CN111032030B

CN111032030B - Application of niclosamide ethanolamine salt in preparing medicine for treating systemic lupus erythematosus and complications thereof

Info

Publication number: CN111032030B
Application number: CN201980000560.8A
Authority: CN
Inventors: 韩鹏勋; 孙惠力; 邵牧民; 余学问; 翁文慈
Original assignee: Shenzhen Traditional Chinese Medicine Hospital
Current assignee: Shenzhen Traditional Chinese Medicine Hospital
Priority date: 2019-04-28
Filing date: 2019-04-28
Publication date: 2023-02-17
Anticipated expiration: 2039-04-28
Also published as: WO2020220170A1; CN111032030A

Abstract

Application of niclosamide ethanolamine salt in preparing medicine for treating systemic lupus erythematosus and its complication is disclosed. Niclosamide Ethanolamine (NEN) can reduce the level of serum anti-ds-DNA antibody, reduce the proteinuria excretion of MRL/lpr mice, reduce the levels of blood creatinine and blood urea nitrogen, improve glomerulosclerosis and tubulointerstitial injury, reduce splenomegaly, reduce lymphadenectasis swelling and reduce skin damage.

Description

Application of niclosamide ethanolamine salt in preparation of medicines for treating systemic lupus erythematosus and complications thereof

Technical Field

The application relates to the technical field of medicines, in particular to application of niclosamide ethanolamine salt in preparing medicines for treating systemic lupus erythematosus and complications thereof.

Background

Systemic Lupus Erythematosus (SLE) is an autoimmune disease, the onset of disease is slow, the occurance of insidious, the clinical manifestations are various, change many, it is an autoimmune disease related to many systems and viscera, because of the cellular and humoral immune dysfunction, produce many autoantibodies, can involve skin, serosa, joint, kidney and central nervous system, and regard autoimmune as the characteristic, there are many autoantibodies in the patient, not only influence humoral immunity, also influence cellular immunity, complement system also changes. The pathogenesis is mainly due to immune complex formation, the exact cause is unknown, and the disease condition is in an alternate process of repeated attack and remission.

Systemic lupus erythematosus can cause skin damage, lupus nephritis, immune system dysfunction, lymphadenectasis, splenomegaly, blood system damage, serositis, nervous system damage and other complications; wherein, lupus nephritis is a common cause of secondary glomerulonephritis, the cause of lupus nephritis is complex, the treatment difficulty is large, the disease condition is lingering and difficult to cure, proteinuria, progressive increase of blood creatinine, glomerulosclerosis and renal tubular injury are the main clinical characteristics of the early stage of lupus nephritis; at present, no effective medicine for delaying the progression of kidney diseases exists clinically.

Niclosamide is a molluscicide and an anthelmintic recommended by WHO, niclosamide Ethanolamine Salt (NEN) is a derivative of Niclosamide Ethanolamine Salt, has better water solubility, and researches in recent years show that the medicine has obvious antitumor and blood sugar reducing effects.

Disclosure of Invention

The application aims to provide application of niclosamide ethanolamine salt in preparing medicines for treating systemic lupus erythematosus and complications thereof.

In order to realize the purpose, the application provides the application of niclosamide ethanolamine salt in preparing the medicines for treating systemic lupus erythematosus and complications thereof.

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

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

In some embodiments of the present application, the complication is lymphadenectasis.

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

In some embodiments of the present application, the complication is a skin lesion.

In some embodiments of the present application, the medicament further comprises a pharmaceutical excipient.

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

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

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

The beneficial effect of this application:

the research of the application finds that: niclosamide Ethanolamine (NEN) can reduce the level of serum anti-ds-DNA antibody, reduce the proteinuria excretion of MRL/lpr mice, reduce the levels of blood creatinine and blood urea nitrogen, improve glomerulosclerosis and tubulointerstitial injury, reduce splenomegaly, reduce lymphadenectasis swelling and reduce skin damage. From the above findings it can be concluded that: the niclosamide ethanolamine salt has obvious curative effect on immune system dysfunction, lupus nephritis, splenomegaly, lymph node hyperplasia, skin damage and other complications caused by systemic lupus erythematosus.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below.

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

FIG. 2 shows the effect of NEN on 24h urinary protein excretion rate in MRL/lpr lupus mice;

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

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

FIG. 5a is a graph showing the effect of NEN on glomerulosclerosis in MRL/lpr mice;

FIG. 5b is a photograph of PAS staining of the effect of NEN on MRL/lpr mouse glomerulosclerosis;

FIG. 6a is a graph showing the effect of NEN on tubulointerstitial injury in MRL/lpr mice;

FIG. 6b is a photograph of PAS staining of the effect of NEN on the tubulointerstitial injury in MRL/lpr mice;

FIG. 7a is the results of the effect of NEN on spleen weight in MRL/lpr mice;

FIG. 7b is a graph showing the effect of NEN on the apparent size of the spleen in MRL/lpr mice;

FIG. 8a is the results of the NEN effect on lymph node weight in MRL/lpr mice;

FIG. 8b is the results of the effect of NEN on the apparent size of lymph nodes in MRL/lpr mice;

FIG. 9a is a graph showing the effect of NEN on skin lesions in MRL/lpr mice;

FIG. 9b is a photograph of the appearance of the effect of NEN on skin damage in MRL/lpr mice.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples were carried out under the conventional conditions, unless otherwise specified. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

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

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

2. the influence of NEN on 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 influence of NEN on the serum urea nitrogen of MRL/lpr lupus mice is researched;

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

6. the effect of NEN on tubulointerstitial injury in MRL/lpr mice was studied;

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

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

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

1. Experimental methods

1. Animal model: female SPF-grade MRL/lpr lupus mice were purchased from Shanghai Style laboratory animals, limited liability company, and the animal experiments were strictly performed according to ethical-related guidelines and regulations of animals, and were bred in the animal center of graduate institute in Shenzhen, beijing university. The experimental animals were controlled at constant room temperature 21-23 deg.C under 12-hour light and 12-hour dark cycles with free access to food and water. Mice were randomly divided into a model group (SLE group in the figure) and a NEN treatment group (SLE + NEN group in the figure) at 12 weeks of age, and female C57BL/6J mice were used as healthy normal control groups (control group in the figure), in which the control group and SLE group were fed with normal standard diet, the SLE + NEN group was fed with medicated diet (NEN/diet: 10 g/kg), and drug intervention was started at 0 week point for 8 weeks. NEN was purchased from hebei shengtian biotechnology limited (china).

2. 24h urine was retained from each group of mice at the 0-week and 8-week point of administration using mouse metabolism cages (Techniplast, italy), and total protein quantification was performed for 24h urine from each group of mice using a Bradford protein assay kit (Bio-Rad, USA).

3. Preparing an organization: semi-quantitative scoring is carried out on skin injury of each group of mice after 8 weeks of NEN drug intervention, each group of mice is killed and materials are taken, whole blood and serum samples are reserved, kidneys are picked up, the mice are photographed and weighed, a certain amount of kidney tissues are cut along a longitudinal section and fixed by 10% formalin, then dehydrated paraffin embedding is carried out, and the rest kidney tissues are immediately frozen in liquid nitrogen and stored at-80 ℃ for subsequent experimental study. The spleen was peeled, photographed and weighed. Subcutaneous major lymph nodes (5-8) were dissected, photographed and weighed, and the average lymph node weight per mouse was calculated. Serum creatinine and urea nitrogen were measured using a fully automated biochemical analyzer (Roche, switzerland). Serum anti-ds-DNA antibody content was determined using ELISA method.

4. Glomerular sclerosis index evaluation method: PAS staining is carried out on the paraffin sections of the kidney tissues, 20-30 glomeruli in the kidney cortex area are randomly shot on each section under a 40 Xobjective, and glomerular sclerosis is mainly manifested by mesangial stromal expansion, capillary vessel occlusion, transparent degeneration and sacculus adhesion.

If the glomeruli are basically normal (or the lesion is less than 5%), 0 is integrated, if the lesion accumulates in 5 to 25% of the glomeruli, 1 is integrated, 25 to 50% is integrated into 2, 50 to 75% is integrated into 3, and 75 to 100% is integrated into 4, and then the average value of each section is calculated.

5. Tubulointerstitial injury index evaluation method: PAS staining is carried out on the paraffin sections of the kidney tissues, 10-20 tubular areas of the cortex of the kidney are randomly photographed on each section under a 20 x objective, and the tubular interstitial injuries are mainly characterized by atrophy, expansion and interstitial inflammatory cell infiltration.

The mean value for each section was calculated by scoring 0 if the tubules were substantially normal (or lesions < 5%), scoring 1 if lesions accumulated in 5-25% of the area of the tubules, scoring 2 from 25-50%, and scoring 3 from 50-100%.

6. Skin lesion assessment method

And (3) observing the skin injury conditions of the neck and the back of each group of mice, and scoring, wherein the scoring standard of the skin injury index of the neck and the back is as follows:

no skin damage: 0;

a small amount of hair is lost, the skin becomes red, and a little is scattered on the skin breakage: 1;

flaking skin redness, scabbing, hair loss: 2;

extensive skin ulceration, hair loss: 3.

7. statistical analysis

The metrology data are expressed as mean ± standard deviation. Comparisons between sets of samples were performed using one-way anova, statistical differences between the two sets of samples were analyzed using independent sample t-test, and statistical analysis was performed using SPSS 22.0 statistical software. Differences were considered statistically significant when P < 0.05.

2. Results of the experiment

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

FIG. 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; in comparison to the SLE group, ^# P<0.05。

as shown in fig. 1, at the 8-week point, serum anti-ds-DNA antibodies were significantly elevated in the SLE group compared to the control group, and significantly decreased in the SLE + NEN group compared to the SLE group after 8 weeks of NEN treatment.

The results in FIG. 1 show that niclosamide ethanolamine salt (NEN) can reduce the level of serum anti-ds-DNA antibodies, thereby ameliorating immune system dysfunction caused by systemic lupus erythematosus.

2. Effect of NEN on 24h urinary protein excretion Rate in MRL/lpr Lupus mice

Figure 2 is the effect of NEN on 24h urinary protein excretion rate in MRL/lpr lupus mice (n =6 groups). Compared with the control group, ^* P<0.05; in comparison to the set of SLEs, ^# P<0.05。

as shown in FIG. 2, at the initiation of treatment (i.e., at the 0-week point), the SLE group had a significantly increased urinary protein excretion rate at 24h as compared to the control group, and the SLE + NEN group had no significant difference from the SLE group. At 8 weeks, the 24h urinary proteinuria excretion rate of the SLE group is obviously increased and still obviously higher than that of the control group, and after 8 weeks of treatment by NEN, SLE + NEN urinary protein is obviously reduced compared with that of the SLE group.

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

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

as shown in fig. 3, serum creatinine levels were significantly increased in the SLE group compared to the control group at the 8-week point, and significantly decreased in the SLE + NEN group compared to the SLE group 8 weeks after NEN treatment.

4. 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; in comparison to the SLE group, ^# P<0.05。

as shown in fig. 4, at the 8-week point, serum urea nitrogen levels were significantly elevated in the SLE group compared to the control group, and significantly decreased in the SLE + NEN group compared to the SLE group 8 weeks after NEN treatment.

5. Effect of NEN on glomerulosclerosis in MRL/lpr mice

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

as shown in fig. 5a, at 8 week point, the glomerular sclerosis index of SLE group mice was significantly increased compared to control group, and significantly decreased compared to SLE group SLE + NEN group.

FIG. 5b is a photograph of PAS staining of the effect of NEN on the glomerular sclerosis in MRL/lpr mice (20 μm ruler length in FIG. 5 b), as shown in FIG. 5b, at 8 weeks point, there was significant sclerosis in SLE group glomeruli and significantly reduced glomerular sclerosis after NEN treatment (SLE + NEN group) compared to control group.

6. Effect of NEN on tubulointerstitial injury in MRL/lpr mice

Figure 6a is the effect of NEN on renal tubulointerstitial injury in MRL/lpr mice (n =6 groups). Compared with the control, the control can be compared with the control, ^* P<0.05; in comparison to the SLE group, ^# P<0.05。

as shown in fig. 6a, at 8 weeks, the indices of tubulointerstitial injury in SLE group mice were significantly increased compared to control group, and the indices of SLE + NEN group tubulointerstitial injury were significantly decreased compared to SLE group.

Fig. 6b is a PAS staining image of the effect of NEN on the tubulointerstitial injury in MRL/lpr mice (ruler length 50 μm in fig. 6 b), as shown in fig. 6b, significant tubulointerstitial injury was present in SLE group and significantly improved in the tubulointerstitial injury after NEN treatment (SLE + NEN group) compared to control group at 8 week point.

The results in fig. 2 to 6b above show that niclosamide ethanolamine salt (NEN) can reduce proteinuria excretion, decrease blood creatinine and blood urea nitrogen levels, and improve glomerulosclerosis and tubulointerstitial injury in MRL/lpr mice, and from the results of the above studies, it can be concluded that: niclosamide Ethanolamine (NEN) can improve systemic lupus erythematosus nephritis, reduce urinary protein excretion rate, improve glomerulosclerosis and tubulointerstitial injury, and delay renal failure.

7. Effect of NEN on spleen size in MRL/lpr mice

Fig. 7a and 7b are the results of the effect of NEN on spleen weight and apparent size, respectively, in MRL/lpr mice (n =6 per group). Compared with the control, the control can be compared with the control, ^* P<0.05; in comparison to the SLE group, ^# P<0.05。

as shown in FIGS. 7a and 7b, at 8 weeks, both spleen weight and apparent size were significantly increased in SLE group mice compared to control group and significantly decreased in SLE + NEN group compared to SLE group.

The results in FIGS. 7a and 7b show that niclosamide ethanolamine salt (NEN) can improve splenomegaly caused by systemic lupus erythematosus.

8. Effect of NEN on lymph node size in MRL/lpr mice

Fig. 8a and 8b show the effect of NEN on lymph node weight and apparent size, respectively, in MRL/lpr mice (n =6 per group). In comparison to the SLE group, ^# P<0.05。

as shown in fig. 8a and 8b, at the 8 week point, both the weight and apparent size of the lymph nodes in the SLE + NEN group were significantly smaller compared to the SLE group.

The results in FIGS. 8a and 8b show that niclosamide ethanolamine salt (NEN) can improve lymphadenectasis caused by systemic lupus erythematosus.

9. Effect of NEN on skin lesions in MRL/lpr lupus mice

FIG. 9a is the effect of NEN on skin lesions in MRL/lpr lupus mice (n =6 per group)). In comparison to the SLE group, ^# P<0.05. as shown in figure 9a, the SLE + NEN group had significantly reduced skin damage at the 8 week point compared to the SLE group.

FIG. 9b is a photograph showing the appearance of the effect of NEN on skin lesions in MRL/lpr mice, as shown in FIG. 9b, at the 8 week point, severe skin lesions were present in the SLE group and the skin lesions were significantly improved after NEN treatment (SLE + NEN group).

The results in fig. 9a and 9b show that niclosamide ethanolamine salt (NEN) can improve the skin damage caused by systemic lupus erythematosus.

The experimental results show that niclosamide ethanolamine salt (NEN) can reduce the level of serum anti-ds-DNA antibodies, reduce the proteinuria excretion of MRL/lpr mice, reduce the levels of blood creatinine and blood urea nitrogen, improve glomerulosclerosis and tubulointerstitial injury, reduce splenomegaly, reduce lymphadenectasis swelling and reduce skin damage. From the above findings it can be concluded that: the niclosamide ethanolamine salt has obvious curative effect on immune system dysfunction, lupus nephritis, splenomegaly, lymph node hyperplasia, skin damage and other complications caused by systemic lupus erythematosus.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Claims

1. The application of niclosamide ethanolamine salt as the only active component in preparing medicine for treating systemic lupus erythematosus.

2. The application of niclosamide ethanolamine salt as the only active ingredient in preparing the medicine for treating the systemic lupus erythematosus complication is lymphadenectasis or splenomegaly.

3. The use of claim 1 or 2, wherein the medicament further comprises a pharmaceutical excipient.

4. The use according to claim 1 or 2, wherein the medicament is an oral, injectable, mucosal or transdermal formulation.

5. The use of claim 4, wherein the medicament is a tablet, capsule, granule, oral liquid, patch or gel.