Application of anti-periostin antibody in preparation of medicine for preventing and treating pterygium

Abstract

The invention relates to the technical field of medicaments, in particular to application of an anti-periostin antibody in preparation of a medicine for preventing and treating pterygium. The invention provides an application of an anti-periostin antibody in preparation of a medicine for preventing and treating pterygium. The invention also provides the anti-periostin antibody eye drops, auxiliary materials are screened, the dosage is optimized, and the obtained eye drops have good stability and still have good titer after being stored for a long time. In animal experiments, the compound also shows good physiological activity, and can have a good treatment effect on pterygium.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to application of an anti-periostin antibody in preparing a medicine for preventing and treating pterygium. Background Art

[0002] Pterygium is a proliferative disease in which local conjunctival tissue proliferates in a triangular shape and invades the cornea. It is one of the most common ophthalmic diseases. At present, most scholars believe that pterygium is caused by the damage of corneal limbal stem cells by external factors, such as ultraviolet radiation, inflammatory stimulation, etc., which causes active cell proliferation, inflammatory response, vascular proliferation, fibrous tissue proliferation, connective tissue remodeling, and corneal "conjunctivalization" to form pterygium. The main pathological characteristics include: disordered expression of certain cytokines that regulate proliferation in pterygium tissue, such as transforming growth factor, basic fibroblast growth factor, etc.; active proliferation of pterygium fibroblasts; active proliferation of pterygium epithelial cells, excessive keratinization, and thickening of epithelial cells; in addition, the number of proteins related to cell apoptosis increases, such as increased expression of p53 protein and increased expression of bcl-2. The above pathological changes indicate that the occurrence of pterygium is related to inflammation, tissue plasticity, vascularization and fibrosis.

[0003] At present, the treatment of pterygium includes surgical treatment and drug treatment. Among them, pterygium surgery is an effective method, but the postoperative recurrence rate is high (more than 20%). This is mainly due to the destruction of the blood-eye barrier caused by surgery and trauma, the increase of vascular permeability, the migration of fibrinogen, inflammatory cells, and fibroblasts into the eye, and then converted into fibrin under the action of thrombin, so that pterygium surgery relapses, which seriously affects the therapeutic effect and success of the surgery. Therefore, inhibiting the fibrosis of postoperative tissues and reducing scar formation are the key to preventing recurrence. At present, auxiliary treatment methods are required to control the recurrence of pterygium after surgery. For example: after pterygium resection, short-term use of glucocorticoids may prevent the recurrence of pterygium by inhibiting local inflammatory response; or by giving drugs that inhibit cell proliferation, such as mitomycin, which inhibits cell DNA replication and inhibits cell growth. However, the effects of these drugs are currently very limited.

[0004] With the maturity and application of antibody technology, it provides a strong guarantee for the precise treatment of various diseases and brings hope of cure to many intractable diseases. It has the advantages of strong specificity, high sensitivity, and large-scale preparation. In the fibrosis process of tissues and organs, many cytokines are involved, such as connective tissue growth factor, fibroblast growth factor, epidermal growth factor, etc. Therefore, trying to use antibodies to inhibit or block these cytokines or their receptors has become a research hotspot for the treatment of pterygium. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the use of anti-periostin antibodies in the preparation of drugs for preventing and treating pterygium.

[0006] Application of anti-periostin antibodies in the preparation of drugs for preventing and treating pterygium.

[0007] The prevention and treatment include inhibiting the migration of retinal pigment epithelial cells, inhibiting the formation of new blood vessels or the vascularization of corneal tissue.

[0008] Pterygium is one of the most common eye diseases in ophthalmology. Its clinical manifestation is the invasion of the transparent cornea by the proliferative fibrovascular tissue. Its main pathological characteristics include active fibroblast proliferation, active pterygium epithelial cell proliferation, hyperkeratosis, and thickening of epithelial cells. The present invention verifies through RPE cell scratch inhibition test and inhibition test of rabbit pterygium caused by alkali burn that Periostin humanized monoclonal antibody eye drops have the effect of preventing and treating pterygium.

[0009] Further, the present invention provides a drug for preventing and treating pterygium, comprising:

[0010] Anti-periostin humanized monoclonal antibody 0.1-5 mg / ml;

[0011] Viscosity lubricant 0.5%~2%;

[0012] Zeta potential regulator 0.01%~3%;

[0013] Prosam 1% to 8%;

[0014] Cyclodextrin 0.1%~4%;

[0015] The balance is water for injection.

[0016] In the present invention, the viscosity lubricant is selected from any one of hyaluronic acid, gelatin, HPMC, polyethylene glycol, and propylene glycol.

[0017] In the present invention, ploxamer is ploxamer 407, 124, 188, 237 or 338.

[0018] In the present invention, the zeta potential regulator is modified chitosan.

[0019] The modified chitosan is selected from acylated modified chitosan, carboxymethyl modified chitosan, carboxymethyl deacetylated chitosan, quaternized modified chitosan, hydroxypropyl modified chitosan, cyclodextrin grafted modified chitosan, crown ether modified chitosan or blended modified chitosan. In the embodiment of the present invention, the modified chitosan is carboxymethyl deacetylated chitosan, which has better effect than other Zeta potential regulators.

[0020] The present invention screens and combines the auxiliary materials in the medicine, and the prepared eye drops can have good stability, so that the anti-periostin antibody can be applied to the ocular surface as a medicine, and the effect of preventing and treating pterygium can be achieved through the administration method of eye drops. In addition, the optimized formula can enable the antibody to play a good role, does not interfere with the physiological activity of the anti-periostin antibody, and is more suitable for tissue absorption of pterygium, further ensuring the prevention and treatment effect.

[0021] In some embodiments, the drug is prepared from the following components:

[0022] Anti-periostin humanized monoclonal antibody 0.1-1 mg / ml;

[0023] Hyaluronic acid 1%;

[0024] Modified chitosan 0.5%~1.5%;

[0025] Prosam 407 2%~5%;

[0026] Cyclodextrin 0.5%~1%;

[0027] The balance is water for injection.

[0028] In other embodiments, the drug is prepared from the following components:

[0029] Anti-periostin humanized monoclonal antibody 0.1-5 mg / ml;

[0030] Polyethylene glycol 0.5% to 2%;

[0031] Modified chitosan 0.01%~3%;

[0032] Prosam 237 1%~8%;

[0033] Cyclodextrin 0.1%~4%;

[0034] The balance is water for injection.

[0035] In other embodiments, the drug is prepared from the following components:

[0036] Anti-periostin humanized monoclonal antibody 0.1-5 mg / ml;

[0037] Polyethylene glycol 0.5% to 2%;

[0038] Modified chitosan 0.01%~3%;

[0039] Prosam 188 1%~8%;

[0040] Cyclodextrin 0.1%~4%;

[0041] The balance is water for injection.

[0042] In a specific embodiment, the drug is prepared from the following components:

[0043] Anti-periostin humanized monoclonal antibody 0.1mg / ml, hyaluronic acid 1%, modified chitosan 1.5%, ploxamer 407 5%, cyclodextrin 0.5%, the balance is water for injection;

[0044] or anti-periostin humanized monoclonal antibody 1mg / ml, hyaluronic acid 1%, modified chitosan 0.5%, ploxamer 407 2%, cyclodextrin 1%, the balance being water for injection;

[0045] or anti-periostin humanized monoclonal antibody 2 mg / ml, polyethylene glycol 0.4%, modified chitosan 1%, ploxamer 237 3%, cyclodextrin 1.5%, the balance being water for injection;

[0046] Or anti-periostin humanized monoclonal antibody 5 mg / ml, polyethylene glycol 0.5%, modified chitosan 0.1%, ploxamer 188 5%, cyclodextrin 0.8%, and the balance is water for injection.

[0047] In the present invention, the percentages are all mass fractions. In the present invention, the degree of polymerization of polyethylene glycol is screened, and preferably, the polyethylene glycol is polyethylene glycol 400.

[0048] In the present invention, the preparation method of the drug comprises:

[0049] The zeta potential regulator and anti-periostin humanized monoclonal antibody were dissolved in PBS and stirred for 0.5 to 1 hour; then cyclodextrin and viscosity lubricant were added in sequence, and after mixing evenly, ploxamer was added, and then water for injection was added.

[0050] In the present invention, after adding water for injection, the preparation method further comprises the steps of adjusting the pH value to 7.0-7.4 and filtering through a 0.45 μm filter membrane.

[0051] The eye drops prepared by the invention have a Zeta potential of +20 to 55 mV and can maintain good stability during storage.

[0052] The present invention provides the use of the above-mentioned drug or the drug prepared by the above-mentioned preparation method in preparing a drug for preventing the occurrence of pterygium, inhibiting the excessive growth of pterygium and / or inhibiting the recurrence of pterygium after surgery.

[0053] In the present invention, the pterygium is a pterygium formed after ocular surface tissue surgery; the ocular surface tissue surgery includes pterygium resection, corneal transplantation, eyelid repair and / or orbital trauma repair.

[0054] The present invention also provides a method for preventing the occurrence of pterygium, inhibiting the excessive growth of pterygium and / or inhibiting the recurrence of pterygium after surgery, which comprises administering the eye drops as described above. The administration method comprises dripping the eye drops into the eye.

[0055] The present invention provides an application of an anti-periostin antibody in the preparation of a drug for preventing and treating pterygium. The present invention also provides an anti-periostin antibody eye drop, which is obtained by screening auxiliary materials and optimizing the dosage, has good stability and still has good potency after long-term storage. It also shows good physiological activity in animal experiments and can produce a good therapeutic effect on pterygium. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The results of humanized monoclonal antibody purification are shown, where: lane MW is a protein marker; lanes 1 and 2 are humanized monoclonal antibodies;

[0057] Figure 2 The present invention shows the scratch inhibition effect of Periostin humanized monoclonal antibody eye drops on human RPE, wherein H802 is Periostin humanized monoclonal antibody, H802-1 is Periostin humanized monoclonal antibody eye drops prescription 1, and H802-2 is Periostin humanized monoclonal antibody eye drops prescription 2;

[0058] Figure 3 The present invention shows the inhibitory effect of Periostin humanized monoclonal antibody eye drops on corneal neovascularization in rabbits, wherein H802 is Periostin humanized monoclonal antibody, H802-1 is Periostin humanized monoclonal antibody eye drops prescription 1, and H802-2 is Periostin humanized monoclonal antibody eye drops prescription 2;

[0059] Figure 4 Figure 2 shows the slit lamp test results after treatment with Periostin humanized monoclonal antibody eye drops, where Figure A is the model control group, Figure B is the effect after treatment with Periostin humanized monoclonal antibody, Figure C is the effect after treatment with Periostin humanized monoclonal antibody eye drops prescription 1, and Figure D is the effect after treatment with Periostin humanized monoclonal antibody eye drops prescription 2. DETAILED DESCRIPTION

[0060] The present invention provides the use of anti-periostin antibodies in the preparation of drugs for preventing and treating pterygium. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0061] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0062] The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

[0063] The term "and / or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the corresponding term".

[0064] The prevention and treatment described in the present invention include both treatment and prevention, and refer to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as pterygium. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects who already have a condition or disease, as well as subjects who are susceptible to a condition or disease or subjects who intend to prevent a condition or disease. When referring to terms such as mitigation, alleviation, attenuation, alleviation, and relief, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0065] The numerical ranges and parameters involved in the present invention have been presented in the specific embodiments as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have certain reasonable deviations within a certain range, for example: within ±10%, ±5%, ±1% or ±0.5%.

[0066] Some cases are recorded in the examples of the present invention. The examples show some implementation methods of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases. In fact, any concentration of each component between the two endpoint values ​​shown in the examples can achieve good stability, and the cases with better effects are Example 3 and Example 5. In addition to the examples, many preliminary experiments were carried out during the research and development process, such as using different components, different ratios or concentrations, but the effects of these attempts were not as good as those of Examples 2 to 5, for example:

[0067] Viscous lubricants were tried: hyaluronic acid, gelatin, HPMC, polyethylene glycol, propylene glycol, with dosages between 0.5% and 2%. However, only polyethylene glycol and hyaluronic acid showed good stability maintenance effects, especially when gelatin and HPMC were used as lubricants, the stability of periostin antibody was not protected.

[0068] Zeta potential is a core factor affecting antibody stability. Zeta potential between +20 and 55 mV can keep the antibody stable as much as possible. In the present invention, the Zeta potential regulator attempts to use: chitosan, modified chitosan, polylactic acid or povidone K, with a dosage between 0.01 and 3%. Among them, chitosan and povidone K have better effects, but polylactic acid cannot maintain the stability of periostin antibodies.

[0069] Ploxamer is used to increase viscosity. In preliminary experiments, Ploxamer 407, Ploxamer 124, Ploxamer 188, Ploxamer 237 or Ploxamer 338 were tried, and only Ploxamer 124 had a poor effect.

[0070] The test materials used in the present invention are all common commercial products and can be purchased on the market. The preparation of periostin humanized monoclonal antibody refers to the Chinese patent application number 202280054341.X.

[0071] The present invention will be further described below in conjunction with embodiments:

[0072] Example 1 Purity determination of periostin humanized monoclonal antibody

[0073] The purity of the raw material of periostin humanized monoclonal antibody was detected by SDS-PAGE method, and the results showed that the purity of periostin humanized monoclonal antibody could reach more than 95% ( Figure 1 ).

[0074] Example 2 Preparation of periostin humanized monoclonal antibody eye drops

[0075] The formula of the periostin humanized monoclonal antibody preparation is as follows: 0.1 mg / ml anti-periostin humanized monoclonal antibody, 1% hyaluronic acid, 1.5% modified chitosan, 5% ploxamer 407, 0.5% cyclodextrin, and the balance is water for injection, pH 7.0.

[0076] The preparation method comprises: dissolving the modified chitosan and the antibody drug in PBS according to a proportion, mixing evenly and stirring for 0.5 to 1 hour; adding cyclodextrin and hyaluronic acid in sequence according to the prescribed amount, mixing evenly and then adding ploxamer, adding water for injection to the prescribed amount, adjusting the pH to 7.0, filtering through a 0.45um filter membrane and then packaging, and the zeta potential is +35mV.

[0077] The stability data of the periostin humanized monoclonal antibody preparation prepared in this example is shown in Table 1.

[0078] Table 1 Changes in OD values ​​after storage of preparations

[0079]

[0080] Example 3 Preparation of periostin humanized monoclonal antibody eye drops

[0081] The formula of the periostin humanized monoclonal antibody preparation is as follows: 1 mg / ml anti-periostin humanized monoclonal antibody, 1% hyaluronic acid, 0.5% modified chitosan, 2% ploxamer 407, 1% cyclodextrin, and the balance is water for injection, pH 7.0.

[0082] The preparation method is the same as that of Example 2, and the zeta potential is +40 mV. The stability data of the periostin humanized monoclonal antibody preparation prepared in this example is shown in Table 2.

[0083] Table 2 Changes in OD values ​​after storage of preparations

[0084]

[0085] Example 4 Preparation of periostin humanized monoclonal antibody eye drops

[0086] The formula of the periostin humanized monoclonal antibody preparation is as follows: 2 mg / ml anti-periostin humanized monoclonal antibody, 0.4% polyethylene glycol, 1% modified chitosan, 3% ploxamer 237, 1.5% cyclodextrin, and the balance is water for injection, pH 7.2.

[0087] The preparation method is the same as that of Example 2, and the zeta potential is +40 mV. The stability data of the periostin humanized monoclonal antibody preparation prepared in this example is shown in Table 3.

[0088] Table 3 Changes in OD values ​​after storage of preparations

[0089]

[0090] Example 5 Preparation of periostin humanized monoclonal antibody eye drops

[0091] The formula of the periostin humanized monoclonal antibody preparation is as follows: 5 mg / ml anti-periostin humanized monoclonal antibody, 0.5% polyethylene glycol, 0.1% modified chitosan, 5% puroxamer 188, 0.8% cyclodextrin, and the balance is water for injection, pH 7.2.

[0092] The preparation method is the same as that of Example 2, and the zeta potential is +35 mV. The stability data of the periostin humanized monoclonal antibody preparation prepared in this example is shown in Table 4.

[0093] Table 4 Changes in OD values ​​after storage of preparations

[0094]

[0095] Example 6 Inhibitory effect of Periostin humanized monoclonal antibody eye drops on scratching of human retinal pigment epithelial cells

[0096] The eye drops in Example 3 with better stability performance are recorded as Prescription 1, and the eye drops in Example 5 are recorded as Prescription 2.

[0097] RPE cells were routinely cultured and inoculated in 6-well plates. When the cells reached 90% fusion, a line was drawn in each group of cells with a pipette tip, and the cells were washed with PBS to prepare the scratch model and grouped:

[0098] Blank control group;

[0099] Periostin group: 10 ng / ml periostin;

[0100] 10ng / ml periostin + 200ug / ml Periostin humanized monoclonal antibody group;

[0101] 10ng / ml periostin + 200ug / ml Periostin humanized monoclonal antibody eye drops prescription group 1;

[0102] 10ng / ml periostin + 200ug / ml Periostin humanized monoclonal antibody eye drops prescription group 2;

[0103] 10ng / ml periostin + 200ug / ml Periostin humanized monoclonal antibody eye drops group.

[0104] The serum-free medium was changed and culture was continued. The scratch area was observed and photographed at 0h, 6h, 24h and 48h. The results showed that at 24h and 48h, the relative migration distance of RPE cells in the humanized monoclonal antibody group, humanized monoclonal antibody eye drops prescription group 1, and humanized monoclonal antibody eye drops prescription group 2 were significantly reduced compared with the control group and periostin group (P < 0.01). The results showed that periostin humanized monoclonal antibody eye drops had a significant inhibitory effect on the migration of retinal pigment epithelial cells, thus having the potential to prevent and treat pterygium ( Figure 2 ).

[0105] Example 7 Inhibitory effect of Periostin humanized monoclonal antibody eye drops on rabbit pterygium model caused by alkali burns

[0106] Alkali burns can damage the ocular surface tissue, causing a series of pathological changes such as inflammatory response, limbal stem cell dysfunction, and subconjunctival fibrosis. Therefore, alkali burns to the ocular surface are used as an animal model of pterygium.

[0107] All animals were modeled after basic index testing. On the day of modeling, alkaline solution of a certain concentration was instilled into both eyes and grouped. They were divided into five groups: blank control group, model control group, humanized monoclonal antibody group, prescription group 1, and prescription group 2. Drug administration began on the day of grouping (D1), and was administered twice a day from D1 to D21, with a dose of 50ug / eye. The anterior segment of the eye was examined by slit lamp and photographed. All animals were photographed once on D0 (the day before administration), D4, D7, D14, and D21. Photos were taken on D21 and the area of ​​new blood vessels was counted. The results showed that compared with the model control group, the humanized monoclonal antibody group, prescription group 1, and prescription group 2 all had the effect of inhibiting rabbit corneal neovascularization, and no excessive proliferation of fibrovascular tissue was found.

[0108] The results of slit lamp examination showed that: Model control group: 7 days after alkali burn, the limbus of the cornea began to dilate and become congested, and gradually grew toward the center of the cornea, until 21 days when the corneal neovascularization was significant, interweaving into a network, showing the state of pterygium; H802, H802-1, H802-2: At each time point, the blood vessels were smaller, short and sparse, mostly concentrated at the limbus of the cornea, growing slowly and not interweaving into a network, and the development of pterygium was inhibited. Since pterygium is a lesion caused by the proliferation and degeneration of conjunctival tissue, the bulbar conjunctiva and fibrovascular tissue invade the shallow layer of the cornea in a wing shape, and humanized monoclonal antibody eye drops can inhibit the excessive proliferation of fibrovascular tissue to develop into pterygium, providing a scientific basis for the treatment of fibrotic lesions with humanized monoclonal antibodies.

[0109] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of anti-periostin antibodies in the preparation of drugs for the prevention and treatment of pterygium.

2. The use according to claim 1, characterized in that: The prevention and treatment include inhibiting the migration of retinal pigment epithelial cells, inhibiting the formation of new blood vessels or the vascularization of corneal tissue.

3. A drug for preventing and treating pterygium, characterized in that Anti-periostin humanized monoclonal antibody 0.1-5 mg / ml; Viscosity lubricant 0.5%~2%; Zeta potential regulator 0.01%~3%; Prosam 1% to 8%; Cyclodextrin 0.1%~4%; The balance is water for injection.

4. The drug according to claim 3, characterized in that The viscosity lubricant is selected from any one of hyaluronic acid, gelatin, HPMC, polyethylene glycol, and propylene glycol. The zeta potential regulator is modified chitosan; The Ploxam is Ploxam 407, 124, 188, 237 or 338.

5. The drug according to claim 3 or 4, characterized in that The modified chitosan is carboxymethyl deacetylated chitosan.

6. The drug according to claim 5, characterized in that Made from the following components: Anti-periostin humanized monoclonal antibody 0.1mg / ml, hyaluronic acid 1%, modified chitosan 1.5%, ploxamer 4075%, cyclodextrin 0.5%, the balance is water for injection; or anti-periostin humanized monoclonal antibody 1mg / ml, hyaluronic acid 1%, modified chitosan 0.5%, ploxamer 4072%, cyclodextrin 1%, the balance being water for injection; or anti-periostin humanized monoclonal antibody 2mg / ml, polyethylene glycol 0.4%, modified chitosan 1%, ploxamer 237 3%, cyclodextrin 1.5%, the balance being water for injection; Or anti-periostin humanized monoclonal antibody 5 mg / ml, polyethylene glycol 0.5%, modified chitosan 0.1%, ploxamer 188 5%, cyclodextrin 0.8%, and the balance is water for injection.

7. A method for preparing the drug according to any one of claims 3 to 6, comprising: Dissolve the zeta potential regulator and anti-periostin humanized monoclonal antibody in PBS and stir for 0.5 to 1 h; Then add cyclodextrin and viscosity lubricant in sequence, mix well, add ploxamer, and then add water for injection.

8. The preparation method according to claim 7, characterized in that: After the injection water is added, the method further comprises the steps of adjusting the pH value to 7.0-7.4 and filtering through a 0.45 μm filter membrane.

9. Use of the drug according to any one of claims 3 to 6 or the drug prepared by the preparation method according to claim 7 or 8 in the preparation of a drug for preventing the occurrence of pterygium, inhibiting the excessive growth of pterygium and / or inhibiting the recurrence of pterygium after surgery.

10. The use according to claim 9, characterized in that: The pterygium is a pterygium formed after ocular surface tissue surgery; the ocular surface tissue surgery includes pterygium resection, corneal transplantation, eyelid repair and / or orbital trauma repair.

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