Application of chenodeoxycholic acid in preparation of medicine for treating acne

Abstract

The invention relates to application of chenodeoxycholic acid or pharmaceutically acceptable salt thereof in preparation of a medicine for treating acne. The chenodeoxycholic acid disclosed by the invention can be a natural organic compound or a synthetic organic compound, has relatively strong propionibacterium acnes inhibitory activity and a pharmacodynamic effect of resisting lipid secretion, and is used as an active component to be prepared into a pharmaceutical composition for treating acnes together with a pharmaceutically acceptable carrier.

Description

Technical Field

[0001] The invention relates to use of chenodeoxycholic acid or a pharmaceutically acceptable salt thereof in preparing a medicine for treating acne. Background Art

[0002] Acne is a common chronic inflammatory skin disease that occurs mainly on the face, neck, chest, back, shoulders, etc. It can occur in people of all ages, but the highest incidence rate is in adolescents. More than 85% of adolescents will be troubled, and permanent scars and skin lesions will occur, affecting their appearance. In 2019, there were 117 million cases of acne worldwide, and 231 million prevalent cases, an increase of about 47% from 1990.

[0003] The pathogenesis of acne involves multiple factors and is caused by the combined action of multiple factors, mainly excessive sebum secretion, obstruction of the sebaceous gland ducts, bacterial infection and inflammatory response.

[0004] At present, the main drug treatment method for acne is to use retinoic acid drugs, whose mechanism of action is to reduce inflammation and keratinization and inhibit sebum secretion, and the side effects are dry skin, inflammation, desquamation, etc. In addition, Chinese patent CN109475564A discloses a composition containing water-soluble ursodeoxycholic acid for preventing or treating inflammatory skin diseases or severe pruritus, which is used to prevent or treat atopic skin diseases, acne, psoriasis, urticaria, inflammatory dermatitis, seborrheic dermatitis and contact dermatitis, but the therapeutic effect of ursodeoxycholic acid (UDCA) on acne is still unsatisfactory.

[0005] Drugs for treating acne that meet clinical needs need to have anti-inflammatory, anti-keratinization, antibacterial and other effects, be able to inhibit sebum secretion, regulate sebaceous gland homeostasis and improve the microenvironment, and be safe with few side effects. Therefore, it is very meaningful to explore and find drugs with good therapeutic effects.

[0006] The main known use of chenodeoxycholic acid (CDCA) is to reduce the cholesterol saturation in bile, but existing evidence has not reported that chenodeoxycholic acid has an inhibitory effect on Propionibacterium acnes and can be used to treat acne. Summary of the invention

[0007] 1. Technical issues to be resolved The technical problem to be solved by the present invention is to solve the problem that the compounds in the prior art have poor therapeutic effects on acne and have large side effects.

[0008] (II) Technical solution In order to solve the above problems existing in the prior art, the present invention establishes three classic models by simulating the three main pathological factors of acne in vitro: (1) excessive proliferation of Propionibacterium acnes; (2) excessive proliferation and inflammatory response of epidermal cells induced by Propionibacterium acnes; (3) excessive sebum secretion of sebaceous gland cells. In these models, the anti-inflammatory, antibacterial and anti-lipid secretion effects of ursodeoxycholic acid and chenodeoxycholic acid were compared. The experimental results show that chenodeoxycholic acid has a lower effective concentration than ursodeoxycholic acid and exhibits good concentration dependence. Studies have shown that chenodeoxycholic acid, as a core active ingredient for the treatment of acne, exhibits significant therapeutic effects in anti-inflammatory, antibacterial and lipid secretion inhibition, which is significantly better than ursodeoxycholic acid, thereby providing the use of chenodeoxycholic acid or its pharmaceutically acceptable salt in the preparation of acne treatment drugs, and the use of chenodeoxycholic acid in the preparation of Propionibacterium acnes inhibitors.

[0009] The object of the present invention is to provide use of chenodeoxycholic acid or a pharmaceutically acceptable salt thereof in preparing a drug for treating acne.

[0010] According to the above-mentioned use, the pharmaceutically acceptable salt is selected from sodium salt, potassium salt, magnesium salt, calcium salt or ammonium salt.

[0011] According to the above-mentioned use, the chenodeoxycholic acid or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier are prepared into a pharmaceutical composition.

[0012] According to the above-mentioned use, the pharmaceutical composition is selected from tablets, capsules, pills or injections.

[0013] Another object of the present invention is to provide use of chenodeoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of a Propionibacterium acnes inhibitor.

[0014] According to the above-mentioned use, the pharmaceutically acceptable salt is selected from sodium salt, potassium salt, magnesium salt, calcium salt or ammonium salt.

[0015] According to the above-mentioned use, the chenodeoxycholic acid or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier are prepared into a pharmaceutical composition.

[0016] According to the above-mentioned use, the pharmaceutical composition is selected from tablets, capsules, pills or injections.

[0017] (III) Beneficial effects The above technical solution of the present invention has the following advantages: (1) The chenodeoxycholic acid of the present invention can effectively inhibit the excessive proliferation of Propionibacterium acnes, inhibit the excessive proliferation and inflammatory response of epidermal cells induced by Propionibacterium acnes, and inhibit the excessive secretion of sebum by sebaceous gland cells, with less side effects.

[0018] (2) Compared with ursodeoxycholic acid, the chenodeoxycholic acid of the present invention has a lower effective concentration and exhibits good concentration dependence, indicating that chenodeoxycholic acid, as a core active ingredient for treating acne, exhibits significant therapeutic effects in anti-inflammatory, antibacterial and lipid secretion inhibition, and is significantly superior to ursodeoxycholic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the effect of UDCA and CDCA on the proliferation of epidermal cells under the stimulation of P. acnes; Figure 2 It is the inhibitory effect of CDCA and UDCA on the growth of Propionibacterium acnes; Figure 3 is the effect of CDCA and UDCA on lipid secretion of human primary sebaceous gland cells induced by linoleic acid; Figure 4 The effects of CDCA and UDCA on the expression of TNF-α and IL-1α in epidermal cells. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 are within the scope of protection of the present invention.

[0021] The chenodeoxycholic acid of the present invention may be a naturally occurring compound or an artificially synthesized compound.

[0022] The chemical formula of chenodeoxycholic acid is C 24 H 40 O4, chemical name 3α,7α-dihydroxy-5β-cholanoic acid, abbreviated as CDCA in English, chemical structure is as follows: The main known use of chenodeoxycholic acid is to reduce the saturation of cholesterol in bile, and it is widely used in fat accumulation and lipid metabolism diseases. The present invention makes an in-depth study on the effect of chenodeoxycholic acid in treating acne, and provides a basis for screening highly effective drugs for treating acne.

[0023] The present invention established three classic models by simulating the three main pathological factors of acne in vitro: (1) excessive proliferation of Propionibacterium acnes; (2) excessive proliferation and inflammatory response of epidermal cells induced by Propionibacterium acnes; and (3) excessive sebum secretion of sebaceous gland cells. The anti-inflammatory, antibacterial and anti-lipid secretion effects of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) were compared in these models. Specific embodiments

[0024] In order to better understand the present invention, the present invention is further explained or illustrated by specific examples below, but these examples should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1: Comparative study of the effects of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) on anti-inflammatory and abnormal proliferation of epidermal cells in vitro Experimental methods: (1) Bacterial culture: Propionibacterium acnes (P. acnes) was inoculated into brain heart infusion (BHI) broth medium and placed in an anaerobic incubator for 3 days at 37°C. After the incubation, the OD600 value was measured at a wavelength of 600 nm, and the bacterial solution was diluted with BHI medium to OD600 = 0.1 (approximately 1×10 8 CFU / mL), and set aside.

[0026] (2) Cell culture: Human keratinocytes (HaCaT cells) were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. During the logarithmic growth phase of the cells, the cells were made into a single cell suspension and the concentration was adjusted to 1×10 6 Pieces / mL.

[0027] (3) Group treatment: set up the control group CTRL (HaCaT cells only), P. acnes group (HaCaT cells co-cultured with P. acnes), P. acnes + UDCA / CDCA (HaCaT cells co-treated with P. acnes and UDCA or CDCA, with a concentration gradient of 100 μmol / L-500 μmol / L). Prepare the corresponding concentration of P. acnes suspension in DMEM medium in advance, and add the corresponding concentration of UDCA and CDCA according to the experimental requirements.

[0028] (4) CCK8 detection of cell proliferation activity: After 24 hours of culture, add 10 μl of CCK-8 solution and 90 μl of serum-free culture medium to each well, and gently shake the culture plate to ensure that the CCK-8 solution is evenly distributed. Then, incubate at 37°C and 5% CO2 for 30 minutes, and measure the absorbance at 450 nm using a microplate reader. Finally, calculate the effect of the drug on cell proliferation according to the following formula: Proliferation rate (cell viability) = [(experimental group - blank control) / (negative control - blank control)] × 100%.

[0029] Experimental results: In the in vitro model of acne, epidermal cells were stimulated with P. acnes, successfully simulating the pathological process of abnormal proliferation of epidermal cells during acne. The experimental results showed that after adding P. acnes, epidermal cells showed obvious abnormal proliferation. On this basis, UDCA and CDCA were added respectively, and it was found that both could inhibit the abnormal proliferation of epidermal cells induced by P. acnes, and the effect of CDCA was significantly better than that of UDCA. The results are shown in Figure 1 . Propionibacterium acnes (P. acnes) stimulates excessive proliferation of epidermal cells, and the effect of adding different concentrations (0-500 μmol / L) of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) after 24 hours of culture. Cell proliferation activity was detected by CCK-8 method, and the results are shown in line graph ( Figure 1 A) and histogram ( Figure 1 B). The data were statistically tested using two-way analysis of variance (ANOVA), with a sample size of n=5. Significant differences were noted as follows: , , , .

[0030] CDCA can significantly inhibit epidermal cell proliferation at a concentration of 100 μmol / L, with an inhibition rate of 45.14%. In contrast, UDCA only starts to take effect at 140 μmol / L, with an inhibition rate of 9.1%.

[0031] In addition, at the same drug concentration, the inhibition rate of CDCA was 1.74 to 9.39 times higher than that of UDCA, especially at 160 μmol / L, which was as high as 9.93 times. The results are shown in Table 1.

[0032] Table 1 Inhibitory effects of UDCA and CDCA on P. acnes-induced epidermal cell hyperproliferation Note: (1) Cell viability (%): obtained by CCK-8 assay, indicating the relative viability of cells after treatment. (2) Inhibition rate (%): calculated as follows: inhibition rate = (1-cell viability of experimental group / cell viability of negative control group) × 100%; (3) CDCA and UDCA inhibition ratio: indicates the inhibitory effect of CDCA relative to UDCA at the same or similar concentration; (4) "——": indicates no comparability or no data; (5) Onset inhibitory concentration: CDCA showed significant inhibitory effect at 100 μmol / L, with an inhibition rate of 45.14%; while UDCA began to take effect at 140 μmol / L, with an inhibition rate of 9.10%; (6) Maximum inhibition ratio: at a concentration of 160 μmol / L, the inhibition rate of CDCA was 9.39 times that of UDCA, showing a stronger inhibitory effect.

[0033] The results in Table 1 indicate that CDCA has a higher activity in inhibiting abnormal proliferation of epidermal cells induced by P. acnes and can be an effective ingredient for the treatment of acne.

[0034] Example 2: Comparative study of in vitro inhibition of Propionibacterium acnes by chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) Experimental method: (1) Bacterial culture: The method is the same as Example 1.

[0035] (2) Grouping and treatment: A control group (CTRL, containing only P. acnes), P. acnes + UDCA, and CDCA (P. acnes co-cultured with different concentrations of CDCA or UDCA, ranging from 0 μmol / L to 500 μmol / L) were set up. All groups were cultured in an anaerobic incubator for 24 hours.

[0036] Antibacterial efficacy index detection: After 24 hours of culture, the OD600 value of each group was measured at a wavelength of 600nm to observe the antibacterial effect of the drug and compare the efficacy.

[0037] Experimental results: In the in vitro experiment, CDCA and UDCA were used to treat P. acnes. The results showed that both had antibacterial effects, and the effect of CDCA was significantly better than that of UDCA. Figure 2 ,in Figure 2 A is a line graph of the optical density (OD600) measured at a wavelength of 600 nm after Propionibacterium acnes was treated with CDCA and UDCA at different concentrations (1-500 μmol / L) for 24 hours, reflecting the bacterial growth; Figure 2B is the corresponding OD600 value bar graph, showing the quantitative comparison of bacterial growth at each concentration. Data are expressed as mean ± standard deviation, sample number n = 5. Significant differences were evaluated by two-way analysis of variance (ANOVA), and significant marks are: , , , .

[0038] CDCA showed obvious antibacterial effect at 40 μmol / L, while UDCA showed a more significant antibacterial effect at 100 μmol / L. At the same concentration, the antibacterial rate of CDCA was 2.16 to 5.97 times that of UDCA, among which the difference was the largest at 60 μmol / L, and the antibacterial effect of CDCA was 5.97 times that of UDCA. The results are shown in Table 2.

[0039] Table 2 The inhibition rate and multiple ratio of CDCA and UDCA on the growth of Propionibacterium acnes Note: (1) OD600: optical density value measured at a wavelength of 600 nm, used to estimate the cell density in bacterial culture medium; (2) inhibition rate (%): calculated as follows: inhibition rate = (control group OD600 − treatment group OD600 / control group OD600) × 100%; (3) CDCA and UDCA inhibition rate ratio: indicates the inhibitory effect of CDCA relative to UDCA at the same or similar concentration; (4) “——”: indicates no inhibitory effect or no data; (5) effective inhibitory concentration: CDCA showed significant inhibitory effect at 40 μmol / L, while UDCA showed a more obvious inhibitory effect at 100 μmol / L; (6) maximum inhibition rate ratio: at a concentration of 60 μmol / L, the inhibitory effect of CDCA was 5.97 times that of UDCA, showing a stronger inhibitory effect.

[0040] The results in Table 2 indicate that CDCA can effectively inhibit the growth of P. acnes at relatively low concentrations, demonstrating its potential advantage as an anti-acne therapeutic agent.

[0041] Example 3: Comparative study of the anti-lipid secretion effects of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) in vitro Experimental methods: (1) Cell culture: First, primary human sebaceous gland cells were cultured using a dedicated epithelial cell culture medium. During the logarithmic growth phase of the cells, the cells were prepared into a single cell suspension and the concentration was adjusted to 1×10 6 Pieces / mL.

[0042] (2) Grouping and treatment: To induce lipid oversecretion, cells were treated with linoleic acid (LA), and the following experimental groups were set up: control group (CTRL), containing sebaceous gland cells only; LA group, sebaceous gland cells treated with LA; LA + CDCA / UDCA group, sebaceous gland cells treated with LA and UDCA / CDCA.

[0043] (3) Nile red staining: After 24 hours of culture, cell samples from each group were collected and Nile red staining was used to mark intracellular lipids. The fluorescence intensity of Nile red was first detected using an ELISA reader with an excitation wavelength of 528 nm and an emission wavelength of 576 nm, and then the intracellular lipid droplet secretion was photographed under a microscope. The lipid secretion status of each group of cells was comprehensively evaluated using the above method.

[0044] Experimental results: In the lipid over-secretion model established by LA-stimulated human primary sebaceous gland cells, CDCA and UDCA were used for treatment respectively. The results showed that both CDCA and UDCA could inhibit the lipid secretion of sebaceous gland cells. Figure 3 ,in Figure 3 A shows the establishment of a lipid hypersecretion model by stimulating primary human sebaceous gland cells with linoleic acid. After the cells were treated with CDCA and UDCA, they were stained with Nile red to observe lipid accumulation. The image shows lipids stained with Nile red (red) and cell nuclei stained with DAPI (blue), taken under a 20x objective; Figure 3 B is the fluorescence intensity measured on a microplate reader after Nile red staining (excitation wavelength 528 nm, emission wavelength 576 nm) to quantitatively analyze the intracellular lipid content. Data are expressed as mean ± standard deviation, n = 5. Significant differences were evaluated by two-way analysis of variance (ANOVA), and significant marks are: , , , .

[0045] At low concentrations (1 μmol / L and 2 μmol / L), the inhibitory effect of UDCA was slightly better than that of CDCA; however, at higher concentrations (4 μmol / L and 8 μmol / L), the inhibitory effect of CDCA significantly exceeded that of UDCA, which was approximately 1.19 to 2.14 times that of UDCA, and CDCA showed good concentration dependence. The results are shown in Table 3.

[0046] Table 3 Inhibitory effects of CDCA and UDCA on lipid secretion of human sebaceous gland cells induced by linoleic acid Note: (1) RFU (Relative Fluorescence Units): Relative fluorescence unit, which indicates the fluorescence intensity measured after Nile red staining and is used to quantitatively evaluate the intracellular lipid content; (2) RFIP (Reduced Fluorescence Intensity Percentage): The percentage of fluorescence intensity reduction, calculated as follows: RFIP = (fluorescence intensity of linoleic acid group − fluorescence intensity of drug group fluorescence intensity of linoleic acid group) × 100%. The higher the RFIP value, the stronger the inhibitory effect of the drug on lipid secretion. The significant multiple ratio relationship of RFIP between CDCA and UDCA at high concentrations is marked (marked in red); (3) RFIP multiple ratio of CDCA to UDCA: indicates the multiple of RFIP of CDCA relative to UDCA at the same concentration; (4) “——”: indicates no data or not applicable; (5) Comparison of low concentration effects: At 1 μmol / L and 2 μmol / L concentrations, the RFIP of UDCA was slightly higher than that of CDCA, showing a stronger inhibitory effect on lipid secretion; (6) Comparison of high concentration effects: At 4 μmol / L and 8 At μmol / L concentration, the RFIP of CDCA was significantly higher than that of UDCA, and the multiple ratio reached 1.19 to 2.14 times, indicating that CDCA had a stronger inhibitory effect at high concentrations.

[0047] The results in Table 3 show that CDCA has a stronger inhibitory effect on the lipid secretion of sebaceous gland cells at high concentrations, demonstrating its potential application value in the treatment of sebaceous gland-related diseases (such as acne).

[0048] Example 4: Comparative study on the protein expression levels of inflammatory factors TNF-α and IL-1α in epidermal cells by chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) Experimental methods: (1) Bacterial culture: Propionibacterium acnes (P. acnes) was inoculated into brain heart infusion (BHI) broth medium and placed in an anaerobic incubator for 3 days at 37°C. After the culture was completed, the OD600 value was measured at a wavelength of 600 nm, and the bacterial solution was diluted with BHI medium to OD600 = 0.1 (approximately 1×10 8 CFU / mL), and set aside.

[0049] (2) Cell culture: Human keratinocytes (HaCaT cells) were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. During the logarithmic growth phase of the cells, the cells were made into a single cell suspension and the concentration was adjusted to 1×10 8 Pieces / mL.

[0050] (3) Grouping treatment: set up the control group CTRL (HaCaT cells only), P. acnes group (HaCaT cells co-cultured with P. acnes), P. acnes + UDCA / CDCA (HaCaT cells co-treated with P. acnes and UDCA or CDCA, the concentration is 100 μmol / L). Prepare the corresponding concentration of P. acnes suspension in DMEM medium in advance, and add the corresponding concentration of UDCA and CDCA according to the experimental requirements.

[0051] (4) Detection of inflammatory indicators: After the culture is completed, cell samples from each group are collected, and the cellular expression of key factors such as TNF-α and IL-1α is observed using immunofluorescence technology. First, the collected cell samples are fixed with 4% paraformaldehyde, and then permeabilized with 0.1% TritonX-100 so that the fluorescent dye can enter the cells. Next, a specific primary antibody (such as anti-TNF-α or anti-IL-1α antibody) is added and incubated under appropriate conditions to allow it to specifically bind to the target protein. Subsequently, a fluorescently labeled secondary antibody is used for staining, and unbound antibodies and non-specific binding substances are removed by washing. Finally, the localization and expression level of inflammatory factors in cells can be observed and analyzed using a fluorescence microscope.

[0052] Experimental results: At a concentration of 100 μmol / L, CDCA was not only superior to UDCA in inhibiting abnormal proliferation of epidermal cells, but also significantly reduced the protein expression of TNF-α and IL-1α, as shown by the decrease in fluorescence intensity. Figure 4 .in Figure 4 A is the immunofluorescence staining of TNF-α in epidermal cells. Green represents TNF-α, and blue represents the cell nucleus stained with DAPI, taken under a 20x objective: Figure 4 B is immunofluorescence staining of IL-1α in epidermal cells. Green represents IL-1α, blue represents DAPI-stained cell nuclei, photographed under a 20x objective lens; Figure 4 C is a bar graph showing the quantitative analysis of the fluorescence intensity of TNF-α and IL-1α. Data are presented as mean ± standard deviation, n = 5. Significant differences were evaluated by one-way analysis of variance (ANOVA), with significant markers: , , , .

[0053] The results suggest that CDCA has a stronger potential therapeutic effect in reducing acne-related inflammatory responses.

[0054] The above experimental results show that chenodeoxycholic acid is superior to ursodeoxycholic acid in antibacterial, anti-inflammatory and inhibition of abnormal proliferation of keratinocytes, showing a lower effective concentration and more significant effect. In addition, in terms of inhibiting sebum secretion, chenodeoxycholic acid shows a strong concentration dependence, and its inhibitory effect at high concentrations is significantly higher than that of ursodeoxycholic acid.

[0055] In summary, the in vitro experimental results show that chenodeoxycholic acid has better efficacy than ursodeoxycholic acid in the key link of acne treatment, showing its application prospect as an effective ingredient for the treatment of acne.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of chenodeoxycholic acid or its pharmaceutically acceptable salt as the sole active ingredient in the preparation of a drug for treating acne.

2. The use according to claim 1, characterized in that The pharmaceutically acceptable salt is selected from sodium salt, potassium salt, magnesium salt, calcium salt or ammonium salt.

3. The use according to claim 1 or 2, characterized in that: The chenodeoxycholic acid or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier are prepared into a pharmaceutical composition.

4. The use according to claim 3, characterized in that The pharmaceutical composition is selected from tablets, capsules, pills or injections.

5. Use of chenodeoxycholic acid or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a Propionibacterium acnes inhibitor.

6. The use according to claim 5, characterized in that The pharmaceutically acceptable salt is selected from sodium salt, potassium salt, magnesium salt, calcium salt or ammonium salt.

7. The use according to claim 5 or 6, characterized in that The chenodeoxycholic acid or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier are prepared into a pharmaceutical composition.

8. The use according to claim 7, characterized in that The pharmaceutical composition is selected from tablets, capsules, pills or injections.

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