An ophthalmic active peptide and its eye drop formulation

Abstract

This invention belongs to the field of ophthalmic peptides and their pharmaceuticals, and provides an ophthalmic fusion peptide that simultaneously relieves dry eye syndrome and has antibacterial effects, as well as an ophthalmic formulation containing said peptide. The ophthalmic fusion peptide of this invention has its own antibacterial properties, and its use in formulations can reduce or eliminate the need for antibacterial agents, resulting in formulations that are less irritating and safer and more effective.

Description

Technical Field

[0001] This invention relates to the field of ophthalmic polypeptide drugs, specifically to an ophthalmic fusion polypeptide and its formulation for relieving dry eye syndrome. Background Technology Dry eye syndrome is a common eye condition, also known as keratoconjunctivitis sicca. Main symptoms include dryness, fatigue, a foreign body sensation, burning, photophobia, and blurred vision. In severe cases, redness, swelling, pain, and corneal epithelial damage may occur. These symptoms can worsen with prolonged eye strain, exposure to dry environments, or use of electronic devices.

[0002] Studies have shown that dry eye syndrome is closely related to insufficient tear production. When tear production is insufficient, the eyes do not receive enough lubrication, easily leading to symptoms such as dryness, fatigue, and a foreign body sensation. Nowadays, people's lives and work are increasingly inseparable from electronic devices; staring at computer, mobile phone, and tablet screens for extended periods significantly reduces blinking frequency. Normally, a person blinks about 15 to 20 times per minute, but when focusing on electronic screens, the blinking frequency may drop to 5 to 10 times per minute or even less. Blinking evenly distributes tears across the surface of the eye, lubricating and protecting the eyes. Reduced blinking prevents tears from adequately lubricating the eyes, causing faster tear evaporation and easily triggering dry eye syndrome. Furthermore, prolonged exposure to dry environments, excessive eye strain, insufficient sleep, and poor eye habits can all easily lead to reduced tear production and cause dry eye syndrome.

[0003] Artificial tears and other eye drops are one of the main clinical treatments for dry eye syndrome. Artificial tears primarily mimic the body's natural tears, replenishing moisture to the eyes and lubricating the ocular surface, thereby relieving dry eye symptoms. Artificial tears can also form a protective film on the ocular surface, reducing tear evaporation and increasing eye comfort. Different types of artificial tears have different compositions; some contain moisturizing ingredients such as sodium hyaluronate, while others contain electrolytes that mimic those found in natural tears.

[0004] Epidermal growth factor (EGF) eye drops are also a commonly used eye drop preparation for treating dry eye syndrome. EGF can promote the proliferation, differentiation, and repair of ocular surface cells. For patients with dry eye syndrome, ocular surface cells may be damaged due to long-term dryness and inflammation. EGF eye drops can stimulate the growth and repair of corneal epithelial cells, conjunctival epithelial cells, etc., helping to restore the normal structure and function of the ocular surface. At the same time, it can also promote the repair and regeneration of tear secretion gland cells, thereby increasing tear secretion to a certain extent.

[0005] However, most eye drop formulations contain small-molecule antibacterial agents, and long-term use of these agents can cause irritation and damage to the ocular surface, exacerbating dry eye symptoms. Therefore, researching and developing eye drop formulations that do not use small-molecule antibacterial agents has broad clinical value. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides an ophthalmic active peptide, which is a fusion peptide comprising epidermal growth factor and a broad-spectrum antimicrobial peptide, linked by a linker without affecting each other's function. The active peptide of this invention possesses antimicrobial activity itself, and its use can reduce or eliminate small-molecule antimicrobial agents in formulations, exhibiting low irritation and greater safety and effectiveness.

[0007] Therefore, in one aspect, the present invention provides an ophthalmic active peptide, the amino acid sequence of which is shown in SEQ ID NO:1. The active peptide is formed by linking modified epidermal growth factor and a broad-spectrum antimicrobial peptide via a linker. The modified epidermal growth factor is a partially truncated and mutated epidermal growth factor, the sequence of which is shown in SEQ ID NO:2. The amino acid sequence of the broad-spectrum antimicrobial peptide is shown in SEQ ID NO:3.

[0008] The ophthalmic active peptides of this invention retain a tear-promoting effect similar to wild-type epidermal growth factor, while also possessing excellent broad-spectrum antibacterial properties. Therefore, when using ophthalmic active peptide eye drops of this invention, there is no need to add antibacterial agents, effectively reducing the irritation of the eye drop formulation, making it safer, more effective, and more suitable for long-term use.

[0009] On the other hand, the present invention provides a nucleotide fragment encoding the above-mentioned ophthalmic active peptide; preferably, the sequence of the nucleotide fragment is shown in SEQ ID NO:4.

[0010] On the other hand, the present invention provides an expression vector comprising a polynucleotide fragment encoding the ophthalmic active peptide described herein; preferably, the expression vector comprises the nucleotide fragment shown in SEQ ID NO:4. The expression vector of the present invention may be a recombinant pET-28a(+), pET-30a(+), or pET-32a(+) plasmid, and preferably a recombinant pET-30a(+) expression vector.

[0011] On the other hand, the present invention provides a recombinant engineered Escherichia coli strain, wherein the recombinant engineered Escherichia coli strain comprises the above-mentioned recombinant expression vector; preferably, the Escherichia coli is selected from BL21(DE3) Escherichia coli. The recombinant expression vector is constructed according to the following method: (1) Synthesize a polynucleotide fragment encoding the ophthalmic active peptide of the present invention, as shown in SEQ ID NO.4; (2) The polynucleotide fragment was cloned into plasmid pET-30a(+) to construct the expression vector; (3) The expression vector was transformed into Escherichia coli BL21(DE3) to obtain recombinant engineered Escherichia coli.

[0012] On the other hand, the present invention provides a method for preparing the ophthalmic active peptide, the method comprising the following steps: culturing the recombinant Escherichia coli engineered strain of the present invention to express the ophthalmic active peptide; and subjecting it to refolding, denaturation and purification treatments to obtain the purified ophthalmic active peptide.

[0013] On the other hand, the present invention provides an ophthalmic formulation comprising the ophthalmic active peptide, preferably, the formulation further comprising sodium hyaluronate, a pH adjuster, and an osmotic pressure adjuster.

[0014] Preferably, the formulation of the present invention is an eye drop.

[0015] Preferably, the concentration of the ophthalmic active peptide in the formulation is 0.003%-0.005%, and more preferably, the concentration of the ophthalmic active peptide is 0.004%.

[0016] Preferably, the pH adjuster is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium phosphate; most preferably, the pH adjuster is sodium dihydrogen phosphate; preferably, the preparation adjusts the pH value to 6.9-7.0.

[0017] Preferably, the osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, potassium chloride, and glycerol; more preferably, the osmotic pressure regulator is sodium chloride. Preferably, the amount of sodium chloride used is 0.7%-1% (w / v), more preferably 0.75%, 0.8%, 0.85%, or 0.9%.

[0018] Preferably, the sodium hyaluronate content is 0.05%-0.2%, more preferably, it is 0.05%, 0.1% or 0.15%.

[0019] On the other hand, the present invention provides a method for preparing an eye drop containing the aforementioned ophthalmic active peptide, the method comprising the following steps: (1) Weigh the ophthalmic active peptide, sodium hyaluronate and sodium chloride described in this invention, dissolve them in water for injection and mix them evenly; (2) Use a pH adjuster to adjust the pH value to 6.9-7.0; (3) Filter through a 0.22μm microporous membrane and use separately.

[0020] Thanks to the fact that the ophthalmic active peptide of the present invention possesses both epidermal growth factor activity and antimicrobial peptide activity, it is safer and more effective as an eye drop. Therefore, the beneficial effects of the present invention are as follows: (1) The ophthalmic active peptide of the present invention can significantly increase tear secretion and can be used to relieve eye discomfort, such as eye fatigue and dry eye symptoms. (2) The ophthalmic active peptide of the present invention has broad-spectrum antibacterial activity. The preparation does not require the addition of antibacterial agents, is non-irritating, and is safer and more effective. (3) The formulation of the present invention contains sodium hyaluronate, which can further prolong the time the drug stays in the eye and enhance the therapeutic effect. Detailed Implementation

[0021] The present invention will now be clearly and completely described in conjunction with specific embodiments. Those skilled in the art will understand that the embodiments described below are some, but not all, embodiments of the present invention, and are only used to illustrate the present invention, and should not be regarded as a limitation on the scope of protection of the present invention.

[0022] Example 1: Preparation of ophthalmic bioactive peptides The polynucleotide fragment described in SEQ ID NO:4 was constructed and inserted into the prokaryotic expression plasmid pET-30a(+) via the NdeI and XhoI sites. Sequencing verification yielded the expression plasmid for transformation. The expression plasmid was transformed into BL21 competent cells and cooled on ice for 25-30 min; after heat activation at 42℃ for 90 sec, it was immediately placed on ice for 3-5 min. 900 μL of sterile LB liquid medium (antibiotic-free) was added to a centrifuge tube, mixed well, and incubated at 37℃, 200-250 rpm for 1 hour to revive the engineered bacteria. 100 μL was plated onto LB agar plates containing kanamycin resistance. The plates were inverted and incubated overnight at 37℃. The next day, single colonies were picked and inoculated into sterile LB liquid medium (antibiotic-containing), and cultured at 37℃, 200-230 rpm on a shaker until the OD600 reached approximately 4. The culture was then stored as a seed culture.

[0023] Add 100 μL of the above-preserved seed culture to 100 mL of LB liquid medium containing kanamycin (50 μg / mL), and incubate overnight at 37°C with shaking at 220 rpm until OD600 > 4.0, to obtain secondary seed culture. Take 50 mL of the secondary seed culture and inoculate it into fermentation medium at a ratio of 1:10. Induce with IPTG (1 mmol / L) and then culture. After the culture is completed, collect the bacterial cells by centrifugation.

[0024] Weigh 100g of bacterial cells and resuspend them in 500mL of lysis buffer. Sonicate the cells for 25-30 minutes using an ultrasonic cell disruptor to break them up. Centrifuge the resulting homogenate at 4°C to collect the precipitate. Dissolve the precipitate in a urea-containing refolding solution, filter it through a filter membrane, and then purify it using nickel column chromatography and ion chromatography. Freeze-dry the eluent to obtain an ophthalmic active peptide with a purity of 93.2%.

[0025] Example 2: Antibacterial susceptibility test of ophthalmic active peptides The drug susceptibility test was performed using the classic continuous two-fold dilution method to determine the minimum inhibitory concentration of the ophthalmic active peptide of the present invention against a variety of Gram-positive and Gram-negative bacteria.

[0026]

[0027] Example 3: Preparation of eye drops containing ophthalmic active peptides Weigh out a fixed amount of ophthalmic active peptide, sodium hyaluronate, and sodium chloride, and dissolve them in water for injection, wherein the concentration of the ophthalmic active peptide is 0.004% (w / v), the concentration of the sodium hyaluronate is 0.1% (w / v), and the concentration of the sodium chloride is 0.75% (w / v). Use disodium hydrogen phosphate as a pH adjuster to adjust the pH value to 6.9-7.0. Filter the solution through a 0.22μm membrane for sterilization and aseptically dispense it.

[0028] Example 4: Tear secretion test Normal 3-4 month old male New Zealand rabbits were used. Each rabbit was subcutaneously injected with 1 mL (5 mg / mL) of scopolamine twice a day for 10 consecutive days. After successful model construction, the model was used for experiments.

[0029] Rabbits successfully modeled were randomly divided into an experimental group and a control group (using commercially available human epidermal growth factor eye drops), with 5 rabbits in each group; 5 New Zealand rabbits successfully modeled were used as negative controls. The negative control group received no treatment. The experimental group and control group were given 2 drops of the eye drops formulation described in Example 3 and the commercially available formulation, respectively, three times daily for 7 consecutive days. Afterward, the amount of tear secretion in the rabbits was measured using Schirmer test strips. The method involved inserting the Schirmer test strip into the conjunctival sac near the junction of the middle and outer thirds of the lower eyelid of the rabbit eye, and reading the length of the wet strip after 5 minutes. The experimental results are as follows:

[0030] During the testing of tear secretion, the eyes of both the experimental and control groups were visually examined, and no obvious abnormalities were found. No obvious congestion, edema, or other irritating symptoms were observed in the cornea, conjunctiva, or iris. This indicates that the eye drop formulation of the present invention has good safety.

Claims

1. An ophthalmic active peptide, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID NO:

1.

2. A nucleotide fragment for encoding the ophthalmic active peptide of claim 1.

3. The nucleotide fragment according to claim 2, characterized in that: The nucleotide fragment sequence is shown in SEQ ID NO:

4.

4. An expression vector comprising the nucleotide fragment of any one of claims 2-3.

5. Recombinant engineered bacteria comprising the expression vector of claim 4.

6. An ophthalmic formulation comprising the ophthalmic active peptide of claim 1.

7. The ophthalmic preparation according to claim 6, characterized in that: The formulation further comprises sodium hyaluronate, a pH adjuster, and an osmotic pressure adjuster.

8. The ophthalmic preparation according to claim 7, characterized in that: The ophthalmic preparation is an eye drop preparation.

9. The ophthalmic formulation according to claim 8, characterized in that: The pH adjuster in the ophthalmic preparation is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium phosphate, and the pH value of the preparation is 6.9-7.

0.

10. The ophthalmic formulation according to claim 9, characterized in that: In the ophthalmic preparation, the osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, potassium chloride, and glycerin.

11. The ophthalmic preparation according to any one of claims 6-10, characterized in that, The concentration of the ophthalmic active peptide is 0.003%-0.005%.

12. The ophthalmic preparation according to claim 11, characterized in that, The content of sodium hyaluronate is 0.05%-0.2%.

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