WO2024078515A1

WO2024078515A1 - Ophthalmic pharmaceutical composition containing levamisole, preparation method therefor, and use thereof

Info

Publication number: WO2024078515A1
Application number: PCT/CN2023/123885
Authority: WO
Inventors: 刘祖国; 黄彩虹; 郑小香
Original assignee: 厦门上佳源生物科技有限公司
Priority date: 2022-10-12
Filing date: 2023-10-11
Publication date: 2024-04-18
Also published as: CN115518036A

Abstract

The present invention relates to an ophthalmic pharmaceutical composition containing levamisole, a preparation method therefor, and a use thereof, and in particular relates to an ophthalmic preparation. The ophthalmic preparation comprises levamisole or a pharmaceutically-acceptable salt thereof as an active ingredient and water, the concentration of levamisole or the pharmaceutically-acceptable salt thereof being 0.01%-10% (w/v). The ophthalmic preparation can promote tear secretion and increase the number of conjunctival goblet cells, can effectively improve dry eye syndrome, corneal epithelium injury and tear film stability of a patient, and can alleviate and/or improve dry eye diseases and/or treat dry eyes, including but not limited to ocular diseases such as dry eye-related ocular surface inflammation, meibomian gland dysfunction, lacrimal gland inflammation, fibrosis, conjunctivitis, etc.

Description

An ophthalmic pharmaceutical composition containing levamisole, and preparation method and application thereof

Technical Field

The present invention relates to the field of medicine, and in particular to an ophthalmic pharmaceutical composition comprising levamisole, and a preparation method and application thereof.

Background technique

Dry eye is the most common chronic ophthalmic disease that affects vision and quality of life, in addition to refractive error. Mild and moderate dry eye mainly affects the quality of life and work efficiency, while severe dry eye can cause visual impairment and even blindness. Epidemiological studies show that the global incidence of dry eye is about 5%-50%, and the incidence of dry eye in my country is even higher, reaching 21-30%. The number of patients with dry eye has accounted for more than 30% of the total number of patients in ophthalmology clinics in my country, becoming one of the largest groups of patients in ophthalmology clinics in my country. In recent years, due to changes in lifestyle, environment and other factors and the acceleration of population aging, the incidence of dry eye continues to increase, and the medical expenses of the whole society for dry eye will also increase year by year. In 2015, the prescription cost of artificial tears in hospitals in my country alone reached 2 billion yuan, and it has increased at a rate of about 30% per year, while the average annual cost of treating dry eye in the United States has reached 3.84 billion US dollars. As a chronic disease, dry eyes have a significant impact on patients' work efficiency and quality of life. The indirect cost of dry eyes in the United States is as high as 55.4 billion U.S. dollars each year. Considering the number of patients with dry eyes in my country, the indirect economic impact will be enormous.

There are many factors that affect the occurrence and development of dry eyes. Factors that induce dry eyes include aging, long-term wearing of corneal contact lenses, meibomian gland dysfunction, long-term use of video terminals, and other immune factors. It is currently believed that the main mechanism of dry eye is insufficient tear secretion or rapid tear evaporation caused by various reasons, which increases the tear osmotic pressure, thereby causing a series of damages to the ocular epithelium, including destruction of the corneal epithelial barrier function, decreased density of conjunctival goblet cells, reduced mucin expression, squamous metaplasia of the ocular epithelium, and ocular surface inflammation. Insufficient tear secretion is the most important cause of dry eyes. Tears are mainly secreted by the lacrimal glands, and conjunctival goblet cells and meibomian glands are also involved. The treatment of dry eyes includes drug therapy, physical therapy, and adjuvant therapy, among which drug therapy is the most common treatment method. Commonly used topical drugs in clinical practice mainly include artificial tears, glucocorticoids, nonsteroidal anti-inflammatory drugs, and immunosuppressants such as cyclosporine A (CsA). However, artificial tears are only a tear substitute and have no therapeutic effect themselves. Although glucocorticoids and CsA have good anti-inflammatory effects, long-term use has certain toxic side effects on the ocular surface.

Therefore, there is an urgent need to develop a drug for the treatment of dry eyes that can effectively promote tear secretion, repair corneal epithelial damage, and control ocular surface inflammation, and that can be used for a long time without obvious local and systemic toxic side effects.

Summary of the invention

Levamisole is a common anthelmintic in clinical practice. It can inhibit succinate dehydrogenase, a key enzyme in the glycolysis pathway of intestinal worms, thereby affecting the energy metabolism of intestinal worms, paralyzing the muscles of intestinal worms, and finally excreting them from the body. Recent studies have found that levamisole has a good immune regulation function and has good effects in the adjuvant treatment of infectious diseases, skin diseases, tumors and allergic diseases. It has also been reported that oral levamisole can assist in the treatment of infectious eye diseases such as infectious keratitis. However, at present, on the one hand, the application of levamisole in the treatment of dry eyes has not been reported. On the other hand, levamisole has pleiotropic effects. Oral levamisole can not only affect multiple tissues and organs throughout the body, but also due to the existence of the blood-brain barrier, oral levamisole cannot be delivered to the ocular surface in a timely and effective manner, and high doses are required to reach effective levels in the eye. However, high doses of levamisole are associated with nephrotoxicity.

Based on the above problems, the inventors of the present application have provided an ophthalmic preparation containing levamisole or a pharmaceutically acceptable salt thereof after a large number of experimental studies. The ophthalmic preparation, as a topical ophthalmic drug, can not only achieve an effective therapeutic concentration of levamisole on the ocular surface, but also reduce its effects and toxic side effects on the systemic system. The ophthalmic preparation can promote tear secretion, increase the number of conjunctival goblet cells, effectively improve patients' dry eye symptoms, corneal epithelial damage and tear film stability, and can alleviate and/or improve dry eye symptoms and/or treat dry eyes, including but not limited to dry eye-related ocular surface inflammation, meibomian gland dysfunction, lacrimal gland inflammation and fibrosis, conjunctivitis and other eye diseases.

To this end, in a first aspect of the present invention, an ophthalmic preparation is provided, comprising: an active ingredient levamisole or a pharmaceutically acceptable salt thereof, and water, wherein the concentration of the levamisole or a pharmaceutically acceptable salt thereof is 0.01%-10% (w/v), for example, the concentration is 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v), 0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v). (w/v), 0.1% (w/v), 0.15% (w/v), 0.2% (w/v), 0.25% (w/v), 0.3% (w/v), 0.35% (w/v), 0.4% (w/v), 0.45% (w/v), 0.5% (w/v), 0.55% (w/v), 0.6% (w/v), 0.65% (w/v), 0.7% (w/v), 0.75% (w/v), 0.8% (w/v), 0.85% (w/v), 0.9% (w/v), 0.95% %, 1% (w/v), 1.5% (w/v), 2% (w/v), 2.5% (w/v), 3% (w/v), 3.5% (w/v), 4% (w/v), 4.5% (w/v), 5% (w/v), 5.5% (w/v), 6% (w/v), 6.5% (w/v), 7% (w/v), 7.5% (w/v), 8% (w/v), 8.5% (w/v), 9% (w/v), 9.5% (w/v) or 10% (w/v), or 0.01% to 0.0 2% (w/v), 0.02%-0.05% (w/v), 005%-0.1% (w/v), 0.1%-0.2% (w/v), 0.2%-0.25% (w/v), 0.25%-0.5% (w/v), 0.5%-1% (w/v), 1%-2% (w/v), 2%-3% (w/v), 3%-4% (w/v), 4%-5% (w/v), 5%-6% (w/v), 6%-7% (w/v), 7%-8% (w/v) or 8%-10% (w/v).

In some embodiments, the levamisole or a pharmaceutically acceptable salt thereof is the only active ingredient.

In some embodiments, the concentration of levamisole or a pharmaceutically acceptable salt thereof is 0.2% (w/v).

In some embodiments, the ophthalmic formulation further comprises an isotonicity agent.

In some embodiments, the isotonic agent is selected from sodium chloride, potassium chloride, boric acid, borax, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, sodium acetate, mannitol, glycerol, propylene glycol, 2-(4-n-octylphenethyl)-2-aminopropanediol hydrochloride, glucose, or any combination thereof.

In some embodiments, the isotonic agent is selected from sodium chloride, potassium chloride, or any combination thereof.

In some embodiments, the isotonic agent is sodium chloride.

In some embodiments, the concentration of the isotonic agent is 0.1%-1% (w/v), for example, 0.1% (w/v), 0.15% (w/v), 0.2% (w/v), 0.25% (w/v), 0.3% (w/v), 0.35% (w/v), 0.4% (w/v), 0.45% (w/v), 0.5% (w/v), 0.55% (w/v), 0.6% (w/v), 0.65% (w/v), 0.7% (w/v), 0.75% (w/v), 0.8% (w/v), 0.85% (w/v), 0.9% (w/v), 0.95% (w/v) or 1% (w/v).

In some embodiments, the concentration of the isotonic agent is 0.7% (w/v).

In some embodiments, the ophthalmic formulation further comprises a bacteriostatic agent.

In some embodiments, the antibacterial agent is selected from benzalkonium chloride, benzalkonium bromide, chlorhexidine acetate, chlorhexidine gluconate, chlorobutanol, phenoxyethanol, methylparaben, ethylparaben, propylparaben, or any combination thereof.

In some embodiments, the antibacterial agent is selected from benzalkonium chloride, benzalkonium bromide, ethylparaben, or any combination thereof.

In some embodiments, the bacteriostatic agent is ethylparaben.

In some embodiments, the concentration of the antibacterial agent is 0.001%-0.1% (w/v), for example, 0.001% (w/v), 0.005% (w/v), 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v), 0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v) or 0.1% (w/v).

In some embodiments, the concentration of the bacteriostatic agent is 0.03% (w/v).

In some embodiments, the ophthalmic formulation further comprises a pH adjusting agent.

In some embodiments, the pH adjuster is selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, or any combination thereof.

In some embodiments, the pH adjuster is selected from sodium carbonate, sodium bicarbonate, sodium hydroxide, or any combination thereof.

In some embodiments, the pH adjuster is sodium hydroxide.

In some embodiments, the content of the pH adjuster is an appropriate amount.

In some embodiments, the pH of the ophthalmic formulation is 5.0-6.0.

In some embodiments, the pH of the ophthalmic formulation is 5.5.

In some embodiments, the water is water for injection.

In some embodiments, the formula of each 100 mL of the ophthalmic preparation is selected from the following:

Recipe 1:

Recipe 2:

Recipe 3:

Recipe 4:

Recipe 5:

Recipe 6:

Recipe 7:

Recipe 8:

Recipe 9:

Recipe 10:

Recipe 11:

Recipe 12:

Recipe 13:

Recipe 14:

Recipe 15:

Recipe 16:

In some preferred embodiments, the formula of each 100 mL of the ophthalmic preparation is:

In some embodiments, the ophthalmic preparation is eye drops.

In a second aspect of the present invention, there is provided a method for preparing the aforementioned ophthalmic preparation, comprising:

Levamisole or a pharmaceutically acceptable salt thereof is mixed with water, dissolved, and sterilized to obtain the ophthalmic preparation.

In some embodiments, the method comprises:

1) Mix the antibacterial agent with water and heat to 80-100°C (e.g. 90°C) to dissolve;

2) adding an isotonic agent to the solution obtained in step 1) and cooling the solution to below 50° C.;

3) adding levamisole or a pharmaceutically acceptable salt thereof to the solution obtained in step 2);

4) adding an optional pH adjuster to the solution obtained in step 3) to adjust the pH to 5.0-6.0 (e.g., 5.5), and adding water to make up to 100 mL;

5) sterilizing the solution obtained in step 4) to obtain the ophthalmic preparation.

In some embodiments, the concentration of levamisole or a pharmaceutically acceptable salt thereof is 0.01%-10% (w/v), for example, 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v), 0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v), 0.1% (w/v), 0.15% (w/v), 0.2% (w/v), 0.25% (w/v), 0.3% (w/v), 0.35% (w/v), 0.4% (w/v), 0.45% (w/v), 0.5% (w/v), 0.6% (w/v), 0.7% (w/v), 0.8% (w/v), 0.9% (w/v), 10% (w/v), 11% (w/v), 12% (w/v), 13% (w/v), 14% (w/v), 15% (w/v), 16% (w/v), 17% (w/v), 18% (w/v), 19% (w/v), 20% (w/v), 21% (w/v), 22% (w/v), 23% (w/v), 24% (w/v), 25% (w/v), 26% (w/v), 27% (w/v), 28% (w/v), 29% (w/v), 30% (w/v), 31% (w/v), 32% (w/v), 33% (w/v), 34% (w/v), 35% (w/v), 36% (w .55% (w/v), 0.6% (w/v), 0.65% (w/v), 0.7% (w/v), 0.75% (w/v), 0.8% (w/v), 0.85% (w/v), 0.9% (w/v), 0.95% (w/v), 1% (w/v), 1.5% (w/v), 2% (w/v), 2.5% (w/v), 3% (w/v), 3.5% (w/v), 4% (w/v), 4.5% (w/v), 5% (w/v), 5.5% (w/v), 6% (w/v), 6.5% (w/v), 7% (w/v), 7.5% (w/v), 8% (w/v), 8.5% (w/v), 9% (w/v), 9.5% (w/v) or 10% (w/v), or 0.01%-0.02% (w/v), 0.02%-0.05% (w/v), 005%-0.1% (w/v), 0.1%-0.2% (w/v), 0.2%-0.25% (w/v), 0.25%-0.5% (w/v), 0.5%-1% (w/v), 1%-2% (w/v), 2%-3% (w/v), 3%-4% (w/v), 4%-5% (w/v), 5%-6% (w/v), 6%-7% (w/v), 7%-8% (w/v) or 8%-10% (w/v).

In some embodiments, the isotonic agent is sodium chloride.

In some embodiments, the concentration of the isotonic agent is 0.7% (w/v).

In some embodiments, the bacteriostatic agent is ethylparaben.

It should be noted that in the above preparation method, the concentration of each added component refers to the concentration of each component in 100 mL of the ophthalmic preparation finally obtained.

In some embodiments, the pH adjuster is sodium hydroxide.

In some embodiments, the content of the pH adjuster is an appropriate amount.

In some embodiments, the water is water for injection.

In a third aspect of the present invention, there is provided a use of levamisole or a pharmaceutically acceptable salt thereof in the preparation of an ophthalmic preparation, wherein the ophthalmic preparation is used for one or more selected from the following (1)-(5):

(1) For the treatment and/or prevention of dry eye or its related diseases;

(2) Used to improve corneal epithelial damage;

(3) Used to improve tear film stability;

(4) Used to promote tear secretion;

(5) Used to increase the number of conjunctival goblet cells.

In a fourth aspect of the present invention, the present invention provides an ophthalmic preparation comprising levamisole or a pharmaceutically acceptable salt thereof, which is used for one or more selected from the following (1)-(5):

(1) For the treatment and/or prevention of dry eye or its related diseases;

(2) Used to improve corneal epithelial damage;

(3) Used to improve tear film stability;

(4) Used to promote tear secretion;

(5) Used to increase the number of conjunctival goblet cells.

In a fifth aspect of the present invention, the present invention provides a method for treating and/or preventing dry eye or a disease related thereto, comprising administering to a subject an effective amount of an ophthalmic preparation comprising levamisole or a pharmaceutically acceptable salt thereof.

In a sixth aspect of the present invention, the present invention provides a method for improving corneal epithelial damage, comprising administering to a subject an effective amount of an ophthalmic preparation, wherein the ophthalmic preparation comprises levamisole or a pharmaceutically acceptable salt thereof.

In a seventh aspect of the present invention, the present invention provides a method for improving tear film stability, comprising administering to a subject an effective amount of an ophthalmic preparation, wherein the ophthalmic preparation comprises levamisole or a pharmaceutically acceptable salt thereof.

In an eighth aspect of the present invention, the present invention provides a method for promoting tear secretion, comprising administering to a subject an effective amount of an ophthalmic preparation, wherein the ophthalmic preparation comprises levamisole or a pharmaceutically acceptable salt thereof.

In a ninth aspect of the present invention, the present invention provides a method for increasing the number of conjunctival goblet cells, comprising administering to a subject an effective amount of an ophthalmic preparation, wherein the ophthalmic preparation comprises levamisole or a pharmaceutically acceptable salt thereof.

The technical solutions of the third to ninth aspects above may further include the following additional technical solutions: At least one of the technical characteristics.

In some embodiments, the dry eye-related disease is selected from ocular surface inflammation, meibomian gland dysfunction, lacrimal gland inflammation and fibrosis, and conjunctivitis.

In some embodiments, the levamisole or a pharmaceutically acceptable salt thereof is the only active ingredient in the ophthalmic formulation.

In some embodiments, the concentration of levamisole or a pharmaceutically acceptable salt thereof is 0.01%-10% (w/v), for example, 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v), 0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v), 0.1% (w/v), 0.15% (w/v), 0.2% (w/v). /v), 0.25% (w/v), 0.3% (w/v), 0.35% (w/v), 0.4% (w/v), 0.45% (w/v), 0.5% (w/v), 0.55% (w/v), 0.6% (w/v), 0.65% (w/v), 0.7% (w/v), 0.75% (w/v), 0.8% (w/v), 0.85% (w/v), 0.9% (w/v), 0.95% (w/v), 1% (w/v), 1.5% (w/v) ), 2% (w/v), 2.5% (w/v), 3% (w/v), 3.5% (w/v), 4% (w/v), 4.5% (w/v), 5% (w/v), 5.5% (w/v), 6% (w/v), 6.5% (w/v), 7% (w/v), 7.5% (w/v), 8% (w/v), 8.5% (w/v), 9% (w/v), 9.5% (w/v) or 10% (w/v), or 0.01% - 0.02% (w/v), 0.0 2%-0.05% (w/v), 005%-0.1% (w/v), 0.1%-0.2% (w/v), 0.2%-0.25% (w/v), 0.25%-0.5% (w/v), 0.5%-1% (w/v), 1%-2% (w/v), 2%-3% (w/v), 3%-4% (w/v), 4%-5% (w/v), 5%-6% (w/v), 6%-7% (w/v), 7%-8% (w/v) or 8%-10% (w/v).

In some embodiments, the isotonic agent is sodium chloride.

In some embodiments, the concentration of the isotonic agent is 0.7% (w/v).

In some embodiments, the bacteriostatic agent is ethylparaben.

In some embodiments, the pH adjuster is sodium hydroxide.

In some embodiments, the content of the pH adjuster is an appropriate amount.

In some embodiments, the pH of the ophthalmic formulation is 5.0-6.0.

In some embodiments, the pH of the ophthalmic formulation is 5.5.

In some embodiments, the ophthalmic formulation further comprises water. In some embodiments, the water is water for injection.

In the tenth aspect of the present invention, there is provided a use of the ophthalmic preparation of the first aspect in the preparation of a medicament, wherein the medicament is used for one or more selected from the following (1)-(5):

(1) For the treatment and/or prevention of dry eye or its related diseases;

(2) Used to improve corneal epithelial damage;

(3) Used to improve tear film stability;

(4) Used to promote tear secretion;

(5) Used to increase the number of conjunctival goblet cells.

In the eleventh aspect of the present invention, the present invention provides an ophthalmic preparation according to the first aspect, which is used for one or more selected from the following (1)-(5):

(1) For the treatment and/or prevention of dry eye or its related diseases;

(2) Used to improve corneal epithelial damage;

(3) Used to improve tear film stability;

(4) Used to promote tear secretion;

(5) Used to increase the number of conjunctival goblet cells.

In the twelfth aspect of the present invention, the present invention provides a method for treating and/or preventing dry eye or a disease related thereto, comprising administering to a subject an effective amount of the ophthalmic preparation of the first aspect.

In a thirteenth aspect of the present invention, the present invention provides a method for improving corneal epithelial damage, comprising administering an effective amount of the ophthalmic preparation of the first aspect to a subject.

In a fourteenth aspect of the present invention, the present invention provides a method for improving tear film stability, comprising administering to a subject an effective amount of the ophthalmic preparation of the first aspect.

In a fifteenth aspect of the present invention, the present invention provides a method for promoting tear secretion, comprising administering an effective amount of the ophthalmic preparation of the first aspect to a subject.

In a sixteenth aspect of the present invention, the present invention provides a method for increasing the number of conjunctival goblet cells, comprising administering to a subject an effective amount of the ophthalmic preparation of the first aspect.

Definition of Terms

In the present invention, "w/v" means mass volume percentage, and the concentration of "X% (w/v)" means that the number of grams (g) of the solute contained in 100 mL of the liquid preparation is X g. For example, "the concentration of levamisole or a pharmaceutically acceptable salt thereof is 0.2% (w/v)" means that 100 mL of the ophthalmic preparation contains 0.2 g of levamisole or a pharmaceutically acceptable salt thereof.

In the present invention, the term "pharmaceutically acceptable salt" refers to a salt formed by a basic functional group present in the compound provided by the present invention and an appropriate inorganic or organic anion (acid), and includes, but is not limited to, hydrohalide salts, such as hydrofluoride, hydrochloride, hydrobromide, hydroiodide, etc.; inorganic acid salts, such as nitrate, perchlorate, sulfate, phosphate, etc.; lower alkane sulfonate salts, such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, etc.; aryl sulfonate salts, such as benzenesulfonate, p-toluenesulfonate, etc.; organic acid salts, such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc.; amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, aspartate, etc. For example, the pharmaceutically acceptable salt of levamisole is levamisole hydrochloride.

In the present invention, the term "appropriate amount" refers to a suitable amount, which can be zero or any value other than zero. For example, the content of pH regulator in an ophthalmic preparation is an appropriate amount, which means that the amount of pH regulator contained can make the pH value of the ophthalmic preparation reach the target requirement. If the pH value reaches the target requirement without adding the pH adjuster, the content of the pH adjuster can be zero.

In the present invention, "treatment" generally refers to obtaining the desired pharmacological and/or physiological effect. The effect can be preventive, based on the complete or partial prevention of the disease or its symptoms; and/or can be therapeutic, based on the partial or complete stabilization or cure of the disease and/or side effects caused by the disease. As used herein, "treatment" covers any treatment of a patient's disease, including: (a) preventing the disease or symptoms from occurring in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) inhibiting the symptoms of the disease, i.e., preventing its development; or (c) alleviating the symptoms of the disease, i.e., causing the disease or symptoms to regress.

In the present invention, "subject" refers to a vertebrate. In certain embodiments, a vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, a mammal refers to a human.

In the present invention, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, a preventive effective amount refers to an amount that can effectively prevent, prevent or delay the occurrence of a disease. A therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc. It is further understood that for any particular individual, the specific dosage regimen can be adjusted over time according to the individual needs and the mode of administration or the professional judgment of the person supervising the administration.

Beneficial Effects

The ophthalmic preparation provided by the present invention has therapeutic activity for ocular surface diseases of non-organic lesions (organic lesions such as infectious keratoconjunctivitis, retinal hemorrhage, cataracts, etc.), especially dry eye and its related diseases (dry eye related diseases such as ocular surface inflammation, meibomian gland dysfunction, lacrimal gland inflammation and fibrosis, conjunctivitis, etc.). The ophthalmic preparation of the present invention can promote tear secretion, increase the number of conjunctival goblet cells, and can effectively improve patients' dry eye symptoms, corneal epithelial damage and tear film stability.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the statistics of tear secretion in mice, where Ctrl represents the normal group, BAC represents the BAC model group, BAC+Vehicle represents the solvent control group, and BAC+LM represents the treatment group; where *P<0.05; **P<0.01; ***P<0.001;

FIG2 shows the statistics of the number of goblet cells in the conjunctiva of mice, wherein: Ctrl represents the normal group, BAC represents the BAC model group, BAC+Vehicle represents the solvent control group, and BAC+LM represents the treatment group; wherein, *P<0.05;**P<0.01;***P<0.001;

FIG3 shows the corneal epithelial defect of mice, wherein FIG3A is a typical picture of mouse corneal OGD staining, and FIG3B is the fluorescence intensity statistics of mouse corneal OGD staining, wherein: Ctrl represents the normal group, BAC represents the BAC model group, BAC+Vehicle represents the solvent control group, and BAC+LM represents the treatment group; wherein, *P<0.05; **P<0.01; ***P<0.001;

Figure 4 shows the changes in the patient's OSDI score before and after 2 weeks;

Figure 5 shows the changes in OSDI scores of patients who completed 2 follow-up visits;

FIG6 shows the changes in the corneal fluorescein sodium staining scores of the patients before and after 2 weeks;

FIG7 shows the changes in corneal fluorescein sodium staining scores in patients who completed 2 follow-up visits;

Figure 8 shows the changes in the patient's FBUT before and after 2 weeks;

FIG9 shows the changes in FBUT in patients who completed 2 follow-up visits.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

Preparation Example 1

1. Prescription:

Levamisole hydrochloride, 0.01g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. Preparation method:

Weigh the prescribed amount (i.e. 0.03 g) of ethylparaben, add about 60 ml of water, heat to about 90°C to dissolve, add sodium chloride, cool to below 50°C, add the prescribed amount (i.e. 0.01 g) of levamisole hydrochloride, stir to dissolve, add sodium hydroxide to adjust the pH to about 5.5, add water to 100 ml, sterilize and filter, and aseptically package.

Preparation Example 2

1. Prescription:

Levamisole hydrochloride, 0.02g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 3

1. Prescription:

Levamisole hydrochloride, 0.05g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 4

1. Prescription:

Levamisole hydrochloride, 0.10g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 5

1. Prescription:

Levamisole hydrochloride, 0.20g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 6

1. Prescription:

Levamisole hydrochloride, 0.25g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 7

1. Prescription:

Levamisole hydrochloride, 0.50g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 8

1. Prescription:

Levamisole hydrochloride, 1.00g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 9

1. Prescription:

Levamisole hydrochloride, 2.00g; NaCl, 0.7g; ethylparaben, 0.03g, plus injection Add water to 100 ml and adjust the pH to about 5.5 with 0.5 M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 10

1. Prescription:

Levamisole hydrochloride, 3.00g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 11

1. Prescription:

Levamisole hydrochloride, 4.00g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 12

1. Prescription:

Levamisole hydrochloride, 5.00g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 13

1. Prescription:

Levamisole hydrochloride, 6.00g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 14

1. Prescription:

Levamisole hydrochloride, 7.00g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 15

1. Prescription:

Levamisole hydrochloride, 8.00 g; NaCl, 0.7 g; ethylparaben, 0.03 g, add water for injection to 100 ml, and adjust the pH to about 5.5 with 0.5 M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Preparation Example 16

1. Prescription:

Levamisole hydrochloride, 10.00g; NaCl, 0.7g; ethylparaben, 0.03g, add water for injection to 100ml, and adjust the pH to about 5.5 with 0.5M NaOH.

2. The preparation method is the same as that in Preparation Example 1.

Experimental Example 1: Effect of Levamisole Hydrochloride Eye Drops on Dry Eyes - Animal Experiment

Animal Experimental Methods:

(1) Establishment of mouse dry eye model and its grouping treatment

Healthy female C57BL/6 mice aged 9-10 weeks were selected and randomly divided into 4 groups: normal group (Ctrl), BAC model group (BAC), vehicle control group (BAC+Vehicle) and levamisole hydrochloride treatment group (BAC+LM)

Method for establishing the benzalkonium chloride model in mice: The mice were housed in standard IVC cages (relative humidity: 50-60%, temperature: 21-23°C) and 0.075% benzalkonium chloride solution (BAC) was administered eye drops (5 μl/eye) twice a day (9:00am, 17:00pm) for 7 consecutive days to induce mice with dry eye symptoms, and the model was successfully established.

Treatment method of normal group mice: The normal group consisted of normal mice, which were not treated with eye drops and were kept in a standard IVC environment with a temperature of (21-23)°C and a relative humidity of 50-60%.

Treatment method for model group mice: The mice in this group were given 0.075% benzalkonium chloride solution (BAC) eye drops (5 μl/eye) twice a day (9:00am, 17:00pm) for 7 consecutive days and were kept in a standard IVC environment with a temperature of (21-23)°C and a relative humidity of 50-60%.

Treatment method for mice in the solvent control group: 0.075% benzalkonium chloride solution (BAC) was administered to the eyes of the mice in this group (5 μl/eye), twice a day (9:00 am, 17:00 pm), and the preparation of the present invention without levamisole hydrochloride was administered to the eyes of the mice in this group (1 drop each time, four times a day, 4 hours apart each time, and the treatment was continued for 7 days).

Treatment method for mice in the treatment group: 0.075% benzalkonium chloride solution (BAC) was administered eye drops (5 μl/eye) twice a day (9:00 am, 17:00 pm), and the ophthalmic preparation prepared in Preparation Example 1-16 was administered to the eyes of the mice in this group (1 drop each time, four times a day, with an interval of 4 hours between each time, and the treatment was continued for 7 days).

The treatment of animals during the experiment complies with the "Guiding Opinions on the Care of Experimental Animals" issued by the Ministry of Science and Technology.

(2) Determination of relevant indicators

After each group of mice was treated, the mice were examined. Each examination was completed by the same handler, and the time, location, lighting, and temperature of each examination were the same.

The tear secretion, corneal OGD staining and conjunctival goblet cell number of the above groups of mice were measured.

1) Phenol red cotton thread test to detect tear secretion:

Phenol red cotton (Zone-Quick; Lacrimedics, Eastsound, WA) was used to detect the tear secretion of mice. Under the slit lamp, ophthalmic forceps were used to place the phenol red cotton thread in the conjunctival fornix below the outer canthus of the mouse eye. After 15 seconds, the length of the phenol red cotton thread dyed was measured and recorded using a millimeter ruler. The results are shown in Table 1-1.

Table 1-1 Tear secretion volume (mm)

2) OGD staining to detect corneal epithelial barrier function:

0.5 μl Oregon green-dextran (OGD, 50 mg/ml, 70,000 molecular weight; Invitrogen) was dripped into the lower conjunctival sac of mice using a capillary pipette. The mice were killed and rinsed with 1 mL of saline. Then, the corneal epithelial fluorescent staining was observed and photographed under an in vivo fluorescence microscope (AZ100, Nikon). The staining fluorescence intensity was measured and recorded using NIS-element software. The results are shown in Table 1-2.

Table 1-2 Staining fluorescence intensity (au)

3) Goblet cell number measurement:

Eye tissue specimens were fixed with 10% formalin and embedded in paraffin. They were stained with periodic acid-Schiff (PAS) reagent, photographed with Nikon Eclipse 50i, and conjunctival goblet cell counts were performed. The results are shown in Tables 1-3.

Table 1-3 Number of conjunctival goblet cells

Analysis of results:

In addition, with respect to the above experiments, the present invention also analyzes the data of Preparation Examples 1, 4, 5, 7, 8, 12, and 16, as shown in FIGS. 1 to 3 , respectively, wherein:

The results in Figure 1 show that the tear secretion of BAC group mice decreased significantly after modeling, and the tear secretion of the treatment group was significantly higher than that of the solvent group, and the effect of the 0.2% levamisole hydrochloride treatment group (BAC+0.2% LM, i.e. Preparation Example 5) was the most significant, ***P<0.001, **P<0.01, *P<0.05.

The results in Figure 2 show that the number of conjunctival goblet cells in the BAC group mice decreased significantly after modeling, and the number of goblet cells in the treatment group was significantly higher than that in the solvent group, and the effect of the treatment group with 0.2% levamisole hydrochloride concentration (BAC+0.2% LM, i.e. Preparation Example 5) was the most significant, ***P<0.001, **P<0.01, *P<0.05.

The results in Figure 3 show that after modeling, the cornea of mice in the dry eye group (i.e., BAC group) showed obvious OGD staining, and the OGD staining in the treatment group was significantly reduced compared with the solvent group, and the effect of the treatment group with 0.2% levamisole hydrochloride concentration (BAC+0.2% LM, i.e., Preparation Example 5) was the most significant, ***P<0.001.

Experimental Example 2: Clinical trial of the effect of levamisole hydrochloride eye drops in the treatment of dry eyes

experimental method:

A self-control study was conducted, and 34 patients with dry eye were included.

Inclusion criteria:

1. Fully understand this study and voluntarily sign the ICF; willing to follow and be able to complete all trial procedures.

2. 18 years old ＜ age ＜ 80 years old, regardless of gender.

3. Patients clinically diagnosed with dry eyes according to the "Chinese Dry Eye Expert Consensus" issued by the Corneal Disease Group of the Ophthalmology Branch of the Chinese Medical Association in 2020. The diagnostic criteria are as follows:

① The patient complained of one of the subjective symptoms such as dry eyes, foreign body sensation, burning sensation, fatigue, discomfort, red eyes, and visual fluctuations, and the Chinese Dry Eye Questionnaire Scale was ≥ 7 points or ocular surface Dry eye can be diagnosed if the ocular surface disease index (OSDI) is ≥13 points; at the same time, the patient's FBUT ≤5s or NIBUT <10s or Schirmer I test (without anesthesia) ≤5mm/5min.

② If the patient has symptoms related to dry eyes, with a Chinese Dry Eye Questionnaire score of ≥7 or an OSDI score of ≥13; at the same time, if the patient's FBUT is >5s and ≤10s or the NIBUT is 10-12s, and the Schirmer I test (without anesthesia) is >5mm/5min and ≤10mm/5min, then sodium fluorescein staining should be used to examine the cornea and conjunctiva, and positive staining (≥5 points) can be used to diagnose dry eyes.

Exclusion criteria: organic eye diseases, such as infectious keratoconjunctivitis, retinal hemorrhage, cataracts, etc.; pregnant and lactating women; other situations that are not suitable for participation.

treatment method:

Patients with dry eyes used the ophthalmic preparation (0.2%) prepared in Preparation Example 5 for 6 weeks, 4 times a day, and were followed up at the second and sixth weeks.

Evaluation indicators and operation methods:

Evaluation indicators: OSDI questionnaire score; corneal fluorescence staining score; FBUT; Schirmer I; conjunctival congestion score.

How to do it:

1. The above evaluation indicators were examined on the patients and included as dry eye patients.

2. Apply levamisole hydrochloride eye drops 4 times a day for 6 weeks.

3. Follow-up visits were conducted at the 2nd and 6th weeks respectively, and the above-mentioned evaluation indicators were collected again and compared with the results of the previous collection.

Table 2-1: OSDI scores

In the past week, have you had any of the following symptoms?

In the past week, have your eyes limited the following aspects of your life?

In the past week, did you experience any eye discomfort when you were in the following environments?

Corneal fluorescein sodium staining score

5-10ul of sodium fluorescein was instilled into the lower eyelid conjunctiva or a commercial fluorescein test strip was used. The patient was asked to blink 3 or 4 times. Under the cobalt blue filter, the corneal epithelium was observed to see if it was stained. Positive staining indicated that the integrity of the corneal epithelial cells was damaged. The corneal epithelium was stained with fluorescein. The score after staining was scored using the National Eye Institute (NEI) corneal scoring scale, with 0.5 points as an increment and a score range of 0-4 (0 = no punctate staining, 4 = severe diffuse or combined with large punctate staining). The scores of each area of the cornea of both eyes were recorded, and the scores of each eye were summed.

Fluorescein tear breakup time (FBUT)

Instill 5-10 μl of sodium fluorescein into the lower eyelid conjunctiva or use commercial fluorescein test strips, and ask the patient to blink 3 or 4 times. The time is calculated from the time the eyes open naturally after the last blink to the time the first black spot appears on the cornea.

Experimental results and conclusions:

A total of 34 patients completed the 2-week follow-up, and 16 patients completed both follow-ups.

The OSDI questionnaire scores before and after treatment showed that the 2-week follow-up results showed that the total OSDI score of the patient before treatment was 47.72±22.75 (mean±standard deviation), and the total score of the dry eye questionnaire of the patient after 2 weeks of treatment was 36.70±21.53. The difference before and after treatment was statistically significant (P＜0.01) (Figure 4); The results of the two follow-up visits showed that with the extension of treatment time, OSDI gradually decreased from 50.06±20.49 to 33.26±21.28 (2 weeks, P<0.001), and then to 27.87±19.17 (6 weeks, P<0.001). The differences before and after treatment were statistically significant (Figure 5). It can be considered that the dry eye symptoms of patients were significantly improved before and after treatment.

The corneal fluorescein sodium staining scores before and after treatment showed that the 2-week follow-up results showed that the corneal fluorescein sodium staining score of the patient before treatment was 4.59±4.52, and the score of the patient after 2 weeks of treatment was 2.62±4.11. The difference before and after treatment was statistically significant (P<0.01) (Figure 6); the results of the two follow-up visits showed that as the treatment time prolonged, the corneal fluorescein sodium staining score gradually decreased from 4.38±4.41 to 2.75±5.09 (2 weeks), and then to 2.25±3.53 (6 weeks, P<0.05). The difference between the sixth week of treatment and the baseline was statistically significant (Figure 7). It can be considered that the corneal epithelial damage of the patient was significantly improved before and after treatment.

FBUT before and after treatment showed: The 2-week follow-up results showed that the FBUT of the patient before treatment was 3.15±1.19, and the FBUT of the patient after 2 weeks of treatment was 4.22±1.47. The difference before and after treatment was statistically significant (P<0.001) (Figure 8); The results of the two follow-up showed that as the treatment time prolonged, FBUT gradually increased from 3.31±1.20 to 4.26±1.37 (2 weeks), and then to 4.75±2.51 (6 weeks, P<0.05). The difference between the sixth week of treatment and the baseline was statistically significant (Figure 9). It can be considered that the tear film stability of the patient was significantly improved before and after treatment.

Therefore, based on the above clinical trial results, the topical eye drops of levamisole hydrochloride of the present invention can effectively improve patients' dry eye symptoms, corneal epithelial damage and tear film stability.

It should be understood that the invention described herein is not limited to specific methodology, experimental protocols or reagents, as these may vary. The discussion and examples provided herein are presented only to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the claims.

Claims

An ophthalmic preparation comprises: an active ingredient levamisole or a pharmaceutically acceptable salt thereof, and water, wherein the concentration of the levamisole or the pharmaceutically acceptable salt thereof is 0.01%-10% (w/v).
The ophthalmic preparation according to claim 1, wherein the levamisole or a pharmaceutically acceptable salt thereof is the only active ingredient.
The ophthalmic preparation according to any one of claims 1 to 2, further comprising one or more selected from the following (1) to (3):

(1) Isotonic agents;

Preferably, the isotonic agent is selected from sodium chloride, potassium chloride, boric acid, borax, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, sodium acetate, mannitol, glycerol, propylene glycol, 2-(4-n-octylphenethyl)-2-aminopropanediol hydrochloride, glucose, or any combination thereof;

More preferably, the isotonic agent is selected from sodium chloride, potassium chloride, or any combination thereof;

Most preferably, the isotonic agent is sodium chloride;

Preferably, the concentration of the isotonic agent is 0.1%-1% (w/v);

More preferably, the concentration of the isotonic agent is 0.7% (w/v);

(2) Antibacterial agents;

Preferably, the antibacterial agent is selected from benzalkonium chloride, benzalkonium bromide, chlorhexidine acetate, chlorhexidine glucose, chlorobutanol, phenoxyethanol, methylparaben, ethylparaben, propylparaben, or any combination thereof;

More preferably, the antibacterial agent is selected from benzalkonium chloride, benzalkonium bromide, ethylparaben, or any combination thereof;

Most preferably, the antibacterial agent is ethylparaben;

Preferably, the concentration of the antibacterial agent is 0.001%-0.1% (w/v);

More preferably, the concentration of the antibacterial agent is 0.03% (w/v);

(3) pH adjuster;

Preferably, the pH regulator is selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, or any combination thereof;

More preferably, the pH regulator is selected from sodium carbonate, sodium bicarbonate, sodium hydroxide, or any combination thereof;

Most preferably, the pH adjusting agent is sodium hydroxide;

Preferably, the content of the pH regulator is an appropriate amount;

Preferably, the pH of the ophthalmic preparation is 5.0-6.0;

More preferably, the pH of the ophthalmic preparation is 5.5.
The ophthalmic preparation according to any one of claims 1 to 3, wherein the formula of each 100 mL of the ophthalmic preparation is selected from the following:

Recipe 1:

Recipe 2:

Recipe 3:

Recipe 4:

Recipe 5:

Recipe 6:

Recipe 7:

Recipe 8:

Recipe 9:

Recipe 10:

Recipe 11:

Recipe 12:

Recipe 13:

Recipe 14:

Recipe 15:

Recipe 16:

Preferably, the formula of each 100 mL of the ophthalmic preparation is:
A method for preparing the ophthalmic preparation according to any one of claims 1 to 4, comprising:

Mixing, dissolving and sterilizing levamisole or a pharmaceutically acceptable salt thereof with water to obtain the ophthalmic preparation;

Preferably, the method comprises:

1) Mix the antibacterial agent with water and heat to 80-100°C (e.g. 90°C) to dissolve;

2) adding an isotonic agent to the solution obtained in step 1) and cooling the solution to below 50° C.;

3) adding levamisole or a pharmaceutically acceptable salt thereof to the solution obtained in step 2);

4) adding an optional pH adjuster to the solution obtained in step 3) to adjust the pH to 5.0-6.0 (e.g., 5.5), and adding water to make up to 100 mL;

5) sterilizing the solution obtained in step 4) to obtain the ophthalmic preparation;

Preferably, the levamisole or a pharmaceutically acceptable salt thereof, the antibacterial agent, the isotonic agent or the pH regulator is as defined in any one of claims 1 to 4.
Use of levamisole or a pharmaceutically acceptable salt thereof in the preparation of an ophthalmic preparation, wherein the ophthalmic preparation is used for one or more selected from the following (1)-(5):

(1) For the treatment and/or prevention of dry eye or its related diseases;

Preferably, the dry eye-related disease is selected from ocular surface inflammation, meibomian gland dysfunction, lacrimal gland inflammation Disease and fibrosis, conjunctivitis;

(2) Used to improve corneal epithelial damage;

(3) Used to improve tear film stability;

(4) Used to promote tear secretion;

(5) Used to increase the number of conjunctival goblet cells.
The use according to claim 6, wherein the levamisole or a pharmaceutically acceptable salt thereof is the only active ingredient.
The use according to any one of claims 6 to 7, wherein the concentration of levamisole or a pharmaceutically acceptable salt thereof in the ophthalmic preparation is 0.01% to 10% (w/v).
Use of the ophthalmic preparation according to any one of claims 1 to 4 in the preparation of a medicament, wherein the medicament is used for one or more selected from the following (1) to (5):

(1) For the treatment and/or prevention of dry eye or its related diseases;

Preferably, the dry eye-related disease is selected from ocular surface inflammation, meibomian gland dysfunction, lacrimal gland inflammation and fibrosis, and conjunctivitis;

(2) Used to improve corneal epithelial damage;

(3) Used to improve tear film stability;

(4) Used to promote tear secretion;

(5) Used to increase the number of conjunctival goblet cells.