WO2021142815A1 - Berberine and coptisine, or active metabolites thereof, and application of salts thereof in medication for preventing and/or treating uric acid nephropathy - Google Patents

Abstract

Disclosed are berberine represented by structural formula (I), coptisine represented by structural formula (II), berberine active metabolites represented by structural formulas (III), (IV), (V), (VI), (VII), (VIII), and (Ⅸ), including jatrorrhizine, dihydroberberine, thalifendine, berberrubine, demethyleneberberine, palmatine, and columbamine, and application of pharmaceutically acceptable salts thereof in preparation of a medication for preventing and/or treating uric acid nephropathy (gouty nephropathy).

Description

Application of berberine, berberine or their active metabolites, and their salts in the prevention and/or treatment of uric acid nephropathy drugs Technical field

The invention relates to the application of berberine, berberine or their active metabolites, and pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention and/or treatment of uric acid nephropathy; it belongs to the technical field of medicine.

Background technique

Uric acid nephropathy is kidney damage caused by hyperuricemia due to excessive blood uric acid production or decreased excretion. It is also called gout nephropathy, which may have elevated uric acid, uric acid stones, proteinuria, edema, nocturia, and high blood pressure. , Hematuria and renal tubular function damage. The disease has no obvious seasonality. The incidence of obesity, carnivorous and alcoholics is high. The incidence of men is higher than that of women, and 85% of the affected population are middle-aged and elderly people. In patients with uric acid nephropathy, the concentration of urate in the blood increases to a supersaturated state, and urate crystals deposit in the kidneys and cause lesions. The histological features are the appearance of urate crystals in the renal interstitium and renal tubules, resulting in damage to the nephron , And easily lead to secondary bacterial infections. If the disease can be diagnosed early and given appropriate treatment (control hyperuricemia and protect renal function), the development of kidney disease can be reduced or stopped. If treatment is delayed or improperly treated, the condition can worsen and develop into end-stage renal failure. Need dialysis treatment. The current treatment methods are mainly low-purine diet, drinking more water, maintaining urine output, alkalizing urine, and applying drugs that inhibit uric acid production and promote uric acid excretion. However, clinical medications often have significant adverse reactions and deserve our in-depth study.

Berberine (BBR) is a natural compound isolated from many different plants, such as Coptis, Eryngium, Ranunculus, Phellodendron, etc. For decades, berberine has been used as an over-the-counter drug to treat diarrhea without obvious adverse reactions. Since 2004, we have discovered that berberine can be used as a new drug to treat hyperlipidemia and type I diabetes. Its clinical efficacy has been confirmed by many domestic and foreign research groups. At the same time, in the experiments of rodent models, we proved that berberine has a good effect on reducing blood uric acid content and treating uric acid nephropathy. This patent describes that oral and intraperitoneal injection of berberine can reduce the blood uric acid content of SD rats with uric acid nephropathy induced by yeast powder and adenine and significantly improve the renal function of SD rats. The berberine analogues jatrorrhizine, palmatine and berberine and the mixture of berberine total alkalis (the extract of berberine medicinal materials, mainly containing berberine, berberine, jatrorrhizine, etc.) can also reduce hyperuricemia after oral administration The content of uric acid in the blood of ICR mice.

Oral or intraperitoneal injection of berberine reduced the blood uric acid content of SD rats with uric acid nephropathy and significantly improved renal function. However, most clinical medications for uric acid nephropathy have different degrees of side effects. Berberine reduces the deposition of uric acid crystals in the kidney by reducing the content of uric acid, and significantly improves renal function. Therefore, it is described in this patent that oral or intraperitoneal injection of berberine, berberine or its active metabolites, and pharmaceutically acceptable salts thereof can reduce the content of uric acid in the blood of SD rats or ICR mice, thereby improving the kidneys. The function suggests that berberine, berberine or its active metabolites, and pharmaceutically acceptable salts thereof have applications in the prevention and/or treatment of uric acid nephropathy.

Summary of the invention

The technical problem to be solved by the present invention is to provide a new class of drugs for preventing and/or treating uric acid nephropathy.

In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

The present invention provides berberine represented by structural formula (I), berberine represented by structural formula (II) or active metabolites of both or both, and pharmaceutically acceptable salts thereof in the preparation of prevention and/or treatment of uric acid The application of drugs for sexual nephropathy (gout nephropathy).

Among them, the application according to claim 1, characterized in that the active metabolism of berberine as shown in the structural formulas (III), (IV), (V), (VI), (VII), (VIII), (Ⅸ) Products include jatrorrhizine, dihydroberberine, Thalictrum, berberine, normethylene berberine, palmatine, and tetrandrine.

Wherein, the pharmaceutically acceptable salt includes hydrochloride, sulfate, hydrobromide, hydroiodide, formate, acetate, and oxalate.

Further, the uric acid nephropathy is caused by the increase in blood uric acid content and the deposition of uric acid crystals in the kidney.

Beneficial technical effect

The SD rat model of uric acid nephropathy induced by yeast powder and adenine is one of the important experiments to evaluate uric acid nephropathy.

In SD rats induced with yeast powder and adenine, the blood uric acid concentration was significantly higher than the normal control group, and the creatinine and urea nitrogen were significantly higher than the normal control group; these results indicate that the uric acid metabolism of SD rats with uric acid nephropathy model An abnormality occurs, a large amount of uric acid is produced and kidney damage occurs. The blood uric acid content of SD rats after oral or intraperitoneal injection of berberine was significantly lower than that of the model group, and creatinine and urea nitrogen were significantly lower than that of the model group, indicating that oral or intraperitoneal injection of berberine can significantly improve uric acid metabolism and improve kidney Function, further can prevent/treat uric acid nephropathy.

Description of the drawings:

Figure 1. Blood uric acid in SD rats with uric acid nephropathy after berberine treatment

Figure 2. Serum creatinine in SD rats with uric acid nephropathy after berberine treatment

Figure 3. Blood urea nitrogen in SD rats with uric acid nephropathy after berberine treatment

Figure 4. Left kidney weight index in SD rats with uric acid nephropathy after berberine treatment

Figure 5. Kidney appearance of SD rats with uric acid nephropathy after berberine treatment

Figure 6. HE staining of rat kidney in SD rats with uric acid nephropathy treated with berberine (*200)

Figure 7. Blood uric acid in ICR mice with hyperuricemia after oral administration of palmatine

Figure 8. Blood uric acid in ICR mice with hyperuricemia after oral administration of rhizoline

Figure 9. Blood uric acid in ICR mice with hyperuricemia after oral administration of berberine

Figure 10. Blood uric acid in ICR mice with hyperuricemia after oral administration of total alkalis of Coptis

Detailed ways

Example 1. The therapeutic effect of berberine in SD rats with uric acid nephropathy induced by yeast powder and adenine.

The blood uric acid content, creatinine content, urea nitrogen content, left kidney weight index and the pathological comparison of rat kidney HE are important indicators to evaluate the therapeutic effect of uric acid nephropathy in rats.

1. Laboratory animals, instruments and pharmaceutical reagents

SD rats (6 weeks old) were purchased from Beijing Weitong Lihua Animal Technology Co., Ltd., and the animals were kept in an SPF environment (21±2°C, 12-hour photoperiod), and they were free to eat and drink during the experiment. Berberine was purchased from Bailingwei Company. Creatinine, uric acid, febuxostat, adenine, 4% tissue cell fixative and yeast powder were purchased from Beijing Soleibao Technology Company. The urea nitrogen test kit (urease method) was purchased from Nanjing Jiancheng Institute of Biological Engineering. Pathological section technical support is provided by the pathological section research group of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

2. Experimental instruments and analytical methods

High performance liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS 8050, Shimadzu Corporation) was used to quantitatively determine the content of blood uric acid and creatinine. The experiment uses a Waters HSS T3 (3.5μm, 2.1x100mm) chromatographic column, and the column temperature is kept at 30°C. The mobile phases for the determination of uric acid and creatinine are water and acetonitrile. Using gradient elution (A:B, 0min, 95:5; 2.0min, 80:20; 2.01min, 5:95; 5min, 5:95; 5.01min, 95:5; 9min 95:5), the flow rate is 0.2mL/min. The quantification adopts multiple reaction monitoring (MRM) mode, the quantitative ion pair of uric acid is 167.00→96.05 (m/z), and the quantitative ion pair of creatinine is 114.35→86.15.

The urea nitrogen (BUN) test kit (urease method) is used for the determination of urea nitrogen, and the specific method is detailed in the instructions.

After the rats were sacrificed, the kidneys were removed, the left kidney capsule was removed, washed with physiological saline, and the remaining water was absorbed by the filter paper. The weight was accurately weighed on an electronic balance to calculate the left kidney weight index, which is the weight of the left kidney (mg)/body weight (g).

The right kidney was fixed in 4% tissue cell fixative (4% paraformaldehyde) for 24 hours, dehydrated by an automatic dehydrator for 16 hours, embedded in a paraffin embedding machine, and then stained with HE and observed under a microscope.

3. Experimental design and animal grouping

Experimental design: The experimental animals were divided into 6 groups, including a control group, a model group, a positive drug oral treatment group, a model intraperitoneal administration treatment group, a model low-dose oral treatment group, and a model high-dose oral treatment group. The establishment of the model started from 6-week-old SD rats. After one week of adaptive feeding with the modeled feed, the model was administered at the same time for 6 weeks. The model feed is a normal feed containing 10% yeast and 0.15% adenine.

Animal grouping:

(1) Control group: normal feed feeding + oral normal saline 1mL;

(2) Model group: Modeling feed feeding + oral normal saline 1mL;

(3) Positive drug oral treatment group: model feed feeding + oral febuxostat (10mg/kg/day);

(4) Model low-dose treatment group: model feed + oral berberine (100mg/kg/day);

(5) Model high-dose treatment group: model feed + oral berberine (200mg/kg/day);

(6) Model intraperitoneal administration treatment group: modeling feed + intraperitoneal injection of berberine (10mg/kg/day).

4. Results

As shown in Figure 1, Table 1, after 6 weeks of modeling, the blood uric acid content of SD rats in the model group was significantly higher than that in the normal control group, indicating that the model was successfully established. At the same time of model building, uric acid in the blood of SD rats after 6 weeks of oral berberine treatment was significantly reduced, and the results of the two groups of oral berberine 100 and 200 mg/kg/day were dose-dependent. The blood uric acid content of the intraperitoneal injection treatment group (10mg/kg/day) decreased significantly, indicating that the intraperitoneal injection treatment was effective, and the positive drug oral group had a significant decrease in blood uric acid, and berberine had the same therapeutic effect.

As shown in Figure 2 and Table 2, the blood creatinine content of the model group was significantly higher than that of the normal control group, indicating that the model was successfully established. Similarly, creatinine in the blood of SD rats treated with berberine for 6 weeks at the same time as the model was significantly reduced and was dose-dependent. The blood creatinine in the intraperitoneal injection treatment group decreased significantly, indicating that the intraperitoneal injection treatment is effective.

As shown in Figure 3 and Table 3, the blood urea nitrogen content of the model group was significantly higher than that of the normal control group, indicating that the model was successfully established. Similarly, the blood urea nitrogen in SD rats after 6 weeks of oral berberine treatment at the same time as the model was significantly reduced and was dose-dependent. The blood urea nitrogen in the intraperitoneal injection treatment group decreased significantly, indicating that the intraperitoneal injection treatment is effective.

As shown in Figure 4 and Table 4, the left kidney index of the model group was significantly higher than that of the normal control group, indicating that the model was successfully established. Similarly, SD rats treated with berberine for 6 weeks at the same time as the model, the left kidney index decreased significantly, and was dose-dependent. The left kidney index decreased significantly in the intraperitoneal injection treatment group, indicating that the intraperitoneal injection treatment is effective.

As shown in Figure 5, it can be seen by naked eye observation that the kidneys of the normal group are light brown, while the kidneys of the rats in the model group are pale, significantly enlarged, with white spots scattered on the surface; while the kidneys of the rats in the berberine group were taken orally at the same time as the model Significant improvement, light brown, and only a few white spots; the kidney in the intraperitoneal injection treatment group also improved significantly, indicating that the intraperitoneal injection treatment is effective.

As shown in Figure 6, the results of HE staining showed that the structure of the kidney tissue in the normal group was normal without urate crystals; while in the renal cortex and medulla tubules and interstitium of the model group, there were obvious urate crystals. Needle-like arrangement, vacuolar degeneration of renal tubules, markedly widened interstitium. However, the kidneys of the rats in the model-making and oral berberine group were significantly improved, with a small amount of urate crystals, and the vacuolar degeneration was lighter; the kidneys in the intraperitoneal injection treatment group also improved significantly, indicating that the intraperitoneal injection treatment is effective.

Table 1 The concentration of uric acid in the blood of SD rats with uric acid nephropathy after berberine treatment (μg/mL, n=9)

Table 2 Blood creatinine concentration in SD rats with uric acid nephropathy after berberine treatment (μg/mL, n=9)

Table 3 Blood urea nitrogen concentration in SD rats with uric acid nephropathy after berberine treatment (μg/mL, n=9)

Table 4 Left kidney weight index of SD rats with uric acid nephropathy after berberine treatment (mg/g, n=9)

Example 2. Palmatine reduces uric acid in ICR mice.

The content of uric acid in blood is an important index to evaluate the therapeutic effect of uric acid nephropathy in rats.

1. Laboratory animals, instruments and pharmaceutical reagents

ICR mice (male, 20±2g) were purchased from Beijing Weitonglihua Animal Technology Co., Ltd., and the animals were kept in an SPF environment (21±2°C, 12-hour photoperiod), and they were free to eat and drink during the experiment. Uric acid and yeast powder were purchased from Beijing Soleibao Technology Co., Ltd., and palmatine was purchased from Chengdu Mansite Biotechnology Co., Ltd.

2. Experimental design and animal grouping

Experimental design: The experimental animals were divided into 3 groups, including the control group, the model group, and the oral palmatine 200mg/kg group. The model was fed with model feed. After one month of feeding, the model was successful. After 14 days of continuous administration, the blood uric acid content was measured. The model feed is 10% yeast powder added to the normal feed.

Animal grouping:

(1) Control group: normal feed feeding + 0.2mL oral normal saline;

(2) Model group: Modeling feed feeding + oral normal saline 0.2mL;

(3) Oral palmatine 200 mg/kg group: model feed feeding + oral palmatine 200 mg/kg.

3. Experimental results

As shown in Figure 7, Table 5, the blood uric acid content of the model group was significantly higher than that of the normal control group, indicating that the model was successfully established. After 14 days of oral administration of palmatine in ICR mice, uric acid in the blood decreased significantly, indicating that palmatine can improve the metabolism of uric acid and has the effect of treating uric acid nephropathy.

Table 5 Blood uric acid content of ICR mice with hyperuricemia after palmatine treatment (μg/mL, n=10)

Example 3. Jatrorrhizine reduces uric acid in ICR mice.

The content of uric acid in blood is an important index to evaluate the therapeutic effect of uric acid nephropathy in rats.

1. Laboratory animals, instruments and pharmaceutical reagents

ICR mice (male, 20±2g) were purchased from Beijing Weitonglihua Animal Technology Co., Ltd., and the animals were kept in an SPF environment (21±2°C, 12-hour photoperiod), and they were free to eat and drink during the experiment. Uric acid and yeast powder were purchased from Beijing Soleibao Technology Co., Ltd., and jatrorrhizine was purchased from Chengdu Mansite Biotechnology Co., Ltd.

2. Experimental design and animal grouping

Experimental design: The experimental animals were divided into 3 groups, including the control group, the model group, and the oral jatrorrhizine 200 mg/kg group. The model was fed with model feed. After one month of feeding, the model was successful. After 14 days of continuous administration, the blood uric acid content was measured. The model feed is 10% yeast powder added to the normal feed.

Animal grouping:

(1) Control group: normal feed feeding + 0.2mL oral normal saline;

(2) Model group: Modeling feed feeding + oral normal saline 0.2mL;

(3) Oral rhizoline 200mg/kg group: model-fed feed + oral rhizoline 200mg/kg.

3. Experimental results

As shown in Figure 8 and Table 6, the blood uric acid content of the model group was significantly higher than that of the normal control group, indicating that the model was successfully established. After 14 days of oral administration of jatrorrhizine in ICR mice, the blood uric acid decreased significantly, indicating that jatrorrhizine can improve the metabolism of uric acid and has the effect of treating uric acid nephropathy.

Table 6 Blood uric acid content of ICR mice with hyperuricemia after being treated with jatrorrhizine (μg/mL, n=10)

Example 4. The effect of berberine on reducing uric acid in ICR mice.

The content of uric acid in blood is an important index to evaluate the therapeutic effect of uric acid nephropathy in rats.

1. Laboratory animals, instruments and pharmaceutical reagents

ICR mice (male, 20±2g) were purchased from Beijing Weitonglihua Animal Technology Co., Ltd., and the animals were kept in an SPF environment (21±2°C, 12-hour photoperiod), and they were free to eat and drink during the experiment. Uric acid and yeast powder were purchased from Beijing Soleibao Technology Co., Ltd., and Coptis was purchased from Chengdu Mansite Biotechnology Co., Ltd.

2. Experimental design and animal grouping

Experimental design: The experimental animals were divided into 3 groups, including the control group, the model group, and the oral berberine 200 mg/kg group. The model was fed with model feed. After one month of feeding, the model was successful. After 14 days of continuous administration, the blood uric acid content was measured. The model feed is 10% yeast powder added to the normal feed.

Animal grouping:

(1) Control group: normal feed feeding + 0.2mL oral normal saline;

(2) Model group: Modeling feed feeding + oral normal saline 0.2mL;

(3) Oral berberine 200mg/kg group: model-fed feed + oral berberine 200mg/kg.

3. Experimental results

As shown in Figure 9 and Table 7, the blood uric acid content of the model group was significantly higher than that of the normal control group, indicating that the model was successfully established. After 14 days of oral administration of berberine in ICR mice, uric acid in the blood decreased significantly, indicating that berberine can improve the metabolism of uric acid and has the effect of treating uric acid nephropathy.

Table 7 Blood uric acid content in ICR mice with hyperuricemia after treatment with berberine (μg/mL, n=10)

Example 5. The effect of total alkalis of Coptis in reducing uric acid in ICR mice.

The content of uric acid in blood is an important index to evaluate the therapeutic effect of uric acid nephropathy in rats.

1. Laboratory animals, instruments and pharmaceutical reagents

ICR mice (male, 20±2g) were purchased from Beijing Weitonglihua Animal Technology Co., Ltd., and the animals were kept in an SPF environment (21±2°C, 12-hour photoperiod), and they were free to eat and drink during the experiment. Uric acid and yeast powder were purchased from Beijing Soleibao Technology Company. Coptis total alkali was purchased from Suzhou Siyuan Natural Products Research and Development Co., Ltd. It is a Chinese medicine Coptis extract, which mainly contains natural products such as berberine, berberine, and jatrorrhizine.

2. Experimental design and animal grouping

Experimental design: The experimental animals were divided into 3 groups, including a control group and a model group, a group of 100 mg/kg of total alkaloids of berberine or 200 mg/kg of total alkaloids of berberine. The model was fed with model feed. After one month of feeding, the model was successful. After 14 days of continuous administration, the blood uric acid content was measured. The model feed is 10% yeast powder added to the normal feed.

Animal grouping:

(1) Control group: normal feed feeding + 0.2mL oral normal saline;

(2) Model group: Modeling feed feeding + oral normal saline 0.2mL;

(3) Oral Coptis total Alkaloids 100mg/kg group: Model feed feeding + Oral Coptis Coptis total Alkaloids 100mg/kg;

(4) Oral Coptis total alkali 200mg/kg group: Modeling feed feeding + oral Coptis total alkali 200mg/kg.

3. Experimental results

As shown in Figure 10 and Table 8, the blood uric acid content of the model group was significantly higher than that of the normal control group, indicating that the model was successfully established. After 14 days of oral administration of Coptis chinensis total alkali in ICR mice, blood uric acid decreased significantly and was dose-dependent, indicating that Coptis chinensis total alkali can improve the metabolism of uric acid and have the effect of treating uric acid nephropathy.

Table 8 Blood uric acid content in ICR mice with hyperuricemia after treatment with Coptis total alkali (μg/mL, n=10)

Claims (4)

1. As the berberine represented by the structural formula (I), the berberine represented by the structural formula (II) or the active metabolites of both, and

   Application of its pharmaceutically acceptable salt in preparing medicine for preventing and/or treating uric acid nephropathy

   
2. As shown in the structural formulas (III), (IV), (V), (VI), (VII), (VIII), (Ⅸ), the active metabolites of berberine, jatrorrhizine, dihydroberberine, and Thalictrum Application of berberine, berberine, normethylene berberine, palmatine, tetrandrine and pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention and/or treatment of uric acid nephropathy

   
3. The use according to claim 1 or 2, wherein the pharmaceutically acceptable salt comprises hydrochloride, sulfate, hydrobromide, hydroiodide, formate, acetate or oxalic acid Salt.
4. The application according to claim 1 or 2, wherein the uric acid nephropathy is caused by the increase of uric acid in the blood and the deposition of uric acid crystals in the kidney.

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