WO2025223198A1 - Use of danshensu or pharmaceutically acceptable salt thereof as pi3k/akt/mtor signaling pathway inhibitor in preparation of drug for treating psoriasis - Google Patents

Abstract

Disclosed is use of danshensu or a pharmaceutically acceptable salt thereof as a PI3K/AKT/mTOR signaling pathway inhibitor in the preparation of a drug for treating psoriasis. Pharmacological experiments reveal that danshensu can inhibit the phosphorylation of the PI3K/AKT/mTOR signaling pathway to significantly ameliorate psoriasis-like skin lesions on mice and reduce the excessive proliferation and abnormal differentiation of epidermal cells and the expression of CCL20mRNA in skin lesions. In vitro experiments show that danshensu can inhibit TNF-α-induced HaCaT cell proliferation and inhibit the generation and secretion of the pro-inflammatory factors IL-6, IL-8, and IFN-γ and the chemokine CCL20. The use of danshensu to prepare a drug for treating psoriasis opens up a new field of application for danshensu.

Description

Application of tanshinone or its drug-acceptable salts as inhibitors of the PI3K/AKT/mTOR signaling pathway in the preparation of drugs for treating psoriasis. Technical Field

This invention belongs to the field of medicinal chemistry technology related to tanshinone, specifically relating to the application of tanshinone or its drug-acceptable salt as an inhibitor of the PI3K/AKT/mTOR signaling pathway in the preparation of drugs for treating psoriasis.

Background Technology

Psoriasis is an immune-mediated, chronic, relapsing, inflammatory, systemic disease induced by both genetic and environmental factors, affecting approximately 3% of the global population. It is characterized by abnormal differentiation (parakeratosis), epidermal hyperplasia (acanthosis), and dermal inflammatory infiltration. Patients may also experience multi-system involvement, including the joints, nails, eyes, cardiovascular system, and gastrointestinal tract.

Psoriasis primarily affects young adults and has a prolonged course. Currently, there is no cure, causing significant impact on patients, their families, and society. While the pathogenesis of psoriasis is not fully understood, the immune-inflammatory circuit involving the interaction between keratinocytes and immune cells, particularly T cells, is crucial to its development.

Therefore, developing drugs to treat psoriasis has become one of the technical challenges in this field.

Danshensu (Salvianic acid A, DSS), or D(+)-β-(3,4-dihydroxyphenyl)lactic acid, is an important water-soluble active ingredient in tanshinone and also the basic structural unit of salvianolic acid compounds. DSS has shown good therapeutic potential in cardiovascular diseases (such as MI/RI, atherosclerosis, hypertension, hyperlipidemia, and myocardial infarction), brain injury and neurodegenerative diseases (such as cerebral ischemia, Alzheimer's disease, Parkinson's disease, and anxiety), and other health problems (such as hypoxic pulmonary hypertension, acute pneumonia, liver fibrosis, and vision protection).

Studies have shown that tanshinone exerts its pharmacological effects primarily through improving microcirculation, anti-oxidation, anti-apoptosis, improving energy metabolism, regulating inflammation, reducing calcium ion influx, and promoting angiogenesis, as well as regulating signaling pathways such as PI3K/Akt/ERK1/2/Nrf2/HO-1, HIF-1α/STA T3/NLRP3, eNOS, mTOR, PKA-CREB, and SIRT1/ROS. However, it remains unclear whether tanshinone has a therapeutic effect on psoriasis.

Summary of the Invention

This invention relates to tanshinone or a pharmaceutically acceptable salt thereof, and also to the use of pharmaceutical compositions comprising tanshinone or a pharmaceutically acceptable salt thereof as inhibitors of the PI3K/AKT/mTOR signaling pathway in the preparation of medicaments for the treatment of psoriasis.

This invention, based on previous network pharmacology and molecular docking technology predictions of DSS targets, uses TNF-α-stimulated HaCaT cells as a cell model and an IMQ-induced psoriasis-like mouse model to verify whether tanshinone has anti-psoriasis effects and to explore its molecular mechanism of action. While discovering potential new drugs for psoriasis treatment, this invention also broadens the medical applications and fields of tanshinone, possessing significant clinical and scientific research value.

This invention provides the use of tanshinone or its pharmaceutically acceptable salt as an inhibitor of the PI3K/AKT/mTOR signaling pathway in the preparation of medicaments for the treatment of psoriasis.

Furthermore, the tanshinone has a structure as shown in formula (1):

Furthermore, the tanshinone or its drug-acceptable salt inhibits the production and secretion of pro-inflammatory factors IL-6, IL-8, IFN-γ and chemokine CCL20 by inhibiting TNF-α-induced HaCaT cell proliferation and apoptosis.

Furthermore, the tanshinone or its drug-acceptable salt improved the psoriasis-like PASI score in mice.

Furthermore, the tanshinone or its drug-acceptable salt improves psoriasis-like skin lesions in mice by inhibiting phosphorylation of the PI3K/AKT/mTOR signaling pathway, reducing excessive proliferation and abnormal differentiation of epidermal cells, and the expression of CCL20 mRNA in the lesions.

Furthermore, the effective dose of the tanshinone or its pharmaceutically acceptable salt is 1–1000 mg/kg.

Furthermore, the drug is a clinically acceptable formulation made by mixing tanshinone or its pharmaceutically acceptable salt as the active ingredient with a pharmaceutically acceptable excipient or carrier.

Furthermore, the preparation is an oral preparation or an injectable preparation.

Furthermore, the oral preparation is an oral tablet.

In this invention, tanshinone or its pharmaceutically acceptable salts refer to basic and/or acidic salts formed by tanshinone with inorganic bases and/or acids, organic bases and/or acids, including zwitterionic salts (internal salts), and quaternary ammonium salts, such as alkyl ammonium salts. The salts described in this invention are selected from: sodium tanshinone, potassium tanshinone, calcium tanshinone, lithium tanshinone, magnesium tanshinone, ammonium tanshinone, meglumine tanshinone, amine tanshinone, arginine tanshinone, and lysine tanshinone.

The advantages of this invention compared to the prior art are as follows:

Pharmacological experiments revealed that tanshinone significantly improved psoriatic-like skin lesions in mice by inhibiting phosphorylation of the PI3K/AKT/mTOR signaling pathway, reducing excessive proliferation and abnormal differentiation of epidermal cells, and decreasing the expression of CCL20 mRNA in the lesions. In vitro experiments showed that tanshinone inhibited TNF-α-induced HaCaT cell proliferation and suppressed the production and secretion of pro-inflammatory factors IL-6, IL-8, IFN-γ, and the chemokine CCL20. The use of tanshinone in the preparation of drugs for treating psoriasis opens up new application areas for tanshinone.

Attached Figure Description

Figure 1 shows the trend of PASI scores in each group of mice.

Figure 2 shows that DSS inhibits TNF-α-induced keratinocyte proliferation; ****P<0.001, vs TNF-α group. ^#### P<0.001, vs Ctrl group.

Figure 3 shows the induction of apoptosis and the quantification of apoptotic cells by Annexin V-FITC/PI staining after 12 hours of treatment of HaCaT cells with TNF-α damage using different doses of DSS.

Figure 4 shows the inhibition of TNF-α-induced CCL20, IL-6, IFN-γ and IL-8 mRNA expression levels in keratinocytes by DSS, as well as the secretion of CCL20, IL-6, IFN-γ and IL-8 in cell supernatant; *P<0.05,**P<0.01,vs TNF-α group, ##P<0.01,vs Ctrl group.

Figure 5 shows the effect of different concentrations of DSS on phosphorylation of the PI3K/AKT/mTOR signaling pathway in an IMQ-induced psoriasis model; *P<0.05,**P<0.01,vs Model group.##P<0.01,vs Ctrl group.

Figure 6 shows the cellular thermal displacement analysis of AKT1 bound to DSS.

Detailed Implementation

The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

As used herein, the term "pharmaceutically acceptable salt of tanshinone" refers to basic and/or acidic salts formed by tanshinone with inorganic bases and/or acids, organic bases and/or acids, including zwitterionic salts (internal salts), and quaternary ammonium salts, such as alkyl ammonium salts. The salts described in this invention are selected from: sodium tanshinone, potassium tanshinone, calcium tanshinone, lithium tanshinone, magnesium tanshinone, ammonium tanshinone, meglumine tanshinone, amine tanshinone, arginine tanshinone, and lysine tanshinone.

The tanshinone involved in this invention can be administered to patients in the form of a pharmaceutically acceptable salt or pharmaceutical composition. A complex needs to be mixed with a suitable carrier or excipient to form a pharmaceutical composition to ensure an effective therapeutic dose. "Effective therapeutic dose" refers to the dose necessary for the tanshinone derivative to achieve a therapeutic effect.

Tanshinone or its pharmaceutically acceptable salts can be formulated into various dosage forms, including solid, semi-solid, liquid, and aerosol formulations (Remington’s Pharmaceutical Sciences, Mack Publishing Company (1995), Philadelphia, PA, 19th ed.). Specific dosage forms within these categories include tablets, pills, sugar tablets, granules, gels, ointments, solutions, suppositories, injections, inhalers, and sprays. These dosage forms can be used for both local and systemic administration, as well as for immediate-release or sustained-release administration.

When tanshinone or its pharmaceutically acceptable saline is administered by injection, these compounds can be formulated into solutions, suspensions, and emulsions using water-soluble or lipid-soluble solvents. Lipid-soluble solvents specifically include vegetable oils and similar oils, synthetic fatty acid glycerides, higher fatty acid esters, and proylene glycol esters. These compounds are more readily soluble in ethanol solutions and trace amounts of DMSO solutions.

When tanshinone or its pharmaceutically acceptable saline is administered orally, it can be compounded with pharmaceutically acceptable excipients using common techniques. These excipients can formulate these compounds into various dosage forms that can be used by patients, such as tablets, pills, suspensions, and gels. There are several methods for formulating oral dosage forms, such as first mixing the compound and solid excipients, thoroughly grinding the mixture, adding appropriate excipients, and processing it into granules. Excipients that can be used to formulate oral dosage forms include: sugars such as lactose, sucrose, mannitol, or sorbitol; and celluloses such as corn starch, wheat starch, potato starch, gelatin, taro gum, methylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone.

The tanshinone or its pharmaceutically acceptable salts involved in this invention can also be formulated as a spray, which is achieved via a pressurizer and a sprayer or a dry powder inhaler. Suitable propellants that can be used in the sprayer include dichlorodifluoromethane, chloroform, dichlorotetrafluoroethane, carbon dioxide, and dimethyl ether. The dosage of the aerosol can be adjusted via a valve on the sprayer.

The various dosage forms involved in this invention relate to the effective therapeutic dose of tanshinone or its pharmaceutically acceptable salts. The effective therapeutic dose of these compounds depends on the patient receiving treatment. In determining the appropriate dose, the patient's weight, condition, method of administration, and the prescribing physician's subjective judgment must be taken into account. The therapeutically effective dose of tanshinone derivatives and compositions containing these compounds should be determined by a competent and experienced prescribing physician.

Although the effective therapeutic dose of tanshinone or its pharmaceutically acceptable salts can vary depending on the patient’s condition, the usual appropriate dosage range is 1–1000 mg/kg.

Example 1

1. Materials

1.1 Animals

Male C57BL/6 mice (6-8 weeks old; 18-22g) were purchased from Shanghai Regen Biotech Co., Ltd. These mice were fed a standard diet, followed a 12-hour circadian rhythm, had free access to water, and were housed in the SPF-grade animal experimental center of Shanghai University School of Medicine. All experiments were approved by the Shanghai University Science and Technology Ethics Committee.

1.2 Drugs and Reagents

Biological reagents and consumables were purchased commercially (Shanghai Titan Technology Co., Ltd., Sinopharm Chemical Reagent Co., Ltd., Sigma-Aldrich, Beyotime Biotechnology Co., Ltd., Suzhou Xinsaimei Biotechnology Co., Ltd., Wuhan Aiboteke Biotechnology Co., Ltd., Tuoran Biotechnology Co., Ltd., etc.). Western blot and IP cell lysis buffer, Annexin V-FITC apoptosis detection kit, and other reagents were purchased from Beyotime Biotechnology Co., Ltd., while antibodies were purchased from Wuhan Aiboteke Biotechnology Co., Ltd., Tuoran Biotechnology Co., Ltd., etc.

2 Methods

2.1 Experimental grouping, modeling, and drug administration

IMQ was used to induce psoriasis: 50 mg of 5% imiquimod (IMQ) cream was applied topically to the shaved area (3 × 2.5 cm) on the back of mice for 7 consecutive days. C57BL/6 mice were randomly divided into 5 groups of 6 mice each, as follows: (1) control group and IMQ group were given saline orally; (2) IMQ + low-dose DSS group were given 25 mg/kg DSS orally; (3) IMQ + medium-dose DSS group were given 50 mg/kg DSS orally; (4) IMQ + high-dose DSS group were given 100 mg/kg DSS orally. IMQ cream was administered 4 hours before each day for 14 days.

2.2 Sample Collection

The mice's weight and skin condition were recorded. On day 15, mice were anesthetized with isoflurane, blood was collected and centrifuged, tissue samples were fixed and subjected to hematoxylin and eosin staining, Western blotting analysis, and real-time quantitative PCR analysis.

2.3 Psoriasis-like skin lesions and disease severity index (PASI) scores in mice

The Psoriasis Area and Severity Index (PASI) (measuring skin erythema, scaling, and thickness) was used to assess the status of psoriasis-like lesions daily for 14 consecutive days. Skin erythema severity was rated on a 5-point scale (0, none; 1, mild; 2, moderate; 3, significant; 4, severe). A PASI score trend graph was plotted.

2.4 Cell Culture

The HaCaT cell line was obtained from the Shanghai Academy of Sciences Cell Bank (Shanghai, China). Cells were grown in DMEM medium supplemented with 10% FBS and incubated at 37°C in a humidified atmosphere of 5% _CO2 .

2.5 MTT Reduction Assay for Cell Viability

An in vitro model was established using TNF-α treatment of HaCaT cells, and cell viability was assessed using the MTT assay. Cells were seeded at a density of 5000 cells per well in 96-well plates containing DMEM + 10% FBS and treated with 20 ng/mL TNF-α. After 12 hours of incubation, different concentrations of DSS or DMEM (control) were added to the wells, followed by 12 hours of incubation. 10 μL of MTT solution was added to each well, and the plates were incubated for 4 hours. Finally, cells were lysed with 0.04 N HCl in isopropanol solution, and the absorbance at 570 nm was assessed in each well.

2.6 Flow Cytometry Analysis

HaCaT cells were harvested to assess apoptosis. After washing twice with PBS, cells were resuspended in 200 μL of annexin V-FITC binding buffer (10 mM HEPES, 140 mM NaCl, ₂ mM MgCl₂, 5 mM KCl, 2.5 mM _CaCl₂ ; pH 7.4) and 10 μL of binding buffer. Annexin V was added to each tube according to the manufacturer's instructions. After incubating in the dark at room temperature for 15 minutes, 10 μL of PI and 200 μL of binding buffer were added to each tube. Finally, the samples were analyzed using flow cytometry.

2.7 Real-time quantitative PCR

After adding TNF-α or a control for 12 hours, DSS or DMEM (control) was added to the wells for co-incubation. After 12 hours, the treated HaCaT cells were harvested to analyze the expression of IFN-γ, CCL20, IL-6, IL-8, and GAPDH mRNA. Total RNA was isolated using Trizol reagent. RNA purity and concentration were determined using a NanoDrop 2000 device (Thermo Scientific, Wilmington, DEUSA). The mRNA was then directly reverse transcribed into cDNA using an RT-PCR kit according to the manufacturer's instructions. PCR amplification conditions were 35 cycles of initial denaturation at 95°C for 15 seconds, followed by 5 seconds of denaturation at 95°C and 15 seconds of annealing at 61°C. Target gene primer sequences are shown in the table below (from 5' to 3'). Relative mRNA counts were determined using the 2ΔΔCt method, and data normalization was performed using the GAPDH housekeeping gene as an internal control for qPCR.

Table 1 Primer sequences for target genes

2.8 Western blot analysis of pathway protein expression levels in mouse skin lesions

Cell samples were lysed using RIPA buffer containing phosphatase and protease inhibitors, and protein quantification was performed using a BCA kit. Protein denaturation, gel preparation, electrophoresis, electroporation, and blocking were performed as before. Primary antibody was prepared using antibody dilution buffer, incubated overnight at 4°C, and then washed three times with TBST for 10 minutes each time. Secondary antibody was then incubated for one hour, followed by three washes with TBST and development with ECL chemiluminescence buffer.

2.9 Interaction analysis between AKT1 and tanshinone

Based on previous network pharmacology and molecular docking studies, AKT1 was identified as one of the key target proteins of tanshinone. Cellular thermal shift assay (CETSA) was used to assess the interaction between AKT1 and tanshinone. For intracellular CETSA experiments, a corresponding volume of 100 mM tanshinone stock solution was transferred to complete culture medium, diluted to 100 μM, and used for cell incubation for 12 h and 24 h; another dish of cells was left untreated as a negative control. After incubation, the culture medium was removed, and the cells were washed twice with 2 mL PBS to remove excess compound. Then, 2 mL PBS was added to scrape the cells and collect them into 1.5 mL centrifuge tubes. The cells were centrifuged at 5000 rpm for 3 min at 4 °C to harvest cell pellets. These pellets were then resuspended in 1 mL PBS (pre-added with PMSF and a phosphatase inhibitor), carefully mixed, and aliquoted into nine 1.5 mL centrifuge tubes, 100 μL of cell suspension per tube. The centrifuge tubes were then heated on a heating block at the previously specified temperature for 3 min. After heating, remove the centrifuge tubes and equilibrate at 25°C for 3 minutes. Then, repeatedly freeze and thaw the cell suspension three times at 25°C and liquid nitrogen (vortexing briefly after each thawing to ensure uniform temperature). Centrifuge at 15,000 rpm for 30 minutes at 4°C. Take 80 μL of protein supernatant from each cell lysate tube, add 20 μL of 5×SDS loading buffer, and boil at 95°C for 10 minutes to fully denature the proteins. The samples obtained after the above steps are ready for subsequent Western blot analysis.

2.10 Statistical Analysis

Statistical analysis was performed using GraphPad Prism 8 software. All experiments were conducted in at least three replicates. Data are expressed as mean ± SEM. Statistical differences were analyzed using Student's t-test. A statistically significant difference was defined as P < 0.05, and P < 0.01 was considered statistically significant.

Example 2

1. Effects of DSS on IMQ-induced psoriatic lesions in mice

After 14 days of intervention in a predetermined manner, the skin on the backs of mice in the blank control group was pink and smooth, while the skin lesions on the backs of mice in the model group resembled psoriasis, including erythema, desquamation, and localized infiltration and thickening. High, medium, and low doses of IFA (IH, IM, and IL groups) and MTX treatment all alleviated these symptoms to varying degrees, resulting in smoother skin, less erythema, and sparser scales.

The PASI score was used for assessment, as shown in Figure 1, revealing the same trend as these changes. The results indicate that IFA can improve IMQ-induced psoriatic-like skin lesions in mice, with high and medium doses of IFA achieving better improvement.

2. Tanshinone inhibits cell proliferation and induces apoptosis in a psoriasis cell model.

HaCaT cells were treated with DSS solution obtained by serial dilution of DMEM for 24 hours, and the MTT assay was used to detect the DSS concentration for pre-screening subsequent experiments. Based on the experimental results, 10 μM, 50 μM, and 100 μM solutions were selected for subsequent in vitro experiments. TNF-α stimulation of 20 ng/mL was used to mimic the state of keratinocytes in psoriasis patients.

As shown in Figure 2, TNF-α can significantly induce the proliferation of HaCaT cells, while DSS can inhibit TNF-α-induced HaCaT cell proliferation in a dose-dependent manner.

3. Effects of DSS on the expression of IFN-γ, CCL20, IL-6, and IL-8 mRNA in HaCaT cell model

Keratinocytes, upon stimulation by initial triggering factors, can produce a variety of chemokines, among which CCL20 is a key chemokine for recruiting CCR6+Th17 cells and three groups of innate lymphoid cells (ILC3). In addition, several pro-inflammatory genes (IL-1β, IL-6, and IL-8, etc.) are involved in amplifying the IL-23/IL-17A axis and generating a "pre-feedback" inflammatory loop. Therefore, the effects of DSS on the expression of IFN-γ, CCL20, IL-6, and IL-8 mRNA were detected using real-time quantitative PCR.

As shown in Figure 4, the results indicate that DSS treatment can dose-dependently inhibit the expression of IFN-γ, CCL20, IL-6, and IL-8 mRNA. Similarly, IFA also inhibits TNF-α-induced secretion of IFN-γ, CCL20, IL-6, and IL-8 in HaCaT cells.

4. DSS improves psoriatic-like skin lesions in mice by inhibiting phosphorylation of the PI3K/AKT/mTOR signaling pathway.

Based on preliminary network pharmacology screening results, AKT1 was identified as one of the key target proteins of tanshinone. Furthermore, given that the classic PI3K/AKT/mTOR pathway is closely involved in the regulation of autophagy in the psoriasis process, the PI3K/AKT/mTOR signaling pathway in IMQ-induced psoriatic mice was investigated.

As shown in Figure 5, DSS treatment inhibited the phosphorylation of PI3K-p85α, Akt, and mTOR in IMQ-induced psoriatic mice and reduced the expression of the chemokine CCL20 mRNA. Therefore, DSS may induce autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway, thereby improving psoriatic-like skin lesions in mice.

5. The CETSA experiment on the binding of AKT1 protein to tanshinone showed a significant temperature shift.

The binding effect of AKT1 protein with tanshinone was verified. As shown in Figure 6, compared with the control group, after co-incubating the active compound tanshinone with HaCaT cell lysate for 1 h or with live cells for 12 h, AKT1 protein showed certain stability under gradient temperatures, with the thermal melting curve shifting significantly to the right and the maximum dissolution temperature difference ΔTm > 14℃. However, in the thermal melting curve after co-incubating tanshinone with live cells for 24 h, the thermal stability of AKT1 protein showed the same trend as the control group.

The results showed that tanshinone may have significantly improved the thermal stability of AKT1 protein by binding to it; however, after the incubation time was extended to 24 h, tanshinone may have been metabolized and transformed by cells, and the unbound AKT1 protein showed similar thermal stability to the control group.

This document describes the compounds, their preparation methods, and applications of the present invention in conjunction with specific embodiments and examples, and also sets forth and explains many details. However, it should be understood that the specific embodiments and examples provided herein are merely exemplary and do not limit the scope of protection of the present invention. In fact, those skilled in the art will recognize that the present invention can be implemented in other specific ways, and any modifications, alterations, or adjustments made accordingly do not depart from the spirit and intent of the present invention, and therefore should all be considered to be included within the scope of the present invention.

Claims (7)

The use of tanshinone or its drug-acceptable salts as inhibitors of the PI3K/AKT/mTOR signaling pathway in the preparation of drugs for the treatment of psoriasis. According to the application of claim 1, the characteristic is that: the tanshinone has the structure shown in formula (1):
According to the application of claim 1, the characteristic is that: the tanshinone or its drug-acceptable salt inhibits the production and secretion of pro-inflammatory factors IL-6, IL-8, IFN-γ and chemokine CCL20 by inhibiting TNF-α-induced HaCaT cell proliferation and apoptosis. According to the application of claim 1, the characteristic is that: the tanshinone or its drug-acceptable salt improves psoriasis-like skin lesions in mice by inhibiting phosphorylation of the PI3K/AKT/mTOR signaling pathway, reducing excessive proliferation and abnormal differentiation of epidermal cells and the expression of CCL20 mRNA in the skin lesions. According to claim 1, the effective dose of the tanshinone or its pharmaceutically acceptable salt is 1 to 1000 mg/kg. According to the application of claim 1, the drug is characterized in that it is a clinically acceptable formulation prepared by mixing tanshinone or its pharmaceutically acceptable salt as the active ingredient with a pharmaceutically acceptable excipient or carrier. According to claim 6, the application is characterized in that the preparation is an oral preparation or an injectable preparation.