CN104546819A - Use of dicaffeoylquinic acid in treatment of systemic autoimmune disease - Google Patents

Abstract

The invention discloses the use of dicaffeoylquinic acid and dicaffeoylquinic acid derivatives represented by formula 1 in treating systemic autoimmune diseases. Preferred systemic autoimmune diseases are selected from systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren syndrome, polymyositis and the like.

Description

Use of dicaffeoylquinic acid in the treatment of systemic autoimmune diseases

technical field

The invention relates to a new application of dicaffeoylquinic acid (DCQA) in the treatment of systemic autoimmune diseases such as systemic lupus erythematosus and a pharmaceutical composition containing dicaffeoylquinic acid.

Background technique

Systemic autoimmune disease refers to the body's immune response to its own components to produce autoantibodies, which combine to form antigen-antibody complexes and deposit on blood vessel walls, etc., resulting in damage or dysfunction of multiple tissues and organs throughout the body. Including systemic lupus erythematosus (Systemic Lupus Erythematosus, SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), Sjogren's syndrome (SS), etc.

Take the more serious systemic lupus erythematosus as an example. SLE is an autoimmune disease that can affect the skin and multiple systems of the body, and the exact etiology is still unclear. The disease is more common in young women. The prevalence rate in my country is about 70/100,000, and more than 50% of the confirmed cases have damage to important organs such as liver and kidney. At present, it is considered that the excessive activation and proliferation of B lymphocytes, the production of autoantibodies and the formation of immune complex deposition are one of the important pathogenic factors of this disease. Currently commonly used therapeutic drugs are: 1) hydroxychloroquine; 2) glucocorticoids; 3) immunosuppressants. Although treatment can effectively alleviate the condition, it cannot be cured. Moreover, the existing drugs also have certain deficiencies, such as the long-term use of the existing immunosuppressants will cause bone marrow suppression, reduce the body's anti-infection ability, and often affect glucose and lipid metabolism to varying degrees. Targeted biotherapeutics such as relatively safe monoclonal antibodies are expensive. Therefore, new small molecule therapeutic drugs with clear mechanism of action, high efficiency, low toxicity and low price have obvious clinical value.

Contents of the invention

The purpose of the present invention is to provide a new medicine for treating autoimmune diseases, which has excellent curative effect and low toxic and side effects.

Dicaffeoylquinic acid (DCQA) has entered phase II clinical trials as an antiviral drug. When the present inventors conducted extensive and in-depth research, they found that DCQA has a significant inhibitory effect on the proliferation of B lymphocytes, can significantly reduce the level of anti-double-stranded DNA antibodies in the serum of systemic lupus erythematosus syndrome-like mice, and reduce the level of immune complexes. Deposition in kidney tissue improves pathological damage of kidney tissue. The present invention has been accomplished based on the above findings.

The first object of the present invention relates to the use of dicaffeoylquinic acid derivatives and analogues of formula 1 (including both enantiomers if there are enantiomers) in the preparation of medicaments for the treatment of systemic autoimmune diseases , the systemic autoimmune diseases include but not limited to systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, polymyositis.

in:

Q ₁ is a monovalent bond, or CH ₂ , CHOH, CH ₂ CH ₂ , CH ₂ CHOH, CHOHCH ₂ , CHCOOH, CH ₂ CH ₂ CHOH, CHOHCH ₂ CH ₂ , CH ₂ CHOHCH ₂ , CH ₂ CHOHCHOH or CHOHCHOHCH ₂ ;

Q ₂ and Q ₃ can be H respectively or together represent CH ₂ CH ₂ , CH ₂ CHOH, CHOHCH ₂ , CH ₂ CH ₂ CHOH, CHOHCH2CH2, CH ₂ CHOHCH ₂ , CH ₂ CHOHCHOH, CHOHCHOHCH ₂ , CH ₂ COH ( COOH)CH ₂ , CH ₂ CH ₂ COH(COOH)CH ₂ or CH ₂ COH(COOH)CH ₂ CH ₂ , thereby forming a ring structure,

_X is O, NH, _CH or CHCOOH;

X ₂ is O, NH, CH ₂ or CHCOOH;

Y ₁ , Y ₂ , Y ₃ and Y ₄ are each a monovalent bond, O or NH;

When each of Y ₁ , Y ₂ , Y ₃ and Y4 is a monovalent bond, R ₁ , R ₂ , R ₃ , and R ₄ are CH ₂ COOH, CH ₂ CH ₂ COOH or CH ₂ CH ₂ CH ₂ COOH;

When each of Y ₁ , Y ₂ , Y ₃ and Y ₄ is O or NH, R ₁ , R ₂ , R ₃ , and R ₄ are H, CH ₃ , CH ₃ CO, CH ₃ CH ₂ CO or CH ₃ CH _{2 CH2CO} ;

Y ₅ , Y ₆ , Y ₇ , Y ₈ , Y ₉ , Y ₁₀ are H, Cl, Br or I;

Z ₁ and Z ₂ are H, COOH, COOM, wherein M represents H, C _1-6 alkyl, benzyl, alkali metal or alkaline earth metal.

When two enantiomers of Formula 1 exist, both enantiomers are included.

Preferred dicaffeoylquinic acid derivatives are selected from 1,3-di-O-caffeoylquinic acid, 1,4-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid quinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, their acetylation derivatives and their corresponding salts.

Another object of the present invention relates to a pharmaceutical composition containing the compound of formula 1, which has a significant inhibitory effect on systemic lupus erythematosus or related diseases. According to the present invention, various pharmaceutical excipients, additives and carriers can be added in the pharmaceutical composition.

According to the present invention, the pharmaceutical composition of the present invention can be made into preparations for enteral or parenteral administration according to methods known in the art, such as tablets, capsules, granules, injections, sprays, creams, nasal drops wait.

Description of drawings

Figure 1 1,5-dicaffeoylquinic acid reduces renal tissue damage in SLE syndrome-like mice

A1 normal group animal kidney tissue; A2～A3 model group animal kidney tissue diffuse injury, glomerular cell proliferation, capillary necrosis, crescent formation, moderate renal tubular lesion, renal interstitial mononuclear, lymphocyte infiltration, Renal vascular fibrosis; B1 prednisolone 5mg/Kg group, renal tissue damage was effectively improved; B2～B31,5-dicaffeoylquinic acid 0.22mg/Kg group and 1.1mg/Kg group, renal tissue damage was obvious improve

Figure 2 1,5-dicaffeoylquinic acid reduces IgG immune complex deposition in kidney tissue of SLE syndrome-like mice

A1 normal animal group, A2～A3 model animal group, diffuse renal tissue injury, a large amount of IgG deposited in the glomerular mesangium, renal tubular basement membrane and renal interstitial infiltrating inflammatory cells; B1 prednisolone 5mg/Kg group, Slight fluorescence in glomeruli and renal tubules; IgG immune complex deposition significantly decreased in B2～B31,5-dicaffeoylquinic acid 0.22mg/Kg and 1.1mg/Kg groups

Detailed ways

The present invention will be further illustrated by the found related effects of dicaffeoylquinic acid.

Embodiment 1. Dicaffeoylquinic acid significantly inhibits the proliferation of mouse splenic B lymphocytes cultured in vitro

1.1 Materials and methods

Clean-grade BALB/c mice, male, weighing (19±2) g, were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences. LPS, ConA, DMSO, MTT Sigma products. RPMI1640 complete medium contains penicillin 100U/L, streptomycin 100U/L, glutamine 2mM and 10% fetal bovine serum. Mice were sacrificed by decapitation, spleen was aseptically removed, lymphocyte suspension was prepared routinely, cells were adjusted to 4×10 ⁹ /L with RPMI1640 complete medium, and 0.1ml/well was inoculated in 96-well plate. Add LPS (final concentration is 10mg/L), and 1,5-dicaffeoylquinic acid or 1,3-dicaffeoylquinic acid (final concentration is 1×10 ^-13 -1×10 ^-6 mol/ L), 6 replicate wells for each concentration. cultured in a 37°C, 5% CO ₂ incubator. After 44 hours, 10 μL of MTT (5 g/L) was added to each well, and the incubation was continued for 4 hours. After centrifugation, the supernatant was gently discarded, 120 μL of acidified isopropanol was added, and the absorbance at 540 nm was measured with a microplate reader.

1.2 Test results

(1) 1,5-dicaffeoylquinic acid has an inhibitory effect on the proliferation of LPS-induced mouse B lymphocytes, and the inhibitory effect is at a concentration of 1×10 ^-13 -10 ^-12 mol/L and 1×10 ^-6 Significantly (p<0.05, Table 1).

Table 11, the effect of 5-dicaffeoylquinic acid on the proliferative response of mouse spleen B-lymphocytes induced by LPS

(*p<0.05, p<0.01 vs control group)

(2) 1,3-dicaffeoylquinic acid has an inhibitory effect on LPS-induced mouse B lymphocyte proliferation, and the inhibitory effect is obvious at the concentration of 1×10 ^-12 and 1×10 ^-7 mol/L (p< 0.05, Table 2).

Table 21, Effect of 3-dicaffeoylquinic acid on LPS-induced proliferation of mouse spleen B-lymphocytes

(*p<0.05, p<0.01 vs control group)

Example 2. Dicaffeoylquinic acid significantly reduces the antibody level and immune complex deposition in active DNA-induced systemic lupus erythematosus syndrome-like mice, and improves renal tissue damage

2.1 Materials and methods

Activation of splenic lymphocytes and extraction of genomic DNA BALB/c mice were sacrificed, spleens were taken aseptically, spleen cell suspensions were routinely prepared, counted with trypan blue (survival rate > 95%), and cells were adjusted with RPMI1640-20% fetal bovine serum 2×10 ⁹ /L, add Con A to make the final concentration 3mg/L, add 20mL per bottle into culture flask, culture upright in 37℃, 5% CO2 incubator, remove after 48h, and collect activated cells by centrifugation. Active DNA was prepared according to the operation steps of the kit by using a large number of rapid extraction kits for animal genome DNA (Beijing Biotech Biogene Technology Co., Ltd.). Establishment of systemic lupus erythematosus syndrome-like mouse model BALB/c mice, 6wk, female. The mice were divided into random groups, 6 in each group, the model group and the drug group were immunized, and the blank group did not receive any treatment. The immunization program is: intradermal multi-point injection, once every 2 weeks, 0.1mg/0.2mL each time, 3 times in total. For the first immunization, the dry DNA powder was added with normal saline to 2 mg/mL, fully emulsified with an equal volume of Freund's complete adjuvant and injected. The second immunization was injected with DNA and Freund's incomplete adjuvant emulsifier; the third immunization was DNA suspension. During the sensitization process, blood was drawn from the orbit to detect the level of anti-ds-DNA antibody in serum. On the eighth day after the third immunization, daily gavage administration was started, 0.2 mL/10 g body weight, 15 times in total. The dosages were 0.22, 1.1, 3.3 and 10 mg/Kg body weight of 1,5-dicaffeoylquinic acid respectively; the positive control drug prednisolone acetate was 5 mg/Kg; the model group and the normal control group were given 0.5% carboxymethyl cellulose. After the last administration, the animals were sacrificed by taking blood; the serum to be tested was collected; the thymus, spleen and liver were weighed to calculate the visceral index; the kidneys were fixed in 4% formaldehyde solution for pathological examination. Anti-ds-DNA Antibody Detection 96-well ELISA plate (COSTAR), add 100 μL salmon dsDNA (Sigma, 50 mg/L) to each well, coat overnight at 4°C, wash the plate with PBS-0.5% Tween20, 2 mg/mL BSA-PBS Adsorb, wash the plate and add 100 μL of diluted serum, use goat-anti-mouse IgG-HRP (eBioscience) and enzyme-linked immunosorbent assay (ELISA) detection kit (Wuhan Boster), and measure the 450nm OD value according to the kit operation method ( Microplate reader, Labsystems). Kidney Histomorphology and Immunopathological ExaminationMouse kidneys were fixed with 4% formaldehyde solution and sectioned in 5μm paraffin. Routine section processing, 1) HE staining, light microscope observation of renal tissue morphological changes, blind pathological scoring: 0 = normal tissue; 1 = mild glomerular lesion; 2 = glomerular mesangial hyperplasia (<30% ), mild renal tubular lesions, mild interstitial mononuclear, lymphocyte infiltration; 3 = a certain proportion (<50%) of the glomeruli showed activity and sclerosis, such as mesangial cell proliferation, capillary necrosis, Microthrombosis, crescent formation, moderate renal tubulopathy, moderate interstitial mononuclear, lymphocytic infiltration, renal vascular fibrosis; 4=>50% glomerular involvement, moderate-severe renal tubular lesions and renal vascular fibrosis Interstitial mononuclear, lymphocytic infiltration, renal vascular fibrous necrosis. 2) Use goat-anti-mouse IgG-FITC (eBioscience) to perform fluorescent examination of IgG immune complexes in kidney tissue, and blind pathological scoring: 0=basically no fluorescence; 1=slightly fluorescent individual glomeruli; 2=a certain number Mild to moderate fluorescence in glomeruli, mild to moderate fluorescence in <25% of tubules; 3 = a large number of glomeruli, 25-50% of tubules, moderate intensity fluorescence in infiltrating inflammatory cells; 4 = large area of glomeruli Strong fluorescence, IgG immune complex deposition in >50% of renal tubules.

test results

(1) On the 7th day after the third immunization, the serum anti-ds-DNA antibody levels of mice in all immunized groups were significantly higher than those in the blank control group, indicating that the model was established successfully.

(2) After 15 days of administration, the serum anti-ds-DNA antibody levels of mice in the 0.22 mg/Kg body weight dicaffeoylquinic acid administration group were 0.270±0.048, which was significantly lower than that of the model group (0.445±0.110) (p<0.05 ,table 3).

Table 31, Effect of 5-dicaffeoylquinic acid on serum anti-ds-DNA antibody of SLE syndrome-like mice

(*p<0.05, **p<0.01 vs model control group)

(3) The routine pathological examination results of HE showed that the kidney tissue of the mice in the model group was diffusely damaged, and lupus nephritis occurred obviously (Table 4, Figure 1A2, A3). 1,5-dicaffeoylquinic acid (0.22-10mg/Kg body weight) and prednisolone (5mg/Kg body weight) administered for 15 days can effectively improve renal pathological damage, in which 1,5-dicaffeoylquinic acid Quinic acid 0.22, 1.1mg/Kg body weight improved significantly (Table 4, Figure 1B1-3).

Table 41, Effect of 5-dicaffeoylquinic acid on renal tissue injury in SLE syndrome-like mice

(*p<0.05 vs model control group)

(4) The results of fluorescent examination of IgG immune complexes in kidney tissue showed that the immunopathological score of kidney tissue in the model group was significantly higher than that in the normal control group (p<0.05, Table 5, Figure A2-A3), prednisolone ( 5mg/Kg body weight) and 1,5-dicaffeoylquinic acid (0.22, 1.1mg/Kg body weight) were administered continuously for 15 days, which could significantly reduce the deposition of IgG immune complexes in kidney tissue (p<0.05, Table 5, Fig. 2).

Table 51, Effect of 5-dicaffeoylquinic acid on immune complex deposition in kidney tissue of SLE syndrome-like mice

(*p<0.05 vs model control group)

The above results show that 1,5-dicaffeoylquinic acid can significantly reduce the serum anti-ds-DNA antibody level of active DNA-induced systemic lupus erythematosus syndrome-like mice, and significantly reduce the deposition and deposition of IgG immune complexes in kidney tissue. Kidney tissue damage.

In summary, dicaffeoylquinic acid can significantly inhibit the proliferation of mouse splenic B lymphocytes cultured in vitro, significantly reduce the serum anti-ds-DNA antibody level of active DNA-induced systemic lupus erythematosus syndrome-like mice, and reduce the IgG The deposition of immune complexes in renal tissue can improve the pathological damage of lupus nephritis. Dicaffeoylquinic acid can be used as a new drug or drug combination for the treatment of autoimmune diseases such as systemic lupus erythematosus.

Claims (4)

1. the use of dicaffeoylquinic acid derivatives as shown in formula 1 in the preparation of medicines for systemic autoimmune diseases, Wherein, Q ₁ is a monovalent bond, or CH ₂ , CHOH, CH ₂ CH ₂ , CH ₂ CHOH, CHOHCH ₂ , CHCOOH, CHOHCH ₂ CH ₂ , CH ₂ CHOHCH ₂ , CH ₂ CH ₂ CHOH, CH ₂ CHOHCHOH or CHOHCHOHCH ₂ ; Q ₂ and Q ₃ can be H respectively or together represent CH ₂ CH ₂ , CH ₂ CHOH, CHOHCH ₂ , CH ₂ CH ₂ CHOH, CHOHCH ₂ CH ₂ , CH ₂ CHOHCH ₂ , CH ₂ CHOHCHOH, CHOHCHOHCH ₂ , CH ₂ COH(COOH)CH ₂ , CH ₂ CH ₂ COH(COOH)CH ₂ or CH ₂ COH(COOH)CH ₂ CH ₂ , thus forming a ring structure, _X is O, NH, _CH or CHCOOH; X ₂ is O, NH, CH ₂ or CHCOOH; Y ₁ , Y ₂ , Y ₃ and Y ₄ are independently selected from monovalent bonds, O or NH; When Y ₁ , Y ₂ , Y ₃ and Y ₄ are independently selected from monovalent bonds, R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from CH ₂ COOH, CH ₂ CH ₂ COOH or CH ₂ H _{2 CH2COOH} ; When Y ₁ , Y ₂ , Y ₃ and Y ₄ are independently selected from O or NH, R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from H, CH ₃ , CH ₃ CO, CH ₃ CH ₂ CO _{or CH3CH2CH2CO ;} Y ₅ , Y ₆ , Y ₇ , Y ₈ , Y ₉ , Y ₁₀ are independently selected from H, Cl, Br or I; Z ₁ and Z ₂ are independently selected from H, COOH, COOM, wherein M represents H, C _1-6 alkyl, benzyl, alkali metal or alkaline earth metal. 2. The use of claim 1, when formula 1 has two enantiomers, both enantiomers are included. 3. purposes according to claim 1, it is characterized in that, described dicaffeoylquinic acid derivative is selected from 1,3-di-O-caffeoylquinic acid, 1,4-di-O-caffeoyl quinic acid, 1,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5- Di-O-caffeoylquinic acids, their acetylated derivatives and their corresponding salts. 4. The use according to claim 1, characterized in that the systemic autoimmune disease is selected from systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, and polymyositis.