CN110960485A - Medicine for treating keratitis and preparation method thereof - Google Patents

Abstract

The invention discloses a medicament for treating keratitis, which comprises (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide and a pharmaceutically acceptable ophthalmic excipient. The invention also relates to a preparation method of the medicine and application of the medicine in preparing a medicinal preparation for treating keratitis, in particular bacterial keratitis, and more particularly bacterial keratitis caused by staphylococcus aureus infection.

Description

Medicine for treating keratitis and preparation method thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a medicine for treating keratitis and a preparation method thereof.

Background

The cornea is a completely transparent thin film at the very front of the eye, in the shape of a transverse oval, approximately 1mm thick. The cornea covers the iris, pupil, and anterior chamber, providing the eye with most of its refractive power. With the refractive power of the crystal, the light can be focused on the retina accurately to form an image. Structurally, the cornea is composed mainly of avascular connective tissue with very sensitive nerve endings, and if foreign objects contact the cornea, eyelids may close involuntarily to protect the eye. In order to maintain transparency, the cornea has no blood vessels, but acquires nutrients and oxygen through the tear fluid and aqueous humor. These characteristics of the cornea determine that the cornea is easily affected by factors such as trauma, environmental pollution, chemical stimulation, microorganisms and the like to generate inflammatory reaction, thereby causing keratitis.

Keratitis is a common disease in ophthalmology, and is also an important blinding eye disease, and is usually clinically manifested by strong inflammation symptoms, such as photophobia, lacrimation, pain, blepharospasm and the like, and corneal transparency reduction, gloss disappearance, ulcer formation and the like can be seen in ophthalmic examination, and serious patients can have secondary intraocular infection, corneal perforation and even blindness. There are many factors that cause keratitis, and common causes are infections with microorganisms such as bacteria, fungi, viruses, chlamydia, trauma, autoimmune diseases, and spread of adjacent tissues, among others.

Clinically common treatments for keratitis include the administration of sensitive antibacterial, antifungal or antiviral ophthalmic preparations, topical heat application, ocular cleansing and the like. However, these approaches have poor clinical efficacy and cannot meet the clinical medication requirements. Therefore, the development of new drugs for treating keratitis has great clinical significance in the field of ophthalmology.

Disclosure of Invention

The invention aims to enrich clinical medication choices of keratitis and provide a medicament for treating keratitis and a preparation method thereof.

The present inventors have unexpectedly found that (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridinyl ] amino ] ethyl ] pent-2-enamide (which will hereinafter be referred to interchangeably as "the compound of the present invention") is effective in ameliorating the symptoms of bacterial keratitis caused by staphylococcus aureus infection, and thus can be expected to be useful in treating keratitis, particularly bacterial keratitis caused by staphylococcus aureus infection.

To this end, the present invention provides a medicament for the treatment of keratitis, said medicament comprising (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridinyl ] amino ] ethyl ] pent-2-enamide and a pharmaceutically acceptable ophthalmic excipient.

The active ingredient of the drug for treating keratitis used in the present invention is (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridinyl ] amino ] ethyl ] pent-2-enamide, which has the following structural formula:

the synthesis of this compound is described in patent document WO2012/041873a1 (see example 10). The patent literature generally relates to novel N-heteroaryl compounds useful as pharmaceuticals, the preparation of such compounds and the use of such compounds. The medicament may preferably be used for the treatment of parasitic infections, such as helminth infections, in particular for the treatment of parasitic diseases, such as those caused by helminth infections. The biological examples of this patent document have in part evaluated the anti-ascaris galli (ascaris galli) and meloidogyne denticola (oesophagogalus) activities of several of the example compounds of the invention and found that many of the example compounds show at least some activity against one or more nematodes (MEC of 50 μ M or less). However, this patent document does not mention the use of said compounds for the treatment of keratitis, in particular bacterial keratitis, which constitutes an unexpected finding of the present invention.

In one aspect, the medicament of the present invention further comprises a pharmaceutically acceptable ophthalmic excipient, such as, but not limited to, one or more of an ophthalmic solvent, a base material, a film-forming material, a pH modifier, an osmotic pressure modifier, a preservative, a bacteriostatic agent, a viscosity modifier.

In one aspect, the medicament of the present invention is prepared into an ophthalmic preparation, such as one of eye drops, eye washes, eye ointments, eye films, eye gels, eye sustained release preparations and eye implants.

In a preferred aspect, the medicament of the present invention is prepared into an ophthalmic gel, wherein the ophthalmic gel is an in-situ ophthalmic gel which is in a liquid state during storage but in a gel state after being dropped into the eye.

Preferably, the in-situ forming eye gel is prepared from (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridinyl ] amino ] ethyl ] pent-2-enamide, poloxamer 407, Gellan gum (Gellan gum), hydroxypropyl- β -cyclodextrin, chlorobutanol, mannitol, triethanolamine and water for injection.

Further preferably, per 1000mL of the in-situ eye gel contains 2.5-10g of (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide, 4073-12g of poloxamer, 2-8g of gellan gum, 1-4g of hydroxypropyl- β -cyclodextrin, 0.8-3.2g of chlorobutanol, 1.5-6g of mannitol, 1-4g of triethanolamine and the balance of water for injection, for example, per 1000mL of the in-situ eye gel contains (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide, 4076g of poloxamer, 4g of gellan gum, 2g of hydroxypropyl- β -cyclodextrin, 1.6g of chlorobutanol, 1.6g of triethanolamine and the balance of water for injection.

Preferably, the preparation method of the in-situ forming eye gel comprises the following steps:

(1) taking a proper amount of water for injection, adding poloxamer 407, gellan gum and chlorobutanol, heating and boiling for 10-20 minutes under stirring, cooling, standing for 18-30 hours, and filtering;

(2) adding hydroxypropyl- β -cyclodextrin, mannitol and triethanolamine into the mixture prepared in the step (1) under stirring, uniformly mixing, and then adding (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide;

(3) filtering the mixture prepared in the step (2), adding water for injection to a sufficient amount, passing through a 0.22 micron microporous filter membrane, and aseptically subpackaging into aseptic vials with appropriate volume to obtain the injection.

The invention also provides application of the medicine in preparing a medicinal preparation for treating keratitis.

In one aspect, the keratitis is bacterial keratitis, particularly bacterial keratitis caused by a staphylococcus aureus infection.

Preferred embodiments of the present invention and effects thereof will be described below with reference to specific examples. It should be understood, however, that the description is for illustrative purposes only and is not intended to limit the claims of the present invention in any way.

Detailed Description

Example 1

In-situ forming eye gel 1

The prescription of the drugs described in this example is shown in the following table:

components	Dosage of
		Compounds of the invention	2.5g
Poloxamer 407	6g
		Gellan gum	4g
Hydroxypropyl- β -cyclodextrin	2g
		Chlorobutanol	1.6g
Mannitol	3g
		Triethanolamine	2g
Water for injection	Adding to 1000ml

The preparation method comprises the following steps:

(1) taking 700mL of water for injection, adding poloxamer 407, gellan gum and chlorobutanol, heating and boiling for 15 minutes under stirring, cooling, standing for 24 hours, and filtering;

(2) adding hydroxypropyl- β -cyclodextrin, mannitol and triethanolamine into the mixture prepared in the step (1) under stirring, uniformly mixing, and then adding (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide;

(3) filtering the mixture prepared in the step (2), adding water for injection to 1000mL, passing through a 0.22 micron microporous membrane, and aseptically subpackaging into sterile vials (5mL each) to obtain the injection.

Example 2

In-situ forming eye gel 2

The prescription of the drugs described in this example is shown in the following table:

components	Dosage of
		Compounds of the invention	5g
Poloxamer 407	6g
		Gellan gum	4g
Hydroxypropyl- β -cyclodextrin	2g
		Chlorobutanol	1.6g
Mannitol	3g
		Triethanolamine	2g
Water for injection	Adding to 1000ml

The preparation method comprises the following steps:

(1) taking 700mL of water for injection, adding poloxamer 407, gellan gum and chlorobutanol, heating and boiling for 15 minutes under stirring, cooling, standing for 24 hours, and filtering;

(2) adding hydroxypropyl- β -cyclodextrin, mannitol and triethanolamine into the mixture prepared in the step (1) under stirring, uniformly mixing, and then adding (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide;

(3) filtering the mixture prepared in the step (2), adding water for injection to 1000mL, passing through a 0.22 micron microporous membrane, and aseptically subpackaging into sterile vials (5mL each) to obtain the injection.

Example 3

In situ forming eye gel 3

The prescription of the drugs described in this example is shown in the following table:

components	Dosage of
		Compounds of the invention	10g
Poloxamer 407	6g
		Gellan gum	4g
Hydroxypropyl- β -cyclodextrin	2g
		Chlorobutanol	1.6g
Mannitol	3g
		Triethanolamine	2g
Water for injection	Adding to 1000ml

The preparation method comprises the following steps:

(1) taking 700mL of water for injection, adding poloxamer 407, gellan gum and chlorobutanol, heating and boiling for 15 minutes under stirring, cooling, standing for 24 hours, and filtering;

(2) adding hydroxypropyl- β -cyclodextrin, mannitol and triethanolamine into the mixture prepared in the step (1) under stirring, uniformly mixing, and then adding (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide;

(3) filtering the mixture prepared in the step (2), adding water for injection to 1000mL, passing through a 0.22 micron microporous membrane, and aseptically subpackaging into sterile vials (5mL each) to obtain the injection.

Comparative example 1

Tobramycin in situ forming eye gel

The formulation of the drug described in this comparative example is shown in the table below:

the preparation method comprises the following steps:

(1) taking 700mL of water for injection, adding poloxamer 407, gellan gum and chlorobutanol, heating and boiling for 15 minutes under stirring, cooling, standing for 24 hours, and filtering;

(2) adding hydroxypropyl- β -cyclodextrin, mannitol and triethanolamine into the mixture prepared in the step (1) under stirring, uniformly mixing, and then adding tobramycin;

(3) filtering the mixture prepared in the step (2), adding water for injection to 1000mL, passing through a 0.22 micron microporous membrane, and aseptically subpackaging into sterile vials (5mL each) to obtain the injection.

Experimental examples pharmacodynamic examination of the drug of the present invention

Purpose of the experiment:

the purpose of this experimental example was to examine whether the in-situ-forming ophthalmic gel prepared according to the examples of the present invention had the desired efficacy against bacterial keratitis.

Experimental animals:

healthy New Zealand rabbits weighing between 2.6 and 3.0kg (purchased from the medical laboratory animal center in Guangdong province) were used in this experiment.

The experimental process comprises the following steps:

1. establishing a rabbit bacterial keratitis model:

method for slowly injecting 20% urethane solution (5mL/kg) through auricular edge veinNew Zealand rabbits were anesthetized. After the rabbit was unconscious, the New Zealand rabbit was fixed on the operating table and 0.10mL of Staphylococcus aureus (ACTT25923, bacterial concentration 2.5X 10)⁹CFU/mL) was injected into the parenchymal layer of rabbit cornea near the central portion and left to stand for 5min, resulting in white plaque with a diameter of about 2.5mm (only one eye of each new zealand rabbit was selected as the experimental eye and the other eye was the negative control). After the new zealand rabbits come back from the anaesthesia state, the new zealand rabbits are sent back to the laboratory animal room to be raised in a single cage mode.

2. The administration scheme is as follows:

all the bacterial keratitis model rabbits were randomly divided into 5 groups of 12 rabbits each, specifically: blank control group, positive control group and examples 1, 2 and 3. Dosing for each group was initiated 24 hours after molding for 1 week, with the following dosing schedule:

blank control group: physiological saline was administered to the affected eyes of New Zealand rabbits 2 drops at a time (about 0.05mL per drop), twice daily.

Positive control group: the in-situ forming ophthalmic gel of comparative example 1 was applied to the affected eye of a new zealand rabbit 2 drops (about 0.05mL per drop) twice daily.

Example 1 group: the in-situ-forming eye gel of example 1 was administered to the affected eyes of New Zealand rabbits 2 drops each time (about 0.05mL each drop), twice daily.

Example 2 group: the in-situ-forming eye gel of example 2 was administered to the affected eyes of New Zealand rabbits 2 drops each time (about 0.05mL each drop), twice daily.

Example 3 group: the in-situ-forming eye gel of example 3 was administered to the affected eyes of New Zealand rabbits 2 drops each time (about 0.05mL each drop), twice daily.

3. The method for evaluating the drug effect comprises the following steps:

since the end of the 1-week-old experiment, the progression of bacterial keratitis in new zealand rabbits was scored according to the international general corneal pathological change scoring table (table 1):

table 1: cornea pathological change scoring table

Finally, the corneal pathological change score values of each experimental group are summarized, and the total score value is divided by the number of animals in each group (12), so as to obtain the average value of the scores.

The experimental results are as follows:

the specific experimental results are shown in table 2.

TABLE 2 pathological corneal Change scores for the respective groups

Note: results are expressed as mean ± standard deviation; indicates comparison to the blank control group: p < 0.05.

The results in table 2 indicate that the corneal pathological changes of the new zealand rabbits of the blank control group are obvious, indicating that the modeling of the rabbit bacterial keratitis model is successful. The corneal pathological change scores were significantly reduced in the positive control group of new zealand rabbits compared to the data for the blank control group, indicating that tobramycin was effective for treating bacterial keratitis caused by staphylococcus aureus infection.

Further, the results in Table 2 suggest that the administration of the in-situ eye gel of examples 1-3 can significantly reduce the corneal pathological change score of New Zealand rabbits, and the effect is significantly better than that of the positive control tobramycin (e.g., the corneal pathological change score of the positive control group is 2.54 + -0.37, while that of example 2 is 1.68 + -0.29 at the same dose), thus indicating that (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide is significantly more effective for treating bacterial keratitis caused by Staphylococcus aureus infection than tobramycin.

In conclusion, the above results suggest that the medicament of the present invention can be used in clinical treatment as an effective therapeutic medicament for the treatment of bacterial keratitis, particularly bacterial keratitis caused by staphylococcus aureus infection.

Claims (10)

1. A medicament for the treatment of keratitis, said medicament comprising (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridinyl ] amino ] ethyl ] pent-2-enamide and a pharmaceutically acceptable ophthalmic excipient.

2. The medicament of claim 1, wherein the pharmaceutically acceptable ophthalmic excipient is one or more of an ophthalmic solvent, a matrix material, a film-forming material, a pH modifier, an osmotic pressure modifier, a preservative, a bacteriostatic agent, a viscosity modifier.

3. The medicament of claim 1 or 2, wherein the medicament is prepared as an ophthalmic formulation.

4. The medicament of claim 3, wherein the ophthalmic formulation is one of an eye drop, an eye lotion, an eye ointment, an eye film, an eye gel, an eye sustained release formulation, and an eye implant.

5. The medicament according to claim 1 or 2, wherein the medicament is prepared as an ophthalmic gel, wherein the ophthalmic gel is an ophthalmic in-situ gel which is in a liquid state during storage but in a gel state after instilling into the eye.

6. The medicament according to claim 5, wherein the ophthalmic in-situ gel is prepared from (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridinyl ] amino ] ethyl ] pent-2-enamide, poloxamer 407, gellan gum, hydroxypropyl- β -cyclodextrin, chlorobutanol, mannitol, triethanolamine and water for injection.

7. The medicament according to claim 6, wherein the in-situ eye gel contains 2.5 to 10g of (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide, 4073 to 12g of poloxamer, 2 to 8g of gellan gum, 1 to 4g of hydroxypropyl- β -cyclodextrin, 0.8 to 3.2g of chlorobutanol, 1.5 to 6g of mannitol, 1 to 4g of triethanolamine and the balance of water for injection per 1000 mL.

8. The medicament of claim 7, wherein the preparation method of the in-situ eye gel comprises the following steps:

(1) taking a proper amount of water for injection, adding poloxamer 407, gellan gum and chlorobutanol, heating and boiling for 10-20 minutes under stirring, cooling, standing for 18-30 hours, and filtering;

(2) adding hydroxypropyl- β -cyclodextrin, mannitol and triethanolamine into the mixture prepared in the step (1) under stirring, uniformly mixing, and then adding (E) -4,4,5, 5-tetrafluoro-N- [2- [ [ 2-methyl-6- (2-methylbutoxy) -4-pyridyl ] amino ] ethyl ] pent-2-enamide;

(3) filtering the mixture prepared in the step (2), adding water for injection to a sufficient amount, passing through a 0.22 micron microporous filter membrane, and aseptically subpackaging into aseptic vials with appropriate volume to obtain the injection.

9. Use of a medicament according to any one of claims 1 to 8 in the preparation of a pharmaceutical formulation for the treatment of keratitis.

10. Use according to claim 9, wherein the keratitis is bacterial keratitis, in particular bacterial keratitis caused by a staphylococcus aureus infection.