A pharmaceutical composition containing pyrrolidine dithiocarbamate (PDTC) as an active ingredient and having an effect of treating or preventing skin inflammation

Abstract

A pharmaceutical composition having a therapeutic or preventive effect against skin inflammation is provided, comprising pyrrolidine dithiocarbamate (PDTC) as an active ingredient.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition having a therapeutic or preventive effect against skin inflammation, comprising pyrrolidine dithiocarbamate (PDTC) as an active ingredient. Background Technology

[0002] Acne is a common inflammatory skin condition caused by excessive sebum. It develops during puberty, leaves scars on the face, and persists for a lifetime. Acne is caused by various factors, including environmental pollution, stress, genetic causes, bacterial infections, colonization of Cutibacterium acnes (C. acnes), and Gram-positive anaerobic bacteria in sebaceous cysts. C. acnes stimulates macrophages to secrete pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α.

[0003] Antibiotics such as tetracycline, clindamycin, and erythromycin are primarily used as treatments for acne. However, long-term, inappropriate use of antibiotics leads to problems such as skin irritation, redness, and hyperpigmentation of the skin, as well as the problem of antibiotic-resistant bacteria.

[0004] Isotretinoin, benzoyl peroxide (BPO), and synthetic sulfones are effective methods for treating acne, but response and tolerance vary from person to person. Therefore, the development of treatments with diverse mechanisms of action remains necessary. The problem to be solved

[0005] Therefore, the problem that the present invention aims to solve is to provide an acne treatment with a novel mechanism of action. means of solving the problem

[0006] To solve the above problem, the present invention provides a pharmaceutical composition having a therapeutic or preventive effect against skin inflammation, comprising pyrrolidine dithiocarbamate (PDTC) as an active ingredient.

[0007] In one embodiment of the present invention, the skin inflammation is skin inflammation caused by Cutibacterium acnes (C. acnes).

[0008] In one embodiment of the present invention, the pyrrolidine dithiocarbamate (PDTC) inhibits the secretion of IL-1β by inhibiting NF-κB activation and inflammatory activation induced by Cutibacterium acnes (C. acnes).

[0009] In one embodiment of the present invention, the skin inflammation is acne.

[0010] In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Effects of the invention

[0011] PDTC (Pyrrolidine dithiocarbamate) according to the present invention is effective as a treatment for skin inflammation caused by Cutibacterium acnes (C. acnes). Brief explanation of the drawing

[0012] (A) Chemical structure of PDTC in Fig. 1. (B) Results of treating mouse BMDM with 10 μM PDTC and 40 μM PDTC for the indicated times, (CH) Results of pretreating mouse BMDM with PDTC (10 μM and 40 μM) for 30 minutes and then incubating with heat-killed C. acnes for 6 hours. Figure 2 (A) is the result of quantifying the relative levels of protein bands (B, C) from Western analysis for Pro-IL-1β, COX-2, and β-actin. Figure 3 shows the results of measuring the phosphorylation of NF-κB(p65) by Western blot analysis. Figure 4 shows the inhibitory effect (AD) of PDTC on inflammasome activation induced by C. acnes, with mouse BMDM primed with heat-killed C. acnes (1 x 10⁶ CFU / mL) for 3 hours, followed by treatment with PDTC (10μM and 40μM) for 1 hour for 30 minutes prior to incubation with ATP (5mM) or nigerisin (10μM). Figure 5 shows the results of analyzing the inhibitory effect of PDTC in a mouse acne model. Figure 6 is a diagram illustrating the mechanism of PDTC for inhibiting skin inflammation induced by C. acnes. Specific details for implementing the invention

[0013] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings.

[0014] Before describing the present invention in detail, the terms and words used in this specification should not be interpreted as being unconditionally limited to their ordinary or dictionary meanings, and the inventor of the present invention may appropriately define and use the concepts of various terms to best describe their invention.

[0015] Furthermore, it should be understood that these terms or words should be interpreted in a meaning and concept consistent with the technical spirit of the present invention.

[0016] In other words, the terms used in this specification are used merely to describe preferred embodiments of the invention and are not intended to specifically limit the content of the invention.

[0017] It should be noted that these terms are defined in consideration of the various possibilities of the present invention.

[0018] Additionally, in this specification, singular expressions may include plural expressions unless the context clearly indicates a different meaning.

[0019] In addition, you should be aware that even if it is expressed in the plural, it may contain a singular meaning.

[0020] Throughout this specification, where it is stated that a component "includes" another component, unless specifically stated otherwise, this may mean that it does not exclude any other component but may include any other component.

[0021] Furthermore, in cases where it is stated that a component "exists inside or is installed in connection with" another component, this component may be installed in direct connection with or in contact with the other component.

[0022] To solve the aforementioned problem, the present invention provides PDTC (Pyrrolidine dithiocarbamate) as a treatment for skin inflammation caused by Cutibacterium acnes (C. acnes), particularly as a treatment for acne.

[0023] FIG. 1A is the chemical structural formula of pyrrolidine dithiocarbamate (PDTC) according to one embodiment of the present invention.

[0024] In one embodiment of the present invention, PDTC and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0025] The acne treatment effect of pyrrolidine dithiocarbamate (PDTC) according to the present invention will be explained in more detail through the following experimental examples.

[0026] Experimental Example

[0027] Experimental Example 1

[0028] To explain the effect of PDTC on Cutibacterium acnes (C. acnes)-induced inflammatory signals, the non-cytotoxic concentration of PDTC in BMDM was first determined.

[0029] (A) Chemical structure of PDTC in Fig. 1. (B) Results of treating mouse BMDM with 10 μM PDTC and 40 μM PDTC for the indicated times, (CH) Results of pretreating mouse BMDM with PDTC (10 μM and 40 μM) for 30 minutes and then incubating with heat-killed C. acnes for 6 hours.

[0030] Here, BMDM was treated with 10 and 40 μM PDTC, and cell viability was measured by an MTT assay for up to 24 hours.

[0031] Referring to the results in Figure 1, PDTC did not induce any inflammation in BMDM up to 24 hours. Based on these results, 10 and 40 μM PDTC were used in all subsequent experiments (see Figure 1B).

[0032] Next, the effect of PDTC on mRNA expression in inflammation induced by C. acnes was analyzed, and for this purpose, quantitative real-time PCR (qPCR) analysis was performed (see Fig. 1C-H).

[0033] As shown in Figures 1C-H, pretreatment with PDTC can be seen to reduce the mRNA levels of C. acnes-induced inflammation regulators such as pro-IL-1β, IL-6, TNF-α, iNOS, COX-2, and NLRP3 in a concentration-dependent manner.

[0035] Experimental Example 2

[0036] Next, protein expression of pro-inflammatory mediators, such as pro-IL-1β and COX-2 induced by PDTC in C. acnes, was analyzed using Western blot analysis. For this purpose, mouse BMDMs were pretreated with PDTC (10 and 40 μM) for 30 minutes, and then co-cultured for 6 hours with heat-killed C. acnes (1 × 10⁶ CFU / mL).

[0037] Figure 2 (A) is the result of quantifying the relative levels of protein bands (B, C) from Western analysis for Pro-IL-1β, COX-2, and β-actin.

[0038] Referring to Figure 2, it can be seen that pretreatment with PDTC inhibits the protein expression of pro-IL-1β induced by C. acnes in a dose-dependent manner, and that the level of COX-2 protein expression is consistently inhibited by PDTC treatment according to the present invention. Therefore, it can be seen that PDTC according to the present invention has an anti-inflammatory effect on the C. acnes-induced inflammatory signaling pathway.

[0040] Experimental Example 3

[0041] The signaling pathways involved in PDTC-induced anti-anxiety against C. acnes were analyzed.

[0042] To this end, the phosphorylation of NF-κB(p65) was measured by Western blot analysis, and this is shown in Figure 3.

[0043] Referring to Fig. 3, it can be seen that BMDM containing C. acnes induces phosphorylation of NF-κB, and that the level of phosphorylation is significantly inhibited by PDTC in a dose-dependent manner (see Fig. 3A,B).

[0044] From these results, it can be seen that the phosphorylation of IκB, which transports NF-κB to the nucleus, is also inhibited by PDTC treatment according to the present invention (see Figs. 3A and C).

[0045] Furthermore, the inhibitory effect of PDTC on NF-κB activation induced by C. acnes was confirmed and measured, as illustrated in Fig. 3D. Referring to Fig. 3D, it can be seen that PDTC continuously inhibits NF-κB-dependent gene transcription and exhibits an inhibitory effect of PDTC on C. acnes-induced inflammation.

[0047] Experimental Example 4

[0048] The secretion of active IL-1β is promoted by the inflammasome pathway. Therefore, this experiment analyzed whether the inflammasome complex is involved in PDTC-mediated inhibition.

[0049] Figure 4 shows the inhibitory effect (AD) of PDTC on inflammasome activation induced by C. acnes, in which mouse BMDMs were primed with heat-killed C. acnes (1 x 10⁶ CFU / mL) for 3 hours and then treated with PDTC (10μM and 40μM) for 1 hour for 30 minutes prior to incubation with ATP (5mM) or nigerisin (10μM).

[0050] For 1 hour, the culture supernatant (Sup) and cell lysate (Lys) were immunoblotted with anti-IL-1β, anti-caspase-1, and anti-NLRP3 antibodies (A,D), and secreted IL-1β levels were measured using ELISA (B,C).

[0051] In addition, (EH) of Fig. 4 shows that mouse BMDM was primed with heat-killed C. acnes (1 × 10⁶ CFU / mL) for 3 hours, then treated with PDTC (10 μM and 40 μM) for 30 minutes before poly(dA:dT) (10 μg / mL) transfection, and then treated with Lipofectamine 3000 or flagellin (5 μg / mL) for 4 hours, and the culture supernatant (Sup) and cell lysate (Lys) were immunoblotted with the indicated antibodies (E,G).

[0052] Referring to the results in Figure 4, in the NLRP3 inflammasome assay for C. acnes, mouse BMDM was primed with C. acnes with or without PDTC and then treated with ATP (see Figures 4A and 4B). The application of ATP induced IL-1β activation and the secretion of cleaved caspase-1 in the supernatant, while PDTC inhibited this (see Figure 4A). Additionally, a decrease in the secretion of active IL-1β protein was further confirmed. The decrease in the secretion of active IL-1β protein was further confirmed using ELISA (Figure 4B).

[0053] Based on these results, it can be seen that PDTC inhibits caspase-1 secretion induced by the NLRP3 inflammasome activator nigerisin, which replaces IL-1β (Fig. 4C,D).

[0054] To investigate the effects of PDTC on other inflammasomes, C. acnes-primed BMDM was treated with AIM2 inflammasome activator poly(dA:dT).

[0055] It was found that PDTC inhibits poly(dA:dT)-induced IL-1β and caspase-1 secretion (Figs. 4E,F). However, PDTC did not alter the levels of active IL-1β and caspase-1 secretion in flagellin-treated BMDM (flagellin NLRC4 inflammasome activator) (Figs. 4G,H). These results suggest that PDTC inhibits NLRP3 and AIM2 inflammasomes but not NLRC4 inflammasomes.

[0057] Experimental Example 5

[0058] Figure 5 shows the results of analyzing the inhibitory effect of PDTC in a mouse acne model.

[0059] In Fig. 5, (A) live C. acnes (1 × 10⁸ CFU / 20 μL in PBS) was injected into the ears of mice with or without PDTC (100 mg / kg), and the mouse ears were photographed 24 hours after injection. (B) the ear tissue was excised and stained with H&E. (C) IL-1β and β-actin protein levels were detected in the ear tissue using Western blot. (D) the relative levels of the protein bands were quantified. (EH) mRNA (IL-6, pro-IL-1β, NLRP3, and TSLP) levels were measured in the mouse ear tissue using quantitative real-time PCR. (I) Immunofluorescence tissue images of F4 / 80 (red) and IL-6 (green) in acne lesions.

[0060] Referring to Fig. 5, it can be seen that inflammatory cells increase and penetrate the dermis following inoculation with C. acnes. However, it can be seen that this reaction is significantly weakened by PDTC (see Fig. 5B).

[0061] Ear IL-1β protein expression levels were significantly increased by C. acnes injection, but it was found that this was reduced by PDTC treatment (Fig. 5C,D).

[0062] mRNA analysis results showed that PDTC lowers the mRNA level of TSLP, a cytokine known to be upregulated in skin inflammation (Fig. 5H).

[0063] The prevalence of F4 / 80+ macrophages, a well-established mouse macrophage marker, was observed in C. acne-treated ear tissues compared to control ear tissues, but it was found that the number of F4 / 80+ macrophages decreased when treated with PDTC (see Fig. 5I,J). In addition, IL-6-expressing F4 / 80+ macrophages were consistently low in PDTC-treated acne lesions.

[0064] Collectively, these results suggest that PDTC is effective in treating or preventing in vivo skin inflammation induced by C. acnes.

[0065] Figure 6 is a diagram illustrating the mechanism of PDTC for inhibiting skin inflammation induced by C. acnes.

[0066] Referring to Figure 6, PDTC inhibits the secretion of IL-1β by inhibiting NF-κB activation and inflammatory activation induced by C. acnes.

[0067] The present invention provides a pharmaceutical composition comprising the above-described PDTC as an active ingredient.

[0068] In this specification, the term “comprising as an active ingredient” means that the PDTC of the present invention is included in an amount sufficient to achieve efficacy against skin inflammation induced by C. acnes.

[0069] The pharmaceutical composition for treating skin inflammation of the present invention may further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0070] Pharmaceutically acceptable carriers in the composition of the present invention are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0071] The pharmaceutical composition of the present invention may be administered orally or parenterally. In the case of parenteral administration, it may be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. Alternatively, it may be applied to the skin in the form of a cream.

[0072] The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion, and response sensitivity, and a physician who is normally skilled can easily determine and prescribe a dosage effective for the desired treatment or prevention.

Claims

Claim 1 A pharmaceutical composition having an effect of treating or preventing skin inflammation, comprising pyrrolidine dithiocarbamate (PDTC) as an active ingredient. Claim 2 A pharmaceutical composition having a therapeutic or preventive effect against skin inflammation, characterized in that, in claim 1, the skin inflammation is skin inflammation induced by Cutibacterium acnes (C. acnes). Claim 3 A pharmaceutical composition having a therapeutic or preventive effect against skin inflammation, wherein the pyrrolidine dithiocarbamate (PDTC) inhibits the secretion of IL-1β by inhibiting NF-κB activation and inflammatory activation induced by Cutibacterium acnes (C. acnes). Claim 4 A pharmaceutical composition having a therapeutic or preventive effect against skin inflammation, characterized in that, in claim 1, the skin inflammation is acne. Claim 5 A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

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