CN119606982A

CN119606982A - Use of chenodeoxycholic acid in preparing acne treatment drugs

Info

Publication number: CN119606982A
Application number: CN202510157546.6A
Authority: CN
Inventors: 王毅
Original assignee: EXPERIMENTAL RESEARCH CENTER CHINA ACADEMY OF CHINESE MEDICAL SCIENCES
Current assignee: EXPERIMENTAL RESEARCH CENTER CHINA ACADEMY OF CHINESE MEDICAL SCIENCES
Priority date: 2025-02-13
Filing date: 2025-02-13
Publication date: 2025-03-14

Abstract

The present invention relates to the use of chenodeoxycholic acid or a pharmaceutically acceptable salt thereof in preparing a drug for treating acne. The chenodeoxycholic acid of the present invention can be a naturally occurring organic compound or a synthetic organic compound, has a strong inhibitory activity against Propionibacterium acnes and a pharmacological effect against lipid secretion, and is used as an active ingredient and a pharmaceutically acceptable carrier to prepare a pharmaceutical composition for treating acne.

Description

Application of chenodeoxycholic acid in preparing medicine for treating acne

Technical Field

The invention relates to application of chenodeoxycholic acid or pharmaceutically acceptable salt thereof in preparing a medicine for treating acne.

Background

Acne is a common chronic inflammatory skin disease mainly occurring on the face, neck, chest, back, shoulders and the like, and can occur in all age groups, but the incidence rate is highest in teenagers. Over 85% of teenagers suffer from permanent scars and skin damage, which affect the appearance. The global acne cases in 2019 are 1.17 hundred million, the epidemic cases reach 2.31 hundred million, and the increase is about 47% compared with 1990.

The pathogenesis of acne involves a number of factors, which are caused by the co-action of a number of factors, among which mainly sebum hypersecretion, obstruction of the pilosebaceous ducts, bacterial infection and inflammatory response.

The main treatment method of the acne at present adopts tretinoin medicines, and the action mechanism of the tretinoin medicines is to reduce inflammation and keratosis and inhibit sebum secretion, and the side effect is that skin dryness, inflammation, desquamation and the like can be caused. In addition, chinese patent CN109475564a discloses a composition for preventing or treating inflammatory skin diseases or severe pruritus containing water-soluble ursodeoxycholic acid for preventing or treating atopic skin diseases, acne, psoriasis, urticaria, inflammatory dermatitis, seborrheic dermatitis, and contact dermatitis, but ursodeoxycholic acid (UDCA) is still unsatisfactory for the treatment effect of acne.

The medicine for treating acne meeting clinical requirements needs to have the effects of anti-inflammatory, anti-keratinization, antibacterial and the like, can inhibit sebum secretion, regulate sebaceous gland homeostasis and improve microenvironment, and has small safety side effects. Therefore, it is very interesting to explore and find drugs with good therapeutic effects.

Chenodeoxycholic Acid (CDCA) is known as a major use for reducing the saturation of cholesterol in bile, but there is no evidence that chenodeoxycholic acid has been reported to have an inhibitory effect on propionibacterium acnes and is useful for treating acne.

Disclosure of Invention

First, the technical problem to be solved

The invention aims to solve the technical problems of bad effect and large side effect of the compound in the prior art on acne treatment.

(II) technical scheme

In order to solve the problems in the prior art, the invention establishes three classical models by simulating three main pathological factors of acne in vitro, namely (1) hyperproliferation of propionibacterium acnes, (2) hyperproliferation and inflammatory reaction of epidermal cells induced by propionibacterium acnes and (3) sebum hypersecretion of sebaceous gland cells. The anti-inflammatory antibacterial and anti-lipid secretion effects of ursodeoxycholic acid and chenodeoxycholic acid were compared in these models. Experimental results show that chenodeoxycholic acid has lower effective concentration compared with ursodeoxycholic acid and shows good concentration dependence. Research shows that chenodeoxycholic acid is used as a core effective component for treating acne, has remarkable curative effects on anti-inflammatory, antibacterial and lipid secretion inhibition, and is obviously superior to ursodeoxycholic acid, so that the application of chenodeoxycholic acid or pharmaceutically acceptable salt thereof in preparing a medicine for treating acne and the application in preparing propionibacterium acnes inhibitor are provided.

The invention aims to provide application of chenodeoxycholic acid or pharmaceutically acceptable salt thereof in preparing a medicine for treating acne.

According to the above use, the pharmaceutically acceptable salt is selected from sodium, potassium, magnesium, calcium or ammonium salts.

According to the above use, the chenodeoxycholic acid or pharmaceutically acceptable salt thereof and pharmaceutically acceptable carrier are prepared into pharmaceutical compositions.

According to the above use, the pharmaceutical composition is selected from the group consisting of tablets, capsules, pills or injections.

It is another object of the present invention to provide the use of chenodeoxycholic acid or a pharmaceutically acceptable salt thereof for the preparation of propionibacterium acnes inhibitors.

(III) beneficial effects

The technical scheme of the invention has the following advantages:

(1) The chenodeoxycholic acid disclosed by the invention can well inhibit the hyperproliferation of propionibacterium acnes, inhibit the hyperproliferation and inflammatory reaction of epidermal cells induced by propionibacterium acnes, and inhibit the excessive secretion of sebum of sebaceous gland cells, so that the side effect is smaller.

(2) Compared with ursodeoxycholic acid, the chenodeoxycholic acid has lower effective concentration and good concentration dependence, and shows that the chenodeoxycholic acid is used as a core effective component for treating acne, has obvious curative effects on anti-inflammatory, antibacterial and lipid secretion inhibition, and is obviously superior to the ursodeoxycholic acid.

Drawings

FIG. 1 is the effect of UDCA and CDCA on epithelial cell proliferation under P. acnes stimulation;

FIG. 2 is the inhibition of Propionibacterium acnes growth by CDCA and UDCA;

FIG. 3 is the effect of CDCA and UDCA on linoleic acid induced lipid secretion from human sebaceous gland primary cells;

FIG. 4 is the effect of CDCA and UDCA on TNF- α and IL-1α expression in epidermal cells.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Chenodeoxycholic acid of the present invention may be a naturally occurring compound or an artificially synthesized compound.

Chenodeoxycholic acid has chemical formula of C ₂₄H₄₀O₄, chemical name of 3α,7α -dihydroxy-5β -cholanic acid, and is abbreviated as CDCA, and chemical structural formula is shown as follows:

Chenodeoxycholic acid is known to be mainly used for reducing the saturation of cholesterol in bile and is widely applied to fat accumulation and lipid metabolism diseases. The invention makes intensive research on the effect of chenodeoxycholic acid on treating acne, and provides basis for screening high-efficiency medicines for treating acne.

The invention establishes three classical models by simulating three main pathological factors of acne in vitro, namely (1) hyperproliferation of propionibacterium acnes, (2) hyperproliferation and inflammatory response of epidermal cells induced by propionibacterium acnes, and (3) sebum hypersecretion 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.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

For a better understanding of the present invention, the following examples are set forth to further illustrate or describe the invention, but are not to be construed as limiting its scope.

Example 1 comparative study of in vitro anti-epidermal cell inflammation and abnormal proliferation of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA)

The experimental method comprises (1) culturing Propionibacterium acnes (P. acnes) in Brain Heart Infusion (BHI) broth, and anaerobic culturing at 37deg.C for 3 days. After the completion of the culture, the OD600 was measured at a wavelength of 600nm, and the bacterial liquid was diluted to OD 600=0.1 (about 1X 10 ⁸ CFU/mL) with BHI medium for use.

(2) Cell culture human keratinocytes (HaCaT cells) were cultured using DMEM medium containing 10% fetal bovine serum and 1% diabody. Cells were prepared as single cell suspensions during the logarithmic growth phase of the cells, and the concentration was adjusted to 1X 10 ⁶/mL after counting.

(3) Grouping treatment, namely setting a control group CTRL (only containing HaCaT cells), a P. acnes group (the HaCaT cells are co-cultured with P. acnes), and P. acnes +UDCA/CDCA (the HaCaT cells are co-treated with P. acnes and UDCA or CDCA), wherein the concentration gradient is 100 mu mol/L-500 mu mol/L. P. acnes suspensions of the corresponding concentrations were prepared in advance using DMEM medium, and UDCA and CDCA of the corresponding concentrations were added according to experimental requirements.

(4) CCK8 assay cell proliferation viability after 24 hours incubation, 10. Mu.l of CCK-8 solution and 90. Mu.l of serum-free medium were added to each well and the plates were gently shaken to ensure uniform distribution of the CCK-8 solution. Then, the samples were incubated at 37℃under 5% CO2 for 30 minutes, and absorbance was measured at 450 nm using a microplate reader. Finally, the effect of the drug on cell proliferation was calculated according to the following formula:

Proliferation rate (cell viability) = [ (experimental group-blank control)/(negative control-blank control) ]x100%.

The experimental result shows that the P. acnes is used for stimulating the epidermal cells in an in-vitro model of the acne, so that the pathological process of the abnormal proliferation of the epidermal cells in the acne process is successfully simulated. Experimental results show that after P. acnes is added, obvious abnormal proliferation of the epidermal cells occurs. Based on this, it was found that both of them can inhibit P. acnes-induced abnormal proliferation of epidermal cells by adding UDCA and CDCA, respectively, and the effect of CDCA was significantly better than that of UDCA, and the result is shown in FIG. 1. Propionibacterium acnes (P. acnes) stimulated excessive proliferation of epidermal cells, and effects of culturing for 24 hours after addition of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) at various concentrations (0-500. Mu. Mol/L). Cell proliferation activity was measured by CCK-8, and the results were presented in the form of a line graph (FIG. 1A) and a bar graph (FIG. 1B). Data were statistically tested using two-way analysis of variance (ANOVA) with a sample size of n=5. The significance differences are noted as follows:,,,。

CDCA can obviously inhibit the proliferation of epidermal cells at the concentration of 100 mu mol/L, and the inhibition rate reaches 45.14%. In contrast, UDCA started to act only at 140. Mu. Mol/L, with an inhibition of 9.1%.

Furthermore, at the same drug concentration, the inhibition rate of CDCA was 1.74 to 9.39 times higher than that of UDCA, especially 160. Mu. Mol/L, and up to 9.93 times, and the results are shown in Table 1.

TABLE 1 UDCA inhibition of P. acnes-induced epidermal cell hyperproliferation by CDCA

Note that (1) cell viability (%) was obtained by CCK-8 assay and indicated the relative viability of the treated cells. (2) The inhibition ratio (%) is calculated as inhibition ratio= (1-cell viability of experimental group/cell viability of negative control) ×100%, (3) inhibition ratio of CDCA to UDCA is multiple of inhibition effect of CDCA relative to UDCA at the same or similar concentration, (4) "-" represents no comparability or no data, (5) effective inhibition concentration is that CDCA shows remarkable inhibition effect at 100 μmol/L, inhibition ratio is 45.14%, and UDCA starts to act at 140 μmol/L, inhibition ratio is 9.10%, and (6) maximum inhibition ratio is 9.39 times of inhibition ratio of CDCA at 160 μmol/L, and stronger inhibition effect is shown.

The results in Table 1 show that CDCA has higher activity in inhibiting the abnormal proliferation of epidermal cells induced by P. acnes, and can be used as an effective component for treating acne.

EXAMPLE 2 comparative study of in vitro inhibition of Propionibacterium acnes by chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA)

Experimental methods (1) bacterial cultures were performed in the same manner as in example 1.

(2) Grouping and treatment control groups (CTRL, P. acnes only), P. acnes + UDCA and CDCA (P. acnes co-cultured with CDCA or UDCA at different concentrations ranging from 0. Mu. Mol/L to 500. Mu. Mol/L) were established. All groups were incubated in anaerobic incubator for 24 hours.

And (3) detecting antibacterial efficacy indexes, namely measuring OD600 values of each group at 600nm wavelength after culturing for 24 hours, observing antibacterial conditions of the medicines, and comparing the efficacy.

Experimental results in vitro experiments, in which p. acnes was treated with CDCA and UDCA, the results showed that both had antibacterial effects and that the effect of CDCA was significantly better than UDCA. The results are shown in FIG. 2, wherein FIG. 2A is a plot of optical density (OD 600) measured at 600 nm wavelength after 24 hours of treatment with CDCA and UDCA at different concentrations (1-500. Mu. Mol/L) reflecting bacterial growth, and FIG. 2B is a bar chart of corresponding OD600 values showing quantitative comparisons of bacterial growth at each concentration. Data are expressed as mean ± standard deviation, number of samples n=5. Significant differences were assessed by two-way analysis of variance (ANOVA), significance signature:,,,。

CDCA shows remarkable antibacterial effect at 40 mu mol/L, and UDCA shows remarkable antibacterial effect at 100 mu mol/L. At the same concentration, the bacteriostatic efficiency of CDCA was 2.16 to 5.97 times that of UDCA, with the greatest difference at 60. Mu. Mol/L concentration, and the bacteriostatic effect of CDCA was 5.97 times that of UDCA, and the results are shown in Table 2.

Table 2 CDCA and the inhibition ratio and the multiplication ratio relation of UDCA to Propionibacterium acnes

Note that (1) OD600 is an optical density value measured at 600 nm wavelength for estimating cell density in bacterial culture, (2) inhibition (%) is calculated as inhibition= (control OD 600-control OD 600) ×100%, (3) inhibition ratio of CDCA to UDCA is 5.97 times that of UDCA at 60 μmol/L, and (4) inhibition ratio of CDCA to UDCA is shown at the same or similar concentration, no inhibition effect or no data is shown, (5) effective inhibition concentration is shown by CDCA at 40 μmol/L, but rather remarkable inhibition effect is shown by UDCA at 100 μmol/L, and (6) maximum inhibition ratio is shown by CDCA at 5.97 times that of UDCA at 60 μmol/L.

The results in table 2 demonstrate that CDCA is effective at inhibiting the growth of p. acnes at lower concentrations, showing its potential advantage as an anti-acne therapeutic.

EXAMPLE 3 in vitro anti-lipid secretion comparative study of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA)

Experimental methods (1) cell culture, primary human sebaceous gland cells are first cultured using a dedicated epithelial cell culture medium. Cells were prepared as single cell suspensions during the logarithmic growth phase of the cells, and the concentration was adjusted to 1X 10 ⁶/mL after counting.

(2) Grouping and treatment to induce lipid over-secretion cells were treated with Linoleic Acid (LA) and the following experimental groups were set, control group (CTRL), containing only sebaceous gland cells, LA group, sebaceous gland cells co-treated with LA, la+cdca/UDCA group, sebaceous gland cells co-treated with LA and UDCA/CDCA.

(3) Nile red staining, namely after the culture is finished for 24 hours, collecting each group of cell samples, marking the intracellular lipid by using a Nile red staining method, firstly detecting the fluorescence intensity of the Nile red by using excitation wavelength and emission wavelength of 528 nm and 576 nm under an enzyme-labeling instrument, and shooting the secretion condition of the intracellular lipid droplets under a microscope. By the above method, the lipid secretion status of each group of cells was comprehensively evaluated.

The experimental results show that in the lipid hypersecretion model established by stimulating the human primary sebaceous gland cells with LA, the treatment is carried out by using CDCA and UDCA respectively, and the results show that the CDCA and the UDCA can inhibit the lipid secretion of the sebaceous gland cells, and the results are shown in figure 3, wherein figure 3A is the linoleic acid for stimulating the human sebaceous gland primary cells, and the lipid hypersecretion model is established. After cells were treated with CDCA and UDCA, nile red staining was performed to observe lipid accumulation. The images show nile red stained lipids (red) and DAPI stained nuclei (blue), photographed under a 20-fold objective, and fig. 3B shows fluorescence intensity (excitation wavelength 528 nm, emission wavelength 576 nm) measured on a microplate reader after nile red staining, and the intracellular lipid content was quantitatively analyzed. Data are expressed as mean ± standard deviation, n=5. Significant differences were assessed by two-way analysis of variance (ANOVA), significance signature:,,,。

At low concentrations (1. Mu. Mol/L and 2. Mu. Mol/L), the inhibition effect of UDCA was slightly better than that of CDCA, however, at higher concentrations (4. Mu. Mol/L and 8. Mu. Mol/L), the inhibition effect of CDCA was significantly more than that of UDCA, about 1.19 to 2.14 times that of UDCA, and CDCA exhibited a better concentration dependence, and the results are shown in Table 3.

TABLE 3 CDCA inhibition of linoleic acid-induced lipid secretion of human sebaceous cells by UDCA

Note that (1) RFU (Relative Fluorescence Units) represents relative fluorescence units, which are measured after staining with nile red, for quantitative assessment of intracellular lipid content, (2) RFIP (Reduced Fluorescence INTENSITY PERCENTAGE) represents percent decrease in fluorescence intensity, calculated as RFIP = (linoleic acid group fluorescence intensity-administration group fluorescence intensity) ×100%, with a higher RFIP value representing stronger inhibition of lipid secretion by the drug, RFIP marked with CDCA and UDCA at higher concentration is significantly higher than CDCA (marked with red), RFIP marked with the same concentration is significantly higher than CDCA with respect to UDCA by RFIP (4) "," representing no data or inapplicability, (5) RFIP of UDCA is slightly higher than CDCA at 1 μmol/L and 2 μmol/L concentrations, and (6) RFIP is significantly higher than CDCA at 4 μmol/L and 8 μmol/L concentrations, and has a significantly higher inhibition of lipid secretion by 1.19 times higher than CDCA at 1.19.

The results in table 3 show that CDCA has a stronger inhibition effect on lipid secretion of sebaceous gland cells at high concentration, and shows potential application value in treating sebaceous gland related diseases (such as acne).

EXAMPLE 4 comparative study of protein expression levels of inflammatory factors TNF-alpha and IL-1 alpha of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) in epidermal cells

The experimental method comprises the following steps:

(1) Bacterial culture propionibacterium acnes (p. acnes) were inoculated into Brain Heart Infusion (BHI) broth, placed in an anaerobic incubator, and anaerobically cultured at 37 ℃ for 3 days. After the completion of the culture, the OD600 was measured at a wavelength of 600nm, and the bacterial liquid was diluted to OD 600=0.1 (about 1X 10 ⁸ CFU/mL) with BHI medium for use.

(2) Cell culture human keratinocytes (HaCaT cells) were cultured using DMEM medium containing 10% fetal bovine serum and 1% diabody. Cells were prepared as single cell suspensions during the logarithmic growth phase of the cells, and the concentration was adjusted to 1X 10 ⁸/mL after counting.

(3) Grouping treatment, CTRL (containing only HaCaT cells), P. acnes (HaCaT cells co-cultured with P. acnes), and P. acnes + UDCA/CDCA (HaCaT cells co-treated with P. acnes and UDCA or CDCA at a concentration of 100. Mu. Mol/L) were set. P. acnes suspensions of the corresponding concentrations were prepared in advance using DMEM medium, and UDCA and CDCA of the corresponding concentrations were added according to experimental requirements.

(4) And detecting inflammation indexes, namely collecting cell samples of each group after the culture is finished, and observing cell expression conditions of key factors such as TNF-alpha, IL-1 alpha and the like by adopting an immunofluorescence technology. First, the collected cell samples were fixed using 4% paraformaldehyde and then permeabilized using 0.1% triton x-100 so that the fluorescent dye could enter the cell interior. Next, a specific antibody (e.g., an anti-TNF- α or anti-IL-1α antibody) is added and incubated under appropriate conditions to allow specific binding to the target protein. Subsequently, staining is performed using a fluorescent-labeled diabody, and unbound antibodies and non-specific binding substances are removed by washing, and finally localization and expression levels of inflammatory factors in cells can be observed and analyzed by a fluorescent microscope.

The experimental result shows that CDCA is superior to UDCA in inhibiting the abnormal proliferation of epidermal cells at the concentration of 100 mu mol/L, and simultaneously, the protein expression of TNF-alpha and IL-1 alpha is obviously reduced, and the result is shown in figure 4. Wherein FIG. 4A is immunofluorescent staining of TNF- α in epidermal cells. Green for TNF-alpha, blue DAPI stained nuclei, 20-fold objective, fig. 4B immunofluorescent staining of IL-1 alpha in epidermal cells. Green represents IL-1 a, blue DAPI stained nuclei, photographed under 20 x objective, and fig. 4C is a histogram of quantitative analysis of TNF-a and IL-1 a fluorescence intensities. Data are expressed as mean ± standard deviation, n=5. Significant differences were assessed by one-way analysis of variance (ANOVA), significance signature:,,,。

The results indicate that CDCA has a greater potential efficacy in reducing the inflammatory response associated with acne.

The experimental results show that the chenodeoxycholic acid is superior to the ursodeoxycholic acid in the aspects of resisting bacteria, resisting inflammation and inhibiting the abnormal proliferation of keratinocytes, and shows lower effective concentration and more remarkable effect. In addition, chenodeoxycholic acid shows strong concentration dependence in terms of sebum secretion inhibition, and the inhibition effect at high concentration is obviously superior to that of ursodeoxycholic acid.

In summary, in vitro experimental results show that chenodeoxycholic acid has better efficacy in key links of acne treatment, is better than ursodeoxycholic acid, and shows application prospect as an effective component for treating acne.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution 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 a pharmaceutically acceptable salt thereof 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 a pharmaceutically acceptable salt thereof 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.