Oral mucosal adhesive formulation and method of preparation thereof

Abstract

The application discloses an oral mucosa adhesive preparation and a preparation method thereof. The oral mucosa adhesive preparation comprises 5 mL of a polyethylene imine aqueous solution, 5 mL of a polyacrylic acid aqueous solution, 100 muL of a tannic acid aqueous solution and 83 mg of GSK126. The polyethylene imine aqueous solution, the polyacrylic acid aqueous solution and the tannic acid aqueous solution are mixed to prepare a first mixed solution; the first mixed solution is mixed with GSK126 to prepare a second mixture; the second mixture is frozen in liquid nitrogen for 10 min, and then freeze-dried for 24 h to remove water, so as to obtain a powder, namely the oral mucosa adhesive preparation. The prepared oral mucosa adhesive preparation can be used for local administration, has strong adhesion, can continuously and slowly release the carried drug in and out of the body, has high bioavailability of the drug, 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] This invention relates to the field of pharmaceutical technology, specifically to an oral mucosal adhesion preparation and its preparation method. Background Technology

[0002] Adhesive hydrogels, due to their strong adhesion, good biocompatibility, good permeability, high deformability, and tunable mechanical properties, are widely used in wound dressings, hemostatic agents, wearable devices, drug delivery systems, and tissue engineering. Bioadhesion refers to the state where at least one of the interfaces is biologically viable, and the two adhere firmly and tightly together under external force. Bioadhesive drug delivery systems refer to materials with adhesive properties that prolong the residence time of the drug delivery system at a specific site, thereby promoting drug absorption, entry into the circulatory system, or for the treatment of local diseases. To achieve adhesion on wet tissue, two main hydrogel design strategies are currently employed: one is to adhere a pre-fabricated adhesive hydrogel to wet tissue, and the other is to form an adhesive hydrogel in situ on wet tissue based on a precursor solution. For the first strategy, the polymer containing adhesive functional groups in the hydrogel network needs to diffuse through water at the tissue interface to bind with the tissue. However, most adhesive hydrogels are stabilized by chemical cross-linking, which limits polymer diffusion, resulting in weaker contact and binding of the tissue hydrogel. For the second strategy, monomers or macromolecules containing adhesion 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 possesses a moist environment containing saliva, and the flow of saliva, along with the movement of the tongue and muscles, makes it difficult for drugs to maintain their local adhesion for extended periods. However, the short hydrogel time and chemically cross-linked hydrogel networks of existing adhesive formulations hinder effective contact and binding between the hydrogel and tissue. Therefore, the lack of polymer kinetics in the tissue interface water and hydrogel network is a key obstacle to achieving strong adhesion between hydrogels and moist tissue. Oral mucosal precancerous lesions, such as oral leukoplakia, carry a significant risk of malignant transformation. Local administration offers advantages such as ease of use, high local concentration, low systemic toxicity, and good anti-cancer effects. However, a topical formulation with good adhesion to slowly release the drug is currently lacking. Achieving good adhesion of bioadhesives to moist tissue remains a significant challenge due to the influence of interface water and limited adhesive-tissue interactions. There is an urgent clinical need for novel formulations with good mucosal adhesion to achieve local drug delivery.

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

[0005] In view of the above shortcomings, the present invention provides a local treatment oral mucosal adhesive formulation that delivers high concentrations of drug locally by means of local administration rather than oral administration, avoiding the first-pass effect of the drug in the body, and has strong adhesion.

[0006] This invention is achieved through the following technical means:

[0007] This invention first discloses an oral mucosal adhesion 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] Further, the polyethyleneimine aqueous solution has a number-average molecular weight (Mn: the statistical average of the molecular weights of all polymer chains in the sample) of 1800 and a mass concentration of 10%; the polyacrylic acid aqueous solution has a Mn of 240,000 and a mass concentration of 10%; and the tannic acid aqueous solution has a molar concentration of 0.1M.

[0010] This invention also discloses a method for preparing the above-mentioned oral mucosal adhesion preparation, comprising:

[0011] (1) Mix the aqueous solutions of polyethyleneimine, polyacrylic acid and tannic acid to prepare a first mixed solution for later use;

[0012] (2) Mix the first mixed solution and GSK126 and stir to obtain the second mixture;

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

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

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

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

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

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

[0019] The beneficial effects of this invention are as follows:

[0020] The oral mucosal adhesive formulation prepared by this invention is simple to produce and exhibits strong adhesion. This oral mucosal adhesive formulation can continuously and slowly release the loaded drug in vitro and in vivo, thereby improving drug bioavailability. Local administration, rather than oral administration, avoids the first-pass effect in vivo and can be used for the local treatment of oral mucosal diseases such as precancerous lesions of the oral cavity (e.g., oral leukoplakia). Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below.

[0022] Figure 1 The ultrastructures of the PPT hydrogel in Comparative Example 1 and the PPT@GSK126 hydrogel in Example 1 are shown.

[0023] Figure 2 In vitro and in vivo adhesion assays of PPT hydrogel in Comparative Example 1 and PPT@GSK126 hydrogel in Example 1.

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

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

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

[0027] Figure 6 This is an in vitro biocompatibility test of the hydrogel in Example 1;

[0028] Figure 7 For the in vivo biosafety test of the present invention, (a) is the result of routine blood tests, (b) is a representative image of TNFα and IL-6 expression in the mouse tongue based on immunohistochemical staining analysis, and (c) is a representative image of HE in important mouse organs.

[0029] Figure 8 The present invention provides a statistical analysis of the therapeutic effect, pathological severity improvement, and cancer prevention effect of oral leukoplakia in animals. (a) is a representative image of tongue lesions in each group of mice after drug administration, and (b) is a statistical analysis of the differences in the number of lesions at different pathological grades in each group of mice.

[0030] Figure 9 This is a flowchart of the process preparation of the present invention. Detailed Implementation

[0031] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0032] Example 1

[0033] An oral mucosal adhesion formulation (PPT@GSK126 hydrogel) and its preparation method, comprising:

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

[0035] (2) Mix the first mixed solution with 83 mg GSK126 and stir to form a gel to obtain the second mixture;

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

[0037] Comparative Example 1

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

[0039] Compared to Example 1, this PPT hydrogel does not contain GSK126. All other steps and methods are the same as in Example 1.

[0040] Experimental Example 1

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

[0042] Figure 1 The SEM images show the ultrastructure of the lyophilized PPT hydrogel (i.e., the sample prepared in Comparative Example 1) and the PPT@GSK126 hydrogel (i.e., the sample prepared in Example 1), respectively. The lyophilized PPT hydrogel and PPT@GSK126 hydrogel exhibit vesicle and membrane structures. The properties of the original PPT hydrogel were not significantly changed after loading GSK126, suggesting that loading the drug does not interfere with the adhesion properties of the latter.

[0043] Experimental Example 2

[0044] In vitro and in vivo adhesion performance testing

[0045] The adhesion forces of drug-loaded hydrogels and simple hydrogels were qualitatively measured using the oscillation method and the flow-through method, respectively. The oscillation method was performed as follows: two powders were applied topically to isolated mouse skin, followed by an appropriate amount of water. Ten parallel points were set, and the skin was then immersed in neutral PBS. The mixture was oscillated at 100 rpm at 37°C, and the gel retention rate was assessed within a predetermined time. The flow-through method was performed as follows: equal amounts of the two powders were applied topically to the dorsum of a rat's tongue. Water was added to form a gel, and PBS was titrated to simulate saliva flow. The flow rate was set to 1 ml / min, and the retention time of the gel on the rat's tongue under flow-through conditions was recorded.

[0046] The adhesion of the two hydrogels was tested using a rheometer. The PPT hydrogel and PPT@GSK126 hydrogel met the clinical adhesion requirements and did not damage the oral mucosa during peeling. Figure 2 The oscillation method results showed that both PPT hydrogel and PPT@GSK126 hydrogel exhibited good wet adhesion properties to porcine skin and porcine buccal mucosa. Both remained intact on the tissue and showed good adhesion after oscillation at 450 rpm for 48 hours at 37°C. Furthermore, both hydrogels could adhere to airborne plastics, glass, and magnetic metals (see details). Figure 3 Similar to in vitro results, both hydrogels formed a film on the rat tongue when PBS flowed at 1 mL / min, adhering closely and comfortably to the tongue tissue. Compared to the PPT hydrogel, the PPT@GSK126 hydrogel maintained a more complete volume after four consecutive washes.

[0047] Experimental Example 3

[0048] In vitro drug sustained release in Example 1

[0049] 12 mg of PPT@GSK126 powder prepared in Example 1 was dissolved in 200 μL of water. After complete gelation, excess water was removed, and 200 μL of PEG mixture (50% PEG + 50% water) was added to form a sustained-release system. 200 μL of the upper layer mixture was collected at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h (200 μL of PEG mixture was added after each collection). The release of the active ingredient GSK126 was quantitatively analyzed using a UV sustained-release method. Finally, the concentration of GSK126 released at different time points was calculated, and a drug sustained-release curve was plotted.

[0050] The results showed that the concentration of GSK126 in solution increased rapidly within the first 6 hours. In the subsequent 72 hours, the drug concentration of GSK126 steadily increased, reaching a cumulative drug release concentration of 0.197 mg / mL (see details). Figure 4 This indicates that GSK126 can be released sustainably during the dosing cycle to achieve therapeutic purposes.

[0051] Test Example 4

[0052] In vivo drug sustained release in Example 1

[0053] Under isoflurane inhalation anesthesia, the tongue of C57BL / 6 mice was pulled back, 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. At the same time, the lyophilized powder was moistened into a gel with a moistened cotton swab. The drugs were administered at predetermined time points (1 h, 6 h, 2 w), and the mice in the 2-week group were administered the drugs daily. Blood was collected from the eyeballs of the mice after anesthesia. Three mice in each group were used at each time point. Serum was collected and the blood GSK126 drug concentration was determined. The mice were sacrificed, and the tongues were preserved 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 tongue of mice at different time points (see details). Figure 5 This indicates that PPT@GSK126 hydrogel can slowly release drugs into the local tongue tissue to achieve local therapeutic purposes.

[0055] Experimental Example 5

[0056] In vitro biosafety testing in Example 1

[0057] First, the supernatant of the extract 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. L92 cells were cultured for 24 h and 48 h using blank control, hydrogel supernatant, equal volume of GSK126 supernatant alone, and GSK126-loaded hydrogel supernatant. The differences in growth between groups were detected by cell viability staining. The method for live / dead staining was as follows: cells were seeded in 12-well plates (1*10^5 cells / well) and adhered for 6 hours. The medium was then replaced with 500 μL of extract and cultured at 37°C for 24 hours and 48 hours. In addition, cells were placed in RPMI-1640 medium containing 10 μg GSK126 and RPMI-1640 medium without GSK126 as controls. The culture medium was aspirated and the cells were washed twice with PBS. 500 μL of Calcein AM / PI detection working solution was added to each well and incubated at 37°C in the dark for 30 minutes. After incubation, cell morphology was observed under a fluorescence microscope, and images were taken and statistically analyzed.

[0058] The results showed no difference in the percentage of viable cells among the four groups (see details). Figure 6 This indicates that both PPT and PPT@GSK126 gels have good biocompatibility.

[0059] Experimental Example 6

[0060] In vivo biosafety of Example 1

[0061] Tissues, serum, and plasma from mice in the local drug-loaded hydrogel and simple hydrogel groups (as described in Experiment 4 above) were collected for in vivo drug release capability testing. The tissues were fixed, dehydrated, embedded, and sectioned in 4% paraformaldehyde solution. HE staining was used to observe changes in tissue microstructure. IHC was used to detect TNFα and IL-6 expression in the tongue to reflect local inflammatory status. Complete blood count was performed to assess toxicity. Results are as follows: Figure 7 As shown, no obvious inflammatory response was observed in the mouse blood routine and important organs. The size and morphology of tissue cells were normal, and no abnormal nuclear division was observed, suggesting that it has good long-term biocompatibility and has the potential for clinical application.

[0062] Application Example 1

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

[0064] Mice were divided into 8 different treatment groups. For detailed procedures, please refer to [link to procedure details]. Figure 8 (a) The groups were: 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 intraperitoneal injection group (25 mg / kg), and GSK126 intraperitoneal injection group (50 mg / kg). The local application method for the drug-loaded hydrogel was as follows: after isoflurane inhalation anesthesia, the mouse tongue was pulled back, 12 mg of PPT@GSK126 lyophilized powder was placed on the tongue and pressed for 1 minute. The lyophilized powder was moistened into a gel with a moistened cotton swab. This was done once a day for 6 weeks. The local injection method for PD-1 monoclonal antibody was as follows: 100 μg of 100 μg was injected obliquely into the middle of the tongue margin of each mouse at a depth of 3-4 mm. PD-1 monoclonal antibody was administered once a week, three times a week. At the end of the experiment, tongue tissue was collected, stained with hematoxylin and eosin (HE), and assessed by an oral pathologist for epithelial dysplasia or carcinogenesis. The differences in pathological severity and carcinogenesis rate among the groups were statistically compared.

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

[0066] In conclusion, topical 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 formulation prepared by this invention is convenient and non-invasive to use. The PPT hydrogel preparation method is simple and has strong adhesion and good biocompatibility. The PPT@GSK126 hydrogel makes it easier for the drug to accumulate locally on the lesion to exert its therapeutic effect, requiring a smaller drug dosage and having higher safety. It is a topical drug formulation 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, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An oral mucosal adhesion preparation, comprising: Apply the oral mucosal adhesion preparation evenly to the affected area and press the preparation for 1 minute. Simultaneously, use a moistened cotton swab to moisten the preparation into a gel. Among other things: The method for preparing this oral mucosal adhesion preparation includes: (1) Mix 5 mL of polyethyleneimine aqueous solution, 5 mL of polyacrylic acid aqueous solution and 100 μL of tannic acid aqueous solution to prepare the first mixed solution for later use. (2) Mix the first mixed solution with 83 mg GSK126 and stir to obtain the second mixture; (3) The second mixture is placed in liquid nitrogen and frozen, then freeze-dried to remove water and made into powder, thus obtaining an oral mucosal adhesion preparation.

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

3. The oral mucosal adhesive according to claim 1, wherein: The freezing time in liquid nitrogen is 10 minutes.

4. The oral mucosal adhesive according to claim 1, wherein: The freeze-drying dehydration includes: The water was removed by freeze drying at -90℃ and 5.8Pa for 24 hours using a freeze dryer.

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