] In one embodiment of the invention, the injectable formulation is administered into the joint cavity within 30 minutes after the first and second agents are mixed.

[0036] In one embodiment of the invention, the injectable formulation is administered once after the first and second agents are mixed.

[0037] In one embodiment of the invention, the injectable formulation is an injectable formulation in which, when the contents of the compound of Chemical Formula 2 and the compound of Chemical Formula 3 are measured within 30 minutes after mixing the first agent and the second agent, the content of the compound of Chemical Formula 2 is 95% or more of the total content of the compound of Chemical Formula 2 and the compound of Chemical Formula 3. [ka]

[0038] In one embodiment of the present invention, the injectable formulation includes a first agent containing Chemical Formula 1 and a second agent which is a buffer solution of pH 7 to 11 for dissolving the compound of Chemical Formula 1, and when the contents of the compound of Chemical Formula 2 and the compound of Chemical Formula 3 are measured within 30 minutes after mixing the first agent and the second agent, the content of the compound of Chemical Formula 2 is 95% or more of the total content of the compound of Chemical Formula 2 and the compound of Chemical Formula 3.

[0039] The injectable formulation of the present invention has the advantage of having a high content of active API since it is prepared by mixing the first agent in powder form with the second agent acting as an injection solvent just before administration to a patient and then administering the drug to a patient within 30 minutes.

[0040] Since the compound of the present invention represented by Chemical Formula 1 is almost insoluble in water, the solubility was analyzed using buffer solutions of various pH values ​​in order to find a solvent capable of dissolving the compound of Chemical Formula 1 at a high content.

[0041] As a result, it was confirmed that the compound of Chemical Formula 1 is almost insoluble in an acidic solvent of pH 4 or less, but begins to dissolve in a weakly acidic solvent of pH 6 or more, and is highly soluble in a basic solvent of pH 7.5 or more. It was also confirmed that the compound of Chemical Formula 1 has the property of decreasing the pH of the solution while dissolving in the solvent.

[0042] In addition, it was found that when the compound of Chemical Formula 1 is dissolved in an aqueous solvent, the lactone ring is cleaved and the compound is converted to the compound of Chemical Formula 2 below. At this time, the compound of Chemical Formula 2 is in an active form, but the compound of Chemical Formula 2 is quickly and irreversibly converted to the structural isomer, the compound of Chemical Formula 3, in an aqueous solution, and the compound of Chemical Formula 3 is in an inactive form that does not exhibit caspase inhibitory activity, unlike the compound of Chemical Formula 2. [ka]

[0043] In addition, the conversion rate of the compound of Chemical Formula 2 to the compound of Chemical Formula 3 was measured depending on the pH of the solvent. As a result, it was confirmed that the compound of Chemical Formula 3 was hardly produced in an acidic solvent, but was rapidly produced in a neutral or more basic solvent.

[0044] Therefore, in order to dissolve the compound of Chemical Formula 1, a basic solvent with a high pH must be used, but it was found that not only does the compound of Chemical Formula 1 dissolve, lowering the pH of the solution to reduce the solubility of the undissolved compound, but also the active form of the dissolved compound is rapidly converted from the basic solvent to an inactive form. This mechanism not only reduces the purity of the active ingredient in an injectable formulation in which the compound of Chemical Formula 1 is an API, thereby affecting the efficacy, but also causes a problem in that it is difficult to adjust the pH when preparing a final drug for administration to an individual, since the compound of Chemical Formula 1 lowers the pH of the solution as it dissolves.

[0045] In order to solve such a pH adjustment problem, the injectable formulation of the present invention contains a buffer as the second agent for dissolving the first agent containing the API.

[0046] The buffer contained in the second agent may be a common buffer that can be used in an injection solvent, and specific examples include, but are not limited to, phosphate buffer, Tris buffer, MES buffer, etc. When a phosphate buffer is used, it may be selected from the group consisting of sodium phosphate dibasic, sodium phosphate monobasic, potassium phosphate monobasic, potassium phosphate dibasic, and mixtures thereof. When a Tris buffer is used, TRIZMA® buffer may be used.

[0047] The buffer has a concentration of 100 to 130 mM based on the second agent, specifically, 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, 100 to 120 mM, 110 to 120 mM, 100 to 110 mM, 110 to 120 mM, or 120 to 130 mM.

[0048] In the injectable formulation of the present invention, the second agent uses NaOH as a pH adjusting agent.

[0049] The NaOH has a concentration of 12 to 42 mM based on the second agent.

[0050] In the injectable preparation of the present invention, the pH of the second agent is adjusted to pH 7 to 11 using a buffer and a pH adjuster, specifically, 7 to 11, 7.5 to 11, 8 to 11, 8.5 to 11, 9 to 11, 9.5 to 11, 10 to 11, 10.5 to 11, 7 to 10.5, 7.5 to 10.5, 8 to 10.5, 8.5 to 10.5, 9 to 10.5, 9. Adjust to 5~10.5, 10~10.5, 7~10, 7.5~10, 8~10, 8.5~10, 9~10, 9.5~10, 7~9.5, 7.5~9.5, 8~9.5, 8.5~9.5, 9~9.5, 7~9, 7.5~9, 8~9, 8.5~9, 7~8.5, 7.5~8.5, 8~8.5, 7~8, or 7.5~8.

[0051] The injectable formulation of the present invention includes a surfactant in the second agent. The surfactant can help dissolve the compound of Chemical Formula 1. The surfactant can be a common surfactant that can be used in an injection solvent, and can be a cyclodextrin-based or polysorbate-based surfactant. Specific examples of polysorbate-based surfactants include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80.

[0052] The concentration of the surfactant is 0.2 to 5.0 (w / v)% based on the second agent, specifically, 0.2 to 5.0 (w / v)%, 0.8 to 5.0 (w / v)%, 1.2 to 5.0 (w / v)%, 1.6 to 5.0 (w / v)%, 2.0 to 5.0 (w / v)%, 2.4 to 5.0 (w / v)%, 2.8 to 5.0 (w / v)%, 3.2 to 5.0 (w / v)%, 3.6 to 5.0 (w / v)%, 4.0 to 5.0 (w / v)%, 4.4 to 5.0 (w / v)%, 0.2 to 4.4 (w / v), 0.8 to 4.4 (w / v), 1.2 to 4.4 (w / v), 1.6 to 4 .4(w / v)%, 2.0~4.4(w / v)%, 2.4~4.4(w / v)%, 2.8~4.4(w / v)%, 3.2~4.4(w / v)%, 3.6~4.4(w / v)%, 4.0~4.4(w / v)%, 0.2~4.0(w / v)%, 0.8~4.0(w / v)%, 1.2~4.0(w / v)%, 1.6~4.0(w / v)%, 2.0~4.0(w / v)%, 2.4~4.0(w / v)%, 2.8~4 .0(w / v)%, 3.2~4.0(w / v)%, 3.6~4.0(w / v)%, 0.2~3.6(w / v)%, 0.8~3.6(w / v)%, 1.2~3.6(w / v)%, 1.6~3.6(w / v)%, 2.0~3.6(w / v)%, 2.4~3.6(w / v)%, 2.8~3.6(w / v)%, 3.2~3.6(w / v)%, 0.2~3.2(w / v)%, 0.8~3.2(w / v)%, 1.2~3 .2(w / v)%, 1.6~3.2(w / v)%, 2.0~3.2(w / v)%, 2.4~3.2(w / v)%, 2.8~3.2(w / v)%, 0.2~2.8(w / v)%, 0.8~2.8(w / v)%, 1.2~2.8(w / v)%, 1.6~2.8(w / v)%, 2 .0~2.8(w / v)%, 2.4~2.8(w / v)%, 0.2~2.4(w / v)%, 0.8~2.4(w / v)%, 1.2~2 .4(w / v)%, 1.6~2.4(w / v)%, 2.0~2.4(w / v)%, 0.2~2.0(w / v)%, 0.8~2.0(w / v)%, 1.2~2.0(w / v)%, 1.6~2.0(w / v)%, 0.2~1.6(w / v)%, 0.8~1.6(w / v)%, 1 .2~1.6(w / v)%, 0.2~1.2(w / v)%, 0.8~1.2(w / v)%, or 0.2~0.8(w / v)%.

[0053] When the injectable formulation of the present invention is mixed with the first and second agents for administration to a patient, the compound of Formula 1 contained in the first agent is dissolved in the second agent, lowering the pH of the solution. However, if the pH of the solution obtained by mixing the first and second agents is too low, it may cause pain to the patient. Therefore, the pH of the solution obtained by mixing the first and second agents must be 6 to 8.

[0054] Specifically, the pH of the solution obtained by mixing the first agent and the second agent is 6 to 8, 6 to 7.7, 6 to 7.4, 6 to 7.1, 6 to 6.8, 6 to 6.5, 6 to 6.2, 6.3 to 8, 6.3 to 7.7, 6.3 to 7.4, 6.3 to 7.1, 6.3 to 6.8, 6.3 to 6.5, 6.6 to 8, 6.6 to 7.7, 6.6 to 7.4, 6.6 to 7.1, 6.6 to 6.8, 6.9 to 8, 6.9 to 7.7, 6.9 to 7.4, 6.9 to 7.1, 7.2 to 8, 7.2 to 7.7, 7.2 to 7.4, 7.5 to 8, or 7.5 to 7.7.

[0055] In the injectable preparation of the present invention, the osmotic pressure of the solution in which the first agent and the second agent are mixed is 280 to 330 mOsm / kg. If the osmotic pressure is lower or higher than the above range, it is not preferable because it may cause pain to the patient.

[0056] The injectable preparation of the present invention has a concentration of 5 to 20 mg / ml of Chemical Formula 1 based on the mixture of the first and second agents.

[0057] Specifically, the content of Chemical Formula 1 is 5-20 mg / ml, 10-20 mg / ml, 15-20 mg / ml, 5-15 mg / ml, 10-15 mg / ml, 5-10 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, or 20 mg / ml based on the preparation in which the first agent and the second agent are mixed, and a single dose is preferable.

[0058] In the injectable formulation of the present invention, when the compound of Chemical Formula 1 contained in the first agent is mixed with the second agent, the compound is completely dissolved and the solution obtained by mixing the first agent and the second agent is transparent.

[0059] The injectable formulation of the present invention does not further contain excipients for preservation such as antioxidants and preservatives, since the first and second agents are mixed together immediately before administration to a patient.

[0060] The present invention provides a method for treating or preventing a caspase-related disease using the injectable formulation of the present invention.

[0061] The term "treatment" means stopping or delaying the progression of a disease when used on a subject who shows symptoms of the disease, and the term "prevention" means stopping or delaying the onset of the disease when used on a subject who does not show symptoms of the disease but is at high risk of such symptoms.

[0062] The "caspase-related disease" of the present invention refers to a disease that can be treated or prevented by inhibiting caspase, and examples thereof include osteoarthritis, rheumatoid arthritis, degenerative arthritis, destructive bone disorders, liver disease caused by hepatitis viruses, acute hepatitis, liver sclerosis, brain damage caused by hepatitis viruses, sudden human liver failure, sepsis, organ transplant rejection, ischemic heart disease, dementia, stroke, brain damage caused by AIDS, diabetes, and gastric ulcers, but are not limited to the above diseases.

[0063] The injectable formulation of the present invention can be administered to an individual in need of treatment or prevention to treat or prevent a caspase-related disease in the individual.

[0064] The term "administration" or "administering" as used herein refers to a method of administering a dose of the injectable formulation of the present invention to a vertebrate or invertebrate animal, including a mammal, a bird, a fish, or an amphibian, by injection. A preferred administration method can be selected from common injection methods such as intravascular injection, subcutaneous injection, transdermal injection, intramuscular injection, spinal injection, and intradermal injection depending on the type of disease to be treated, the site of the disease, and the severity of the disease, and in particular, in the case of osteoarthritis, rheumatoid arthritis, degenerative arthritis, or destructive bone disorders, which are diseases related to bone and joints, intra-articular injection can be selected.

[0065] When the injectable preparation of the present invention is injected into a joint cavity, it can be guided by using an imaging method such as ultrasound, or a local anesthetic can be administered before or in combination with the injection.

[0066] The term "individual" as used herein refers to a human or a non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human ape, or bird. In some embodiments, the individual is a human.

[0067] The injectable formulation of the present invention is administered to an individual within 30 minutes after the first agent and the second agent are mixed to prepare a mixed solution. Thus, the injectable formulation of the present invention is for single administration for injection or administration to a single patient.

[0068] In addition, the injectable preparation of the present invention can be administered in combination with drugs such as painkillers, anti-inflammatory drugs, steroids, etc. Specifically, it can be administered in combination with drugs such as ibuprofen, naproxen, aspirin, acetaminophen, indomethacin, diclofenac, meloxicam, celecoxib, piroxicam, etodolac, nabumetone, lumiracoxib, valdecoxib, etoricoxib, parecoxib, fenoprofen, oxaprozin, mefenamic acid, diflunisal, flurbiprofen, ketoprofen, and other nonsteroidal anti-inflammatory drugs (NSAIDs); corticoid drugs such as prednisone and methylprednisolone, narcotic painkillers such as codeine, pentanyl, morphine, and meperidine, or injections of hyaluronic acid derivatives.

[0069] The present invention provides a kit including the injectable formulation of the present invention. In one embodiment of the present invention, the kit may include an injectable formulation including a first agent including the compound of Formula 1 of the present invention and a second agent which is an aqueous solution for dissolving the compound of Formula 1; an administration system such as one or more syringes for administering the formulation, a method for mixing the first agent and the second agent, and instructions for using the kit including instructions for treating a patient.

[0070] In one embodiment of the present invention, the kit may include a label stating that the formulation and contents described herein are administered to a patient suffering from osteoarthritis, rheumatoid arthritis, degenerative arthritis, or destructive bone disorders, which are diseases associated with bone and joints, wherein selecting an appropriate dosage for a patient is within the knowledge of a person of ordinary skill in the art, taking into account the symptoms, age, etc., of the patient.

[0071] Unless otherwise indicated, all numbers used in the specification and claims should be understood to be modified in all cases, whether stated or not, by the term "about". Also, the precise numerical values ​​used in the specification and claims should be understood to form additional embodiments of the present disclosure. Efforts have been made to ensure the accuracy of the numerical values ​​disclosed in the examples. However, all measured numerical values ​​inherently contain certain error values ​​generated from the standard deviations observed with the respective measuring techniques.

[0072] Various evaluations in the examples and comparative examples were carried out as follows. EXAMPLES

[0073] Manufacturing Example In the present invention, the compound of formula 1 used in the API, (R)-N-((2S,3S)-2-(fluoromethyl)-2-hydroxy-5-oxotetrahydrofuran-3-yl)-5-isopropyl-3-(isoquinoline-1-yl)-4,5-dihydroisooxazol-5-carboxamide) (hereinafter referred to as "API"), was prepared according to KR10-0774999.

[0074] Test Example 1: Confirmation of API solubility depending on the pH of the solvent (Examples 1 to 10) In order to determine the optimal pH for dissolving the API, the API was dissolved to the maximum extent in sodium phosphate buffer (PB) (Examples 1 to 9) and distilled water (Example 10) having various pH values, each in 10 ml, for 24 hours, and then the pH, concentration of active substance (compound of formula 2), and concentration of inactive substance (compound of formula 3) were analyzed by HPLC.

[0075] As a result, the API was more soluble in basic solvents (Examples 8 and 9), and was more soluble in phosphate buffer (Example 4) than in distilled water (Example 10) at similar pH. Also, it was found that the API was not as soluble in acidic solvents as in neutral or basic solvents, but the conversion of active substances to inactive substances was lower than in basic solvents (Example 1).

[0076] In addition, after dissolving the API in a solvent, the change in the content of Chemical Formula 2, which is the active form, was measured over time. As a result, in the case of Example 1, the content of the active form was maintained for up to 25 hours after dissolution, but as the pH increased, the content of the active form rapidly decreased, and in particular, in the cases of Examples 3 to 8, 20-30% was converted to the inactive form within 5 hours (FIG. 1).

[0077] Therefore, it can be seen that the API is highly soluble in a basic solvent, and after dissolution, the conversion rate from the active compound of Formula 2 to the inactive compound of Formula 3 is higher in a basic solvent than in an acidic solvent. It can also be seen that the pH of the solution is lower than the pH of the solvent in all of the solvents in Examples 1 to 10, and particularly in the case of Example 10, distilled water has no buffering effect, so the pH drops rapidly. [Table 1]

[0078] Test Example 2: Confirmation of dissolution of API, dissolution time, and pH depending on the solvent composition (Examples 11 to 20) Solvents were prepared to prepare 10 mg / ml solutions by dissolving APIs according to the compositions shown in Table 2. As shown in Table 2, solvents were prepared with sodium phosphate buffer (PB) concentrations of 0 to 10 mM, NaOH concentrations of 23 to 50 mM, Tween® 80 concentrations of 0 to 0.4 (w / v)%, and hydroxypropyl beta cyclodextrin (HPβCD) concentrations of 0 to 20 (w / v)%. Since APIs are highly soluble in basic solutions, solvents with pH values ​​of 11 to 12 were prepared using NaOH and / or PB.

[0079] Sodium phosphate buffer was prepared at pH 7.4 by mixing sodium phosphate dibasic heptahydrate and sodium phosphate monobasic monohydrate. HPβCD was a parenteral grade reagent with a molar substitution range of 0.81–0.99 and a molecular weight of approximately 1500 g / mol. [Table 2]

[0080] The solvents (1 ml) prepared according to Examples 11 to 20 according to the composition were placed in a container with a lid, and API was added at a concentration of 10 mg / ml, and then the lid was closed and the container was shaken by hand for up to 10 minutes to mix. At this time, the dissolution of the API, the time required for the API to be completely dissolved (dissolution time), and the pH of the solvent after dissolving the API were measured and are shown in Table 3.

[0081] The solvents prepared in Examples 11 to 13 contained 25 to 50 mM NaOH, and no other excipients were used. Although Example 1, which contained 50 mM NaOH, and Example 2, which contained 30 mM NaOH, dissolved 10 mg / ml of API well, the pH of the solution in which the API was dissolved was 10 or higher, which could induce pain in the patient when the dosage form was administered into the joint cavity, so Examples 11 and 12 were not suitable as solvents for dissolving the API. Also, Example 13, which contained 25 mM NaOH, was not suitable as a solvent because it could not completely dissolve the API. Therefore, the results of Examples 11 to 13 show that a solvent containing only NaOH is not suitable as a solvent for dissolving the API.

[0082] Examples 14 and 15 are solvents prepared by adding polysorbate 80 (hereinafter referred to as "Tween (registered trademark) 80") or HPβCD as a solubilizer to 25 mM NaOH, respectively. It can be seen that the API is not completely dissolved in each solvent, and that the solvents further containing a solubilizer in addition to NaOH are also not suitable as solvents for dissolving the API.

[0083] The solvents prepared in Examples 16 to 20 were identical in that they contained 10 mM PB and 0.4 (w / v)% Tween® 80, and only the NaOH content was different, but the API was well dissolved in all of the solvents. However, while the pH of Examples 16 and 17 was still high at 11.3 and 9.4 after the API was dissolved, the pH of Examples 18 to 20 was all 7.0 to 7.3, indicating that these solvents are suitable for use as injection dosage forms.

[0084] In addition, in Example 19, 10 mM PB was added to the composition of Example 14, but 10 mg / ml of the API that was not dissolved was dissolved, and the pH of the solution after dissolution was appropriately adjusted to 7.3. Therefore, it can be seen that the presence of PB, which acts as a pH buffer, is very important in selecting a solvent for dissolving the API of the present invention.

[0085] The HPβCD surfactant did not show a significant effect even at 20% compared to 0.4% Tween® 80, and Tween® 80 was selected as the surfactant because the osmotic pressure became too high when the HPβCD content was increased. [Table 3]

[0086] Test Example 3: Confirmation of osmotic pressure depending on solvent composition (Examples 21 to 25) Solvents in Examples 21 to 25 were prepared according to the compositions shown in Table 4, and the osmotic pressure of each solvent was measured using an osmometer. Then, 10 mg / ml of API was dissolved in each solvent, and the osmotic pressure of the resulting solution (mixture) was measured. [Table 4]

[0087] The solvents of Examples 21 to 25 all dissolved 10 mg / ml of API well. Table 5 shows the osmotic pressure of the solvent, the osmotic pressure of the solution in which the API was dissolved, and the difference between the two osmotic pressures.

[0088] In Examples 21 to 25, the osmotic pressure increased after dissolving 10 mg / ml compared to the osmotic pressure of the solvent. In particular, in the case of Examples 21 to 23, in which the osmotic pressure was increased with PB, the difference in osmotic pressure was 22 to 23 mOsm / kg, which indicates that the API was completely dissolved in Examples 21 to 23, since the molar concentration is 24 mM when 10 mg / ml of API is dissolved. However, in Examples 24 and 25, in which the osmotic pressure of the solvent was adjusted with NaCl, not only was the API not completely dissolved, but the difference in osmotic pressure was also 11 to 13 mOsm / kg, reflecting this.

[0089] Furthermore, the solvents in Examples 24 and 25 have a higher pH than those in Examples 21 to 23, but are unable to completely dissolve the API, indicating that a certain degree of buffer capacity is essential to dissolve the API of the present invention.

[0090] In addition, the preferred osmolality range for a dosage form used as an injection is 280-330mOsm / kg, and if it does not reach or exceed this range, the patient may feel pain. Therefore, when applying this standard, it can be seen that the dosage form of Example 23, which has a final solution osmolality of 318mOsm / kg, is preferred as an injection dosage form. [Table 5]

[0091] Test Example 4: Confirmation of API stability in solution Since the API (active form) of Chemical Formula 1 is quickly hydrolyzed in an aqueous solution and turns into an inactive form, in order to measure the content of the active form in the solution after preparation of the injection, 10 mg / ml of the API was dissolved using the solvent of Example 23, and the concentration and purity of the active form API (compound of Chemical Formula 2) that changed over time were measured using HPLC up to 60 minutes after dissolution.

[0092] As shown in Table 6 below, 10 mg / ml of API was mixed in the solvent of Example 23 to prepare an API solution, and the concentration (total amount of active and inactive API) of the compound of Chemical Formula 2 (hereinafter referred to as "active API") and the compound of Chemical Formula 3 (hereinafter referred to as "inactive API") reached a concentration of 9.69 mg / ml within 10 minutes after mixing, and maintained a similar concentration until 30 minutes, but showed a tendency to decrease after 60 minutes after mixing.

[0093] However, in the case of the active API, the content continued to decrease after mixing. After 30 minutes of mixing, the content of the active API was about 95.5% and the content of the inactive API was 4.33%, while after 60 minutes, the content of the active API was 94.18% and the content of the inactive API was 5.59%, which is below the normal API specification of 95% or more of the active API. Therefore, it was confirmed that the API of the present invention should be used within 30 minutes after mixing with the solvent. [Table 6]

[0094] Test Example 5: Preparation of API solutions with various contents As shown in Table 7, based on the solvent of Example 23, the solvents of Examples 26 to 28 were prepared by changing the composition of PB and NaOH in consideration of the property that the API of the present invention is highly soluble in alkaline solution and the osmotic pressure of the final solution. [Table 7]

[0095] After measuring the pH and osmolality of the solvents of Examples 26 to 28, 2 mg / ml, 5 mg / ml, and 15 mg / ml of API were dissolved therein, and the pH and osmolality of the mixtures were measured. As a result, as shown in Table 8, when the API is dissolved in the solvents of Examples 26 to 28, the pH and osmolality of the mixtures are suitable for administration into the joint cavity of a patient. [Table 8]

[0096] Next, the API concentration and purity of the injections prepared using the solvents of Examples 26 to 28 were measured immediately after preparation, 30 minutes after preparation, and 60 minutes after preparation, and the results are shown in Table 9. The purity immediately after mixing was about 97%, the purity after 30 minutes mixing was 95-96%, and the purity after 60 minutes mixing was 93-94%. Therefore, the finished products of all doses maintained appropriate purity up to 30 minutes of mixing, and after 60 minutes of mixing, the inactive API was generated above the specification (inactive API less than 5%), making them unsuitable for administration to patients. Therefore, it was confirmed that all finished products should be prepared immediately before use and injected within 30 minutes after mixing. [Table 9]

Claims

1. A first agent comprising a compound of formula 1; and 【Chemistry 1】 a second agent which is an aqueous solution having a pH of 7 to 11 and which contains a phosphate buffer for dissolving the compound of formula 1, a surfactant, and NaOH as a pH adjusting agent;

2. The injectable formulation of claim 1 , wherein the first agent is in powder form.

3. The injectable preparation according to claim 1, wherein the content of the compound of Chemical Formula 1 is 5 to 20 mg / ml based on a mixed solution of the first agent and the second agent.

4. The injectable preparation according to claim 1, wherein the solution in which the first agent and the second agent are mixed has a pH of 6 to 8.

5. The injectable preparation according to claim 1, wherein the osmotic pressure of the solution in which the first agent and the second agent are mixed is 280 to 330 mOsm / kg.

6. 2. The injectable formulation of claim 1, wherein the phosphate buffer is a mixture of sodium phosphate dibasic heptahydrate and sodium phosphate monobasic monohydrate.

7. The injectable preparation according to claim 6, wherein the content of the phosphate buffer is 100 to 130 mM based on the second agent.

8. 2. The injectable formulation of claim 1, wherein the surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

9. The injectable formulation of claim 8, wherein the surfactant is polysorbate 80.

10. The injectable preparation according to claim 9, wherein the content of the surfactant is 0.2 to 5.0 (w / v)% based on the second agent.

11. The injectable preparation according to claim 1, wherein the content of NaOH is 12 to 42 mM based on the second agent.

12. The injectable formulation according to claim 1, which is for treating or preventing a disease selected from osteoarthritis, rheumatoid arthritis, degenerative arthritis, destructive bone disorders, liver disease caused by hepatitis viruses, acute hepatitis, liver sclerosis, brain damage caused by hepatitis viruses, sudden human liver failure, sepsis, organ transplant rejection, ischemic heart disease, dementia, stroke, brain damage caused by AIDS, diabetes, and gastric ulcers.

13. The injectable preparation according to claim 12, wherein the disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, degenerative arthritis, and destructive bone disorders.

14. The injectable preparation according to claim 1 , wherein the injectable preparation is used in a single dose in the form of a solution in which the first agent and the second agent are mixed.

15. The injectable preparation according to claim 14 , wherein the injectable preparation is administered into an articular cavity after the first agent and the second agent are mixed.

16. The injectable preparation according to claim 15, wherein the injectable preparation is administered into the joint cavity within 30 minutes after the first agent and the second agent are mixed.

17. The injectable formulation according to claim 16, wherein the injectable formulation is administered once after the first and second agents are mixed.

18. A first agent comprising a compound of formula 1; and 【Chemistry 2】 a second agent which is a buffer solution having a pH of 7 to 11 for dissolving the compound of formula 1; The injectable formulation is an injectable formulation, in which when the contents of the compound of Chemical Formula 2 and the compound of Chemical Formula 3 are measured within 30 minutes after mixing the first and second agents, the content of the compound of Chemical Formula 2 is 95% or more of the total content of the compound of Chemical Formula 2 and the compound of Chemical Formula 3. 【Chemistry 3】 【Chemistry 4】

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