Eye drops containing recombinant type III humanized collagen and preparation method thereof

Abstract

The present application relates to the technical field of eye care liquid, and specifically provides an eye care liquid containing recombinant type III humanized collagen; according to the total mass of the components, the components of the eye care liquid include 0.02-1% sodium hyaluronate, 0.01-0.5% buffer salt, 0.5-1.5% osmotic pressure regulator, 0.001-0.1% recombinant type III humanized collagen, and 100% solvent supplementation. By optimizing the molecular weight of sodium hyaluronate, the pH of the system, etc. and the recombinant type III humanized collagen, the eye care liquid of the present application has a long-lasting moisturizing and soothing effect, and can also effectively stay around the eyeball, and basically does not cause irritation and discomfort to the eyeball.

Description

Technical Field

[0001] The present invention relates to the technical field of eye drops, and in particular to an eye drop containing recombinant type III humanized collagen and a preparation method thereof. Background Art

[0002] With the increase of work pressure, the continuous updating of electronic products, and the longer and longer use of electronic products such as computers and mobile phones, dry eyes, eye fatigue, eye inflammation and other symptoms are particularly common in people's daily lives. Most of the artificial tears on the market for the above eye symptoms include sodium hyaluronate, vitamins, taurine, borneol, mint, etc. This kind of artificial tears can provide eye relief and moisturizing, reduce red bloodshot and fatigue, etc. However, these artificial tears cannot fundamentally change the pathological conditions of eye cell tissues to solve the above problems; in addition, these artificial tears are easily washed out and cannot effectively stay around the eyeball to achieve long-term moisturizing, soothing and therapeutic effects.

[0003] Chinese patent CN105214071A discloses a fibronectin eye drop and its preparation method. The main components of this patent are fibronectin, sodium hyaluronate and adjuvants. The content of fibronectin is 50μg / mL to 500μg / mL, the content of sodium hyaluronate is 0.1%-0.3%, and the adjuvants are isotonic regulators, pH regulators, and injection water. This patent uses fibronectin and sodium hyaluronate together to prolong the drug action time by synergistic effect, greatly enhancing the repair and protection of the cornea. Chinese patent CN107802597B discloses a sodium hyaluronate composition, including high molecular weight sodium hyaluronate with an average molecular weight of 400,000-2 million, low molecular weight sodium hyaluronate with an average molecular weight of 5,000-20,000, excipients and injection water. The synergistic effect of low molecular weight sodium hyaluronate and high molecular weight sodium hyaluronate is more effective in treating dry eyes. However, the above patents are still limited in their therapeutic effect on the above eye symptoms. Summary of the invention

[0004] In order to solve the above technical problems, the present invention first provides an eye lotion containing recombinant humanized type III collagen; based on the total mass of the components, the components of the eye lotion include 0.02-1% sodium hyaluronate, 0.01-0.5% buffer salt, 0.5-1.5% osmotic pressure regulator, 0.001-0.1% recombinant humanized type III collagen, and the solvent supplement balance is 100%.

[0005] Furthermore, based on the total mass of the components, the components of the eye lotion include 0.05-0.5% sodium hyaluronate, 0.02-0.1% buffer salt, 0.5-1.2% osmotic pressure regulator, 0.005-0.05% recombinant type III humanized collagen, and the solvent supplement balance is 100%.

[0006] In a preferred embodiment, the components of the eye lotion include, based on the total mass of the components, 0.07-0.12% sodium hyaluronate, 0.02-0.05% buffer salt, 0.5-1.0% osmotic pressure regulator, 0.005-0.02% recombinant type III humanized collagen, and the solvent supplement balance is 100%.

[0007] Furthermore, the buffer salt includes but is not limited to at least one of phosphate, carbonate, bicarbonate, borate, citrate and tartrate.

[0008] Furthermore, the phosphate includes but is not limited to at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

[0009] Furthermore, the carbonate includes but is not limited to at least one of sodium carbonate and potassium carbonate.

[0010] Furthermore, the bicarbonate includes but is not limited to at least one of sodium bicarbonate, potassium bicarbonate, and ammonium bicarbonate.

[0011] Furthermore, the buffer salt includes phosphate; the phosphate includes disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0012] Furthermore, based on the total mass of the components, the phosphate includes 0.01-0.05% of disodium hydrogen phosphate and 0.005-0.02% of sodium dihydrogen phosphate.

[0013] In a preferred embodiment, the phosphate comprises 0.01-0.05% of anhydrous disodium hydrogen phosphate and 0.005-0.02% of sodium dihydrogen phosphate monohydrate, based on the total mass of the components.

[0014] Furthermore, the osmotic pressure regulator includes but is not limited to at least one of sodium nitrate, potassium nitrate, sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, glycerol, propylene glycol, mannitol, sorbitol, and glucose.

[0015] Furthermore, the osmotic pressure regulator includes sodium chloride.

[0016] Furthermore, the average molecular weight of the sodium hyaluronate is 1-2 million, preferably 1.2-1.8 million.

[0017] More preferably, the average molecular weight of the sodium hyaluronate is 1.4-1.6 million, and can be selected from at least one of 1.4 million, 1.45 million, 1.5 million, 1.55 million, 1.6 million, etc.

[0018] Sodium hyaluronate has high hydrophilicity. Each hyaluronic acid molecule can absorb 500-1000 times its own weight of water. Using it in eye protection solution can provide a good wetting effect on the eyes. However, there are obvious differences in the water retention of sodium hyaluronate with different molecular weights. This application stipulates that when the average molecular weight of sodium hyaluronate is 1.4-1.6 million, it can not only improve the water retention and retention of eye moisturizing solution, but also further maintain the osmotic pressure of eye moisturizing solution in the range of 270-310mOsmol / kg; this is because 1.4-1.6 million square The molecular chain length of sodium hyaluronate with average molecular weight is appropriate. The hydrogen bonding and electrostatic effects formed between molecules through hydroxyl, carboxyl and amide groups can ensure that water and drugs can be locked in its three-dimensional cross-linked structure, and stay around the eyeball for a longer time, and play a continuous moisturizing and therapeutic effect; more specifically, under this average molecular weight condition, sodium hyaluronate can be guaranteed to have a specific number of molecules, and further work together with sodium chloride to ensure that the osmotic pressure of the eye solution is maintained at 270-310mOsmol / kg, making the eye solution basically non-irritating to the eyes. Generally speaking, when the average molecular weight of sodium hyaluronate is too high, the viscosity of the eye solution is too high to be easy to operate and use, and the eye solution is prone to precipitation or flocculation during storage. When the average molecular weight is too low, the osmotic pressure of the eye solution will increase, which may cause dryness and discomfort of the eye tissue.

[0019] Furthermore, the eye drops also contain 0.002-0.1% of other ingredients.

[0020] Furthermore, the other ingredients include but are not limited to at least one of vitamins, edetate disodium, erythromycin, ofloxacin, norfloxacin, and bromfenac sodium.

[0021] Preferably, the other ingredients include vitamins and edetate disodium.

[0022] Further preferably, based on the total mass of the components, the other ingredients include 0.005-0.03% of vitamins and 0.002-0.01% of edetate disodium.

[0023] Furthermore, the vitamins include but are not limited to at least one of vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B9, and vitamin B12.

[0024] Preferably, the vitamins include vitamin B12.

[0025] Furthermore, the solvent is medically acceptable water, including but not limited to purified water, water for injection, etc.; preferably water for injection.

[0026] Secondly, the present application also provides a method for preparing the eye drops containing recombinant humanized type III collagen, comprising the following steps:

[0027] (1) placing the components except the recombinant humanized type III collagen in a sterilized container, mixing and stirring, and sterilizing to obtain a matrix;

[0028] (2) Cooling the substrate and adjusting the pH of the substrate to 6-8;

[0029] (3) Add recombinant humanized type III collagen and mix well.

[0030] Furthermore, in step (1), sterilization is performed by heating, the heating temperature is 121-134° C., and the heating time is 5-30 min.

[0031] In a preferred embodiment, the heating temperature is 121° C. and the heating time is 15-20 min.

[0032] Furthermore, in step (2), the pH value of the matrix is ​​6.5-7.5, and can be selected as 6.5, 6.8, 7.0, 7.2, 7.5, etc. The intermolecular force of sodium hyaluronate is regulated by pH. When the pH is 6-8, the dynamic viscosity of the sodium hyaluronate aqueous solution is relatively stable. However, at a pH of 6.5-7.5, the electrostatic force generated by the surface charge of the molecule can produce the best synergistic effect with hydrogen bonds, etc., stabilizing the drug ingredients and recombinant type III humanized collagen to enter the cross-linked network and be stably released, thereby increasing the retention of the active ingredients in the eye solution around the eye and enhancing the eye-moistening effect. When the pH is too high or too low, the viscosity of the sodium hyaluronate aqueous solution will be abnormal, which is not only not conducive to the dynamic viscosity of the eye solution, but also not conducive to its therapeutic effect.

[0033] Furthermore, the osmotic pressure of the recombinant type III humanized collagen eye solution is 270-310 mOsm / kg.

[0034] Furthermore, the recombinant humanized type III collagen eye drops are used for cleaning, lubricating and moisturizing the eyeball, daily care of dry eyes, and repairing corneal injury wounds.

[0035] Furthermore, the eye drops can be used by instillation or injection.

[0036] Beneficial Effects

[0037] 1. This application utilizes the characteristics of recombinant humanized type III collagen, which is fully human, highly water-soluble, highly biologically active, effectively supplements the defective collagen, and promotes tissue cell regeneration, to fundamentally improve the eyeball environment, and works together with sodium hyaluronate and other ingredients to achieve the long-lasting moisturizing and soothing effects of eye drops;

[0038] 2. The average molecular weight of sodium hyaluronate optimized in this application is 1.4-1.6 million, which improves the wetting, moisturizing and retention effects of the eye drops, and keeps the osmotic pressure of the eye drops at 270-310 mOsmol / kg, so that the eye drops of this application basically do not cause irritation and discomfort to the eyeball;

[0039] 3. The pH of the matrix optimized in this application is 6.5-7.5, which can further control the cross-linking strength of sodium hyaluronate, increase the retention of the active ingredients in the eye solution around the eye, and enhance the eye-moistening effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Attached Figure 1 : Cell migration test results after the eye drops were diluted to different protein concentrations in Example 4;

[0041] Attached Figure 2 : Results of cell migration experiments after the eye drops were diluted to different protein concentrations in Example 1;

[0042] Attached Figure 3 : Cell adhesion test results after the eye drops were diluted to different protein concentrations in Example 4;

[0043] Attached Figure 4 : Cell adhesion test results after the eye drops were diluted to different protein concentrations in Example 1. DETAILED DESCRIPTION

[0044] Example

[0045] Example 1

[0046] This embodiment provides an eye-lubricating liquid containing recombinant humanized type III collagen; based on the total mass of the components, the components of the eye-lubricating liquid include 0.1% sodium hyaluronate, 0.03% buffer salt, 0.85% osmotic pressure regulator, 0.01% recombinant humanized type III collagen, 0.015% vitamin B12, 0.005% disodium edetate, and the solvent supplement balance is 100%.

[0047] The average molecular weight of the sodium hyaluronate was 1.5 million and was purchased from Zhongshan Biotechnology Co., Ltd.

[0048] The buffer salt is 0.02% anhydrous disodium hydrogen phosphate and 0.01% sodium dihydrogen phosphate monohydrate.

[0049] The osmotic pressure regulator is sodium chloride.

[0050] The recombinant humanized type III collagen is produced by the company and disclosed in the Chinese patent application number ZL201811438582.6 on November 22, 2019.

[0051] The solvent is water for injection.

[0052] A method for preparing an eye-lubricating liquid containing recombinant type III humanized collagen comprises the following steps:

[0053] (1) placing sodium hyaluronate, buffer salt, osmotic pressure regulator, vitamin B12, disodium edetate and a solvent in a sterilized container, mixing and stirring them evenly, and sterilizing them at 121° C. for 15 minutes to obtain a matrix;

[0054] (2) cooling the substrate and adjusting the pH of the substrate to 7.2;

[0055] (3) Add recombinant humanized type III collagen and mix evenly, and then test the pH and osmotic pressure to see if they are qualified.

[0056] Example 2

[0057] This embodiment provides an eye lotion containing recombinant humanized type III collagen; based on the total mass of the components, the components of the eye lotion include 0.07% sodium hyaluronate, 0.02% buffer salt, 0.5% osmotic pressure regulator, 0.005% recombinant humanized type III collagen, 0.005% vitamin B12, 0.002% disodium edetate, and the solvent supplement balance is 100%.

[0058] The average molecular weight of the sodium hyaluronate was 1.6 million and was purchased from Zhongshan Biotechnology Co., Ltd.

[0059] The buffer salt is 0.015% of anhydrous disodium hydrogen phosphate and 0.005% of sodium dihydrogen phosphate monohydrate.

[0060] The osmotic pressure regulator is sodium chloride.

[0061] The recombinant humanized type III collagen is produced by the company and disclosed in the Chinese patent application number ZL201811438582.6 on November 22, 2019. The solvent is water for injection.

[0062] A method for preparing an eye-lubricating liquid containing recombinant type III humanized collagen comprises the following steps:

[0063] (1) placing sodium hyaluronate, buffer salt, osmotic pressure regulator, vitamin B12, disodium edetate and a solvent in a sterilized container, mixing and stirring them evenly, and sterilizing them at 121° C. for 15 minutes to obtain a matrix;

[0064] (2) Cooling the substrate and adjusting the pH of the substrate to 7.5;

[0065] (3) Add recombinant humanized type III collagen and mix evenly, and then test the pH and osmotic pressure to see if they are qualified.

[0066] Example 3

[0067] This embodiment provides an eye lotion containing recombinant humanized type III collagen; based on the total mass of the components, the components of the eye lotion include 0.12% sodium hyaluronate, 0.05% buffer salt, 1.0% osmotic pressure regulator, 0.02% recombinant humanized type III collagen, 0.03% vitamin B12, 0.01% disodium edetate, and the solvent supplement balance is 100%.

[0068] The average molecular weight of the sodium hyaluronate was 1.4 million and was purchased from Zhongshan Biotechnology Co., Ltd.

[0069] The buffer salt is 0.04% anhydrous disodium hydrogen phosphate and 0.01% sodium dihydrogen phosphate monohydrate.

[0070] The osmotic pressure regulator is sodium chloride.

[0071] The recombinant humanized type III collagen is produced by the company and disclosed in the Chinese patent application number ZL201811438582.6 on November 22, 2019. The solvent is water for injection.

[0072] A method for preparing an eye-lubricating liquid containing recombinant type III humanized collagen comprises the following steps:

[0073] (1) placing sodium hyaluronate, buffer salt, osmotic pressure regulator, vitamin B12, disodium edetate and a solvent in a sterilized container, mixing and stirring them evenly, and sterilizing them at 121° C. for 15 minutes to obtain a matrix;

[0074] (2) Cooling the substrate and adjusting the pH of the substrate to 6.5;

[0075] (3) Add recombinant humanized type III collagen and mix evenly, and then test the pH and osmotic pressure to see if they are qualified.

[0076] Example 4

[0077] This embodiment provides an eye lotion containing recombinant humanized type III collagen; based on the total mass of the components, the components of the eye lotion include 0.1% sodium hyaluronate, 0.03% buffer salt, 0.85% osmotic pressure regulator, 0.01% recombinant humanized type III collagen, and the solvent supplement balance is 100%.

[0078] The average molecular weight of the sodium hyaluronate was 1.5 million and was purchased from Zhongshan Biotechnology Co., Ltd.

[0079] The buffer salt is 0.02% anhydrous disodium hydrogen phosphate and 0.01% sodium dihydrogen phosphate monohydrate.

[0080] The osmotic pressure regulator is sodium chloride.

[0081] The recombinant humanized type III collagen is produced by the company and disclosed in the Chinese patent application number ZL201811438582.6 on November 22, 2019. The solvent is water for injection.

[0082] A method for preparing an eye-lubricating liquid containing recombinant type III humanized collagen comprises the following steps:

[0083] (1) placing sodium hyaluronate, buffer salt, osmotic pressure regulator and solvent in a sterilized container, mixing and stirring, and sterilizing at 121° C. for 15 minutes to obtain a matrix;

[0084] (2) cooling the substrate and adjusting the pH of the substrate to 7.2;

[0085] (3) Add recombinant humanized type III collagen and mix evenly, and then test the pH and osmotic pressure to see if they are qualified.

[0086] Performance testing method:

[0087] 1. Physical and chemical property test items: According to the general rules of the four parts of the "Pharmacopoeia of the People's Republic of China" (2020 edition), including relative density, osmotic pressure, dynamic viscosity (third method rotational viscometer determination method), pH value, trace elements, ignition residue, exogenous DNA residue, sterility, bacterial endotoxin (gel method), pyrogen, etc.; the schemes in the embodiments all meet the indicators in Table 1.

[0088] Table 1

[0089]

[0090] 2. Cell migration test: (1) Experimental grouping: basal culture medium (or serum concentration not higher than 2%) as control, recombinant type III humanized collagen content 0.05mg / mL, 0.1mg / mL, 0.5mg / mL, 1mg / mL eye drops as test products. (2) Marking and positioning: Use a marker pen to draw lines every 0.5cm on the back of the 24-well plate to facilitate subsequent photography and positioning. Seed about (5-15)×104 cells per well, and the specific seeding number can be adjusted according to the different cells. Each group has 3 replicate wells. (3) Cell scratching: After 24 hours of cell culture, use a toothpick to measure the ruler perpendicular to the horizontal line (you can also use a special cell culture dish for scratching), gently push down the longitudinal line to form a scratch, rinse the cells with PBS 3 times, remove the scratched cells, and add 500L of serum-free culture medium prepared test sample and control solution. (4) Cell photography: Cultured in a 37°C, 5% CO2 incubator, photographed under a 10x microscope at 0h, 12h, and 24h, with the intersection of the horizontal and vertical lines as the core. (5) Data processing: Use ImageJ software to measure the scratch area and calculate the cell migration rate of each group of cells, or use the scratch width to select the cell migration rate. The formula is as follows:

[0091] Cell migration rate = total area of ​​migrating cells in the fixed scratch area / initial scratch area × 100%; cell migration rate = (0h scratch width - scratch width after culture) / 0h scratch width × 100%; relative cell migration rate = experimental group cell migration rate / control group cell migration rate; (6) Statistical analysis: Two-tailed t-test was used to statistically analyze the statistical differences between the target recombinant humanized collagen and the negative control bovine, where *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The test results are shown in the attached Figure 1 and attached Figure 2 .

[0092] Results: Attached Figure 1 The results of the cell migration experiment with the eye drops of Example 4 as the test sample at different protein concentrations are shown. Compared with the negative group, the relative cell migration rates of the eye drops of Example 4 at the experimental concentrations of 0.5 mg / mL and 1 mg / mL of recombinant type III humanized collagen were 113.0% and 119.9%, respectively; this proves that the eye drops of Example 4 of the present application have a promoting effect on cell migration. Figure 2 The results of the cell migration experiment with the eye drops of Example 1 as the test sample at different protein concentrations are shown. Compared with the negative group, the relative cell migration rates of the eye drops at experimental concentrations of 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL and 1 mg / mL are 107.8%, 108.7%, 123.6% and 125.5%, respectively, proving that the eye drops of Example 1 of the present application have a more obvious promoting effect on cell migration.

[0093] 3. Cell adhesion: (1) Sample preparation: Use D-PBS to dilute the eye drops of the example to the concentration of recombinant type III humanized collagen of 0.01 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL for use; prepare the positive control eye drops containing bovine type I collagen (PC) to 1 mg / mL according to the above method for use; the negative control is D-PBS buffer (NC). (2) Coating: Add different concentrations of collagen and positive and negative controls to the ELISA plate, 100 μL per well, set 5 replicates per group, and incubate at 4°C overnight. (3) Blocking: Discard the supernatant, add 100 μL 1% BSA (heat inactivated at 56°C for 30 min), and incubate at 37°C for 60 min. Discard the supernatant and wash 3 times with D-PBS solution. (4) Cell inoculation: Add 105 3T3 / NIH cells in good culture state resuspended in D-PBS to each well and incubate at 37°C for 120 minutes. Wash each well three times with D-PBS solution. (5) Detection: Use CCK8 detection kit to detect the absorbance at OD450nm. Calculate the degree of cell adhesion according to the following formula (the cell adhesion rate can reflect the cell adhesion ability of collagen; the higher the cell adhesion ability, the better the external environment can be provided to the cells in a short time to help the cells adhere to the wall):

[0094]

[0095] Where:

[0096] P: relative cell adhesion ratio;

[0097] OD1: average UV absorbance of each replicate well of the test collagen sample at 450 nm; OD2: average UV absorbance of each replicate well of the control collagen sample at 450 nm; OD0: average UV absorbance of each replicate well of the blank control group at 450 nm.

[0098] (6) Statistical analysis: Two-tailed t-test was used to analyze the statistical difference between the target recombinant humanized collagen and the negative control, where *, P < 0.05; **, P < 0.01; ***, P < 0.001. The results are shown in the attached Figure 3-4 .

[0099] Results: Attached Figure 3 and attached Figure 4 The cell adhesion test results of the eye drops of Example 4 and Example 1, and the eye drops diluted to different protein concentrations, are shown in the attached Figure 3-4 It can be seen that compared with the negative group, the eye drops of Example 1 and Example 4 at different concentrations all have a promoting effect on cell adhesion.

Claims

1. An eye drops containing recombinant humanized type III collagen, characterized in that: According to the total mass of the components, the components of the eye drops include 0.05-0.5% sodium hyaluronate, 0.02-0.1% buffer salt, 0.5-1.2% osmotic pressure regulator, 0.005-0.05% recombinant type III humanized collagen, and the solvent supplement balance is 100%; the buffer salt is anhydrous disodium hydrogen phosphate and sodium dihydrogen phosphate monohydrate; the osmotic pressure regulator is sodium chloride; The average molecular weight of the sodium hyaluronate is 1.4-1.6 million; The method for preparing the eye drops containing recombinant type III humanized collagen comprises the following steps: (1) placing the components except the recombinant humanized type III collagen in a sterilized container, mixing and stirring, and sterilizing to obtain a matrix; (2) Cool the substrate and adjust the pH of the substrate to 6.5-7.5; (3) Add recombinant humanized type III collagen and mix well.

2. The eye-lubricating solution according to claim 1, characterized in that: In step (1), sterilization is performed by heating, the heating temperature is 121-134° C., and the heating time is 5-30 min.

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