Preparation and application of intelligent response type lycium barbarum L. glycopeptide nasal temperature-sensitive in-situ gel

Abstract

The application discloses preparation and application of a smart response type medlar glycopeptide nasal temperature-sensitive in-situ gel, and belongs to the technical field of pharmaceutical preparations. The gel comprises the following components: medlar glycopeptide, poloxamer 407, poloxamer 188, sulfobutyl-beta-cyclodextrin, nipagin ethyl ester, and the rest is water. The preparation is in a liquid state at room temperature, and is rapidly converted into a semi-solid gel state at a physiological temperature of 29-34 DEG C in a nasal cavity. The gel is prepared by a cold solution method, has suitable gelation temperature and gelation time, has a cumulative release degree of 78-85% in vitro for 48 hours, has good sustained-release characteristics, and has no irritation to a nasal mucosa. Pharmacodynamic research shows that the gel can significantly improve nasal allergic symptoms of allergic rhinitis mice after nasal administration, repair nasal mucosa damage, regulate Th1 / Th2 immune balance, and has a treatment effect superior to that of a medlar glycopeptide solution at the same dose.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, and particularly relates to the preparation and application of a smart responsive wolfberry glycopeptide nasal thermosensitive in situ gel. Background Technology

[0002] Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa mediated by immunoglobulin E (IgE). Its pathogenesis involves the participation of various immune-active cells and cytokines. AR has become a global public health problem, affecting more than 500 million people, with a global prevalence of up to 40%. Clinical manifestations include red eyes, itchy nose, nasal congestion, runny nose, sneezing, and decreased sense of smell. Severe cases can induce asthma, anxiety, depression, cognitive impairment, and sleep disorders, significantly reducing patients' quality of life. Influenced by industrialization and environmental changes, the incidence of AR is increasing year by year, and currently, there is no cure.

[0003] Allergens causing allergic reactions (AR) are numerous, mainly including pollen, dust mites, animal dander, and mold. Among them, Artemisia pollen is the most prevalent allergen in northern China and the most common inhaled allergen for AR patients in Ningxia. Modern medicine believes that AR results from the interaction between allergic and inflammatory responses induced by allergens. Its core pathological mechanism lies in the disorder or deficiency of the body's immune function. When the nasal mucosa's defense function is impaired, allergens can easily invade and trigger the disease process.

[0004] Goji berries (Lycium barbarum) are a traditional Chinese medicine used in both food and medicine. Its active ingredient, Lycium barbarum polysaccharides (LBP), possesses pharmacological activities including antioxidant, anti-inflammatory, immunomodulatory, neuroprotective, and hypoglycemic / lipid-lowering effects. Previous studies have reported that LBP can exert immunomodulatory effects and alleviate symptoms in ovalbumin-induced atopic dermatitis (AR) animal models by correcting helper T cell (Th) imbalance. Lycium barbarum glycopeptide (LbGP) is an active component obtained through further isolation and purification of LBP, removing inactive components such as inorganic salts and monosaccharides. Its immunomodulatory activity is 10–20 times higher than that of LBP, exhibiting significant immune-enhancing effects and demonstrating benign regulatory effects on both cellular and humoral immunity. Existing literature also suggests that LbGP has potential for symptom relief and immunomodulation in pollen-induced AR models.

[0005] In terms of administration route, local administration has advantages over oral administration, as it allows the drug to accumulate in the nasal lesions, improving bioavailability and reducing systemic toxicity. Given that AR involves not only local immune inflammation of the nasal mucosa but also imbalances in the systemic immune system, the ideal treatment strategy should be a local nasal administration regimen that combines direct action on the local lesions with systemic immune modulation.

[0006] Currently, commonly used nasal preparations include antihistamines, corticosteroids, anticholinergics, and decongestants. However, nasal corticosteroids are limited to local anti-inflammatory and anti-allergic effects; antihistamines and anticholinergics often require combination therapy; decongestants only provide temporary relief from nasal congestion, and long-term use can easily induce drug-induced rhinitis. Existing nasal drug delivery formulations such as nasal drops, sprays, films, and ordinary gels also have significant drawbacks: nasal drops and sprays are easily affected by the physiological clearance of nasal cilia, resulting in short local retention time and low bioavailability; although films have sustained-release properties, they cause significant foreign body sensation and poor patient compliance; and although ordinary gels can prolong contact time, the dosage is difficult to control and inconvenient to use.

[0007] In-situ gels, also known as readily available gels, are intelligent responsive drug delivery systems. After administration in solution form, they undergo a reversible phase transition at the administration site under physiological stimuli such as temperature, pH, or ionic strength, forming a semi-solid gel. These formulations combine ease of use (liquid at room temperature for precise drug delivery) with good tissue compatibility, bioadhesion, and sustained-release properties. Temperature-sensitive in-situ gels, in particular, are suitable for nasal, ocular, and intracavitary mucosal administration because the physiological temperature of the nasal cavity (29–34°C) triggers their liquid-gel phase transition. This significantly prolongs drug retention time, controls drug release, and improves bioavailability. Given the good water solubility of LbGP, it is suitable for preparing aqueous temperature-sensitive gel systems.

[0008] However, there are currently no reports on the preparation of LbGP as a thermosensitive in-situ nasal gel. Existing thermosensitive gel formulations (such as the poloxamer system) often face technical bottlenecks when applied to specific active ingredients, including mismatch between gelation temperature and the nasal physiological environment, uncontrollable release behavior, insufficient mucosal permeability, and poor long-term stability. Therefore, there is an urgent need to develop a smart, responsive, thermosensitive in-situ LbGP nasal gel formulation with a suitable phase transition temperature, good sustained-release properties, and mucosal safety to solve the problems of short duration of action, low bioavailability, and inconvenient administration of existing nasal formulations, providing a new formulation direction for the clinical treatment of AR. Summary of the Invention

[0009] Therefore, this invention proposes the preparation and application of a smart responsive nasal thermosensitive in-situ gel containing wolfberry glycopeptide, aiming to solve the technical problems of short local action time, low bioavailability, and inconvenient administration of existing nasal preparations, and to provide a novel nasal drug delivery system for the treatment of allergic rhinitis.

[0010] To achieve the above objectives, the present invention provides the following technical solution: A smart responsive nasal thermosensitive in-situ gel containing goji berry glycopeptide (LbGP) 5% (w / v, g / mL), poloxamer 407 (P407) 19% (w / v), poloxamer 188 (P188) 2% (w / v), sulfobutyl-β-cyclodextrin (SBE-β-CD) 7% (w / v), ethylparaben 0.02% (w / v), and the balance being water.

[0011] Furthermore, the intelligent responsive wolfberry glycopeptide nasal thermosensitive in-situ gel is liquid at room temperature and transforms into a semi-solid gel state at the physiological temperature of the nasal cavity of 29-34°C. The contents of rhamnose, glucose and arabinose in the wolfberry glycopeptide were determined by pre-column derivatization HPLC using PMP.

[0012] This invention also provides a method for preparing a smart responsive goji berry glycopeptide nasal thermosensitive in-situ gel, comprising the following steps: (1) Dissolve the wolfberry glycopeptide in water, add sulfobutyl-β-cyclodextrin and ethylparaben, and sonicate until completely dissolved; (2) Under ice-water bath conditions, slowly add poloxamer 407 and poloxamer 188 in sequence, and continue to add water and stir evenly; (3) Place the solution obtained in step (2) at 4°C for 24 hours to allow it to fully swell, and you will get the intelligent responsive wolfberry glycopeptide nasal thermosensitive in situ gel.

[0013] Furthermore, the wolfberry glycopeptide is hydrolyzed and pre-column derivatized using PMP before step (1) for content determination. The hydrolysis conditions are: 1 mol·L⁻¹ -1 Trifluoroacetic acid was hydrolyzed at 100℃ for 6 h; the derivatization conditions were: 0.3 mol·L⁻¹ -1 NaOH and 0.7 mol·L -1 PMP-methanol solution, reacted at 70℃ for 1 h.

[0014] Furthermore, the sulfobutyl-β-cyclodextrin is used as an absorption enhancer at an amount of 7% (w / v).

[0015] Furthermore, the mass ratio of poloxamer 407 to poloxamer 188 is 19:2.

[0016] The present invention also provides the application of a smart responsive wolfberry glycopeptide nasal thermosensitive in situ gel in the preparation of a drug for treating allergic rhinitis.

[0017] Furthermore, the allergic rhinitis mentioned is allergic rhinitis induced by artemisia annua pollen.

[0018] Furthermore, the intelligent responsive nasal thermosensitive in-situ gel containing goji berry glycopeptides is administered via the nasal cavity, transforming into a gel state at nasal temperature, thus prolonging drug retention time and achieving sustained and continuous release. At the same dosage, the intelligent responsive nasal thermosensitive in-situ gel containing goji berry glycopeptides exhibits superior therapeutic effects compared to goji berry glycopeptide solution, demonstrating more significant improvement in nasal allergy symptoms, more effective restoration of immune organ indices, better repair of nasal mucosal pathological damage, and stronger regulation of Th1 / Th2 cell balance.

[0019] The administration method is nasal instillation, and the dosage is 150 mg / kg based on the amount of wolfberry glycopeptide.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects: (1) The present invention uses P407 and P188 as thermosensitive gel matrix. By optimizing the ratio, a suitable gelation temperature is obtained, so that the preparation remains liquid at room temperature for accurate drug administration. At nasal cavity temperature, it is rapidly transformed into a semi-solid gel, prolonging the drug retention time and improving bioavailability.

[0021] (2) The present invention uses 7% SBE-β-CD as an absorption enhancer, which significantly improves the permeation rate of wolfberry glycopeptide through the nasal mucosa and promotes drug absorption.

[0022] (3) The present invention uses a cold dissolution method to avoid the destruction of the activity of wolfberry glycopeptides by high temperature and ensure the stability of the preparation quality.

[0023] (4) The cumulative release rate of the formulation of the present invention reaches 78-85% after 48 hours, which has good sustained-release characteristics and can reduce the frequency of administration.

[0024] (5) The formulation of the present invention has no obvious irritation to the nasal mucosa, has good safety, and can be used for nasal administration.

[0025] (6) The therapeutic effect of the preparation of the present invention is significantly better than that of the wolfberry glycopeptide solution at the same dosage, providing a new and effective drug delivery system for the treatment of allergic rhinitis. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1The images show the morphology of the smart-responsive LbGP thermosensitive in-situ gel prepared in Example 1; where, left: blank thermosensitive in-situ gel; right: smart-responsive LbGP thermosensitive in-situ gel. Figure 2 The in vitro cumulative release curve is shown in Figure A (rhamnose; B (glucose); C (arabinose). Figure 3 The erosion rate curve of the intelligent responsive LbGP thermosensitive in-situ gel (n=3); Figure 4 The cumulative release rate curve of the intelligent responsive LbGP thermosensitive in-situ gel (n=3); Figure 5 The graph shows the dissolution rate versus cumulative release rate (n=3). Figure 6 Images of rat nasal stimulation (A: saline group; B: 1% sodium deoxycholate solution group; C: intelligent responsive LbGP thermosensitive in situ gel group; D: blank thermosensitive in situ gel group). Figure 7 Observation of rat nasal mucosa pathological sections (×400, scale bar=20 μm) (A: physiological saline group; B: 1% sodium deoxycholate solution group; C: smart responsive LbGP thermosensitive in situ gel group; D: blank thermosensitive in situ gel group). Figure 8 The effect of LbGP-TSG on nasal symptom scores in AR mice (x±s, n=6); where, compared with the normal control group: ### P <0.001; compared to the AR model group: * P <0.05, ** P <0.01, *** P <0.001; Figure 9 Effects of LbGP-TSG on immune function in AR mice (x±s, n=6) (A: spleen index; B: thymus index); compared with the normal control group: ### P <0.001; compared to the AR model group: * P <0.05, ** P <0.01, *** P <0.001; Figure 10Effects of LbGP-TSG on histopathological changes in nasal mucosa of allergic rhinitis (AR) mice (×400, scale bar = 20 μm) (A: normal control group; B: AR model group; C: LbGP group; D: LbGP-TSG group; E: positive drug Dex group). Figure 11 The effects of LbGP-TSG on peripheral blood cytokine expression in AR mice (x±s, n=3) (A: effect of LbGP-TSG on IgE; B: effect of LbGP-TSG on IL-4; C: effect of LbGP-TSG on IFN-γ); compared with the normal control group: ## P <0.01, ### P <0.001; compared to the AR model group: * P <0.05, ** P <0.01, *** P <0.001. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0031] As used herein, the terms "comprise", "include", "have", "contain" and the like are open-ended terms, which mean including but not limited to.

[0032] The technical solution of the present invention is further illustrated by the following examples.

[0033] I. Experimental Materials 1.1 Main experimental reagents and instruments Lycium barbarum glycopeptide (LbGP, purity 95±3%), supplied by Ningxia Tianren Lycium barbarum Biotechnology Co., Ltd.; Poloxamer 407 and Poloxamer 188, supplied by BASF, Germany; sulfobutyl-β-cyclodextrin (SBE-β-CD), supplied by Shanghai Macklin Biochemical Technology Co., Ltd.; ethylparaben, supplied by Shanghai Aladdin Biochemical Technology Co., Ltd.; methanol and acetonitrile (chromatographic grade), supplied by Thermo Fisher Scientific (China) Co., Ltd.; high performance liquid chromatograph (Agilent 1260), supplied by Agilent Technologies, USA; electronic analytical balance (EL104), supplied by Mettler-Toledo Instruments (Shanghai) Co., Ltd.; drug transdermal diffusion tester (RYJ-12B), supplied by Shanghai Huanghai Pharmaceutical Testing Instrument Co., Ltd.; high-speed refrigerated centrifuge (5430R), supplied by Eppendorf, Germany; heating magnetic stirrer (S025), supplied by IKA, Germany; pH meter (METTLER TOLEDO), supplied by Mettler-Toledo Instruments (Shanghai) Co., Ltd.; ultrasonic cleaner, supplied by Shenzhen Fuyang Technology Group Co., Ltd.; electric heating constant temperature water bath (DK-S24), supplied by Shanghai Jinghong Experimental Equipment Co., Ltd.

[0034] 1.2 Animal treatment SPF-grade BALB / c female mice, body weight 15-20 g, supplied by Chongqing Tengxin Biotechnology Co., Ltd., production license No.: SCXK (Jing) 2019-0008; SPF-grade SD rats, body weight 200-250 g, half male and half female, provided by Laboratory Animal Center of Ningxia Medical University, production license No.: SCXK (Ning) 2021-0001. Animal feeding environment: temperature (23±2)°C, humidity 40-65%, 12 h light / dark cycle, free access to food and water, adaptive feeding for 3-7 days.

[0035] II. Experimental Procedure (I) Example 1: Preparation of intelligent responsive Lycium barbarum glycopeptide (LbGP) temperature-sensitive in-situ gel The prescription for a 10 mL preparation is as follows: Lycium barbarum glycopeptide 0.5 g, poloxamer 407 1.9 g, poloxamer 188 0.2 g, SBE-β-CD 0.7 g, ethylparaben 0.002 g, and purified water added to 10 mL.

[0036] The preparation method is cold melting, and the specific steps are as follows: (1) Dissolve 0.5 g LbGP in an appropriate amount of purified water, add 0.7 g SBE-β-CD and 0.002 g ethylparaben, and sonicate to dissolve completely; (2) Under ice-water bath conditions, slowly add 1.9 g P407 and 0.2 g P188 in sequence, add purified water to make up to 10 mL, and stir well; (3) Place the solution obtained in step (2) at 4°C for 24 hours to allow it to fully swell, and you will get a clear, bubble-free, and clumpy smart responsive LbGP thermosensitive in situ gel (LbGP-TSG).

[0037] (II) Example 2: Quality evaluation of intelligent responsive LbGP thermosensitive in-situ gel agent 1. Appearance inspection Three batches of smart-responsive LbGP thermosensitive in-situ gel were prepared in parallel according to the preparation process in Example 1. Their color and appearance were visually inspected under bright light conditions, but not direct sunlight. The results are as follows: Figure 1 As shown, the prepared LbGP-TSG is a dark brown, uniform and delicate liquid with good fluidity at room temperature (25 ℃); at the physiological temperature of the nasal cavity (32 ℃), it rapidly transforms into a dark brown semi-solid gel state, which is uniformly dispersed and free of lumps.

[0038] 2. Gelation temperature determination The stir bar method was used for determination: An appropriate amount of LbGP-TSG and a stir bar were placed in a vial, a thermometer with an accuracy of 0.1℃ was inserted, and the vial was placed in a cold water bath. The gel was stirred at 200 rpm at a rate of 1–2℃·min. -1 The temperature was increased slowly, and the temperature at which the stir bar completely stopped rotating was recorded.

[0039] The results are shown in Table 1. The gelation temperatures of the three batches of samples were 31.7 ℃, 31.4 ℃, and 32.1 ℃, respectively, with an average value of 31.73±0.35 ℃ (n=3), which is consistent with the physiological temperature range of the nasal cavity (29~34 ℃).

[0040] Table 1. Results of gelation temperature determination of LbGP thermosensitive in-situ gelling agent (n=3) 3. Gelation time determination Take 5 mL of LbGP-TSG and place it in a 10 mL vial. Preheat the sample to 20 °C and place it in a 32 °C constant temperature water bath. Stir continuously at 32 °C (200 rpm) and record the time when the magnetic particle stops rotating due to gelation.

[0041] As shown in Table 2, the gelation times of the three batches of samples were 29.3 s, 28.7 s, and 28.9 s, respectively, with an average value of 28.97 ± 0.31 s (n=3), which meets the requirements for nasal preparations.

[0042] Table 2. Results of gelation time determination of LbGP thermosensitive in-situ gelling agent (n=3) 4. pH value measurement The pH values ​​of three batches of LbGP-TSG were directly measured using a pH meter.

[0043] The results are shown in Table 3. The pH values ​​of the three batches of LbGP-TSG were 5.25, 5.26 and 5.21, respectively, with an average value of 5.24 ± 0.03 (n=3), which meets the pH requirements for nasal preparations (5.0 to 7.0) and will not cause irritation to the nasal mucosa.

[0044] Table 3. pH value determination results of LbGP thermosensitive in-situ gel (n=3) 5. Determination of expansion coefficient Take 1 mL of LbGP-TSG into a 5 mL centrifuge tube. Record the volume in the 25℃ water bath as V0 and the volume in the 32℃ water bath as V. Calculate the expansion coefficient S (%) = (V-V0) / V0×100%.

[0045] The results showed that the expansion coefficient of LbGP-TSG was 1.6%, indicating that there was no significant expansion in volume after being instilled into the nasal cavity, and the patient compliance was good.

[0046] 6. Mechanical stability test LbGP-TSG was driven at 3500 r·min -1 After centrifugation for 30 min, the preparation was found to be a dark brown, uniformly dispersed solution with no stratification or flocculation, and good mechanical stability.

[0047] 7. Temperature stability test LbGP-TSG was stored at 4 ℃ and 25 ℃ respectively, and samples were taken at 0, 7, 15 and 30 days to observe its appearance and determine its gelation temperature, pH and LbGP content.

[0048] The results are shown in Table 4. After 30 days at 4 ℃, there were no significant changes in appearance, gelation temperature, pH value, and content; however, the changes in each indicator were more significant at 25 ℃. This indicates that LbGP-TSG should be stored at 4 ℃.

[0049] Table 4 Temperature stability of smart responsive LbGP thermosensitive in-situ gelling agent (III) Example 3: Study on the in vitro release behavior of intelligent responsive LbGP thermosensitive in-situ gel 1. Dialysis bag method Method: One end of the pretreated dialysis bag (molecular weight cutoff 8000–14000 Da) was clamped, and 2 mL of LbGP-TSG and LbGP aqueous solution (equivalent to 100 mg LbGP) were added respectively. The other end was clamped and immersed in 150 mL of release medium (32℃, pH 6.8 artificial nasal solution) in a 250 mL Erlenmeyer flask. The flask was then placed in a constant temperature shaker at (32±0.5)℃ (100 r·min). -1 ) Take 2 mL samples at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, and 48 h, and immediately add an equal volume of fresh medium. After filtering the samples through a 0.45 μm microporous membrane, determine the drug concentration by HPLC and calculate the cumulative release rate.

[0050] The results are as follows Figure 2 As shown, LbGP aqueous solution was rapidly and completely released within 8 hours (rhamnose 100.77%, glucose 98.49%, arabinose 100.95%), while LbGP-TSG was released slowly, with cumulative release rates of 84.91% for rhamnose, 78.40% for glucose, and 81.56% for arabinose after 48 hours, indicating that LbGP-TSG has a good sustained-release effect.

[0051] The release curves were fitted using zero-order, first-order, and Higuchi equations, and the results are shown in Table 5. The Higuchi release model showed the highest fit (R²) for all three monosaccharides. 2 >0.97), indicating that the in vitro release of LbGP-TSG is mainly due to gel skeleton erosion.

[0052] Table 5. Fitting of the drug release mechanism of intelligent responsive LbGP thermosensitive in-situ gel 2. Membrane-free dissolution method Method: 2 mL of LbGP-TSG was placed in a 5 mL pre-weighed centrifuge tube, heated to 32 °C to allow complete gelation, weighed, and 1 mL of release medium (32 °C, pH 6.8 artificial nasal solution) was added. The tube was then placed in a 32 °C constant temperature shaking incubator (100 r·min). -1 The release medium was poured out at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 h. The centrifuge tubes were quickly dried and weighed. 1 mL of fresh medium was added, and the process was repeated until the gel was completely dissolved. The gel dissolution rate Y was calculated, and the drug concentration in the poured-out liquid was measured to calculate the cumulative release rate Q. The correlation between Y and Q was established.

[0053] The results are as follows Figure 3 As shown, the in vitro dissolution rate of LbGP-TSG reached 94.49% after 12 hours. Figure 4 As shown, the cumulative release rates of rhamnose, glucose, and arabinose over 12 hours were 82.49%, 87.70%, and 77.0%, respectively. Figure 5 As shown, the linear regression equations for dissolution rate and cumulative release rate are as follows: Rhamnose: y = 0.8436x + 0.8048 (R² + π / 2)² 2 =0.9983); Glucose: y=0.8756x+2.3162 (R 2 =0.9968); Arabinose: y=0.7689x+0.9207 (R 2 =0.9952), indicating that the gel dissolution rate is a key factor affecting the in vitro drug release rate.

[0054] (iv) Example 4: Local safety study of smart responsive LbGP thermosensitive in-situ gel. Experimental animals: SD rats, 200–250 g, half male and half female, provided by the Experimental Animal Center of Ningxia Medical University, production license number: SCXK (Ning) 2021-0001. Rats were housed under 12-hour light / dark alternation at a suitable temperature for 3–7 days for acclimatization. Sixteen rats were randomly divided into four groups (n=4): Negative control group: physiological saline; Positive control group: 1% sodium deoxycholate solution; Blank thermosensitive in situ gel group (without LbGP); Drug-loaded thermosensitive in situ gel assembly (LbGP-TSG).

[0055] Administration method: Rats were immobilized and 50 μL of the corresponding solution was instilled into the nasal cavity once daily for 7 consecutive days. After administration, the rats were placed in a supine position for 15 minutes to prevent drug loss. Nasal edema, necrosis, and congestion were observed. The rats were sacrificed 24 hours after the last administration.

[0056] Mucosal hyperemia, edema and secretions were scored according to the scoring standard, and the total score was calculated: 0-3 points mean no irritation, 4-6 points mean mild irritation, 7-9 points mean moderate irritation, and 10-12 points mean severe irritation.

[0057] The results are shown in Table 6. The total score of the 1% sodium deoxycholate solution group was 8 points (moderate irritation), and the total scores of both the LbGP-TSG group and the blank gel group were 1 point (no irritation). The physical diagram is shown in Figure 6 .

[0058] Table 6 Evaluation results of nasal irritation in rats (n=4) Histopathological examination: After the rats were decapitated and sacrificed, nasal tissues were collected, fixed with 4% paraformaldehyde, decalcified with 10% EDTA, dehydrated, embedded, sectioned, stained with H&E, and observed under a 400-fold microscope.

[0059] The results are shown in Figure 7 . In the normal saline group, the nasal mucosal epithelial cells were arranged neatly, the cilia were continuous and intact, and no mucosal swelling, tissue necrosis or shedding were observed; in the 1% sodium deoxycholate solution group, the nasal mucosal epithelial structure was destroyed, the cilia were broken, and edema, thickening, shedding and necrosis were observed; in the LbGP-TSG group and the blank gel group, the epithelial cells were closely arranged with regular shape, and no obvious pathological changes were observed. This indicates that LbGP-TSG causes no obvious damage to the nasal mucosa, has good safety, and can be used for intranasal administration.

[0060] (V) Example 5: Pharmacodynamic study of intelligent responsive LbGP temperature-sensitive in-situ gel on allergic rhinitis Experimental animals: Female BALB / c mice, weighing 15-20 g, were purchased from Chongqing Tengxin Biotechnology Co., Ltd., with production license number: SCXK (Jing) 2019-0008. They were housed under 12 h light / dark alternation at suitable temperature, and fed adaptively for 3-7 days. The mice were randomly divided into normal control group, AR model group, AR+LbGP group (150 mg / kg, intragastric administration), AR+LbGP-TSG group (intranasal administration, containing 150 mg / kg of LbGP), and AR+dexamethasone positive drug group (3 mg / kg, intragastric administration) Establishment of AR model: On the 1st, 3rd, 5th, 7th, 9th, 11th and 13th days, the model group and each administration group were intraperitoneally injected with 200 μL of artemisia annua pollen protein sensitizer (50 μg artemisia annua pollen protein + 2 mg aluminum hydroxide adjuvant, adjusted to a constant volume of 200 μL with PBS), and the normal control group was injected with the same volume of PBS. From day 15 to day 21, the model group and each administration group were instilled with 20 μL of artemisia annua pollen protein challenge (400 μg artemisia annua pollen protein) into the nasal cavity, and the normal control group was given the same volume of PBS.

[0061] Administration method: Administered 30 minutes before drug challenge on days 15–21. The LbGP group was given 150 mg / kg LbGP solution by gavage; the LbGP-TSG group was given an equal volume of thermosensitive gel containing 150 mg / kg LbGP by nasal instillation; the dexamethasone group was given 3 mg / kg dexamethasone by gavage; the normal control group and the model group were given an equal volume of physiological saline.

[0062] 1. Behavioral assessment of nasal symptoms Within 10 minutes after the last challenge, count the number of sneezes and nose scratches in mice and score them according to the following criteria: Nasal itching: 1 point (mild nose scratching <5 times), 2 points (frequent nose scratching 5-10 times), 3 points (continuous nose scratching >10 times); Sneezing: 1 point (1-3 times), 2 points (4-10 times), 3 points (>11 times). The results are as follows Figure 8 As shown, compared with the normal control group, the AR model group mice exhibited significantly increased nasal scratching and sneezing behaviors (P<0.001). Compared with the AR model group, LbGP intervention (150 mg / kg) significantly alleviated nasal symptoms (P<0.05), and the LbGP-TSG group showed a more significant effect (P<0.01), indicating that at the same dose, LbGP-TSG is superior to LbGP solution in improving nasal allergy symptoms in AR mice.

[0063] 2. Immune organ index measurement After the behavioral assessment, the mice were euthanized by cervical dislocation, and the spleen and thymus were removed and weighed. The immune organ index was calculated as organ weight (g) / body weight (g) × 100%.

[0064] The results are as follows Figure 9 As shown, compared with the normal control group, the spleen index and thymus index of the AR model group were significantly decreased (P<0.001). Both LbGP and LbGP-TSG interventions increased the spleen index in AR mice (P<0.01, P<0.001), and LbGP-TSG also significantly increased the thymus index (P<0.05), while there was no significant difference in the LbGP solution group. This indicates that LbGP-TSG has a better therapeutic effect on improving immune dysfunction in AR mice at the same dose.

[0065] 3. Histopathological observation of nasal mucosa (H&E staining) Mouse nasal tissue was collected, fixed with 4% paraformaldehyde, decalcified with 10% EDTA, dehydrated, embedded, sectioned, stained with H&E, and observed under a 400x microscope.

[0066] The results are as follows Figure 10As shown, in the normal control group, the nasal mucosal epithelial cells were neatly arranged, the cilia were continuous and intact, and no mucosal swelling, tissue necrosis or shedding was observed; in the AR model group, the nasal mucosal epithelial cells were structurally disordered, the cilia were broken, and edema, shedding, necrosis and a large number of inflammatory cell infiltrations were observed; after LbGP and LbGP-TSG intervention, the tissue structure was significantly improved, with only a small number of cilia being disordered and the cilia density increasing, and the improvement effect of the LbGP-TSG group was better than that of the LbGP group.

[0067] 4. Serum cytokine level measurement (ELISA) Peripheral blood was collected 24 hours after the last stimulation via enucleation and stored at 4°C and 8000 r·min. -1 Centrifuge for 10 min, collect serum, and store at -80℃. ELISA kits are used to detect serum IgE, IL-4, and IFN-γ levels.

[0068] The results are as follows Figure 11 As shown, serum IgE and IL-4 levels in the AR model group were significantly higher than those in the normal control group (P<0.01, P<0.001), while IFN-γ levels were lower (P<0.001). After intervention with LbGP and LbGP-TSG, IgE and IL-4 levels in AR mice decreased, while IFN-γ levels increased (P<0.05, P<0.01, P<0.001). The intervention effect was more significant in the LbGP-TSG group, indicating that LbGP-TSG has a stronger regulatory effect on Th1 / Th2 cell balance at the same dose.

[0069] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A smart responsive goji berry glycopeptide nasal thermosensitive in-situ gel, characterized in that, It contains the following components: Lycium barbarum glycopeptide 5% (w / v), poloxamer 407 19% (w / v), poloxamer 188 2% (w / v), sulfobutyl-β-cyclodextrin 7% (w / v), ethylparaben 0.02% (w / v), and the balance being water.

2. The intelligent responsive wolfberry glycopeptide nasal thermosensitive in-situ gel according to claim 1, characterized in that, The intelligent responsive wolfberry glycopeptide nasal thermosensitive in-situ gel is liquid at room temperature, but transforms into a semi-solid gel state at the physiological temperature of the nasal cavity of 29-34℃.

3. A method for preparing a smart responsive goji berry glycopeptide nasal thermosensitive in-situ gel as described in any one of claims 1 to 2, characterized in that, Includes the following steps: (1) Dissolve the wolfberry glycopeptide in water, add sulfobutyl-β-cyclodextrin and ethylparaben, and sonicate until completely dissolved; (2) Under ice-water bath conditions, slowly add poloxamer 407 and poloxamer 188 in sequence, and continue to add water and stir evenly; (3) Place the solution obtained in step (2) at 4°C for 24 hours to allow it to fully swell, and you will get the intelligent responsive wolfberry glycopeptide nasal thermosensitive in situ gel.

4. The use of the intelligent responsive wolfberry glycopeptide nasal thermosensitive in-situ gel as described in any one of claims 1 to 2 in the preparation of a drug for treating allergic rhinitis.

5. The application according to claim 4, characterized in that, The allergic rhinitis mentioned is allergic rhinitis induced by Artemisia annua pollen.

6. The application according to claim 4, characterized in that, The intelligent responsive wolfberry glycopeptide nasal thermosensitive in-situ gel is administered through the nasal cavity. It transforms into a gel state at the nasal cavity temperature, prolonging the drug retention time and achieving sustained and continuous release.

7. The application according to claim 6, characterized in that, The nasal administration method is nasal instillation, and the dosage is 150 mg / kg based on the amount of wolfberry glycopeptide.

Images