WO2021134935A1 - Use of cannabidiol in preparation of drugs for prompting healing of oral mucosa - Google Patents

Abstract

Use of cannabidiol or a pharmaceutically acceptable salt, ester and solvate thereof in preparation of drugs for prompting healing of oral mucosa. Applying the cannabidiol or the pharmaceutically acceptable salt, ester and solvate thereof to the preparation of drugs for prompting the healing of oral mucosa has significant effects of prompting proliferation and migration of oral mucosa keratinocytes and suppressing excessive pyroptosis of oral mucosa keratinocytes, has a significant effect of prompting the healing of oral mucosa of mice of an oral ulcer model, and has a downregulation effect, discovered by analysis using the transcriptome sequencing technology, on most genes in keratinocytes that induce an inflammation, and especially has an obvious downregulation effect on genes related to activation and pyroptosis of NLRP3 inflammasomes.

Description

Application of cannabidiol in preparing medicine for promoting oral mucosa healing Technical field

This application relates to the field of drug applications, in particular to a new strategy for promoting the healing of the oral mucosa, and in particular to the application of cannabidiol in the preparation of drugs for promoting the healing of the oral mucosa.

Background technique

Inflammatory lesions of the oral mucosa, involving the epithelium and subepithelial connective tissue (lamina propria), are severely painful, and can seriously affect the patient's quality of life. It is the result of multiple factors with unknown etiology. There is no stable and effective treatment at present. The clinical treatment is mostly symptomatic treatment, mainly to relieve pain and promote healing, such as oral cavity washing, local or systemic use of analgesics, hormones, etc. . In recent years, scholars have successively proposed the use of growth factors, anti-inflammatory substances, immunomodulators, anti-radiation drugs, etc. for prevention and treatment, most of which are still in the preclinical stage, and most growth factors have the potential to promote the growth of cancer cells.

CN101468039B discloses a preparation method of a compound double-layer sustained-release medicinal film for the treatment of oral mucosal diseases, which is based on borneol, artificial bezoar, tinidazole, dyclonine hydrochloride, tween-80, glycerin, sodium saccharin, and carboxymethyl It is made of raw materials such as sodium cellulose and polyvinyl alcohol. The compound double-layer sustained-release medicinal film overcomes the shortcomings of single-layer and single-layer films of Chinese and Western medicine, is durable and has obvious curative effect, and has the effect of promoting the healing of oral lesions. Compared with the single-layer film, the double-layer film changes from a single-layer drug release to a double-layer drug release, so that the drug is limited to local contact with the diseased part, reducing the drug outflow and reducing the peculiar smell of oral drugs.

CN104189165A discloses a traditional Chinese medicine preparation for oral mucosal ulcers. The mass ratio of each component in the formula is: rhubarb 500-700g; Glauber's salt 500-700g; licorice 500-700g; gardenia 200-400g; scutellaria 200-400g ; Mint 200～400g; Forsythia 1100～1300g. It acts directly on the mucous membranes, has fast drug absorption, and has obvious curative effects. It has the characteristics of good biocompatibility, prevention of exudate overflow, hemostasis and pain relief, anti-inflammatory and antibacterial, and promotes the formation of granulation tissue and mucosal tissue, and fast healing and repair of skin and mucosal tissue.

CN106692201A discloses a composition for preventing and treating radiation oral mucositis and its application. The composition includes mussel soft tissue extract and fungal polysaccharide. The composition for preventing and treating radiation oral mucositis of the present application can block radiation damage caused by exogenous factors, has a super anti-radiation effect, prevents oral mucositis, promotes the regeneration and repair of oral mucosal tissues, shortens wound healing time, and reduces inflammation Pain relief. This application is mild, non-irritating, non-toxic and side-effect, and non-use-dependent.

The main active ingredient in cannabis leaf extract is cannabinoids. The cannabinoids isolated from the cannabis plant are tetrahydrocannabinol (THCV), cannabidiol (CBD), cannabidiol (CBDV), and cannabidiol ( CBG) and so on. Among them, cannabidiol, as a non-psychological component, has medicinal value in anti-tumor, nervous system protection, immune regulation, anti-inflammatory and antioxidant properties. In recent years, researchers have made great progress in the pharmacological research of cannabinoids and the development and utilization of drugs, but there is very little research in the field of oral cavity, especially in promoting the healing of oral mucosa.

Summary of the invention

In view of the shortcomings of the prior art, the purpose of this application is to provide a new strategy for promoting the healing of the oral mucosa, in particular to provide the application of cannabidiol in the preparation of medicines for promoting the healing of the oral mucosa.

In order to achieve the purpose of this application, this application adopts the following technical solutions:

In the first aspect, this application provides the use of cannabidiol or its pharmaceutically acceptable salts, esters, and solvates in the preparation of drugs for promoting oral mucosal healing.

This application creatively discovers that cannabidiol or its pharmaceutically acceptable salts, esters, and solvates can be used in the preparation of drugs that promote oral mucosal healing, through the exploration of the effect of cannabidiol on the biological behavior of human oral keratinocytes The investigation of the therapeutic effect of oral ulcer model mice found that it has a significant effect on promoting proliferation and migration of oral mucosal keratinocytes and inhibiting excessive pyrolysis of oral mucosal keratinocytes, and these three effects are concentration and time dependent; It has a significant effect on promoting the healing of oral mucosa of oral ulcer model mice; analysis of transcriptome sequencing technology found that it has a tendency to down-regulate most of the genes in keratinocytes that induce inflammation, especially the activation of NLRP3 inflammasomes and pyrolysis All related genes have a significant down-regulation effect.

Preferably, the drug promotes proliferation of oral keratinocytes.

Preferably, the drug promotes the migration of oral keratinocytes.

Preferably, the drug inhibits oral keratinocyte pyrolysis.

Preferably, the drug inhibits the NLRP3 inflammasome activation pathway.

In the second aspect, this application provides the use of cannabidiol or its pharmaceutically acceptable salts, esters, and solvates in the preparation of oral keratinocyte proliferation promoters.

In the third aspect, this application provides the use of cannabidiol or its pharmaceutically acceptable salts, esters, and solvates in the preparation of oral keratinocyte migration promoters.

In the fourth aspect, this application provides the use of cannabidiol or its pharmaceutically acceptable salts, esters, and solvates in the preparation of oral keratinocyte pyrolysis inhibitors.

The oral keratinocyte pyrolysis inhibitor here means that cannabidiol or its pharmaceutically acceptable salts, esters, and solvates can inhibit the excessive pyrolysis of oral keratinocytes in the process of relieving inflammation and promoting healing, rather than Refers to the complete suppression of scorch.

In the fifth aspect, this application provides the use of cannabidiol or its pharmaceutically acceptable salts, esters, and solvates in the preparation of NLRP3 inflammasome activation pathway inhibitors.

In this application, the medicine may also include pharmaceutically acceptable excipients.

Preferably, the auxiliary material includes any one or a combination of at least two of diluents, carriers, flavoring agents, binders or fillers, such as a combination of binders and diluents, The combination of carrier and flavoring agent, combination of binder and filler, etc., and other arbitrary combinations will not be repeated here.

Preferably, the carrier includes liposomes, micelles, dendrimers, microspheres or microcapsules.

The cannabidiol or its pharmaceutically acceptable salts, esters, and solvates described in this application can be loaded on common medicinal carriers as drugs to promote oral mucosal healing to achieve better biocompatibility, targeting, Biological safety and drug delivery effect.

The cannabidiol or its pharmaceutically acceptable salts, esters, and solvates described in this application can also be used in combination with other drugs to achieve a better effect of promoting oral mucosal healing.

Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, injections, sprays, films, suppositories, nose drops or pills, preferably oral sprays.

Drugs using cannabidiol or its pharmaceutically acceptable salts, esters, or solvates as the active ingredient can be prepared into any of the above-mentioned pharmaceutical dosage forms according to actual needs, and the drugs of each dosage form can be carried out according to conventional methods in the pharmaceutical field. preparation.

In this application, the route of administration of the drug can be selected from oral administration, sublingual administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral spray administration or transdermal administration according to actual needs. Kind of way.

Compared with the prior art, this application has the following beneficial effects:

This application creatively discovers that cannabidiol or its pharmaceutically acceptable salts, esters, and solvates can be used in the preparation of drugs that promote oral mucosal healing, through the exploration of the effect of cannabidiol on the biological behavior of human oral keratinocytes The investigation of the therapeutic effect of oral ulcer model mice found that it has a significant effect on promoting proliferation and migration of oral mucosal keratinocytes and inhibiting excessive pyrolysis of oral mucosal keratinocytes, and these three effects are concentration and time dependent; It has a significant effect on promoting the healing of oral mucosa of oral ulcer model mice; analysis of transcriptome sequencing technology found that it has a tendency to down-regulate most of the genes in keratinocytes that induce inflammation, especially the activation of NLRP3 inflammasomes and pyrolysis All related genes have a significant down-regulation effect. It provides a theoretical basis for studying the treatment strategy of oral mucosal injury, and provides an embedding point for the preparation of new drugs that promote oral mucosal healing.

Description of the drawings

Figure 1 is the result of the proliferation-promoting effect of cannabidiol on human oral keratinocytes (in the figure, CBD-0, CBD-0.5, CBD-2, CBD-10, CBD-50 represent the concentration of 0, 0.5, 2, 10. The result of 50μmol/L cannabidiol culture medium);

Figure 2 is the result of the effect of cannabidiol on the migration promotion of human oral keratinocytes (from top to bottom, from left to right in the figure represents the administration of cannabidiol at concentrations of 0, 0.5, 2, 10, and 50 μmol/L Scratch cell migration after 0h, 24h, 48h);

Figure 3A is a graph showing the results of the inhibitory effect of cannabidiol on human oral keratinocytes on pyrolysis (Figure 3A represents the ratio of pyrolysis after treatment with cannabidiol and 50μmol/L cannabidiol);

Figure 3B is the result of the inhibitory effect of cannabidiol on human oral keratinocytes on pyrolysis (Figure 3B is the result of statistical analysis of the results of Figure 3A);

Figure 4 is a graph showing the effect of cannabidiol on the body weight of mice;

Figure 5 is a graph showing the effect of cannabidiol on the healing of oral mucosa in mice;

Figure 6 is the results of HE staining of mouse oral tissues (where (a), (b), (c) represent the HE staining results of the negative control group, low concentration drug group, and high concentration drug group, respectively);

Figure 7 is the results of EDU staining of mouse oral tissue (where (a), (b), (c) represent the EDU staining results of the negative control group, the low-concentration drug group, and the high-concentration drug group, respectively);

Figure 8 is the results of immunofluorescence staining of mouse oral tissue (where (a), (b), (c) represent the immunofluorescence staining results of the negative control group, low concentration drug group, and high concentration drug group, respectively);

Figure 9A is a graph of the results of transcriptome sequencing analysis (A is a graph of differential gene expression in cells after administration of cannabidiol);

Figure 9B is a graph of the results of transcriptome sequencing analysis (B is a graph of the enrichment results of NLRP3 inflammasome activation and pyrolysis-related gene sets).

Detailed ways

The technical solutions of the present application will be further explained through specific implementations below. It should be understood by those skilled in the art that the described embodiments are merely to help understand the application and should not be regarded as specific limitations to the application.

The process, conditions, reagents, experimental methods, etc. of the implementation of this application, except for the content specifically mentioned below, are all common knowledge and common knowledge in the field, and this application has no special restrictions on the content. The experimental methods for which specific conditions are not indicated in each embodiment are usually in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer.

Unless otherwise specified, the meanings of all professional terms and scientific terms used in this specification are the same as those generally understood by those skilled in the technical field to which this application belongs. However, in case of conflict, the specification including definitions shall prevail.

The source of the drug cannabidiol is: Yunnan Hanmeng Pharmaceutical Co., Ltd. self-made sample;

Experimental animal SPF grade C57BL/6 mice were purchased from Shanghai Southern Model Biotechnology Co., Ltd., 250-350g, male, all operations on mice were performed in a sterile laminar flow chamber;

Oral keratinocytes (immortalized human oral keratinocytes HOK-16B cultured in SFM medium containing keratinocyte growth factor) were purchased from the No-Hee Park laboratory of the University of California, Los Angeles.

Example 1

Evaluation of Cannabidiol Promoting Proliferation of Oral Keratinocytes

The CCK-8 method is used to detect the viability of oral keratinocytes. The operation method is: prepare a cell suspension, count the cells; inoculate into a 96-well plate, the number of cells per well is about 5000/mL, each group has 3 auxiliary wells, each well Add 100μL of culture fluid; incubate in a 37°C incubator for 24 hours. Then change to a medium containing different concentrations of cannabidiol (0, 0.5, 2, 10, 50 μmol/L) after culturing for 24, 48, 72, and 96 hours, and then use the CCK8 kit to detect the proliferation of oral keratinocytes in different groups (The specific operation steps are carried out in accordance with the kit instructions).

The results are shown in Fig. 1. It can be seen from Fig. 1 that cannabidiol has an effect of significantly promoting the proliferation of oral keratinocytes, and the effect of cannabidiol on the proliferation of oral keratinocytes is significantly concentration-dependent and time-dependent.

Example 2

Evaluation of the effect of cannabidiol on the migration of oral keratinocytes

The cell scratch test detects the migration behavior of oral keratinocytes. The operation method is: prepare a cell suspension and count the cells; inoculate oral keratinocytes HOK-16B in a 12-well plate, the number of cells per well is about 1×10 ⁵ mL, each Set up 3 auxiliary wells, add 800μL of culture medium to each well; after culturing overnight in a 37°C incubator, the bottom of the well plate is covered with a single layer of keratinocytes. Use a 200μl pipette tip to make cell scratches perpendicular to the bottom of the well plate (try to ensure that the width of each scratch is the same). The original culture medium was aspirated, washed three times with PBS, and replaced with serum-free medium containing different concentrations of cannabidiol (0, 0.5, 2, 10, 50 μmol/L), and photographed and recorded. Incubate in a 5% CO2, 37°C incubator, and take pictures every 24 hours.

The results are shown in Figure 2. It can be seen from Figure 2 that cannabidiol has the effect of significantly promoting the migration of oral keratinocytes, and its promotion effect is positively correlated with the concentration.

Example 3

Evaluation of the inhibitory effect of cannabidiol on oral keratinocytes

Annexin V-FITC detects oral keratinocyte pyrolysis, the operation method is: prepare cell suspension, cell count; inoculate oral keratinocyte HOK-16B in 6-well plate, add different concentrations (0, 50μmol/L) The culture medium of cannabidiol is incubated in a 5% CO2, 37°C incubator. After 48 hours of inoculation, the Annexin V-FITC cell pyroptosis detection kit was used to detect the pyroptosis rate (the specific operation steps were carried out in accordance with the kit instructions).

The results are shown in Fig. 3A and Fig. 3B: the pyrolysis rate of HOK-16B cells after 50μmol/L cannabidiol was lower than that of the adjuvant control group without cannabidiol (the Q2 and Q3 quadrants in Fig. 3A represent the early and late pyroptosis的 cells), and it can be seen from Figure 3B that the difference between the two is statistically significant.

Example 4

In vivo evaluation test

The specific operation method is as follows:

(1) Establish a mouse oral ulcer model by mechanical damage method

Adult male mice (250-350g) were placed in a plastic cage and fed normally for 5 days to adapt to the environment of the animal experiment center. Randomly divide into 3 groups, each with 30 animals. After the three groups of mice were anesthetized by intraperitoneal injection, a 5mm inner diameter circumcision drill was used to directly drill the mucosal tissue of the back of the tongue, resulting in a defect of about 5mm in diameter and about 1mm in depth, and continued to be given a high-viscosity diet. Ulcers formed one day after modeling .

(2) Set up negative control group (1mL saline instead of medicine), low-concentration drug group (1mg/mL) and high-concentration drug group (10mg/mL), starting from the day of oral ulcer formation (1 day after modeling) The three groups of mice were sprayed orally at the ulcer on the back of the tongue, once a day. Samples were collected for observation 3 days after administration (4 days after modeling).

Evaluation indicators include the following:

(1) Weight change monitoring

The method of operation is as follows: from the day of oral ulcer modeling, daily monitoring of the changes in body weight of mice in each group. The results are shown in Figure 4: As the course of the disease progresses, the weight of the mice first drops and then rises. There is no significant difference in weight change between the low-concentration administration group and the control group, and the weight recovery of the high-concentration administration group is faster.

(2) Monitoring of changes in ulcer size

The method of operation is as follows: From the day of oral ulcer modeling, measure the maximum diameter (D) and minimum diameter (d) of the oral ulcer lesion every day, and record the ulcer size as the average of the two. The results are shown in Figure 5. It can be seen from the figure that the size of the ulcer is significantly reduced after the local spray treatment of cannabidiol, that is, the oral mucosa is promoted after administration, and the healing effect of the high concentration group is more significant than that of the low concentration group.

(3) HE staining

The operation method is: after routine dewaxing of the paraffin sections of the tongue tissue, staining with hematoxylin, washing with water and staining with eosin, dehydrating the section with gradient alcohol, sealing the slide with neutral gum after transparency, observing and taking pictures under an inverted microscope.

The results are shown in Figure 6. It can be seen from the figure (the dotted line is the defect): the epithelial barrier of the high concentration and low concentration drug groups has been basically restored after three days of drug administration (Figure 6-b, c). There was a little inflammatory cell infiltration under the skin (Figure 6-b), while the epithelial layer of the negative control group was not fully recovered, there were still obvious defects, and the defect and its surrounding inflammatory cell infiltration were obvious (Figure 6-a). It can be seen that the administration of cannabidiol can significantly promote the healing of oral mucosal ulcers.

(4) EDU dyeing

The operation method is: two hours before the collection of the sample, the mouse is injected with EDU into the abdominal cavity, two hours later, the sample is collected, and the tissue is made into paraffin sections. After routine deparaffinization, 0.5% TritonX-100 ruptures the tissue. After EDU is incubated at room temperature for 30 minutes in the dark, DAPI recovers Stain cell nuclei, mount the slides, observe and take pictures under a fluorescence microscope.

As shown in Figure 7, it can be seen from the figure (the dotted line is the defect): Three days after the drug was administered, the epithelial and subepithelial connective tissue cells around the defect in the high-concentration drug group proliferated significantly (Figure 7-c), and the low-concentration drug group was only in the defect There was significant proliferation of surrounding epithelial cells (Figure 7-b), while the negative control group had no significant cell proliferation at or around the defect (Figure 7-a). It can be seen that the administration of cannabidiol can significantly promote the rapid proliferation of epithelial and subepithelial connective tissue cells at the edge of the lesion, and the promotion effect is positively correlated with drug concentration.

(5) Immunofluorescence staining

The method of operation is: fix the tongue tissue after sampling, make tissue paraffin sections, after routine deparaffinization, sodium citrate antigen heat repair, hydrogen peroxide inhibits endogenous peroxisomes, incubate the primary antibody overnight at 4°C. Incubate the secondary antibody at 37°C for 1 hour, counter-stain the cell nucleus with DAPI, mount the slide, observe under a fluorescent microscope, and take pictures.

As shown in Figure 8, it can be seen from the figure (the dotted line is the defect): Three days after the drug was administered, the defect in the negative control group and the subepithelial connective tissue around the defect had obvious inflammatory factor IL-6 infiltration (Figure 8-a), In the low-concentration drug group, there was only a small amount of inflammatory factor IL-6 infiltration in the subepithelial connective tissue of the defect (Figure 8-b), while the high-concentration drug group had no obvious inflammatory infiltration at or around the defect (Figure 8-c) . It shows that the administration of cannabidiol can inhibit the release of inflammatory factor IL-6, and the effect is positively correlated with the administration concentration.

Example 5

Transcriptome sequencing analysis

Transcriptome sequencing technology was used to analyze the changes in related genes after inflammatory-stimulated oral keratinocytes were treated with cannabidiol. The specific operation method is as follows: prepare a cell suspension and count the cells; inoculate oral keratinocytes HOK-16B in a 6-well plate, incubate in a 5% CO2, 37°C incubator for 18 hours, and give LPS (100ng/mL) for 6 hours After adding different concentrations of (0, 50μmol/L) cannabidiol for 2h, and finally adding ATP (4mM) for 30min, the cells were collected, and total RNA was extracted for transcriptome sequencing analysis.

The results are shown in Figures 9A and 9B. It can be seen from the figure that most of the genes in the keratinocytes that induce inflammation in vitro are down-regulated after treatment with cannabidiol (the point in Figure 9A is that the cannabidiol treatment group has a higher expression than the control group). Different genes, the abscissa represents the multiple of the difference, plus or minus represents up and down, the larger the absolute value, the greater the multiple of the difference; the ordinate represents the adjusted P value, the larger the value, the more significant the difference, so the horizontal and vertical in the figure The area separated by the dotted line, that is, the upper left grid and the upper right grid are considered to be genes that are significantly down-regulated and significantly up-regulated after treatment with cannabidiol). Compared with the control group, the administration group listed in Figure 9B has significantly down-regulated genes related to NLRP3 inflammasome activation and pyrolysis (CK1-3 is the administration group, NC1-3 is the control group, and the color ranges from light to Deep is the expression from low to high).

The applicant declares that this application uses the above examples to illustrate the application of the cannabidiol of this application in the preparation of drugs for promoting oral mucosal healing, but this application is not limited to the above examples, which does not mean that this application must rely on the above The embodiment can be implemented. Those skilled in the art should understand that any improvement to this application, the equivalent replacement of each raw material of the product of this application, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

The preferred embodiments of the present application are described in detail above. However, the present application is not limited to the specific details in the foregoing embodiments. Within the scope of the technical concept of the present application, a variety of simple modifications can be made to the technical solution of the present application. These simple modifications All belong to the protection scope of this application.

In addition, it should be noted that the various specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application provides various possible combinations. The combination method will not be explained separately.

Claims (13)

1. Use of cannabidiol or its pharmaceutically acceptable salts, esters and solvates in the preparation of drugs for promoting oral mucosal healing.
2. The use according to claim 1, wherein the drug promotes proliferation of oral keratinocytes.
3. The use according to claim 1, wherein the medicine promotes the migration of oral keratinocytes.
4. The use according to any one of claims 1 to 3, wherein the drug inhibits oral keratinocyte pyrolysis.
5. The use according to any one of claims 1 to 4, wherein the drug inhibits the NLRP3 inflammasome activation pathway.
6. Use of cannabidiol or its pharmaceutically acceptable salts, esters and solvates in the preparation of oral keratinocyte proliferation promoters.
7. Use of cannabidiol or its pharmaceutically acceptable salts, esters and solvates in the preparation of oral keratinocyte migration promoters.
8. Use of cannabidiol or its pharmaceutically acceptable salts, esters and solvates in the preparation of oral keratinocyte pyrolysis inhibitors.
9. Use of cannabidiol or its pharmaceutically acceptable salts, esters and solvates in the preparation of NLRP3 inflammasome activation pathway inhibitors.
10. The use according to any one of claims 1-9, wherein the medicine further comprises pharmaceutically acceptable excipients.
11. The application according to claim 10, wherein the auxiliary material comprises any one or a combination of at least two of diluents, carriers, flavoring agents, binders or fillers.
12. The use according to claim 11, wherein the carrier comprises liposomes, micelles, dendrimers, microspheres or microcapsules.
13. The application according to any one of claims 1-12, wherein the dosage form of the drug includes tablets, capsules, granules, powders, injections, sprays, films, suppositories, nasal drops or pills , Preferably oral spray.

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