TW202404603A - Composition including cftr activator - Google Patents

Abstract

Provided is a composition including a cystic fibrosis transmembrane conductance regulator (CFTR) activator as an active pharmaceutical ingredient. More particularly, provided is a composition having improved solubility, excellent storage stability, and stable appearance.

Description

Compositions containing CFTR activators

The present invention relates to pharmaceutical compositions, which contain compounds as active pharmaceutical ingredients that increase the activity of cystic fibrosis transmembrane conductance regulator (CFTR), and more particularly to pharmaceuticals that improve solubility and physicochemical stability. composition.

CFTR refers to the membrane protein and chloride ion (Cl ^- ) channel encoded by the CFTR gene. When CFTR present in cell membranes is activated, several diseases mediated by loss and degeneration of CFTR function can be treated. For example, substances that activate CFTR can activate ^Cl channels to increase tear production. Taken together, it can be expected to correct the abnormal tear film associated with dry eye syndrome.

However, since the eye is composed of a variety of complex tissues, various obstructive factors can block drug delivery between tissues, and active pharmaceutical ingredients cannot be effectively delivered to the eye tissue only by instilling pharmaceutical ingredients. In addition, considering that the eye is one of the most sensitive organs in the body, in order to deliver drugs to the eye, eye drops must have physical and chemical stability, and the osmotic pressure or pH value of the eye drops must be similar to that of tears. , and it must be ensured that microorganisms will not contaminate or corrode the eye drops. Most importantly, the solubility of the active pharmaceutical ingredients must be ensured. Under this consideration, the development of eye drops is very difficult. Furthermore, due to the low original solubility of the CFTR activator contained in the active pharmaceutical ingredient in the composition of the present invention, many formulation studies are required to develop eye drops for delivery to the eye.

The inventor of the present invention devoted himself to developing a substance that activates CFTR, and further developed the substance into an eye drop formulation with improved solubility and physicochemical stability to complete the present invention.

Prior technical literature patent documents Korean patent application number: 10-2018-0102590.

technical issues

One embodiment provides a pharmaceutical composition, which includes a CFTR activator as an active pharmaceutical ingredient and has improved solubility and physicochemical stability.

Another embodiment provides a composition for use in the eye, which includes a CFTR activator as an active pharmaceutical ingredient and has improved solubility and physicochemical stability.

Another embodiment requires a pharmaceutical composition or ophthalmic composition to prevent, improve, or treat eye-related diseases or symptoms.

Other objects and advantages of the present invention can be more clearly explained from the following detailed content and patent scope. Those with ordinary knowledge in the technical field of the present invention or similar technical fields can fully understand and infer that content that is not explained here will be omitted here.

problem solving methods

An embodiment provides a pharmaceutical composition including a CFTR activator as an active pharmaceutical ingredient; and any selected from the group consisting of polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, and combinations thereof. A surfactant.

Another embodiment provides an ophthalmic composition including a CFTR activator as an active pharmaceutical ingredient; selected from the group consisting of polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, and combinations thereof. Any kind of surfactant.

Another embodiment provides eye drops including ophthalmic compositions to prevent, improve, or treat eye-related diseases or symptoms.

Advantageous effects of the present invention

The present invention provides pharmaceutical compositions that ensure improved solubility and physicochemical stability and ensure physiological isotonicity. The pharmaceutical composition of the present invention can improve the solubility of a CFTR activator with poor solubility and good stability, so its quality changes slightly over time. In addition, the pharmaceutical composition can be in the form of eye drops for treating eye diseases, so patients have high drug compliance and can be effectively used to prevent or treat eye diseases related to CFTR activity. In addition, the present invention simplifies the manufacturing process and scales it up to the scale of industrial production, thereby achieving economical pharmaceutical production.

The invention is described in further detail below.

Unless otherwise defined, all technical terms used in this specification generally have the same meanings as understood by those with ordinary knowledge in the relevant fields. In addition, the instructions describe suitable methods or samples, but similar or identical methods and samples also fall within the scope of the instructions.

The terms "CFTR activator" or "compound that increases CFTR activity" as used in this specification refer to compounds that act to increase CFTR activity. In other words, this compound refers to a compound that has the effect of activating the chloride ion (Cl ^- ) channel of the cell membrane by promoting the production and movement of the membrane protein encoded by the CFTR gene or by targeting CFTR. For example, the "CFTR activator" can be a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. The compound represented by Formula 1 may include its racemate, enantiomer, diastereomer, solvate, hydrate, isomer, or pharmaceutically acceptable salts. For example, the compound represented by Formula 1 can be any compound represented by Formula 1a, 1b, and 1c.

The pharmaceutical composition provided by one embodiment of the present invention includes active pharmaceutical ingredients such as the compound represented by Formula 1 or its racemate, enantiomer, diastereomer, solvate, hydrate, isomer, or pharmaceutically acceptable salts; and

Select from polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, and any surfactant in combination with the above:

Formula 1

Among them, in equation 1,

R ¹ and R ² may each be independently selected from H, optionally substituted C ₁ -C ₁₀ alkyl, optionally substituted C ₁ -C ₁₀ alkenyl, optionally substituted aryl, optionally substituted heteroaryl base, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted monocyclic or bicyclic carbocyclic ring, and optionally substituted monocyclic or bicyclic heterocycle ,or

R ¹ and R ² may be cyclically linked together with the nitrogen atom to which R ¹ and R ² are attached to form an optionally substituted monocyclic or bicyclic heterocycle.

In one embodiment, the compound represented by Formula 1 can be selected from any compound represented by Formulas 1a, 1b, and 1c.

Formula 1a

The compound of formula 1a can also be regarded as (S)-(4-benzoyl-3-methylpiperazin-1-yl)(7-(3,4-dimethoxyphenyl)pyrazolo[ 1,5-a]pyrimidin-2-yl)methanone;

Formula 1b

The compound of formula 1b can also be regarded as 7-(3,4-dimethoxyphenyl)-N-(4-hydroxyphenyl)pyrazolo[1,5-a]pyrimidine-2-methamide; and

Formula 1c

The compound of formula 1 can also be regarded as 7-(3,4-dimethoxyphenyl)-N-(5-(4-methylpiperazin-1-yl)pyridin-2-yl)pyrazolo[1 ,5-a]pyrimidine-2-methamide hydrochloride.

In this specification, unless otherwise stated, the main pharmaceutical ingredient (API) refers to the compound of formula 1, or the compound of formula 1a, formula 1b, or formula 1c contained therein.

The compounds of the present invention may contain asymmetric or chiral centers and thus may exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, such as diastereomers, enantiomers, and racemic mixtures, are considered to form part of the present invention.

The term "enantiomer" as used herein refers to a compound of two stereoisomers that are non-superimposable mirror images of each other. The term "diastereomer" as used herein refers to two or more stereoisomers having two or more chiral centers whose molecules are not mirror images of each other. Additionally, a 50:50 mixture of enantiomers is called a "racemate" or "racemic mixture."

The term "pharmaceutically acceptable salts" in the instructions refers to salts commonly used in the pharmaceutical industry. Examples include inorganic ionic salts prepared from calcium, potassium, sodium, magnesium, or the like; salts prepared from hydrochloric acid, nitric acid, phosphoric acid, hydrogen Inorganic acid salts prepared from bromic acid, iodic acid, perchloric acid, tartaric acid, sulfuric acid, or the like; from acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid , mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydrogen Organic acid salts prepared from iodic acid, or the like; sulfonates prepared from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, or the like; glycine, arginine, Amino acid salts prepared from lysine, or the like; or amine salts prepared from trimethylamine, triethylamine, ammonia, pyridine, picoline, or the like. However, the embodiment is not specifically limited thereto.

The pharmaceutical composition of one embodiment may include (S)-(4-benzyl-3-methylpiperazin-1-yl)(7-(3,4-dimethoxyphenyl)pyrazolo [1,5-a]pyrimidin-2-yl)methanone as the active pharmaceutical ingredient; and selected from polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, and the above Any combination of surfactants.

The pharmaceutical composition of one embodiment may include 7-(3,4-dimethoxyphenyl)-N-(4-hydroxyphenyl)pyrazolo[1,5-a]pyrimidine-2-methamide As an active pharmaceutical ingredient; and any surfactant selected from the group consisting of polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, and combinations of the above.

The pharmaceutical composition of one embodiment may include 7-(3,4-dimethoxyphenyl)-N-(5-(4-methylpiperazin-1-yl)pyridin-2-yl)pyrazolo [1,5-a]pyrimidine-2-methamide hydrochloride as the active pharmaceutical ingredient; and selected from polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, Any surfactant combined with the above.

The pharmaceutical composition according to an embodiment of the present invention may include a CFTR activator such as an active pharmaceutical ingredient, and its content may be less than or equal to 1% (w/v) based on the total amount of the pharmaceutical composition. For example, the amount of active pharmaceutical ingredient may be greater than or equal to 0.00001% (w/v) and less than or equal to 1% (w/v), greater than or equal to 0.00005% (w/v) and less than or equal to 1% (w/ v), greater than or equal to 0.0001 % (w/v) and less than or equal to 1 % (w/v), greater than or equal to 0.0005 % (w/v) and less than or equal to 1 % (w/v), or greater than or Equal to 0.001 % (w/v) and less than or equal to 1 % (w/v).

For example, the content of the CFTR activator such as the compound of Formula 1 (such as the compound of Formula 1a, 1b, or 1c) based on the total amount of pharmaceutical ingredients can be less than or equal to 1% (w/v), 0.001% (w/ v) to 1 % (w/v), 0.005 % (w/v) to 1 % (w/v), 0.005 % (w/v) to 0.9 % (w/v), 0.005 % (w/v) to 0.8 % (w/v), 0.005 % (w/v) to 0.7% (w/v), 0.005 % (w/v) to 0.6 % (w/v), 0.005 % (w/v) to 0.5 % (w/v), 0.005 % (w/v) to 0.4 % (w/v), 0.005 % (w/v) to 0.3 % (w/v), 0.005 % (w/v) to 0.2 % ( w/v), 0.01 % (w/v) to 0.9 % (w/v), 0.01 % (w/v) to 0.8 % (w/v), 0.01 % (w/v) to 0.7 % (w/ v), 0.01 % (w/v) to 0.6 % (w/v), 0.01 % (w/v) to 0.5 % (w/v), 0.01 % (w/v) to 0.4 % (w/v) , 0.01 % (w/v) to 0.3 % (w/v), 0.01 % (w/v) to 0.2 % (w/v), 0.03 % (w/v) to 0.5 % (w/v), 0.03 % (w/v) to 0.4 % (w/v), 0.03 % (w/v) to 0.3 % (w/v), 0.03 % (w/v) to 0.2 % (w/v), 0.05 % ( w/v) to 0.9 % (w/v), 0.05 % (w/v) to 0.5 % (w/v), 0.05 % (w/v) to 0.4 % (w/v), 0.05 % (w/ v) to 0.3 % (w/v), 0.05 % (w/v) to 0.2 % (w/v), 0.1 % (w/v) to 0.9 % (w/v), 0.1 % (w/v) to 0.8 % (w/v), 0.1 % (w/v) to 0.7 % (w/v), 0.1 % (w/v) to 0.6 % (w/v), 0.1 % (w/v) to 0.5 % (w/v), or 0.1 % (w/v) to 0.4 % (w/v). The compound of formula 1 of the present invention, such as the compound of formula 1a, 1b, or 1c, has poor solubility, but within the content range described here, it can improve the stability and storage of the drug without causing precipitation of the active pharmaceutical ingredient. or alter the appearance of the active pharmaceutical ingredient.

Furthermore, within the scope described herein, the pharmaceutical compositions of the present invention can provide CFTR control effects, such as the effects of preventing, ameliorating, or treating dry eye or conjunctivitis without side effects.

The term "surfactant" used in the specification refers to a substance dissolved in water or an aqueous solution to reduce interfacial tension or surface tension.

The surfactant in one embodiment can be any one selected from polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, and combinations of the above, and it is confirmed that the surfactant can Further improve the solubility of the compound represented by Formula 1 (such as the compound of Formula 1a, 1b, or 1c) (the solubility in phosphate buffered saline is 34.1 μg/mL), and improve the physical and chemical stability of the active pharmaceutical ingredient, because Surfactants do not change appearance after storage for a period of time.

"Polysorbate 80" in the specification refers to a nonionic surfactant obtained by the addition polymerization of ethylene oxide to the free hydroxyl group of sorbitan oleate, and includes the commercially available product "Tween 80 (manufacturer : Croda)". However, the present invention is not limited to such products.

The "polyoxyethylene-35 castor oil" in the instructions mainly contains polyethylene glycol ricinoleate and triricinoleate of free glycol and ethoxylated glycerin. It can be obtained, for example, from the reaction between ethylene oxide and ricinolein. It is usually the commercially available "Kolliphor EL (manufacturer: BASF)" or "Kolliphor ELP (manufacturer: BASF)", but is not limited to this product.

In one embodiment, the surfactant may be polyoxyethylene-35 castor oil.

In the pharmaceutical composition of one embodiment, the content of the surfactant is sufficient to reduce the interfacial tension of the active pharmaceutical ingredient in the aqueous solution and stably maintain the dissolved state of the active pharmaceutical ingredient, that is, the amount is sufficient to improve the solubility of the active pharmaceutical ingredient. Specifically, even using a small amount of polysorbate 80, polyethylene glycol 40-stearate, and/or polyoxyethylene-35 castor oil can improve the solubility of the active pharmaceutical ingredient to a level sufficient to exhibit pharmacological effects. degree.

The pharmaceutical composition of an embodiment of the present invention may include a surfactant, the amount of which may be less than or equal to 9% (w/v) or less than or equal to 7% (w/v) based on the total composition. Specifically, the amount of surfactant in the composition of the present invention can be 1% (w/v) to 9% (w/v), 2% (w/v) to 9% (w/v), 4 % (w/v) to 9 % (w/v), 1 % (w/v) to 7 % (w/v), or 2 % (w/v) to 7 % (w/v). When the content of the surfactant is within the above range, the pharmaceutical composition of the present invention can maintain a stable appearance and fully maintain the solubility of the active pharmaceutical ingredients in the liquid phase, and therefore can be effectively used as a formula for eye drops.

The pharmaceutical composition according to an embodiment of the present invention may include a surfactant, and its amount based on the total composition may be less than or equal to 9% (w/v), less than or equal to 7% (w/v), 1% (w/ v) to 9 % (w/v), 2 % (w/v) to 9 % (w/v), 4 % (w/v) to 9 % (w/v), 1 % (w/v) to 7% (w/v), or 1% (w/v) to 9% (w/v); the CFTR activator compound of Formula 1 (such as the compound of Formula 1a, 1b, or 1c), the amount may be Less than or equal to 1% (w/v), 0.001 % (w/v) to 1 % (w/v), 0.005 % (w/v) to 1 % (w/v), 0.005 % (w/v) to 0.9 % (w/v), 0.005 % (w/v) to 0.8 % (w/v), 0.005 % (w/v) to 0.7 % (w/v), 0.005 % (w/v) to 0.6 % (w/v), 0.005 % (w/v) to 0.5 % (w/v), 0.005 % (w/v) to 0.4 % (w/v), 0.005 % (w/v) to 0.3 % ( w/v), 0.005 % (w/v) to 0.2 % (w/v), 0.01 % (w/v) to 0.9 % (w/v), 0.01 % (w/v) to 0.8 % (w/ v), 0.01 % (w/v) to 0.7 % (w/v), 0.01 % (w/v) to 0.6 % (w/v), 0.01 % (w/v) to 0.5 % (w/v) , 0.01 % (w/v) to 0.4 % (w/v), 0.01 % (w/v) to 0.3 % (w/v), 0.01 % (w/v) to 0.2 % (w/v), 0.03 % (w/v) to 0.5 % (w/v), 0.03 % (w/v) to 0.4 % (w/v), 0.03 % (w/v) to 0.3 % (w/v), 0.03 % ( w/v) to 0.2 % (w/v), 0.05 % (w/v) to 0.9 % (w/v), 0.05 % (w/v) to 0.5 % (w/v), 0.05 % (w/ v) to 0.4% (w/v), 0.05% (w/v) to 0.3% (w/v), 0.05% (w/v) to 0.2% (w/v), 0.1% (w/v) to 0.9 % (w/v), 0.1 % (w/v) to 0.8 % (w/v), 0.1 % (w/v) to 0.7 % (w/v), 0.1 % (w/v) to 0.6 % (w/v), 0.1 % (w/v) to 0.5 % (w/v), or 0.1 % (w/v) to 0.4 % (w/v).

In the pharmaceutical composition according to an embodiment of the present invention, the surfactant can be used as a component to form nanocells to facilitate corneal penetration and increase the absorption of nanometer-sized drugs entering the eye. Pharmaceutical compositions of embodiments of the present invention may include a CFTR activator and any interface activity selected from the group consisting of polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, and combinations thereof. Nanocell formula formed by the agent.

The pharmaceutical composition prepared based on the nanocell formulation technology of the present invention contains CFTR activator and surfactant, and has improved solubility.

The term "nanocells" in the instructions refers to the spontaneous nanoparticles generated by dropping surfactant at a critical cell concentration or higher into an aqueous medium and stirring. Its size is usually less than or equal to 200 nm.

In one embodiment, the average particle diameter of the nanocell particles of the present invention can be less than or equal to 100 nm, less than or equal to 90 nm, less than or equal to 80 nm, less than or equal to 70 nm, less than or equal to 60 nm, less than Or equal to 50 nm, less than or equal to 40 nm, less than or equal to 30 nm, or less than or equal to 20 nm.

In one embodiment, the average particle diameter of the nanocell particles can be 0.1 nm to 100 nm, 1 nm to 100 nm, 5 nm to 50 nm, 10 nm to 30 nm, 10 nm to 20 nm, 10 nm to 15 nm, 12 nm to 14 nm, 11 nm to 14 nm, 10.5 nm to 13 nm, or 11 nm to 13 nm. In the nanocell particles in the composition of an embodiment of the present invention, even during production or under harsh storage conditions (such as refrigeration for 5 days, 60˚C for 5 days, and 60˚C for 10 days), the particles The diameter change can be maintained within ±10%.

When the pharmaceutical composition is applied in the form of eye drops, the advantage of the pharmaceutical composition containing nanocells in one embodiment of the present invention is excellent corneal permeability and increased intraocular absorption of nano-sized drugs. The ophthalmic composition provided by this embodiment of the present invention includes: CFTR activator; and selected from the group consisting of polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, and combinations of the above. Any kind of surfactant.

In one embodiment, the composition may further include at least one additive selected from buffers, isotonic agents, thickeners, pH adjusters, preservatives, and antioxidants.

The term "buffer" in the specification refers to a substance used to stabilize the pH value within a specific range.

In one embodiment, the buffering agent may be any one selected from the group consisting of phosphate, trishydroxymethylaminomethane, citrate, and combinations thereof.

For example, the phosphate can be a buffer solution of disodium phosphate (disodium hydrogen phosphate, Na ₂ HPO ₄ ) and monosodium phosphate (sodium dihydrogen phosphate, NaH ₂ PO ₄ ). Phosphates can be provided as anhydrides or hydrates. In one embodiment, the buffer may be a mixture of Na ₂ HPO ₄ ·7H ₂ O and NaH ₂ PO ₄ ·2H ₂ O.

In one embodiment, the buffering agent may be citrate. For example, the buffer may be a mixture of citric acid and citrate salts. For example, the citrate salt may be sodium citrate. The citrate salt may be in the form of an anhydride or a hydrate (such as sodium citrate dihydrate). For example, the citrate salt may be a mixture of sodium citrate (eg its dihydrate) and citric acid. In one embodiment, when a composition containing citric acid as a buffer needs to be stored for a long time, the pH change is significantly reduced and the generation of impurities is reduced, which can improve the physical and chemical stability of the composition.

In one embodiment of the pharmaceutical composition, the content of the buffering agent is sufficient to stabilize the pH value of the composition containing the active pharmaceutical ingredient.

In the pharmaceutical composition of one embodiment, the content of the buffer can be 0.1% (w/v) to 0.5% (w/v), 0.15% (w/v) to 0.4% (w/v), 0.2% (w/v) to 0.4 % (w/v), 0.2 % (w/v) to 0.3 % (w/v), 0.25 % (w/v) to 0.28 % (w/v), 0.27 % (w /v) to 0.28 % (w/v), 0.1 % (w/v), 0.2 % (w/v), 0.25 % (w/v), 0.3 % (w/v), or 0.35 % (w/ v).

The term "isotonic agent" used in the instructions refers to a substance used to relieve pain or discomfort and prevent red blood cells from lysing and shrinking when administered as eye drops, injections, sprays, or the like.

In one embodiment, the isotonic agent can be selected from mannitol, sorbitol, sodium chloride, glycerin, polyethylene glycol, propylene glycol, and any combination of the above, but is not limited thereto.

Polyethylene glycol is a relatively stable and non-toxic substance, and its shape and properties can change with molecular weight at room temperature. For example, polyethylene glycol 200 (PEG 200), polyethylene glycol 300 (PEG 300), polyethylene glycol 400 (PEG 400), and polyethylene glycol 600 (PEG 600) are transparent at room temperature. Liquid form. For example, polyethylene glycol 1000 (PEG 1000) is a pasty, opaque white solid at room temperature. For example, polyethylene glycol 4000 (PEG 4000) is an opaque solid at room temperature such as flakes, granules, or powder.

The polyethylene glycol that can be used in the composition of the present invention is in the form of a transparent liquid, which can be evenly mixed with other pharmaceutical ingredients to be used as eye drops, and the average molecular weight of the polyethylene glycol is 190 to 630. In one embodiment, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, or polyethylene glycol 600 may be used.

In one embodiment, the polyethylene glycol may be polyethylene glycol 400.

In one embodiment, the isotonicity agent may be sodium chloride, propylene glycol, or a combination thereof.

In one embodiment, the composition includes sodium chloride as a buffer for long-term storage, which can significantly reduce pH changes and the generation of impurities, thereby improving the physical and chemical stability of the composition.

In one embodiment, the pharmaceutical composition contains an isotonic agent in an amount sufficient to reduce pain or discomfort when the active pharmaceutical ingredient composition is administered as eye drops.

The pharmaceutical composition of an embodiment may include 0% (w/v) to 10% (w/v), such as 0% (w/v) to 5.0% (w/v) or 0% (w/v) to 1.0 % (w/v) isotonic agent. For example, the amount of isotonic agent in the pharmaceutical composition can be 0% (w/v) to 9% (w/v), 0% (w/v) to 7% (w/v), 0% (w/v) /v) to 5 % (w/v), 0 % (w/v) to 0.8 % (w/v), 0.00001 % (w/v) to 9 % (w/v), 0.00001 % (w/v ) to 8 % (w/v), 0.0001 % (w/v) to 0.8 % (w/v), 0.001 % (w/v) to 0.8 % (w/v), 0.01 % (w/v) to 0.8 % (w/v), 0.1 % (w/v) to 0.8 % (w/v), 0.2 % (w/v) to 0.8 % (w/v), 0.3 % (w/v) to 0.8 % (w/v), 0.5 % (w/v) to 0.8 % (w/v), 0.5 % (w/v) to 0.75 % (w/v), 0.7 % (w/v) to 0.8 % (w /v), 0.1% (w/v) to 0.75% (w/v), 0.2% (w/v), 0.3% (w/v), or 0.75% (w/v). The composition of one embodiment may contain no isotonicity agent.

The term "thickener or viscosity-increasing agent" in the instructions refers to substances used to increase the viscosity of liquid pharmaceutical products.

In one embodiment, the thickener may be any one selected from the group consisting of hydroxypropyl methylcellulose, polyvinyl alcohol, sodium carboxymethyl cellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, and combinations thereof. , but not limited to this.

In one embodiment, when a thickener is used, the physical and chemical stability of the active pharmaceutical ingredient can be improved. For example, the thickening agent may be hydroxypropyl methylcellulose.

The term "hydroxypropyl methylcellulose" in the instructions can also be regarded as "hypromellose" and the trade name "Pharmacoat". However, the thickener of the present invention is not limited to this.

The substance referred to by the term "polyvinylpyrrolidone" in the instructions can be regarded as the trade name "Kollidon 25". However, the thickener of the present invention is not limited to this.

In one embodiment, the thickener may be hydroxypropyl methylcellulose or sodium carboxymethylcellulose.

The pharmaceutical compositions of other embodiments may contain no thickening agent.

The pharmaceutical composition of one embodiment may include an appropriate amount of thickener to make the viscosity of the composition containing active pharmaceutical ingredients suitable for applying eye drops and/or to maintain the appearance and physical and chemical stability of the drug.

The pharmaceutical composition of one embodiment may include a thickening agent in an amount of 0% (w/v) to 2.0% (w/v). For example, the amount of thickener in the pharmaceutical composition can be 0.00001% (w/v) to 2.0% (w/v), 0.0001% (w/v) to 2.0% (w/v), 0.1% ( w/v) to 2.0 % (w/v), 0.05 % (w/v) to 1.0 % (w/v), 0.1 % (w/v) to 1.0 % (w/v), 0.1 % (w/ v) to 0.6% (w/v), 0.1% (w/v), 0.6% (w/v), or 0.015% (w/v).

The term "antioxidants" used in the instructions refers to substances used to prevent the deterioration of drug quality due to oxidation.

In one embodiment, the antioxidant may be any one selected from the group consisting of ethylenediaminetetraacetic acid, sodium sulfite, sodium bisulfite, sodium metabisulfite, and combinations thereof, but is not limited thereto.

In one embodiment, the antioxidant may be sodium sulfite or sodium bisulfite.

In the pharmaceutical composition of one embodiment, antioxidants can be selected as appropriate components to prevent oxidation of the drug.

In the pharmaceutical composition of one embodiment, the content of antioxidants is sufficient to prevent oxidation of the drug.

The pharmaceutical composition of an embodiment may include an antioxidant in an amount of 0% (w/v) to 0.3% (w/v) or 0% (w/v) to 0.2% (w/v). For example, the antioxidant content in the composition may be 0.1% (w/v). The composition of one embodiment may not contain antioxidants.

The pharmaceutical composition of one embodiment may not contain antioxidants. In one embodiment, a composition that does not contain antioxidants can reduce pH changes and impurities to improve the effect.

The pharmaceutical composition of one embodiment provides optimized ingredients and compositions for preparing eye drop formulations.

The pharmaceutical composition of one embodiment has excellent mixing compatibility with active pharmaceutical ingredients, which can improve the physical and chemical stability in appearance, quantity, pH, particle size, and the evolution of impurities, and improve solubility and physiological isotonicity. , and eye drops containing compounds of formula 1 (such as compounds of formula 1a, formula 1b, or formula 1c) can be used as active pharmaceutical ingredients.

In one embodiment, the pharmaceutical composition may include 0.1% (w/v) to 0.4% (w/v) of the active pharmaceutical ingredient. For example, the amount of active pharmaceutical ingredient in the composition may be 0.2% (w/v) to 0.3% (w/v), or 0.25% (w/v).

The quantity unit "% (w/v)" used in this specification refers to the quantity based on the total amount of the composition. For example, when the total base of the composition is 100 mL, the weight (g) of the ingredients contained in it.

In one embodiment, the pharmaceutical composition may include an active pharmaceutical ingredient at a concentration of less than or equal to 10 mg/mL. In a more specific example, the compositions of the present invention may include an active pharmaceutical ingredient at a concentration of less than or equal to 2.7 mg/mL. In one embodiment, the composition of the present invention may include an active pharmaceutical ingredient at a concentration of 0.001 mg/mL to 10 mg/mL or 0.001 mg/mL to 2.7 mg/mL.

The pharmaceutical composition of one embodiment may have a clear and transparent appearance.

In the pharmaceutical composition of one embodiment, the nanocells remain as they were when they were formed, and the pharmaceutical composition does not change its appearance over time.

The osmotic pressure of the pharmaceutical composition in one embodiment is 150 mOsm/kg to 450 mOsm/kg or 200 mOsm/kg to 400 mOsm/kg, such as 250 mOsm/kg to 343 mOsm/kg or 260 mOsm/kg to 360 mOsm/ kg.

In one or more embodiments, the pharmaceutical composition may further include additives such as diluents, solvents, photo-opaque agents, or the like as needed. For example, the composition may be an aqueous composition using water as a solvent (base), such as aqueous eye drops.

In addition to the active pharmaceutical ingredients and the additives described in this specification, those with ordinary knowledge in the art can further select suitable additives according to the purpose of the present invention.

Another embodiment provides an ophthalmic composition including a CFTR activator as an active pharmaceutical ingredient, and is selected from the group consisting of polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil, and combinations thereof. any kind of surfactant. Ophthalmic compositions are intended to include all compositions in which substances are administered topically via the eye. The ophthalmic composition may also be an eye drop composition. For example, ophthalmic compositions can be used to prevent, improve, or treat eye diseases, dry eye syndrome, burning sensation, itching, foreign body sensation, or roughness.

In one embodiment, the ophthalmic composition may include an active pharmaceutical ingredient (a compound represented by Formula 1 such as a compound of Formula 1a, a compound of Formula 1b, or a compound of Formula 1c), and a compound selected from the group consisting of polysorbate 80, polyethylene Glycol 40-stearate, polyoxyethylene-35 castor oil, and any surfactant in combination with the above.

In one embodiment, the pharmaceutical composition includes an active pharmaceutical ingredient in an amount less than or equal to 1% (w/v), 0.00001% (w/v) to 1% (w/v), 0.00001% (w/v) to 0.9 % (w/v), 0.00001 % (w/v) to 0.8 % (w/v), 0.00001 % (w/v) to 0.7 % (w/v), 0.00001 % (w/v) to 0.6 % (w/v), 0.00001 % (w/v) to 0.5 % (w/v), 0.00001 % (w/v) to 0.4 % (w/v), 0.00001 % (w/v) to 0.3 % ( w/v), 0.00001 % (w/v) to 0.2 % (w/v), 0.00005 % (w/v) to 1 % (w/v), 0.00005 % (w/v) to 0.9 % (w/ v), 0.00005 % (w/v) to 0.8 % (w/v), 0.00005 % (w/v) to 0.7 % (w/v), 0.00005 % (w/v) to 0.6 % (w/v) , 0.00005 % (w/v) to 0.5 % (w/v), 0.00005 % (w/v) to 0.4 % (w/v), 0.00005 % (w/v) to 0.3 % (w/v), 0.00005 % (w/v) to 0.2 % (w/v), 0.0001 % (w/v) to 1 % (w/v), 0.0001 % (w/v) to 0.9 % (w/v), 0.0001 % ( w/v) to 0.8 % (w/v), 0.0001 % (w/v) to 0.7 % (w/v), 0.0001 % (w/v) to 0.6 % (w/v), 0.0001 % (w/ v) to 0.5 % (w/v), 0.0001 % (w/v) to 0.4 % (w/v), 0.0001 % (w/v) to 0.3 % (w/v), 0.0001 % (w/v) to 0.2 % (w/v), 0.0005 % (w/v) to 1 % (w/v), 0.0005 % (w/v) to 0.9 % (w/v), 0.0005 % (w/v) to 0.8 % (w/v), 0.0005 % (w/v) to 0.7 % (w/v), 0.0005 % (w/v) to 0.6 % (w/v), 0.0005 % (w/v) to 0.5 % ( w/v), 0.0005 % (w/v) to 0.4 % (w/v), 0.0005 % (w/v) to 0.3 % (w/v), 0.0005 % (w/v) to 0.2 % (w/ v), 0.001 % (w/v) to 1 % (w/v), 0.005 % (w/v) to 1 % (w/v), 0.005 % (w/v) to 0.9 % (w/v) , 0.005 % (w/v) to 0.8 % (w/v), 0.005 % (w/v) to 0.7 % (w/v), 0.005 % (w/v) to 0.6 % (w/v), 0.005 % (w/v) to 0.5 % (w/v), 0.005 % (w/v) to 0.4 % (w/v), 0.005 % (w/v) to 0.3 % (w/v), 0.005 % ( w/v) to 0.2 % (w/v), 0.01 % (w/v) to 0.9 % (w/v), 0.01 % (w/v) to 0.8 % (w/v), 0.01 % (w/ v) to 0.7 % (w/v), 0.01 % (w/v) to 0.6 % (w/v), 0.01 % (w/v) to 0.5 % (w/v), 0.01 % (w/v) to 0.4 % (w/v), 0.01 % (w/v) to 0.3 % (w/v), 0.01 % (w/v) to 0.2 % (w/v), 0.03 % (w/v) to 0.5 % (w/v), 0.03 % (w/v) to 0.4 % (w/v), 0.03 % (w/v) to 0.3 % (w/v), 0.03 % (w/v) to 0.2 % ( w/v), 0.05 % (w/v) to 0.9 % (w/v), 0.05 % (w/v) to 0.5 % (w/v), 0.05 % (w/v) to 0.4 % (w/ v), 0.05 % (w/v) to 0.3 % (w/v), 0.05 % (w/v) to 0.2 % (w/v), 0.1 % (w/v) to 0.9 % (w/v) , 0.1 % (w/v) to 0.8 % (w/v), 0.1 % (w/v) to 0.7 % (w/v), 0.1 % (w/v) to 0.6 % (w/v), 0.1 % (w/v) to 0.5 % (w/v), or 0.1 % (w/v) to 0.4 % (w/v). In the cell-based activity test, it was confirmed that the pharmaceutical composition containing the compound of Formula 1 of the present invention exhibited activity at a concentration of 0.00001% (w/v). For example, the amount of active pharmaceutical ingredient in the composition may be 0.2% (w/v) to 0.3% (w/v), or 0.25% (w/v).

In one embodiment of the present invention, the ophthalmic composition includes an amount of main pharmaceutical ingredients that is less than or equal to 1% (w/v), 0.00001% (w/v) to 1% (w/v), 0.00001% ( w/v) to 0.9 % (w/v), 0.00001 % (w/v) to 0.8 % (w/v), 0.00001 % (w/v) to 0.7 % (w/v), 0.00001 % (w/ v) to 0.6 % (w/v), 0.00001 % (w/v) to 0.5 % (w/v), 0.00001 % (w/v) to 0.4 % (w/v), 0.00001 % (w/v) to 0.3 % (w/v), 0.00001 % (w/v) to 0.2 % (w/v), 0.00005 % (w/v) to 1 % (w/v), 0.00005 % (w/v) to 0.9 % (w/v), 0.00005 % (w/v) to 0.8 % (w/v), 0.00005 % (w/v) to 0.7 % (w/v), 0.00005 % (w/v) to 0.6 % ( w/v), 0.00005 % (w/v) to 0.5 % (w/v), 0.00005 % (w/v) to 0.4 % (w/v), 0.00005 % (w/v) to 0.3 % (w/ v), 0.00005 % (w/v) to 0.2 % (w/v), 0.0001 % (w/v) to 1 % (w/v), 0.0001 % (w/v) to 0.9 % (w/v) , 0.0001 % (w/v) to 0.8 % (w/v), 0.0001 % (w/v) to 0.7 % (w/v), 0.0001 % (w/v) to 0.6 % (w/v), 0.0001 % (w/v) to 0.5 % (w/v), 0.0001 % (w/v) to 0.4 % (w/v), 0.0001 % (w/v) to 0.3 % (w/v), 0.0001 % ( w/v) to 0.2 % (w/v), 0.0005 % (w/v) to 1 % (w/v), 0.0005 % (w/v) to 0.9 % (w/v), 0.0005 % (w/ v) to 0.8 % (w/v), 0.0005 % (w/v) to 0.7 % (w/v), 0.0005 % (w/v) to 0.6 % (w/v), 0.0005 % (w/v) to 0.5 % (w/v), 0.0005 % (w/v) to 0.4 % (w/v), 0.0005 % (w/v) to 0.3 % (w/v), 0.0005 % (w/v) to 0.2 % (w/v), 0.001 % (w/v) to 1 % (w/v), 0.005 % (w/v) to 1 % (w/v), 0.005 % (w/v) to 0.9 % ( w/v), 0.005 % (w/v) to 0.8 % (w/v), 0.005 % (w/v) to 0.7 % (w/v), 0.005 % (w/v) to 0.6 % (w/ v), 0.005 % (w/v) to 0.5 % (w/v), 0.005 % (w/v) to 0.4 % (w/v), 0.005 % (w/v) to 0.3% (w/v) , 0.005 % (w/v) to 0.2 % (w/v), 0.01 % (w/v) to 0.9 % (w/v), 0.01 % (w/v) to 0.8 % (w/v), 0.01 % (w/v) to 0.7 % (w/v), 0.01 % (w/v) to 0.6 % (w/v), 0.01 % (w/v) to 0.5 % (w/v), 0.01 % ( w/v) to 0.4% (w/v), 0.01% (w/v) to 0.3% (w/v), 0.01% (w/v) to 0.2% (w/v), 0.03% (w/ v) to 0.5 % (w/v), 0.03 % (w/v) to 0.4 % (w/v), 0.03 % (w/v) to 0.3 % (w/v), 0.03 % (w/v) to 0.2 % (w/v), 0.05 % (w/v) to 0.9 % (w/v), 0.05 % (w/v) to 0.5 % (w/v), 0.05 % (w/v) to 0.4 % (w/v), 0.05 % (w/v) to 0.3 % (w/v), 0.05 % (w/v) to 0.2 % (w/v), 0.1 % (w/v) to 0.9 % ( w/v), 0.1 % (w/v) to 0.8 % (w/v), 0.1 % (w/v) to 0.7 % (w/v), 0.1 % (w/v) to 0.6 % (w/ v), 0.1 % (w/v) to 0.5 % (w/v), or 0.1 % (w/v) to 0.4 % (w/v). In the cell-based activity test, it can be confirmed that the pharmaceutical composition containing the compound of Formula 1 of the present invention exhibits activity at a concentration of 0.00001% (w/v).

In one embodiment, the ophthalmic composition may be a formulation based on nanocell technology. The ophthalmic composition of the present invention may include nanocells.

In one embodiment, the average particle size of the nanocells can be less than or equal to 100 nm, such as less than or equal to 90 nm, less than or equal to 80 nm, less than or equal to 70 nm, less than or equal to 60 nm, less than or equal to 50 nm. nm, less than or equal to 40 nm, less than or equal to 30 nm, or less than or equal to 20 nm.

In one embodiment, the average particle diameter of the nanocell particles can be 0.1 nm to 100 nm, 1 nm to 100 nm, 5 nm to 50 nm, 10 nm to 30 nm, 10 nm to 20 nm, 10 nm to 15 nm, 12 nm to 14 nm, 11 nm to 14 nm, 10.5 nm to 13 nm, or 11 nm to 13 nm. In one embodiment of the present invention, the particle size variation of the nanocell particles in the composition can be maintained within ±10%, even during manufacturing and under harsh storage conditions (such as refrigeration for 5 days, at 60˚C 5 days, and 10 days at 60˚C).

In one embodiment, the pH value of the ophthalmic composition may be 6.0 to 8.0.

The ophthalmic composition may be a composition for use by an ocular route of administration. Ophthalmic compositions may be used for pharmaceutical or veterinary administration purposes and may serve as pharmaceuticals as well as quasi-drugs.

In one embodiment, the ophthalmic composition may be a topically absorbed or systemically absorbed ophthalmic composition. For example, an ophthalmic composition can be a composition for topical application to the eye.

In one embodiment, the ophthalmic composition can be any type of formulation used in ophthalmology according to a doctor's prescription (or can be used without a doctor's prescription).

In one embodiment, the ophthalmic composition may be a liquid composition or a semi-solid composition. In one embodiment, the ophthalmic composition may be in the form of an eye drop solution, ointment, gel, cream, lotion, emulsion, suspension, or spray. For example, the ophthalmic composition may be in the form of eye drops or ointment. The main advantage of the eye drops of one embodiment is that it is simple and convenient to use. The eye drops may be in the form of solution or suspension. An advantage of the eye ointment of one embodiment may be that it remains in the eyeball for a long time. Additionally, the ophthalmic composition may be provided in the form of artificial tears or eye cleansers.

The ophthalmic composition of an embodiment of the present invention can be prepared according to known methods in the art. A person with ordinary knowledge in the art can easily confirm the appropriate excipients and/or carriers and their dosage according to the formulation to be prepared.

Ophthalmic compositions may be contained or stored in containers made of a variety of materials. For example, the container may be composed of polyethylene or polypropylene (including low density polyethylene (LDPE) or the like), or a glass container. When the composition disclosed here is an eye drop, it is contained in an eye drop container, more specifically, a multi-dose eye drop container or a unit dose eye drop container.

The multi-dose eye drop container has a container body and a lid attached to the container body, wherein the lid can be freely opened and resealable. Multi-dose eye drop containers typically contain multiple doses of eye drop solution for use over a set period of time. Unit dose eye drop containers refer to eye drop containers with a fused and sealed cap at the mouth of the bottle. During use, pull apart the fused part between the cap and the bottle body. Unit-dose eye drop containers contain eye drops for single or multiple use. Additionally, the eye drops contained in unit dose eye drop containers may be free or substantially free of preservatives such as benzalkonium chloride.

The ophthalmic composition of one embodiment may have the following characteristics. When directly observed with the naked eye, the ophthalmic composition according to an embodiment of the present invention may be a colorless or light yellow transparent liquid.

The ophthalmic composition according to one embodiment of the present invention is suitable for testing based on HPLC-PDA and HPLC testing methods.

The pH value of the ophthalmic composition according to an embodiment of the present invention can be from 6.0 to 8.0, and the measurement method can be the pH measurement method in the general test methods of the United States Pharmacopeia.

The osmotic pressure of the ophthalmic composition according to an embodiment of the present invention may be 260 mOsm/kg to 360 mOsm/kg, and the measurement method may be the osmotic pressure measurement method in the general test methods of the United States Pharmacopoeia.

When tested according to the insoluble particle test method used for eye drops in the general test methods of the United States Pharmacopoeia, in the ophthalmic composition of one embodiment of the present invention, the content of particles with a particle size greater than or equal to 10 μm is less than or equal to 50 particles/mL. , the content of particles with a particle size greater than or equal to 25 μm is less than or equal to 5 particles/mL, and the content of particles with a particle size greater than or equal to 50 μm is less than or equal to 2 particles/mL.

When tested according to the osmotic pressure measurement method in the general test methods of the United States Pharmacopoeia, no microbial growth was observed in the ophthalmic composition of one embodiment of the present invention.

In addition, unless otherwise clearly defined, descriptions of pharmaceutical compositions in the instructions may also apply to ophthalmic compositions. Conversely, descriptions given for ophthalmic compositions may also apply to pharmaceutical compositions.

Another embodiment uses the pharmaceutical composition or ophthalmic composition to prevent, improve, or treat eye-related diseases or symptoms. In one embodiment, the eye-related disease or symptom may include dry eye, keratoconjunctivitis, dry eyes, burning sensation, itching, or any foreign body or rough sensation in the eyeball. The pharmaceutical composition or ophthalmic composition may contain a CFTR activator, that is, a compound of Formula 1 such as a compound of Formula 1a, Formula 1b, or Formula 1c, in an amount suitable for the aforementioned purposes (such as a therapeutically effective amount).

The dosage of the composition may be appropriately changed depending on the formulation, severity of symptoms, age and weight of the patient to be treated, physician's judgment, or the like. For example, when the formula of the composition is eye drops, each dose applied to the eyes can be 1 drop to 5 drops, preferably 1 drop to 3 drops. In addition, when using this composition, the composition can be applied from 1 to several times a day, such as from 1 to 4 times a day, from 1 to 3 times a day, from 1 to 2 times a day, or once a day, or as often as needed. or applied irregularly.

The above content is only used to illustrate the present invention. For those with ordinary knowledge in the art, the embodiments disclosed herein can be easily adjusted into other specific forms without changing the technical spirit or basic characteristics. Accordingly, the embodiments described herein are in all respects illustrative and not restrictive. All numbers used herein to express amounts of ingredients, properties such as molecular weight, reaction conditions, or the like, are to be understood to be modified in all instances by the term "about" unless otherwise stated. In summary, the above numbers in the specification are approximations and may vary depending on the properties required for the invention.

The term "about" in the specification refers to a value within 5% of a given value or range, preferably a value within 1% to 2%. For example, "about 10%" refers to 9.5% to 10.5%, preferably 9.8% to 10.2%. In another example, "about 100˚C" refers to a temperature between 95˚C and 105˚C, preferably between 98˚C and 102˚C.

The expressions "has", "can have", "includes", "can include", or similar terms in the specification refer to the presence of corresponding features (such as numerical values or components), and are not used to exclude the presence of additional features. For example, when a structure is described, it is understood that all stereoisomers and tautomeric forms, as applicable, are included unless otherwise stated.

Furthermore, ranges stated in the specification include the endpoints unless expressly defined otherwise. All publications cited in the specification are incorporated by reference in their entirety.

The present invention will be described in detail below with reference to the following examples. However, these examples are for illustrative purposes only and do not limit the scope of the invention. [Example]

1. Evaluation criteria

(1)Device information

The device information used is as follows.

HPLC: Shimadzu HPLC/model NO.L20204908166 CD

Zeta particle size analyzer: Malvern Zeta particle size analyzer NS/model NO.MAL1045766

Osmometer: Fiske Model 210 micro osmometer/model NO.1402195D

pH meter: Mettler Toledo pH meter/model NO.DLCCCL0411C

(2)-1 Analysis conditions

Compositions 1, 2, 8, 9, 10, and 12 were analyzed under the following numerical analysis conditions.

① HPLC analysis conditions

Depending on the active pharmaceutical ingredient compound, analysis can be performed under the following conditions.

A. Compound 1a

Column: Shiseido C18 4.6 x 150 nm, 5 μm

Temperature (°C): 40

Mobile phase: isobaric elution (acetonitrile: deionized water: trifluoroacetic acid = 40:60:0.05)

Flow rate (mL/min): 1.0

Injection volume (μl): 20

Wavelength (nm): 240

Residence time (min): 5.0

B. Compound 1b

Column: Shiseido C18 4.6 x 150 nm, 5 μm

Temperature (°C): 40

Mobile phase: isobaric elution (acetonitrile: deionized water: trifluoroacetic acid = 40:60:0.05)

Flow rate (mL/min): 1.0

Injection volume (μl): 20

Wavelength (nm): 240

Residence time (min): 4.1

C. Compound 1c

Column: Shiseido C18 4.6 x 150 nm, 5 μm

Temperature (°C): 40

Mobile phase: isobaric elution (acetonitrile: deionized water: trifluoroacetic acid = 25:75:0.05)

Flow rate (mL/min): 1.0

Injection volume (μl): 20

Wavelength (nm): 240

Residence time (min): 4.6

②Prepare stock solution

Weigh 25 mg of sample and add it to a 100 mL quantitative flask. Prepare a diluting solvent (deionized water: methanol = 2:8), and add approximately 50 mL of the diluting solvent to the quantitative flask and vortex for 10 minutes to dissolve the sample. Add additional dilution solvent to the volumetric flask to accurately align with the 100 mL mark (concentration: 250 μg/mL).

③ Prepare standard solution

After diluting the stock solution with acetonitrile or a mixed solution of deionized water and methanol (deionized water:methanol=2:8), perform a final dilution of 2 to 10 times with the mobile phase, and dilute it with 0 μg/mL to 50 μg/mL. The concentration in mL was analyzed.

④Preparing samples

After diluting the sample with acetonitrile or a mixed solution of deionized water and methanol (deionized water:methanol=2:8), perform a final dilution of 2 to 10 times with the mobile phase, and dilute it with 0 μg/mL to 50 μg/mL. concentration was analyzed.

(2)-2 Analysis conditions

Compositions 3, 4, 5, 6, 7, and 11 were analyzed according to the following analysis conditions.

① HPLC analysis conditions

The HPLC analysis conditions within the 10-minute analysis time are as follows:

Column: Shiseido C18 4.6 x 150 nm, 5 μm

Temperature (°C): 40

Mobile phase: isobaric elution (acetonitrile: deionized water: trifluoroacetic acid = 33:67:0.05)

Flow rate (mL/min): 1.0

Injection volume (μl): 20

Wavelength (nm): 240

Residence time (min): 8.7

②Prepare stock solution

Weigh 10 mg of sample and add it to a 10 mL quantitative flask. Prepare a diluting solvent (deionized water: methanol = 2:8), and add approximately 5 mL of the diluting solvent to the quantitative flask and vortex for 10 minutes to dissolve the sample. Add additional dilution solvent to the quantitative flask to accurately align with the 10 mL mark (concentration: 1.0 mg/mL).

③Prepare standard solution

The stock solution was diluted with a mixed solution of deionized water and acetonitrile (deionized water:acetonitrile = 67:33, containing 0.05% trifluoroacetic acid) and analyzed at a concentration of 100 μg/mL.

④Preparing samples

Samples were diluted in mobile phase and analyzed at a concentration of 100 μg/mL.

⑤ Impurity calculation

Impurities for each relative retention time (RRT) are expressed as % relative to the major component used for comparison. All signal peaks other than the signal peak of the main component are considered impurities, and not every signal peak area is assigned a weighting factor.

(3) Assessment of physical and chemical properties

①Amount: Measure the amount in each formula according to the aforementioned HPLC conditions.

② Osmotic pressure: Measure osmotic pressure based on the freezing point depression theory.

③Particle size: Place each sample in a tube and measure its particle size with Zeta Particle Size Analyzer NS equipment.

④ pH: Measure the pH value of each formula with a pH meter.

(4) Evaluate stability at 60˚C

To evaluate the chemical stability of the drug over a short period of time, the samples were stored in an oven at 60˚C for at least 3 days and their appearance, pH, and quantity were evaluated.

(5) Evaluate stability under centrifugation/freeze-thaw conditions

Formulations with guaranteed chemical stability were evaluated for physical stability over short periods of time (such as precipitation, aggregation, foreign body formation, or similar properties) as follows.

① Centrifugation: After centrifugation at 13,000 rpm for 5 minutes, the particle size is evaluated by Zeta particle size analyzer.

②Freezing-thawing: After repeating the freezing-thawing process 5 times at 4-hour intervals, measure the particle size with a Zeta particle size analyzer.

(6) Evaluate the compatibility of LDPE materials

Fill the drug into the LDPE eye drop container, and evaluate the drug's appearance, amount, impurities, particle size, pH, osmotic pressure, and insoluble foreign matter under harsh conditions (60˚C).

(7)Assessment items

The respective evaluation criteria for the tests are shown in Table 1.

Test their respective assessment criteria

Table 1 Assessment project Applicable standards Initial immediate assessment Appearance Microcell: clear and transparent Emulsion: uniform milk color quantity 80% to 120% of labeled amount pH 7.0±0.5 Osmotic pressure 250 mOsm/kg to 343 mOsm/kg particle size ＜100nm After storage at 60˚C Appearance Same as initial appearance quantity Greater than or equal to 90% of the initial amount pH Initial pH ± 1.0 Centrifuge/freeze-after thawing Appearance Same as initial appearance particle size Less than or equal to 2 times the initial particle size After sterile filtration quantity ±5% of initial amount particle size Less than or equal to 2 times the initial particle size

2. Preparation of eye drop formula (compositions 2 to 12)

1) Contains thickener (compositions 6 and 11)

(1) Prepare solution A (drug solution)

A. Place the magnetic rod in the glass scintillation vial and add 6 mL of sterile water, then heat the glass scintillation vial to 60˚C in a water bath.

B. Add excipients other than solubilizers and thickeners (such as buffers, isotonic agents, antioxidants, or the like) to the solution in step A according to the amounts shown in the composition table for each composition, Then stir at 300 rpm for 30 minutes at 60˚C. Here, only half the amount of the buffer described in the composition table of each composition is added to the solution in Step A, and the other half is added to the solution in Step B in (2) Preparing Solution B.

C. According to the composition table of each component, use a viscosity pipette to drop the solubilizer into the solution in step B, then heat it to 60˚C in a water bath, and stir at 300 rpm for 90 minutes.

D. Add the active pharmaceutical ingredient (i.e., the compound of formula 1a, 1b, or 1c) to the solution in step C according to the amount in the composition table of each composition, and then stir at 400 rpm at a water bath temperature of 60˚C for 180 minutes.

(2) Prepare solution B (thickener solution)

A. Place the magnetic rod in the glass scintillation vial and add 6 mL of sterile water, then heat the glass scintillation vial to 60˚C in a water bath.

B. Add half the amount of the buffer described in the composition table for each composition to the solution in step A, and then stir at 300 rpm in a water bath at 60˚C for 30 minutes.

C. Add the thickener of each composition in the composition table to the solution in step B, then maintain it in a water bath at 60˚C and stir at 300 rpm for 5 minutes, and stir thoroughly through a multi-vortexer at room temperature. Dissolve.

(3) Mix and sterile filter solutions A and B

A. Mix 5 mL of solution A and 5 mL of solution B, and stir thoroughly using a multi-vortexer at room temperature for greater than or equal to 2 hours.

B. Then drop in 1N HCl and 1N NaOH to bring the pH value of the solution to about 7.0.

C. On a clean workbench, use a syringe to draw the solution from step B, slowly pass it through a syringe filter composed of 0.20 μm mixed cellulose ester (MCE) material, and dispense it into autoclaved vials for storage.

2) No thickener (compositions 2 to 5 and compositions 7 to 10)

A. Place the magnetic rod into the glass scintillation vial, add 10 mL of sterile water, and heat the glass scintillation vial to 60°C in a water bath.

B. According to the amount of each component in the composition table, add excipients other than solubilizers (such as buffers, isotonic agents, antioxidants, and the like) to the solution in step A, and then incubate at 60˚ Stir at 300 rpm for 30 minutes at a temperature of C.

C. According to the composition table of each component, use a viscosity pipette to drop the solubilizer into the solution in step B, then heat it to 60˚C in a water bath and stir at 300 rpm for 90 minutes.

D. According to the composition table of each composition, add the pharmaceutically active ingredient (ie, the compound of formula 1a, 1b or 1c) to the solution in step C, and then maintain it in a water bath at 60˚C and stir at 400 rpm for 180 minutes. .

E. Then drop in 1N HCl and 1N NaOH to adjust the pH of the solution to about 7.0.

F. On a clean workbench, use a syringe to draw the solution from step E, slowly pass it through a syringe filter composed of 0.20 μm mixed cellulose ester (MCE) material, and dispense it into autoclaved vials for storage.

3. Preparation and Evaluation of Compositions 1 to 11

1) Preparation of composition 1 and solubility evaluation of each surfactant

(1) Prepare 10 mM phosphate buffer solution

1.549 g of Na ₂ HPO ₄ ·7H ₂ O and 0.659 g of NaH ₂ PO ₄ ·2H ₂ O were weighed and added to 1 L of distilled water, and stirred by a magnetic stirrer for 4 hours.

1N NaOH and 1N HCl were added dropwise to adjust the pH to 7.0.

(2) Prepare Composition 1 (Compositions 1A to 1E) and evaluate the solubility of each surfactant

The composition of the surfactant was varied to prepare a composition containing the compound of formula 1a (solubility in phosphate buffered saline 34.1 μg/mL). Compositions can be prepared based on the amounts of surfactants in Table 2 and their solubility can be evaluated.

Specifically, 5 mL of phosphate buffered saline was added to a 20 mL scintillation vial, and the surfactant of Tween 80 was dissolved in it (concentration: 1%, composition 1A). In addition, add 5 mL of the previously prepared 10 mM phosphate buffer solution to a 20 mL scintillation vial, and add Tween 80, polyethylene glycol 40-stearate, Kolliphor EL, and Tween 80 + Kolliphor (concentrations respectively). 4%, 7%, 5%, 4% + 5%, compositions 1B to 1E). Here, an excess of the compound of formula 1a is added dropwise to each sample and stirred with a multivortexer for greater than or equal to 6 hours. When the pH changes, add IN NaOH/HCl dropwise to adjust the pH to approximately 7.0 again.

Store samples under refrigeration and room temperature for more than 12 hours. Then 1 mL of each sample was added to the electropolishing tubes and centrifuged at 13,000 rpm for 5 min. Take the clarified supernatant after centrifugation and dilute it with the diluting solvent (acetonitrile/deionized water + methanol/mobile phase), and the calibration curve concentration is 0 μg/mL to 50 μg/mL. The results were then placed into HPLC vials and analyzed by HPLC (n=2).

The solubility of the composition containing each surfactant was then evaluated, and the results are shown in Table 2.

Results of solubility assessment

Table 2 Composition 1A 1B 1C 1D 1E surfactant Tween 80 Tween 80 Polyethylene glycol 40-stearate Kolliphor EL Tween 80 + Kolliphor EL 1%(w/v) 4%(w/v) 7%(w/v) 5%(w/v) 4%(w/v) + 5%(w/v) Solubility(mg/mL) 1.478 3.320 ± 0.16 4.780 ± 0.24 3.041 ± 0.03 5.813 ± 0.005

As shown in Table 2, it is confirmed that the use of surfactants (i.e., polysorbate 80, polyethylene glycol 40-stearate, polyoxyethylene-35 castor oil (Kolliphor EL), or a combination of the above) can improve formula 1a the solubility of the compound. In addition, summarizing the amounts in Table 4, polysorbate 80, polyethylene glycol 40-stearate, and polyoxyethylene-35 castor oil (Kolliphor EL) can be used as surfactants to reduce changes in appearance and improve physical chemistry. Stability.

2) Prepare composition 2 and evaluate the compatibility of each surfactant

(1) Preparation of composition 2 (compositions 2A to 2D)

The composition of the surfactant was changed to prepare a composition containing the compound of formula 1a, as shown in Table 3. In terms of details, the composition can be prepared according to the method described in the aforementioned paragraph 2. Preparing the Eye Drop Formula. Prepare eye drop formulation. The solubility of each surfactant in Table 2 can be considered to set the active pharmaceutical ingredient amount (mg) of each composition.

Composition table for surfactant screening study (1)

table 3 mixed purpose Material name Composition 2A 2B 2C 2D active pharmaceutical ingredient Compound of formula 1a (mg) 10.0 15.0 10.0 20.0 Buffer Na ₂ HPO ₄ ·7H ₂ O (mg) 7.745 7.745 7.745 7.745 NaH ₂ PO ₄ ·2H ₂ O (mg) 3.295 3.295 3.295 3.295 surfactant Polysorbate 80 (%)(w/v) 4 Polyethylene glycol 40-stearate (%) (w/v) 7 Polyoxyethylene-35 castor oil (%) (w/v) 5 Polysorbate(%)(w/v) + Polyoxyethylene-35 Castor Oil(%)(w/v) 4+5 pH adjuster NaOH/HCl (pH 7.0) Appropriate amount Appropriate amount Appropriate amount Appropriate amount Total volume (mL) 5 5 5 5

(2) Results of compatibility assessment

The compatibility of the compositions containing each surfactant was evaluated, and the results are shown in Table 4. Compositions 2A to 2C were confirmed to be suitable because of their clear and transparent appearance. Composition 2D was confirmed to be suitable as it had a uniform milky appearance (see Table 1). In compositions 2A, 2B, and 2C, the compatibility of the compound of formula 1a can be ensured.

Results of compatibility assessment

Table 4 Composition 2A 2B 2C 2D surfactant Tween 80 (polysorbate 80) Polyethylene glycol 40-stearate Kolliphor EL (polyoxyethylene-35 castor oil) Tween 80 + Kolliphor EL (Polysorbate 80 + Polyoxyethylene-35 Castor Oil) Concentration of active pharmaceutical ingredient (compound of formula 1a) (mg/mL) 2.0 3.0 2.0 4.0 Appearance initial suitable suitable suitable suitable Refrigerate for 1 day suitable suitable suitable suitable 3 days at 60˚C suitable suitable suitable Not suitable (color change) quantity(%) initial 89.0 97.7 89.5 77.7 Refrigerate for 1 day 88.5 89.0 93.0 79.3 3 days at 60˚C 100.5 99.3 114.0 120 pH initial 6.8 6.9 6.9 6.9 Refrigerate for 1 day 7.0 7.0 7.0 7.2 3 days at 60˚C 6.6 6.7 6.5 7.15

As shown in Table 4, Composition 2D used a combination of polysorbate 80 and polyoxyethylene-35 castor oil as the surfactant, which was confirmed to have an inappropriate appearance (due to color change).

In summary, it is confirmed that polysorbate 80, polyethylene glycol 40-stearate, or polyoxyethylene-35 castor oil (Kolliphor EL) is used as the surfactant (such as compositions 2A, 2B, and 2C) , ensuring the compatibility of the compound of formula 1a.

3) Prepare composition 3 and evaluate the compatibility of each surfactant

(1) Preparation of composition 3 (compositions 3A to 3C)

Compositions containing the compound of formula 1a were prepared using surfactants of the same concentration but different compositions, as shown in Table 5.

Composition table for surfactant screening research (2)

table 5 Element Material name Composition 3A 3B 3C Active pharmaceutical ingredient (mg) Compounds of formula 1a 25 25 25 Buffer (mg) Sodium citrate dihydrate 24.1 24.1 24.1 citric acid 3.47 3.47 3.47 Surfactant(%)(w/v) Kolliphor ELP 5.0 Tween 80 5.0 Ethylene glycol 40-stearate 5.0 Isotonicity agent(%)(w/v) NaCl 0.75 0.75 0.75 pH adjuster NaOH or HCl Appropriate amount Appropriate amount Appropriate amount Total volume (mL) 10 10 10

(2) Results of compatibility assessment

The compatibility of the compositions containing each surfactant was then evaluated, and the results are shown in Table 6.

Results of compatibility assessment (after 10 days of storage at 60˚C)

Table 6 Composition 3A 3B 3C Appearance suitable suitable A small increase in foreign matter quantity(%) initial 97.55 ± 0.52 93.52 ± 2.46 88.84 ± 3.60 10 days at 60˚C 95.93 ± 0.76 93.07 ± 0.22 90.98 ± 0.80 pH initial 6.97 7.00 6.98 10 days at 60˚C 6.92 6.55 6.68

As shown in Table 6, the pH value of composition 3B using Tween 80 as the surfactant changed. In addition, composition 3C using polyethylene glycol 40-stearate as the surfactant produced foreign matter in appearance and changed the pH value. The appearance, amount, and pH value of composition 3A using Kollinphor ELP as the surfactant were all suitable.

(3) Impurity evaluation results

The impurities in the compositions containing each surfactant were evaluated and the results are shown in Table 7.

Impurity evaluation results (10 days at 60˚C)

Table 7 relative residence time active pharmaceutical ingredients Composition 3A 3B 3C 0.22 0±0 0±0 0±0 0±0 0.24 0±0 0.01±0 0.03±0 0±0 0.26 0±0 0±0 0±0 0±0 0.28 0±0 0±0 0±0 0.01±0.01 0.31 0±0 0±0 0±0 0±0 0.41 0±0 0.1±0 0.07±0 0.07±0 0.51 0±0 0±0 0±0 0±0 0.6 0±0 0±0 0±0 0±0 0.63 0±0 0±0 0±0 0±0 0.66 0±0 0±0 0±0 0±0 0.74 0±0 0±0 0±0 0±0 0.79 0±0 0±0 0±0 0±0 0.87 0.03±0.01 0.04±0 0.04±0.02 0.03±0.02 1 100±0 100±0 100±0 100±0 1.26 0±0 0±0 0±0 0±0 1.38 0.03±0.02 0.03±0.01 0.03±0 0±0 total impurities 0.06±0.02 0.18±0.01 0.18±0.02 0.11±0.04

Evaluate the stability of the composition containing each surfactant under harsh conditions, and summarize the evaluation results of appearance, amount, pH value, and impurities to confirm that the composition using Kolliphor ELP as the surfactant has the best compatibility.

4) Prepare composition 4 and evaluate the compatibility of each buffer

(1) Preparation of composition 4 (compositions 4A to 4D)

The composition of the buffer was changed to prepare a composition containing the compound of Formula 1a, as shown in Table 8. The amount of each buffer can be set for evaluation comparison where the pH is 7.0, the drug concentration is 2.5 mg/mL, and the buffer concentration is 10 mM.

Buffer screening study composition table

Table 8 Element Material name Composition 4A 4B 4C 4D Active pharmaceutical ingredient (mg) Compounds of formula 1a 17.5 17.5 17.5 17.5 Buffer (mg) Na ₂ HPO ₄ ·7H ₂ O (mg) 10.84 NaH ₂ PO ₄ ·2H ₂ O (mg) 4.61 trihydroxymethylaminomethane 8.49 Sodium citrate dihydrate 20.24 citric acid 2.91 Surfactant (mg) Kolliphor ELP 350 350 350 350 pH adjuster NaOH or HCl Appropriate amount Appropriate amount Appropriate amount Appropriate amount Total volume (mL) 7.0 7.0 7.0 7.0

(2) Compatibility evaluation results

After 10 days of storage at 60˚C, the composition compatibility of each buffer was evaluated and the results are shown in Table 9.

Table 9 Composition 4A 4B 4C 4D Buffer deionized water Phosphate (Na ₂ HPO ₄ ·7H ₂ O NaH ₂ PO ₄ ·2H ₂ O trihydroxymethylaminomethane Citrate (sodium citrate 2H ₂ O, citric acid) Target concentration (mg/mL) 2.5 2.5 2.5 2.5 Appearance suitable suitable suitable suitable quantity(%) initial 96.2 97.3 101.3 94.4 10 days at 60˚C 95.4 95.1 99.9 93.9 pH initial 7.01 7.21 7.14 7.09 10 days at 60˚C 3.55 6.47 4.62 6.98 Particle size (nm) (polymer dispersion index) initial 11.98 (0.062) 12.07 (0.037) 12.05 (0.055) 12.12 (0.044) 10 days at 60˚C 12.03 (0.023) 12.1 (0.027) 11.92 (0.024) 11.95 (0.024)

As shown in Table 9, it was confirmed that the appearance and similar properties of the composition were suitable regardless of the type of buffer. In particular, the pH change of composition 4D using citric acid as a buffer was significantly reduced.

(3) Impurity evaluation results

The results of the evaluation of impurities in the composition for each buffer are shown in Table 10.

Impurity assessment results (10 days at 60°C)

Table 10 relative residence time active pharmaceutical ingredient Composition 4A 4B 4C 4D 0.22 0±0 0.04±0 0.03±0 0.02±0 0.01±0 0.24 0.08±0 0.06±0 0.07±0 0.03±0 0.01±0 0.26 0±0 0.05±0 0.05±0 0.03±0 0±0 0.28 0±0 0±0 0±0 0.02±0 0±0 0.31 0±0 0.07±0 0.07±0 0.03±0 0±0 0.33 0±0 0.05±0 0.02±0 0.03±0.01 0±0 0.34 0±0 0±0 0.04±0 0.03±0.01 0±0 0.40 0.52±0 0.67±0 0.69±0 0.57±0 0.57±0 0.50 0±0 0.08±0.08 0.02±0 0.01±0 0±0 0.54 0±0 0.01±0 0±0 0±0 0±0 0.57 0±0 0.02±0 0±0 0±0 0±0 0.60 0±0 0.02±0.01 0±0 0±0 0±0 0.63 0±0 0.06±0.01 0.07±0 0.05±0 0.08±0 0.66 0±0 0.07±0.01 0.09±0 0.06±0.01 0.08±0 0.70 0±0 0.01±0 0±0 0±0 0±0 0.74 0±0 0.08±0 0.1±0 0.05±0 0±0 0.79 0±0 0.05±0 0.06±0 0.03±0 0±0 0.85 0.01±0 0.01±0 0.02±0 0±0 0.03±0 0.95 0±0 0.05±0 0.13±0 0.03±0 0±0 1 100±0 100±0 100±0 100±0 100±0 1.25 0.11±0 0.1±0.01 0.09±0 0.1±0 0.1±0 1.36 0.09±0.01 0.22±0.16 0.06±0 0.09±0.02 0.05±0.01 total impurities 0.77±0.04 1.75±0.24 1.65±0.05 1.25±0.03 0.86±0.03

As shown in Table 10, in the composition (4D) using citric acid as the buffering agent, the impurities produced were significantly reduced.

Each buffer was evaluated for its stability under harsh conditions. Summarizing the evaluation results of appearance, quantity, pH, particle size, and impurities, it was confirmed that the composition using citric acid as the buffer agent has the best compatibility.

5) Prepare the composition and evaluate the compatibility of each isotonic agent

(1) Preparation of composition 5 (compositions 5A to 5F)

The composition of the isotonic agent was changed to prepare a composition containing the compound of formula 1a, as shown in Table 11. The pH was fixed at 7.0, the drug concentration was fixed at 2.5 mg/mL, and a phosphate buffer solution of 10 mM was used for comparative evaluation.

Composition table for isotonic agent screening studies

Table 11 Element Material name Composition 5A 5B 5C 5D 5E 5F Active pharmaceutical ingredient (mg) Compounds of formula 1a 17.5 17.5 17.5 17.5 17.5 17.5 Buffer(mg) Na ₂ HPO ₄ ·7H ₂ O 10.843 10.843 10.843 10.843 10.843 10.843 NaH ₂ PO ₄ ·2H ₂ O 4.613 4.613 4.613 4.613 4.613 4.613 Surfactant(%)(w/v) Kolliphor ELP 5.0 5.0 5.0 5.0 5.0 5.0 Isotonicity agent(%)(w/v) NaCl 0.75 - - - - - glycerin 2 - - - - propylene glycol - - 2 - - - Polyethylene glycol 400 - - - 7 - - Mannitol - - - - 5 - Sorbitol - - - - 5 pH adjuster NaO or HCl Appropriate amount Appropriate amount Appropriate amount Appropriate amount Appropriate amount Appropriate amount Total volume (mL) 7.0 7.0 7.0 7.0 7.0 7.0

(2) Results of compatibility assessment

After 10 days of storage at 60˚C, the compatibility of the compositions containing each isotonicity agent was evaluated and the results are shown in Table 12.

Table 12 Composition 5A 5B 5C 5D 5E 5F Isotonic agent NaCl glycerin propylene glycol Polyethylene glycol 400 Mannitol Sorbitol Appearance suitable suitable suitable suitable suitable suitable quantity(%) initial 99.6 96.1 98.8 95.2 100.5 99.8 10 days at 60˚C 95.6 92.4 94.8 92.1 94.5 96.2 pH initial 6.92 7.18 7.23 7.05 7.07 7.10 10 days at 60˚C 6.75 6.62 6.62 5.84 6.46 6.32 Particle size (nm) (polymer dispersion index) initial 12.12 (0.036) 12.67 (0.024) 12.7 (0.025) 16.49 (0.046) 13.79 (0.047) 13.80 (0.046) 10 days at 60˚C 12.07 (0.016) 12.67 (0.037) 12.73 (0.029) 16.27 (0.046) 13.62 (0.041) 13.58 (0.034)

As shown in Table 12, it was confirmed that the appearance, particle size, or similar properties of the composition were suitable regardless of the type of isotonic agent. Specifically, in compound 5A using NaCl as the isotonic agent, the pH change was significantly reduced.

(3) Results of impurity assessment

The impurities in the compositions containing each renal agent were evaluated and the results are shown in Table 13.

Impurity evaluation results (10 days at 60˚C)

Table 13 relative residence time Active ingredients Composition 5A 5B 5C 5D 5E 5F 0.22 0±0 0±0 0.03±0 0.03±0 0.03±0 0.03±0 0.04±0 0.24 0.08±0 0.01±0 0.06±0 0.03±0.04 0.06±0 0.06±0.01 0.08±0 0.26 0±0 0±0 0.04±0 0.04±0 0.03±0 0.04±0.01 0.05±0 0.28 0±0 0±0 0.01±0.02 0±0 0±0 0±0 0±0 0.31 0±0 0±0 0.05±0.01 0.06±0 0.05±0 0.06±0.01 0.07±0 0.33 0±0 0±0 0.02±0 0.02±0 0.02±0 0.02±0 0.02±0 0.34 0±0 0±0 0.03±0 0.03±0 0.02±0 0.03±0.01 0.03±0 0.40 0.52±0 0.58±0 0.63±0 0.71±0 0.63±0 0.7±0 0.67±0 0.50 0±0 0±0 0.02±0 0.02±0 0.02±0 0.02±0 0.02±0 0.54 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0.57 0±0 0±0 0±0 0.01±0 0±0 0±0 0±0 0.60 0±0 0±0 0.01±0 0±0 0.01±0 0±0 0.01±0 0.63 0±0 0±0 0.03±0.01 0.06±0 0.07±0 0.05±0 0.08±0 0.66 0±0 0±0 0.06±0.01 0.1±0.03 0.11±0.03 0.09±0.02 0.11±0 0.70 0±0 0±0 0±0 0±0 0.01±0 0±0 0±0 0.74 0±0 0.01±0 0.07±0 0.09±0 0.07±0 0.08±0 0.1±0 0.79 0±0 0.02±0 0.05±0 0.06±0 0.03±0 0.05±0 0.05±0 0.85 0.01±0 0.02±0.01 0.01±0 0.01±0 0.01±0.01 0.01±0 0.01±0 0.95 0±0 0±0 0.09±0 0.11±0 0.09±0 0.1±0 0.12±0 1.00 100±0 100±0 100±0 100±0 100±0 100±0 100±0 1.25 0.11±0 0.1±0 0.08±0.01 0.1±0 0.09±0.02 0.1±0.01 0.09±0.01 1.36 0.09±0.01 0.07±0.01 0.05±0 0.07±0.02 0.25±0.03 0.16±0.2 0.19±0.08 total impurities 0.77±0.04 0.85±0.04 1.43±0.01 1.62±0.01 1.68±0.07 1.68±0.18 1.83±0.09

As shown in Table 13, in the composition 5A using NaCl as the isotonic agent, the amount of impurities generated is significantly reduced.

The stability of the composition containing each isotonic agent can be evaluated under harsh conditions. By summarizing the evaluation results of appearance, amount, pH, particle size, and impurities, it can be confirmed that the composition using NaCl as the isotonic agent has the best compatibility.

6) Prepare composition 6 and evaluate the compatibility of each thickener

(1) Preparation of composition 6 (compositions 6A to 6F)

The composition of the thickener was changed to prepare a composition containing the compound of Formula 1a, as shown in Table 14. The pH was fixed at 7.0, the drug concentration was fixed at 2.5 mg/mL, and a phosphate buffer solution of 10 mM was used for comparative evaluation.

Composition table for thickener screening studies

Table 14 Element Material name Composition 6A 6B 6C 6D 6E 6F Active pharmaceutical ingredient (mg) Compounds of formula 1a 17.5 17.5 17.5 17.5 17.5 17.5 Buffer (mg) Na ₂ HPO ₄ ·7H ₂ O 10.843 10.843 10.843 10.843 10.843 10.843 NaH ₂ PO ₄ ·2H ₂ O 4.61 4.61 4.61 4.61 4.61 4.61 Surfactant(%)(w/v) Kolliphor ELP 5.0 5.0 5.0 5.0 5.0 5.0 Isotonicity agent(%)(w/v) NaCl 0.75 0.75 0.75 0.75 0.75 0.75 Thickener(%) EG-30P (polyvinyl alcohol) 1.4 Pharmacoat 606 (HPMC) 0.6 Na.CMC 0.2 Natrosol 250L Pharm (hydroxyethyl cellulose) 0.35 Kollidon 25 2 pH adjuster NaOH or HCl Appropriate amount Appropriate amount Appropriate amount Appropriate amount Appropriate amount Appropriate amount Total volume (mL) 7 7 7 7 7 7

The thickeners used in the above table are as follows: polyvinyl alcohol (GOHSENOL EG-30P, Mitsubishi Chemical), hydroxypropyl methylcellulose (HPMC, Pharmacoat 606, Shin-Etsu), sodium carboxymethylcellulose (Na. CMC), hydroxyethylcellulose (Natrosol 250L Pharm, Ashland), and polyvinylpyrrolidone (PVP, Kollidon 25, BASF).

(2) Results of compatibility assessment

After 10 days of storage at 60˚C, the compatibility of the compositions containing each thickener was evaluated as shown in Table 15.

Table 15 Composition 6A 6B 6C 6D 6E 6F Appearance suitable suitable suitable suitable Foreign body Foreign body quantity(%) initial 89.80 ± 1.30 93.22 ± 6.62 86.70 ± 0.33 86.17 ± 0.41 94.96 ± 2.89 93.59 ± 2.30 10 days at 60˚C 89.63 ± 1.59 97.45 ± 2.02 89.21 ± 1.43 88.86 ± 1.92 93.75 ± 0.62 91.62 ± 0.88 pH initial 6.95 6.99 7.07 7.03 7.00 6.94 10 days at 60˚C 7.06 6.01 7.13 6.89 7.04 6.90 Particle size (nm) (polymer dispersion index) initial 12.01 (0.026) 29.37 (0.488) 20.33 (0.261) 19 (0.369) 16.91 (0.237) 14.66 (0.151) 10 days at 60˚C - 31.59 (0.513) 20.61 (0.255) 19.99 (0.387) 16.54 (0.228) 14.96 (0.152)

As shown in Table 15, it was confirmed that compositions 6A to 6D were suitable. Specifically, composition 6C using hydroxypropyl methylcellulose (HPMC) as the thickener or compound 6D using sodium carboxymethylcellulose (Na.CMC) as the thickener has better stability.

7) Prepare composition 7 and evaluate the compatibility of each antioxidant

(1) Preparation of composition 7 (compositions 7A and 7B)

The composition of the antioxidant was changed to prepare a composition containing the compound of Formula 1a, as shown in Table 16. The pH was fixed at 7.0, the drug concentration was fixed at 2.5 mg/mL, and a phosphate buffer solution of 10 mM was used for comparative evaluation.

Composition table for antioxidant screening studies

Table 16 Element Material name Composition 7A 7B 7C 7D Active pharmaceutical ingredient (mg) Compounds of formula 1a 25 25 25 25 Buffer (mg) Sodium citrate dihydrate 24.1 24.1 24.1 24.1 citric acid 3.47 3.47 3.47 3.47 Surfactant(%)(w/v) Kolliphor ELP 5 5 5 5 Isotonicity agent(%)(w/v) NaCl 0.75 0.75 0.75 0.75 Antioxidant(%)(w/v) Disodium ethylenediaminetetraacetate 0.1 sodium sulfite 0.1 sodium metabisulfite 0.1 pH adjuster NaOH or HCl Appropriate amount Appropriate amount Appropriate amount Appropriate amount Total volume (mL) 10 10 10 10

(2) Results of compatibility evaluation (after 10 days of storage at 60˚C)

Table 17 Composition 7A 7B 7C 7D Appearance (initial, 10 days) suitable suitable suitable suitable quantity(%) initial 89.57 ± 2.95 90.70 ± 0.20 89.56 ± 0.11 87.77 ± 1.31 10 days at 60˚C 92.66 ± 1.49 92.88 ± 3.47 93.60 ± 0.11 87.04 ± 2.94 pH initial 7.00 6.95 6.97 6.95 10 days at 60˚C 6.93 6.33 6.61 6.25 Particle size (nm) (polymer dispersion index) initial 11.92 (0.015) 11.93 (0.025) 11.97 (0.036) 11.88 (0.027)

As shown in Table 17, composition 7A without antioxidants can significantly reduce pH changes.

(3) Impurity assessment results

The impurities in the compositions containing each antioxidant were evaluated and the results are shown in Table 18.

Results of impurity assessment (10 days at 60˚C)

Table 18 relative residence time active pharmaceutical ingredient Composition 7A 7B 7C 7D 0.22 0±0 0±0 0.03±0 0.03±0 0.06±0 0.24 0±0 0.01±0 0.04±0 0.04±0 0.06±0 0.26 0±0 0±0 0.05±0.01 0±0 0±0 0.28 0±0 0±0 0±0 0.01±0 0.05±0 0.31 0±0 0±0 0.02±0.02 0.02±0 0.06±0 0.41 0±0 0.09±0 0.08±0 0.1±0 0.14±0 0.51 0±0 0±0 0.02±0 0±0 0.01±0 0.6 0±0 0±0 0.02±0 0.05±0.01 0.02±0.01 0.63 0±0 0±0 0.03±0.01 0.03±0 0.02±0.01 0.66 0±0 0±0 0.02±0 0.01±0.01 0.02±0 0.74 0±0 0±0 0.04±0 0.02±0 0.03±0 0.79 0±0 0±0 0.05±0 0.04±0 0.06±0 0.87 0.03±0.01 0.04±0.01 0.03±0.01 0.03±0.01 0.03±0.01 1 100±0 100±0 100±0 100±0 100±0 1.26 0±0 0±0 0±0 0±0 0.1±0.02 1.38 0.03±0.02 0.03±0 0.05±0.01 0.02±0.01 0.03±0.01 total impurities 0.06±0.02 0.18±0.01 0.48±0.05 0.40±0.03 0.68±0.02

As shown in Table 18, composition 7A without antioxidants can significantly reduce the generation of impurities.

Evaluate the stability of the composition containing each buffer under harsh conditions, and summarize the evaluation results of appearance, amount, pH, particle size, and impurities to confirm that the composition without antioxidants has the best compatibility.

8) Preparation of composition 8 (compositions 8A and 8B) and stability evaluation (1)

(1) Preparation of composition 8 (compositions 8A and 8B)

The compounds of Formula 1a and 1b were prepared for use in eye drops containing the composition shown in Table 19, and their physicochemical stability under various conditions was evaluated.

Table 19 mixed purpose Material name Composition 8A Composition 8B active pharmaceutical ingredients Compound of formula 1a (mg) 10 Compound of formula 1b (mg) 0.5 Buffer Na ₂ HPO ₄ ·7H ₂ O (mg) 15.49 15.49 NaH ₂ PO ₄ ·2H ₂ O (mg) 6.59 6.59 surfactant Kolliphor EL (μl) 500 500 Isotonic agent NaCl (mg) 20 20 Propylene glycol (μl) 120 120 Preservatives Benzalkonium chloride (mg) 1 1 pH adjuster 1N NaOH or HCl (pH 7.0) Appropriate amount Appropriate amount Solvent water for injection Appropriate amount Appropriate amount total volume - 10mL 10mL

(2) Experimental results under refrigeration and 60˚C

Compositions 8A and 8B were subjected to storage tests under refrigeration and 60˚C conditions, and the results are shown in Table 20.

Table 20 Evaluation items/storage conditions Composition 8A Composition 8B Appearance initial suitable suitable Refrigerate for 5 days suitable suitable 5 days at 60°C suitable suitable 10 days at 60˚C suitable suitable quantity(%) initial 95.3 96.5 Refrigerate for 5 days 96.9 97.2 5 days at 60°C 98.8 96.0 10 days at 60˚C 94.9 95.0 pH initial 6.88 6.85 Refrigerate for 5 days 6.94 6.95 5 days at 60°C 6.79 6.88 10 days at 60˚C 6.42 6.80 Particle size (nm) (polymer dispersion index) initial 12.65 (0.083) 12.25 (0.014) Refrigerate for 5 days 12.7 (0.103) 12.22 (0.028) 60°C, 5 days 12.47 (0.048) 12.26 (0.028) 10 days at 60°C 12.64 (0.052) 12.52 (0.037)

As shown in Table 20, the physical and chemical stability of the composition under refrigeration and 60˚C conditions was confirmed.

(3) Stability evaluation results under centrifugation/freeze-thaw conditions

Compositions 8A and 8B can be tested for stability under centrifugation and freeze-thaw conditions, and the results are shown in Table 21.

Table 21 Project/storage conditions Composition 8A Composition 8B Appearance initial suitable suitable After centrifugation suitable suitable Freeze-after thawing suitable suitable Particle size (nm) (polymer dispersion index) initial 12.65 (0.083) 12.25 (0.014) After centrifugation 12.83 (0.081) 12.31 (0.026) Freeze-after thawing 12.83 (0.076) 12.49 (0.025)

As shown in Table 21, the physical and chemical stability of the composition under centrifugation and freezing-thawing conditions was confirmed.

(4) Evaluation of results of filtration sterilization

Compositions 8A and 8B were evaluated for filtration sterilization, and the results are shown in Table 22.

Table 22 Evaluation items/storage conditions Composition 8A Composition 8B Appearance Measure immediately after manufacturing suitable suitable After filtration of mixed cellulose esters suitable suitable quantity(%) Measure immediately after manufacturing 95.5 100.95 After filtration of mixed cellulose esters 94.2 99.9 Particle size (nm) (polymer dispersion index) Measure immediately after manufacturing 12.58 (0.048) 12.29 (0.025) After filtration of mixed cellulose esters 12.29 (0.025) 12.37 (0.02)

Summarizing the above overall results, it can be confirmed that the eye drops of composition 8 are physicochemically stable at 60˚C, refrigeration, centrifugation, or freeze-thaw conditions, and on this basis, it can be used in the formulation for preclinical evaluation. .

9) Preparation of composition 9 (compositions 9A, 9B, and 9C) and stability evaluation (2)

(1) Preparation of composition 9 (compositions 9A, 9B, and 9C)

As shown in Table 23, compounds of formulas 1a, 1b, and 1c were prepared for eye drops of composition 9 (such as composition 9A (concentration: 1.0 mg/mL), composition 9B (concentration: 0.05 mg/mL) ), and composition 9C (concentration: 0.05 mg/mL)).

Table 23 mixed purpose Material name Composition 9A Composition 9B Composition 9C active pharmaceutical ingredients Compound of formula 1a (mg) 15 Compound of formula 1b (mg) 0.75 Compound of formula 1c (mg) 7.5 Buffer Na ₂ HPO ₄ ·7H ₂ O (mg) 23.235 23.235 23.235 NaH ₂ PO ₄ ·2H ₂ O (mg) 9.885 9.885 9.885 surfactant Kolliphor EL (μl) 750 750 750 Isotonic agent NaCl (mg) 30 30 30 Polyethylene glycol (μl) 165 165 165 Preservatives Benzalkonium chloride(mg) 1.5 1.5 1.5 pH adjuster 1N NaOH or HCl (pH 7.0) Appropriate amount Appropriate amount Appropriate amount Solvent water for injection Appropriate amount Appropriate amount Appropriate amount total volume - 15mL 15mL 15mL

(2)Quality control results

The quality control results of compositions 9A, 9B, and 9C are shown in Table 24.

Table 24 Assessment project standard Composition 9A Composition 9B Composition 9C Bottle 1 Bottle 2 Bottle 3 Bottle 1 Bottle 2 Bottle 3 Bottle 1 Bottle 2 Bottle 3 Appearance clear and transparent suitable suitable suitable suitable suitable suitable suitable suitable suitable quantity(%) 80% to 120% of labeled amount 96.5 99.5 95.2 95.2 94.2 97.1 107.7 111.8 108.8 pH 7.0±0.5 7.05 7.04 7.05 7.10 7.11 7.09 7.06 7.05 7.05 Osmotic pressure (mOsm/kg) 250 mOsm/kg to 343 mOsm/kg 266 265 265 281 271 271 269 267 265 Particle size (nm) (polymer dispersion index) ＜100nm 12.49 (0.039) 12.62 (0.028) 12.44 (0.026) 12.73 (0.039) 12.58 (0.021) 12.47 (0.021) 12.49 (0.028) 12.74 (0.054) 12.69 (0.054)

As shown in Table 24, it was confirmed that composition 9 (such as compositions 9A, 9B, and 9C) was suitable for all items of the preclinical stimulation test.

10) Preparation of composition 10 and stability evaluation (3)

(1) Preparation of composition 10

For preclinical stimulation testing, a formulation of composition 10 (concentration: 0.2 mg/mL) was prepared as shown in Table 25.

Table 25 mixed purpose Material name quantity active pharmaceutical ingredient Compound of formula 1a (mg) 3 Buffer Na ₂ HPO ₄ ·7H ₂ O (mg) 23.235 NaH ₂ PO ₄ ·2H ₂ O (mg) 9.885 surfactant Kolliphor EL (μl) 750 Isotonic agent NaCl (mg) 30 Polyethylene glycol (μl) 165 Preservatives Benzalkonium chloride (mg) 1.5 pH adjuster 1N NaOH or HCl (pH 7.0) Appropriate amount Solvent water for injection Appropriate amount total volume - 15mL

(2)Quality control results

The quality control results of Composition 10 are shown in Table 26.

Table 26 Assessment project standard Experimental results Bottle 1 Bottle 2 Bottle 3 Appearance clear and transparent suitable suitable suitable quantity(%) 80% to 120% of display volume 97.2 93.0 97.9 pH 7.0±0.5 7.05 7.03 7.05 Osmotic pressure (mOsm/kg) 250 mOsm/kg to 343 mOsm/kg 264 264 265 Particle size (nm) (polymer dispersion index) ＜100nm 12.49 (0.028) 12.44 (0.038) 12.62 (0.055)

As shown in Table 26, it can be confirmed that the formula of composition 10 is suitable for all items of the preclinical stimulation test.

11) Prepare composition 11 and evaluate the stability under accelerated conditions, and evaluate the compatibility of LDPE containers.

(1) Preparation of composition 11 (compositions 11A to 11C)

The compositions of the preparation compositions 11A to 11C are shown in Table 27.

Table 27 Element Material name Composition 11A 11B 11C Active pharmaceutical ingredient (mg) Compounds of formula 1a 25 25 25 Buffer (mg) Sodium citrate dihydrate 24.1 24.1 24.1 citric acid 3.5 3.5 3.5 Surfactant(mg) Kolliphor ELP 500.0 500.0 500.0 Isotonic agent(mg) NaCl 75.0 75.0 75.0 Thickener(mg) Pharmacoat 606 (HPMC) 60 10 0 pH adjuster NaOH or HCl Appropriate amount Appropriate amount Appropriate amount Total volume (mL) 10 10 10

(2) Stability evaluation results of Composition 11A

The results of the stability evaluation and impurity evaluation of Composition 11A are shown in Tables 28 to 30.

Stability evaluation results of composition 11A

Table 28 initial 5 weeks at room temperature (glass) 3 weeks at 40˚C (LDPE) Stress Stability Assessment (10 days at 60˚C) LDPE Glass Appearance small particles observed small particles observed small particles observed small particles observed small particles observed quantity(%) 101.85 ± 1.58 100.59 ± 1.85 102.47 ± 0.23 101.21 ± 1.06 98.21 ± 1.20 Impurities 0.0±0.0 0.02±0.00 0.1±0.0 0.46 ± 0.00 0.3 ± 0.01 pH 7.01 - - - 6.75 Osmotic pressure (mOsm/kg) 304 - - 323 293 Particle size (nm) (polymer dispersion index) 12.01 (0.026) - - 17.55 (0.181) 13.02 (0.073)

Impurity evaluation results of composition 11A

Table 29 relative residence time standard 5 weeks at room temperature (glass) 3 weeks at 40˚C (LDPE) 0.24 0.0±0.0 0.01±0.0 0.01±0.0 0.41 0.0±0.0 0.01±0.0 0.09±0.0 1 100±0.0 100±0.0 100±0.0 total impurities 0.0±0.0 0.02±0.0 0.1±0.0

Impurity evaluation results of Composition 11A (stability evaluation under harsh conditions such as 60˚C for 10 days)

Table 30 relative residence time standard LDPE Glass 0.23 0.0±0.0 0.01±0.0 0.0±0.0 0.39 0.0±0.0 0.45±0.0 0.3±0.0 1 100±0.0 100±0.0 100±0.0 total impurities 0.0±0.0 0.46±0.0 0.3±0.0

(3) Stability evaluation results of composition 11B (LDPE)

The results of the stability evaluation and impurity evaluation of Composition 11B are shown in Tables 31 and 32.

Stability evaluation results of composition 11B

Table 31 initial 12 weeks at room temperature 16 weeks at 40˚C Appearance clear and transparent clear and transparent clear and transparent quantity(%) 98.99 ± 0.93 101.20 ± 0.76 112.37 ± 0.47 Impurities 0.0±0.0 0.0±0.0 0.07±0.0 pH 7.06 6.80 6.93 Osmotic pressure (mOsm/kg) 288 289 325 Particle size (nm) (polymer dispersion index) 12.01 (0.026) - -

Impurity evaluation results of composition 11B

Table 32 relative residence time standard 12 weeks at room temperature 16 weeks at 40˚C 0.37 0.0±0.0 0.0±0.0 0.07±0.0 1 100±0.0 100±0.0 100±0.0 total impurities 0.0±0.0 0.0±0.0 0.07±0.0

(4) Stability evaluation results of composition 11C (glass bottle)

The results of the stability evaluation and impurity evaluation of composition 11C are shown in Tables 33 and 34.

Stability evaluation results of composition 11C

Table 33 initial 19 weeks at room temperature 27 weeks at room temperature Appearance clear and transparent clear and transparent clear and transparent quantity(%) 89.63 ± 1.59 90.50 ± 0.94 86.86 ± 0.28 Impurities 0.0±0.0 0.0±0.0 0.0±0.0 pH 6.95 7.01 7.1 Osmotic pressure (mOsm/kg) 288 295 296 Particle size (nm) (polymer dispersion index) 12.01 (0.026) - -

Impurity evaluation results of composition 11C

Table 34 relative residence time standard 19 weeks at room temperature 27 weeks at room temperature 1 100±0.0 100±0.0 100±0.0 total impurities 0.0±0.0 0.0±0.0 0.0±0.0

As shown in the above results, it was confirmed that the prepared eye drops have physical and chemical stability in both LDPE containers and glass containers. Furthermore, reducing the thickening dose improves the physicochemical stability of the eye drops.

4. Preparation of eye drop formula (composition 12) and stability evaluation under harsh conditions

(1) As shown in Table 35 for preparing composition 12, prepare the compounds of formulas 1b and 1c as eye drop formulations of composition 12 (such as compositions 12A to 12H).

Composition of composition 12 (compositions 12A to 12H)

Table 35 mixed purpose Material name Composition 12A 12B 12C 12D 12E 12F 12G 12H Active pharmaceutical ingredient (mg) Compounds of formula 1b 3.5 3.5 3.5 3.5 Compounds of formula 1c 3.5 3.5 3.5 3.5 Surfactant(%)(w/v) Kolliphor EL 5 5 5 5 5 5 5 5 Isotonicity agent(%)(w/v) NaCl 0.7 0.2 0.2 0.2 0.7 0.2 0.2 0.2 Co-solvent(%)(w/v) glycerin 1.5 1.5 propylene glycol 1 1 Polyethylene glycol 400 7 7 Preservative(%)(w/v) benzalkonium chloride 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 pH adjuster NaOH or HCl Appropriate amount Appropriate amount Appropriate amount Appropriate amount Appropriate amount Appropriate amount Appropriate amount Appropriate amount total amount 7.0mL 7.0mL 7.0mL 7.0mL 7.0mL 7.0mL 7.0mL 7.0mL

(2) Evaluate stability under harsh conditions (at 60˚C)

Among the evaluation results of stability under harsh conditions (60˚C), the evaluation results of compositions 12A to 12D are shown in Table 36, and the evaluation results of compositions 12E to 12H are shown in Table 37.

Stability evaluation results of eye drop formulations under harsh conditions (1): Compositions 12A to 12D (i.e. compounds of formula 1b)

Table 36 Composition 12A 12B (glycerin) 12C (propylene glycol) 12D (polyethylene glycol 400) Active pharmaceutical ingredient concentration (mg/mL) 0.05 0.05 0.05 0.05 Appearance initial suitable suitable suitable suitable Refrigerate for 5 days suitable suitable suitable suitable 10 days at 60˚C suitable suitable suitable suitable quantity(%) initial 95.9 97.5 96.5 104.4 Refrigerate for 5 days 95.6 96.5 97.2 102.4 5 days at 60˚C 94.8 94.1 96.0 99.7 pH initial 6.63 6.84 6.85 6.91 Refrigerate for 5 days 6.74 6.96 6.95 6.93 5 days at 60˚C 6.59 6.78 6.88 6.74 Particle size (nm) (polymer dispersion index) initial 12.05 (0.017) 12.41 (0.023) 12.25 (0.014) 16.09 (0.031) Refrigerate for 5 days 12.04 (0.022) 12.43 (0.02) 12.22 (0.028) 16.08 (0.039) 5 days at 60˚C 12.07 (0.03) 12.47 (0.024) 12.26 (0.028) 16.17 (0.041)

In this way, the stability of the amount, pH, and particle size of the compound of formula 1b can be ensured, and the formula of composition 12C (propylene glycol) is particularly confirmed to be the most stable.

Stability evaluation results of eye drop formulation (2) under harsh conditions: compositions 12E to 12H (i.e. compounds of formula 1c)

Table 37 Composition 12E 12F (glycerin) 12G (propylene glycol) 12H (polyethylene glycol 400) Active pharmaceutical ingredient concentration (mg/mL) 0.05 0.05 0.05 0.05 Appearance initial suitable suitable suitable suitable Refrigerate for 5 days suitable suitable suitable suitable 10 days at 60˚C suitable suitable suitable suitable quantity(%) initial 107.9 104.7 112.4 93.2 Refrigerate for 5 days 102.2 100.9 110.7 91.3 5 days at 60˚C 74.9 69.9 76.7 63.5 pH initial 6.35 6.54 6.47 6.62 Refrigerate for 5 days 6.42 6.68 6.63 6.79 5 days at 60˚C 6.34 6.52 6.46 6.43

It will be understood by those of ordinary skill in the art that the present invention can be implemented in modified forms without departing from the essential characteristics of the invention. The disclosed embodiments should therefore be considered illustrative rather than limiting. The scope of the present invention should be defined by the patent application scope rather than the above description, and differences equivalent to the scope should be included in the present invention.

without

without.

without.

Claims (22)

A pharmaceutical composition, including: a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active pharmaceutical ingredient; and selected from the group consisting of polysorbate 80, polyethylene glycol 40-stearate, and polyoxyethylene -35 castor oil, any surfactant combined with the above: Formula 1 Wherein, in formula 1, R ¹ and R ² are each independently selected from H, optionally substituted C ₁ -C ₁₀ alkyl, optionally substituted C ₁ -C ₁₀ alkenyl, optionally substituted aryl, Optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted monocyclic or bicyclic carbocyclic ring, and optionally substituted A monocyclic or bicyclic heterocycle, or R ¹ and R ² and the nitrogen atom to which R ¹ and R ² are attached are cyclically linked together to form an optionally substituted monocyclic or bicyclic heterocycle. The pharmaceutical composition of claim 1, wherein the compound represented by formula 1 is any one of the compounds represented by formulas 1a, 1b, and 1c: Formula 1a Formula 1b Formula 1c . The pharmaceutical composition of claim 1, wherein the surfactant is polyoxyethylene-35 castor oil. For example, the pharmaceutical composition of claim 1, wherein the content of the surfactant is less than or equal to 9% (w/v). The pharmaceutical composition of claim 1 further includes at least one additive selected from the group consisting of a buffer, an isotonicity agent, a thickener, a pH adjuster, a preservative, and an antioxidant. The pharmaceutical composition of claim 5, wherein the buffering agent is selected from the group consisting of phosphate, trishydroxymethylaminomethane, citrate, and any combination of the above. The pharmaceutical composition of claim 5, wherein the buffering agent is citrate. For example, the pharmaceutical composition of claim 5, wherein the isotonicity agent is selected from mannitol, sorbitol, sodium chloride, glycerin, polyethylene glycol, propylene glycol, and any combination of the above. For example, the pharmaceutical composition of claim 5, wherein the isotonicity agent is sodium chloride, propylene glycol, or a combination of the above. Such as the pharmaceutical composition of claim 5, wherein the thickener is selected from the group consisting of hydroxypropyl methylcellulose, polyvinyl alcohol, sodium carboxymethylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, and combinations of the above any of. The pharmaceutical composition of claim 5, wherein the thickener is hydroxypropyl methylcellulose or sodium carboxymethylcellulose. For example, the pharmaceutical composition of claim 5, wherein the pH adjusting agent is an acid or an alkali, which respectively adjusts the pH value of the pharmaceutical composition to 6.0 to 8.0. For example, the pharmaceutical composition of claim 5, wherein the preservative is selected from the group consisting of benzalkonium chloride, benzethonium chloride, phenyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, and the above. Any combination. Such as the pharmaceutical composition of claim 5, wherein the antioxidant is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), sodium sulfite, sodium bisulfite, sodium metabisulfite, and any combination of the above. For example, the pharmaceutical composition of claim 1, wherein the content of the active pharmaceutical ingredient is greater than or equal to 0.00001% (w/v) and less than or equal to 1% (w/v). For example, the pharmaceutical composition of claim 5, wherein the content of the buffer is about 0.2% (w/v) to 0.4% (w/v). For example, the pharmaceutical composition of claim 5, wherein the content of the isotonic agent is about 0% (w/v) to 1.0% (w/v). For example, the pharmaceutical composition of claim 5, wherein the content of the thickener is about 0% (w/v) to 2.0% (w/v). Such as the pharmaceutical composition of claim 1, wherein the pharmaceutical composition is used to prevent, improve, or treat eye-related diseases. An ophthalmic composition, including: a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active pharmaceutical ingredient; and a compound selected from the group consisting of polysorbate 80, polyethylene glycol 40-stearate, and polyoxyethylene Ethylene-35 castor oil, any surfactant combined with the above: Formula 1 Wherein, in Formula 1, R ¹ and R ² are each independently selected from H, optionally substituted C ₁ -C ₁₀ alkyl, optionally substituted C ₁ -C ₁₀ alkenyl, optionally substituted aryl, Optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted monocyclic or bicyclic carbocyclic ring, and optionally substituted A monocyclic or bicyclic heterocycle, or R ¹ and R ² and the nitrogen atom to which R ¹ and R ² are attached are cyclically linked together to form an optionally substituted monocyclic or bicyclic heterocycle. For example, the ophthalmic composition of claim 20, wherein the compound represented by Formula 1 is any one of the compounds represented by Formula 1a, 1b, and 1c: Formula 1a Formula 1b Formula 1c . Such as the ophthalmic composition of claim 20, wherein the form of the composition is an eye drop solution, ointment, gel, cream, lotion, emulsion, suspension, or spray.