Platelet-derived growth factor mutant eye drops

Abstract

The invention provides a liquid composition containing a platelet-derived growth factor mutant. The composition comprises the following components: polyvinyl alcohol, hydroxypropyl methyl cellulose, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, mannitol and the like. The invention also provides a preparation method of the composition and application of the composition in corneal injury treatment medicines. The eye drops prepared from the composition disclosed by the invention have good stability and safety, and have the technical effects of small dosage and good repairing effect in repairing corneal injury.

Description

Technical Field

[0001] The present invention discloses a pharmaceutical preparation, belonging to the field of biological products. Background Art

[0002] Platelet-derived growth factor is an important component of platelets and is released during the blood clotting process at the site of injury. Platelet-derived growth factor is synthesized by platelets, smooth muscle cells, activated macrophages, and activated endothelial cells, and can promote cell mitosis and enhance chemotaxis to promote tissue repair and regeneration. Recombinant human platelet-derived growth factor is an important mitogen that can promote the proliferation of cells such as skin, cornea, osteoblasts, and periodontal ligament fibroblasts, exert chemotactic effects, promote cell migration, promote the synthesis of blood vessels and matrix, and achieve the effects of tissue regeneration and wound repair. PDGFs have four monomers, A, B, C, and D, which are connected by disulfide bonds to form homodimers or heterodimers (PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, PDGF-AB). After PDGF binds to its receptor PDGFR, it causes two receptor molecules to form a dimer and become activated, phosphorylating the transmembrane tyrosine kinase domain, thereby transmitting signals into the cell and regulating cell division, proliferation, and other life activities through cascade amplification.

[0003] The main biological characteristics of PDGF are as follows: ① Chemotaxis: Stimulating the chemotactic migration of fibroblasts, smooth muscle cells, neutrophils, and monocytes, which is extremely important for the repair of body injuries; ② Mitogenicity: Promoting the DNA synthesis, cell lysis, and proliferation of various cells; ③ Increasing the synthesis and accumulation of extracellular matrix (ECM): Promoting the synthesis of various ECM components such as collagen fibers, integrins, fibronectin (FN), laminin (LN), and mucopolysaccharides, inhibiting the degradation of ECM, and participating in the occurrence and development of tissue and organ fibrosis and sclerosis; ④ Vasoactive effect: Capable of inducing the contraction of different types of blood vessels.

[0004] A large number of studies have found that the heterogeneity, biological activity, stability, and physicochemical properties of platelet-derived growth factor are affected by different cleavage forms formed by specific-site protease degradation, post-translational glycosylation modification, and complex dimer structures of platelet-derived growth factor, posing a huge challenge to the drugability of platelet-derived growth factor. Site-directed mutagenesis of some cleavage sites of platelet-derived growth factor can improve the post-translational cleavage and modification forms of platelet growth factor dimers, and further affect the biological activity, homogeneity, and related physicochemical properties of platelet-derived growth factor. In particular, modifying the amino acid sequence in the binding region of platelet-derived growth factor to its receptor directly affects the activation of its downstream signaling pathway by platelet-derived growth factor and the execution of its biological functions.

[0005] The cornea is an important refractive medium of the eye. When the injury is severe, such as stromal layer injury or relatively severe corneal laceration, the repair time may be as long as 1 - 3 months. This long - term passive repair is likely to cause corneal infection, ulcer, and at the same time form scars of varying degrees or changes in corneal curvature, seriously affecting eyesight. Platelet - derived growth factor, as an effective mitogen, can promote the proliferation, division, and chemotaxis of various cells into fibroblasts and immune cells, accelerating the repair of damaged tissues and organs. These characteristics of platelet - derived growth factor make it have great application potential in the repair of corneal injury in the eye and is expected to become a good therapeutic drug for corneal injury. Summary of the Invention

[0006] Based on the above - mentioned purpose, the present invention first provides a liquid composition containing a platelet - derived growth factor mutant. The amino acid sequence of the platelet - derived growth factor (PDGF - B) mutant is shown as SEQ ID NO.3, and the composition contains: Platelet - derived growth factor mutant: 800 - 7000 IU / ml, Thickening agent: 0.1 - 2% (g / ml), Protein protectant: 0.05 - 3% (g / ml), Phosphate buffer: 10 - 30 mM, Osmotic pressure regulator: 0.25 - 0.75% (g / ml), The pH of the liquid composition is 6.8 - 7.4, Among them, the thickening agent is selected from one or more of polyvinyl alcohol, sodium hyaluronate, hydroxypropyl methylcellulose, and methylcellulose; the protein protectant is selected from one or more of glycine, glutamic acid, lysine, histidine, arginine, sucrose, trehalose, polyethylene glycol 400, and mannitol; the osmotic pressure regulator is selected from one or more of sodium chloride, glucose, or sorbitol.

[0007] In a preferred embodiment, the dosage of recombinant human platelet - derived growth factor in the composition is 1000 - 6000 IU / ml.

[0008] In a preferred embodiment, the thickening agent is polyvinyl alcohol, and the dosage of polyvinyl alcohol is 0.5% - 1.5%.

[0009] In a preferred embodiment, the thickening agent is hydroxypropyl methylcellulose, and the dosage of hydroxypropyl methylcellulose is 0.1% - 0.5%.

[0010] In a preferred embodiment, the protein protectant is mannitol, and the dosage of mannitol is 0.05% - 3%.

[0011] In a preferred embodiment, the osmotic pressure regulator is sodium chloride, and the amount of sodium chloride used is 0.25% - 0.75%.

[0012] In a more preferred embodiment, the amounts of the components in the composition are as follows: Platelet-derived growth factor mutant 3000 IU / ml, Polyvinyl alcohol 1% (g / ml), Hydroxypropyl methylcellulose 0.1% (g / ml), Mannitol 1% (g / ml), Sodium chloride 0.56% (g / ml), Disodium hydrogen phosphate dodecahydrate 0.52% (g / ml), Sodium dihydrogen phosphate monohydrate 0.08% (g / ml), Phosphate buffer is 20 mM, and the pH is 6.8 - 7.4.

[0013] Secondly, the present invention provides a method for preparing the above liquid composition, and the method includes the following steps: (1) Disperse the thickener polyvinyl alcohol in water for injection, sterilize by autoclaving at 121 °C, and reserve after cooling; (2) Dissolve the platelet-derived growth factor mutant, hydroxypropyl methylcellulose, phosphate buffer, osmotic pressure regulator, and protein protectant in water for injection, and filter aseptically with a 0.22 μm filter membrane; (3) Under aseptic conditions, mix the solution obtained in step (1) and the solution obtained in step (2) evenly, make up the volume with sterile water for injection, and then immediately package and reserve.

[0014] In a specific embodiment, the method for preparing the above liquid composition includes the following steps: (1) Uniformly disperse 0.1 - 2% of polyvinyl alcohol in an appropriate amount of water for injection, sterilize by autoclaving at 121 °C, and reserve after cooling; (2) Dissolve 800 - 7000 IU / ml of recombinant human platelet-derived growth factor, 0.1% - 0.3% of hydroxypropyl methylcellulose, 0.05 - 3% of protein protectant, 10 - 30 mM of phosphate buffer, and 0.25 - 0.75% of osmotic pressure regulator in an appropriate amount of water for injection, and filter aseptically with a 0.22 μm filter membrane; (3) Under aseptic conditions, mix the solution obtained in step (1) and the solution obtained in step (2) evenly, make up the volume with sterile water for injection, and then immediately package, thus obtaining the product.

[0015] Finally, the present invention provides the application of the above liquid composition in the preparation of a drug for treating corneal injury.

[0016] In a preferred embodiment, the corneal injury includes chemical burns and mechanical injuries.

[0017] The present invention has the following advantages compared with the prior art: 1. This product is fast and efficient, capable of repairing various corneal injuries, such as mechanical injuries, mild to moderate chemical burns, corneal surgeries and poor postoperative healing, mild to moderate dry eye, etc. It is supplemented with excipients to produce a synergistic effect between the excipients and the platelet-derived growth factor mutant, increasing the biological activity and stability of recombinant human platelet-derived growth factor in the eye drops.

[0018] 2. By designing the prescription and selecting excipients for the human platelet-derived growth factor mutant, this product has good safety, little irritation, no preservatives, avoiding the damaging effects of preservatives on the eye, such as corneal ulcers, etc.; the production process is simple, with broad industrialization prospects and great application potential.

[0019] 3. The present invention has screened an eye drop formulation of a human platelet-derived growth factor mutant, which solves the eye discomfort and visual obstruction caused by eye gels and ointments, improves the residence time of platelet-derived growth factor in the cornea, and increases its bioavailability. In addition, this eye drop prescription also improves the stability of PDGF. Instead of affecting the biological activity of PDGF, it can increase its biological activity, forming an enhancing effect, enabling a lower dose of PDGF to achieve better therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 . Schematic diagram of the PDGF-B protein structure. The precursor protein has a total of 241 amino acids. Among them, the 1st - 20th amino acids are the signal peptide, the 21st - 81st amino acids are the pro-sequence preceding the growth factor domain, the 82nd - 190th amino acids are the mature PDGF-B peptide segment consisting of 109 amino acids, and the 191st - 241st amino acids are also peptide segments that will be cut off during the protein maturation process.

[0021] Figure 2 . SDS-PAGE electrophoresis diagram of different purified recombinant platelet-derived growth factor BB mutants. As shown in the figure, lane 5 is the Marker, lane 6 is the electrophoresis diagram of the purified mutant G0, and lane 7 is the electrophoresis diagram of the purified mutant G1. It can be seen from the electrophoresis diagram that the purity of the two mutants after purification is 100%.

[0022] Figure 3 . Statistical chart of the biological activities of different platelet-derived growth factor B mutants after purification, where ST is the international standard of platelet-derived growth factor BB (NIBSC, 94 / 728); G0 is the biological activity of platelet-derived growth factor BB with 5 amino acids deleted from the N-terminus, and G1 is the biological activity of platelet-derived growth factor BB with 5 amino acids deleted from the N-terminus and the 32nd amino acid mutated to proline.

[0023] Figure 4 . Curve graph of corneal epithelial healing rate at different times.

[0024] Figure 5 . Diagram of the results of fluorescein sodium staining of rabbit corneas on day 0 of drug administration.

[0025] Figure 6 . Diagram of the results of fluorescein sodium staining of rabbit corneas on day 7 of drug administration. Detailed implementation manners

[0026] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the protection scope defined by the claims of the present invention. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0027] CN1661011A constructs a platelet-derived growth factor mutant of 104 amino acids. Compared with the wild-type platelet-derived growth factor, this mutant has 5 amino acids deleted from its N-terminus and better homogeneity, while its biological activity is not lost. Compared with the wild-type platelet-derived growth factor, this mutant has great advantages. Therefore, the mutant research in the present invention is all based on this mutant, and the platelet-derived growth factor with 5 amino acids deleted from the N-terminus is named G0. All descriptions of the site positions are based on the wild-type PDGF-B (109 amino acids). The Genbank number of the amino acid sequence of wild-type PDGF-B is NM-002608.4.

[0028] Based on the platelet-derived growth factor B mutant G0, the present invention further modifies its amino acid sequence to further optimize the platelet-derived growth factor, especially its biological activity, and then provides an eye drop composed of this mutant.

[0029] Preparation Example 1. Preparation of PDGF-B mutant 1. Modification of the PDGF-B mutant sequence The amino acid sequence of platelet-derived growth factor mutant G0 is as follows: TIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVT (SEQ ID NO: 1) The amino acid sequence of platelet-derived growth factor mutant G1 is as follows: TIAEPAMIAECKTRTEVFEISRRLIDPTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVT (SEQ ID NO: 3) The nucleotide sequence of platelet-derived growth factor mutant G0 is as follows: 5’-ACCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGGTGTTCGAGATCTCCCGGCGCCTCATAGACCGCACCAACGCCAACTTCCTGGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAACAACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTGTCCAGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGCCACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGACAGTGGCAGCTGCACGGCCTGTGACC-3’ (SEQ ID NO: 2) The nucleotide sequence of platelet-derived growth factor mutant G1 is as follows: 5’-ACCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGGTGTTCGAGATCTCCCGGCGCCTCATAGACCCCACCAACGCCAACTTCCTGGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAACAACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTGTCCAGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGCCACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGACAGTGGCAGCTGCACGGCCTGTGACC-3’ (SEQ ID NO: 4) 2. Construction of expression vector 2.1 Construction of recombinant expression clone Figure 1 It is a schematic diagram of the PDGF-B protein structure. The precursor protein has a total of 241 amino acids. Among them, the 1st - 20th amino acids are the signal peptide, the 21st - 81st amino acids are the pro-sequence preceding the growth factor domain (PRO), the 82nd - 190th amino acids are the mature PDGF-B peptide segment consisting of 109 amino acids (Mature peptide), and the 191st - 241st amino acids are the peptide segment that will also be cleaved during protein maturation.

[0030] Two kinds of PDGF mutants were constructed in this invention. Among them, the 104 - amino acid sequence lacking the 5 N-terminal amino acids is G0 (CN105085652A); the one lacking the 5 N-terminal amino acids and with the 32nd arginine mutated to proline is G1. The above mutant DNA sequences were all synthesized by Tsingke Biotechnology, and the synthesized sequences were inserted into the pPICZαA plasmid and named pPICZαA-G0 and pPICZαA-G1 respectively. After the obtained plasmid sequences were confirmed correct by sequencing, they were amplified by Escherichia coli and the plasmids were extracted.

[0031] 2.2 Preparation of genetically engineered bacteria The two plasmids containing the PDGF-B mutant genes were linearized by digestion with SacⅠ enzyme, and the digestion system is shown in Table 1.

[0032] Table 1. SacⅠ enzyme digestion system

[0033] After complete digestion, the obtained linearized plasmid was purified and stored at -20 °C.

[0034] 2.3 Electroporation of linearized plasmid The linearized plasmids pPICZαA-G0 and pPICZαA-G1 were transformed into the competent cells of the Pichia pastoris expression strain GS115 by electroporation. The specific steps are as follows: ① Thaw the prepared competent cells and the purified linearized plasmid on ice, and pre-cool the YPDS medium and the electroporation cuvette on ice. ② Put 900 μl of competent cells and 100 μl of linearized plasmid into a 1.5 ml sterile EP tube and mix well. After mixing, transfer it into a pre-cooled electroporation cuvette (0.2 cm). ③ Wipe the electroporation cuvette clean, put it into the electroporator, and select the Pichia pastoris electroporation program (Pic-2kv 5ms) for electroporation. ④ Immediately after electroporation, add 900 μl of YPDS medium to the electroporation cuvette, mix well, and transfer it to a 1.5 ml sterile EP tube.

[0035] 2.4 Screening of electroporated strains Put the 1.5 ml sterile centrifuge tube containing the electroporated strains into a 30 °C constant temperature incubator for 1.5 - 2 h; then put it into a constant temperature shaker and culture it at 30 °C and 220 rpm for 1.5 - 2 h. Then, perform primary screening of the strains on YPD (Yeast Extract Peptone Dextrose Medium) containing Zecion (bleomycin) resistance, and screen monoclonal with high expression efficiency and identify positive recombinant yeast strains by PCR method.

[0036] 3. Induced expression of recombinant protein 3.1 Initial culture of bacteria Inoculate the monoclonal of the screened recombinant yeast strain into YPD liquid medium for seed culture, and then perform amplification culture. Then, inoculate it into the fermentation medium (yeast extract 10 g / L, tryptone: 20 g / L, potassium dihydrogen phosphate: 16 g / L, glycerol: 20 g / L, sodium chloride: 10 g / L, YNB (yeast nitrogen base medium): 12 g / L, pH value 5.5) at an inoculation ratio of 10 - 15 v / v%. The initial culture conditions are 30 - 32 °C, aeration rate 0.5 - 1.5 vvm. As the bacteria grow continuously, control the dissolved oxygen above 40% by increasing the rotation speed and aeration rate. When the dissolved oxygen value quickly rebounds above 80%, enter the glycerol supplementation stage.

[0037] 3.2 Glycerol supplementation After the initial cultivation of the bacterial cells, a glycerol solution of about 50 v / v% is supplemented at an exponential feeding rate starting from 8 - 12 ml / L / h. During the cultivation process, the rotation speed and aeration volume are adjusted to control the dissolved oxygen at 30 - 60%. When the wet cell weight reaches 150 ± 5 g / l, the addition of glycerol is stopped.

[0038] 3.3 Induction expression After the glycerol supplementation stage, without starvation, the cultivation temperature is reduced to 24 - 28 °C, and the induction culture is started by supplementing 100% methanol solution. During the induction process, the rotation speed and aeration volume are adjusted to control the dissolved oxygen at 35 - 55%; After the induction is completed, the obtained bacterial solution is centrifuged at 6000 rpm for 10 min, and then the fermentation supernatant is clarified with a 0.22 - µm filter membrane.

[0039] 4. Purification of recombinant protein The fermentation supernatant obtained after centrifugation and filtration is successively passed through cation exchange chromatography such as the SP Sepharose FF cation exchange chromatography column of Cytiva, whose packing material is a sulfopropyl (SP) strong cation exchange chromatography packing material for rapid protein purification; gel filtration chromatography such as the Superdex™ 75 Increase gel filtration chromatography column of Cytiva, whose packing material is a cross-linked agarose and dextran complex; strong anion composite mode chromatography such as Capto adhere and Capto adhere ImpRes of Cytiva, whose ligand is N-benzyl-n-methylethanolamine, which can efficiently remove various negatively charged impurities such as endotoxin, HCP (host cell protein), and nucleic acid in the sample, and has higher purification efficiency and stronger impurity removal ability than single anion exchange chromatography. The three chromatography modes endow the purification of platelet-derived growth factor mutant with advantages such as high throughput, high purity, easy operation, more economical, and safer.

[0040] 4.1 Ion exchange chromatography includes the following steps: 4.1.1. Take the culture supernatant containing the mutant of platelet-derived growth factor B and clarify the fermentation broth by centrifugation and tandem sterilization filtration with a depth filter. 4.1.2. Connect the chromatography column to the chromatography system, set the linear flow rate to 300 cm / h, and equilibrate the chromatography column with the equilibration buffer for 3 - 10 column volumes (CV). The equilibration buffer is 20 mmol / L PB, 0.15 mol / L sodium chloride, pH 7.2. 4.1.3. After the sample is loaded onto the column, wash the column with the equilibration buffer. 4.1.4. Elute the miscellaneous proteins on the column using a washing buffer. The washing solution is 20 mmol / L PB, 0.5 mol / L sodium chloride, pH 7.2; 4.1.5. Elute the target protein using an elution buffer and collect the target protein. The elution solution is 20 mmol / L PB, 1 mol / L sodium chloride, pH 7.2.

[0041] 4.2 Gel filtration chromatography includes the following steps: 4.2.1. Connect the chromatography column to the chromatography system and set the linear flow rate at 30 cm / h. First, wash the chromatography column with 0.5 M NaOH for 1 - 2 column volumes (CV), and then soak it for at least 30 min; 4.2.2. Equilibrate the chromatography column with an equilibration buffer for 2 - 3 column volumes (CV). The equilibration solution is 20 mmol / L PB, 0.15 mol / L NaCl, pH 7.2; 4.2.3. Divide the target protein eluate from the previous step into two equal parts and load the samples in two times. The volume of each sample loading does not exceed 7% of the column volume (CV), and the interval between the two sample loadings is at least 1 column volume (CV); 4.2.4. Continue to wash the column with the equilibration buffer and collect the target protein, namely the purified platelet-derived growth factor B or its mutant.

[0042] 4.3 Anion complex mode chromatography includes the following steps: 4.3.1. Connect the chromatography column to the chromatography system and set the linear flow rate at 300 cm / h. First, wash the chromatography column with 0.5 M NaOH for 5 column volumes (CV), and then soak it for at least 30 min. Then, regenerate the chromatography column with 1 M NaCl for 5 column volumes (CV); 4.3.2. Equilibrate the chromatography column with an equilibration buffer for 10 column volumes (CV). The equilibration solution is 20 mmol / L PB, 0.15 mol / L NaCl, pH 7.2; 4.3.3. Load the eluate from the previous step onto the chromatography column and collect the flow-through, namely the purified mutant of platelet-derived growth factor B.

[0043] 5. SDS-PAGE detection of recombinant protein Take 10 μg of the purified PDGF-B mutant stock solution and add it separately to the precast electrophoresis gel containing SDS (SurePAG, Bis-Tris, 15%, 12 wells). Among them, the loading buffer is 4X LDS Sample Buffer. Place the loaded electrophoresis gel into the Tris-MOPS-SDS Running Buffer electrophoresis buffer for SDS-PAGE electrophoresis (180 V, 50 min). Stain and decolorize the electrophoresis gel with an eStain L1 protein staining instrument, and finally analyze the electrophoresis purity with a gel imager. Figure 2 Figure Figure 2 shows the SDS-PAGE electrophoresis diagrams of different recombinant platelet-derived growth factor BB mutants after purification. As shown in the figure, lane 5 is the Marker, lane 6 is the electrophoresis diagram of the purified mutant G0, and lane 7 is the electrophoresis diagram of the purified mutant G1. It can be seen from the electrophoresis diagram that the purity of the two mutants after purification is 100%.

[0044] 6. Detection of recombinant protein content Use a Lowry kit (Solarbio, PC0030) for detection. Add samples to a 96-well plate, make a protein standard curve, read the light absorption value with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the protein concentration.

[0045] 6.1. Reagent preparation Take appropriate amounts of Folin phenol reagent A and B and mix them at a ratio of 50:1. Prepare and use immediately. Similarly, take an appropriate amount of BSA standard and dilute it 10 times with PBS to a concentration of 0.5 mg / ml.

[0046] 6.2. Add the standard products to the 96-well plate at 0, 2, 4, 6, 8, 12, 16, 20 μl, and then supplement with 20, 18, 16, 14, 12, 8, 4, 0 μl of PBS respectively. The total volume of each well is 20 μl.

[0047] 6.3. Take 20 μl of the diluted sample to be tested and add it to the sample wells of the 96-well plate.

[0048] 6.4. Add 200 μl of the prepared Folin phenol reagent A to each well, gently shake and mix well, and let it stand at room temperature for 10 minutes.

[0049] 6.5. Add 20 μl of Folin phenol reagent B to each well, mix quickly, and let it stand at 37°C for 30 minutes. Measure A650 with an ELISA reader to calculate the protein concentration.

[0050] The experimental data is calculated by the linear regression calculation method, and the content of the protein sample is calculated according to the regression equation of the standard product.

[0051] Table 2. Comparison of the content of protein samples of two recombinant proteins

[0052] Two PDGF-B mutants G0 and G1 were fermented under the same conditions and scale, and purified by three-step chromatography under the same conditions. The volumes of the final harvested stock solutions of the target proteins were basically the same. However, the final protein concentration was the highest for mutant G1, and the yield of mutant G1 was also the highest in the end. It was found that the yield of mutant G1 was about 20% higher than that of G0 under the same conditions.

[0053] 7. Detection of the biological activity of recombinant proteins BALB / c 3T3-SRE-Luc cells (obtained by transfecting pGL4.33 plasmid into BALB / c 3T3 cells using electroporation) were cultured in DMEM complete medium containing 10% FBS and 1% double antibiotics (penicillin-streptomycin) in an incubator at 37°C and 5% carbon dioxide. After digesting the cells with 0.25% trypsin (0.02% EDTA), collecting and centrifuging them, and counting the cells with a cell counter, a cell suspension of 5x10 4 cells / ml was prepared with DMEM complete medium and inoculated into a white-bottom transparent 96-well cell culture plate. 100 μl of the cell suspension was added to each well and cultured in an incubator at 37°C and 5% carbon dioxide for 8 h; then it was replaced with DMEM basal medium and continued to be cultured in an incubator at 37°C and 5% carbon dioxide overnight (15 h - 16 h); the original medium was discarded, and the pre-gradient diluted recombinant PDGF-BB solution was added, 100 μl to each well, and duplicate wells were set for each concentration; it was continued to be cultured in an incubator at 37°C and 5% carbon dioxide for 4 h; the cell proliferation activity was detected by the reporter gene method; 100 μl of One-Lite Luciferase Assay System fluorescence enzyme activity detection reagent (DD1203, Novozymes) was added to each well, and after incubating at room temperature in the dark for 20 min, the fluorescence signal was detected with SpectraMax Paradigm (Agilent Biotek). The experimental data were calculated by the four-parameter regression calculation method, and the EC50 value of the sample to be tested was calculated according to the EC50 luminescence value of the standard protein sample. Figure 3 The statistical chart of the biological activity after purification of different platelet-derived growth factor B mutants is given. The specific biological activity ratio is shown in Table 3. Compared with mutant G0, the biological activity of G1 was increased by more than 2 times and was 5.2 times that of the international standard product.

[0054] Table 3. Comparison of the biological activities of two recombinant proteins

[0055] It can be seen from the activity results that the biological activity of mutant G1 is about twice that of G0.

[0056] Preparation Example 2: Prescription Screening of Recombinant Platelet-Derived Growth Factor Mutant Eye Drops According to the principle of Quality by Design, the present invention screens the key excipients in the prescription of recombinant human platelet-derived growth factor mutant eye drops, such as thickeners, protein protectants, buffer systems, osmotic pressure regulators, etc., to form the optimal prescription. The following further illustrates with the preferred embodiments of the present invention, and these embodiments should be understood not to limit the scope of implementation of the present invention, and their variations and equivalent methods are all covered within the protection scope of the present invention.

[0057] 1. Prescription Design Prescription F1: Platelet-derived growth factor mutant 1,200,000 IU, Sodium hyaluronate 0.4 g, Mannitol 4 g, Sodium dihydrogen phosphate monohydrate 0.32 g, Disodium hydrogen phosphate dodecahydrate 2.08 g, Sodium chloride 2.24 g, Add water for injection to 400 ml, Specific preparation method: Dissolve recombinant human platelet-derived growth factor mutant, sodium hyaluronate, mannitol, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dodecahydrate and sodium chloride in an appropriate amount of water for injection, and filter sterilize with a 0.22 μm filter membrane; under aseptic conditions, mix the solutions obtained in the above two steps evenly, make up the volume with sterile water for injection and immediately dispense to obtain the product.

[0058] Prescription F2: Platelet-derived growth factor mutant 1,200,000 IU, Polyvinyl alcohol 4 g, Mannitol 4 g, Sodium dihydrogen phosphate monohydrate 0.32 g, Disodium hydrogen phosphate dodecahydrate 2.08 g, Sodium chloride 2.24 g, Add water for injection to 400 ml, Specific preparation method: Disperse polyvinyl alcohol in an appropriate amount of water for injection, sterilize at 121 °C for 30 min, and cool for later use; dissolve recombinant human platelet-derived growth factor mutant, mannitol, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dodecahydrate and sodium chloride in an appropriate amount of water for injection, and filter sterilize with a 0.22 μm filter membrane; under aseptic conditions, mix the solutions obtained in the above two steps evenly, make up the volume with sterile water for injection and immediately dispense to obtain the product.

[0059] Prescription F3 Platelet-derived growth factor mutant 1,200,000 IU, Polyvinyl alcohol 4 g, Hydroxypropyl methylcellulose 0.4 g, Mannitol 4 g, Sodium dihydrogen phosphate monohydrate 0.32 g, Disodium hydrogen phosphate dodecahydrate 2.08 g, Sodium chloride 2.24 g, Add water for injection to make 400 ml, Specific preparation method: Disperse polyvinyl alcohol in an appropriate amount of water for injection, sterilize at 121 °C for 30 min, and set aside after cooling; dissolve recombinant human platelet-derived growth factor mutant, hydroxypropyl methylcellulose, mannitol, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dodecahydrate and sodium chloride in an appropriate amount of water for injection, and filter aseptically through a 0.22-μm filter membrane; mix the solutions obtained in the above two steps evenly under aseptic conditions, make up the volume with sterile water for injection, and then aliquot immediately to obtain the product.

[0060] Prescription F4 Platelet-derived growth factor mutant 1,200,000 IU, Polyvinyl alcohol 4 g, Methylcellulose 0.4 g, Mannitol 4 g, Sodium dihydrogen phosphate monohydrate 0.32 g, Disodium hydrogen phosphate dodecahydrate 2.08 g, Sodium chloride 2.24 g, Add water for injection to make 400 ml, Specific preparation method: Disperse polyvinyl alcohol in an appropriate amount of water for injection, sterilize at 121 °C for 30 min, and set aside after cooling; dissolve recombinant human platelet-derived growth factor mutant, methylcellulose, mannitol, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dodecahydrate and sodium chloride in an appropriate amount of water for injection, and filter aseptically through a 0.22-μm filter membrane; mix the solutions obtained in the above two steps evenly under aseptic conditions, make up the volume with sterile water for injection, and then aliquot immediately to obtain the product.

[0061] 2. Prescription screening: Perform accelerated tests on the eye drops of platelet-derived growth factor and its mutants prepared with different prescriptions under different conditions such as high temperature and high humidity, and light, and screen the best prescription of platelet-derived growth factor mutant eye drops through the biological activities of the eye drops with different prescriptions.

[0062] (1)High temperature and high humidity test The eye drops of different preparation examples were filled into colorless plastic bottles, capped and sealed, and then placed under the conditions of 40 °C and 75% relative humidity for accelerated testing. The biological activities of the eye drops were detected at 0, 1, 2, and 3 months respectively. The results are shown in Table 4: Table 4. Changes in biological activities of each formulation in the high temperature and high humidity test

[0063] (2)Accelerated light test The eye drops of different preparation examples were filled into colorless plastic bottles, sealed and placed under 3000 Lx light for testing. The biological activities of the eye drops were detected at 0, 1, 2, and 3 months respectively. The results are shown in Table 5: Table 5. Changes in biological activities of each formulation in the accelerated light test

[0064] Through the accelerated test, the biological activities of the above four formulations were investigated, and it was found that the biological activity stability of Formulation 3 was better. When accelerated at 40 °C for three months, the biological activity of Formulation 3 was 5750 IU / ml, significantly higher than 1800 IU / ml of Formulation 1, 5000 IU / ml of Formulation 2, and 5100 IU / ml of Formulation 4; when accelerated under light conditions for three months, the biological activity of Formulation 3 was 5600 IU / ml, significantly higher than 1600 IU / ml of Formulation 1, 5100 IU / ml of Formulation 2, and 5050 IU / ml of Formulation 4. Therefore, Formulation 3 was selected as the preferred formulation for the recombinant platelet-derived growth factor mutant eye drops.

[0065] Stability investigation (3)High temperature and high humidity test The eye drops of Formulation 3 were filled into colorless plastic bottles, capped and sealed, and then placed under the conditions of 40 °C and 75% relative humidity for accelerated testing. The content, appearance, pH, osmotic pressure, sterility, and biological activity of the eye drops were detected at 0, 1, 2, and 3 months respectively. The results of the high temperature and high humidity test are shown in Table 6: Table 6. Table of changes in technical parameters of Formulation 3 in the high temperature and high humidity test

[0066] The test results show that the quality of the eye drops of the present invention is stable in the high temperature and high humidity test.

[0067] (4)Long-term low temperature test The eye drops of Prescription 3 were filled into colorless plastic bottles, sealed and tested at 4°C. The content, appearance, pH, osmotic pressure, sterility and bioactivity of the eye drops were detected at 0, 1, 3, 6 and 12 months respectively. The results of the low-temperature test are shown in Table 7: Table 7. Variation Table of Technical Parameters for Long-Term Low-Temperature Test of Prescription 3

[0068] The test results show that the quality of the eye drops of the present invention is stable in the long-term low-temperature test.

[0069] (5) Accelerated Light Test The eye drops of Prescription 3 were filled into colorless plastic bottles, sealed and tested under 3000 Lx light. The content, appearance, pH, osmotic pressure, sterility and bioactivity of the eye drops were detected at 0, 1, 2 and 3 months respectively. The results of the accelerated light test are shown in Table 8: Table 8. Variation Table of Technical Parameters for Accelerated Light Test of Prescription 3

[0070] The test results show that the quality of the eye drops of the present invention is stable in the accelerated light test.

[0071] Application Experimental Example 1: Treatment of Rabbit Corneal Injury Model with PDGF-B Mutant Eye Drop Formula The eye drops of Prescription 3 were prepared using recombinant platelet-derived growth factor G0 and G1, with the content of PDGF being 3000 IU / ml. Since the bioactivity of G1 is 3.94E+06 IU / mg, the concentration of G1 in the G1 eye drops is 0.63 μg / ml, while the bioactivity of G0 is 1.90E+06 IU / mg, so the concentration of G0 in the G0 eye drops is 1.31 μg / ml. The pharmacodynamic evaluation of these two eye drops was carried out in animal experiments.

[0072] Test animals: Healthy New Zealand white rabbits, weighing 2 - 3 kg, both males and females were used. The anterior segment was examined by slit lamp and no abnormalities were found. 15 animals were randomly divided into 3 groups, with 5 animals in each group.

[0073] Rabbit corneal alkali burn model: For animals with normal external eye examination, the right eye was selected as the experimental eye. The animals were anesthetized systemically by intraperitoneal injection of chloral hydrate. At the same time, the right eye was washed twice with normal saline, and the eye was anesthetized by instilling 50 μl of dicaine eye drops twice. A filter paper disc with a diameter of 8 mm was soaked with 1 M sodium hydroxide, and the excess sodium hydroxide in the filter paper disc was blotted with a dry filter paper. Then, the rabbit's eyelids were opened with an eyelid retractor, and the excess moisture on the ocular surface was wiped off with a cotton swab. The filter paper disc was carefully placed in the center of the right cornea of the rabbit for 40 s, and then the ocular surface and conjunctival sac were thoroughly rinsed with excess normal saline for 1 min, thus completing the experimental chemical burn of the rabbit ocular surface.

[0074] Test samples: The negative control was the model control group without any drug administration, the test group was the recombinant human platelet-derived growth factor mutant G1 eye drops of Prescription F3, and the positive control group was the recombinant human platelet-derived growth factor G0 eye drops of Prescription F3. 1 - 2 drops were administered each time, 4 times a day, and the administration was continued for two weeks.

[0075] Evaluation indicators: At 1, 3, 5, 7, 10, and 14 days of treatment respectively, the damaged corneas of rabbits were stained with sodium fluorescein, and then standard photographs of the corneas were taken with a slit lamp fluorescence microscope. The corneal epithelial healing rates of each group at different times were analyzed through a computer image analysis system. The higher the corneal epithelial healing area and the corneal epithelial healing rate, the better the treatment effect.

[0076] The statistical results of the test are shown in Table 9 and Figure 4 as follows: Table 9: Corneal epithelial healing area at different times of each group (%,X±S)

[0077] Since only the damaged cornea can be stained green by sodium fluorescein under the microscope, the darker the green stain of sodium fluorescein in the rabbit corneal alkali burn model, the more severe the damage, and the lighter the green, the better the repair effect.

[0078] The results of animal experiments showed (as Figure 5 and Figure 6 ) that the recombinant platelet-derived growth factor eye drops prepared with G1 and G0 in Prescription 3 had good repair effects in the rabbit corneal alkali burn injury model, significantly better than the model injury group. Both G1 and G0 were basically repaired after seven days of drug administration, and the model control group was still not completely repaired at 14 days. At the same time, there was no significant difference in the repair effects between the two recombinant platelet-derived growth factor eye drops of G1 and G0, and the drug effects were equivalent.

[0079] The drug effects of the recombinant platelet-derived growth factor eye drops prepared with G0 and G1 according to Prescription 3 were equivalent, but the protein concentration of G0 in the G0 eye drops was 1.31 ug / ml, while the protein content of G1 in the G1 eye drops was 0.63 ug / ml. It can be seen from this that using G1 to prepare the recombinant platelet-derived growth factor eye drops saves half of the protein usage compared with using G0, greatly reduces the protein usage, saves production costs, and has great economic benefits.

[0080] Application Experimental Example 2: Irritation test The eye drops prepared with the eye drop prescription in Application Experiment Example 1 and blank excipients were selected to observe the irritation responses of the conjunctiva, cornea, and iris after the test samples were contacted with the eyes of animals. Grouping for administration: Nine healthy rabbits with no eye damage in the eye examination (the cornea was not cloudy, the conjunctiva was not congested, edematous, or secreting, the pupils were round, equal in size on both sides, and the light reflex was good) were randomly divided into 3 groups, with 3 rabbits in each group. In the first group, normal saline was given to both the left and right eyes. In the second group, normal saline was given to the left eye as a self-control, and blank excipient eye drops were given to the right eye. In the third group, normal saline was also given to the left eye as a self-control, and the eye drops prepared with Prescription 3 were given to the right eyes respectively. Administration method: The medicinal solution was dropped into the conjunctival sac of the rabbits' eyes respectively, the nasolacrimal duct was compressed, and the eyes were passively closed for about 5 - 10 seconds. 4 times a day, 1 - 2 drops (about 50 μl) each time, and eye drops were instilled continuously for 14 days. Before each administration and at 1, 2, 4, 24, 48, and 72 hours after the last administration, the cornea, iris, and conjunctiva were directly observed with the naked eye or with a magnifying glass for irritation, and the irritation was scored. Fluorescein sodium 2% was stained in a timely manner to observe whether there was coloring in each part. The irritation response scores were carried out according to Table 10. The irritation response scores of the cornea, iris, and conjunctiva of each rabbit at each observation time were added up to obtain the total score. The total score of a group was divided by the number of animals to obtain the final average score, which was compared with the left eye. Then, the eye irritation of the sample was judged according to the evaluation criteria in Table 11. When the irritation intensities of the cornea, iris, and conjunctiva were inconsistent, evaluations should be made separately. After all the observations were completed, the rabbits were sacrificed, the eyes and the upper and lower eyelids were cut, and any abnormal changes such as redness and swelling were observed with the naked eye. Then, they were fixed in 10% formaldehyde solution, embedded in paraffin, sectioned, and stained with HE for histopathological examination. Irritation results: See Table 12.

[0081] Table 10: Standards for Eye Irritation Response Scores

[0082]

[0083] Table 11: Evaluation Criteria for Eye Irritation

[0084] Table 12: Results of the Irritation Test of Recombinant Human Platelet-Derived Growth Factor Eye Drops

[0085] From the results of the animal eye irritation test, it can be seen that the eye drops prepared with Prescription 3 have no irritation to the blank excipients and normal saline.

[0086] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the protection scope of the present invention should be defined by the scope of the claims. For those of ordinary skill in the art, several improvements can be made without departing from the spirit and scope of the present invention, and these improvements should also be regarded as within the protection scope of the present invention.

Claims

1. A liquid composition containing a platelet-derived growth factor mutant, characterized in that, The amino acid sequence of the platelet-derived growth factor mutant is as shown in SEQ ID NO.3, and the composition contains: Platelet-derived growth factor mutant 800 - 7000 IU / ml, Thickener 0.1 - 2% (g / ml), Protein protectant 0.05 - 3% (g / ml), Phosphate buffer 10 - 30 mM, Osmotic pressure regulator 0.25 - 0.75% (g / ml), The pH of the liquid composition is 6.8 - 7.4, wherein, the thickener is selected from one or more of polyvinyl alcohol, sodium hyaluronate, hydroxypropyl methylcellulose, and methylcellulose; the protein protectant is selected from one or more of glycine, glutamic acid, lysine, histidine, arginine, sucrose, trehalose, polyethylene glycol 400, and mannitol; the osmotic pressure regulator is selected from one or more of sodium chloride, glucose, or sorbitol.

2. The liquid composition according to claim 1, characterized in that, The dosage of recombinant human platelet-derived growth factor in the composition is 1000 - 6000 IU / ml.

3. The liquid composition according to claim 1, characterized in that, The thickener is polyvinyl alcohol, and the dosage of polyvinyl alcohol is 0.5% - 1.5%.

4. The liquid composition according to claim 1, wherein The thickener is hydroxypropyl methylcellulose, and the dosage of hydroxypropyl methylcellulose is 0.1% - 0.5%.

5. The liquid composition according to claim 1, characterized in that, The protein protectant is mannitol, and the dosage of mannitol is 0.05% - 3%.

6. The liquid composition according to claim 1, wherein The osmotic pressure regulator is sodium chloride, and the dosage of sodium chloride is 0.25% - 0.75%.

7. The liquid composition according to claim 1, characterized in that, The dosages of each component in the composition are respectively: Platelet-derived growth factor mutant 3000 IU / ml, Polyvinyl alcohol 1% (g / ml), Hydroxypropyl methylcellulose 0.1% (g / ml), Mannitol 1% (g / ml), Sodium chloride 0.56% (g / ml), Phosphate buffer is 20 mM, and the pH is 6.8 - 7.

4.

8. A method for preparing the liquid composition according to any one of claims 1 to 7, characterized in that, The method includes the following steps: (1) Disperse polyvinyl alcohol in water for injection, sterilize by autoclaving at 121 °C, and reserve after cooling; (2) Dissolve the platelet-derived growth factor mutant, hydroxypropyl methylcellulose, phosphate buffer, osmotic pressure regulator, and protein protectant in water for injection, and filter aseptically with a 0.22 μm filter membrane; (3) Under aseptic conditions, mix the solution obtained in step (1) and the solution obtained in step (2) evenly, make up the volume with sterile water for injection, and immediately dispense and reserve.

9. Use of the liquid composition according to any one of claims 1 - 7 in the preparation of a drug for treating corneal injury.

10. The application according to claim 9, characterized in that, The corneal injury includes chemical burn and mechanical injury.

Images