Use of a composition comprising neojuncan and astaxanthin in the preparation of a formulation for the treatment of dry eye and a method of preparing an in situ gel eye drop

Abstract

This invention belongs to the technical field of biomedicine and discloses the application of a composition comprising neo-agar oligosaccharides and astaxanthin in the preparation of formulations for the treatment of dry eye syndrome, as well as the formulation of in-situ gel eye drops and its preparation method. The composition provided by this invention specifically comprises neo-agar oligosaccharides and astaxanthin, which together constitute an organic whole, synergistically contributing to excellent ocular surface retention time and penetration efficiency. It can effectively inhibit the activation of core inflammatory pathways at low doses, reduce inflammatory cell infiltration in ocular surface tissues, promote goblet cell proliferation and epithelial repair, increase tear secretion, and improve corneal epithelial integrity. This achieves multiple effects, including anti-inflammatory properties, increased tear secretion, reduced corneal damage, promotion of ocular surface tissue repair, and improvement of corneal epithelial integrity. Furthermore, the composition exhibits excellent ocular surface biocompatibility and has very promising application prospects in the preparation of formulations for the treatment of dry eye syndrome.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, and particularly relates to the application of a composition including neoazooligosaccharide and astaxanthin in the preparation of formulations for the treatment of dry eye syndrome, and the preparation method of compound in situ gel eye drops. Background Technology

[0002] Dry eye syndrome is a chronic ocular surface disease caused by insufficient tear secretion or excessive tear evaporation, leading to decreased tear film stability, accompanied by ocular surface inflammation, tissue damage, and neurosensory abnormalities. It falls under the category of multifactorial ocular surface dysfunction. The core pathological mechanism of dry eye syndrome is a vicious cycle of "inflammation-damage" triggered by tear hyperosmolarity—increased tear film osmolarity directly damages ocular surface epithelial cells, triggering an innate immune response centered on NLRP3 inflammasome activation. This releases key pro-inflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), resulting in ocular surface inflammation, goblet cell loss, corneal epithelial barrier disruption, and impaired lacrimal gland secretion. Clinical manifestations mainly include dry eyes, foreign body sensation, burning sensation, fluctuating vision, and sensitivity to irritants. Continued progression can lead to corneal epithelial defects and conjunctival squamous metaplasia, severely affecting visual function and quality of life.

[0003] Currently, medications for treating dry eye syndrome are categorized into lubricating replacements, anti-inflammatory drugs, and immunomodulatory drugs. Among lubricating replacements, artificial tears can temporarily replenish tears and relieve dryness, with rapid onset and wide application. However, they only treat the symptoms and cannot halt disease progression; some preservative-containing preparations may worsen ocular surface damage with long-term use. Lubricating eye ointments such as petrolatum provide long-lasting hydration and are suitable for nighttime use, but they can easily cause blurred vision. Among anti-inflammatory and immunomodulatory drugs, topical corticosteroids such as fluorometholone can rapidly and effectively suppress inflammation, suitable for the acute phase of moderate to severe inflammation. However, long-term use can lead to increased intraocular pressure, cataracts, infection, and rebound effects after discontinuation. Cyclosporine A eye drops can inhibit T-cell activation and are suitable for inflammation-related dry eye, but they have a slow onset and can cause ocular surface irritation. Tacrolimus eye drops have a similar mechanism of action but may be less irritating. Secretagogues such as diquafosol sodium can stimulate mucin and tear secretion, improving tear film quality. However, their effectiveness is limited for some patients, and they can cause side effects such as eye discomfort. Regarding natural and nutritional supplements, there are currently studies on the effects of Omega-3 fatty acids, vitamin A, and extracts of traditional Chinese medicine such as wolfberry polysaccharides and Buddleja officinalis extract on dry eye syndrome relief. However, most natural ingredients have dispersed targets and unclear pharmacodynamic material basis, and lack high-quality clinical evidence to support the certainty and stability of their efficacy. Single use is difficult to effectively control the pathological process of moderate to severe dry eye syndrome, which has significant limitations. Summary of the Invention

[0004] The primary objective of this invention is to address the problems of limited therapeutic effects, limited bioavailability, and less than ideal biosafety in existing dry eye treatments due to their single mechanism of action, and to provide the application of the composition in the preparation of formulations for the treatment of dry eye.

[0005] The second objective of this invention is to provide a compound in situ gel eye drop.

[0006] A third objective of this invention is to provide a method for preparing the above-mentioned compound in situ gel eye drops.

[0007] Specifically, in the application of the composition provided by the present invention in the preparation of a formulation for the treatment of dry eye syndrome, the composition includes neoazolam oligosaccharide and astaxanthin.

[0008] Furthermore, the degree of polymerization of the new agar oligosaccharide is 2 to 6.

[0009] Furthermore, the proportion of all-trans isomers in the astaxanthin is not less than 95%.

[0010] Furthermore, the mass ratio of the added neo-agar oligosaccharide to astaxanthin is (1~10):(1~10).

[0011] Furthermore, the dosage form of the dry eye treatment preparation is selected from one or more of the following: solution, gel, ointment, and spray.

[0012] The compound in-situ gel eye drops provided by the present invention specifically include neo-Agar oligosaccharide, composite lipid nanoparticles and pharmaceutically acceptable excipient carrier. The composite lipid nanoparticles are coated with astaxanthin, the neo-Agar oligosaccharide is adsorbed on the surface of the composite lipid nanoparticles, and the mass ratio of neo-Agar oligosaccharide to astaxanthin is (1~10):(1~10).

[0013] Furthermore, the degree of polymerization of the new agar oligosaccharide is 2 to 6.

[0014] Furthermore, the particle size of the composite lipid nanoparticles is 80nm~120nm.

[0015] Furthermore, the preparation of the composite lipid nanoparticles includes: mixing astaxanthin, phospholipids and an amphiphilic nonionic surfactant evenly to obtain an oil phase solution; and subjecting the oil phase solution and water to high-pressure microfluidic nanodispersion treatment to obtain a dispersion containing the composite lipid nanoparticles.

[0016] Furthermore, in the preparation of the composite lipid nanoparticles, the mass ratio of astaxanthin, phospholipids and amphiphilic nonionic surfactant is (1~3):(30~60):(5~15).

[0017] Furthermore, in the preparation of the composite lipid nanoparticles, the phospholipid is soybean lecithin and / or egg yolk lecithin.

[0018] Furthermore, in the preparation of the composite lipid nanoparticles, the amphiphilic nonionic surfactant is selected from one or more of polyoxyethylene ether compounds, polyoxyethylene sorbitan ester compounds, and polyoxyethylene-polyoxypropylene block copolymers.

[0019] Furthermore, in the preparation of the composite lipid nanoparticles, the amphiphilic nonionic surfactant is selected from one or more of poloxamer 124, poloxamer 188, and poloxamer 407.

[0020] Furthermore, in the preparation of the composite lipid nanoparticles, the volume ratio of the oil phase solution to water is (2~5):(95~98).

[0021] Furthermore, the pharmaceutically acceptable excipient carrier is a thermosensitive hydrogel matrix, which is selected from one or more of cellulose and its derivatives, chitosan, poloxamer, and poly(N-isopropylacrylamide).

[0022] Furthermore, in the compound in situ gel eye drops, the concentration of the thermosensitive hydrogel matrix is ​​1wt%~20wt%, the concentration of the neo-agar oligosaccharide is 0.001wt%~1wt%, and the concentration of the composite lipid nanoparticles is 0.005wt%~5wt%.

[0023] The preparation method of the compound in-situ gel eye drops provided by the present invention specifically includes: mixing the new agar oligosaccharide and the composite lipid nanoparticles, and carrying out a self-assembly reaction under a light-protected and inert atmosphere to obtain a nanocomposite; mixing the nanocomposite with a pharmaceutically acceptable excipient carrier evenly to obtain the compound in-situ gel eye drops.

[0024] Furthermore, the temperature of the self-assembly reaction is 30℃~40℃, the stirring speed is 200rpm~400rpm, and the time is 20min~40min.

[0025] Beneficial effects: The composition provided by this invention specifically includes neo-agar oligosaccharides and astaxanthin. Neo-agar oligosaccharides and astaxanthin together constitute an organic whole, working synergistically to endow the composition with excellent ocular surface retention time and penetration efficiency. It can effectively inhibit the activation of core inflammatory pathways at low doses, reduce the infiltration of inflammatory cells in ocular surface tissues, promote goblet cell proliferation and epithelial repair, increase tear secretion, and improve corneal epithelial integrity. Thus, it achieves multiple effects such as anti-inflammation, increased tear secretion, reduced corneal damage, promotion of ocular surface tissue repair, and improvement of corneal epithelial integrity. Moreover, the composition has excellent ocular surface biocompatibility and has very promising application prospects in the preparation of formulations for the treatment of dry eye syndrome.

[0026] In some specific embodiments, a compound in-situ gel eye drop is also provided. This compound in-situ gel eye drop specifically comprises neo-Agar oligosaccharides, composite lipid nanoparticles, and a thermosensitive hydrogel matrix. Astaxanthin is coated within the composite lipid nanoparticles, and neo-Agar oligosaccharides are adsorbed onto the surface of the composite lipid nanoparticles. The introduction of the thermosensitive hydrogel matrix allows the compound in-situ gel eye drop to remain on the cornea in a semi-solid mucosal form, effectively prolonging the ocular surface retention time of the active substance. Furthermore, neo-Agar oligosaccharides not only act as an active substance, playing an anti-inflammatory role and promoting ocular surface tissue repair, but also serve as a carrier promoting ocular surface adhesion and penetration, strengthening the binding interface of the composite lipid nanoparticles and further prolonging the contact time between the composite lipid nanoparticles and corneal tissue. This facilitates better slow release of astaxanthin, allowing neo-Agar oligosaccharides and astaxanthin to better exert a synergistic effect, resulting in excellent ocular retention time, drug penetration efficiency, and controllable sustained release of the compound in-situ gel eye drop, leading to superior treatment efficacy for dry eye. Attached Figure Description

[0027] Figure 1 This is one of the experimental results of the mouse tear film function test provided in Embodiment 2 of the present invention; Figure 2 This is one of the experimental results of the mouse corneal integrity test provided in Embodiment 2 of the present invention (the scales of each sub-figure are consistent); Figure 3 This is the second experimental result diagram of the mouse corneal integrity test provided in Embodiment 2 of the present invention; Figure 4 This is one of the experimental results of the distribution test of the inflammatory factor IL-1β in the ocular surface tissue of mice provided in Example 2 of the present invention (the scales of each sub-figure are consistent); Figure 5 This is the second figure showing the experimental results of the distribution test of the inflammatory factor IL-1β in the ocular surface tissue of mice provided in Example 2 of the present invention; Figure 6This is one of the experimental results of the distribution test of the inflammatory factor IL-6 in mouse ocular surface tissue provided in Example 2 of the present invention (the scales of each sub-figure are consistent); Figure 7 This is the second figure showing the experimental results of the distribution test of the inflammatory factor IL-6 in the ocular surface tissue of mice provided in Example 2 of the present invention; Figure 8 This is one of the experimental results of the distribution test of the inflammatory factor TNF-α in mouse ocular surface tissue provided in Example 2 of the present invention (the scales of each sub-figure are consistent); Figure 9 The second figure shows the experimental results of the distribution test of the inflammatory factor TNF-α in the ocular surface tissue of mice provided in Example 2 of the present invention; Figure 10 This is a graph showing the experimental results of testing the IL-1β content in mouse ocular surface tissue serous fluid provided in Example 2 of the present invention; Figure 11 This is a graph showing the experimental results of testing the IL-6 content in mouse ocular surface tissue serous fluid provided in Example 2 of the present invention; Figure 12 This is a graph showing the experimental results of testing TNF-α content in mouse ocular surface tissue serous fluid as provided in Example 2 of the present invention; Figure 13 This is a graph showing the experimental results of the distribution test of Tunel-positive cells in mouse ocular surface tissue provided in Example 2 of the present invention (the scales of each sub-graph are consistent). Figure 14 This is a diagram showing the experimental results of testing the number of Tunel-positive cells in mouse ocular surface tissue as provided in Example 2 of the present invention; Figure 15 This is a graph showing the experimental results of the distribution test of Ki67 positive cells in mouse ocular surface tissue provided in Example 2 of the present invention (the scales of each sub-graph are consistent). Figure 16 This is a diagram showing the experimental results of testing the number of Ki67-positive cells in mouse ocular surface tissue provided in Example 2 of the present invention; Figure 17 The figure shows the experimental results of H&E staining of mouse ocular surface tissue provided in Example 2 of the present invention (the scales of each sub-figure are consistent). Figure 18 This is a diagram showing the experimental results of testing the number of inflammatory cells in the ocular surface tissue of mice, as provided in Example 2 of this invention. Figure 19 The experimental results of PAS staining of mouse ocular surface tissue provided in Example 2 of the present invention are shown in the figure (the scales of each sub-figure are consistent). Figure 20 This is a diagram showing the experimental results of testing the number of conjunctival goblet cells in mouse ocular surface tissue provided in Embodiment 2 of the present invention. Figure 21 This is an experimental result diagram of F4 / 80 immunofluorescence staining of mouse ocular surface tissue provided in Example 2 of the present invention (the scales of each sub-figure are consistent). Figure 22 This is a diagram showing the experimental results of testing the number of F4 / 80 positive cells in the ocular surface tissue of mice, as provided in Example 2 of this invention. Detailed Implementation

[0028] The application of the composition provided by this invention in the preparation of formulations for treating dry eye syndrome, specifically comprising neoazolam oligosaccharides and astaxanthin, wherein the ratio of neoazolam oligosaccharides to astaxanthin is preferably (1~10):(1~10), specifically 1:1, 2:1, 4:1, 8:1, 10:1, 5:9, 1:10 or any value between them. In this case, the neoazolam oligosaccharides and astaxanthin can synergistically work together to achieve effective retention and penetration on the ocular surface tissue, while simultaneously inhibiting the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α in the ocular surface tissue, alleviating ocular surface inflammation, and promoting conjunctival goblet cell proliferation and corneal epithelial barrier repair, thereby improving tear film function and corneal integrity. This composition shows excellent application prospects in the preparation of formulations for treating dry eye syndrome.

[0029] In this invention, the neo-chitosan oligosaccharide refers to a small molecular weight fragment obtained by chemical or enzymatic degradation of the natural polysaccharide chitosan, with a preferred degree of polymerization of 2 to 6, specifically 2, 3, 4, 5, or 6. In this case, the neo-chitosan oligosaccharide possesses excellent bioactivity, ocular surface adhesion, and penetration-enhancing effects, thereby giving the composition a superior therapeutic effect for dry eye.

[0030] In this invention, the proportion of the all-trans isomer of astaxanthin is preferably not less than 95%, specifically it can be 95%, 96.3%, 97%, 98%, 99%, 99.9999%, 100%, or any value between them. In this case, the astaxanthin exhibits superior bioactivity and bioavailability, thereby giving the composition a more effective treatment for dry eye.

[0031] In this invention, the dry eye treatment formulation refers to a class of drugs or medical device products specifically used to treat dry eye syndrome. The key to treating, improving or alleviating dry eye syndrome lies in the introduction of neo-agar oligosaccharides and astaxanthin. The dosage form of the dry eye treatment formulation can be one of the following existing options, including but not limited to: solutions, gels, ointments and sprays.

[0032] To enhance the therapeutic effect of the aforementioned composition comprising neo-Agar oligosaccharides and astaxanthin in the treatment of dry eye, this invention also provides a compound in-situ gel eye drop. More specifically, the compound in-situ gel eye drop comprises neo-Agar oligosaccharides, composite lipid nanoparticles, and a pharmaceutically acceptable excipient carrier. The composite lipid nanoparticles are coated with astaxanthin, and the neo-Agar oligosaccharides are adsorbed onto the surface of the composite lipid nanoparticles. In this case, the lipid nanoparticles achieve an organic combination of neo-Agar oligosaccharides and astaxanthin. Through the combined effects of the ocular surface adhesion and permeation-enhancing properties of neo-Agar oligosaccharides and the protective and cell transport-promoting properties of the lipid nanoparticles, neo-Agar oligosaccharides and astaxanthin can better exert a synergistic effect, resulting in superior anti-inflammatory, tear secretion-enhancing, corneal damage-reducing, ocular surface tissue repair-promoting, and corneal epithelial integrity-improving therapeutic effects.

[0033] In this invention, the mass ratio of the neo-agar oligosaccharide and astaxanthin in the compound in situ gel eye drops is specifically (1~10):(1~10), and can be 1:1, 2:1, 4:1, 8:1, 10:1, 5:9, 1:10 or any value between them.

[0034] In this invention, the composite lipid nanoparticles refer to shell-shaped nanoparticles constructed using phospholipids as the core membrane matrix, through non-covalent bonding of astaxanthin and amphiphilic nonionic surfactants, and via nanodispersion technology, self-assembly technology, or other methods. More specifically, the particle size of the composite lipid nanoparticles is preferably 80 nm to 120 nm, specifically 80 nm, 90 nm, 100 nm, 105 nm, 110 nm, 120 nm, or any value between them. Specific examples of the phospholipids include, but are not limited to, soybean lecithin and / or egg yolk lecithin. Specific examples of the amphiphilic nonionic surfactants include, but are not limited to, one or more of polyoxyethylene ether compounds, polyoxyethylene sorbitan ester compounds, and polyoxyethylene-polyoxypropylene block copolymers.

[0035] In some specific embodiments, the amphiphilic nonionic surfactant is preferably selected from one or more of poloxamer 124, poloxamer 188, and poloxamer 407.

[0036] In this invention, the preferred preparation method of the composite lipid nanoparticles includes the following steps: mixing astaxanthin, phospholipids, and an amphiphilic nonionic surfactant to obtain an oil phase solution; subjecting the oil phase solution and water to high-pressure microfluidic nanodispersion treatment to obtain a dispersion containing the composite lipid nanoparticles. The high-pressure microfluidic nanodispersion treatment is a conventional technique used in the preparation of existing lipid nanoparticles, and those skilled in the art can make adaptive selections according to actual needs; this invention does not impose any particular limitation.

[0037] In some specific embodiments, the preferred mass ratio of astaxanthin, phospholipids and amphiphilic nonionic surfactant is (1~3):(30~60):(5~15), specifically it can be 1:30:5, 1:40:10, 2:50:8, 3:60:15 or any value between them.

[0038] In some specific embodiments, the preferred volume ratio of the oil phase solution to water is (2~5):(95~98), specifically 2:98, 3:97, 4:96, 5:95 or any value between them.

[0039] In this invention, the pharmaceutically acceptable excipient carrier refers to a pharmaceutical excipient used as a carrier, dispersion, encapsulation, or delivery medium for neo-agar oligosaccharides and complex lipid nanoparticles. Various existing options are available, and this invention does not impose any particular limitation.

[0040] In this invention, the pharmaceutically acceptable excipient carrier is preferably a thermosensitive hydrogel matrix. Specific examples of the thermosensitive hydrogel matrix include, but are not limited to, one or more of cellulose and its derivatives, chitosan, poloxamer, and poly(N-isopropylacrylamide). The use of this thermosensitive hydrogel matrix endows the resulting compound in-situ gel eye drops with the ability to gel and form a film on the ocular surface, effectively prolonging the retention time of neoazolam oligosaccharides and astaxanthin on the ocular surface, thereby achieving better therapeutic effects.

[0041] In this invention, the concentration of the thermosensitive hydrogel matrix in the compound in-situ gel eye drops is preferably 1wt% to 20wt%, specifically 1wt%, 5wt%, 10wt%, 15wt%, 20wt%, or any value between them; the concentration of the neo-agar oligosaccharide is preferably 0.001wt% to 1wt%, specifically 0.001wt%, 0.05wt%, 0.1wt%, 0.5wt%, 0.8wt%, 1wt%, or any value between them; the concentration of the composite lipid nanoparticles is 0.005wt% to 5wt%, specifically 0.005wt%, 0.01wt%, 0.1wt%, 0.5wt%, 1wt%, 2wt%, 3wt%, 5wt%, or any value between them.

[0042] Based on obtaining the above-mentioned compound in-situ gel eye drops, the present invention also provides a method for preparing the compound in-situ gel eye drops. The preparation method specifically includes: mixing the aforementioned neo-agar oligosaccharide and composite lipid nanoparticles, and carrying out a self-assembly reaction under a light-protected, inert atmosphere to obtain a nanocomposite; mixing the nanocomposite with a pharmaceutically acceptable excipient carrier to obtain the compound in-situ gel eye drops.

[0043] In this invention, the mixing of the novel oligosaccharide and the composite lipid nanoparticles can be one of various existing options. Those skilled in the art can make an adaptive choice according to actual needs, and this invention does not impose any particular limitations on it.

[0044] In some specific embodiments, the preferred method for mixing the neo-Agar oligosaccharide and the composite lipid nanoparticles includes: dissolving the neo-Agar oligosaccharide in PBS buffer to obtain a neo-Agar oligosaccharide solution; and adding the composite lipid nanoparticles to the neo-Agar oligosaccharide solution under light-protected, gentle stirring, and inert atmosphere conditions to complete the mixing of the neo-Agar oligosaccharide and the composite lipid nanoparticles. More specifically, the concentration of the PBS buffer is 10 mM to 50 mM, and the pH is 7.2 to 7.6; the concentration of the neo-Agar oligosaccharide solution is 0.1 wt% to 1 wt%; and the stirring speed of the gentle stirring is 200 rpm to 400 rpm.

[0045] In this invention, the conditions for the self-assembly reaction specifically include a temperature preferably of 30°C to 40°C, specifically 30, 32, 34, 35, 38, 40°C or any value between them; a stirring speed preferably of 200 rpm to 400 rpm, specifically 200 rpm, 220 rpm, 240 rpm, 280 rpm, 300 rpm, 350 rpm, 400 rpm or any value between them; and a time preferably of 20 min to 40 min, specifically 20 min, 23 min, 25 min, 28 min, 30 min, 32 min, 35 min, 40 min or any value between them.

[0046] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0047] Example 1 This embodiment illustrates the preparation of a compound in-situ gel eye drop, specifically including the following steps: 1. Preparation of new agar oligosaccharides: Agar powder and Tris-HCl buffer (50 mM, pH=7.0) were mixed evenly at a final concentration of 2 wt% to obtain an agar solution; β-agarase AgaP4383 was added to the agar solution at a final concentration of 0.1 wt%, and enzymatic hydrolysis was performed at 42℃ for 2 h to obtain an enzymatic hydrolysate; the enzymatic hydrolysate was ultrafiltered and fractionated using a 1 kDa~3 kDa ultrafiltration membrane and then freeze-dried to obtain new agar oligosaccharides (NAOs) with a degree of polymerization of 2~6.

[0048] 2. Preparation of Astaxanthin Nanodispersion: Natural astaxanthin crystals (all-trans isomer content ≥95%, Aladdin, catalog number A390859) were mixed evenly with soybean lecithin (Aladdin, catalog number L105732) and poloxamer 188 (MCE, catalog number HY-D1005A) at a mass ratio of 2:50:12 to obtain an oil phase solution with a total solute concentration of 0.1wt%. The oil phase solution and ultrapure water were treated with a high-pressure microfluidic nanodispersion device at a volume ratio of 3:97 to obtain a stable astaxanthin nanodispersion (ASTA-NDs) containing nanoparticles with a particle size of approximately 80nm~120nm.

[0049] 3. Preparation of compound in situ gel eye drops: (1) Dissolve neo-agar oligosaccharide in pre-cooled PBS buffer (30mM, pH=7.4) to obtain a neo-agar oligosaccharide solution with a concentration of 0.4wt%; under the conditions of 300rpm, protection from light and inert gas, according to m 新琼寡糖 :m 虾青素 With an addition ratio of 4:1, astaxanthin nano-dispersion was slowly added dropwise to the neo-agar oligosaccharide solution, and the nanocomposite was obtained by self-assembly at 35℃ for 30 min. (2) According to the addition mass ratio of 1:4, take the nanocomposite and the pre-cooled thermosensitive poloxamer F127 matrix (the concentration of the thermosensitive poloxamer F127 matrix is ​​18wt%, the solvent is PBS buffer (30mM, pH7.4), mix them evenly at 4℃, and filter them through a 0.22μm filter membrane to obtain the compound in situ gel eye drops (NAOs / ASTA-Gel).

[0050] Example 2 This embodiment illustrates the application potential of the compound in-situ gel eye drops provided in Example 1 in the treatment of dry eye syndrome. The specific tests include: 1. Construction of a dry eye model and grouping for drug administration Eight-week-old male C57BL / 6J mice were used as experimental animals. After 7 days of acclimatization, they were randomly divided into groups for the following procedures: i. Blank control group (n=5): PBS buffer (30mM, pH=7.4) was instilled into the conjunctival sacs of both eyes of mice at a dosage of 5μL / mouse, 3 times a day for 7 consecutive days; ii. BAC model group (n=5): 5 μL / mouse was administered of PBS buffer (30 mM, pH=7.4) containing 0.2% benzalkonium chloride into the conjunctival sacs of both eyes of mice, 3 times a day for 7 consecutive days. iii. NAOs group (n=5): Based on the treatment of the BAC model group, the thermosensitive poloxamer F127 matrix containing 0.5% neo-agar oligosaccharide was instilled into the conjunctival sac of both eyes of mice at a dosage of 5 μL / mouse, 3 times a day for 7 consecutive days. iv. ASTA group (n=5): Based on the treatment of the BAC model group, 5 μL / mouse of thermosensitive poloxamer F127 matrix containing 0.5% astaxanthin was instilled into the conjunctival sac of both eyes of mice, 3 times a day for 7 consecutive days. v. NAOs / ASTA combination group (n=5): Based on the treatment of the BAC model group, the combination in situ gel eye drops were instilled into the conjunctival sac of both eyes of mice at a dosage of 5 μL / mouse, 3 times a day for 7 consecutive days.

[0051] 2. Tear film function and corneal integrity (1) Thirty minutes after the last administration, a standardized phenol red cotton thread was placed in the conjunctival sac of the middle and outer third of the lower eyelid of the mouse. The length of the thread infiltrated by tear fluid was measured after 30 seconds. The results are as follows: Figure 1 As shown.

[0052] (2) Thirty minutes after the last administration, 1% sodium fluorescein solution was instilled into the conjunctival sac of both eyes of mice at a dosage of 5 μL / mouse. Five minutes later, the mice were photographed under cobalt blue light and their corneas were scored (the area and density of corneal epithelial defects, on a scale of 0 to 15). The results are as follows: Figure 2 and 3 As shown.

[0053] Depend on Figures 1-3 The results show that benzalkonium chloride treatment significantly reduced tear secretion in mice and caused diffuse punctate staining of the corneal epithelium, successfully establishing a dry eye model. Compared to the NAOs and ASTA groups, the NAOs / ASTA combination in situ gel eye drops significantly improved tear film function and corneal integrity in mice—tear secretion was significantly restored, corneal fluorescein staining scores decreased significantly, and the corneal epithelium was basically restored to its original integrity and smoothness.

[0054] 3. Inflammatory response of ocular surface tissues (1) Thirty minutes after the last administration, mice were sacrificed and their eyeballs were harvested. Frozen sections of the eyeballs were prepared. The frozen sections were stained with anti-IL-1β antibody (Abcam, catalog number ab283818), anti-IL-6 antibody (Abcam, catalog number ab290735), and anti-TNF-α antibody (Abcam, catalog number ab183218) according to the manufacturer's instructions. The fluorescence intensity of the ocular surface tissue was measured by fluorescence microscopy. The results are as follows: Figures 4-9 As shown.

[0055] (2) After homogenizing a portion of the conjunctiva and corneal tissue, tissue serous fluid was obtained. The contents of IL-1β, IL-6, and TNF-α in the tissue serous fluid were tested using the corresponding ELISA kits (Beyotime, catalog numbers PI301, PI326, and PT512) according to the instructions. The results are as follows: Figures 10-12 As shown.

[0056] Depend on Figures 4-12 The results show that, compared with the NAOs group and the ASTA group, the application of the compound in situ gel eye drops in the NAOs / ASTA compound group can significantly reduce the expression of IL-1β, IL-6 and TNF-α in ocular surface tissues and effectively alleviate ocular surface inflammation.

[0057] 4. Apoptosis and proliferation of ocular surface cells The frozen sections of the eyeball were stained using the TUNEL apoptosis detection kit (Beyotime, catalog number C1088) and anti-Ki67 antibody (Abcam, catalog number ab16667) according to the manufacturer's instructions. The number of TUNEL-positive cells and Ki67-positive cells were counted. The results are as follows: Figures 13-16 As shown.

[0058] Depend on Figures 13-16 The results show that benzalkonium chloride treatment led to a significant increase in the number of Tunel-positive and Ki67-positive cells, indicating an increase in pathological apoptosis and abnormal cell proliferation in the ocular surface tissue. Compared with the NAOs group and the ASTA group, the application of the compound in-situ gel eye drops in the NAOs / ASTA compound group significantly reduced the number of apoptotic cells while effectively promoting the proliferative activity of epithelial cells.

[0059] 5. Ocular surface histopathology and inflammatory cell infiltration Frozen sections of the eyeball were stained with H&E, PAS, and F4 / 80 immunofluorescence. The number of conjunctival goblet cells, inflammatory cells, and F4 / 80 positive cells was counted using fluorescence microscopy. The results are as follows: Figures 17-22 As shown.

[0060] Depend on Figures 17-22The results show that, compared with the NAOs group and the ASTA group, the NAOs / ASTA combination group had the most regular arrangement of corneal epithelium, the fewest number of subconjunctival inflammatory cells, and the same level of conjunctival goblet cells as the blank control group. It also had excellent regulatory effect on F4 / 80 positive macrophages and could effectively repair ocular surface tissue structure and cell population.

[0061] Furthermore, it should be noted that in the acute eye irritation test, after administering the compound in situ gel eye drops provided in Example 1 of this invention to rabbits via bilateral intraconjunctival sac administration (3 times daily for 7 consecutive days), no obvious hyperemia, edema, or increased secretions were observed on the rabbit ocular surface. Corneal fluorescein staining was negative, and the conjunctival goblet cell structure remained intact. In the long-term toxicity test, after administering the compound in situ gel eye drops provided in Example 1 of this invention to rabbits via bilateral intraconjunctival sac administration (3 times daily for 28 consecutive days), histopathological examination of the rabbit eye tissue showed normal corneal, conjunctival, iris, and retinal structures, with no pathological changes observed. In the skin sensitization test (guinea pig maximization test), guinea pigs were treated with the compound in situ gel eye drops provided in Example 1 of this invention, and the results were all negative, indicating that the compound in situ gel eye drops have no sensitization risk. The above results confirm that the compound in situ gel eye drops have excellent ocular surface biocompatibility and are suitable for long-term topical use.

[0062] Example 3 This embodiment illustrates the storage stability of the compound in-situ gel eye drops provided in Example 1. The test specifically includes: storing the compound in-situ gel eye drops at 4℃, 25℃, and 40℃ for 6 months, and taking samples at 0, 1, 3, and 6 months of storage. The molecular weight distribution of the samples is obtained by high-performance gel permeation chromatography. At the same time, the total sugar content of the samples is determined by the sulfuric acid-phenol method. The total sugar retention rate and activity retention rate of the neoagar oligosaccharides in the compound in-situ gel eye drops are calculated according to the following formula. The results are shown in Table 1.

[0063]

[0064]

[0065] Table 1.

[0066] As shown in Table 1, the compound in-situ gel eye drops provided in Example 1 of the present invention, after being stored at 4°C and 25°C for 6 months, have a total sugar retention rate of over 93% and an activity retention rate of over 94%, demonstrating excellent storage stability and meeting the stability requirements of pharmaceutical excipients and active ingredients.

[0067] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. The use of the composition in the preparation of formulations for the treatment of dry eye syndrome, characterized in that, The composition includes neoazooligosaccharides and astaxanthin.

2. The use of the composition according to claim 1 in the preparation of a formulation for the treatment of dry eye syndrome, characterized in that, The degree of polymerization of the new agar oligosaccharide is 2 to 6; Optionally, the all-trans isomer accounts for no less than 95% of the astaxanthin; Optionally, the mass ratio of the added neo-agar oligosaccharide to astaxanthin is (1~10):(1~10).

3. The use of the composition according to claim 1 in the preparation of a formulation for the treatment of dry eye syndrome, characterized in that, The dosage form of the dry eye treatment preparation is selected from one or more of the following: solution, gel, ointment, and spray.

4. A compound in-situ gel eye drop, characterized in that, The compound in-situ gel eye drops comprise neo-Agar oligosaccharide, composite lipid nanoparticles, and pharmaceutically acceptable excipient carriers. The composite lipid nanoparticles are coated with astaxanthin, and the neo-Agar oligosaccharide is adsorbed on the surface of the composite lipid nanoparticles. The mass ratio of the neo-Agar oligosaccharide to astaxanthin is (1~10):(1~10).

5. The compound in-situ gel eye drops according to claim 4, characterized in that, The degree of polymerization of the new agar oligosaccharide is 2 to 6.

6. The compound in-situ gel eye drops according to claim 4, characterized in that, The composite lipid nanoparticles have a particle size of 80 nm to 120 nm.

7. The compound in-situ gel eye drops according to claim 4, characterized in that, The preparation of the composite lipid nanoparticles includes: mixing astaxanthin, phospholipids and amphiphilic nonionic surfactants evenly to obtain an oil phase solution; and subjecting the oil phase solution and water to high-pressure microfluidic nanodispersion treatment to obtain a dispersion containing the composite lipid nanoparticles. Optionally, the mass ratio of the astaxanthin, phospholipids and amphiphilic nonionic surfactant added is (1~3):(30~60):(5~15); Optionally, the phospholipid is soybean lecithin and / or egg yolk lecithin; Optionally, the amphiphilic nonionic surfactant is selected from one or more of polyoxyethylene ether compounds, polyoxyethylene sorbitan ester compounds, and polyoxyethylene-polyoxypropylene block copolymers; Optionally, the amphiphilic nonionic surfactant is selected from one or more of poloxamer 124, poloxamer 188, and poloxamer 407; Optionally, the volume ratio of the oil phase solution to water is (2~5):(95~98).

8. The compound in-situ gel eye drops according to claim 4, characterized in that, The pharmaceutically acceptable excipient carrier is a thermosensitive hydrogel matrix, which is selected from one or more of cellulose and its derivatives, chitosan, poloxamer and poly(N-isopropylacrylamide); Optionally, in the compound in situ gel eye drops, the concentration of the thermosensitive hydrogel matrix is ​​1wt%~20wt%, the concentration of the neo-agar oligosaccharide is 0.001wt%~1wt%, and the concentration of the composite lipid nanoparticles is 0.005wt%~5wt%.

9. The method for preparing the compound in-situ gel eye drops according to any one of claims 4 to 8, characterized in that, The preparation method includes: mixing the new agar oligosaccharide and the composite lipid nanoparticles, and carrying out a self-assembly reaction under a light-protected and inert atmosphere to obtain a nanocomposite; mixing the nanocomposite with a pharmaceutically acceptable excipient carrier to obtain the compound in-situ gel eye drops.

10. The preparation method according to claim 9, characterized in that, The self-assembly reaction was carried out at a temperature of 30℃~40℃, a stirring speed of 200rpm~400rpm, and a time of 20min~40min.

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