CN111346217A

CN111346217A - New application of polypeptide TDL23

Info

Publication number: CN111346217A
Application number: CN202010126308.6A
Authority: CN
Inventors: 许元生; 苏博雅; 卢圆; 汤宇晴
Original assignee: Dr August Wolff & CoKg Arzneimittel GmbH
Current assignee: Dr August Wolff & CoKg Arzneimittel GmbH
Priority date: 2020-02-27
Filing date: 2020-02-27
Publication date: 2020-06-30
Anticipated expiration: 2040-02-27
Also published as: CN111346217B

Abstract

The polypeptide TDL23 is found for the first time to be used for treating xerophthalmia and can be developed into a novel medicine for treating xerophthalmia. When the polypeptide TDL23 is used for treating xerophthalmia, the treatment effect is superior to that of the existing treatment medicine (artificial tears). The invention also provides application of the polypeptide with the amino acid sequence consistency of more than 22/23 compared with the polypeptide TDL23 in preparing a medicine for treating dry eye.

Description

New application of polypeptide TDL23

Technical Field

The invention relates to the technical field of dry eye treatment, in particular to a new application of polypeptide TDL23 in preparing a medicine for treating dry eye.

Background

According to the disclosed prior art, the polypeptide TDL23 can be used for the related purposes of bone metabolism regulation, such as promoting bone formation, cartilage formation and the like, and the polypeptide TDL23 is composed of 23 amino acids, and the amino acid sequence of the polypeptide TDL23 is TDLQERGDNDISPFSGDGQPFKD (SEQ ID NO. 1). However, in the prior art, no related technical report that the polypeptide TDL23 is used for treating dry eye is found.

Disclosure of Invention

Based on the above problems, the object of the present invention is to provide a novel use of polypeptide TDL23, namely for the treatment of dry eye.

In order to achieve the purpose, the invention adopts the technical scheme that:

the application of the polypeptide with the amino acid sequence consistency of more than 22/23 compared with the polypeptide TDL23 in the preparation of the medicine for treating the xerophthalmia. It should be noted that, compared with the amino acid sequence shown in SEQ ID No.1, the amino acid sequence of the polypeptide for treating dry eye of the present invention may have a substitution, inversion or deletion of one amino acid at the N-terminal or C-terminal of polypeptide TDL23, and another amino acid at the opposite end, and it falls within the scope of the present invention as long as the converted polypeptide still has the effect of treating dry eye.

As a further optimization of the scheme, the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

As a further optimization of the scheme, the administration concentration of the polypeptide is 0.1-1 mg/ml.

As a further optimization of the above protocol, the polypeptide is administered at a concentration of 1 mg/ml.

In another aspect of the present invention, there is provided a medicament for treating dry eye, which comprises a polypeptide having an amino acid sequence identity of 22/23 or more to TDL 23.

Through a plurality of experiments, the inventor of the application finds that the effect of treating the dry eye is better when the administration concentration of the polypeptide TDL23 is 1 mg/ml.

As a further optimization of the scheme, the medicine contains a solvent, and the solvent is normal saline. It should be noted that the solvent of the drug for treating dry eye of the present invention includes, but is not limited to, physiological saline, and other conventional solvents can be used as long as the therapeutic effect of the polypeptide TDL23 of the present invention for treating dry eye is not affected.

In conclusion, the beneficial effects of the invention are as follows:

the TDL23 polypeptide is found for the first time to be used for treating xerophthalmia and can be developed into a novel medicine for treating xerophthalmia; when the polypeptide TDL23 is used for treating xerophthalmia, the administration concentration of TDL23 is 0.1-1mg/ml, the administration mode is eye drop administration, 2-3 times a day, and the solvent is normal saline; when the polypeptide TDL23 is used for treating xerophthalmia, the treatment effect is superior to that of the existing treatment medicine (artificial tears).

Drawings

FIG. 1 is an HPLC chromatogram of solid phase synthesis of polypeptide TDL 23;

FIG. 2 is a graph of the results of the effect of test samples on mouse dry eye model sodium fluorescein staining scores (7 days, 14 days);

FIG. 3 is a graph showing the effect of a test sample on the amount of tear secretion in a mouse model of dry eye;

FIG. 4 is a graph of the effect of test samples on pathology scores in a mouse model of dry eye;

FIG. 5 is a photomicrograph of a normal control group (× 200);

FIG. 6 is a photomicrograph of a model control group (× 200);

FIG. 7 is a photomicrograph of the low dose group (× 200) of polypeptide TDL 23;

FIG. 8 is a photomicrograph of the high dose group (× 200) of polypeptide TDL 23;

fig. 9 is a photomicrograph of the positive control group (× 200).

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods of the present invention are all conventional methods, and reagents, materials, cells, experimental animals, etc. of the present invention are commercially available or publicly available.

EXAMPLE 1 polypeptide Synthesis

TDL23 polypeptide was synthesized in a total of 500mg with a purity of > 95% by solid phase synthesis, and the HPLC chromatogram of the polypeptide TDL23 solid phase synthesis is shown in FIG. 1.

Example 2 modeling, grouping and administration

1) Molding: 80 male SPF-grade Balb/c mice at the age of 8 weeks are quarantined for 3-7 days. Sucking 5 mu L of 1% fluorescein sodium by using a pipette gun, dripping the fluorescein sodium into the right eye of the mouse, flushing the right eye by using 100 mu L of normal saline after 90s, continuously flushing for three times, slightly sucking away liquid around the canthus by using a cotton swab, observing the right eye of the mouse under the cobalt blue light of a slit lamp, and selecting the mouse with normal surface of the right eye to perform xerophthalmia modeling. 5 mu L of 0.2% benzalkonium chloride solution is dripped into the right eye of the modeled mouse, 2 times a day for 4 weeks, and the model of the xerophthalmia caused by the rapid evaporation of the mouse is induced. Normal control group was given physiological saline, and the other operations were the same.

2) Grouping: after molding for 4 weeks, the animals were randomly divided into 5 groups by score, namely a normal control group, a model control group, a TDL23 low dose group (0.1mg/mL), a TDL23 high dose group (1mg/mL) and a positive control group (artificial tears), and each group contains 10 animals.

3) Administration: after grouping, each group is given the corresponding test drug treatment, and the administration scheme is shown in table 1.

TABLE 1 dosing regimens for various groups of animals

Example 3 Effect detection

The eye surface morphology of the mice of example 2 was observed and recorded under slit lamp cobalt blue light after 3 days, 5 days, 7 days, 10 days, and 14 days of treatment, respectively, with a sodium fluorescein stain score, tear secretion was measured with phenol red cotton thread, and the animals were sacrificed after 14 days of treatment to take the eyeballs and the surrounding eyelids for histopathological observation.

1) Corneal fluorescein sodium staining score

Sucking 5 mu L of 1% liquid fluorescein sodium by using a pipette gun, dripping the liquid fluorescein sodium onto the surface of a mouse eye, closing an eyelid to uniformly coat the fluorescein sodium on the surface of a cornea, flushing the cornea surface for 3 times by using 200 mu L of physiological saline after 90s, slightly sucking the liquid around the eye by using a cotton swab, and observing the staining condition of the corneal epithelium fluorescein sodium under cobalt blue light of a slit lamp microscope. The ocular surface is divided into four quadrants, and each quadrant is scored, with the scoring divided into four levels: no dyeing is 0 min; slight punctate staining was 0.5 point; the diffuse dotted dyeing is 1 point; diffuse staining covering less than one third of the cornea was score 2; diffuse staining covering one-third to two-thirds of the cornea for 3 points; diffuse staining covering more than two thirds of the cornea was divided into 4 points, and the four quadrants were divided into 12 points in total, with higher scores indicating more severe dry eye.

2) Measurement of tear secretion amount

Anaesthetizing and fixing the mouse, gently pulling the lower eyelid of the mouse to expose the lower conjunctival sac by using the micro conjunctival forceps, placing one end of the phenol red cotton thread in the inner conjunctival sac of the lower eyelid for a period of time, taking out the phenol red cotton thread, and measuring the total length of the red part of the phenol red cotton thread soaked by tears.

3) Pathological observation

After the experiment, the mice are killed by anesthesia, the whole eyeballs including the upper eyelid and the lower eyelid are rapidly taken down, the eyeball stationary liquid is fixed and dyed, pathological sections are manufactured, and the pathological changes of conjunctiva and corneal tissues are observed under an optical microscope.

Data processing: experimental data were statistically processed by GraphPad Prism 7.0 biometrics software: the metric data are expressed as Mean ± SD and analyzed using two-way ANOVA combined with Dunnett's multiple comparisons; analysis is carried out by combining variance analysis with Dunnett's multiple comparison method; the counting data is analyzed by Kruskal-Wallis rank sum test; one-way ANOVA was used in conjunction with Dunnett's multiple comparison method for analysis.

4) Results and analysis of the experiments

4.1) general State Observation and body weight

The general state of each group of mice has no obvious abnormality; compared with a normal control group, the body weight of the mice in the model control group is not statistically different (P > 0.05); the mice weight average of each treatment group was not statistically different (P >0.05) compared to the model control group, and the results are shown in table 2.

Table 2 effect of test samples on body weight of mouse model with dry eye syndrome (n 10, Mean ± SD)

Time/group classification	Normal control group	Model control group	TDL23 Low dose group	TDL23 high dose	Positive control group
						Before administration	27.03±1.35	27.34±1.67	26.84±2.76	27.13±1.69	27.68±2.49
The administration is carried out for 7 days	27.59±1.68	27.85±1.40	27.38±2.85	27.98±1.85	27.46±1.98
						The administration is carried out for 14 days	28.42±1.79	28.68±1.45	28.96±2.97	28.67±2.81	28.28±2.23

Note: compared to the model control group,: p < 0.05; **: p < 0.01.

4.2) corneal fluorescein sodium staining score

Compared with a normal control group, the fluorescein staining score of the model control group is obviously increased, and the statistical difference (P <0.05 or <0.01) is provided, which indicates that the molding is successful. The treatment groups tended to decrease the sodium fluorescein ocular surface score compared to the model control group, with the TDL23 high dose group (1mg/mL) being the most significant and statistically different (P <0.01), with the results shown in table 3 and fig. 2.

Table 3 effect of test samples on sodium fluorescein ocular scoring in a mouse model of dry eye (n 10, Mean ± SD)

Note: compared to the model control group,: p < 0.05; **: p < 0.01.

4.3) measurement of tear secretion amount

Compared with the normal control group, the length of the phenol red cotton thread of the mouse of the model control group is obviously shortened, and the lacrimal secretion amount is obviously reduced (P <0.01), which indicates that the model building is successful. Compared with the model control group, each treatment group has the tendency of increasing the length of the phenol red cotton thread and increasing the amount of the lacrimal fluid secretion, and the TDL23 high dose group (1mg/mL) and the positive control group are the most significant and have significant statistical difference (P <0.01), and the results are shown in Table 4 and figure 3.

Table 4 influence of test sample on lacrimal secretion of mouse xerophthalmia model (n 10, Mean ± SD)

Note: compared to the model control group,: p < 0.05; **: p < 0.01.

4.4) pathological changes

Compared with the normal control group, the pathological score of the model control group is obviously increased (P < 0.01). Each treatment group had a tendency to decrease pathology scores compared to the model control group, with the TDL23 high dose group (10)

mg/mL) and positive control group are most obvious and have significant statistical significance (P <0.01), and the results are shown in table 5 and fig. 4. FIGS. 5-9 show micrographs of various sets of conjunctival and corneal tissue.

Table 5 effect of test samples on pathology in a mouse model of dry eye (n 10, Mean ± SD)

Note: compared to the model control group,: p < 0.05; **: p < 0.01.

As can be seen from FIG. 5, the conjunctival epithelium of the normal control group was intact, the epithelial cell morphology was normal, no goblet cell was seen, the corneal front surface epithelium was uniform in thickness, the cell morphology was normal, no cutin was present, the stroma was uniform in thickness, no blood vessel and inflammation were present, a large number of artificial fissures were seen, the corneal endothelium was intact, the thickness was uniform, and no obvious abnormality was seen.

As can be seen from FIG. 6, the conjunctival epithelium of the model control group was intact and normal, a small number of goblet cells were observed, the epithelium on the anterior surface of the cornea had uneven thickness and narrowed locally, and had fissures, and the stroma was heavily swollen, which was manifested as fissure dilatation, the corneal endothelium was intact and uniform in thickness, and no significant abnormality was observed.

As can be seen from fig. 7, the low dose TDL23 group had intact conjunctival epithelium, a small amount of nucleus of epithelium was deeply contracted and fixed, more goblet cells were visible, a small amount of inflammatory cell infiltration was visible in the connective tissue under the epithelium, the epithelium on the anterior surface of the cornea was uniform in thickness, the cell morphology was normal, there was no cutin, the stroma was uniform in thickness, there was no blood vessel, inflammation, a large amount of artificial fissures were visible, the corneal endothelium was intact, the thickness was uniform, and no obvious abnormality was visible; a small amount of inflammatory cell infiltration was seen at the corneal-conjunctival junction.

As can be seen from fig. 8, the high dose of TDL23 showed complete conjunctival epithelium with normal epithelial cell morphology, a small amount of goblet cells, a small amount of inflammatory cell infiltration in the subepithelial connective tissue, uniform thickness of the epithelium on the anterior surface of the cornea, normal cell morphology, no cutin, uniform thickness of the stroma, no blood vessels, no inflammation, a large amount of artificial fissures, complete corneal endothelium, uniform thickness, no obvious abnormality.

As can be seen from fig. 9, the positive control group had intact conjunctival epithelium, normal epithelial cell morphology, no visible goblet cells, a small amount of inflammatory cell infiltration in the subepithelial connective tissue, uniform thickness of the epithelium on the anterior surface of the cornea, normal cell morphology, no cutin, uneven stroma thickness, small crack enlargement, intact corneal endothelium, uniform thickness, and no other visible abnormalities.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Guangzhou Zhicheng medical science and technology Limited

New application of <120> polypeptide TDL23

<130>2020

<160>1

<170>PatentIn version 3.5

<210>1

<211>23

<212>PRT

<213> Intelligent people

<400>1

Thr Asp Leu Gln Glu Arg Gly Asp Asn Asp Ile Ser Pro Phe Ser Gly

1 5 10 15

Asp Gly Gln Pro Phe Lys Asp

20

Claims

1. The application of the polypeptide with the amino acid sequence consistency of more than 22/23 compared with the polypeptide TDL23 in the preparation of the medicine for treating the xerophthalmia.

2. The use according to claim 1, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID No. 1.

3. The use according to claim 2, wherein the polypeptide is administered at a concentration of 0.1 to 1 mg/ml.

4. The use according to claim 3, wherein the polypeptide is administered at a concentration of 1 mg/ml.

5. A drug for treating dry eye, characterized by containing a polypeptide having an amino acid sequence identity of 22/23 or more to the polypeptide TDL 23.

6. The medicament of claim 5, wherein the amino acid sequence of the polypeptide is shown as SEQ ID No. 1.

7. The medicament of claim 6, wherein the polypeptide is administered in a dose of 0.1 to 1 mg/ml.

8. The medicament according to claim 7, wherein the polypeptide is administered in a dose of 1 mg/ml.

9. The medicament according to any one of claims 5 to 8, wherein the medicament contains a solvent, and the solvent is normal saline.