CN106692949B

CN106692949B - Medicine for treating eye diseases and composition thereof

Info

Publication number: CN106692949B
Application number: CN201611204462.0A
Authority: CN
Inventors: 马素永; 许松山; 聂李亚; 徐立华; 马杉姗; 汤晓闯; 徐宏伟
Original assignee: Beijing Northland Biotech Co Ltd
Current assignee: Beijing Northland Biotech Co Ltd
Priority date: 2016-12-23
Filing date: 2016-12-23
Publication date: 2022-03-15
Anticipated expiration: 2036-12-23
Also published as: CN106692949A

Abstract

The invention provides an application of a pharmaceutical composition containing recombinant human thymosin beta4 in preparing ophthalmic disease drugs, in particular to an application in the aspect of dry eye, which is characterized in that the concentration of the recombinant human thymosin beta4 is 1 mu g/mL-1000 mu g/mL. The invention also provides a pharmaceutical composition containing a pharmaceutically acceptable carrier.

Description

Medicine for treating eye diseases and composition thereof

Technical Field

The invention relates to the technical field of biochemistry and protein engineering, in particular to a medicine capable of treating dry eye syndrome and a composition thereof.

Background

Dry eye refers to a general term for a variety of diseases characterized by abnormal quality or quantity of tear fluid or abnormal kinetics, resulting in decreased tear film stability, and associated ocular discomfort and/or ocular surface tissue pathology, from any cause. Also known as keratoconjunctival xerosis. Common symptoms comprise dry eyes, easy tiredness, itching eyes, foreign body sensation, burning pain sensation, viscous secretion, intolerance of wind, photophobia and sensitivity to external stimulation, frequent lacrimation is caused sometimes by insufficient basic tears and excessive dryness of eyes, reflective tears secretion is stimulated instead, and eyes of severe people can have red swelling, congestion and keratinization, corneal epithelium is stripped off and filiform substance is adhered, and the injury can cause keratoconjunctival lesion and influence vision for a long time. The international dry eye working group defined dry eye in 2007 as a tear and ocular surface disease caused by a variety of factors, including ocular surface discomfort symptoms, visual changes and tear film instability with potential ocular surface damage, with increased tear osmolality and ocular surface inflammatory responses. The dry eye syndrome is manifested by dry eyes, red eyes, easy fatigue, burning sensation, foreign body sensation, photophobia, asthenopia and pain, and serious patients can cause vision loss and even blindness.

The pathogenesis of dry eye is mainly inflammation, apoptosis, sex hormone level disorder and the like, wherein the inflammation is an important factor for causing the dry eye to develop. The ultimate goal of dry eye therapy is to protect the visual function of the patient, inhibit the inflammatory response of the ocular surface, and restore the normal structure and function of the ocular surface. The current dry eye treatment methods include drug treatment and surgical treatment, and mainly comprise drug treatment. The common medicines include artificial tears or autologous serum, anti-inflammatory medicines and the like, and mainly comprise eye drops, such as recombinant bovine basic fibroblast growth factor eye drops, sodium hyaluronate eye drops and the like. Although many drugs are clinically used, satisfactory treatment effect cannot be achieved.

Thymosin beta4 (hereinafter referred to as T beta 4) is a small peptide substance naturally existing in human bodies, and the T beta4 has the highest content in human spleen, thymus, lung and peritoneal macrophages, and then is brain, liver, kidney, testis and heart. Native T beta4 contains 43 amino acids, has a molecular weight of about 5kD, and an isoelectric point of 5.1.

Thymosin beta4 (T β 4) was a hotspot for drug research in recent years. Extensive research has demonstrated that: tbeta 4 is a multifunctional molecule and plays an important role in promoting healing of wounds and corneal injuries, repairing cardiac muscle, promoting tissue regeneration, angiogenesis, resisting apoptosis, resisting inflammation and the like.

CN100360174C discloses the use of thymosin beta4 (T beta 4) in medicaments for promoting reversal of or inhibiting eye degeneration. Sosne G et al reported that thymosin beta4 reduced dry eye symptoms in a mouse model of dry eye (Expert Opinion on Biological Therapy, 15: sup1, 155-161) at a test dose of 0.1% in animals.

Thymosin beta4 has been reported to be synthesized by chemical synthesis, and its structure is identical to natural T beta4, and all contains 43 amino acids. The method has the defects of low chemical synthesis purity, organic impurities and inorganic impurities with similar structures and toxic and side effects. Meanwhile, the chemically synthesized thymosin beta4 has large clinical application dose and general clinical effect. Meanwhile, the chemically synthesized polypeptide has low yield and great difficulty in industrial production, and cannot meet the clinical application. The inventor adopts a genetic engineering method to perform site-specific modification on the N-terminal of the natural thymosin beta4, is easy for large-scale preparation, has low cost, does not cause pollution to the environment, and can meet the clinical requirement. Meanwhile, the activity of the obtained recombinant human thymosin beta4 in the aspect of treating dry eye is unexpectedly found to be obviously higher than that of natural thymosin beta 4.

Disclosure of Invention

The recombinant human thymosin beta4 is a polypeptide protein drug produced by a genetic engineering technology, has a structure different from a natural structure, contains 44 amino acids, has an N-terminal sequence different from that of the natural amino acids, removes acetylation of the N-terminal of the natural thymosin beta4, and adds a Gly or Ala to obtain a natural thymosin beta4 mutant, so that the in vivo biological activity is increased. The recombinant human thymosin beta4 is shown as a sequence 1 and a sequence 2, namely Gly-rh T beta4 and Ala-rh T beta 4. CN100582121C discloses the derivatives and their amino acid sequences.

The invention aims to provide application of a pharmaceutical composition containing recombinant human thymosin beta4 in preparing medicines for treating ophthalmic diseases, preferably dry eye, and is characterized in that the concentration of the recombinant human thymosin beta4 is 1-1000 [ mu ] g/mL.

In some embodiments, the recombinant human thymosin beta4 is at a concentration of 1 μ g/mL to 500 μ g/mL.

In some embodiments, the recombinant human thymosin beta4 is at a concentration of 10 μ g/mL to 100 μ g/mL.

It is another object of the present invention that the pharmaceutical composition contains a pharmaceutically acceptable carrier. The drug carrier comprises one or more of pH regulator, osmotic pressure regulator, absorption enhancer and sterile water for injection.

The pH regulator comprises one or more of hydrochloric acid, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium phosphate.

The osmotic pressure regulator is a reagent for regulating osmotic pressure, and comprises one or more of sodium chloride, glucose, sorbitol, glycerol, PEG, propylene glycol, mannitol, boric acid, borax and sodium sulfate.

The absorption promoting agent provided by the invention comprises but is not limited to one or more of sulfoxides, pyrrolidones, fatty acids and esters thereof, surfactants, propylene glycol, glycerol and the like, and preferably one or more of propylene glycol, glycerol, azepine, cyclodextrin, lecithin, soybean lecithin, phosphatidylglycerol and phosphatidylethanolamine.

It is another object of the invention that the medicament is for topical administration.

Another object of the present invention is that the pharmaceutical composition can be in the form of eye drops, gel, solution, cream, ointment, lotion, lyophilized powder, preferably eye drops and gel.

Drawings

FIG. 1: photographic results of rhT beta4 on anterior segment of eye after treatment of rat model for dry eye

FIG. 2: results of staining of corneal HE and PAS by rh-T beta4 after treatment of rat xerophthalmia model

The following examples are intended to illustrate the invention but are not intended to limit it.

Examples

Example one determination of the Effect of rh T β 4 on the expression of MUC5AC and MUC1 mRNA genes by RT-PCR

The source of the drug is as follows: the natural beta4 in the experiment is obtained by chemical synthesis, the recombinant human thymosin beta4 is provided by Beijing Nuo Si Lande biotechnology GmbH, and is Gly-rh T beta4 and Ala-rh T beta4 respectively, and each sample has two specifications (50,100 mu g/mL).

Conjunctival cells are separated from donated eyeballs from an eye bank, conjunctival goblet cells are cultured and purified in vitro, different thymosin beta4 (Gly-rh T beta 450 mu g/mL, Gly-rh T beta 4100 mu g/mL, Ala-rh T beta 450 mu g/mL, Ala-rh T beta 4100 mu g/mL) are added in the culture process, natural T beta4 is chemically synthesized T beta4 (0.1% is 1mg/mL and is used as a positive control), and PBS is given to a negative control group (negative control). The influence of thymosin beta4 on mRNA of mucin expression related genes MUC5AC and MUC1 is determined by RT-PCR and Western blot.

The in vitro experiment results show that: the rh-T beta4 can promote the proliferation of human goblet cells, promote the expression of MUC5AC and MUC1 mRNA and promote the expression of MUC1 mucin. Compared with the positive control group, the expression of MUC5AC in the four groups of test groups is significantly different, P is less than 0.05, and the expression of MUC1 is obviously higher than that in the positive control group. The Gly-rh T beta4 group and the Ala-rh T beta4 group have similar MUC5AC and MUC1 gene expression results. The results are shown in Table 1.

TABLE 1 RT-PCR assay of MUC5AC, MUC1 Gene expression

Grouping	Dosage form	MUC5AC（2^^ΔΔCT）	MUC1（2^{^ΔΔCT}）
				Negative Control	--	1±0	1±0
Positive control group Tbeta 4	1mg/mL	0.62±0.30	1.32±0.27
				Gly- rh Tβ4	50ug/mL	1.61±0.77*	1.52±0.31
Gly- rh Tβ4	100ug/mL	1.65±0.42	1.57±0.43
				Ala-rh Tβ4	50ug/mL	1.60±0.40*	1.54±0.26
Ala-rh Tβ4	100ug/mL	1.69±0.25*	1.63±0.24

Note: p <0.05 compared to positive control group.

EXAMPLE two results of rhT beta4 on corneal fluorescein sodium scoring in rat model of Dry eye

A rat xerophthalmia model prepared by 0.3% benzalkonium chloride is divided into 6 groups randomly, 6 animals in each group are respectively administered with rh-T beta4 (Gly-rh T beta 450 mu g/mL, Gly-rh T beta 4100 mu g/mL, Ala-rh T beta 450 mu g/mL, Ala-rh T beta 4100 mu g/mL), natural T beta4 (0.1% or 1mg/mL, positive control group) and PBS (negative control group), and the administration is carried out 3 times daily for 7 days continuously. The rh-T beta4 was observed to score for sodium corneal fluorescein. The result shows that the score of the rh-T beta4 and the positive control group is reduced compared with that of the negative control group, and the difference between the Gly-rh T beta 4100 mug/mL dose group and the Ala-rh T beta 4100 mug/mL dose group is obvious (p is less than 0.05) compared with that of the negative control group. Compared with a positive control group, the Ala-rh T beta 4100 mug/mL dose group, the Gly-rh T beta 450 mug/mL dose group and the Gly-rh T beta 4100 mug/mL dose group have obvious difference in result. The results are shown in Table 2.

TABLE 2 corneal fluorescein sodium score

Note: p <0.05 compared to negative control group.

Example results of slit-lamp anterior ocular segment photography and sodium fluorescein staining scoring of triple rh-T beta4 after treatment of rat model for Dry eye disease

A rat xerophthalmia model prepared by 0.3% benzalkonium chloride is adopted, xerophthalmia animals are randomly divided into 6 groups, 6 animals in each group are respectively given different rh-T beta4 (Gly-rh T beta 450 mu g/mL, Gly-rh T beta 4100 mu g/mL, Ala-rh T beta 450 mu g/mL, Ala-rh T beta 4100 mu g/mL), natural T beta4 (0.1% namely 1mg/mL, positive control group) and PBS (negative control group), and the administration is carried out for 7 days by dropping 3 times daily. The results are shown in FIG. 1. The fluorescein sodium staining examination result shows that cornea repair effects of a Gly-rh T beta 4100 mug/mL dose group and an Ala-rh T beta 4100 mug/mL dose group are most obvious and are obviously different from those of a positive control drug, a Gly-rh T beta 450 mug/mL dose group is basically consistent with that of the positive control group, and Ala-rh T beta 450 mug/mL dose group is not obviously different from that of a negative control group.

Example four pathological examination results

The results of corneal HE and PAS staining, i.e., pathological examination, after treatment of the rat dry eye model with rh-T beta4 are shown in FIG. 2.

Normal group: normal tissue structure is visible in the cornea and conjunctival epithelium stained by HE, and goblet cells are visible in PAS staining;

model group: corneal and conjunctival epithelial detachment;

negative control group: epithelial tissues are repaired to a certain extent, and a small amount of goblet cells are visible;

positive control group: epithelial tissue repair is good, and goblet cells can be seen;

ala-rh T β 450 ug/mL panel: epithelial tissue has some degree of repair, but not obvious;

ala-rh T β 4100 ug/mL panel: the epithelial cells are repaired more perfectly, and the goblet cells are distributed uniformly;

gly-rh T beta 450 mug/mL group: epithelial tissue repair is good, and goblet cells can be seen;

gly-rh T beta 4100 mug/mL group: the epithelial cells are repaired more perfectly, and the goblet cells are distributed uniformly;

and (4) conclusion: the epithelial tissue repair of the Gly-rh T beta 4100 ug/mL group and the Ala-rh T beta 4100 mug/mL group is better, basically close to normal and better than that of the positive control group.

<110> Beijing Nuo Si Lande Biotechnology GmbH

<120> a medicine for treating eye diseases and composition thereof

<160> 2

<210> 1

<211> 44

<212> PRT

<213> Artificial sequence

<400> 1

Gly Ser Asp Lys Pro Asp Met Ala Glu Ile Glu Lys Phe Asp Lys

1 5 10 15

Ser Lys Leu Lys Lys Thr Glu Thr Gln Glu Lys Asn Pro Leu Pro

20 25 30

Ser Lys Glu Thr Ile Glu Gln Glu Lys Gln Ala Gly Glu Ser

35 40

<210>2

<211>44

<212>PRT

<213> Artificial sequence

<400>2

Ala Ser Asp Lys Pro Asp Met Ala Glu Ile Glu Lys Phe Asp Lys

1 5 10 15

Ser Lys Leu Lys Lys Thr Glu Thr Gln Glu Lys Asn Pro Leu Pro

20 25 30

Ser Lys Glu Thr Ile Glu Gln Glu Lys Gln Ala Gly Glu Ser

35 40

Claims

1. An application of a pharmaceutical composition containing recombinant human thymosin beta4 in preparing a medicine for treating xerophthalmia, wherein an amino acid sequence of the recombinant human thymosin beta4 is shown as a sequence 1 in a sequence table.

2. The use of claim 1, wherein the recombinant human thymosin β 4 is at a concentration of 1-1000 μ g/mL.

3. The use of claim 1, wherein the recombinant human thymosin β 4 is at a concentration of 1-500 μ g/mL.

4. The use of claim 1, wherein the recombinant human thymosin β 4 is at a concentration of 10-100 μ g/mL.

5. The use of any one of claims 1-4, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

6. The use according to claim 5, wherein the pharmaceutically acceptable carrier comprises one or more of a pH regulator, an osmotic pressure regulator, an absorption enhancer and sterile water for injection.

7. The use according to any one of claims 1 to 4, wherein the medicament is a topically administered medicament.

8. Use according to any one of claims 1 to 4, wherein the pharmaceutical composition is in the form of eye drops, a gel, a solution, a cream, an ointment, a lotion or a lyophilized powder.

9. Use according to claim 8, wherein the pharmaceutical composition is in the form of eye drops.

10. The use according to claim 8, wherein the pharmaceutical composition is in the form of a gel.