Oral mucosa adhesion preparation and preparation method thereof

Abstract

The invention discloses an oral mucosa adhesion preparation and a preparation method thereof, and the oral mucosa adhesion preparation comprises 5 mL of a polyethyleneimine aqueous solution, 5 mL of a polyacrylic acid aqueous solution, 100 [mu] L of a tannic acid aqueous solution, and 3mg of GSK12683. Mixing a polyethyleneimine aqueous solution, a polyacrylic acid aqueous solution and a tannic acid aqueous solution to prepare a first mixed solution for later use; mixing and stirring the first mixed solution and GSK126 to obtain a second mixture; freezing the second mixture in liquid nitrogen for 10 minutes, then freeze-drying for 24 hours to remove water, and preparing into powder, namely the oral mucosa adhesion preparation. The oral mucosa adhesion preparation prepared by the invention can be used for local administration, has strong adhesion, can continuously and slowly release and carry drugs in vivo and in vitro, has high bioavailability of the drugs, and can be used for local treatment of oral mucosa diseases such as oral precancerous lesions (oral leukoplakia and the like).

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an oral mucosal adhesive preparation and a preparation method thereof. Background Art

[0002] Adhesive hydrogels are widely used in wound dressings, hemostats, wearable devices, drug delivery systems, and tissue engineering due to their strong adhesion, good biocompatibility, good permeability, high deformability, and adjustable mechanical properties. Bioadhesion refers to the state in which at least one of the two interfaces is biological and the two are firmly and closely contacted and adhered together under the action of external force. Bioadhesive drug delivery systems refer to materials with adhesive properties that allow drug delivery systems to stay in specific locations for a longer time, thereby promoting drug absorption, entering the circulatory system, or being used for the treatment of local diseases. In order to achieve adhesion on wet tissue, two main hydrogel design strategies are currently used: one is to adhere prefabricated adhesive hydrogels to wet tissue, and the other is to form adhesive hydrogels in situ on wet tissue based on precursor solutions. For the first strategy, polymers containing adhesive functional groups in the hydrogel network need to diffuse through tissue interface water to bind to the tissue. However, most adhesive hydrogels are stabilized by chemical crosslinking, which limits the diffusion of polymers, resulting in weak contact and binding of tissue hydrogels. For the second strategy, monomers or macromolecules containing adhesive functional groups in the precursor solution also need to diffuse through interfacial water to bind to the tissue while undergoing rapid polymerization.

[0003] The oral cavity has a moist environment containing saliva, and the flow of saliva, the movement of the tongue and muscles make it difficult for drugs to be maintained locally for a long time without falling off. However, the short hydrogel time and chemically cross-linked hydrogel network of existing adhesive preparations hinder the effective contact and bonding between the hydrogel and the tissue. Therefore, the lack of polymer dynamics in the tissue interface water and the hydrogel network is a key obstacle to achieving firm adhesion between the hydrogel and the wet tissue. Oral mucosal precancerous lesions such as oral leukoplakia have a greater risk of canceration. Local administration has the advantages of simple operation, high local concentration, low systemic toxicity, and good anti-cancer effect, but there is currently a lack of a local preparation with good adhesion to slowly release the drug to be carried. Due to the influence of interfacial water and limited adhesive-tissue interaction, achieving good adhesion of bioadhesives on wet tissues is still a huge challenge. There is an urgent clinical need for a new preparation with good mucosal adhesion to achieve local delivery of drugs.

[0004] Based on the above analysis, an oral mucosal adhesive preparation that can be administered locally, has strong adhesion, can continuously and slowly release the drug in vivo and in vitro, and has high bioavailability of the drug, and can be used for local treatment of oral mucosal diseases such as oral precancerous lesions (oral leukoplakia, etc.) is urgently needed in the industry. Summary of the invention

[0005] In view of the above-mentioned deficiencies, the present invention provides an oral mucosal adhesive preparation for local treatment, which achieves local delivery of high-concentration drugs through local administration rather than oral administration, avoiding the first-pass effect of drugs in the body, and has strong adhesion.

[0006] The present invention is achieved by the following technical means:

[0007] The present invention first discloses an oral mucosal adhesive preparation, comprising:

[0008] 5 mL of polyethyleneimine aqueous solution, 5 mL of polyacrylic acid aqueous solution, 100 μL of tannic acid aqueous solution, and 3 mg of GSK1268.

[0009] Furthermore, the polyethyleneimine aqueous solution has Mn (number average molecular weight: the statistical average molecular weight of all polymer chains in a sample) = 1800, and a mass concentration of 10%; the polyacrylic acid aqueous solution has Mn = 240,000, and a mass concentration of 10%; and the molar concentration of the tannic acid aqueous solution is 0.1M.

[0010] The present invention also discloses a method for preparing the oral mucosal adhesive preparation, comprising:

[0011] (1) mixing a polyethyleneimine aqueous solution, a polyacrylic acid aqueous solution and a tannic acid aqueous solution to prepare a first mixed solution for standby use;

[0012] (2) mixing the first mixed solution and GSK126 with stirring to obtain a second mixture;

[0013] (3) The second mixture is frozen in liquid nitrogen, then freeze-dried to remove water, and made into powder to obtain an oral mucosal adhesive preparation.

[0014] Furthermore, the freezing time in liquid nitrogen is 10 minutes.

[0015] Furthermore, the freeze-drying and dehydration comprises: using a freeze dryer to freeze-dry for 24 hours to remove water at -90°C and a vacuum degree of 5.8 Pa.

[0016] The invention also discloses an oral mucosal adhesive preparation prepared according to the preparation method.

[0017] The present invention also discloses a method for using any of the above-mentioned oral mucosal adhesive preparations, comprising:

[0018] Take the oral mucosal adhesive preparation and apply it evenly to the affected area and press the preparation for 1 minute. At the same time, use a soaked cotton swab to moisten the preparation into a gel.

[0019] The beneficial effects of the present invention are:

[0020] The oral mucosal adhesive preparation prepared by the present invention is simple and has strong adhesion. The oral mucosal adhesive preparation can continuously and slowly release the loaded drug in vivo and in vitro, and can improve the bioavailability of the drug. By local administration rather than oral administration, the first-pass effect of the drug in the body can be avoided, and it can be used for local treatment of oral mucosal diseases such as oral precancerous lesions (oral leukoplakia, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments.

[0022] Figure 1 The ultrastructures of the PPT hydrogel of Comparative Example 1 and the PPT@GSK126 hydrogel of Example 1;

[0023] Figure 2 The in vitro and in vivo adhesion test of the PPT hydrogel of Comparative Example 1 and the PPT@GSK126 hydrogel of Example 1;

[0024] Figure 3 The adhesion of the hydrogel in Example 1 on different medium surfaces;

[0025] Figure 4 The in vitro sustained drug release of the hydrogel of Example 1;

[0026] Figure 5 The in vivo drug sustained release performance of the hydrogel of Example 1;

[0027] Figure 6 In vitro biosafety test of the hydrogel of Example 1;

[0028] Figure 7 This is the in vivo biosafety test of the present invention, wherein (a) is the result of blood routine test, (b) is a representative picture of the expression of TNFα and IL-6 in the mouse tongue based on immunohistochemical staining, and (c) is a representative picture of HE in important organs of mice;

[0029] Figure 8 The present invention is a statistical analysis of the therapeutic effect of the present invention on oral leukoplakia in animals, the improvement of pathological severity and the prevention of canceration, wherein (a) is a representative picture of tongue lesions in each group of mice after medication, and (b) is a statistical analysis of the difference in the number of lesions of different pathological grades in each group of mice;

[0030] Fig. 9 A flow chart is prepared for the process of the present invention. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical schemes in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0032] Example 1

[0033] An oral mucosal adhesive preparation (PPT@GSK126 hydrogel) and a preparation method thereof, comprising:

[0034] (1) 5 mL of a polyethyleneimine aqueous solution with a mass concentration of 10% and a molar concentration of 1800, 5 mL of a polyacrylic acid aqueous solution with a mass concentration of 10% and a molar concentration of 240,000, and 100 μL of a tannic acid aqueous solution with a molar concentration of 0.1 M were mixed to prepare a first mixed solution for later use;

[0035] (2) mixing the first mixed solution and 83 mg of GSK126 into a gel to obtain a second mixture;

[0036] (3) The second mixture was frozen in liquid nitrogen for 10 minutes, and then freeze-dried for 24 hours in a freeze dryer at -90°C and a vacuum of 5.8 Pa to remove water, and finally the block material was crushed into powder to obtain an oral mucosal adhesive preparation, namely PPT@GSK126 hydrogel. The dosage of GSK126 on the tongue dorsum of each mouse of the PPT@GSK126 hydrogel was 0.5 mg or 1 mg.

[0037] Comparative Example 1

[0038] A method for preparing a PPT hydrogel, comprising:

[0039] Compared with Example 1, the PPT hydrogel does not contain GSK126. The remaining steps and methods are the same as those in Example 1.

[0040] Test Example 1

[0041] Comparison of SEM images of samples obtained in Example 1 and Comparative Example 1

[0042] Figure 1 The SEM images in the figure respectively observe the ultrastructure of the freeze-dried PPT hydrogel (i.e., the sample prepared in Comparative Example 1) and the PPT@GSK126 hydrogel (i.e., the sample prepared in Example 1). The freeze-dried PPT hydrogel and PPT@GSK126 hydrogel present vesicle and membrane structures. The properties of the original PPT hydrogel are not significantly changed after loading with GSK126, indicating that the loaded drug does not interfere with the adhesion performance of the latter.

[0043] Test Example 2

[0044] In vitro and in vivo adhesion performance testing

[0045] The adhesion of drug-loaded hydrogel and simple hydrogel was qualitatively measured by oscillation method and flowing water method. The oscillation method was operated as follows: two groups of powders were applied to the isolated mouse skin, an appropriate amount of water was given, 10 parallel points were set, and then the skin was immersed in neutral PBS, oscillated at 100 rpm at 37°C, and the retention rate of the gel within a predetermined time was evaluated. The flowing water method was operated as follows: equal amounts of two kinds of powders were applied to the back of the rat tongue, and after adding water to form gel, PBS was titrated to simulate the flow of saliva. The flow rate was set to 1 ml / min, and the retention time of the gel on the rat tongue under the flowing water state was recorded.

[0046] The adhesion of the two hydrogels was tested by rheometer. The results showed that PPT hydrogel and PPT@GSK126 hydrogel could meet clinical adhesion requirements and would not damage the oral mucosa when peeled off. Figure 2 The results of the oscillation method in the experiment showed that both PPT hydrogel and PPT@GSK126 hydrogel showed good wet adhesion to pig skin and pig buccal mucosa. They both remained intact on the tissue and had good adhesion to the tissue after oscillation at 450 rpm for 48 hours at 37°C. In addition, both hydrogels could adhere to plastic, glass and magnetic metal in the air (see Figure 3 ). Similar to the in vitro results, when PBS flowed at a rate of 1 mL / min, both hydrogels formed a film with the rat tongue, fitting the tongue tissue tightly and comfortably. Compared with the PPT hydrogel, the PPT@GSK126 hydrogel had a more complete retention volume after 4 consecutive rinses.

[0047] Test Example 3

[0048] In vitro drug sustained release of Example 1

[0049] 12mg of the PPT@GSK126 powder prepared in Example 1 was dissolved in 200uL of water. After complete gelation, the excess water was sucked out, and 200uL of PEG mixed solution (50% PEG + 50% water) was added to form a sustained-release system. 200uL of the upper mixed solution was sucked at 0h, 2h, 4h, 6h, 8h, 12h, 24h, 48, and 72h (200uL of PEG mixed solution was added after each suction). The release of the active ingredient GSK126 was quantitatively analyzed by ultraviolet sustained-release method, and finally the concentration of GSK126 released at different time points was calculated to draw a drug sustained-release curve.

[0050] The results showed that within the first 6 hours, the concentration of GSK126 in the solution increased rapidly. In the subsequent 72 hours, the concentration of GSK126 drug increased steadily, and the cumulative drug release concentration reached 0.197 mg / mL (see Figure 4 ), indicating that GSK126 can be sustained-released during the administration cycle to achieve therapeutic purposes.

[0051] Test Example 4

[0052] In vivo drug sustained release of Example 1

[0053] Under isoflurane inhalation anesthesia, the tongue of C57BL / 6 mice was pulled, and 12 mg of PPT prepared in Comparative Example 1 or 12 mg of PPT@GSK126 lyophilized powder prepared in Example 1 was evenly applied to the back of the tongue and the powder was pressed for 1 minute, and the lyophilized powder was moistened into a gel with a soaked cotton swab; the drugs were administered at predetermined time points (1h, 6h, 2w), and the mice in the 2-week group were administered every day. After the mice were anesthetized, blood was collected from the eyeballs. There were 3 mice in each group at each time point, and the serum was collected and the blood GSK126 drug concentration was determined; the mice were killed, and the tongues of the mice were stored at -20°C for subsequent HPLC determination of the drug concentration in the tongue tissue.

[0054] The results showed that a certain concentration of GSK126 could be detected in the mouse tongue at different time points (see Figure 5 ), indicating that PPT@GSK126 hydrogel can slowly release drugs into the local tongue tissue to achieve local treatment purposes.

[0055] Test Example 5

[0056] In vitro biosafety test of Example 1

[0057] First, the extract supernatant was prepared. 100 mg of PPT prepared in Comparative Example 1 or PPT@GSK126 powder prepared in Example 1 was added to 10 mL of culture medium to form a self-gel. After culturing for 24 h, the supernatant was collected to prepare the extract. After culturing L92 cells for 24 h and 48 h using blank control, hydrogel supernatant, simple equal amount of GSK126 supernatant and GSK126-loaded hydrogel supernatant, the growth difference between the groups was detected by cell live-death staining. The live-dead staining method is as follows: cells are seeded in a 12-well plate (1*10^5 cells / well) and allowed to adhere for 6 hours, and then the culture medium is replaced with 500 μL of the extract, and cultured continuously at 37°C for 24 hours and 48 hours; in addition, the cells are placed in RPMI-1640 culture medium containing 10uM GSK126 and RPMI-1640 culture medium without GSK126 as a control; the culture medium is aspirated and the cells are washed twice with PBS; 500 μL of CalceinAM / PI detection working solution is added to each well, and the cells are incubated at 37°C in the dark for 30 minutes. After the incubation, the cell morphology is observed under a fluorescence microscope, and pictures and statistical analysis are performed.

[0058] The results showed that there was no difference in the percentage of viable cells among the four groups (see Figure 6 ), indicating that both PPT and PPT@GSK126 gels have good biosafety.

[0059] Test Example 6

[0060] In vivo biosafety of Example 1

[0061] The tongue, heart, liver, spleen, lung, kidney, esophagus, stomach tissues, serum and plasma of mice in the local drug-loaded hydrogel and simple hydrogel groups in the in vivo drug sustained-release capacity test in the above-mentioned Experimental Example 4 were collected, and the tissues were fixed, dehydrated, embedded and sliced ​​in a 4% paraformaldehyde solution. HE staining was used to observe the changes in tissue microstructure, and IHC was used to detect the expression of TNFα and IL-6 in the tongue to reflect the local inflammatory state. Blood routine tests were performed to evaluate toxicity. The results are as follows Figure 7 As shown, there was no obvious inflammatory response in the blood routine and important organs of mice, the size and morphology of tissue cells were normal, and no abnormal nuclear division images were observed, indicating that it has good long-term biocompatibility and potential for clinical application.

[0062] Application Example 1

[0063] Application effects of PPT hydrogel and PPT@GSK126 hydrogel in mouse oral leukoplakia model

[0064] The mice were divided into 8 different treatment groups. Figure 8 (a), respectively: PBS local application, PD-1 monoclonal antibody local injection, PPT, PPT@GSK126 (0.5 mg), PPT@GSK126 (1 mg), PPT@GSK126 (1 mg) + PD-1 monoclonal antibody local injection group, GSK126 drug intraperitoneal injection group (25 mg / kg), GSK126 drug intraperitoneal injection group (50 mg / kg); the method of local application of drug-loaded hydrogel is as follows: after isoflurane inhalation anesthesia, pull the mouse tongue, place 12 mg PPT@GSK126 lyophilized powder on the tongue and press the powder for 1 min, use a soaked cotton swab to moisten the lyophilized powder into gel, once a day for 6 weeks; the method of local injection of PD-1 monoclonal antibody is as follows: the needle is inserted obliquely 3-4 mm in the middle of the tongue edge of each mouse, 100 ug PD-1 monoclonal antibody was injected 3 times a week. Tongue tissues were collected at the end of the experiment, and oral pathologists evaluated epithelial abnormal proliferation or cancer after HE staining. The differences in pathological severity and canceration rate among the groups were statistically compared.

[0065] The results are as follows Figure 8(b) As shown: The number of lesions with specific pathological subtypes in different groups was compared. Compared with PPT, the severe abnormal proliferation in the PPT@GSK126 (0.5 mg or 1 mg) group and the PPT@GSK126 (1 mg) + αPD-1 group was significantly reduced; the number of cancerous changes in the PPT@GSK126 (1 mg) group and the PPT@GSK126 (1 mg) + αPD-1 group was significantly reduced, and the number of cancerous changes in the other two groups showed a downward trend relative to PPT; the number of mild abnormal proliferation in the PPT@GSK126 (0.5 mg or 1 mg) group and the PPT@GSK126 (1 mg) + PD-1 monoclonal antibody local injection group was higher than that in the PPT group, but the difference was not statistically significant.

[0066] In summary, the local application of PPT@GSK126 and its combination with αPD-1 showed the best efficacy in reducing the risk of malignant transformation of OLK, especially in reducing its carcinogenesis rate.

[0067] In summary, the oral mucosal adhesive preparation prepared by the present invention is easy to use and non-invasive. The preparation method of PPT hydrogel is simple and has strong adhesion and good biocompatibility. PPT@GSK126 hydrogel makes it easier for drugs to be enriched in the lesion to exert their therapeutic effects, with a smaller drug dosage requirement and higher safety. It is a local drug preparation with great potential for the treatment of oral precancerous lesions in future clinical applications.

[0068] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An oral mucosal adhesive preparation comprising: 5 mL of polyethyleneimine aqueous solution, 5 mL of polyacrylic acid aqueous solution, 100 μL of tannic acid aqueous solution, and 3 mg of GSK1268.

2. The oral mucosal adhesive preparation according to claim 1, wherein: The polyethyleneimine aqueous solution has a Mn of 1800 and a mass concentration of 10%; The polyacrylic acid aqueous solution has Mn=240,000 and a mass concentration of 10%; The molar concentration of the tannic acid aqueous solution is 0.1M.

3. A method for preparing the oral mucosal adhesive preparation according to claim 1 or 2, comprising: (1) mixing a polyethyleneimine aqueous solution, a polyacrylic acid aqueous solution and a tannic acid aqueous solution to prepare a first mixed solution for standby use; (2) mixing the first mixed solution and GSK126 with stirring to obtain a second mixture; (3) The second mixture is frozen in liquid nitrogen, then freeze-dried to remove water, and made into powder to obtain an oral mucosal adhesive preparation.

4. The preparation method according to claim 3, wherein: The freezing time in the liquid nitrogen is 10 minutes.

5. The preparation method according to claim 3, wherein: The freeze-drying dehydration comprises: Use a freeze dryer at -90°C and a vacuum of 5.8 Pa for 24 hours to remove water.

6. An oral mucosal adhesive preparation obtained by the preparation method according to any one of claims 3 to 5.

7. A method for using the oral mucosal adhesive preparation according to any one of claims 1, 2, and 6, comprising: Take the oral mucosal adhesive preparation and apply it evenly to the affected area and press the preparation for 1 minute. At the same time, use a soaked cotton swab to moisten the preparation into a gel.

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