CN111346217B - New application of polypeptide TDL23 - Google Patents
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- CN111346217B CN111346217B CN202010126308.6A CN202010126308A CN111346217B CN 111346217 B CN111346217 B CN 111346217B CN 202010126308 A CN202010126308 A CN 202010126308A CN 111346217 B CN111346217 B CN 111346217B
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Abstract
The polypeptide TDL23 can be used for treating xerophthalmia for the first time, and can be developed into a novel medicine for treating xerophthalmia. When the polypeptide TDL23 is used for treating xerophthalmia, the therapeutic effect of the polypeptide TDL23 is superior to that of the existing therapeutic drug (artificial tear). 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 medicament for treating xerophthalmia.
Description
Technical Field
The invention relates to the technical field of xerophthalmia treatment, in particular to a novel application of polypeptide TDL23 in preparing a medicine for treating xerophthalmia.
Background
According to the disclosed prior art, the polypeptide TDL23 can be used for the purpose related to the regulation of bone metabolism, such as promotion of bone formation, cartilage formation, etc., and the polypeptide TDL23 consists of 23 amino acids, and has the amino acid sequence of TDLQERGDNDISPFSGDGQPFKD (SEQ ID NO. 1). However, in the prior art, no technical report has been found about the use of the polypeptide TDL23 for the treatment of dry eye.
Disclosure of Invention
Based on the above problems, it is an object of the present invention to provide a novel use of polypeptide TDL23, i.e. for the treatment of dry eye.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
application of polypeptide with amino acid sequence consistency of 22/23 or more in preparing medicine for treating xerophthalmia compared with polypeptide TDL 23. 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 xerophthalmia in the invention can be that one of the amino acids is replaced or overturned, or one of the amino acids at the N end or the C end of the polypeptide TDL23 is deleted, and the other amino acid is connected at the opposite end, so long as the polypeptide after the transition still has the effect of treating xerophthalmia, the polypeptide falls into the protection scope of the invention.
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 scheme, the administration concentration of the polypeptide is 1mg/ml.
As another aspect of the present invention, there is also provided a medicament for treating dry eye, which comprises a polypeptide having an amino acid sequence identity of 22/23 or more as compared to the polypeptide TDL 23.
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 scheme, the administration concentration of the polypeptide is 1mg/ml.
The inventors of the present application found that the effect of treating dry eye was better when the administration concentration of the polypeptide TDL23 was 1mg/ml through a plurality of experiments.
As a further optimization of the scheme, the medicine contains a solvent, and the solvent is physiological saline. The solvent of the drug for treating dry eye of the present invention includes, but is not limited to, physiological saline, and may be other conventional solvents as long as the therapeutic effect of the polypeptide TDL23 of the present invention for treating dry eye is not affected.
In summary, the beneficial effects of the invention are as follows:
the invention discovers for the first time that the TDL23 polypeptide can 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 the TDL23 is 0.1-1mg/ml, the administration mode is eye drop administration, 2-3 times a day, and the solvent is physiological saline; when the polypeptide TDL23 is used for treating xerophthalmia, the therapeutic effect of the polypeptide TDL23 is superior to that of the existing therapeutic drug (artificial tear).
Drawings
FIG. 1 is an HPLC chart of solid phase synthesis of polypeptide TDL 23;
FIG. 2 is a graph of results of the effect of test samples on the scoring of sodium fluorescein staining in a dry eye model of mice (7 days, 14 days);
FIG. 3 is a graph showing the effect of a test sample on tear secretion in a mouse dry eye model;
FIG. 4 is a graph showing the effect of a test sample on pathology scores of a dry eye mouse model;
FIG. 5 is a photomicrograph of a normal control group (x 200);
FIG. 6 is a photomicrograph of a model control group (x 200);
FIG. 7 is a photomicrograph of a low dose group (x 200) of polypeptide TDL 23;
FIG. 8 is a photomicrograph of a high dose group (x 200) of polypeptide TDL 23;
fig. 9 is a micrograph of a positive control group (×200).
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise indicated, all methods of experimentation in the present invention are conventional, and reagents, materials, cells, laboratory animals, etc. in the present invention are available from the market or other public sources.
EXAMPLE 1 polypeptide Synthesis
The TDL23 polypeptide is synthesized by adopting a solid phase, the total synthesis is 500mg, the purity is more than 95 percent, and the HPLC chart of the solid phase synthesis of the polypeptide TDL23 is shown in figure 1.
EXAMPLE 2 modeling, grouping and administration
1) And (3) molding: 80 male SPF-class Balb/c mice aged 8 weeks were quarantined for 3-7 days. Sucking 5 mu L of 1% sodium fluorescein by a pipette, dripping the solution into the right eye of a mouse, flushing the right eye with 100 mu L of normal saline after 90s, flushing the solution three times continuously, gently sucking liquid around the canthus by a cotton swab, observing the right eye of the mouse under cobalt blue light of a slit lamp, and selecting the normal mouse on the surface of the right eye for dry eye. The right eye of the molded mice was instilled with 5 μl of 0.2% benzalkonium chloride solution 2 times daily for 4 weeks, inducing the mice to evaporate too rapidly in the dry eye model. Normal control group was given normal saline, and the other operations were the same.
2) Grouping: after molding for 4 weeks, the animals were randomly divided into 5 groups according to scores, namely a normal control group, a model control group, a TDL23 low-dose group (0.1 mg/mL), a TDL23 high-dose group (1 mg/mL) and a positive control group (artificial tears), and each group comprises 10 animals.
3) Administration: the groups were given the corresponding test drug treatments after grouping, and the dosing schedule is shown in table 1.
Table 1 dosing regimen for each group of animals
Example 3 Effect detection
The eye surface morphology of the mice of example 2 was observed and recorded under cobalt blue light of a slit lamp after 3 days, 5 days, 7 days, 10 days, and 14 days, respectively, while the scoring for sodium fluorescein staining was performed, the tear secretion was measured with a phenol red cotton thread, and the animals were sacrificed after 14 days of treatment to take eyeballs and surrounding eyelids for histopathological observation.
1) Corneal fluorescein sodium staining score
The 1% liquid state sodium fluorescein was sucked up to 5. Mu.L by a pipette and dropped onto the surface of the eye of the mouse, and the eyelid was closed to uniformly coat the sodium fluorescein on the surface of the cornea, after 90s, the cornea was rinsed 3 times with 200. Mu.L of physiological saline, and after the liquid around the eye was gently sucked up by a cotton swab, the staining of the corneal epithelium sodium fluorescein was observed under cobalt blue light by a slit lamp microscope. The ocular surface was divided into four quadrants and each quadrant was scored, with the scores being divided into four classes: no staining was 0 minutes; slight spot staining was 0.5 minutes; diffuse punctate staining was 1 minute; diffuse staining covering less than one third of the cornea was 2 minutes; diffuse staining covering one third to two thirds of the cornea was 3 minutes; diffuse staining covering more than two thirds of the cornea was 4 points, with four quadrants being divided into 12 points overall, with higher scores indicating more severe dry eye.
2) Tear secretion assay
The mice were anesthetized and fixed, the lower eyelid of the mice was gently pulled with microscopic conjunctival forceps to expose the lower conjunctival sac, one end of the phenol red cotton wire was placed in the inner conjunctival sac of the lower eyelid for a period of time and then removed, and the total length of the red portion of the phenol red cotton wire wetted by tears was measured.
3) Pathological observation
After the experiment is finished, the mice are anesthetized and sacrificed, the whole eyeball including the upper eyelid and the lower eyelid is rapidly removed, the eyeball fixing solution is fixed and stained, pathological sections are manufactured, and the pathological changes of conjunctiva and cornea tissues are observed under an optical microscope.
And (3) data processing: experimental data were statistically processed by GraphPad Prism 7.0 biometric software: metering data are expressed in mean±sd and analyzed using two-way ANOVA in combination with Dunnett's multiple comparison method; analysis is carried out by adopting a variance analysis combined with Dunnett's multiple comparison method; the count data were analyzed using the Kruskal-Wallis rank sum test; analysis was performed using one-way ANOVA in combination with Dunnett's multiplex comparison.
4) Experimental results and analysis
4.1 General state observation and weight
The general state of each group of mice is free from obvious abnormality; the body weight of the mice in the model control group was not statistically different (P > 0.05) compared to the normal control group; there was no statistical difference in mice weights (P > 0.05) for each treatment group compared to the model control group, and the results are shown in table 2.
Table 2 effect of sample on body weight of dry eye mouse model (n=10, mean±sd)
| Time/group | Normal control group | Model controlGroup of | TDL23 low dose group | TDL23 high dose | Positive control group |
| Before administration of the drug | 27.03±1.35 | 27.34±1.67 | 26.84±2.76 | 27.13±1.69 | 27.68±2.49 |
| 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 |
| 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 that: compared to model control group: p <0.05; * *: p <0.01.
4.2 Corneal fluorescein sodium staining score
The model control group had significantly higher fluorescein staining scores compared to the normal control group, with statistical differences (P <0.05 or < 0.01), indicating successful modeling. Each treatment group had a trend to decrease the fluorescein sodium ocular surface score compared to the model control group, with the TDL23 high dose group (1 mg/mL) being most pronounced and there was a clear statistical difference (P < 0.01), see table 3, fig. 2.
Table 3 effect of sample on fluorescein sodium ocular surface score of dry eye mouse model (n=10, mean±sd)
Note that: compared to model control group: p <0.05; * *: p <0.01.
4.3 Tear secretion amount measurement
Compared with the normal control group, the length of the phenol red cotton wire of the mice in the model control group is obviously shortened, and the tear secretion is obviously reduced (P is less than 0.01), which indicates that the modeling is successful. Each treatment group had a trend to increase the length of phenol red cotton and increase the amount of tear secretion compared to the model control group, and was most pronounced in the TDL23 high dose group (1 mg/mL), the positive control group, and had significant statistical differences (P < 0.01), as the results are shown in table 4, fig. 3.
Table 4 effects of sample on tear secretion in the mouse dry eye model (n=10, mean±sd)
Note that: compared to model control group: p <0.05; * *: p <0.01.
4.4 Pathologic changes
Model control pathology scores were significantly elevated compared to normal control (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), positive control group was most evident and had significant statistical significance (P < 0.01), see table 5, fig. 4. Fig. 5 to 9 show photomicrographs of the conjunctiva and cornea tissue of each group.
Table 5 effects of sample on pathology in dry eye mouse model (n=10, mean±sd)
Note that: compared to model control group: p <0.05; * *: p <0.01.
As can be seen from fig. 5, the normal control group has intact conjunctival epithelium, normal epithelial cell morphology, no goblet cells, uniform thickness of the anterior surface epithelium of cornea, normal cell morphology, no horniness, uniform thickness of matrix, no blood vessel or inflammation, and a large number of artificial fissures, and the cornea endothelium is complete and uniform in thickness, and is not obviously abnormal.
As can be seen from fig. 6, the conjunctival epithelium of the model control group was complete and normal, a small amount of goblet cells were seen, the anterior surface epithelium of the cornea was uneven in thickness and locally narrowed, and fissures were developed, and severe swelling of the stroma was manifested as fissure expansion, the corneal endothelium was complete and uniform in thickness, and no obvious abnormality was seen.
As can be seen from fig. 7, the TDL23 low dose group conjunctiva epithelium is complete, a small amount of epithelial nuclei is densely stained, more goblet cells are visible, a small amount of inflammatory cell infiltration is visible in the connective tissue below the epithelium, the thickness of the epithelium on the front surface of the cornea is uniform, the cell morphology is normal, no horniness, the thickness of the stroma is uniform, no blood vessel and inflammation are avoided, a large amount of artificial fissures are visible, the cornea endothelium is complete, the thickness is uniform, and no obvious abnormality is seen; a small amount of inflammatory cell infiltration is seen at the interface of the cornea and conjunctiva.
As can be seen from fig. 8, the TDL23 high dose conjunctival epithelium is intact, the morphology of the epithelial cells is normal, a small number of goblet cells are visible, a small number of inflammatory cells infiltrate into the connective tissue under the epithelium, the anterior surface epithelium of the cornea is uniform in thickness, the morphology of the cells is normal, no keratin is present, the thickness of the stroma is uniform, no blood vessel or inflammation is present, a large number of artificial fissures are visible, the corneal endothelium is intact, the thickness is uniform, and no obvious abnormality is visible.
As can be seen from fig. 9, the positive control group had intact conjunctival epithelium, normal morphology of epithelial cells, no apparent goblet cells, small inflammatory cell infiltration in the subepithelial connective tissue, uniform thickness of the epithelium at the anterior surface of the cornea, normal morphology of cells, no horniness, uneven thickness of the stroma, as evidenced by small number of enlarged fissures, intact corneal endothelium, uniform thickness, and no other apparent abnormalities.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been 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 to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Guangzhou Zhencheng medical science and technology Co., ltd
New application of <120> polypeptide TDL23
<130> 2020
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> PRT
<213> Chile person
<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 (3)
1. The application of the polypeptide with the amino acid sequence shown as SEQ ID NO.1 in preparing a medicament for treating xerophthalmia.
2. The use according to claim 1, wherein the polypeptide is administered at a concentration of 0.1 to 1mg/ml.
3. The use according to claim 1, wherein the polypeptide is administered at a concentration of 1mg/ml.
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| EP1841444A4 (en) * | 2005-01-07 | 2009-12-09 | Acologix Inc | Formulations of peptides for periodontal and dental treatments |
| ES2542509T3 (en) * | 2007-01-22 | 2015-08-06 | Orthotrophix, Inc. | A peptide composition and a method of stimulating cartilage formation |
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| 干眼症病因及治疗研究分析;黄妍;《求医问药(下半月)》;20121001(第10期);479页左栏第4段 * |
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