A sulfonated albumin derivative, its preparation and use

Abstract

The present application belongs to the field of biological medicine, and particularly relates to sulfonated albumin derivatives and application thereof. The derivatives are introduced with sulfonic groups in the albumin molecular skeleton, so that the derivatives can inhibit secretion of inflammatory factors by M1 macrophages and exhibit anti-inflammatory activity. The protein derivatives can be used as functional materials or drugs for treating and / or preventing macrophage M1 polarization related inflammatory diseases, and have application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to albumin derivatives with sulfonic acid structure modification and application thereof. BACKGROUND

[0002] Macrophages are important regulatory mediators of self-defense and repair of the body, and can be activated to M1 type through the classical pathway and release pro-inflammatory substances to develop and maintain inflammation to resist injury. M2 type macrophages exhibit anti-inflammatory type, and can reduce inflammation to restore normal body function by secreting cell surface molecules and anti-inflammatory active substances such as IL-10. However, when macrophages are in M1 type for a long time, chronic inflammation occurs, which is extremely harmful to the human body, such as rheumatoid arthritis and osteoarthritis. At present, the most widely used drugs in clinic are non-steroidal anti-inflammatory drugs and glucocorticoids, and long-term use has many side effects, such as gastrointestinal damage and central obesity. In addition, immunosuppressive drugs are often used for autoimmune diseases such as rheumatoid arthritis, but systemic immunosuppression and high price often cause great burden to patients and their families. Therefore, there is an urgent need for a safe, effective and low-cost substance to inhibit the polarization of M1 type macrophages, so as to treat and / or prevent the development of diseases related to the polarization of M1 type macrophages.

[0003] Albumin, including human serum albumin, recombinant human serum albumin, bovine serum albumin and porcine serum albumin, etc., has been widely used due to its low immunogenicity, high biocompatibility and low cost. At present, the application of albumin mainly focuses on two aspects: 1) as a carrier to encapsulate drugs; 2) chemical bonding of drugs to reduce drug toxicity and improve bioavailability. However, few studies have been conducted on structural modification of albumin to endow albumin with specific therapeutic effects.

[0004] In view of the above problems, the present application first proposes to modify albumin by sulfonation. Substitution reactions are carried out on carboxyl, amino or sulfhydryl groups on the molecular chain of albumin to obtain a series of albumin derivatives with different degrees of substitution. In vivo and in vitro experiments have shown that the series of albumin derivatives can inhibit M1 type macrophages, thereby inhibiting the release of pro-inflammatory substances, including reactive oxygen species and pro-inflammatory cytokines, and exhibit significant anti-inflammatory activity, which can be applied to various inflammatory diseases and has great application prospect. No literature or patent has reported this kind of albumin derivative. SUMMARY

[0005] One of the purposes of the present application is to provide an albumin derivative with sulfonic acid group structure. The derivative can inhibit M1 type macrophages and exhibit anti-inflammatory activity, and can be used as a functional material or a pharmaceutical excipient for inflammatory diseases related to the polarization of M1 type macrophages.

[0006] Another object of the present application is to provide a method for preparing the sulfonated albumin derivative.

[0007] Still another object of the present application is to provide the use of the sulfonated albumin derivative and the composition thereof in inflammatory diseases.

[0008] The sulfonated albumin derivative, wherein the albumin is selected from the group consisting of, but not limited to, human serum albumin, recombinant human serum albumin, bovine serum albumin and porcine serum albumin.

[0009] The sulfonic group donor is characterized by being a sulfonic derivative, one end of which provides a sulfonic group, and the other end of which has a group for coupling with the albumin, including but not limited to carboxyl, amino or aldehyde group, preferably carboxyl. Its chemical structure is as follows:

[0010]

[0011] wherein n = 0 to 20, preferably n = 0 to 10. R1 is a coupling group for substitution reaction with the albumin, including but not limited to carboxyl, amino or aldehyde group; Y is hydrogen ion, metal ion or a mixture of hydrogen ion and metal ion, preferably sodium ion.

[0012] The sulfonated albumin derivative, wherein the coupling group in the albumin molecule chain for coupling with the sulfonic donor molecule is amino, carboxyl or sulfhydryl, preferably amino. Its structure is schematically shown as follows:

[0013]

[0014] wherein n = 0 to 20, m = 1 to 60. Y is hydrogen ion, metal ion or a mixture of hydrogen ion and metal ion, preferably sodium ion.

[0015] The sulfonated albumin derivative, further preferably by reacting the amino group in the albumin molecule chain with the carboxyl group in the sulfonic group donor molecule to provide a certain number of sulfonic groups, inhibiting M1 macrophages, as schematically shown as follows:

[0016]

[0017] wherein m = 1 to 60.

[0018] The method for preparing the sulfonated albumin derivative comprises the following steps:

[0019] The albumin is dissolved or dispersed in a mixed solvent of water or organic solvent, substitution reaction is carried out between the amino or carboxyl group in the sulfonic group donor molecule and the amino, carboxyl or sulfhydryl in the albumin molecule chain, impurities are removed by dialysis bag, and then lyophilization is carried out to obtain the product. By controlling the reaction time and the ratio of raw materials, a series of albumin derivatives with different substitution degrees can be obtained.

[0020] The method for calculating the degree of substitution is obtained by detecting the content change of free α-amino group in albumin molecule before and after modification by ninhydrin colorimetry. Albumin molecule contains about 60 α-amino groups, and then the number of sulfonic acid groups modified on one albumin molecule, i.e. the value of m, is determined according to the percentage of the degree of substitution obtained by the ninhydrin method.

[0021] The sulfonated albumin derivative is characterized in that it can be used for treating and / or preventing diseases related to M1 polarization of macrophages, preferably treating and / or preventing inflammatory diseases, including but not limited to atopic dermatitis and rheumatoid arthritis, preferably rheumatoid arthritis.

[0022] The substance for treating and / or preventing diseases related to M1 polarization of macrophages includes but is not limited to drugs, sulfonated albumin derivatives, sulfonated albumin derivative preparations or sulfonated albumin derivative pharmaceutical compositions, preferably sulfonated albumin derivative gels.

[0023] The sulfonated albumin derivative gel, preferably the gel matrix is hyaluronic acid, is characterized in that it is a medical grade.

[0024] Beneficial effects

[0025] The present application surprisingly found that BSA itself can promote LPS-activated RAW 264.7 cells to release more active oxygen and pro-inflammatory cytokines, and after sulfonation structural modification, this effect is obviously reversed, i.e. sBSA can significantly reduce the level of active oxygen and pro-inflammatory factor release in the positive model group cells, and shows a dose-dependent characteristic. After incubation with a higher concentration of sBSA (100 μg / mL), the levels of active oxygen and pro-inflammatory factors can be reduced to below the negative group, which further illustrates the importance of sulfonation structural modification. In addition, there is no significant difference between the anti-inflammatory effect of sBSA and sBSA / HA, which may be because the content of HA in the gel system is low, which is not enough to reflect its anti-inflammatory activity, and the inflammation level of the HA matrix control group has no significant difference with the positive group, which also directly proves this point. In addition, sulfonation is a protein substitution method, which has not been reported, and after sulfonation, it is reversed, which may be related to the formation of derivative structure. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Circular dichroism spectrum of sulfonated bovine serum albumin.

[0027] Figure 2 Scanning electron micrographs of sulfonated bovine serum albumin and its hyaluronic acid gel (hyaluronic acid (a), sulfonated bovine serum albumin (b) and sulfonated bovine serum albumin hyaluronic acid gel (c)).

[0028] Figure 3RAW 264.7 cells (Mean ± SD, n = 4, compared with positive group: ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001; compared with negative group: ### p<0.001).

[0029] Figure 4 RAW 264.7 cells (Mean ± SD, n = 4, compared with positive group: ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001; compared with negative group: ## p<0.01, ### p<0.001).

[0030] Figure 5 Clinical index changes (clinical score (a), knee joint diameter (b), paw thickness (c) and ankle joint diameter (d), Mean ± SD, n = 6, compared with disease group: ns p>0.05, # p<0.05, ## p<0.01, ### p<0.001; compared with disease group: * p<0.05, ** p<0.01, *** p<0.001; ## p<0.01, ### p<0.001).

[0031] Figure 6 Histological analysis of knee joint of rheumatoid arthritis rats treated by intra-articular injection of sulfonated albumin derivative and its hydrogel (H&E staining (a) and type II collagen immunohistochemical staining (b)). DETAILED DESCRIPTION

[0032] The present application is further illustrated by the following specific examples, but the present application is not limited to the contents contained in the following examples.

[0033] sHSA: sulfonated human serum albumin;

[0034] Example 1

[0035] Human serum albumin and 3-sulfonic acid benzene carboxylic acid disodium salt monohydrate containing carboxyl group were dissolved in appropriate amount of phosphate buffer (PBS, pH 7.2) respectively, the latter was added dropwise into the human serum albumin solution, and the amide reaction was carried out between the amino group in human serum albumin and the carboxyl group in the sulfonic acid group donor. After 10-30 min, the solution was dialyzed in PBS (pH 7.2) for 48 h, and then freeze-dried to obtain the product.

[0036] Determination of the degree of substitution:

[0037] The degree of substitution of the prepared human serum albumin sulfonic acid group was tested by the following method:

[0038] Human serum albumin was used as a standard substance, and a series of standard protein solutions with different concentrations were prepared by dissolving in purified water. 1 mL of ice acetic acid buffer (pH 5.4), standard protein solution and 2% ninhydrin color developing solution were mixed uniformly, heated at 100°C for 15 min, cooled to room temperature after standing, then diluted with 5 mL of 60% ethanol, and the absorbance A of the solution was measured at 570 nm, repeated for 3 times.

[0039] Ninhydrin and free amino groups in albumin form a blue-violet substance under heating conditions. The content of free amino groups in albumin and sulfonated albumin derivatives can be detected by color reaction, and the degree of substitution can be calculated accordingly.

[0040] The degree of substitution formula (1) is as follows:

[0041]

[0042] According to the calculation of formula (1), the degree of substitution of the sulfonated human serum albumin prepared in Example 1 is 42.89±8.23%.

[0043] The structure of the sulfonated human serum albumin derivative prepared in Example 1 is shown in the following schematic diagram:

[0044]

[0045] According to the measured degree of substitution of 42.89±8.23% and the fact that the unmodified albumin molecule contains 60 α-amino groups, it is determined that m is 21 to 31.

[0046] Example 2

[0047] Human serum albumin and 3-sulfonic acid benzene carboxylic acid disodium salt monohydrate containing carboxyl group were dissolved in appropriate amount of phosphate buffer (PBS, pH 7.2) respectively, the latter was added dropwise into the human serum albumin solution, and the amide reaction was carried out between the amino group in human serum albumin and the carboxyl group in the sulfonic acid group donor. After 10-30 min, the solution was dialyzed in PBS (pH 7.2) for 48 h, and then freeze-dried to obtain the product.

[0048] Determination of the degree of substitution:

[0049] The sulfonated degree of the human serum albumin prepared in Example 1 was 54.19±8.02% as determined by the method described above.

[0050] The structure of the sulfonated human serum albumin prepared in Example 2 is shown in the following:

[0051]

[0052] According to the determined percentage of the substitution degree of 54.19±8.02% and the unmodified albumin molecule containing 60 α-amino groups, m is determined to be 28 to 37.

[0053] Example 3

[0054] The bovine serum albumin and 3-sulfonic acid benzene carboxylic acid disodium salt monohydrate containing carboxyl group were dissolved in an appropriate amount of phosphate buffer (PBS, pH 7.2) respectively, and the latter was added dropwise into the bovine serum albumin solution for amide reaction. After 20-40 min, the solution was dialyzed in PBS (pH 7.2) for 48 h and freeze-dried to obtain the product.

[0055] The sulfonated degree of the bovine serum albumin prepared in Example 3 was 63.37±7.08% as determined by the method described above.

[0056] The structure of the sulfonated bovine serum albumin prepared in Example 3 is shown in the following:

[0057]

[0058] According to the determined percentage of the substitution degree of 63.37±7.08% and the unmodified albumin molecule containing 60 α-amino groups, m is determined to be 34 to 42.

[0059] Example 4

[0060] The bovine serum albumin and 3-sulfonic acid benzene carboxylic acid disodium salt monohydrate containing carboxyl group were dissolved in an appropriate amount of phosphate buffer (PBS, pH 7.2) respectively, and the latter was added dropwise into the bovine serum albumin solution for amide reaction. After 20-40 min, the solution was dialyzed in PBS (pH 7.2) for 48 h and freeze-dried to obtain the product.

[0061] The sulfonated degree of the bovine serum albumin prepared in Example 3 was 63.37±7.08% as determined by the method described above.

[0062] The sulfonated degree of the bovine serum albumin prepared in Example 4 was 70.24±7.29% as determined by the method described above.

[0063] The structure of the sulfonated bovine serum albumin prepared in Example 4 is shown in the following:

[0064]

[0065] wherein the percentage of degree of substitution is determined to be 70.24±7.29%, and the unmodified albumin molecule contains 60 α-amino groups, m is determined to be 38 to 47.

[0066] Example 5

[0067] The bovine serum albumin and the carboxyl-containing 3-sulfonic acid phenylboronic acid disodium salt monohydrate were dissolved in an appropriate amount of phosphate buffer (PBS, pH 7.2) respectively, and the latter was added dropwise into the former. After 60-90 min of reaction, the solution was dialyzed in PBS (pH 7.2) for 48 h, and then lyophilized to obtain the product.

[0068] Determination of degree of substitution:

[0069] The determination method was the same as in Example 1, and the degree of sulfonic acid substitution of the bovine serum albumin prepared in Example 5 was 73.12±6.53%.

[0070] The structure of the sulfonated bovine serum albumin derivative prepared in Example 5 is shown in the following schematic diagram:

[0071]

[0072] wherein the percentage of degree of substitution is determined to be 73.12±6.53%, and the unmodified albumin molecule contains 60 α-amino groups, m is determined to be 40 to 48.

[0073] Example 6

[0074] 37.5 mg of the lyophilized powder of the sulfonated human serum albumin (sHSA) prepared in Example 1 was dissolved in 1.0 g of purified water. 0.1 g of carbomer was added and 9.9 g of purified water was added to swell the carbomer. The pH was adjusted to 6-7 with triethylamine. 0.2 g of the swollen carbomer was taken and 0.8 g of the sHSA solution was added. After stirring, the sHSA carbomer gel was obtained, and the sHSA carbomer gel lyophilizate was obtained by lyophilization.

[0075] Example 7

[0076] 37.5 mg of the lyophilized powder of the sHSA prepared in Example 2 was dissolved in 1.0 g of phosphate buffer (pH 7.2). 0.1 g of poloxamer 407 was added and 0.9 g of phosphate buffer (pH 7.2) was added to swell the poloxamer 407. 0.2 g of the swollen poloxamer 407 was taken and 0.8 g of the sHSA solution was added. After stirring, the sHSA poloxamer gel was obtained, and the sHSA poloxamer gel lyophilizate was obtained by lyophilization.

[0077] Example 8

[0078] According to the mass ratio of 10:1, take the appropriate amount of sulfonated bovine serum albumin (sBSA) freeze-dried powder prepared in Example 3 and hyaluronic acid in a test tube, add phosphate buffer (pH 7.2) to swell, so that the concentration of sBSA is 30 mg / mL, and the concentration of hyaluronic acid is 3 mg / mL, to obtain sBSA hyaluronic acid gel, and freeze-drying to obtain sBSA hyaluronic acid hydrogel lyophilizate.

[0079] Example 9

[0080] According to the mass ratio of 6.7:1, take the appropriate amount of sBSA freeze-dried powder prepared in Example 4 and hyaluronic acid in a test tube, add phosphate buffer (pH 7.2) to swell, so that the concentration of sBSA is 30 mg / mL, and the concentration of hyaluronic acid is 4.5 mg / mL, to obtain sBSA hyaluronic acid gel, and freeze-drying to obtain sBSA hyaluronic acid hydrogel lyophilizate.

[0081] Example 10

[0082] According to the mass ratio of 4.8:1, take the appropriate amount of sBSA freeze-dried powder prepared in Example 5 and hyaluronic acid in a test tube, add phosphate buffer (pH 7.2) to swell, so that the concentration of sBSA is 30 mg / mL, and the concentration of hyaluronic acid is 6.25 mg / mL, to obtain sBSA hyaluronic acid gel, and freeze-drying to obtain sBSA hyaluronic acid hydrogel lyophilizate.

[0083] Example 11

[0084] The following is a part of the cell anti-inflammatory activity determination of a series of sulfonated albumin derivatives and their compositions.

[0085] Experimental method:

[0086] 1. Determination of active oxygen level

[0087] Take RAW 264.7 cells in the logarithmic growth phase, inoculate 2×10 5 cells per well in a 24-well plate, and culture for 24 h to allow the cells to adhere and enter the logarithmic growth phase. Except for the negative control group, the other groups were activated for 4 h by replacing the culture medium with a culture medium containing lipopolysaccharide (LPS) at a concentration of 1 μg / mL. In the experimental group, the culture medium was replaced with complete culture medium containing sulfonated albumin derivative at a concentration of 100 μg / mL. The specific preparation was prepared according to the actual mass ratio by diluting the freeze-dried preparation of sulfonated albumin derivative or its hydrogel with complete culture medium. The positive control group was replaced with complete culture medium. The negative control group only added cells and complete culture medium. After incubation in an incubator for 24 h, the average fluorescence intensity in the cells was detected by flow cytometry using an active oxygen kit to reflect the active oxygen level. The lower the average fluorescence intensity, the lower the active oxygen level in the cells, and each group was repeated 4 times.

[0088] 2. Determination of pro-inflammatory factor expression

[0089] RAW 264.7 cells in logarithmic growth phase were inoculated in a 48-well plate at 2 x 10 5 cells per well, and cultured for 24 h to allow the cells to adhere and enter the logarithmic growth phase. Except for the negative control group, the other groups were replaced with culture medium containing 1 μg / mL of LPS for 4 h. In the experimental groups, the culture medium was replaced with complete culture medium containing 100 μg / mL of sulfonated albumin derivative, which was prepared by diluting the lyophilized preparation of sulfonated albumin derivative or its hydrogel with complete culture medium according to the actual mass ratio. The positive control group was replaced with complete culture medium. The negative control group was only added with cells and complete culture medium. After 24 h of incubation in an incubator, the cell supernatant was taken, and the expression of TNF-α, IL-6 and IL-1β, three pro-inflammatory factors, was detected using an ELISA kit, with 6 repeats for each group.

[0090] The following table is the anti-inflammatory activity data of some sulfonated albumin derivatives and their compositions.

[0091] Table 1 Anti-inflammatory activity of sulfonated albumin derivatives and their compositions (Mean ± SD)

[0092]

[0093] As can be seen from Table 1, sulfonated albumin derivatives with different degrees of substitution all exhibit obvious effects of inhibiting LPS-activated M1 macrophages, showing good antioxidant activity and the ability to inhibit the secretion of pro-inflammatory cytokines, especially the secretion of IL-6, indicating that structural modification of sulfonic groups can endow albumin with the effect of inhibiting M1 macrophages, exhibit significant anti-inflammatory activity, and have great application value.

[0094] In order to explore whether sulfonated albumin derivatives have dose dependence, we randomly selected Example 4 and Example 9 for further study, and added the following groups: BSA solution (100 μg / mL), BSA solution (50 μg / mL), Example 4 and Example 9 equivalent to 50 μg / mL of sBSA, and another hyaluronic acid control group, and detected the intracellular reactive oxygen species and the levels of pro-inflammatory factors in the cell supernatant, the results are shown in Figure 3 and Figure 4 .

[0095] According to the literature, albumin has both "positive" and "negative" effects in disease development, and can promote inflammatory cells to release more pro-inflammatory mediators and exacerbate inflammation [1,2] . Figure 3 and 4The results showed that BSA itself could promote the release of more active oxygen and pro-inflammatory cytokines from LPS-activated RAW264.7 cells. However, after sulfonated structural modification, this effect was obviously reversed, i.e., sBSA could significantly reduce the level of active oxygen and pro-inflammatory cytokine release in the positive model group cells, and showed a dose-dependent characteristic. After incubation with a higher concentration of sBSA (100 μg / mL), the levels of active oxygen and pro-inflammatory cytokines could be reduced to below the negative group, further illustrating the importance of sulfonated structural modification. In addition, there was no significant difference between the anti-inflammatory effect of sBSA and sBSA / HA, which may be because the content of HA in the gel system was low, which was not enough to reflect its anti-inflammatory activity, and the inflammation level of the HA matrix control group had no significant difference with the positive group, which also directly proved this point.

[0096] BALB / c mice aged 6-8 weeks were randomly divided into 4 groups, 6 mice in each group, respectively blank group, model group, sHSA group (Example 2), sHSA gel group (Example 7). A solution of acetone: olive oil = 4:1 (v / v) was configured as a matrix solution, and 1% and 0.5% of dinitrochlorobenzene (DNCB) were configured as atopic dermatitis inducing liquid using the matrix solution. Before the model was established, all mice were shaved on the back (2 cm x 2 cm) using depilatory cream for modeling and drug administration. On the 1st, 3rd and 7th day, 200 μL of 1% DNCB was used to smear the back of the mice, 20 μL of 1% DNCB was used to smear the right ear of the mice, and the blank group was smeared with the matrix solution; from the 8th to the 14th day, the mice were normally fed without treatment; between the 15th and 28th day, two days were selected to use 0.5% 50 μL of DNCB to smear the back of the mice, and 0.5% 10 μL of DNCB to treat the right ear of the mice for maintaining the disease symptoms, and the blank group was smeared with the matrix solution.

[0097] After two weeks from the beginning of modeling, 200 μL of sHSA (30 mg / mL) solution (Example 2), sHSA gel (Example 7) was applied to the back of the mice every two days, and the blank group and the model group were given the same amount of solvent. Until the end of the experiment on the 28th day. The swelling degree of the right ear of the mice was measured on the 14th, 21st and 28th day, and the data was recorded, and at the same time, the degree of skin damage of the mice was scored, and the degree of skin damage of each mouse was evaluated from four symptoms of dryness / desquamation, red rash / bleeding, ulceration, and edema. Each symptom was scored from four levels of no (0 points), mild (1 point, the symptom needs to be identified), moderate (2 points, the symptom is easy to confirm) and severe (3 points, the symptom is obvious), and the scores of the four symptoms were added together, and the total score ranged from 0 to 12 points.

[0098] The following Tables 2 and 3 show the treatment effect of sHSA and sHSA gel on atopic dermatitis mice.

[0099] Table 2 Changes of ear swelling in atopic dermatitis mice (Mean ± SD, n = 6)

[0100]

[0101]

[0102] Table 3 Changes of back skin lesion scores in atopic dermatitis mice (Mean ± SD, n = 6)

[0103]

[0104] From the above Tables 2 and 3, it can be seen that sHSA and its poloxamer gel also have obvious anti-inflammatory activity when used for topical treatment of atopic dermatitis in mice. The symptoms of dryness, red rash, ulceration and even edema on the back of the mice were significantly alleviated. Simple sHSA also has a significant anti-inflammatory effect. The gel may have better anti-inflammatory effect than the solution in the early stage of treatment due to its adhesion. Both the sHSA solution and the gel can alleviate the symptoms of atopic dermatitis to a comparable degree at the end of treatment, further demonstrating the anti-inflammatory activity and clinical application value of sHSA.

[0105] The SD rats were subcutaneously injected twice with a 1:1 (v / v) emulsion of bovine type II collagen (2 mg / mL) and Freund's complete adjuvant at 0.2 mL for the first injection and 0.1 mL for the booster injection one week later, thereby inducing a rat rheumatoid arthritis model. After 14 days of modeling, the rats showed obvious foot swelling, increased ankle and knee joint diameters, and increased clinical scores, indicating successful modeling.

[0106] The sBSA and sBSA / HA lyophilized powder prepared in Example 4 and Example 9 were respectively reconstituted with appropriate amount of PBS (pH 7.2) to prepare a solution of sBSA at a concentration of 30 mg / mL (3% sBSA) and a gel of sBSA / HA at a concentration of 30 mg / mL (3% sBSA / HA), and a gel of sBSA / HA at a concentration of 15 mg / mL (1.5% sBSA / HA). At the same time, a group of HA matrix and a group of methotrexate (MTX) with the same concentration of HA as in the 3% sBSA / HA gel group were set as controls to explore the therapeutic effect on collagen-induced rheumatoid arthritis rats. Specifically, the rats were randomly divided into 7 groups, 6 rats in each group, namely the disease group, the healthy group, the HA matrix group, the 3% sBSA gel group, the 1.5% sBSA gel group, the 3% sBSA solution group and the MTX group (dose of 1 mg / kg), each group was injected with 0.1 mL of the corresponding preparation into the joint cavity every two weeks, and the disease group and the healthy group were injected with phosphate buffer, a total of 2 times of administration. The clinical scores of the rats were recorded every 3 days from the first day of modeling (see Table 4 for scoring rules), and the foot thickness, ankle diameter and knee diameter of the two hind limbs of the rats were recorded and the average values were recorded.

[0107] Table 4 Clinical scores of collagen-induced rheumatoid arthritis rats

[0108]

[0109] From modeling to the end of administration, a total of 6 weeks, the clinical scores, foot thickness, ankle diameter and knee diameter were recorded as shown in Table 5, and the H&E sections of the femur tissues of the rats in each group and the results of immunohistochemical sections of type II collagen (Col II) are shown in Figure 5 . Figure 6

[0110] Table 5 Clinical scores, foot thickness, ankle diameter and knee diameter at the end of treatment in each group (Mean ± SD, n = 6)

[0111]

[0112] ​The results showed that the 3% sBSA / HA gel could significantly improve the disease state of rats, and the clinical score decreased significantly after the first intra-articular injection. The swelling of the foot and the increase of the ankle and knee joint were also significantly reversed. At the end of treatment, there was no significant difference between the healthy rats. The therapeutic effect of sBSA / HA gel showed a dose-dependent manner. The 1.5% sBSA / HA gel group also had a relatively obvious therapeutic effect on arthritis, but there was still mild arthritis at the end of treatment. The anti-inflammatory effect of 3% sBSA solution was lower than that of the gel group, indicating that the viscoelastic properties of the hyaluronic acid hydrogel system provide lubrication and buffering, which is very important in the intra-articular injection treatment of rheumatoid arthritis. Although the HA matrix can alleviate the disease symptoms to some extent, it has no obvious therapeutic effect. MTX is a first-line drug in the clinical treatment guidelines, an anti-rheumatic drug (DMARDs) that needs to be administered continuously for 6 weeks to 6 months to see obvious disease relief [3,4] . In the experiment, MTX was administered in the form of a solution, and the administration interval was long. Due to its short half-life in the body and the need for long-term continuous administration, the therapeutic effect in the experiment was poor. In contrast, the 3% sBSA / HA gel showed a more effective treatment, with a longer anti-inflammatory effect, further directly reflecting the strong anti-inflammatory advantage of sBSA and the treatment prospects of the hydrogel system.

[0113] In addition, H&E staining was performed on the knee joints of each treatment group ( Figure 6 ), which can preliminarily observe the damage degree and osteoclast secretion state. It is obvious that the femoral head tissue of rats in the disease group and the HA matrix group has obvious lesions, and the number of osteoclasts increases ( Figure 6 black arrow). In the 3% sBSA / HA group, the 1.5% sBSA / HA group and the 3% sBSA group, the bone morphology is relatively complete, and there is no obvious up-regulation of osteoclast secretion. The expression amount of Col II in the knee joint bone tissue is positively correlated with the amount of chondrocytes, and the content can reflect the degree of cartilage destruction. The results show that the expression amount of Col II in the disease group and the HA matrix group is significantly lower than that in the healthy group, and the cartilage surface is rough, indicating that the joint cartilage of rats in these two groups has significant lesions. The cartilage surface of the MTX group and the 3% sBSA solution group is rough and uneven, but the cartilage damage degree is slightly lower than that of the disease group and the HA group. Compared with the healthy group, the cartilage of the 3% sBSA / HA group only has mild damage, followed by the 1.5% sBSA / HA group. The degree of cartilage damage in the 3% sBSA solution treatment group further illustrates the strong anti-inflammatory activity of sBSA, which needs to be realized with the aid of the hydrogel system, by relieving the inflammatory state of the joint synovium, preventing the development of the disease, and delaying the degree of cartilage and bone damage.

[0114] It is proved by experiments that the sulfonated albumin derivative and the composition prepared by the application can be widely used for treating and / or preventing macrophage M1 polarization related diseases, preferably treating inflammatory diseases, including but not limited to atopic dermatitis and rheumatoid arthritis, preferably rheumatoid arthritis.

[0115] The above only describes the preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0116] References

[0117] [1]LeVine SM.Albumin and multiple sclerosis.BMC Neurol.2016;16:47.doi:10.1186 / s12883-016-0564-9.

[0118] [2]da Silva NIO,Salvador EA,Rodrigues Franco I,et al.Bovine serumalbumin nanoparticles induce histopathological changes and inflammatory cellrecruitment in the skin of treated mice.Biomed Pharmacother.2018;107:1311-1317.doi:10.1016 / j.biopha.2018.08.106.

[0119] [3]Smolen JS,Aletaha D,McInnes IB.Rheumatoid arthritis.Lancet.2016;388(10055):2023-2038.doi:10.1016 / S0140-6736(16)30173-8.

[0120] [4]Radu AF,Bungau SG.Management ofRheumatoid Arthritis:AnOverview.Cells.2021;10(11):2857.doi:10.3390 / cells10112857.

Claims

1. The use of a product in the preparation of drugs for treating or preventing macrophage M1 polarization-related diseases, characterized in that, The product is a composition formed of sulfonated albumin derivatives, sulfonated albumin derivatives and pharmaceutically acceptable excipients; The sulfonated albumin derivative has the following structure: Where m = 21 to 48; The macrophage M1 polarization-related diseases are atopic dermatitis and rheumatoid arthritis.

2. The application according to claim 1, characterized in that, The albumin mentioned is human serum albumin, bovine serum albumin, or porcine serum albumin.

3. The application according to claim 1, characterized in that, The sulfonated albumin derivative was prepared by the following steps: Albumin is dispersed in an aqueous phase, and sulfonic acid group donor molecules undergo a substitution reaction with amino groups on the albumin molecular chain, resulting in albumin derivatives with sulfonic acid group structures.

4. The application according to claim 1, characterized in that, The excipient mentioned is hyaluronic acid.

Images