CN112472703A

CN112472703A - Use of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate for the preparation of ophthalmic formulations

Info

Publication number: CN112472703A
Application number: CN201910865360.0A
Authority: CN
Inventors: 左建平; 何世君; 唐炜; 杨晓倩; 林泽民
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2019-09-12
Filing date: 2019-09-12
Publication date: 2021-03-12
Anticipated expiration: 2039-09-12
Also published as: WO2021047464A1; CN112472703B

Abstract

The present invention relates to the use of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate for the preparation of ophthalmic preparations, and more particularly, to the use of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate for the preparation of ophthalmic preparations for the prevention or treatment of inflammatory injury of the eye caused by insufficient tear secretion.

Description

Use of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate for the preparation of ophthalmic formulations

Technical Field

The invention relates to a new application of 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyric acid methyl ester, in particular to an application of 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyric acid methyl ester in preparing an ophthalmic external preparation for treating ocular inflammation injury caused by insufficient tear secretion.

Background

Dry eye, also known as keratoconjunctivitis sicca, refers to a general term for a group of diseases including ocular discomfort and ocular surface damage due to abnormalities in the quality or quantity of tears or hydrodynamic abnormalities of tears. The clinical manifestations are dry and astringent, burning, foreign body sensation, itching, photophobia, red eyes, blurred vision, visual fluctuation and asthenopia. Severe dry eye can also cause keratitis, corneal angiogenesis, corneal ulceration, and the like. Xerophthalmia is mainly divided into tear deficiency type and evaporation excess type, and meibomian gland dysfunction, xerosis syndrome, vitamin A deficiency, allergy, pregnancy and drug treatment can cause xerophthalmia. With the development of socio-economic and the improvement of living standard of people, there is a tendency that the incidence of dry eye is increased and the diseased population is younger.

At present, the clinical medicines for treating xerophthalmia are divided into antibacterial drugs, hormones drugs, artificial tears and the like, but the treatment effect on xerophthalmia is very limited due to the problems that eye tissues are sensitive and drug resistance is easy to generate and the like.

Scopolamine hydrobromide is an anticholinergic with strong peripheral action, and can block M choline receptor and inhibit gland secretion^[1]. The method can establish stable and durable tear deficiency type xerophthalmia by inducing the reduction of the tear secretion of rats through subcutaneous injection of scopolamine, and is a recognized tear generation deficiency type xerophthalmia animal model^[2-3]。

S-adenosyl-L-homocysteine hydrolase (SAHH) is an enzyme that is widely present in cells and catalyzes the hydrolysis of S-adenosyl-L-homocysteine (AdoHcy) to adenosine and homocysteine (Hcy). Inhibition of SAHH will result in the accumulation of Adohcy within the cell, thereby producing a feedback inhibition of the transmethylation reaction. SAHH inhibitors are classified into three types, type I, type II and type III, depending on the mechanism of inhibition of the enzyme. The type I and type II SAHH inhibitors are irreversible, and because SAHH is widely present in vivo, the irreversible inhibition of the enzyme can generate toxic and side effects. The inhibitory effect of type iii SAHH enzyme inhibitors on enzymes is reversible, and as the activity of the drug diminishes, the activity of the enzyme can be restored, and thus its toxic effect is less. 4- [9- (6-aminopurine) ] -2(S) -hydroxy methyl butyrate is a type III SAHH inhibitor, the cytotoxicity is low, and 4- [9- (6-aminopurine) ] -2(S) -hydroxy methyl butyrate has good treatment effect on animal models for treating diseases such as Experimental Autoimmune Encephalomyelitis (EAE), Systemic Lupus Erythematosus (SLE), Psoriasis (Psoriasisis, Ps) and the like. 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyric acid methyl ester has the following structural formula:

at present, the application and report of 4- [9- (6-aminopurine) ] -2(S) -methyl hydroxybutyrate for treating eye inflammation injury caused by insufficient tear secretion are not available.

Disclosure of Invention

The present inventors have found for the first time the therapeutic effect of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate on inflammatory injury of the eye caused by insufficient tear secretion and thus have completed the present invention.

Accordingly, an object of the present invention is to provide a use of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate for producing an ophthalmic preparation (particularly, an ophthalmic external preparation) for preventing or treating an inflammatory injury of the eye caused by insufficient secretion of lacrimal fluid.

The inventor finds that the curative effect and the convenience of the methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate can be improved by a local administration mode.

Therefore, according to one aspect, the present invention provides the use of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate for the preparation of an ophthalmic preparation (particularly, an ophthalmic external preparation) for preventing or treating ocular inflammatory injury caused by insufficient tear secretion.

In the present invention, the ocular inflammatory injury caused by insufficient lacrimal secretion includes, but is not limited to, ophthalmic diseases such as insufficient lacrimal secretion, corneal and conjunctival inflammatory injuries caused by diseases or overuse of the eye, and the like. Preferably, the ocular inflammatory injury caused by insufficient tear secretion is dry eye, particularly tear-deficient dry eye.

In the present invention, preferably, the ophthalmic preparation may be in various dosage forms known in the art, including, but not limited to, eye drops, ophthalmic gel, ophthalmic ointment, and the like.

The ophthalmic preparation may comprise a therapeutically effective amount of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate and one or more pharmaceutically acceptable conventional excipients. The pharmaceutically acceptable conventional auxiliary materials can be excipient, filler or diluent and the like.

As used herein, the term "therapeutically effective amount" means that amount which has a therapeutic effect and is useful in the prevention or treatment of the particular disease, disorder, or condition described herein. For example, a "therapeutically effective amount" may refer to the amount required to provide a therapeutic or desired effect in the subject being treated. As will be appreciated by those skilled in the art, a therapeutically effective amount will vary with the route of administration, the use of excipients, and the possibility of co-administration with other therapies.

The invention utilizes a dry eye model with insufficient tear secretion of rats induced by scopolamine hydrobromide, researches find that the ophthalmic external preparation containing 4- [9- (6-aminopurine) ] -2(S) -methyl hydroxybutyrate can increase the tear secretion, reduce corneal inflammation injury, increase the number of conjunctival goblet cells, increase mucin secretion, maintain conjunctival barrier function and the like, thereby treating eye diseases caused by insufficient tear secretion of rats and having good clinical application prospect.

Drawings

FIG. 1 shows the results of the treatment of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate of example 2 to increase the amount of scopolamine-induced tear secretion in rats. Wherein, FIG. 1A is a graph showing the amount of tear secretion in each group of rats during the treatment cycle; FIG. 1B is a graph showing the amount of tear secretion by each group of rats on day 0 of treatment;

FIG. 1C is a graph showing the amount of tear secretion by groups of rats on day 3 of treatment; FIG. 1D is a graph showing the amount of tear secretion by groups of rats on day 5 of treatment; FIG. 1E is a graph showing the amount of tear secretion by groups of rats on day 7 of treatment. Wherein, each group is compared with the model group, P <0.05, P <0.01, P < 0.001.

FIG. 2 shows the results of the relief of scopolamine-induced corneal injury in rats by methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate of example 2. Wherein, fig. 2A is a graph showing the corneal fluorescein sodium staining damage score of groups of rats on day 3 of treatment; FIG. 2B is a graph showing the corneal fluorescein sodium staining damage score for groups of rats at day 5; figure 2C is a graph showing the corneal fluorescein sodium staining damage score for groups of rats on day 7 of treatment. Wherein, each group is compared with the model group, P <0.05, P <0.01, P < 0.001.

FIGS. 3A-C show representative graphs of the relief of scopolamine-induced corneal injury in rats by methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate of example 2. Wherein, fig. 3A is an image of rat corneal fluorescein sodium staining observed under a slit lamp on day 3 of treatment; FIG. 3B is a graph of sodium fluorescein staining observed in rat cornea under slit lamp on day 5 of treatment; figure 3C is an image of rat corneal sodium fluorescein staining observed under a slit lamp on day 7 of treatment.

Fig. 4 is a graph showing the results of Periodic Acid Schiff (PAS) staining of rat conjunctival tissues and counting of conjunctival goblet cells 7 days after the treatment in example 2. Wherein, each group is compared with the model group, P <0.05, P <0.01, P < 0.001.

FIG. 5 is a graph showing the results of Periodic Acid Schiff (PAS) staining of rat conjunctival tissues obtained in example 2.

Detailed Description

The invention is further illustrated by the following specific embodiments. The examples provided herein are merely illustrative or explanatory of embodiments of the invention and are not intended to limit the scope of the invention.

EXAMPLE 1.4 preparation of methyl- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate as an eye drop formulation

Methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate may be prepared by synthetic methods known in the art, for example, as described in relation to CN 1014568680B.

The ophthalmic preparation described in this example was an eye drop solution containing 1g or 0.25g or 0.0625g of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate, 0.8g of sodium chloride, 0.02g of potassium chloride, 0.2924g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium dihydrogen phosphate, and pH 7.37, in 100 ml.

The preparation method comprises the following steps:

dissolving 8g of sodium chloride, 0.2g of potassium chloride, 2.924g of disodium hydrogen phosphate dodecahydrate and 0.2g of potassium dihydrogen phosphate in 1L of distilled water, and mixing at room temperature to obtain a solution with a pH value of 7.39, namely a solution A;

taking 1g or 0.25g or 0.0625g of 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyric acid methyl ester, adding the mixture into 100ml of the solution A, standing at room temperature, fully dissolving, and filtering by a sterile filter membrane to prepare the 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyric acid methyl ester eye drops with the content of 1 w/v%, 0.25 w/v% and 0.0625 w/v%, respectively, and the pH value is 7.37.

Example 2. therapeutic Effect of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate drops prepared in example 1 on ocular inflammatory injury caused by hyposecretion of rat tear induced by scopolamine

1. Main experimental materials and sources

(1) Animals: SPF grade SD rat, female, weight 120-: 20180004003956.

(2) main experimental medicine and reagent

4- [9- (6-aminopurine) ] -2(S) -hydroxybutyric acid methyl ester, property: white powder, and made into eye drops according to example 1.

Reagent: scopolamine hydrobromide, available from national pharmaceutical group chemicals, inc, CAS: 6533-68-2, production batch number: # F1713061. The concentration was 1.5 w/v% and 1.75 w/v% in sterile water. Sodium hyaluronate eye drops are produced by Nentan pharmaceutical Co., Ltd, Shentian pharmaceutical (China) Co., Ltd, and are packaged separately, and the imported drug registration certificate is small package certificate number: h20130583, registration certificate number of imported drugs: h20130584, sub-package approval document No.: the national medicine standard character J20130150. The tear detection phenol red cotton thread is produced by Tianjin Jingming New technology development company Limited, and has a production license number: jin food and medicine supervision mechanical production permit No. 20100040, registration number: jin food medicine supervision instrument (standard) 2014 No. 2410002, production lot no: 20171002. the fluorescein sodium injection is produced by Guangzhou Baiyunshanming Mingxing pharmaceutical Co., Ltd, and has the national medicine standard H44023401 and the product batch number 170303.

(3) Main instrument

KOWA hand-held slit lamp, model SL-17, available from Shanghai Yoseng Tree Biotech, Inc. Fluorescence scanner for morbid sections of hamamatsu (model: Nanozomer 2.0 HT).

2. The experimental method comprises the following steps:

(1) grouping experiments: before modeling, all rats are detected by phenol red cotton thread for lacrimal secretion, and are averagely grouped according to the level of the lacrimal secretion, wherein each group comprises 10 rats and the groups respectively comprise: normal control group, model control group, sodium hyaluronate control group, (1%, 0.25%, 0.0625%) eye drop treatment group.

(2) Model construction

Except for the normal control group, each rat was injected with 0.2ml of scopolamine injection with concentration of 1.5 w/v% subcutaneously at 9:00, 12:00 and 15:00 o 'clock and 0.2ml of scopolamine injection with concentration of 1.75 w/v% subcutaneously at 18:00 o' clock continuously for 7 days.

(3) Method of treatment

The rats in the normal control group and the model control group did not intervene, the rats in the sodium hyaluronate control group were treated with 0.1% sodium hyaluronate eye drop (20. mu.l/eye/time) 4 times a day, and the rats in the eye drop treatment group were treated with 1%, 0.25% and 0.0625% 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyric acid methyl ester eye drop (20. mu.l/eye/time) prepared in example 1, 4 times a day. The eye dropping time is about 9:30, 12:30, 15:30 and 18:30 daily, and is continuously carried out for 7 days.

1) Detecting the amount of lacrimal secretion: testing the lacrimal secretion of rats on 0, 3, 5 and 7 days with phenol red cotton thread;

2) corneal injury scoring: staining the cornea with sodium fluorescein on the 3 rd day, the 5 th day and the 7 th day of treatment, and observing the corneal staining area with a slit lamp to score the injury;

3) conjunctival goblet cell count: at the end of the experiment, rat left eye conjunctiva tissues are taken and placed in 4% formaldehyde for fixation at room temperature, paraffin sections are subjected to Periodic Acid Schiff (PAS) staining, 3 visual fields are taken within a 40-time amplification range of each specimen for positive staining goblet cell counting, and the average value of the 3 visual field counting is the conjunctival goblet counting result of the specimen;

(4) results of the experiment

1) As shown in figure 1, 1% and 0.25% of 4- [9- (6-aminopurine) ] -2(S) -hydroxy butyric acid methyl ester eye drops can obviously improve the tear secretion of rats;

2) as shown in fig. 2 and fig. 3A-C, the results of rat bilateral cornea staining with fluorescein sodium show that 1% and 0.25% of 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyric acid methyl ester eye drops can remarkably improve rat corneal injury, and the relieving effect is superior to that of sodium hyaluronate eye drops;

3) as shown in FIG. 4 and FIG. 5, the rat conjunctiva goblet cell count table indicates that 1%, 0.25% and 0.0625% of the eye drops of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate can maintain the normal number of goblet cells in the rat conjunctiva, wherein 1% and 0.25% of the eye drops have better protection effect on the goblet cells than sodium hyaluronate eye drops.

The results show that the 4- [9- (6-aminopurine) ] -2(S) -methyl hydroxybutyrate has obvious treatment effect on the eye injury caused by insufficient tear secretion of rats, can improve the tear secretion, improve the corneal inflammation injury and maintain the conjunctival goblet cell number, has better effect than sodium hyaluronate eye drops, and can be developed into the medicine for treating the eye inflammation injury caused by insufficient tear secretion.

Reference to the literature

[1]Viau S，Maire MA，Pasquis B，et al.Time course of ocular surface annd lacrimal gland changes in a new scopolamine-induced dry eye model[J].Graefes Arch Clin Exp Ophthalmol，2008.246:857-867.

[2] Establishment of rat Dry eye model and its corneal nerve changes [ J ] New ophthalmic Advance, 2014, 34 (5): 422-427.

[3] Eye drops of Liuxue, Xuwen, Duckweed, atropine sulfate eye drops and subcutaneous injection of scopolamine hydrobromide were used to prepare an aqueous fluid deficient type dry eye rabbit model experimental study [ J ]. J. Clin ophthalmology, 2015 (3): 263-266.

Claims

Use of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate for the preparation of an ophthalmic preparation for the prevention or treatment of ocular inflammatory injury caused by insufficient tear secretion.
2. The use of claim 1, wherein the injury to inflammation of the eye due to insufficient tear secretion comprises an ophthalmic disease of damaged inflammation of the cornea or conjunctiva due to insufficient tear secretion caused by a disease or an overuse of the eye.
3. The use of claim 1, wherein the injury to ocular inflammation due to insufficient tear secretion is dry eye.
4. The use of claim 1, wherein the ocular inflammatory injury caused by insufficient tear secretion is tear-deficient dry eye.
5. The use of claim 1, wherein the ophthalmic formulation comprises eye drops, an ophthalmic gel or an ophthalmic ointment.
6. The use according to claim 1, wherein the ophthalmic preparation comprises a therapeutically effective amount of methyl 4- [9- (6-aminopurine) ] -2(S) -hydroxybutyrate and one or more pharmaceutically acceptable excipients.
7. The use according to claim 6, wherein the pharmaceutically acceptable excipient is selected from an excipient, a filler or a diluent.