WO2020220170A1 - Application of niclosamide ethanolamine salt in preparation of medicament for treating systemic lupus erythematosus and complications thereof - Google Patents

Abstract

An application of niclosamide ethanolamine salt in the preparation of a medicament for treating systemic lupus erythematosus and complications thereof. Niclosamide ethanolamine salt (NEN) may reduce serum anti-ds-DNA antibody levels, reduce proteinuria excretion in MRL/lpr mice, lower blood creatinine and blood urea nitrogen levels, improve glomerulosclerosis and tubular interstitial damage, reduce spleen swelling, reduce lymph node hyperplasia and swelling, and reduce skin damage.

Description

Application of niclosamide ethanolamine salt in preparation of medicaments for treating systemic lupus erythematosus and its complications Technical field

This application relates to the technical field of medicines, in particular to the application of niclosamide ethanolamine salt in the preparation of medicines for treating systemic lupus erythematosus and its complications.

Background technique

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with slow onset, insidious occurrence, diverse clinical manifestations and various changes. It is an autoimmune disease involving many systems and organs. And humoral immune dysfunction, it produces a variety of autoantibodies, which can affect the skin, serosal membrane, joints, kidneys and central nervous system, etc., and is characterized by autoimmunity. There are multiple autoantibodies in the patient, which not only affects humoral immunity, but also affects Cellular immunity, complement system has also changed. The pathogenesis is mainly due to the formation of immune complexes, the exact cause of which is unknown, and the condition is alternating between repeated attacks and remission.

Systemic lupus erythematosus can cause skin damage, lupus nephritis, immune system dysfunction, lymphadenopathy, spleen enlargement, blood system damage, serositis, nervous system damage and many other complications; among them, lupus nephritis is secondary Common causes of glomerulonephritis. Lupus nephritis has complex etiologies, difficult treatment, lingering and difficult to heal, proteinuria, progressive increase in blood creatinine, glomerular sclerosis and renal tubular damage as the main early clinical features; currently There is no effective drug to delay the progression of kidney disease in clinical practice.

Niclosamide is a molluscicide and anthelmintic recommended by the WHO. Niclosamide Ethanolamine Salt (NEN) is its derivative, which has better water solubility. Recent studies have shown that this type of drug has obvious The anti-tumor and blood sugar lowering effect.

Summary of the invention

The purpose of this application includes providing an application of niclosamide ethanolamine salt in the preparation of a medicine for treating systemic lupus erythematosus and its complications.

In order to achieve the above objectives, the present application provides an application of niclosamide ethanolamine salt in the preparation of a medicine for treating systemic lupus erythematosus and its complications.

In some embodiments of the application, the complication is immune system dysfunction.

In some embodiments of the application, the complication is lupus nephritis.

In some embodiments of the present application, the complication is lymph node enlargement.

In some embodiments of the application, the complication is splenomegaly.

In some embodiments of the application, the complication is skin damage.

In some embodiments of the application, the medicine further includes pharmaceutical excipients.

In some embodiments of the application, the drug is an oral administration preparation, an injection administration preparation, a mucosal administration preparation or a transdermal administration preparation.

In some embodiments of the present application, the drug is a tablet, capsule, granule, oral liquid, patch or gel.

In some embodiments of the present application, the oral administration preparation is a tablet, capsule, granule or oral liquid; the mucosal administration preparation is a patch or gel; the transdermal administration preparation is Patch or gel.

The beneficial effects of this application:

The study in this application found that: Niclosamide ethanolamine salt (NEN) can reduce serum anti-ds-DNA antibody levels, reduce proteinuria excretion in MRL/lpr mice, reduce blood creatinine and blood urea nitrogen levels, and improve glomerular sclerosis and kidney Tubular interstitial damage, reduce spleen enlargement, reduce lymph node hyperplasia and enlargement, and reduce skin damage. According to the above research results, it can be concluded that niclosamide ethanolamine salt has a significant effect on the treatment of immune system dysfunction caused by systemic lupus erythematosus, lupus nephritis, splenomegaly, lymph node hyperplasia, skin damage and other complications.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present application more clearly, the following will briefly introduce the drawings needed in the embodiments.

Figure 1 shows the effect of NEN on serum anti-ds-DNA antibodies in MRL/lpr lupus mice;

Figure 2 shows the effect of NEN on the 24h urine protein excretion rate of MRL/lpr lupus mice;

Figure 3 shows the effect of NEN on serum creatinine in MRL/lpr lupus mice;

Figure 4 shows the effect of NEN on serum urea nitrogen in MRL/lpr lupus mice;

Figure 5a shows the effect of NEN on glomerulosclerosis in MRL/lpr mice;

Figure 5b is a PAS staining picture of the effect of NEN on glomerulosclerosis in MRL/lpr mice;

Figure 6a shows the effect of NEN on renal tubular interstitial injury in MRL/lpr mice;

Figure 6b is a PAS staining picture of the effect of NEN on renal tubular interstitial damage in MRL/lpr mice;

Figure 7a shows the effect of NEN on the spleen weight of MRL/lpr mice;

Figure 7b shows the effect of NEN on the appearance and size of the spleen of MRL/lpr mice;

Figure 8a shows the effect of NEN on the weight of lymph nodes in MRL/lpr mice;

Figure 8b shows the effect of NEN on the appearance and size of lymph nodes in MRL/lpr mice;

Figure 9a shows the effect of NEN on the skin damage of MRL/lpr mice;

Figure 9b is an appearance photograph of the effect of NEN on the skin damage of MRL/lpr mice.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with specific examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If specific conditions are not specified in the examples, it shall be carried out according to conventional conditions. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

The following studies were carried out in the embodiments of the present invention:

1. The effect of NEN on the serum anti-ds-DNA antibody of MRL/lpr lupus mice was studied;

2. The effect of NEN on the 24h urine protein excretion rate of MRL/lpr lupus mice was studied;

3. The effect of NEN on serum creatinine of MRL/lpr lupus mice was studied;

4. The effect of NEN on serum urea nitrogen in MRL/lpr lupus mice was studied;

5. The effect of NEN on glomerulosclerosis in MRL/lpr mice was studied;

6. The effect of NEN on renal tubular interstitial injury in MRL/lpr mice was studied;

7. The effect of NEN on the spleen size of MRL/lpr mice was studied;

8. The effect of NEN on the size of lymph nodes in MRL/lpr mice was studied;

9. The effect of NEN on the skin damage of MRL/lpr mice was studied.

1. Experimental method

1. Animal model: Female SPF-grade MRL/lpr lupus mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. The animal experiments were carried out in strict accordance with the relevant animal ethics guidelines and regulations, and they were raised in the Animal Center of Peking University Shenzhen Graduate School. The experimental animals were controlled at a constant room temperature of 21～23℃, under the conditions of 12 hours of light and 12 hours of dark cycle, while freely eating and drinking. At 12 weeks of age, mice were randomly divided into model group (i.e. SLE group in the figure) and NEN treatment group (i.e. SLE+NEN group in the figure). Female C57BL/6J mice were used as healthy normal control group (i.e. control Group), the control group and SLE group were fed with regular standard feed, and the SLE+NEN group was fed with medicated feed (NEN/feed: 10g/kg), and the drug was set to 0 week point when the intervention was started, for a total of 8 weeks of feeding. NEN was purchased from Hubei Shengtian Hengchuang Biotechnology Co., Ltd. (China).

2. Use mouse metabolic cages (Tecniplast, Italy) to collect 24h urine of mice in each group at 0 and 8 weeks of medication, and use Bradford protein assay kit (Bio-Rad, USA) to detect small groups. Quantification of total protein in mouse 24h urine.

3. Tissue preparation: After 8 weeks of NEN drug intervention, the skin damage of each group of mice was scored semi-quantitatively. The mice of each group were sacrificed and samples were taken. Whole blood and serum samples were collected. The kidneys were taken, photographed and weighed. A certain amount of kidney tissue was cut from the longitudinal section and fixed with 10% formalin, followed by dehydration and paraffin embedding. The remaining kidney tissue was immediately frozen in liquid nitrogen and stored at -80°C for subsequent experimental studies. The spleen was peeled off, photographed and weighed. The main subcutaneous lymph nodes (5-8) were stripped, photographed and weighed, and the average lymph node weight of each mouse was calculated. Serum creatinine and urea nitrogen were measured using an automatic biochemical analyzer (Roche, Switzerland). The ELISA method was used to determine the serum anti-ds-DNA antibody content.

4. Evaluation method of glomerular sclerosis index: paraffin sections of kidney tissue were stained with PAS, and 20-30 glomeruli in the renal cortex area were randomly photographed on each section under a 40× objective lens. Glomerular sclerosis was mainly manifested as mesangial matrix expansion. , Capillary occlusion, hyaline degeneration and balloon adhesion.

If the glomerulus is basically normal (or the lesion is less than 5%), then the score is 0, if the lesion accumulates 5 to 25% of the glomerulus, then 1 point, 25 to 50%, 2 points, and 50 to 75%, 3 points. 75-100% is 4 points, and then calculate the average value of each slice.

5. Evaluation method of renal tubulointerstitial injury index: paraffin sections of renal tissue were stained with PAS. Each section was randomly photographed in 10-20 renal tubule areas in the renal cortex under a 20× objective lens. Tubular interstitial injury mainly manifested as atrophy, Expansion and infiltration of interstitial inflammatory cells.

If the renal tubules are basically normal (or the disease is less than 5%), 0 points are accumulated, if the lesions accumulate 5-25% of the renal tubules, 1 point, 25-50%, 2 points, 50-100%, 3 points, and then Calculate the average of each slice.

6. Skin damage assessment method

Observe and score the skin damage on the back of the neck of each group of mice. The scoring standard of the skin damage index on the back of the neck is:

No skin damage: 0;

A small amount of hair loss, redness of the skin, and a little scattered skin damage: 1;

Small patches of skin redness, crusting, and hair loss: 2;

Extensive skin ulcers, hair loss: 3.

7. Statistical analysis

The measurement data is expressed by the mean ± standard deviation. One-way analysis of variance was used for the comparison between multiple groups of samples, the statistical differences between the two groups of samples were analyzed by independent sample t test, and the statistical analysis was processed by SPSS 22.0 statistical software. When P<0.05, the difference is considered to be statistically significant.

2. Experimental results

1. The effect of NEN on serum anti-ds-DNA antibodies in MRL/lpr lupus mice

Figure 1 shows the effect of NEN on serum anti-ds-DNA antibodies in MRL/lpr lupus mice (n=6 per group). Compared with the control group, ^* P<0.05; compared with the SLE group, ^# P<0.05.

As shown in Figure 1, at 8 weeks, compared with the control group, the serum anti-ds-DNA antibody of the SLE group was significantly increased. After 8 weeks of NEN treatment, compared with the SLE group, the SLE+NEN group was significantly decreased.

The results in Figure 1 show that niclosamide ethanolamine salt (NEN) can reduce serum anti-ds-DNA antibody levels, thereby improving immune system dysfunction caused by systemic lupus erythematosus.

2. The effect of NEN on the 24h urine protein excretion rate of MRL/lpr lupus mice

Figure 2 shows the effect of NEN on the 24h urine protein excretion rate of MRL/lpr lupus mice (n=6 per group). Compared with the control group, ^* P<0.05; compared with the SLE group, ^# P<0.05.

As shown in Figure 2, at the beginning of treatment (that is, at week 0), compared with the control group, the 24h urine protein excretion rate of the SLE group was significantly increased, and there was no significant difference between the SLE+NEN group and the SLE group. At the 8th week, the 24-h urine proteinuria excretion rate of the SLE group was significantly increased, and was still significantly higher than that of the control group. After 8 weeks of NEN treatment, the urine protein of the SLE+NEN group was significantly lower than that of the SLE group.

3. The effect of NEN on serum creatinine in MRL/lpr lupus mice

Figure 3 shows the effect of NEN on serum creatinine in MRL/lpr lupus mice (n=6 per group). Compared with the control group, ^* P<0.05; compared with the SLE group, ^# P<0.05.

As shown in Figure 3, at 8 weeks, compared with the control group, the serum creatinine level of the SLE group was significantly increased. After 8 weeks of NEN treatment, compared with the SLE group, the SLE+NEN group decreased significantly.

4. The effect of NEN on serum urea nitrogen in MRL/lpr lupus mice

Figure 4 shows the effect of NEN on serum urea nitrogen in MRL/lpr lupus mice (n=6 per group). Compared with the control group, ^* P<0.05; compared with the SLE group, ^# P<0.05.

As shown in Figure 4, at 8 weeks, compared with the control group, the serum urea nitrogen level in the SLE group was significantly increased. After 8 weeks of NEN treatment, compared with the SLE group, the SLE+NEN group decreased significantly.

5. The effect of NEN on glomerulosclerosis in MRL/lpr mice

Figure 5a shows the effect of NEN on glomerulosclerosis in MRL/lpr mice (n=6 per group). Compared with control, ^* P<0.05; compared with SLE group, ^# P<0.05.

As shown in Figure 5a, at 8 weeks, compared with the control group, the glomerular sclerosis index of mice in the SLE group was significantly increased, and compared with the SLE group, the glomerular sclerosis index of the SLE+NEN group was significantly decreased.

Figure 5b is a PAS staining picture of the effect of NEN on glomerulosclerosis in MRL/lpr mice (the length of the ruler in Figure 5b is 20μm). As shown in Figure 5b, at 8 weeks, compared with the control group, the SLE group has a smaller kidney The glomerular sclerosis was significantly reduced after NEN treatment (SLE+NEN group).

6. The effect of NEN on renal tubular interstitial injury in MRL/lpr mice

Figure 6a shows the effect of NEN on renal tubular interstitial damage in MRL/lpr mice (n=6 per group). Compared with control, ^* P<0.05; compared with SLE group, ^# P<0.05.

As shown in Figure 6a, at 8 weeks, compared with the control group, the renal tubular interstitial injury index of mice in the SLE group increased significantly, and compared with the SLE group, the renal tubular interstitial injury index of the SLE+NEN group was significantly reduced.

Figure 6b is a PAS staining picture of the effect of NEN on renal tubular interstitial damage in MRL/lpr mice (the ruler length in Figure 6b is 50μm). As shown in Figure 6b, at 8 weeks, compared with the control group, the SLE group has significant The renal tubular interstitial injury was significantly improved after NEN treatment (SLE+NEN group).

The results shown in Figure 2 to Figure 6b above show that Niclosamide ethanolamine salt (NEN) can reduce proteinuria excretion in MRL/lpr mice, lower blood creatinine and blood urea nitrogen levels, and improve glomerular sclerosis and tubular interstitial damage. According to the above research results, it can be concluded that niclosamide ethanolamine salt (NEN) can improve systemic lupus erythematosus nephritis, reduce urine protein excretion rate, improve glomerular sclerosis and tubular interstitial damage, and delay renal failure.

7. The effect of NEN on the spleen size of MRL/lpr mice

Figures 7a and 7b are the results of the effect of NEN on the weight and appearance of the spleen of MRL/lpr mice (n=6 per group). Compared with control, ^* P<0.05; compared with SLE group, ^# P<0.05.

As shown in Figure 7a and Figure 7b, at 8 weeks, compared with the control group, the weight and appearance of the spleen of the SLE group increased significantly. Compared with the SLE group, the weight and appearance of the spleen of the SLE+NEN group were both Obviously smaller.

Figures 7a and 7b show that niclosamide ethanolamine salt (NEN) can improve the spleen enlargement caused by systemic lupus erythematosus.

8. The effect of NEN on the size of lymph nodes in MRL/lpr mice

Figures 8a and 8b are the results of the effect of NEN on the weight and appearance of lymph nodes in MRL/lpr mice (n=6 per group). Compared with the SLE group, ^# P<0.05.

As shown in Figure 8a and Figure 8b, at 8 weeks, compared with the SLE group, the weight and appearance of the lymph nodes in the SLE+NEN group were significantly smaller.

The results of Figure 8a and Figure 8b show that niclosamide ethanolamine salt (NEN) can improve lymph node enlargement caused by systemic lupus erythematosus.

9. The effect of NEN on the skin damage of MRL/lpr lupus mice

Figure 9a shows the effect of NEN on the skin damage of MRL/lpr lupus mice (n=6 per group). Compared with the SLE group, ^# P<0.05. As shown in Figure 9a, at the 8th week, compared with the SLE group, the degree of skin damage in the SLE+NEN group was significantly reduced.

Figure 9b is an appearance photograph of the effect of NEN on the skin damage of MRL/lpr mice. As shown in Figure 9b, at 8 weeks, there was severe skin damage in the SLE group, and the skin damage was significantly improved after NEN treatment (SLE+NEN group) .

Figures 9a and 9b show that niclosamide ethanolamine salt (NEN) can improve skin damage caused by systemic lupus erythematosus.

The above experimental results show that niclosamide ethanolamine salt (NEN) can reduce serum anti-ds-DNA antibody levels, reduce proteinuria excretion in MRL/lpr mice, reduce blood creatinine and blood urea nitrogen levels, and improve glomerular sclerosis and kidney Tubular interstitial damage, reduce spleen enlargement, reduce lymph node hyperplasia and enlargement, and reduce skin damage. According to the above research results, it can be concluded that niclosamide ethanolamine salt has a significant effect on the treatment of immune system dysfunction caused by systemic lupus erythematosus, lupus nephritis, splenomegaly, lymph node hyperplasia, skin damage and other complications.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: It is still possible to modify the technical solutions described in the foregoing embodiments, or equivalently replace some or all of the technical features; these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention range.

In addition, those skilled in the art can understand that although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments means that they fall within the scope of the present invention. And form different embodiments. For example, in the above claims, any one of the claimed embodiments can be used in any combination. The information disclosed in the background section is only intended to deepen the understanding of the overall background of the present invention, and should not be regarded as an acknowledgement or any form of suggestion that the information constitutes the prior art known to those skilled in the art.

Claims (10)

Application of niclosamide ethanolamine salt in the preparation of medicines for treating systemic lupus erythematosus and its complications. The use according to claim 1, wherein the complication is immune system dysfunction. The use according to claim 1, wherein the complication is lupus nephritis. The application according to claim 1, wherein the complication is lymph node enlargement. The application according to claim 1, wherein the complication is splenomegaly. The application according to claim 1, wherein the complication is skin damage. The application according to claim 1, wherein the medicine further comprises pharmaceutical excipients. The application according to claim 1, wherein the medicine is an oral administration preparation, an injection administration preparation, a mucosal administration preparation or a transdermal administration preparation. The application according to claim 1, wherein the medicine is a tablet, capsule, granule, oral liquid, patch or gel. The application according to claim 8, wherein the oral administration preparation is a tablet, capsule, granule or oral liquid; the mucosal administration preparation is a patch or gel; the transdermal preparation The administration preparation is a patch or a gel.

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