Pranoprofen temperature-sensitive gel for treating xerophthalmia and preparation method thereof

Abstract

The invention discloses pranoprofen temperature-sensitive gel for treating xerophthalmia and a preparation method, the gel takes poloxamer 407 and poloxamer 188 as matrixes, the optimal ratio of the poloxamer 407 to the poloxamer 188 is 23.5%: 2%, and the ratio is optimized and determined by a central composite design-response surface method. During preparation, a blank gel solution is prepared firstly, and then auxiliary materials such as pranoprofen and the like are added for dissolution. The in-vitro release characteristic is good, the accumulated dissolution rate within 210 min exceeds 90%, and the zero-level pharmacokinetic process is achieved. An SD rat xerophthalmia model is established, and it is proved that the xerophthalmia treatment effect of the composition is better than that of eye drops. In eye tissue distribution and aqueous humor pharmacokinetic studies, higher target organ distribution characteristics are shown, the aqueous humor AUC0-t is 1.85 times that of eye drops, and the bioavailability is high. The invention further provides a gel preparation method, a drug effect evaluation method and a related research method, and a new effective dosage form and a new treatment thought are provided for xerophthalmia treatment.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to a pharmaceutical preparation for treating dry eye disease, its related preparation method, efficacy evaluation method, and pharmacokinetic research method. More specifically, it relates to a thermosensitive gel preparation containing pranoprofen, and involves aspects such as the prescription optimization of this preparation, in vitro release characteristics, efficacy evaluation in the treatment of dry eye disease, and tissue distribution and pharmacokinetic research in the eye. Background Art

[0002] Dry eye disease is an ocular surface multi-factorial disease characterized by the imbalance of the ocular surface microenvironment or the tear film homeostasis, accompanied by ocular symptoms such as unstable tear film, ocular surface inflammation, and damage. The ocular surface microenvironment includes the tear film functional unit composed of the cornea, conjunctiva, lacrimal gland, eyelids, meibomian glands, and sensory and motor nerves, as well as other ocular tissues, and the tear film, immune, and nervous systems. Any disruption of these components may lead to unstable tear film.

[0003] Among them, the abnormality of the tear film functional unit can lead to the occurrence of ocular surface diseases with the imbalance of tear film homeostasis as the core pathogenesis. The role of the tear film functional unit is to secrete basal tears and respond to the effects of endocrine, cerebral cortex, and environment in the body to maintain tear film homeostasis, and the realization of its function depends on the stability of the ocular surface microenvironment. The interaction between the two leads to symptoms such as inflammatory reaction, tissue damage, and nerve abnormality, which in turn cause a decline in visual quality and visual dysfunction. It can be seen that maintaining the ocular surface microenvironment and tear film homeostasis and combining anti-inflammatory drugs are the keys to treating dry eye disease.

[0004] In clinical applications, artificial tears and sodium hyaluronate are mainly used in combination with non-steroidal anti-inflammatory drugs such as pranoprofen, diclofenac sodium, and cyclosporine A to moisten the ocular surface tissue and inhibit ocular surface inflammation. Currently, commercially available eye drop preparations generally contain preservatives to inhibit the growth of microorganisms, but long-term exposure to these substances has been proven to potentially cause damage to the eyes. Benzalkonium chloride is a widely used cationic surfactant preservative in ophthalmic preparations, and its long-term use may cause ocular surface toxicity, accelerate tear evaporation, lead to ocular surface inflammation and unstable tear film, and these factors are all related to the clinical occurrence of dry eye disease.

[0005] In addition, after local administration of eye drops, due to physiological factors such as blinking, tear secretion, and nasolacrimal duct drainage, it is easy to be rapidly cleared from the ocular surface, which limits the residence time of the drug in front of the cornea, reduces drug absorption and intraocular bioavailability, usually not exceeding 5%. At the same time, the permeability of the drug in the cornea is also relatively low, only 5%, and the drainage effect of the nasolacrimal duct may bring the drug to non-target areas, increasing the risk of side effects. Frequent or excessive use of certain ophthalmic preparations may cause ocular discomfort and affect the medication compliance of patients.

[0006] In the prior art, Chinese Patent CN118045040A discloses a single-dose pranoprofen eye drops, which improves the action time of the drug on the cornea by adding sodium polyacrylate as a thickening agent. This eye drops does not contain antioxidants and bacteriostatic agents, reducing the potential irritation to the eyes. However, although this solution increases the viscosity, the form of the eye drops is still difficult to provide a lasting drug concentration on the ocular surface, which may lead to drug loss and frequent administration.

[0007] In addition, although ocular drug delivery systems such as lipid carriers, nanocrystals, nanoemulsions, etc. have their respective advantages, they have different defects; lipid carrier formulations have limited encapsulation efficiency and drug loading capacity, and there are toxic side effects such as extensive irritation clinically. The drug particles in ophthalmic suspensions are generally between 0.5 and 10 μm, which is an unstable heterogeneous liquid dispersion system. During storage, the particles are prone to aggregation and caking, affecting the use and drug efficacy.

[0008] Although nanocrystals have significant advantages in improving drug solubility and bioavailability, they have physicochemical-related stability problems. In the clinical application of nanoemulsions, such as cyclosporine A nanoemulsion, it may cause irritating side effects such as a burning sensation in the eyes and conjunctival congestion. Liposomes such as cyclosporine A liposomes may cause side effects such as eye pain, eye irritation, tearing, eye congestion, and eyelid erythema.

[0009] Therefore, there is still a large room for improvement in the current prior art. Summary of the Invention

[0010] The present invention aims to develop a new drug dosage form, prepare a thermosensitive gel with pranoprofen as the main drug and a specific ratio of poloxamer 407 and poloxamer 188 as the matrix, optimize the prescription to improve the in vitro release efficiency and intraocular bioavailability of the drug, solve the problems of short residence time on the ocular surface, low bioavailability and poor drug efficacy when pranoprofen is used to treat dry eye, and avoid the use of toxic preservatives at the same time, providing a new solution for the clinical treatment of dry eye.

[0011] Based on the first main aspect of the present invention, there is provided a pranoprofen thermosensitive gel for treating dry eye, which is prepared by the following method:

[0012] Weigh poloxamer 407 and poloxamer 188 in the prescribed amount of water, store them in a 4°C refrigerator for 24 hours to swell, and obtain a blank gel solution; accurately weigh pranoprofen, place it in a volumetric flask, add Tween-80, boric acid, borax, an appropriate amount of 0.2% tris(hydroxymethyl)aminomethane solution and the blank gel matrix, make up the volume and sonicate until completely dissolved;

[0013] Among them, the optimal ratio of P407 and P188 is 23.5%:2%.

[0014] As a further preferred embodiment, in the aforementioned pranoprofen thermosensitive gel, the optimal ratio is determined by optimizing the formulation through the central composite design-response surface method. The optimization process includes using the optimal concentration ranges of poloxamer 407 and poloxamer 188 screened as independent variables, conducting a central composite design at multiple experimental levels, using the gel temperature before and after dilution with artificial tears as the response variable, performing multiple linear regression analysis using Design-Expert 13 software, generating three-dimensional response surface plots and two-dimensional contour plots, and determining the optimal matrix ratio based on the deviation between the predicted value and the measured value.

[0015] As a further preferred embodiment, in the aforementioned pranoprofen thermosensitive gel, the in vitro release characteristics of the thermosensitive gel are as follows: When the in vitro release study is carried out using the membrane-free dissolution method, the cumulative erosion rate within 210 minutes is greater than 90%, and the cumulative release rate has a good linear relationship with time, showing a zero-order pharmacokinetic process.

[0016] Based on the second main aspect of the present invention, a method for preparing a pranoprofen thermosensitive gel for treating dry eye is provided, comprising the following steps:

[0017] Prepare a blank gel solution: Weigh poloxamer 407 and poloxamer 188 in the prescribed amount of water, store them in a 4°C refrigerator for 24 hours for swelling;

[0018] Prepare the pranoprofen thermosensitive gel: Weigh pranoprofen precisely, place it in a volumetric flask, add Tween-80, boric acid, borax, an appropriate amount of 0.2% tris(hydroxymethyl)aminomethane solution and the blank gel matrix, make up the volume, and ultrasonicate until completely dissolved;

[0019] Optimize the formulation by the central composite design-response surface method: Use the optimal concentration ranges of poloxamer 407 and poloxamer 188 screened as independent variables, conduct a central composite design at multiple experimental levels, use the gel temperature before and after dilution with artificial tears as the response variable, perform multiple linear regression analysis using Design-Expert 13 software, generate three-dimensional response surface plots and two-dimensional contour plots, and determine the optimal ratio of poloxamer 407 to poloxamer 188 to be 23.5%:2%.

[0020] As a further preferred embodiment, in the preparation method of the aforementioned pranoprofen thermosensitive gel, during the optimization of the formulation, when screening for the optimal concentration of poloxamer 407, gel solutions of different concentrations of poloxamer 407 were placed in ampoules, immersed in an ice-water bath at 4°C, a precision thermometer was inserted, stirred and slowly heated, and the sol-gel phase transition temperature at which the gel solution stopped flowing was recorded to screen out the optimal concentration range; thereafter, the gelling temperature after mixing with different concentrations of poloxamer 188 was measured, and the concentration range of poloxamer 407 was determined to be 20%-26% and the concentration range of poloxamer 188 was 0%-6% during the optimization of the central composite design formulation.

[0021] As a further preferred embodiment, in the preparation method of the aforementioned pranoprofen thermosensitive gel, the concentrations of the gel solutions of different concentrations of poloxamer 407 include 16%, 18%, 20%, 22%, 24%, 26%; the concentrations of different concentrations of poloxamer 188 include 0%, 2%, 4%, 6%, 8%.

[0022] Based on the third main aspect of the present invention, a method for evaluating the efficacy of pranoprofen thermosensitive gel in the treatment of dry eye is provided, including:

[0023] Establishing a dry eye model in SD rats induced by 0.5% benzalkonium chloride;

[0024] Dividing the experimental rats into a normal group, a model group, a thermosensitive gel group without pranoprofen, a pranoprofen eye drop group, and a pranoprofen thermosensitive gel group; 5 days after modeling, each group was administered at a frequency of 20 μL / eye, 4 times a day with an interval of 3 h each time, for 9 consecutive days;

[0025] Detecting and evaluating indicators such as corneal neovascularization, corneal turbidity, conjunctival irritation, tear secretion volume, and conjunctival inflammatory factors on the 7th and 14th days after modeling, and the results showed that the efficacy of pranoprofen thermosensitive gel in the treatment of dry eye was superior to that of pranoprofen eye drops.

[0026] As a further preferred embodiment, in the preparation method of the aforementioned pranoprofen thermosensitive gel, the corneal neovascularization was evaluated by adding up the scores of the four quadrants of the cornea using a slit lamp; the corneal turbidity was evaluated according to a specific slit lamp observation scoring standard; the conjunctival irritation was comprehensively evaluated by scoring conjunctival congestion, edema, and eye discharge respectively; the tear secretion volume was measured by placing a modified tear filter paper in the outer 1 / 3 conjunctival sac of the lower eyelid of the rat for 2 min and measuring the wetting length; the conjunctival inflammatory factors were evaluated by homogenizing the conjunctival tissue of the rat eye and detecting the levels of TNF-α and IL-1β using an ELISA kit.

[0027] Based on the fourth main aspect of the present invention, a method for studying the ocular tissue distribution and aqueous humor pharmacokinetics of pranoprofen thermosensitive gel is provided. In the tissue distribution study, healthy SD rats are divided into a pranoprofen eye drop group and a pranoprofen thermosensitive gel group, and a single dose of 20 μL is given to the left eye. The rats are killed at time points including 0.167, 0.5, 1, 3, 4, and 6 hours, and the aqueous humor, cornea, and vitreous are taken to detect the concentration of pranoprofen; in the aqueous humor pharmacokinetics study, healthy rabbits are divided into a pranoprofen eye drop group and a pranoprofen thermosensitive gel group, and a single dose of 5 μL is given to the left and right eyes, respectively, and the aqueous humor is taken at time points including 0.017, 0.083, 0.167, 0.5, 1, 2, 3, 4, 6, and 8 hours to detect the concentration of pranoprofen and draw a pharmacokinetic curve. The results show that the pranoprofen thermosensitive gel has a higher target organ distribution characteristic, and the aqueous humor AUC0-t is 1.85 times that of the eye drops.

[0028] As a further preferred embodiment, in the aforementioned method for studying the ocular tissue distribution and aqueous humor pharmacokinetics of pranoprofen thermosensitive gel, liquid chromatography-mass spectrometry was used to detect the concentration of pranoprofen, the chromatographic column was a Waters BEH C18 (2.1 mm × 150 mm, 1.7 μm) column, the guard column was a Waters VanGuard BEH C18 (2.1 mm × 5 mm, 1.7 μm), the injection volume was 1 μL, the mobile phase was 0.2% formic acid acetonitrile (B)-0.2% formic acid water (A), and the elution program and other mass spectrometry parameters were performed as set, as follows:

[0029] Elution program: the mobile phase is 0.2% formic acid acetonitrile (B)-0.2% formic acid water (A), and the elution program is 0-1.5 min, 92%-70% A; 1.5-2.0 min, 70%-60% A; 2.0-2.5 min, 60%-10% A; 2.5-3.0 min, 10% A; 3.0-4.0 min, 10%-92% A. Through such an elution program, effective separation of different components in the sample can be achieved.

[0030] Mass spectrometry parameters:

[0031] Ionization source and related parameters: Electrospray ionization source (ESI) was used, capillary ionization voltage was 3 kV, ion source temperature was 120 ° C, spray gas and backflush gas were N2, desolvation gas flow rate was 650 L / hr, and desolvation gas temperature was 350 ° C. The setting of these parameters ensured the ionization effect of the sample and the transmission efficiency of ions.

[0032] Scanning mode and quantitative ion pairs: The scanning mode is multiple reaction monitoring (MRM). The ion pairs used for quantitative analysis are puerarin m / z417 (internal standard), with a cone voltage of 30 V; pranoprofen m / z255.8, with a cone voltage of 30 V, both in positive ion scanning mode. By selecting specific ion pairs and cone voltages, the accuracy and sensitivity of the detection can be improved, enabling the quantitative analysis of pranoprofen and the internal standard.

[0033] Data acquisition and processing software: The mass spectrometry data acquisition and processing software is the MassLynx 4.1 workstation. This software is used to process and analyze the collected data to obtain the required experimental results.

[0034] Advantages and beneficial effects of the present invention:

[0035] Compared with the prior art, the pranoprofen thermosensitive gel of the present invention shows significant effects in multiple aspects in the field of dry eye treatment, greatly improving the treatment effect and the patient's medication experience.

[0036] First of all, through the central composite design-response surface method, the present invention accurately determines the optimal ratio of poloxamer 407 and poloxamer 188 to be 23.5%:2%. This optimization not only endows the thermosensitive gel with unique thermosensitive properties, making it easy to drop into the eyes at room temperature, quickly transform into a gel at body temperature, and prolong the residence time of the drug on the ocular surface, but also significantly improves the bioavailability of the drug. Compared with traditional ophthalmic preparations, its innovative matrix combination provides good wettability and biocompatibility, effectively reducing drug loss and providing a strong guarantee for the efficient delivery of the drug to the ocular lesion site.

[0037] Secondly, when the in vitro release study is carried out by the membrane-free dissolution method, the pranoprofen thermosensitive gel of the present invention has a cumulative erosion rate greater than 90% within 210 min, and the cumulative release rate has a good linear relationship with time, conforming to the zero-order pharmacokinetic process. This characteristic indicates that the gel can achieve the sustained and stable release of the drug, avoiding the problems of rapid drug release and short action time of traditional eye drops, helping to maintain the stability of the drug concentration in the eyes, thus more effectively exerting the therapeutic effect of the drug, reducing the dosing frequency, and improving the patient's medication compliance.

[0038] Thirdly, by establishing a dry eye model of SD rats induced by 0.5% benzalkonium chloride for efficacy evaluation, the results show that the pranoprofen thermosensitive gel is superior to pranoprofen eye drops in terms of improving corneal neovascularization, corneal turbidity, conjunctival irritation, tear secretion volume, and reducing the level of conjunctival inflammatory factors. Specifically, it can significantly reduce corneal damage, reduce neovascularization, relieve inflammatory symptoms such as conjunctival congestion and edema, increase tear secretion, and effectively improve dry eye symptoms, providing a more effective treatment option for dry eye patients.

[0039] Fourth, in the studies on the tissue distribution in the eye and the pharmacokinetics in aqueous humor of the present invention, it was found that pranoprofen thermosensitive gel has a higher characteristic of target organ distribution. In the rat experiment, the drug concentration in the cornea gradually accumulated and increased over time, and a double absorption peak appeared in the aqueous humor; in the rabbit experiment, pranoprofen thermosensitive gel maintained a relatively high drug concentration within the study time, with a shorter peak time, and the area under the concentration-time curve (AUC0-t) was 1.85 times that of the eye drops. This means that the gel can more effectively deliver the drug to the target tissues in the eye, improve the bioavailability of the drug in the aqueous humor, enhance the drug efficacy while reducing the risk of systemic adverse reactions. The present invention established a comprehensive research method for pranoprofen thermosensitive gel, covering prescription optimization, in vitro release study, efficacy evaluation, and tissue distribution and pharmacokinetic studies. These studies adopted scientific and rigorous experimental designs and advanced detection techniques, such as central composite design-response surface methodology, liquid chromatography-mass spectrometry, etc., providing solid data support and theoretical basis for the development and application of the product, ensuring the quality and safety of the product, and laying a good foundation for clinical application.

[0040] Finally, the thermosensitive gel matrix selected in the present invention showed significant reduction in corneal neovascularization and corneal fluorescein staining area in the anti-dry eye rat efficacy experiment, indicating that the sustained-release property of the thermosensitive gel matrix and its protective effect on corneal epithelium may contribute to reducing corneal damage and inhibiting neovascularization, promoting the self-repair of the cornea, and thus alleviating dry eye symptoms. Similar to the effect of sodium hyaluronate in clinical practice, the preparation of the present invention can therefore reduce the need for patients to instill eye drops for treating dry eye and drugs such as sodium hyaluronate for wetting the ocular surface simultaneously. Description of the Drawings

[0041] Figure 1 The two-dimensional contour map (T1) of the gelation temperatures T1 and T2 for optimizing the preparation prescription by the central composite design-response surface methodology in an embodiment of the present invention is shown;

[0042] Figure 2 The two-dimensional contour map (T2) of the gelation temperatures T1 and T2 for optimizing the preparation prescription by the central composite design-response surface methodology in an embodiment of the present invention is shown;

[0043] Figure 3 The three-dimensional response surface map (T1) of the gelation temperatures T1 and T2 for optimizing the preparation prescription by the central composite design-response surface methodology in an embodiment of the present invention is shown;

[0044] Figure 4 The three-dimensional response surface map (T2) of the gelation temperatures T1 and T2 for optimizing the preparation prescription by the central composite design-response surface methodology in an embodiment of the present invention is shown;

[0045] Figure 5Shows the two-dimensional contour overlay diagram of the optimized preparation prescription by the central composite design-response surface method in an embodiment of the present invention;

[0046] Figure 6 Shows the characteristics of the thermosensitive gel in an embodiment of the present invention and the effect of temperature on the viscosity of the formulation in the evaluation. (no: viscosity without STF gel interaction; +S: viscosity of gel interaction with artificial tears);

[0047] Figure 7 Shows the cumulative erosion rate of pranoprofen thermosensitive gel in the characteristics and evaluation of the thermosensitive gel in an embodiment of the present invention;

[0048] Figure 8 Shows the cumulative release rate of pranoprofen thermosensitive gel in the characteristics and evaluation of the thermosensitive gel in an embodiment of the present invention;

[0049] Figure 9 Shows the cumulative release rate of two preparations measured by the dialysis bag method in the characteristics and evaluation of the thermosensitive gel in an embodiment of the present invention;

[0050] Figure 10 Shows the pharmacodynamic evaluation diagram of pranoprofen thermosensitive gel on dry eye model rats in an embodiment of the present invention: (a) corneal turbidity and corneal neovascularization; (b) conjunctival irritation; (c) corneal fluorescein sodium staining;

[0051] Figure 11 Shows the corneal neovascularization score in the pharmacodynamic evaluation of pranoprofen thermosensitive gel on dry eye model rats in an embodiment of the present invention;

[0052] Figure 12 Shows the corneal turbidity score in the pharmacodynamic evaluation of pranoprofen thermosensitive gel on dry eye model rats in an embodiment of the present invention;

[0053] Figure 13 Shows the analysis of the conjunctival irritation score in the pharmacodynamic evaluation of pranoprofen thermosensitive gel on dry eye model rats in an embodiment of the present invention, including redness and swelling and total secretion scores;

[0054] Figure 14 Shows the analysis of the fluorescein sodium staining score in the pharmacodynamic evaluation of pranoprofen thermosensitive gel on dry eye model rats in an embodiment of the present invention;

[0055] Figure 15 Shows the measurement of the tear secretion of rats on the 1st, 4th, 7th, 10th, and 14th days after modeling and administration in the pharmacodynamic evaluation of pranoprofen thermosensitive gel on dry eye model rats in an embodiment of the present invention;

[0056] Figure 16 Shows the analysis of the inflammatory factor TNF-α in the conjunctiva in the pharmacodynamic evaluation of pranoprofen thermosensitive gel on dry eye model rats in one embodiment of the present invention;

[0057] Figure 17 Shows the analysis of the inflammatory factor IL-1β in the conjunctiva in the pharmacodynamic evaluation of pranoprofen thermosensitive gel on dry eye model rats in one embodiment of the present invention;

[0058] Figure 18 Shows the tissue distribution of pranoprofen in the ocular tissues after administration of different preparations to rats and the concentration of pranoprofen in the cornea in the pharmacokinetics of rabbit aqueous humor in one embodiment of the present invention;

[0059] Figure 19 Shows the tissue distribution of pranoprofen in the ocular tissues after administration of different preparations to rats and the concentration of pranoprofen in the vitreous in the pharmacokinetics of rabbit aqueous humor in one embodiment of the present invention;

[0060] Figure 20 Shows the tissue distribution of pranoprofen in the ocular tissues after administration of different preparations to rats and the concentration of pranoprofen in the aqueous humor in the pharmacokinetics of rabbit aqueous humor in one embodiment of the present invention;

[0061] Figure 21 Shows the tissue distribution of pranoprofen in the ocular tissues after administration of different preparations to rats and the concentration-time curve of pranoprofen in the aqueous humor of rabbits after administration of different preparations to rabbits in the pharmacokinetics of rabbit aqueous humor in one embodiment of the present invention. Detailed Description of the Invention

[0062] The preferred embodiments of the present invention will be described in detail below so as to more clearly understand the purpose, features and advantages of the present invention. It should be understood that the following embodiments are not intended to limit the scope of the present invention, but only to illustrate the essential spirit of the technical solution of the present invention.

[0063] In the following description, certain specific details are set forth for the purpose of explaining various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other instances, well-known technologies associated with the present application may not be shown or described in detail so as to avoid unnecessarily obscuring the description of the embodiments.

[0064] References to "an embodiment" or "one embodiment" in the entire specification mean that the specific features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment. Therefore, the appearances of "in one embodiment" or "in an embodiment" at various positions throughout the specification do not necessarily all refer to the same embodiment. Additionally, the specific features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0065] Example 1:

[0066] 1. Preparation of pranoprofen thermosensitive gel

[0067] Weigh poloxamer 407 (P407) and poloxamer 188 (P188) in the prescribed amount of water, place it in a 4°C refrigerator for 24 h to swell, and obtain a blank gel solution. Weigh 10 mg of pranoprofen precisely, place it in a 10 mL volumetric flask, add 0.01 g of Tween-80, 0.1752 g of boric acid, 0.0476 g of borax, an appropriate amount of 0.2% tris(hydroxymethyl)aminomethane (Tris) solution, and the blank gel matrix, make up the volume, and ultrasonicate until completely dissolved to obtain the product.

[0068] 2. Investigation of the gelation temperature of the gel matrix

[0069] Put the gel solutions of 16%, 18%, 20%, 22%, 24%, 26% (w / w) P407 into ampoules, place them in an ice-water bath at 4°C, make a hole in the middle of the ampoule stopper, insert a precision thermometer with an accuracy of 0.1°C, completely immerse the mercury bulb part of the thermometer in the solution, stir the solution with a magnetic stirrer, and slowly increase the temperature (about 1°C / min). Tilt the ampoule quickly by 60° every time the temperature rises by 0.1°C, and record the sol-gel phase transition temperature at which the gel solution stops flowing as the gelation temperature. All results are measured three times. Screen out the optimal concentration of P407, and then determine the gelation temperature after mixing with 0%, 2%, 4%, 6%, 8% of P188. The determination steps are the same as above.

[0070] The results are shown in Table 1. As the concentration of P407 increases, the gelation temperature becomes lower. When the concentration of poloxamer is lower than 16%, the gelation temperature cannot be measured. When the content of poloxamer reaches 18%, the gelation temperature higher than 30°C is not selected either.

[0071] This indicates that the gelling temperature depends on the concentration of the gel matrix. Increasing the concentration of P407 results in a decrease in the sol-gel phase transition temperature. To increase the viscosity of the gel system and adjust the gelling temperature to a physiological value, P188 is added to the formulation. Screening of the gelling temperature of P188 at a fixed concentration of 22% of P407 found that the gelling temperature increased with increasing concentration of P188. The gelling temperature of P407 at a concentration of 20-26% was less than 30 °C, and the addition of P188 at a concentration of 0-8% increased the gel temperature. The addition of 6% and 8% of P188 had a comparable effect on increasing the gelling temperature. Therefore, when optimizing the central composite design prescription, the concentration range of P407 was selected as 20%-26%, and the concentration range of P188 was selected as 0%-6%.

[0072] Table 1. Effects of P407 and P188 at different concentrations on the gel temperature.

[0073]

[0074] Note: "-" represents that the gelling temperature is higher than 60 °C or gelation cannot occur.

[0075] 3. Optimization and verification of the prescription of thermosensitive gel

[0076] The central composite design-response surface method was used to optimize the prescription. Central composite design was carried out according to the selected optimal concentration ranges of P407 and P188 as independent variables (X1 and X2) at five experimental levels: -1.414, -1, 0, 1, and 1.414. The response variables T1 (°C) and T2 (°C) selected in the study were the gel temperatures T1 and T2 before and after dilution with artificial tears.

[0077] Multivariate linear regression analysis was performed using Design-Expert 13 software to generate three-dimensional response surface plots and two-dimensional contour plots to visualize the changes in T1 and T2 before and after dilution with artificial tears with different ratios of P407 and P188. Gels were prepared according to the prescription optimization results, and the gelling temperatures T1 and T2 before and after dilution with artificial tears were measured respectively. The measurement steps were the same as above, and the deviation was calculated based on the predicted and measured values to determine the optimal matrix ratio.

[0078] The results are shown in Table 2. Multivariate linear regression analysis was performed on the central composite design data using Expert Designer software. Taking the concentration of P407 (X1) and the concentration of P188 (X2) as independent variables, and the gelling temperatures T1 and T2 before and after dilution with artificial tears as dependent variables, the following binomial equations were obtained respectively:

[0079] T1 = 28.70 - 3.81X1 + 3.72X2 - 0.9250X1X2 + 0.8063X1 2 + 0.6563X2 2(r = 0.9950, P < 0.001)

[0080] T2 = 37.20 - 4.25X1 + 4.69X2 + 0.7000X1X2 + 0.6000X1 2 -1.15X2 2 (r = 0.9967, P < 0.001)

[0081] According to the r value of the above equation, the fitting result is good. Based on this, the two-dimensional contour map and three-dimensional response surface map are drawn using Expert Designer software, and the results are shown in Figures 1-4 . When the mass fraction of P188 is constant, as the mass fraction of P407 increases, the gelation temperature gradually decreases; when the mass fraction of P407 is constant, as the mass fraction of P188 increases, the gelation temperature gradually increases.

[0082] Overlay the two-dimensional contours of T1 and T2 in Figure 1 and Figure 2 . The grid part in the figure obtained is the optimized part (27°C ≥ T1 ≥ 25°C, 35°C ≥ T2 ≥ 33°C).

[0083] At Figure 5 Select a representative prescription at the upper, middle, and lower positions in the optimized region, that is, Prescription 1 is P407:P188 (23.5%:2%); Prescription 2 is P407:P188 (24.0%:2.5%); Prescription 3 is P407:P188 (24.5%:2.5%).

[0084] Prepare gels according to the above matrix ratios, measure the gelation temperatures T1 and T2 before and after dilution with artificial tears respectively, and calculate the deviation based on the predicted values and actual values to determine the optimal matrix ratio.

[0085] As can be seen from Table 3, the deviations of the three groups of prescriptions are all less than 3%, indicating that the established non-linear regression equation has high accuracy and good predictability. Among them, the deviation of Prescription 1 is the smallest, and the dosages of P407 and P188 are the least. At the same time, it can also meet the usage requirements of thermosensitive gels in ophthalmic preparations. Therefore, the optimal ratio of P407 to P188 in the pranoprofen thermosensitive gel prescription is determined to be 23.5%:2%.

[0086] Table 2. Results of central composite design.

[0087]

[0088] Note: T1 is the gel temperature without adding artificial tears; T2 is the gel temperature with added artificial tears

[0089] 3. Central composite design - Response surface method to verify and optimize the prescription

[0090]

[0091] 4 Characterization of Optimized Pranoprofen Thermosensitive Gel

[0092] Prepare pranoprofen thermosensitive gel according to the optimal matrix ratio of P407 and P188, observe the physical appearance and clarity of the gel; determine the gelation temperatures T1 and T2 before and after dilution with artificial tears; measure the viscosity of the gel using a rotational viscometer under non-physiological conditions (25 °C, not mixed with artificial tears) and physiological conditions (34 °C, mixed with artificial tears); use pH test paper to determine the pH value of the pranoprofen thermosensitive gel formulation.

[0093] The results showed that the pranoprofen thermosensitive gel prepared according to the optimal formulation ratio (23.5%: 2%) was a clear and transparent liquid before dilution with artificial tears (25 °C) and a clear and transparent semi-solid after dilution with artificial tears (34 °C); the gel pH value was 7.2 - 7.4, and the gelation temperatures T1 and T2 were 26.2 °C and 33.6 °C respectively, meeting the requirements for ophthalmic drug use.

[0094] The viscosity results are as Figure 6 shown. The viscosities of the gel before and after dilution with artificial tears at 25 °C were (217 ± 6.43) mPa·s and (203 ± 6.08) mPa·s respectively, and the viscosities of the gel before and after dilution with artificial tears at 34 °C were (39721 ± 3180.12) mPa·s and (5678 ± 136.18) mPa·s respectively. The phase transition characteristics of the thermosensitive gel enable the formation of a stable drug delivery system in the eye. The gel exhibits a low viscosity at 25 °C, which helps it to spread and distribute easily when dropped into the eye; while at 34 °C, the viscosity of the gel increases, which helps to form a well-adherent gel layer in the eye, thereby prolonging the residence time of the drug and improving the bioavailability of the drug

[0095] 5. In vitro Release Study of Pranoprofen Thermosensitive Gel

[0096] The in vitro release study of pranoprofen eye drops and thermosensitive gel was carried out by the membrane-free dissolution method. Taking the cumulative erosion rate and cumulative release rate as the ordinate and time as the abscissa to plot a graph, the cumulative erosion curve and cumulative drug release curve of the thermosensitive gel group over time were obtained. The results are as Figure 7 shown. The cumulative erosion rate of pranoprofen thermosensitive gel within 210 min was greater than 90%, and the cumulative release rate had a good linear relationship with time (y = 0.5090x - 9.593, r = 0.9976), showing a zero-order pharmacokinetic process; as Figure 8, the drug cumulative release rate of pranoprofen thermosensitive gel showed a good linear relationship with time (y = 0.4677x - 3.813, r = 0.9963), which was a zero-order pharmacokinetic process.

[0097] In addition, there was a good linear relationship between the in vitro cumulative erosion rate and the cumulative release rate of pranoprofen thermosensitive gel (y = 0.9193x + 4982, r = 0.9995), indicating that the release of pranoprofen in the ophthalmic thermosensitive gel depended on the erosion of the gel.

[0098] The in vitro release studies of pranoprofen eye drops and thermosensitive gel were carried out using dialysis bags, and the results were as Figure 9 shown. The relationship between the drug cumulative release rate of pranoprofen eye drops and time conforms to the first-order kinetic model (y = 96.0158(1 - e 0.0694 ), R 2 = 0.9992); the relationship between the drug cumulative release rate of pranoprofen thermosensitive gel and time conforms to the first-order kinetic model (y = 90.9942(1 - e 0.0115 ), R 2 = 0.9994).

[0099] In addition, the drug release rates of both groups reached 90%. The drug release of pranoprofen eye drops was basically complete at 60 min, and that of pranoprofen thermosensitive gel was basically complete at 300 min. Compared with pranoprofen eye drops, pranoprofen thermosensitive gel had a good sustained-release effect, prolonged the residence time of the drug in the eye, and effectively avoided drug loss, which was of great significance for reducing the administration frequency and dosage frequency of drug preparations.

[0100] 5. Pharmacodynamic evaluation of pranoprofen thermosensitive gel on dry eye model rats

[0101] A dry eye model of rats was induced by 0.5% benzalkonium chloride to evaluate the pharmacodynamics of pranoprofen thermosensitive in-situ gel on dry eye model rats (corneal neovascularization, corneal turbidity, conjunctival irritation, tear secretion, conjunctival inflammatory factors).

[0102] 5.1 Establishment and grouping of dry eye model

[0103] After 30 SD rats were appropriately fed for one week and no abnormalities were found through ocular surface examination, they were randomly divided into a normal group, a model group, a thermosensitive gel group (without pranoprofen), a pranoprofen eye drop group, and a pranoprofen thermosensitive gel group, with 6 rats in each group. Except for the normal group, all were given 0.5% benzalkonium chloride - normal saline solution for eye drops, 2 times a day (9:00 and 21:00), 50 μL / eye, to prepare a dry eye model. After 5 days of modeling, each group was given the corresponding drug, 20 μL / eye, 4 times a day, with an interval of 3 h each time (10:00, 13:00, 16:00, 19:00), for 9 consecutive days. Partial pharmacodynamic experiments were carried out on the 7th and 14th days of modeling respectively. On the 14th day, the rats were sacrificed, the conjunctival tissues were taken, blotted dry with filter paper, placed in a 1.5 mL Eppendorf tube and weighed and recorded; they were stored in a -80°C refrigerator for ELISA experiments.

[0104] 5.2 Measurement of corneal neovascularization

[0105] The corneal neovascularization was examined under general anesthesia and scored using a slit lamp. Briefly, the cornea was divided into four quadrants and scored separately. The scoring for the area of neovascularization was as follows: 0, no blood vessels growing into the clear cornea; 1, blood vessels growing in one - quarter (or less) of the cornea but not zero; 2, blood vessels growing from one - quarter to one - half of the cornea; 3, blood vessels growing from one - half to three - quarters of the cornea; 4, blood vessels growing in three - quarters of the entire corneal surface. The final score of corneal neovascularization was calculated by adding the scores of the four quadrants (total score, 16 points).

[0106] 5.3 Measurement of corneal opacity

[0107] According to the previously described method, the corneal opacity was analyzed using a slit lamp and scored as follows: 1, scattered or diffuse areas where iris details are visible; 2, semi - transparent areas that are easily distinguishable and the iris details are slightly blurred; 3, milky white areas where iris details are not visible and the pupil size can hardly be seen; 4, opaque, iris not visible. The scoring for the opaque area was as follows: 1, one - quarter (or less) of the area is opaque but not zero; 2, more than one - quarter of the area is opaque but less than one - half; 3, more than half of the area is opaque but less than three - quarters; 4, more than three - quarters up to the entire area is opaque.

[0108] 5.4 Conjunctival irritation score

[0109] Conjunctival congestion, edema, and ocular secretions were observed under a slit lamp. The conjunctival congestion was scored as follows: 0 points: normal blood vessels; 1 point: blood vessels congested and bright red; 2 points: blood vessels congested and dark red, with blood vessels difficult to distinguish; 3 points: diffuse congestion and purple-red. The conjunctival edema was scored as follows: 0 points: no edema; 1 point: mild edema; 2 points: obvious edema with partial eyelid ectropion; 3 points: edema to almost half-closure of the eyelid; and 4 points: edema until more than half of the eyelid is closed. The conjunctival secretion was scored as follows: 0 points: no secretion; 1 point: a small amount of secretion; 2 points: the secretion moistens or makes the eyelids and eyelashes sticky; 3 points: the secretion moistens or makes the entire ocular area sticky.

[0110] The results showed that a slit lamp was used to evaluate corneal turbidity, corneal neovascularization, and conjunctival irritation in the benzalkonium chloride-induced dry eye model. The results were as Figures 10-13 , after 14 days of eye drop administration, the corneas of the normal group were clear and transparent, with few corneal neovascularizations and conjunctivas; compared with the normal group, the benzalkonium chloride-induced model group showed severe corneal turbidity and a large number of neovascularizations, and severe damage to the conjunctiva, including conjunctival congestion, edema, and secretions.

[0111] Compared with the model group, the scores of neovascularization and corneal turbidity in the thermosensitive gel group were reduced to varying degrees, but there was no significant difference; after treatment with pranoprofen eye drops and pranoprofen thermosensitive gel, especially local treatment with pranoprofen thermosensitive gel, significantly improved the dry eye symptoms induced by benzalkonium chloride; among them, the effect after treatment with pranoprofen thermosensitive gel was more significant than that of pranoprofen eye drops (P < 0.05), and the results showed that pranoprofen thermosensitive gel had a significant effect in improving the dry eye symptoms induced by benzalkonium chloride.

[0112] 5.5 Corneal fluorescein staining

[0113] A 0.1% sodium fluorescein solution was introduced into the lower conjunctival sac using a pipette. Blink three to five times, and the test was performed under cobalt blue light using a surgical microscope, and photos were taken and scored. Each cornea was divided into four quadrants and scored separately. A 4-point system was used to calculate the intensity of corneal fluorescein staining: 0, absent; 1, superficial punctate micro-punctate staining, with <30 spots; 2, punctate staining with >30 spots, but no diffuse staining; 3, severe diffuse staining, but no positive plaque / plaque; 4, positive fluorescein plaque / patch. The scores of the four fields were added up to obtain the final score, ranging from 0 to 16.

[0114] 5.6 Measurement of tear secretion volume

[0115] The tear secretion volume was measured using tear secretion filter paper on the 1st, 4th, 7th, 10th, and 14th days respectively. The modified tear filter paper (1 mm × 30 mm) was placed in the conjunctival sac of the outer 1 / 3 of the lower eyelid of the rats, and the wetting length was measured after timing for 2 minutes. The data obtained were the average values of both eyes to evaluate the tear secretion situation.

[0116] The distribution and degree of corneal injury were observed by corneal fluorescein sodium staining under a cobalt blue lamp. The results were as Figure 10 and Figure 14 , compared with the normal group, the stained area was significantly increased in the model rats induced by benzalkonium chloride, indicating corneal epithelial injury; compared with the model group, the stained area was significantly reduced after treatment with pranoprofen eye drops and pranoprofen thermosensitive gel, and the result of the pranoprofen thermosensitive gel group was the best. The results showed that pranoprofen thermosensitive gel could improve corneal epithelial injury.

[0117] By observing the tear secretion volume of rats after administration of benzalkonium chloride, the results were as Figure 15 , with the prolongation of the modeling time, the tear secretion volume of rats in each group gradually decreased; compared with the normal group, on the 10th and 14th days of modeling, the tear secretion volume of rats in the model group was significantly reduced; after treatment with pranoprofen eye drops and pranoprofen thermosensitive gel, the tear secretion of dry eye rats induced by benzalkonium chloride was significantly improved, and pranoprofen thermosensitive gel performed better in increasing the tear secretion volume.

[0118] 5.7 ELISA analysis

[0119] The rat ocular conjunctiva was taken. After tissue homogenization, the supernatant was taken, and the levels of inflammatory factors TNF-α and IL-1β in the supernatant were quantified by ELISA kit according to the enzyme-linked immunosorbent assay.

[0120] The results were as Figure 16 and Figure 17 , compared with the normal group, the expressions of TNF-α and IL-1β in the conjunctival tissue of the model group were significantly increased; compared with the model group, after treatment with pranoprofen eye drops and pranoprofen thermosensitive gel, pranoprofen eye drops only significantly reduced the expression of TNF-α, while pranoprofen thermosensitive gel more significantly reduced the expressions of TNF-α and IL-1β than pranoprofen eye drops. The results showed that pranoprofen thermosensitive gel could significantly reduce the levels of conjunctival inflammatory factors and alleviate the inflammatory response.

[0121] 6. Study on the tissue distribution and aqueous humor pharmacokinetics of pranoprofen thermosensitive gel

[0122] 6.1 LC and MS conditions and parameters

[0123] Chromatographic column: Waters BEH C18 column (2.1 mm × 150 mm, 1.7 μm), guard column: Waters Van Guard BEH C18 (2.1 mm × 5 mm, 1.7 μm), injection volume: 1 μL. Mobile phase: 0.2% formic acid in acetonitrile (B) - 0.2% formic acid in water (A), elution program: 0 - 1.5 min, 92% - 70% A; 1.5 - 2.0 min, 70% - 60% A; 2.0 - 2.5 min, 60% - 10% A; 2.5 - 3.0 min, 10% A; 3.0 - 4.0 min, 10% - 92% A; flow rate: 0.35 mL / min, column temperature: 40 °C.

[0124] Electrospray ionization source (ESI) was used. Capillary ionization voltage: 3 kV, ion source temperature: 120 °C, spray gas and counter gas; N2 desolvation gas flow rate: 650 L / hr, desolvation gas temperature: 350 °C. The scanning mode was multiple reaction monitoring (MRM). The ion pairs for quantitative analysis were puerarin m / z 417 (internal standard), cone voltage: 30 V; pranoprofen m / z 255.8, cone voltage: 30 V. Both were in positive ion scanning mode. The mass spectrometry data acquisition and processing software was MassLynx 4.1 workstation.

[0125] 6.2 Distribution study of pranoprofen thermosensitive gel in rat eye tissues

[0126] Forty-eight healthy SD rats were randomly divided into a pranoprofen eye drop group and a pranoprofen thermosensitive gel group, with 24 rats in each group. Four eyes were used at the same time point. The left eye was administered once with a dose of 20 μL. The rats were sacrificed at 0.167, 0.5, 1, 3, 4, and 6 h. 10 μL of aqueous humor was taken into a 1.5 mL centrifuge tube; the cornea and vitreous body were taken out, the surface blood of the tissue was washed with normal saline, blotted dry with filter paper, placed in a 1.5 mL EPP tube and weighed and recorded; stored at -80 °C in the refrigerator for later use.

[0127] The results showed that after local ocular administration, the time-course changes of pranoprofen in various tissues of rats at different times are shown in Figures 18-20 . The drug concentrations in the cornea and aqueous humor of the eye drops gradually decreased with time. The peak contents in the cornea and aqueous humor reached (2.55 ± 0.44) ng / mL and (439.51 ± 94.45) ng / mL at 0.167 h respectively, and were completely eliminated after 6 h.

[0128] The drug concentration of thermosensitive gel in the cornea gradually accumulates and increases with time, reaching the peak content of (1.85±0.75) ng / mL at 6 h; and the maximum absorption peaks appear in the aqueous humor at 0.167 h and 4 h, with the concentrations of (527.18±100.61) ng / mL and (310.25±75.06) ng / mL respectively, showing a double absorption peak.

[0129] The drug concentrations of the two dosage forms in the vitreous body are at a relatively low level, indicating that pranoprofen is not easily absorbed into the posterior eye tissues, effectively improving the local drug concentration; from the perspective of drug distribution content, both dosage forms can be rapidly distributed in the eye tissues, and the content from high to low is aqueous humor > cornea > vitreous body.

[0130] 6.3 Pharmacokinetic study of pranoprofen thermosensitive gel in the aqueous humor of rats and rabbits

[0131] Eight healthy rabbits were randomly divided into the pranoprofen eye drop group and the pranoprofen thermosensitive gel group, with 4 rabbits in each group and 8 eyes at the same time point. The left and right eyes were administered once respectively, with a dosage of 50 μL. At the time points of 0.017, 0.083, 0.167, 0.5, 1, 2, 3, 4, 6, and 8 h, 100 μL of aqueous humor was taken into a 1.5 mL centrifuge tube, centrifuged at 14000 r / min for 10 min, and the supernatant was taken and stored at -80 °C in the refrigerator for later use.

[0132] In vivo pharmacokinetic studies were carried out to study the release behavior of the drug in the local eye tissues. The concentration of pranoprofen in the aqueous humor was detected, and a function curve was plotted with the average blood drug concentration C and time t. The results Figure 21 showed that compared with the eye drops, the pranoprofen thermosensitive gel maintained a higher drug concentration during the study period, and a double-peak phenomenon appeared in the aqueous humor of rabbits after eye drop administration, and the results of the aqueous humor of rats were similar.

[0133] In addition, Table 4 provides the pharmacokinetic parameters of the two groups of pranoprofen. Compared with the eye drop group, the peak time T max (0.02±0.00 h) of the pranoprofen thermosensitive gel was shorter than that of the eye drops T max (0.06±0.03 h), indicating that the pranoprofen thermosensitive gel can be rapidly distributed in the eye tissues.

[0134] The peak concentration C max of the pranoprofen thermosensitive gel (670.43±54.47 ng / mL) was lower than that of the eye drops C max (1304.80±893.73 ng / mL), indicating that the pranoprofen thermosensitive gel has a sustained-release property in the eye tissues, can maintain the drug concentration for a long time, and reduce the dosing frequency.

[0135] The area under the curve AUC of the thermosensitive gel group 0-t(455.63 ± 212.94 h·ng / mL) was significantly higher than the AUC of the eye drops 0-t (245.34 ± 102.22 h·ng / mL), which was 1.85 times that of the eye drops (P < 0.05), indicating that pranoprofen thermosensitive gel improved the bioavailability of pranoprofen in aqueous humor after topical administration and enhanced the compliance of clinical medication.

[0136] 4. Pharmacokinetic parameters of pranoprofen in the two groups

[0137]

[0138] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A pranoprofen thermosensitive gel for treating dry eyes, characterized in that: The thermosensitive gel is prepared by the following method: Weigh poloxamer 407 and poloxamer 188 in the prescribed amount of water, store in a 4°C refrigerator for 24 hours to swell, and obtain a blank gel solution; accurately weigh pranoprofen, place in a volumetric flask, add Tween-80, boric acid, borax, an appropriate amount of 0.2% tris(hydroxymethyl)aminomethane solution and blank gel matrix, adjust to volume, and sonicate until completely dissolved; The optimal ratio of P407 to P188 is 23.5%:2%.

2. The pranoprofen thermosensitive gel according to claim 1, characterized in that The optimal ratio is determined by optimizing the prescription through central composite design-response surface method. The optimization process includes taking the optimal concentration range of poloxamer 407 and poloxamer 188 screened as independent variables, performing central composite design at multiple experimental levels, taking the gel temperature before and after artificial tears dilution as the response variable, and performing multiple linear regression analysis using Design-Expert 13 software to generate a three-dimensional response surface map and a two-dimensional contour map, and determining the optimal matrix ratio based on the deviation between the predicted value and the measured value.

3. The pranoprofen thermosensitive gel according to claim 1, characterized in that The in vitro release characteristics of the thermosensitive gel are as follows: when the membraneless dissolution method is used for in vitro release research, the cumulative dissolution rate within 210 minutes is greater than 90%, and the cumulative release rate has a good linear relationship with time, which is a zero-order pharmacokinetic process.

4. A method for preparing pranoprofen thermosensitive gel for treating dry eye, characterized in that: The following steps are involved: Prepare blank gel solution: weigh poloxamer 407 and poloxamer 188 in the prescribed amount of water and store in a 4°C refrigerator for 24 hours to swell; Preparation of pranoprofen thermosensitive gel: accurately weigh pranoprofen, place in a volumetric flask, add Tween-80, boric acid, borax, appropriate amount of 0.2% tris(hydroxymethyl)aminomethane solution and blank gel matrix, dilute to volume, and sonicate until completely dissolved; The prescription was optimized by central composite design-response surface methodology: the optimal concentration ranges of poloxamer 407 and poloxamer 188 were selected as independent variables, and central composite design was performed at multiple experimental levels. The gel temperature before and after artificial tears dilution was used as the response variable. Multiple linear regression analysis was performed using Design-Expert 13 software to generate three-dimensional response surface plots and two-dimensional contour plots. The optimal ratio of poloxamer 407 to poloxamer 188 was determined to be 23.5%:2% based on the deviation between the predicted and measured values.

5. The preparation method of pranoprofen thermosensitive gel according to claim 4, characterized in that, In the process of optimizing the prescription, when screening the best concentration of poloxamer 407, poloxamer 407 gel solutions of different concentrations were placed in a syringe bottle, placed in a 4°C ice water bath, inserted with a precision thermometer, stirred and slowly heated, and the sol-gel phase transition temperature at which the gel solution stopped flowing was recorded to screen out the best concentration range; then the gelation temperature after mixing with poloxamer 188 of different concentrations was measured, and it was determined that the concentration range of poloxamer 407 was 20%-26%, and the concentration range of poloxamer 188 was 0%-6% when the central composite design prescription was optimized.

6. The method for preparing the pranoprofen thermosensitive gel according to claim 4, wherein: The concentrations of the poloxamer 407 gel solutions of different concentrations include 16%, 18%, 20%, 22%, 24%, and 26%; the concentrations of the poloxamer 188 of different concentrations include 0%, 2%, 4%, 6%, and 8%.

7. A method for evaluating the efficacy of pranoprofen thermosensitive gel in treating dry eye, characterized in that: include: The dry eye model of SD rats was established by inducing 0.5% benzalkonium chloride; The experimental rats were divided into a normal group, a model group, a thermosensitive gel group without pranoprofen, a pranoprofen eye drops group, and a pranoprofen thermosensitive gel group; 5 days after modeling, each group was given medication at a frequency of 20 μL / eye, 4 times / day with an interval of 3 hours each time, for 9 days; Corneal neovascularization, corneal opacity, conjunctival irritation, tear secretion, conjunctival inflammatory factors and other indicators were tested and evaluated on the 7th and 14th days of modeling. The results showed that the efficacy of pranoprofen thermosensitive gel in the treatment of dry eye was better than that of pranoprofen eye drops.

8. The method for evaluating the efficacy of pranoprofen thermosensitive gel in treating dry eye according to claim 7, characterized in that: The corneal neovascularization is evaluated by scoring the four quadrants of the cornea separately with a slit lamp and then adding up the total score; the corneal opacity is evaluated according to a specific slit lamp observation and scoring standard; the conjunctival irritation is evaluated by scoring the conjunctival congestion, edema and eye secretions separately and then comprehensively evaluating; the tear secretion is evaluated by placing a modified tear filter paper strip in the outer 1 / 3 conjunctival sac of the rat lower eyelid and measuring the wet length for 2 minutes; the conjunctival inflammatory factors are evaluated by taking the rat conjunctival tissue homogenate and detecting the levels of TNF-α and IL-1β with an ELISA kit.

9. A method for studying the distribution of pranoprofen thermosensitive gel in ocular tissues and the pharmacokinetics of aqueous humor, characterized in that: In the tissue distribution study, healthy SD rats were divided into pranoprofen eye drops group and pranoprofen thermosensitive gel group. A single dose of 20 μL was given to the left eye. The rats were killed at time points including 0.167, 0.5, 1, 3, 4, and 6 h, and the aqueous humor, cornea, and vitreous were taken to detect the concentrations of pranoprofen. In the aqueous humor pharmacokinetics study, healthy rabbits were divided into pranoprofen eye drops group and pranoprofen thermosensitive gel group. A single dose of 5 μL was given to the left and right eyes, respectively. Aqueous humor was taken at time points including 0.017, 0.083, 0.167, 0.5, 1, 2, 3, 4, 6, and 8 h to detect the concentration of pranoprofen and draw pharmacokinetic curves. The results showed that pranoprofen thermosensitive gel had higher target organ distribution characteristics, and the aqueous humor AUC0-t was 1.85 times that of eye drops.

10. The method for studying the distribution of pranoprofen thermosensitive gel in ocular tissues and aqueous humor pharmacokinetics according to claim 9, characterized in that: The concentration of pranoprofen was detected by liquid chromatography-mass spectrometry, the chromatographic column was a Waters BEHC18 (2.1 mm×150 mm, 1.7 μm) column, the guard column was a Waters VanGuard BEH C18 (2.1 mm×5 mm, 1.7 μm), the injection volume was 1 μL, the mobile phase was 0.2% formic acid acetonitrile (B)-0.2% formic acid water (A), and the elution program and other mass spectrometry parameters were performed as set.

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