Lipid liquid crystal precursor eye drops as well as preparation method and application thereof

Abstract

The invention relates to a drug sustained release system, in particular to lipid liquid crystal precursor eye drops as well as a preparation method and application thereof. The lipid liquid crystal precursor eye drops comprise a matrix material, a cosolvent and a raw material medicine, wherein the matrix material is an amphiphilic substance and an oil phase; the lipid liquid crystal precursor eye drops are prepared from the following components in percentage by weight: 0 to 35 percent of amphiphilic substance, 40 to 80 percent of oil phase, 15 to 20 percent of cosolvent and 0.001 to 5 percent of raw material medicine. According to the constructed drug-loaded lipid liquid crystal precursor eye drops, when the drug-loaded lipid liquid crystal precursor makes contact with water in tears, the drug-loaded lipid liquid crystal precursor can instantly become drug-loaded lipid liquid crystal gel, compared with common eye drops, the retention time of the drug-loaded lipid liquid crystal gel on the ocular surface is longer, the lipid liquid crystal has a phospholipid bilayer structure, and the drug-loaded lipid liquid crystal gel can be used for preparing the drug-loaded lipid liquid crystal precursor eye drops. Insoluble raw material medicines can be distributed in the lipid layer, so that the solubility of the raw material medicines is greatly improved, the raw material medicines can be slowly released by gelatinizing after meeting water, the administration frequency is reduced, the bioavailability and patient compliance are greatly improved, and the defects of the eye drops in treatment of glaucoma are overcome.

Description

Technical Field

[0001] This invention relates to a drug sustained-release system, and more specifically to a lipid liquid crystal precursor eye drop, its preparation method, and its application. Background Technology

[0002] In recent years, with the widespread use of electronic products and the accelerating global aging process, the number of patients with eye diseases has continued to increase, with the number of glaucoma patients worldwide exceeding 76 million. As the world's most populous country, my country has the largest number of dry eye and glaucoma patients globally, and the affected population is showing a trend towards younger ages. Overall, the global patient population for eye diseases is enormous.

[0003] Glaucoma is a chronic eye disease characterized by progressive damage to the optic nerve and visual field defects. High intraocular pressure (IOP) is a major modifiable risk factor. Lowering IOP is currently the primary treatment for glaucoma. Latanoprost, a potent prostaglandin F2α analog, significantly lowers IOP by increasing aqueous humor outflow through the uveal-scleral pathway and is a first-line drug for treating open-angle glaucoma and ocular hypertension.

[0004] Currently, eye drops, such as latanoprost eye drops (Xalatan), are commonly used to treat glaucoma. However, eye drops have several drawbacks. These include: the corneal barrier and nasolacrimal duct drainage result in a short residence time of the drug on the ocular surface, poor penetration at the lesion site, and an absorption rate typically below 5%. Furthermore, preservatives in eye drops (such as benzalkonium chloride) and the drug itself can cause adverse reactions such as conjunctival hyperemia, burning sensation in the eye, foreign body sensation, eyelash hyperplasia, and increased iris pigmentation. Eye drops usually need to be administered once daily, which can lead to poor patient compliance, and missed doses can affect efficacy. Latanoprost is sensitive to light and heat, and is prone to degradation and has low solubility. These factors contribute to the low bioavailability of latanoprost.

[0005] In recent years, various novel sustained-release matrix materials have been widely used in the treatment of ocular diseases, such as suspensions, emulsions, in-situ gels, and nanoparticles. Lipid liquid crystalline (LLC) is a substance state between liquid and solid, formed by one or more structurally suitable amphiphilic compounds, an oil phase, and a solvent. Under solvent induction, amphiphilic molecules at specific concentrations self-assemble to form micelles; as the concentration continues to increase, the micelles further associate to form liquid crystals, and this phase transition process can be triggered or enhanced by the increase of water content. Due to its unique structural characteristics, it can provide a broad carrier platform for hydrophilic, lipophilic, and amphiphilic drugs, and also possesses biomembrane-like permeability, thereby improving drug bioavailability. However, its applications are currently limited to injection, skin, and mucous membranes. Due to its high viscosity and limitations in drug loading, its compatibility with other dosage forms requires further research. Summary of the Invention

[0006] To address the shortcomings of eye drops in the treatment of glaucoma, the present invention aims to provide a lipid liquid crystal precursor eye drop, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A lipid liquid crystal precursor eye drop comprises a matrix material, a cosolvent, and a drug substance; wherein the matrix material is an amphiphilic substance or an amphiphilic substance and an oil phase; the lipid liquid crystal precursor eye drop, by weight percentage, comprises 0%-35% amphiphilic substance, 40%-80% oil phase, 15-20% cosolvent, and 0.001%-5% drug substance.

[0008] The matrix material is an amphiphilic substance and an oil phase; wherein, the amphiphilic substance is selected from one or more of lecithin, phosphatidylcholine, and phosphatidylethanolamine; the oil phase is selected from monooleylglycerol, dioleylglycerol, phytanetriol, and tocopherol, and oleic acid is preferably one or more of dioleylglycerol and monooleylglycerol.

[0009] The co-solvent is selected from one or more of propylene glycol, ethylene glycol, and dimethyl sulfoxide; The active pharmaceutical ingredient is one or more of the following: pilocarpine, acetazolamide, timarolol, brinzolamide, prostaglandin latanoprost, travoprost, or coenzyme Q10.

[0010] The lipid liquid crystal precursor eye drops, by weight percentage, consist of 16%-32% amphiphilic substance, 48%-64% oil phase, 15-20% cosolvent and 0.001%-5% active pharmaceutical ingredient.

[0011] A method for preparing the lipid liquid crystal precursor eye drops, wherein the matrix is ​​dissolved in a solvent according to the above proportion to obtain a blank gel precursor; then, the active pharmaceutical ingredient and a co-solvent are added to the blank gel precursor, and under sealed conditions, the solvent used to dissolve the matrix is ​​removed by blowing with N2 to obtain the lipid liquid crystal precursor eye drops.

[0012] The solvent is ethanol.

[0013] An application of the aforementioned lipid liquid crystal precursor eye drops, wherein the lipid liquid crystal precursor eye drops are used as eye drops for the treatment of acute ocular hypertension glaucoma.

[0014] An application of the aforementioned lipid liquid crystal precursor eye drops, wherein the lipid liquid crystal precursor eye drops are used as eye drops for the treatment of chronic ocular hypertension glaucoma.

[0015] An application of the aforementioned lipid liquid crystal precursor eye drops, specifically its use as a long-acting eye drop.

[0016] The lipid liquid crystal precursor eye drops of this invention use a lipid liquid crystal structure as a carrier and contain the original drug. When the lipid liquid crystal precursor solution comes into contact with water in the tear fluid, the lipid liquid crystal precursor solution instantly transforms into a lipid liquid crystal gel. Compared with eye drops, it has a longer retention time on the ocular surface. Furthermore, the lipid liquid crystal has a phospholipid bilayer structure, and the poorly soluble latanoprost can be distributed in the lipid layer, which greatly increases the solubility of latanoprost. The gelation upon contact with water can achieve the slow release of latanoprost, reduce the frequency of administration, and greatly increase bioavailability and patient compliance.

[0017] The obtained lipid liquid crystal precursor eye drops were characterized using polarized light microscopy (PLM), small-angle X-ray diffraction (SASX), rheology, and SEM. The sustained release behavior of the lipid liquid crystal gel was measured in vitro, and its cytotoxicity to HCE-T and ARPE-19 cells was evaluated. OCT was used to observe the transformation of the lipid liquid crystal precursor into a lipid liquid crystal gel in the cornea. HPLC was used to measure the content of the active pharmaceutical ingredient (API) in the aqueous humor and cornea after degradation by corneal esterase. In vivo imaging was used to examine the residence time of the lipid liquid crystal gel on the ocular surface. An acute ocular hypertension model was established in vivo using SD rats via microsphere injection, and changes in ocular pressure during treatment were monitored using an ICARE tonometer. HE-stained sections and corneal confocal microscopy were used to observe changes in corneal and retinal morphology and thickness in each group. Finally, slit-lamp staining and sodium fluorescein staining were used to assess the safety of the liquid crystal gel. Attached Figure Description

[0018] Figure 1 The following are phase transition diagrams and PLM diagrams of the lipid liquid crystal precursor eye drops of the present invention and the simulated tear fluid after contact: wherein, a is the lipid liquid crystal precursor solution of the embodiment, b is the lipid liquid crystal gel of the embodiment, c is the lipid liquid crystal precursor solution of the embodiment (with rhodamine), d is the lipid liquid crystal gel of the embodiment (with rhodamine), and PLM is the polarized light microscope image of the lipid liquid crystal gel of the embodiment.

[0019] Figure 2 Rheological characterization results of lipid liquid crystal gels in embodiments of the present invention.

[0020] Figure 3 SASX diagram of lipid liquid crystal gel in an embodiment of the present invention.

[0021] Figure 4 SEM image of lipid liquid crystal gel in an embodiment of the present invention.

[0022] Figure 5 The results of lipid liquid crystal gel release in simulated tears in the embodiments of the present invention.

[0023] Figure 6 Results of experiments on the cytotoxicity of latanoprost solution against HCE-T cells.

[0024] Figure 7 OCT showed that lipid liquid crystal precursor eye drops transformed into lipid liquid crystal gel on the corneal surface.

[0025] Figure 8 Concentration of latanoprostonic acid in the anterior chamber and cornea of ​​the body.

[0026] Figure 9 The results of cytotoxicity experiments on HCE-T and ARPE-19 in this embodiment of the invention.

[0027] Figure 10 Results of live and dead staining experiments on HCE-T and ARPE-19 in this embodiment of the invention.

[0028] Figure 11 Treatment protocol of latanoprost lipocrystalline precursor eye drops in an acute ocular hypertension model.

[0029] Figure 12 Validation of an acute ocular hypertension model by injecting microspheres into the anterior chamber.

[0030] Figure 13 : Changes in intraocular pressure during treatment in each group and comparison of the extent of intraocular pressure reduction in the Xalatan group and the LAT-LLC group.

[0031] Figure 14 HE staining results of cornea and retina in each group.

[0032] Figure 15 Total retinal thickness and corneal epithelial and corneal stroma thickness in each group.

[0033] Figure 16 : Retention time of latanoprost lipid liquid crystal gel on the ocular surface.

[0034] Figure 17 HE staining was used to assess the safety of latanoprost lipid liquid crystal precursor eye drops.

[0035] Figure 18 Statistical analysis of corneal epithelium, corneal stroma, and retinal thickness in each group.

[0036] Figure 19 Slit lamp and corneal fluorescein sodium staining were used to assess the safety of latanoprost lipid liquid crystal precursor eye drops.

[0037] Figure 20 Ocular surface score based on the modified Draize stimulus score.

[0038] Figure 21 Representative images of corneal epithelium, stroma, and endothelial cells in rabbits after treatment with latanoprost lipid liquid crystal precursor eye drops. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available.

[0040] The lipid liquid crystal precursor eye drops of this invention have good biocompatibility and adhesion. The main components are similar to biological membranes, have good affinity for ocular tissues, and can prolong the retention time in the cornea. The eye drops have certain sustained-release properties, with the drug slowly diffused and released from highly ordered nanochannels, providing a sustained drug concentration. They also have improved permeability by interfering with the corneal lipid layer or acting as "channels" for drug penetration, thus promoting drug absorption. At the same time, they have the potential for being preservative-free or low-preservative. The high-viscosity gel state physically isolates microorganisms, which can reduce or avoid the use of preservatives and reduce the risk of irritation.

[0041] The following examples use latanoprost, a prostaglandin, as an example of the original drug.

[0042] Example 1: Latanoprost Lipid Liquid Crystal Progenitor Eye Drops (Formula 1: GDO:SPC 5:0) Latanoprost: 5 mg (0.005% w / w) Soybean phosphatidylcholine (SPC): 0 mg (0.0% w / w) Diglyceride (GDO): 800 mg (80% w / w) Propylene glycol: 195 mg (19.995% w / w) Anhydrous ethanol: 4 ml Preparation method: Accurately weigh 800 mg of diglyceride into a vial, then add 4 mL of anhydrous ethanol. Transfer the vial to an ultrasonic instrument and sonicate for 10 minutes until the diglyceride is completely dissolved. Then, accurately weigh 195 mg of propylene glycol and 5 mg of latanoprost into the above system, vortex for 1 minute, mix well, and finally weigh the net weight of the system to approximately 4120 mg. Place the vial open under nitrogen and blow for 30 minutes to evaporate and remove the remaining ethanol solvent until the net weight of the system is 1000 mg. A clear and transparent lipid liquid crystal precursor solution is obtained. Subsequently, inject 5 mL of water into the vial using a syringe, and equilibrate for 2 days in the dark to obtain latanoprost lipid liquid crystal precursor eye drops.

[0043] Example 2 Latanoprost Lipid Liquid Crystal Progenitor Eye Drops (Formula 2 GDO:SPC 4:1) Latanoprost: 5 mg (0.005% w / w) Soy phosphatidylcholine (SPC): 160 mg (16% w / w) Diglyceride (GDO): 640 mg (64% w / w) Propylene glycol: 195 mg (19.995% w / w) Anhydrous ethanol: 4 ml Preparation method: Accurately weigh 640 mg of diglyceride and 160 mg of soybean phosphatidylcholine into a vial, then add 4 mL of anhydrous ethanol. Transfer the vial to an ultrasonic instrument and sonicate for 10 minutes until the diglyceride and soybean phosphatidylcholine are completely dissolved. Then, accurately weigh 195 mg of propylene glycol into the above system, vortex for 1 minute, mix well, and finally weigh the net weight of the system to 4120 mg. Place the vial open under nitrogen and blow for 30 minutes to evaporate and remove the remaining ethanol solvent until the net weight of the system is 1000 mg. A clear and transparent lipid liquid crystal precursor solution is obtained. Subsequently, inject 5 mL of water into the vial using a syringe, and equilibrate for 2 days in the dark to obtain latanoprost lipid liquid crystal precursor eye drops.

[0044] Example 3 Latanoprost Lipid Liquid Crystal Progenitor Eye Drops (Formula 3 GDO:SPC 3:2) Latanoprost: 5 mg (0.005% w / w) Soybean phosphatidylcholine (SPC): 320 mg (32% w / w) Diglyceride (GDO): 480 mg (48% w / w) Propylene glycol: 195 mg (19.995% w / w) Anhydrous ethanol: 4 ml Preparation method: Accurately weigh 480 mg of diglyceride and 320 mg of soybean phosphatidylcholine into a vial, then add 4 mL of anhydrous ethanol. Transfer the vial to an ultrasonic instrument and sonicate for 10 minutes until the diglyceride and soybean phosphatidylcholine are completely dissolved. Then, accurately weigh 195 mg of propylene glycol and 5 mg of latanoprost into the above system, vortex for 1 minute, mix well, and finally weigh the net weight of the system to 4120 mg. Place the vial open under nitrogen and blow for 30 minutes to evaporate and remove the remaining ethanol solvent until the net weight of the system is 1000 mg. A clear and transparent lipid liquid crystal precursor solution is obtained. Subsequently, inject 5 mL of water into the vial using a syringe, and equilibrate for 2 days in the dark to obtain latanoprost lipid liquid crystal precursor eye drops.

[0045] Comparative Example 1: Latanoprost Lipid Liquid Crystal Progenitor Eye Drops (Formulation 4 GDO:SPC 1:1) Latanoprost: 5 mg (0.005% w / w) Soy phosphatidylcholine (SPC): 400 mg (40% w / w) Diglyceride (GDO): 400 mg (40% w / w) Propylene glycol: 195 mg (19.995% w / w) Anhydrous ethanol: 4 ml Preparation method: Accurately weigh 400 mg of diglyceride and 400 mg of soybean phosphatidylcholine into a vial, then add 4 mL of anhydrous ethanol. Transfer the vial to an ultrasonic instrument and sonicate for 10 minutes until the diglyceride and soybean phosphatidylcholine are completely dissolved. Then, accurately weigh 195 mg of propylene glycol and 5 mg of latanoprost into the above system, vortex for 1 minute, mix well, and finally weigh the net weight of the system to 4120 mg. Place the vial open under nitrogen and blow for 30 minutes to evaporate and remove the remaining ethanol solvent until the net weight of the system is 1000 mg. A clear and transparent lipid liquid crystal precursor solution is obtained. Subsequently, inject 5 mL of water into the vial using a syringe, and equilibrate for 2 days in the dark to obtain latanoprost lipid liquid crystal precursor eye drops.

[0046] Comparative Example 2: Latanoprost Lipid Liquid Crystal Progenitor Eye Drops (Formulation 5 GDO:SPC 0:5) Latanoprost: 5 mg (0.005% w / w) Soy phosphatidylcholine (SPC): 800 mg (80% w / w) Diglyceride (GDO): 0 mg (0% w / w) Propylene glycol: 195 mg (19.995% w / w) Anhydrous ethanol: 4 ml Preparation method: Accurately weigh 800 mg of soybean phosphatidylcholine into a vial, then add 4 mL of anhydrous ethanol. Transfer the vial to an ultrasonic instrument and sonicate for 10 minutes until the dioleate and soybean phosphatidylcholine are completely dissolved. Then, accurately weigh 195 mg of propylene glycol and 5 mg of latanoprost into the above system, vortex for 1 minute, mix well, and finally weigh the net weight of the system to 4120 mg. Place the vial open under nitrogen and blow for 30 minutes to evaporate and remove the remaining ethanol solvent until the net weight of the system is 1000 mg. A clear and transparent lipid liquid crystal precursor solution is obtained. Subsequently, inject 5 mL of water into the vial using a syringe, and equilibrate for 2 days in the dark to obtain latanoprost lipid liquid crystal precursor eye drops.

[0047] The liquid crystal precursor and phase transition diagrams and PLM diagrams after contact with simulated tear fluid were obtained from the above embodiments and comparative examples. After preparing the liquid crystal precursor eye drops in the above embodiments and comparative examples, a small amount of rhodamine solution was added to facilitate observation. An appropriate amount of physiological saline was added to the vial. After equilibration for two days, the vial was inverted to observe whether the gel slipped and whether the physiological saline became cloudy. Photos were taken and recorded using a mobile phone. Figure 1 As shown, the liquid crystal gels obtained in Examples 1-3 all exhibited good gelling properties and remained at the bottom of the vial, resulting in clear and transparent physiological saline. In contrast, Comparative Examples 1 and 2 failed to form liquid crystal gels, sliding off the bottom of the vial and forming a cloudy emulsion with the physiological saline. Small amounts of the liquid crystal gels formed in the examples and comparative examples were placed on glass slides, covered with coverslips, and observed under a polarizing microscope (PLM). Figure 1 As shown, Examples 1-3 exhibit isotropic properties and show no polarization under a polarizing microscope, thus presenting a dark field and indicating a cubic phase. Comparative Examples 1 and 2 are non-liquid crystal morphologies, exhibiting a disordered structure under light.

[0048] The rheological properties of the lipid liquid crystal gels of Examples 1-3 were characterized using a rheometer.

[0049] The temperature was set to 25℃. After zeroing the gap between the upper and lower plates, the liquid crystal gel was placed on the lower plate with a radius of 20 mm and equilibrated for 5 minutes. Then, its storage modulus (G') and loss modulus (G”) were measured in angular frequency scanning mode, and modulus-time curves were plotted. The angular frequency was fixed in the range of 0.01-100 rad / s. The shear rate was set to 0-100 s. -1 Internal changes were recorded, and the viscosity of the liquid crystal gel was measured. The results are as follows: Figure 2 As shown, the elastic modulus of each liquid crystal gel in Examples 1-3 is greater than the viscous modulus, which meets the gel requirements; and the viscosity gradually decreases with the increase of shear rate, which is a shear-thinning gel; as the temperature rises, G' is always greater than G”, that is, it is always in a gel state.

[0050] The lipid liquid crystal gels of Examples 1-3 were characterized by small-angle X-ray diffraction. A small amount of gel from different groups was placed in the sample cell, sealed, and then placed in the instrument's sample holder. After evacuating to 5-10 Pa, small-angle X-ray scattering was measured at 25°C for 20 minutes. The detector recorded the scattering data, ultimately obtaining a one-dimensional scattering curve. Instrument conditions: copper target tube, tube voltage 40 kV, tube current 40 mA, wavelength 0.1542 nm. Detector: image plate (0.07-28 nm). -1 ), Decris Mythen2 1D (0.06-7 nm); Sample cell: Anton Paar Paste Cell (custom-designed window). Small-angle X-ray diffraction results are as follows: Figure 3 As shown, the relative position scattering vector ratio of the Bragg peak is Therefore, it can be determined that the liquid crystal is a double rhombic cubic liquid crystal. With the increase of phosphatidylcholine, the strongest Bragg peak shifts slightly to the right, indicating that the quality parameters of the gel decrease slightly with the increase of the proportion of phosphatidylcholine added.

[0051] SEM images of the lipid liquid crystal gels from Examples 1-3 above. Sample Sampling: Cut small samples from the surface or interior of the liquid crystal gel, keeping the size within a few millimeters to avoid damaging the morphology. Drying Treatment: Frequently use freeze-drying or critical point drying to prevent solvent evaporation from causing gel shrinkage and collapse. Sample Loading: Fix the treated sample onto the sample stage, ensuring stable placement. Electron Microscopy Observation: Place the sample in the SEM sample chamber, evacuate, and adjust parameters such as accelerating voltage and working distance for morphological imaging and analysis. SEM Imaging Display ( Figure 4 The internal phase of the LLC gel consists of many stacked quasi-spherical lattices and water channels of varying sizes. As the SPC ratio increases, the diameter of the water channels gradually increases.

[0052] In summary, Examples 1-3 can all achieve sol-gel phase transition, and thus can be used as long-acting sustained-release eye drops.

[0053] Then, taking the lipid liquid crystal gel of Example 3 as an example, its in vitro release behavior was further investigated in simulated tears.

[0054] Using simulated artificial tears (STF) as the release medium, the preparation method is as follows: 1000g water containing 6.78g NaCl, 2.18g NaHCO3, 0.084g CaCl2·2H2O, 1.38g KCl, and pH 7.40. Accurately weigh 50mg of each sample into a pretreated dialysis bag (molecular weight cutoff: 8000-10000KDa), and tie both ends of the dialysis bag tightly. Then, fix the dialysis bag in 12mL of release medium. Place it in a constant temperature shaker at 35℃ ± 0.5℃ and a rotation speed of 100rpm, with each prescription in triplicate. Take 1mL of sample at 2h, 6h, 8h, 10h, 12h, 24h, 36h, 48h, and 72h, and simultaneously replenish with the same volume of fresh artificial tears to meet the leakage conditions. The concentration of latanoprost was measured by HPLC, and the cumulative release at each time point was calculated, resulting in a release curve. Figure 5 As can be seen, in Example 3, a cumulative release of 60% was achieved within 72 hours.

[0055] Example 4: Cytotoxicity experiment of latanoprost, the active pharmaceutical ingredient used in Examples 1-3, against HCE-T cells. Healthy human corneal epithelial cells (HCE-T) were seeded into 96-well plates at a density of 5000-7000 cells / well. The plates were then placed in a cell culture incubator for 24 hours to allow cell adhesion. Latamoplastin was then dissolved in DMSO to prepare concentration gradients of 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL. 100 μL of latanoprost solution was added to each well, with six replicates for each concentration gradient. The HCE-T cells were incubated for 24 hours. After incubating, the latanoprost solution was removed, and 10 μL of CCK-8 solution was added to each well. Cell viability was then measured at 450 nm using a microplate reader. Results are shown below. Figure 6 As shown, the IC50 value of latanoprost was calculated to be 62.43 ug / mL using Graph prism.

[0056] Example 5: OCT shows that lipid liquid crystal precursor eye drops form a liquid crystal gel on the corneal surface. Nine New Zealand rabbits were divided into three groups (n=3 per group) as follows: Control group, BLLC group (blank liquid crystal precursor, i.e., blank liquid crystal precursor without drug loading as described in Example 3), and LAT-LLC group (Lataprost lipocrystalline liquid crystal precursor eye drops group as described in Example 3). Rabbits were anesthetized by intramuscular injection of 3% sodium pentobarbital (40 mg / kg). Once the rabbits were anesthetized, each group was administered 40 μL of BLLC precursor solution or LAT-LLC precursor eye drops. After 10 seconds, the rabbit's eye was aligned with the lens of an optical coherence tomography (OCT) scanner (Intlight VG200I) to observe and record the formation process of the lipocrystalline liquid crystal gel. A transparent gel conforming to the corneal curvature was observed forming on the corneal surface. Figure 7 .

[0057] Example 6: Concentration of latanoprost in the anterior chamber and cornea Twenty-four New Zealand rabbits were injected with 40 μL of LAT-LLC eye drops (1 mg / mL) into both conjunctival sacs. Eyes were closed for 10 seconds after instillation to prevent leakage. Rabbits were euthanized at 4, 6, 12, 24, 36, 48, 60, and 72 hours post-instillation (n=3 at each time point). Eyeballs were enucleated and dissected to separate the cornea and aqueous humor (AH). Each cornea was weighed individually. All samples (cornea and aqueous humor) were stored in separate tubes, immediately frozen, and maintained at -80°C until analysis. Pharmacokinetic studies of the in vivo samples were performed using high-performance liquid chromatography (HPLC Agilent ZORBAX SB Aq).

[0058] Aqueous humor samples were treated using protein precipitation: 50 μL of aqueous humor sample was mixed with 150 μL of methanol and vortexed for 1 minute. The mixture was then centrifuged at 13000 rpm for 10 minutes, and the supernatant was collected for quantitative analysis. For corneal samples, homogenization was performed in methanol at a ratio of 100 mg sample to 1 mL of methanol, and the mixture was stored at -20°C for 12 hours. Subsequently, the mixture was centrifuged at 4000 rpm for 10 minutes. 50 μL aliquots of the supernatant were further treated using protein precipitation: 150 μL of methanol was added, vortexed for 1 minute, and centrifuged at 13000 rpm for 10 minutes. Finally, 10 μL of the sample was injected into the HPLC system for quantitative analysis. Since latanoprost is broken down into latanoprost acid by corneal esterase in the eye, the effective concentration of latanoprost acid in the aqueous humor and cornea was measured.

[0059] Chromatographic separation was performed on a C18 column (50 mm × 2.1 mm, 1.7 μm) at a flow rate of 0.2 mL / min. Mobile phase A consisted of methanol, and mobile phase B was 0.2% formic acid aqueous solution. The gradient elution program was as follows: 0 to 1.0 min 95% to 80% A, 1.0 to 1.5 min 80% to 95% A, and 1.5 to 2.0 min maintained at 95% A. Results are shown in [Figure number missing]. Figure 8 The concentration of latanoprost acid in the aqueous humor (AH) reached 250 ug / mL, and the concentration in the cornea reached 135 ug / mL.

[0060] Example 7: Example 3 yielded latanoprost lipid liquid crystal precursor eye drops with cytotoxic and live / dead staining effects on HCE-T and ARPE-19 cytotoxicity. Healthy human corneal epithelial cells (HCE-T) and human retinal pigment epithelial cells (ARPE-19) were seeded into different 96-well plates at a density of 5000-7000 cells / well. The 96-well plates were then placed in a cell culture incubator for 24 hours to allow cell adhesion. The liquid crystal gel precursor solutions from formulations 1-3 were then added to serum-free cell culture medium, and after soaking for 24 hours, the extract was filtered through a 0.22 μm filter. The extract was then diluted with culture medium to create concentration gradients of 25%, 50%, 75%, and 100%. 100 μL of each dilution was administered to each well, with six replicates for each concentration gradient. The latanoprost lipid liquid crystal precursor eye drops obtained in Example 3 were incubated with HCE-T and ARPE-19 cells in an incubator for 24 hours. Remove the liquid crystal gel extraction buffer, add 10 μL of cck-8 solution to each well, incubate in an incubator for 30 minutes, and then detect cell viability at 450 nm using a microplate reader. Results are as follows: Figure 9As shown, the cell survival rate of HCE-T cells co-cultured with 100% gel extract in Example 3 was approximately 70%. Cell survival rates in 25%, 50%, and 75% gel extracts were all greater than 80%. Example 3 demonstrated outstanding cell survival. For ARPE-19, using different concentrations of gel extract, Example 3 showed significantly better cell survival and less irritation.

[0061] The HCE-T and ARPE-19 cells that showed good growth in the above experiments were seeded into 96-well plates and incubated for 24 hours to allow them to adhere. Then, 200 μL of liquid crystal gel extraction medium was added to each well. After incubating the cells with the extraction medium for 12 or 24 hours, the extraction medium was removed. 200 μL of PBS was added to each well to wash the cells, and then the PBS was discarded. 100 μL of AM / PI working solution (1:1000 PBS dilution) was added to each well. After incubation for 30 minutes, the AM / PI working solution was removed, and the cells were photographed under a laser confocal microscope. The results are as follows: Figure 10 As shown, Example 3 showed a 24-hour cell survival rate of 92.5% for HCE-T cells. Simultaneously, it showed a 24-hour cell survival rate of 97% for ARPE-19 cells, exhibiting minimal irritation and safety.

[0062] Example 8: Treatment procedure of latanoprost lipocrystalline precursor eye drops in an acute ocular hypertension model, obtained using the method described in Example 3 above. Experimental animals and grouping: Six- to eight-week-old male SD rats (n=6) were used and divided into five groups: I. Control Group II. Saline group III. Blank Liquid Crystal Gel Assembly (BLLC) IV. Xalatan V. Latanoprost Lipid Liquid Crystal Progenitor Eye Drops (LAT-LLC) On the day of surgery, rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (30 mg / kg). The periorbital area was disinfected with povidone-iodine, and local anesthesia was performed by instilling 2% procaine into the ocular surface. Group I received sham surgery, while groups II to V had 9 μm polystyrene microbeads suspended in 0.9% sodium chloride solution. Using a 34G injection needle connected to a microsyringe, 15 μL of the microbead suspension (containing approximately 10 mg / kg of sodium chloride) was injected into the anterior chamber. 7(Microbeads). Remove the needle and apply levofloxacin gel to the injection site to prevent infection. Postoperative assessment: On the second day post-surgery, intraocular pressure (IOP) in rats was measured using a rebound tonometer (TONOLAB, ICARE). An IOP increase of 20 mmHg or more compared to a normal eye was considered a successful model. Groups II and IV received daily saline and Xalatan. Groups III and V received liquid crystal every two days for a total of three times, with daily monitoring of IOP changes. On day 9, eyes were harvested for HE staining to observe corneal and retinal changes. The modeling and treatment procedures are as follows: Figure 11 As shown.

[0063] The mice treated with the above methods were then further validated. 1) Validation of an acute ocular hypertension model by injecting microspheres into the anterior chamber Mice treated according to the above embodiments were euthanized. After euthanasia, both eyes were removed, fixed with ocular fixative, embedded in paraffin, and sectioned. Sections were approximately 4-5 μm thick and stained with hematoxylin and eosin (HE). Histopathological examination was performed using an optical microscope. Figure 12 As shown, compared with the histological microscopic examination of normal mouse eyes, the anterior chamber angle of glaucoma mice has accumulated numerous polystyrene microspheres, which obstruct the outflow of aqueous humor, and the histological examination clearly shows that the model was successfully established.

[0064] 2) Changes in intraocular pressure during treatment in each group and comparison of the extent of intraocular pressure reduction between the Xalatan group and the LAT-LLC group. After injecting polystyrene microspheres into the anterior chamber, intraocular pressure was measured in each group of mice at fixed times daily using an ICARE tonometer. Figure 13 As shown, in groups II-V, intraocular pressure rose to 25 mmHg on the first day and to 30 mmHg on the second day, while the intraocular pressure in the control group remained almost unchanged, ranging from 8 to 12 mmHg.

[0065] Intraocular pressure (IOP) was then monitored daily in each group of SD mice, with six measurements taken from each eye and the average value recorded. Groups II and IV received daily administration of saline and Xalatan. Groups III and V received liquid crystal therapy every two days for a total of three times. IOP changes were monitored daily throughout the period, and a 9-day IOP chart was created. Figure 10As shown, the intraocular pressure (IOP) in the control group remained almost unchanged within 8-12 mmHg over 9 days. In groups II and III, IOP continued to rise within the 21-38 mmHg range over 9 days, indicating that without effective drug treatment, IOP in glaucoma will continue to rise and worsen. Both marketed Xalatan and LAT-LLC showed varying degrees of IOP reduction within 9 days, with Xalatan maintaining IOP at 13-18 mmHg and LAT-LLC maintaining IOP at 8-15 mmHg, almost identical to the control group. Due to insufficient residence time of latanoprost eye drops on the ocular surface, its bioavailability was low, resulting in a less effective IOP-lowering effect compared to LAT-LLC. A comparison between the LAT-LLC and Xalatan groups revealed that the LAT-LLC group exhibited a more sustained and stable IOP-lowering effect because latanoprost in the LAT-LLC group was slowly released during gel dissolution.

[0066] 3) HE staining of eyeball sections from each treatment group (1) After the rats were euthanized, the eyeballs were quickly and completely removed with ophthalmic microforceps. A portion of the optic nerve was left and fixed in the eyeball fixation solution at room temperature for more than 24 hours. The fixed eyeballs were then removed in a ventilated area to remove excess tissue around the eyeballs. Then, the eyeballs were dehydrated with different concentrations of alcohol (75% for 4 hours, 85% for 2 hours, 90% for 2 hours, 95% for 1 hour and anhydrous ethanol for 30 minutes twice). Next, the eyeballs were soaked in benzene for 10 minutes, xylene for 10 minutes, and then in xylene for 10 minutes. The eyeballs were then placed in melted paraffin for 1 hour. The process was repeated twice in fresh melted paraffin.

[0067] (2) Embedding: The tissue impregnated with paraffin was embedded using an embedding machine. After embedding, the tissue was cooled at -20°C.

[0068] (3) Use a paraffin microtome to cut the eyeball into celestial sections, cutting to the optic nerve position in the middle of the eyeball, with a thickness of 10 μm. After cutting, bake the sections in an oven at 60°C.

[0069] (4) Next, the sections were dewaxed and hydrated, and then stained with hematoxylin and eosin according to the steps of the HE staining kit.

[0070] (5) Dehydration and mounting: Place the sections in anhydrous ethanol for 5 min, repeating three times; place in xylene for 5 min, repeating twice; finally, mount with neutral resin. Observe the corneal and retinal morphology under a fluorescence microscope using white light and take photographs. Results are as follows: Figure 14As shown, compared to Control, Saline and BLLC resulted in persistently high intraocular pressure, leading to corneal thinning, corneal epithelial cell vacuolation, reduced cell number, sparse and irregular arrangement, and corneal stromal hyperplasia. However, after treatment with Xalatan and LAT-LLC, the high intraocular pressure was relieved to varying degrees, with no significant changes in corneal thickness or morphology. In Saline, BLLC, and Xalatan-treated mice, the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the retina exhibited disordered cell arrangement and loss of the original dense cellular structure. Furthermore, the GCL showed varying degrees of edema and cell loss, and the overall retinal thickness increased, as shown in the figure. Figure 15 As shown, there were also many dead RGCs, characterized by deeply stained, bluish-black nuclei, rounded cells, and reduced cell size. However, the retinal damage in mice treated with LAT-LLC was relatively mild, with retinal thickness and morphology remaining almost unchanged, indicating that LAT-LLC has a good effect in relieving high intraocular pressure.

[0071] Example 9 Example 3 Obtaining LAT-LLC Ocular Surface Retention SD mice were randomly divided into three groups: the DIR group, the Xalatan group, and the LAT-LLC group obtained in Example 3, with three mice in each group. For the LAT-LLC group, DIR was added to the liquid crystal precursor at a concentration of 50-100 μg / mL, and each mouse was given 10-20 μL of the liquid crystal precursor solution. For the DIR group, a DIR solution of 50-100 μg / mL was prepared, and each mouse was given 10-20 μL of the DIR solution. For the Xalatan group, DIR was added to Xalatan at a concentration of 50-100 μg / mL and mixed thoroughly. The DIR signal intensity in the LAT-LLC was captured using a small animal live imaging system at 0h, 6h, 12h, 24h, and 48h. Figure 16 As shown, the DIR solution showed no residue on the ocular surface after 6 hours; the Xalatan group showed almost no residue on the ocular surface after 12 hours; and LAT-LLC still had a small amount of residue on the ocular surface after 48 hours, indicating good retention. This may be because the liquid crystal precursor solution encounters moisture on the ocular surface, achieving solvent dispersion, and the lipid liquid crystal appears in the form of a liquid crystal gel, which can remain on the ocular surface for a long time.

[0072] Example 10: HE staining assessment of the safety of latanoprost lipocrystalline precursor eye drops SD mice were randomly divided into 5 groups: Control, Saline, BLLC, LAT-LLC (obtained in Example 3), and Xalatan, with 3 mice in each group. Control mice received no treatment; Saline mice received physiological saline, 10-20 μL once daily; BLLC mice received a drug-free liquid crystal precursor solution, 10-20 μL every two days; LAT-LLC mice received a liquid crystal precursor solution containing latanoprost, 10-20 μL every two days; and the Xalatan group received Xalatan eye drops, 10-20 μL once daily. Four days after administration, the rats were sacrificed, and the eyeballs were quickly and completely removed using ophthalmic microforceps, preserving a portion of the optic nerve. The eyes were then fixed in an ocular fixation solution at room temperature for at least 24 hours. The fixed eyeballs were then excised in a ventilated area to remove excess tissue around the eyeballs. The eyes were then dehydrated with different concentrations of alcohol, embedded, sectioned, stained, and mounted. Results are as follows: Figure 17 As shown, in the treatment group, corneal cells were tightly and neatly arranged without vacuoles or infiltration, and there was no significant proliferation of the corneal stroma; the total retinal thickness did not change significantly, optic ganglion cells were tightly and orderly arranged, and there was no infiltration or proliferation of cells in the inner and outer nuclear layers. This indicates that LAT-LLC is not significantly irritating compared to the Control group. Statistical analysis of corneal and retinal thickness between the LAT-LLC and Control groups showed no significant difference. Figure 18 .

[0073] Example 11 Safety evaluation of LAT-LLC using slit lamp and corneal fluorescein staining Different groups of SD rats were administered Saline, BLLC, Xalatan, and LAT-LLC (obtained in Example 3), and observations were conducted on days 1, 2, and 3 after administration. After anesthetizing the SD rats, they were placed under a slit lamp, and corneal imaging was performed under white light. Fluorescein sodium solution (0.5% 10 μL / eye) was then instilled into the animals' eyes. Starting approximately 1.5 minutes after staining, the conjunctival sac was flushed with 1.25 ml of sterile saline every 10 seconds for three consecutive times. After each flush, the saline solution around the eyes was blotted dry with tissue paper. Approximately 5 minutes after staining, the ocular surface was observed and photographed using a slit lamp (with a cobalt blue filter). Results are as follows: Figure 19 As shown, no corneal damage or staining areas were observed under either white light or sodium fluorescein staining, further assessing the safety of the lipid liquid crystal. Ocular surface scoring was rigorously performed by professional ophthalmologists according to the modified Draize irritation score. All scores were less than 3 points. Figure 20 This indicates that LAT-LLC did not cause corneal irritation.

[0074] Example 12: Observation of corneal epithelium, stroma, and endothelial cells in rabbits after treatment with LAT-LLC precursor eye drops using corneal confocal microscopy. Three New Zealand rabbits were anesthetized by intramuscular injection of 3% sodium pentobarbital (40 mg / kg). Local anesthesia of the ocular surface was achieved by instillation of 2% procaine. Once the rabbits reached a stable state of anesthesia, 40 μL of the LAT-LLC precursor solution from Example 3 was administered topically to the treated eye. After a 10-second stabilization period, the treated eye was focused using a corneal confocal microscope lens (Heidelberg Engineering, HRT3). The focus was progressively adjusted to sequentially capture images of the corneal epithelium, corneal stroma, and corneal endothelium. In vivo confocal microscopy of the cornea (… Figure 21 The results showed no morphological changes in corneal epithelial cells, stromal cells, or endothelial cells. Therefore, LAT-LLC gel exhibits long-term biocompatibility, supporting the safety of our treatment system.

[0075] In summary, this invention constructs a drug-loaded lipid liquid crystal precursor eye drop. When the drug-loaded lipid liquid crystal precursor comes into contact with water in the tear fluid, it instantly transforms into a drug-loaded lipid liquid crystal gel. Compared to ordinary eye drops, it has a longer retention time on the ocular surface. Furthermore, the lipid liquid crystal has a phospholipid bilayer structure, allowing poorly soluble active pharmaceutical ingredients to be distributed within the lipid layer, greatly increasing the solubility of the active pharmaceutical ingredient. The gelation upon contact with water enables the slow release of the active pharmaceutical ingredient, reducing the frequency of administration and significantly increasing bioavailability and patient compliance, thus compensating for the shortcomings of eye drops in the treatment of glaucoma.

Claims

1. A lipid liquid crystal precursor eye drop, characterized in that: The lipid liquid crystal precursor eye drops comprise a matrix material, a cosolvent, and a drug substance; wherein the matrix material is an amphiphilic substance and an oil phase; the lipid liquid crystal precursor eye drops, by weight percentage, comprise 0%–35% amphiphilic substance, 40%–80% oil phase, 15–20% cosolvent, and 0.001%–5% drug substance.

2. The lipid liquid crystal precursor eye drops according to claim 1, characterized in that: The matrix material is an amphiphilic substance and an oil phase; wherein, the amphiphilic substance is selected from one or more of lecithin, phosphatidylcholine, and phosphatidylethanolamine; the oil phase is selected from monooleylglycerol, dioleylglycerol, phytanetriol, and tocopherol, and oleic acid is preferably one or more of dioleylglycerol and monooleylglycerol.

3. The lipid liquid crystal precursor eye drops according to claim 1, characterized in that: The co-solvent is selected from one or more of propylene glycol, ethylene glycol, and dimethyl sulfoxide; The active pharmaceutical ingredient is one or more of the following: pilocarpine, acetazolamide, timarolol, brinzolamide, prostaglandin latanoprost, travoprost, or coenzyme Q10.

4. The lipid liquid crystal precursor eye drops according to any one of claims 1-3, characterized in that: The lipid liquid crystal precursor eye drops, by weight percentage, consist of 16%-32% amphiphilic substance, 48%-64% oil phase, 15-20% cosolvent and 0.001%-5% active pharmaceutical ingredient.

5. A method for preparing the lipid liquid crystal precursor eye drops according to claim 1, characterized in that: According to the above proportions, the matrix material is dissolved in a solvent to obtain a blank gel precursor; then, the active pharmaceutical ingredient and cosolvent are added to the blank gel precursor, and under sealed conditions, the solvent that dissolved the matrix is ​​removed by N2 blowing to obtain the lipid liquid crystal precursor eye drops.

6. The method for preparing lipid liquid crystal precursor eye drops according to claim 5, characterized in that: The solvent is anhydrous ethanol.

7. The application of the lipid liquid crystal precursor eye drops according to claim 1, characterized in that: The application of the lipid liquid crystal precursor eye drops as an eye drop for the treatment of acute ocular hypertension glaucoma.

8. The application of the lipid liquid crystal precursor eye drops according to claim 1, characterized in that: The application of the lipid liquid crystal precursor eye drops as an eye drop for the treatment of chronic ocular hypertension glaucoma.

9. The application of the lipid liquid crystal precursor eye drops according to claim 1, characterized in that: The application of the lipid liquid crystal precursor eye drops as a long-acting eye drop.

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