Application of berberine derivative in preparation of medicine for preventing and treating ulcerative colitis

Abstract

The invention belongs to the field of biological medicines, discloses a novel application of a berberine derivative, and particularly relates to an application of the berberine derivative as an NLRP3 inflammasome inhibitor and a mitochondrial chaperonin HSP60 regulator in preparation of medicines for preventing and treating ulcerative colitis. The anti-inflammatory activity of the berberine derivative is obviously improved compared with that of berberine, and the berberine derivative has further research and development value in the aspect of preventing and treating ulcerative colitis. In addition, the NLRP3 and HSP60 double-target action mechanism of the berberine derivative provides a potential value for intervention of other diseases with abnormal NLRP3 and / or HSP60.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and discloses a new use of a berberine derivative, and specifically relates to application of the berberine derivative as an NLRP3 inflammasome inhibitor and a mitochondrial chaperone HSP60 modulator in preparation of a medicine for preventing and treating ulcerative colitis. BACKGROUND

[0002] Ulcerative colitis (UC) is a chronic intestinal inflammatory disease, and is collectively referred to as inflammatory bowel disease (IBD) together with Crohn's disease (CD). UC has become a global health challenge, and the epidemiological trend is constantly evolving. Mitochondrial dysfunction and abnormal activation of NLRP3 inflammasome are important pathogenesis of UC, but there is currently a lack of drugs targeting mitochondria or NLRP3 inflammasome on the market. Current UC treatment drugs mainly include aminosalicylates, glucocorticoids, immunomodulators and biological agents, although some small molecule drugs are on the market, but the current treatment strategy can only achieve clinical remission in 30% to 60% of patients, and biological drugs are often expensive due to stringent manufacturing and quality control standards, which has caused great burden on the economy and psychology of patients. Therefore, it is of great significance to develop more economical and effective drugs for treating UC.

[0003] Berberine (BBR) is a plant alkaloid with isoquinoline structure, which exists widely in nature and can also be obtained by artificial synthesis. Modern pharmacological studies have shown that berberine has various biological activities such as antibacterial, antioxidant, anti-inflammatory, hypoglycemic, lipid-lowering, and anti-tumor activities, and some studies have explored its effective role in the treatment of UC. However, berberine has poor water solubility and extremely low oral bioavailability, which greatly affects its clinical application. Therefore, developing berberine derivatives through structural modification provides a new strategy with more transformation potential for anti-inflammatory and UC prevention and treatment. Here, we found a berberine derivative, which is named Benzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium, 9-(benzoyloxy)-5,6-dihydro-10-methoxy-, which can exist in the form of a chloride salt, and the present application simply refers to it as B4. Its chemical formula is as follows:

[0004]

[0005] So far, there is no record and report in the prior art about B4 directly targeting NOD-like receptor heat protein domain-related protein 3 (NLRP3) and heat shock protein 60 (HSP60). Given the pivotal role of NLRP3 and / or HSP60 in various pathological processes (such as rheumatoid arthritis and other diseases related in the literature "Carnazzo V, Rigante D, Restante G, et al. The entrenchment of NLRP3 inflammasomes in autoimmune disease-related inflammation. Autoimmun Rev. 2025, 24(7): 103815" and the literature "van Eden W, Jansen MAA, Ludwig IS, et al. Heat Shock Proteins Can Be Surrogate Autoantigens for Induction of Antigen Specific Therapeutic Tolerance in Rheumatoid Arthritis. Front Immunol. 2019, 10: 279."), B4 has potential cross-disease application value due to its dual-targeting mechanism. This study focuses on verifying its prevention and treatment efficacy for UC. SUMMARY

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a compound B4 for preventing and treating UC and to clarify the target of the compound, providing a new choice for the prevention and treatment of UC.

[0007] On the one hand, the present application provides the use of B4 in inhibiting inflammatory factor IL-1β, and the half maximal inhibitory concentration (IC 50 ) of B4 for IL-1β inhibition is 0.25 μM, while the IC 50 of BBR is 12.15 μM, and the activity of B4 is increased by 48.6 times compared with BBR.

[0008] On the other hand, the present application provides that B4 has good safety in vivo and in vitro. In vitro cytotoxicity experiments show that the survival rates of macrophages and colon epithelial cells intervened by B4 within 100 μM are all above 80%, and no significant difference is shown compared with BBR; B4 mouse acute oral toxicity shows that the median lethal dose (LD 50 ) is greater than 2000 mg / kg.

[0009] Further, the use of B4 in effectively preventing and treating UC is better than the same dose of berberine and twice the dose of mesalazine (5-ASA).

[0010] Further, the direct target of B4 is mitochondrial chaperone HSP60, which is directly regulated by B4.

[0011] Further, B4 exerts the role of regulating mitochondrial homeostasis through the direct target HSP60, and reduces the further activation of inflammation as damage-associated molecular patterns (DAMPs) of HSP60.

[0012] Further, another direct target of B4 is NLRP3, which is directly inhibited by B4.

[0013] Further, B4 inhibits the activation of the NLRP3 inflammasome pathway through the direct target NLRP3, and reduces the release of pro-inflammatory factors IL-1β and IL-18.

[0014] Further, B4 effectively breaks the vicious positive feedback loop of “mitochondrial dysfunction-NLRP3 inflammasome activation-inflammation exacerbation-mitochondrial damage aggravation” through the dual-target effect.

[0015] The “prevention and treatment” of the present application refers to the use of B4 to alleviate the disease in the case of possible UC or already occurred UC.

[0016] The dual-target mechanism (HSP60 regulation + NLRP3 inhibition) of the drug of the present application provides a new strategy for intervention in the pathological conditions related to abnormal activation of mitochondria and / or NLRP3 inflammasome. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1100uM or less B4 and BBR intervention in THP-1 macrophage cell survival rate

[0018] Figure 2100uM or less B4 and BBR intervention in HIEC cell survival rate

[0019] Figure 3 HE staining of mouse organ tissues after acute gavage of 2000mg / kg B4, observed after 14 days

[0020] Figure 4 B4 prophylactic treatment of DSS-induced UC mice body weight change graph (BBR and 5-ASA as control)

[0021] Figure 5 B4 prophylactic treatment of DSS-induced UC mice disease activity index (DAI) score change graph (BBR and 5-ASA as control)

[0022] Figure 6 B4 prophylactic treatment of DSS-induced UC mice colon length change graph (BBR and 5-ASA as control)

[0023] Figure 7 B4 prophylactic treatment of DSS-induced UC mice colon tissue HE staining chart (BBR and 5-ASA as control)

[0024] Figure 8 Surface plasmon resonance (SPR) verified the sensorgram of direct binding of HSP60 to B4

[0025] Figure 9 Surface plasmon resonance (SPR) verified the sensorgram of direct binding of HSP60 to B4, KD = 0.839 μΜ

[0026] Figure 10 B4 intervention LPS / ATP-induced THP-1 macrophage supernatant HSP60 change chart

[0027] Figure 11 B4 intervention before and after HSP60 knockdown LPS / ATP-induced THP-1 macrophage mitochondrial membrane potential improvement chart

[0028] Figure 12 Surface plasmon resonance (SPR) verified the sensorgram of direct binding of NLRP3 to B4

[0029] Figure 13 Surface plasmon resonance (SPR) verified the sensorgram of direct binding of NLRP3 to B4, KD = 3.52 μΜ

[0030] Figure 14 B4 intervention before and after NLRP3 knockdown LPS / ATP-induced THP-1 macrophage supernatant IL-1β level regulation change chart

[0031] Figure 15 B4 intervention before and after NLRP3 knockdown LPS / ATP-induced THP-1 macrophage supernatant IL-18 level regulation change chart DETAILED DESCRIPTION

[0032] In order to better understand the present application, the above content of the present application is further described in detail by the following specific examples. However, this should not be understood as a limitation of the present application. The following are specific examples of the present application, which further describe the technical solutions of the present application, but the content of the present application is not limited to the scope described in the examples, any changes or equivalent substitutions without departing from the concept of the present application are included in the protection scope of the present application.

[0033] The experimental methods in the following examples are all routine methods, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0034] Example 1: IC of B4 and BBR inhibiting LPS / ATP-induced IL-1β release of macrophages 50 Determination

[0035] Using the laboratory-derived THP-1 human monocyte cell line, which was cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin in a 37°C, 5% CO2 incubator. Before the experiment, the cells were seeded into a 6-well plate (density 5 x 10 5 cells / well), 100 nM phorbol ester (PMA) was added for 24 hours of incubation to induce differentiation into macrophages. After differentiation, the medium was replaced with serum-free medium.

[0036] After 1 μg / mL lipopolysaccharide (LPS) was added to the differentiated macrophages for 3 hours of stimulation, different concentrations (0.1-20 μM) of B4 or BBR were added for 2 hours of continuous incubation. The model group was added with the same volume of DMSO solvent, 5 mM adenosine triphosphate (ATP) was added 30 minutes before the collection of cell supernatant, and the blank control group was not stimulated by LPS / ATP.

[0037] The cell supernatant was aspirated, centrifuged at 3000g at 4°C for 5 minutes to remove cell debris. The human IL-1β ELISA kit was used, and the operation was completed according to the instructions. The absorbance was measured at 450 nm wavelength using a microplate reader, and the IL-1β concentration of each group was calculated according to the standard curve.

[0038] Using GraphPad Prism 9.0 software, the four-parameter logistic model (4PL) was selected to fit the concentration-inhibition rate curve, and the IC 50 of B4 on IL-1β inhibition was calculated to be 0.25 uM, while the IC 50 of BBR was 12.15 uM.

[0039] Example 2: In vitro cytotoxicity determination of B4

[0040] Macrophage model: THP-1 human monocyte cell line was used, and the operation was the same as in Example 1. Before use, it was induced to form adherent macrophages; colon epithelial cell model: HIEC human normal colon epithelial cells were selected, and were cultured in DMEM medium (containing 10% FBS and 1% double-antibiotic). Both cells were cultured at 37°C, 5% CO2, and logarithmic growth phase cells were used for the experiment.

[0041] The experimental group was 6.25, 12.5, 25, 50, 100 μM B4 or BBR, the blank control was complete medium containing 0.1% DMSO, and the cell blank group was cell-free medium. Macrophages and colon epithelial cells were seeded into 96-well plates (density 1 x 10 4After 24 hours of adhesion, the medium was replaced with the medium containing the corresponding concentration of compound, and 3 wells were set for each group. The treatment lasted for 24 hours.

[0042] Cell viability was determined by CCK8 method. The OD value was read by microplate reader at 450 nm wavelength.

[0043] Survival rate (%) = [(experimental group OD 450 - cell blank group OD 450 ) / (blank control group OD 450 - cell blank group OD 450 )] x 100%. The data were expressed as mean ± standard error, and the comparison between groups was analyzed by one-way ANOVA. Survival rate ≥ 80% was considered to have no significant toxicity.

[0044] The results are shown in Figure 2 and Figure 3 , and the survival rates of macrophages and colon epithelial cells treated with B4 within 100 μM were all above 80%.

[0045] Example 3: In vivo acute oral toxicity of B4 in mice

[0046] According to the No. 423 guide of the Organization for Economic Cooperation and Development (OECD), the in vivo toxicological properties of B4 were studied. Considering that the cytotoxicity of B4 is comparable to that of BBR, and the median lethal dose (LD 50 ) of BBR in mice is more than 300 mg / kg; in addition, since the pharmacodynamic evaluation of B4 was performed using C57BL / 6 mice, and considering that females are more sensitive than males in acute toxicity tests, the use of female mice can result in conservative results, B4, DMSO, polyethylene glycol 300 (PEG300) and sterile water were prepared into a suspension with a concentration of 2000 mg / kg, and were administered to 3 randomly selected female mice (6 in total for two tests) by gavage within 24 hours. The control group was given the same volume of solvent. All mice were observed for two weeks, and their condition was recorded daily. After the observation period and a period of fasting, tissue samples of heart, liver, spleen, lung, kidney and colon were collected for HE staining evaluation.

[0047] As shown in Figure 3 , the heart, liver, spleen, lung, kidney and colon of mice in the B4 treatment group showed no obvious pathological changes, indicating that there was no obvious toxicity to these organs at this dose.

[0048] Example 4: Effect of B4 in the prophylactic treatment of DSS-induced UC in mice

[0049] Select 6-8 weeks old male C57BL / 6 mice, body weight 18-22 grams, randomly divided into 6 groups, 6 in each group, respectively, normal control group (NC), DSS model group (DSS), B4 low dose administration group (B4, 10 mg / kg), B4 high dose administration group (B4, 20 mg / kg), BBR administration group (BBR, 20 mg / kg) and 5-ASA administration group (5-ASA, 40 mg / kg). The administration group is given corresponding dose of drug by gavage, and the rest of the group is given the same volume of solvent, once a day, for 7 days. Except for the normal control group, the model group and the administration group freely drink 2.5% DSS water for modeling for seven days. The body weight change and DAI score of mice were recorded every day during the experiment. After the experiment, the mice were sacrificed by cervical dislocation, the colon tissue was collected and the length was measured, and the colon end tissue was stained with HE.

[0050] As shown in Figure 4 , B4 prophylactic treatment can dose-dependently slow down the weight loss of DSS-induced UC mice, and the effect is better than that of the same dose of BBR and twice the dose of 5-ASA.

[0051] As shown in Figure 5 , B4 prophylactic treatment can dose-dependently reduce the disease activity index of DSS-induced UC mice, and the effect is better than that of the same dose of BBR and twice the dose of 5-ASA.

[0052] As shown in Figure 6 , B4 prophylactic treatment dose-dependently protects mice from colon shortening caused by colon tissue damage, and the effect is better than that of the same dose of BBR and twice the dose of 5-ASA.

[0053] As shown in Figure 7 , B4 prophylactic treatment dose-dependently protects DSS-induced UC mice from colon pathological tissue damage, and the effect is better than that of the same dose of BBR and twice the dose of 5-ASA.

[0054] Example 5: B4 regulates mitochondrial homeostasis by directly binding to HSP60 and reduces the release of HSP60 as a DAMP

[0055] The method of SPR was used to verify the direct binding of B4 to HSP60. The ligand protein HSP60 was immobilized on the CM5 sensor chip by amine coupling chemistry (NHS / EDC activation), and the reference channel was set using protein-free acetate buffer. Gradient-diluted analyte B4 was injected at a flow rate of 30 μL / min for 60 seconds, and the chip surface was regenerated by using glycine-HCl solution at pH 2.0 between cycles, and the background refraction interference was corrected by subtracting the reference signal and blank injection. Finally, the Biacore analysis software was used to globally fit the sensorgram with a 1:1 Langmuir binding model to obtain the equilibrium dissociation constant (K D ). AsFigures 8-9 K value of B4 binding to HSP60 was 0.839 μM, indicating that B4 directly bound to HSP60 with high affinity. D

[0056] THP-1 cells were induced to differentiate into macrophages according to the operation of Example 1. After 1 μg / mL lipopolysaccharide (LPS) was added to stimulate the differentiated macrophages for 3 h, 10 μM and 20 μM B4 were added respectively to continue incubation for 15 h. The model group was added with the same volume of DMSO solvent, and 5 mM adenosine triphosphate (ATP) was added 30 min before the supernatant of the cells was collected. The blank control group was not stimulated by LPS / ATP. Figure 10 As shown in the figure, B4 reduced the concentration of HSP60 in the supernatant of the cells in a dose-dependent manner.

[0057] THP-1 cells were inoculated in a 6-well plate (density 5×10 5 macrophage differentiation was induced by adding 100 nM PMA for 24 h. The siHSP60 sequence with the highest knockdown efficiency was used to knock down HSP60 of the THP-1 cells induced into macrophages in the experimental group, and the empty sequence was used as a control. The transfection procedure was as follows:

[0058] siHSP60 or siNC was diluted in 250 μL Opti-MEM to a final concentration of 50 nM, and was allowed to stand for 5 min. Lipofectamine 3000 was diluted in 250 μL Opti-MEM, and was allowed to stand for 5 min. The two solutions were mixed, and were incubated at room temperature for 20 min to form a complex. The cell culture hole (final volume 2 mL) was added, and was transduced for 6 h before the complete culture medium was replaced. After transfection for 48 h, 1 μg / mL LPS was added to the model group to stimulate for 3 h, 20 μM B4 was added to the drug group to continue incubation for 15 h, the model group was added with the same volume of DMSO solvent, and 5 mM ATP was added 30 min before the next experiment. The blank control group (siHSP60 for HSP60 knockdown group, and siNC for empty group) was not stimulated by LPS / ATP.

[0059] ​To detect the change of mitochondrial membrane potential, JC-1 staining working solution was prepared in advance: take appropriate amount of JC-1 (200X), dilute JC-1 according to the ratio of 8ml ultrapure water per 50ul JC-1 (200X). Vortex vigorously to dissolve and mix well. Then add 2ml JC-1 staining buffer (5X), mix well to get JC-1 staining working solution. Remove the culture medium from each group, wash the cells with PBS gently for 2 times. Add 1ml fresh cell culture medium to each well, then add 1ml JC-1 staining working solution and mix well. Incubate in a cell incubator at 37°C for 20 minutes. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) according to the ratio of 4ml distilled water per 1ml JC-1 staining buffer (5X) and place it in ice bath. After 37°C incubation, remove the supernatant and wash with JC-1 staining buffer (1X) for 2 times. Add 2ml cell culture medium and observe under fluorescence microscope.

[0060] As shown in Figure 11 , B4 intervention improves the decline of mitochondrial membrane potential, and this improvement is significantly weakened after knocking down HSP60, indicating that B4 plays this role by targeting HSP60. It is proved that B4 plays a role in regulating mitochondrial homeostasis by directly acting on the target HSP60 and reducing the further activation of inflammation by HSP60 as DAMP.

[0061] Example 6: B4 inhibits inflammasome activation by directly binding to NLRP3 and reduces the release of pro-inflammatory factors IL-1β and IL-18

[0062] The method of SPR was used to verify the direct binding of B4 to NLRP3. The ligand protein NLRP3 was immobilized on the CM5 sensor chip by amine coupling chemistry (NHS / EDC activation), and the reference channel was set using protein-free acetate buffer. Gradient-diluted analyte B4 was injected at a flow rate of 30ul / min for 60 seconds, and the chip surface was regenerated by glycine-HCl solution at pH 2.0 between cycles, and the background refraction interference was corrected by subtracting the reference signal and blank injection. Finally, the Biacore analysis software was used to globally fit the sensorgram with 1:1 Langmuir binding model to obtain the equilibrium dissociation constant (K D ). As shown in Figures 12-13 , the K D value of B4 binding to NLRP3 is 3.52uM, indicating that B4 has direct binding to NLRP3.

[0063] THP-1 cells were seeded in 6-well plates (density 5×10 5Cells were seeded in 6-well plates (5x105cells / well) and induced to differentiate into macrophages by adding 100 nM PMA for 24 hours. The siNLRP3 with the highest knockdown efficiency was used to knock down NLRP3 in THP-1 cells induced into macrophages, and the siNC was used as a control. The transfection procedure was as follows:

[0064] Each group of siNLRP3 or siNC was diluted in 250 μL Opti-MEM to a final concentration of 50 nM and allowed to stand for 5 minutes. Lipofectamine 3000 was diluted in 250 μL Opti-MEM and allowed to stand for 5 minutes. The two solutions were mixed and incubated at room temperature for 20 minutes to form a complex. The complex was added to the cell culture wells (2 mL final volume), and the complete medium was replaced after 6 hours of transfection. After 48 hours of transfection, 1 μg / mL of LPS was added to the model group for 3 hours, and 20 μM of B4 was added to the B4 intervention group for 15 hours. The model group was added with the same volume of DMSO solvent, and 5 mM of ATP was added 30 minutes before the collection of the cell supernatant. The blank control group (siNLRP3 for NLRP3 knockdown group and siNC for empty group) was not stimulated by LPS / ATP. The cell supernatant was aspirated and centrifuged at 3000 g for 5 minutes at 4°C to remove cell debris. Human IL-1β and IL-18 ELISA kits were used to complete the operation according to the instructions. The absorbance was measured at 450 nm using a microplate reader, and the IL-1β and IL-18 concentrations in the supernatant of each group were calculated according to the respective standard curves.

[0065] The results are shown in Figure 14 B4 intervention dose-dependently reduced the IL-1β concentration in the cell supernatant, and knocking down NLRP3 itself also reduced the production of IL-1β under LPS / ATP stimulation, but the down-regulation effect of B4 disappeared, indicating that B4 plays a role in down-regulating the abnormal secretion of IL-1β by targeting NLRP3. As shown in Figure 15 B4 intervention dose-dependently reduced the IL-18 concentration in the cell supernatant, and knocking down NLRP3 itself also reduced the production of IL-18 under LPS / ATP stimulation, but the down-regulation effect of B4 was weakened, indicating that B4 plays a role in down-regulating the abnormal secretion of IL-18 by targeting NLRP3. In general, B4 inhibits the activation of the NLRP3 inflammasome pathway by directly acting on the target NLRP3, reducing the release of pro-inflammatory factors IL-1β and IL-18.

[0066] According to the positive feedback vicious cycle between mitochondrial damage and NLRP3 inflammasome activation, B4 can effectively break the vicious positive feedback cycle of "mitochondrial dysfunction-NLRP3 inflammasome activation-inflammation aggravation-mitochondrial damage aggravation" by double-target action.

[0067] In summary, the present application proves for the first time through a series of examples that berberine derivative B4 blocks the vicious cycle of "mitochondrial damage -> NLRP3 activation -> inflammation amplification -> secondary mitochondrial damage" by double targeting mitochondrial chaperone HSP60 and NLRP3 inflammasome, providing a new choice for the prevention and treatment of UC. In view of the important role of NLRP3 and HSP60 in the pathology of rheumatoid arthritis and the like, this double-target mechanism provides a potential new strategy for intervention in diseases related to mitochondria and / or inflammasome.

Claims

1. Use of a berberine derivative in the preparation of a drug for preventing and treating ulcerative colitis, the structural formula of the compound being as follows: The compound has a better curative effect than an equivalent dose of berberine and twice the dose of mesalazine in a DSS-induced UC mouse model.

2. Use according to claim 1, characterized in that: The compounds inhibit the release of the pro-inflammatory factor IL-1 β with a half maximal inhibitory concentration (IC 50 ) of 0.25 uM.

3. Use according to claim 1, characterized in that: The compounds have good safety in vivo and in vitro: the survival rate of macrophages and colon epithelial cells is maintained at more than 80% at a concentration of 100 μM, and the acute oral median lethal dose (LD 50 ) of mice is greater than 2000 mg / kg.

4. Use according to claim 1, characterized in that: The use is achieved by inhibiting the activation of NLRP3 inflammasome and regulating the function of mitochondrial chaperone HSP60.

5. The use according to claim 1, characterized in that: The compound plays a preventive and therapeutic role through the following mechanisms:

6. Use according to claim 1, characterized in that: (a) directly targeting HSP60 and regulating its function, restoring mitochondrial homeostasis and reducing the release of HSP60 as a DAMP; (b) directly targeting NLRP3, blocking inflammasome assembly and IL-1β / IL-18 mature release; (c) synergistically blocking the vicious cycle of "mitochondrial damage→NLRP3 activation→inflammation amplification→secondary mitochondrial damage". ​

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