Application of bile acid derivative in preparation of radiation prevention and treatment medicine

Abstract

The invention relates to the field of biological pharmacy, in particular to application of bile acid derivatives in preparation of radiation prevention and treatment drugs. The bile acid derivative is one or more of glycodeoxycholic acid and a pharmaceutically acceptable derivative thereof, and glycochenodeoxycholic acid and a pharmaceutically acceptable derivative thereof. The glycodeoxycholic acid and the glycochenodeoxycholic acid both can reduce the sensitivity of epidermal cells to radiation, and are beneficial to improving the cell viability and inhibiting cell apoptosis. The irradiated skin is treated through the glycodeoxycholic acid or the glycochenodeoxycholic acid, damage and inflammation of the skin can be relieved, the area proportion of erythema desquamation in the irradiated area is reduced, and therefore the treatment effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to the use of a bile acid derivative in the preparation of radiation prevention and treatment drugs. Background Art

[0002] Bile acids (BA) are amphoteric steroids derived from cholesterol and are primarily found in the bile of mammals and animals. They are primarily found in the bile of mammals and other vertebrates. Traditional theory suggests that bile acids primarily function in the digestive system, such as promoting the absorption of dietary cholesterol, but bile acid metabolites have also been reported to have multiple functions. Studies have shown that bile acids have therapeutic effects on radiation-induced skin damage. In order to practically apply bile acids to the preparation of radiation prevention and treatment drugs, chemically modifying bile acid derivatives that also have the ability to treat radiation-induced skin damage is of high clinical significance. Summary of the Invention

[0003] The object of the present invention is to provide a bile acid derivative capable of alleviating radiation-induced skin tissue damage.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] A use of a bile acid derivative in the preparation of a radiation prevention and treatment drug, wherein the bile acid derivative is one or more of glycodeoxycholic acid and its pharmaceutically acceptable derivatives, and glycochenodeoxycholic acid and its pharmaceutically acceptable derivatives.

[0006] Optionally, the bile acid derivative is used to reduce the radiosensitivity of irradiated epidermal cells.

[0007] Optionally, the bile acid derivative is used to inhibit the impaired proliferation capacity of irradiated epidermal cells caused by radiation.

[0008] Optionally, the bile acid derivative is used to inhibit radiation-induced apoptosis of irradiated epidermal cells.

[0009] Optionally, the bile acid derivative is used to treat skin erythema caused by radiation exposure.

[0010] Optionally, the bile acid derivative is used to treat skin damage caused by radiation exposure.

[0011] Optionally, the bile acid derivative is used to treat skin inflammation caused by radiation exposure.

[0012] Optionally, the drug is administered by subcutaneous injection into the wound.

[0013] Optionally, the drug further comprises excipients, wherein the excipients comprise excipients and / or carriers, and the carriers comprise one or more of diluents, buffers and suspending agents.

[0014] Optionally, the concentration of the bile acid derivative in the drug is any value between 1 μmol / mL and 15 μmol / mL.

[0015] The beneficial effects of the present invention are that both glycodeoxycholic acid and glycochenodeoxycholic acid can reduce the sensitivity of epidermal cells to radiation, help increase cell activity, and inhibit cell apoptosis. Treating irradiated skin with glycodeoxycholic acid or glycochenodeoxycholic acid helps alleviate skin damage and inflammation, and reduces the proportion of erythema and desquamation in the irradiated area, thereby achieving a therapeutic effect.

[0016] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a staining and quantitative analysis diagram of the plate colony formation experiment shown in Example 1 of the present invention;

[0018] Figure 2 This is a quantitative analysis chart of cell activity and LDH release in the cell injury experiment shown in Example 1 of the present invention;

[0019] Figure 3 The flow cytometry scatter plot and the quantitative analysis graph of cell apoptosis rate in the fluorescence staining experiment shown in Example 1 of the present invention are shown;

[0020] Figure 4 This is a diagram analyzing the expression of apoptosis-related proteins in the immunoblotting experiment shown in Example 1 of the present invention;

[0021] Figure 5 These are diagrams showing changes in skin wounds in rats of each group after irradiation and treatment as shown in Example 1 of the present invention;

[0022] Figure 6 This is a statistical graph of skin injury scores of rats in each group after irradiation and treatment as shown in Example 1 of the present invention;

[0023] Figure 7 These are staining images of skin wound tissue samples of rats in each group after irradiation and treatment as shown in Example 1 of the present invention;

[0024] Figure 8 This is an analysis chart of interleukin-6 expression in rats of each group after irradiation and treatment as shown in Example 1 of the present invention;

[0025] Figure 9 This is an analysis chart of interleukin-10 expression in rats of each group after irradiation and treatment as shown in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0027] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0028] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0029] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] According to the existing technology in this field, different bile acid derivatives have different specific effects. For example, studies have shown that tauro-β-polyphenolic acid (T-β-MCA) and deoxycholic acid (DCA) can induce malignant transformation of cancerous liver cells and promote the development of adenomas to adenocarcinomas. Other studies have shown that the bile acid metabolite isolithocholic acid (isoLCA) can inhibit T helper cells expressing IL-17A, thereby promoting the occurrence of inflammatory bowel disease. And upregulation of taurodeoxycholic acid (TDCA) can induce the autoimmune disease celiac disease, stimulate inflammatory responses in the small intestine, and lead to villous atrophy. In addition, there are reports of the existence of thousands of previously unknown bile acid metabolites in the human body, but the specific functions of most bile acid metabolites are still unclear. In summary, there are many types of bile acid derivatives, and the functions of various bile acid derivatives are different, so they cannot be generalized.

[0031] The present invention relates to the use of a bile acid derivative in the preparation of radiation prevention and treatment drugs. The bile acid derivative in the present invention is one or more of glycodeoxycholic acid and its pharmaceutically acceptable derivatives, and glycochenodeoxycholic acid and its pharmaceutically acceptable derivatives.

[0032] Both glycodeoxycholic acid and glycochenodeoxycholic acid can reduce the sensitivity of epidermal cells to radiation, help increase cell activity, and inhibit apoptosis. Treating irradiated skin with glycodeoxycholic acid or glycochenodeoxycholic acid can help reduce skin damage and inflammation, and reduce the incidence of erythema and desquamation in the irradiated area, thereby achieving a therapeutic effect.

[0033] It should be noted that the chemical formulas of glycodeoxycholic acid and glycochenodeoxycholic acid are both C 26 H 43 NO5, but has a different chemical structure, isomers, so the chemical properties are different, should not be generalized. Pharmaceutically acceptable derivatives can be, for example, pharmaceutically acceptable salts.

[0034] In some embodiments, bile acid derivatives are used to reduce the radiosensitivity of irradiated epidermal cells.

[0035] In some embodiments, bile acid derivatives are used to inhibit radiation-induced impairment of the proliferation capacity of irradiated epidermal cells.

[0036] In some embodiments, bile acid derivatives are used to inhibit radiation-induced apoptosis of irradiated epidermal cells.

[0037] In some embodiments, bile acid derivatives are used to treat skin erythema induced by radiation exposure.

[0038] In some embodiments, bile acid derivatives are used to treat skin damage caused by radiation exposure.

[0039] In some embodiments, bile acid derivatives are used to treat skin inflammation caused by radiation exposure.

[0040] In some embodiments, the drug is administered by subcutaneous injection into the wound.

[0041] In some embodiments, the drug further includes excipients, which include excipients and / or carriers, and carriers include one or more of diluents, buffers, and suspending agents.

[0042] In some embodiments, the concentration of the bile acid derivative in the drug is any value between 1 μmol / mL and 15 μmol / mL, for example, 1 μmol / mL, 3 μmol / mL, 7 μmol / mL, 11 μmol / mL and 15 μmol / mL. Within this concentration range, the bile acid derivative has a higher therapeutic effect on radiation-induced human epidermal cell proliferation inhibition and causes less damage to the cells.

[0043] Please refer to the following examples for details.

[0044] Example 1:

[0045] Glycodeoxycholic acid and glycochenodeoxycholic acid were purchased from MedChemExpress (https: / / www.medchemexpress.cn). The drug ID number for glycodeoxycholic acid is 360-65-6, and the drug ID number for glycochenodeoxycholic acid is 640-79-9. Glycodeoxycholic acid and glycochenodeoxycholic acid were dissolved in phosphate-buffered saline (PBS) to prepare glycodeoxycholic acid solutions and glycochenodeoxycholic acid solutions of varying concentrations.

[0046] Human immortalized epidermal (HaCaT) cells were used for in vitro cell experiments.

[0047] The effects of glycodeoxycholic acid and glycochenodeoxycholic acid on the radiosensitivity of irradiated skin cells were evaluated by plate colony formation assay. The cells were cultured to the logarithmic growth phase, collected and washed to obtain a single cell suspension, counted and seeded in a culture dish containing the same culture medium at a cell density of 2000 cells per dish, and equal amounts of 7.5 μmol / L glycodeoxycholic acid (GDCA) solution, 12.5 μmol / L glycochenodeoxycholic acid (GCDCA) solution and phosphate buffered saline (PBS) were added, respectively. The glycodeoxycholic acid solution and glycochenodeoxycholic acid solution in the present invention are both solutions dissolved in PBS. After continued culture for 24 hours, sham irradiation, 5 mJ / cm 2 Single exposure to ultraviolet light and 10mJ / cm 2The cells were irradiated with ultraviolet light once, collected and washed again to obtain a single cell suspension, counted, and seeded into culture dishes containing the same culture medium at a density of 2,000 cells per dish. After 10 days of culture, visible colonies appeared. The culture medium was discarded, and the cells were washed and fixed. The number of cell colonies was observed after staining with crystal violet and counted under a microscope. Figure 1 It can be seen that pretreatment with glycodeoxycholic acid or glycochenodeoxycholic acid in the culture medium can reduce the decrease in cell clone formation ability caused by ultraviolet irradiation, indicating that glycodeoxycholic acid or glycochenodeoxycholic acid has the effect of reducing the radiosensitivity of irradiated cells.

[0048] The effects of glycodeoxycholic acid and glycochenodeoxycholic acid on the proliferation of irradiated cells were evaluated by cell injury experiments. The cells were cultured to the logarithmic growth phase, and single cell suspensions were collected and washed to obtain suspensions. The suspensions were then seeded at the same density in 96-well plates. After the cells adhered to the plate, the original culture medium was discarded, and culture medium containing equal amounts of glycodeoxycholic acid solution or glycochenodeoxycholic acid solution at different concentrations was added. After culturing for 24 hours, each culture well was exposed to 10 mJ / cm 2 After a single irradiation with ultraviolet light, the culture medium in each well was collected. After the cells were washed with PBS, 100 μl of fresh culture medium and 10 μl of CCK-8 reagent were added to each well for activity detection. The culture medium was used to detect the release of LDH from the cells using a lactate dehydrogenase (LDH) detection reagent. In this experiment, nine concentrations of 1 μmol / L, 5 μmol / L, 7.5 μmol / L, 10 μmol / L, 12.5 μmol / L, 15 μmol / L, 17.5 μmol / L, 20 μmol / L and 22.5 μmol / L were used for both glycodeoxycholic acid solution and glycochenodeoxycholic acid solution. For experimental results, please see Figure 2 , it can be seen that pretreatment with glycodeoxycholic acid or glycochenodeoxycholic acid can reduce the cell proliferation inhibition caused by UV irradiation and promote skin cell proliferation. At the same time, pretreatment with glycodeoxycholic acid or glycochenodeoxycholic acid increases the release of LDH, inducing cell damage, and this induction ability is dose-dependent. Therefore, the concentration of glycodeoxycholic acid or glycochenodeoxycholic acid for treating UV damage should be limited to 1μmol / mL to 15μmol / mL.

[0049] The effects of glycodeoxycholic acid and glycochenodeoxycholic acid on apoptosis of irradiated cells were evaluated by fluorescence staining experiments. The cells were cultured to the logarithmic growth phase, and after washing to obtain a single cell suspension, they were inoculated into culture dishes. After the cells attached to the wall, the original culture medium was discarded, and a culture medium containing equal amounts of 7.5μmol / L glycodeoxycholic acid (GDCA) solution, 12.5μmol / L glycochenodeoxycholic acid (GCDCA) solution, and phosphate buffered saline (PBS) was added. After 24 hours of culture, the two samples with the same treatment solution were sham irradiated and irradiated with 10mJ / cm 2 After irradiation, cells were collected and resuspended in PBS. A portion of the cell suspension was added to a flow cytometer tube and stained with Annexin V (AV) and propidium iodide (PI) using a kit. AV was labeled with fluorescein isothiocyanate (FITC). Flow cytometry was used to generate flow cytometric scatter plots and quantitatively analyze the cell apoptosis rate. Figure 3 It can be seen that pretreatment with glycodeoxycholic acid or glycochenodeoxycholic acid can reduce cell apoptosis caused by ultraviolet irradiation, indicating that glycodeoxycholic acid or glycochenodeoxycholic acid has the effect of reducing the apoptosis of irradiated skin cells.

[0050] HaCaT cells were treated with the same drugs and irradiation as in the fluorescent staining experiments, and immunoblotting (WB) was performed. β-Actin was used as an internal control to detect changes in the expression of apoptosis-related proteins poly(ADP-ribose) polymerase (PARP), cleaved poly(ADP-ribose) polymerase (C-PARP), cleaved cytoplasmic caspase 3 (C-Cas3), cytoplasmic caspase 3 (Cas3), Bcl-2-associated X protein (BAX), and Bcl-2. PARP and Cas3 are both key proteins in apoptosis, and the appearance of their cleaved forms, C-PARP and C-Cas3, is a hallmark of apoptosis. BAX is a pro-apoptotic protein, and Bcl-2 is an anti-apoptotic protein. (See [ 1 ] for details.) Figure 4 It can be seen that pretreatment with glycodeoxycholic acid or glycochenodeoxycholic acid can reduce the cell apoptosis caused by irradiation, further verifying the conclusion of the fluorescence staining experiment.

[0051] Male SD rats aged 6 to 8 weeks were purchased from Chengdu Dashuo Animal Co., Ltd. for in vivo animal experiments.

[0052] After adaptive feeding, rats were randomly divided into three groups, with six rats in each group. After anesthesia with 1% chloral hydrate injected intraperitoneally at 1% of each rat's body weight, the rats' hind limbs were depilated and fixed to a wooden board to reduce accidental movement during irradiation. The rats were irradiated with a 3mW / cm2 UV irradiator generated by a Bio-SUN UV irradiator from Vilber, France.2 Ultraviolet rays were irradiated on the rat buttocks skin at a dose of 2 J / cm 2 The irradiation area was outlined with a marker before irradiation. Immediately after irradiation, the drug was administered by topical application to the irradiated area. The three groups of rats were smeared with 7.5 μmol / L glycodeoxycholic acid solution, 12.5 μmol / L glycochenodeoxycholic acid solution, and phosphate buffer solution, respectively, with a dosage of 1 mL for each. The drug was administered once after each UV irradiation, for a total of 4 times. After treatment, the changes in the rats' buttocks skin were observed daily. Figure 5 , it can be seen that the rats in the groups that received subcutaneous injections of glycodeoxycholic acid solution and glycochenodeoxycholic acid solution experienced slower progression of UV skin damage than the group that received phosphate buffer saline injection, and the area damaged by UV radiation was smaller. Comparison of the skin wounds of the three groups of rats showed that four days after UV irradiation and phosphate buffer saline injection, large areas of erythema and desquamation appeared on the rat skin surface, while only a few areas of erythema and desquamation appeared on the rat skin after treatment with glycodeoxycholic acid or glycochenodeoxycholic acid, indicating that glycodeoxycholic acid or glycochenodeoxycholic acid can reduce UV skin damage and promote wound healing.

[0053] The skin wounds of the rats in each group were scored according to existing techniques in the field and will not be described in detail here. For specific scoring information, please refer to Table 1 below.

[0054] Table 1:

[0055]

[0056]

[0057] Take the mean value of each group as the skin damage score and draw a line graph. Figure 6 It can be seen that treatment with glycodeoxycholic acid or glycochenodeoxycholic acid can effectively reduce UV skin damage and promote wound healing.

[0058] from Figure 6 As can be seen from the figure, the skin damage of rats was most serious on the 10th day after treatment. The tissues at the wound surface of the rat skin on the 10th day after treatment were longitudinally sectioned and stained with hematoxylin-eosin (HE) to observe the damage to the rat skin epidermis. Figure 7 , it can be seen that glycodeoxycholic acid or glycochenodeoxycholic acid treatment can effectively reduce skin epidermal damage caused by ultraviolet rays.

[0059] Skin tissue from the wound surface and unirradiated skin tissue from each group of rats 10 days after irradiation and treatment were collected and fixed in 4% paraformaldehyde. The same sample was divided into two parts and immunofluorescence stained with primary and secondary antibodies against interleukin-6 (IL-6) and interleukin-10 (IL-10), respectively. The sections were observed using a fluorescence microscope, and the fluorescence images were analyzed using image analysis software. The fluorescence intensity of the unirradiated control group samples and the irradiated and treated experimental group samples was measured, and the ratio was calculated to determine the expression of IL-6 and IL-10. Please refer to Figure 8 and Figure 9 It can be seen that compared with PBS treatment, the expression of IL-6 was significantly downregulated and the expression of IL-10 was significantly upregulated after treatment with glycodeoxycholic acid or glycochenodeoxycholic acid, which indicates that glycodeoxycholic acid or glycochenodeoxycholic acid has the effect of alleviating ultraviolet radiation inflammation.

[0060] In summary, glycodeoxycholic acid or glycochenodeoxycholic acid can enhance cell proliferation and repair damaged tissues after ultraviolet radiation damage. It can also reduce skin radiosensitivity and apoptosis. Therefore, the use of glycodeoxycholic acid or glycochenodeoxycholic acid provided in this application in the preparation of a drug for treating radiation-induced inflammatory skin damage has important significance and practical value.

[0061] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0062] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Use of a bile acid derivative in the preparation of radiation prevention and treatment drugs, characterized in that: The bile acid derivatives are one or more of glycodeoxycholic acid and pharmaceutically acceptable derivatives thereof, and glycochenodeoxycholic acid and pharmaceutically acceptable derivatives thereof.

2. The use according to claim 1, characterized in that The bile acid derivatives are used to reduce the radiosensitivity of irradiated epidermal cells.

3. The use according to claim 1, wherein The bile acid derivative is used for inhibiting the impaired proliferation ability of irradiated epidermal cells caused by radiation.

4. The use according to claim 1, wherein The bile acid derivative is used for inhibiting apoptosis of irradiated epidermal cells caused by radiation.

5. The use according to claim 1, characterized in that The bile acid derivative is used for treating skin erythema caused by radiation exposure.

6. The use according to claim 1, wherein The bile acid derivative is used for treating skin damage caused by radiation exposure.

7. The use according to claim 1, wherein The bile acid derivative is used for treating skin inflammation caused by radiation exposure.

8. The use according to any one of claims 1 to 7, characterized in that The drug is administered through subcutaneous injection on the wound surface.

9. The use according to claim 8, characterized in that The medicine further comprises excipients, wherein the excipients comprise excipients and / or carriers, and the carriers comprise one or more of diluents, buffers and suspending agents.

10. The use according to claim 8, characterized in that The concentration of the bile acid derivative in the drug is any value between 1 μmol / mL and 15 μmol / mL.

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