阅读说明：本技术 香豆素衍生物在制备预防和/或治疗痛风药物中的应用 (Application of coumarin derivative in preparation of medicine for preventing and/or treating gout ) 是由 孔令义 夏元铮 许伊婷 边静 罗建光 殷勇 徐文军 张�杰 韦荣缘 张尧尧 韩亮 于 2021-02-09 设计创作，主要内容包括：本发明公开了一类香豆素衍生物在制备预防和/或治疗痛风药物中的应用,所述香豆素衍生物为东莨菪苷、法荜枝苷、异嗪皮啶-7-O-β-D-葡萄糖苷或所述任一化合物药学上可接受的盐、酯或糖基化物。该类衍生物对高尿酸血症小鼠的血清尿酸、肌酐、尿素氮的生成具有显著抑制作用,对尿液尿酸的排泄具有显著促进作用；还对尿酸钠晶体在关节腔聚集所诱导的痛风性关节肿胀大鼠的血清和滑膜中生成的白介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)的释放具有显著抑制作用；此外还能显著抑制肾损伤,促进肾脏功能恢复,为痛风的预防和/或治疗提供了新的药物开发策略。(The invention discloses an application of coumarin derivatives in preparation of medicines for preventing and/or treating gout, wherein the coumarin derivatives are scopolamine, fapiperlongoside, isofraxidin-7-O-beta-D-glucoside or pharmaceutically acceptable salts, esters or glycosylation compounds of any compound. The derivatives have obvious inhibiting effect on the generation of serum uric acid, creatinine and urea nitrogen of a hyperuricemia mouse, and have obvious promoting effect on the excretion of urine uric acid; the release of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) generated in serum and synovium of a rat with gouty joint swelling induced by the aggregation of sodium urate crystals in joint cavities is remarkably inhibited; in addition, the traditional Chinese medicine composition can also obviously inhibit kidney injury and promote the recovery of kidney functions, and provides a new medicine development strategy for preventing and/or treating gout.)

1. The application of the coumarin derivative in preparing the medicine for preventing and/or treating gout is disclosed, wherein the coumarin derivative is scopolamine, fapiperlongoside, isofraxidin-7-O-beta-D-glucoside or pharmaceutically acceptable salts, esters or glycosylation compounds of any one of the compounds.

2. The use according to claim 1, wherein the coumarin derivative is used for the preparation of a medicament for the prevention and/or treatment of hyperuricemia.

3. The use according to claim 2, wherein the coumarin derivative is used for preparing a medicament for the prevention and/or treatment of hyperuricemia-induced gouty arthritis.

4. The use according to claim 2, wherein the coumarin derivative is for use in the manufacture of a medicament for the prevention and/or treatment of renal injury caused by hyperuricemia.

5. The use according to claim 1, wherein the medicament for preventing and/or treating gout comprises scopolamine, fapiperlongoside, isofraxidin-7-O-beta-D-glucoside or pharmaceutically acceptable salts, esters or glycosylation compounds of any of the compounds, and pharmaceutically acceptable excipients.

6. The use according to claim 1, wherein the medicament for preventing and/or treating gout is a medicament for inhibiting uric acid formation.

7. The use according to claim 1, wherein the medicament for preventing and/or treating gout is a medicament for promoting uric acid excretion.

8. The use according to claim 1, wherein the medicament for the prevention and/or treatment of gout is used in combination with other drugs that inhibit uric acid formation.

9. The use according to claim 1, wherein the medicament for preventing and/or treating gout is used in combination with other medicaments for promoting uric acid excretion.

10. The use according to claim 1, wherein the agent for the prophylaxis and/or treatment of hyperuricemia is used in combination with other anti-gouty arthritis agents.

Technical Field

The invention relates to application of coumarin derivatives, in particular to application of the coumarin derivatives in preparation of medicines for preventing and/or treating gout.

Background

The important biochemical basis for gout development is hyperuricemia, which is diagnosed when the serum uric acid concentration rises to 6-8mg/dL when the organism is due to abnormal liver metabolism or dysfunction of renal excretion. The organs of the human body for uric acid metabolism are mainly the liver and the kidney. Purine is oxidized by Xanthine Oxidase (XOR) in the liver to produce uric acid, which is supplied to the body's tissues via blood flow, and then most of the uric acid is excreted from the body via the kidney. Unlike murine animals, human beings lose uricase genes during evolution, and uric acid in murine animals can be further decomposed into allantoin by uricase to promote excretion in the kidney, so that the human beings are more likely to suffer from gout caused by hyperuricemia. Foods such as meat, beer and seafood are considered to contain more purine, when a human body ingests excessive purine foods, uric acid produced by liver metabolism is increased, and high-concentration uric acid can induce kidney injury, so that the uric acid filtering capability of renal tubules is reduced. Uric acid in about 2/3 of a human body is excreted out of the body by the kidney, and a plurality of uric acid transporters exist in a renal tubule basement membrane and a renal apical membrane, so that the uric acid transporters play a role in reabsorbing or excreting uric acid from urine, for example, URAT1 (uric acid transporter 1) participates in the process of transporting uric acid from the kidney to blood, and is an important mechanism for regulating the blood uric acid level; GLUT9, a member of the glucose transporter family, can accelerate uric acid reabsorption by transporting glucose; OAT1 (organic anion transporter 1) plays a role in the secretion of uric acid in the kidney. The kidney uric acid transport protein of a healthy human body has normal expression quantity and limited transport capacity for uric acid, and when the kidney is damaged or the uric acid concentration is too high, uric acid excretion is reduced due to the functional disorder of the uric acid transport protein.

Long-term hyperuricemia can induce diseases such as gouty arthritis, renal failure, essential hypertension, cardiovascular events, atherosclerosis and the like. According to statistics, the incidence rate of hyperuricemia patients in China is 10%, wherein about 1700 thousands of patients with gout have hyperuricemia which becomes the second major metabolic disease in China. The existing drugs for treating hyperuricemia are mainly divided into uric acid generation inhibitors, such as allopurinol and febuxostat; uricosuric agents, such as probenecid, benzbromarone; uric acid dissolution promoting drugs such as labyrinase. However, the medicines clinically used for treating gout and hyperuricemia at present have toxic and side effects of different degrees, such as liver and kidney injury, sensitization, diarrhea, nausea and the like.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide application of coumarin derivatives in preparation of medicines for preventing and/or treating gout.

The technical scheme is as follows: the coumarin derivative can be applied to preparation of medicines for preventing and/or treating gout, wherein the coumarin derivative is scopolamine, fapiperlongoside, isofraxidin-7-O-beta-D-glucoside or pharmaceutically acceptable salts, esters or glycosylation compounds of any one of the compounds.

Wherein, the specific structure of the compound is as follows:

the scopolamine has obvious inhibiting effect on the generation of uric acid, creatinine and urea nitrogen in serum of a hyperuricemia mouse induced by potassium oxonate and hypoxanthine, has obvious promoting effect on urinary uric acid excretion, and has the activity of preventing and/or treating hyperuricemia; scopolamine has obvious inhibition effect on the release of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) generated in serum and synovium of rats with gouty joint swelling induced by sodium urate crystals (MSU), and has obvious activity of resisting gouty arthritis.

Furthermore, the coumarin derivative can be applied to preparation of drugs for preventing and/or treating hyperuricemia.

Furthermore, the coumarin derivative can be applied to preparation of medicaments for preventing and/or treating gouty arthritis and kidney injury caused by hyperuricemia.

Further, the medicament for preventing and/or treating gout comprises scopolamine, fabuloside, isofraxidin-7-O-beta-D-glucoside or pharmaceutically acceptable salts, esters or glycosylation compounds of any one of the compounds and pharmaceutically acceptable auxiliary materials. More specifically, the medicament for preventing and/or treating gout is an injection preparation, an oral preparation, a transdermal absorption preparation or an inhalation preparation; wherein, the carrier comprises a diluent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a transdermal absorbent, a propellant and the like which are conventional in the pharmaceutical field, and the preparation also comprises a carrier material, a drug delivery device and the like which are required by preparation.

Further, the medicament for preventing and/or treating gout is a medicament for inhibiting the formation of uric acid.

Further, the medicament for preventing and/or treating gout is a medicament for promoting uric acid excretion.

Further, the medicament for preventing and/or treating gout can be combined with other medicaments for inhibiting the formation of uric acid, promoting the excretion of uric acid or resisting gouty arthritis.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:

(1) the coumarin derivatives have obvious inhibition effect on the generation of serum uric acid, creatinine and urea nitrogen of a hyperuricemia mouse (compared with a control group, the optimal P is less than 0.0001), have obvious promotion effect on the excretion of urine uric acid (compared with the control group, the optimal P is less than 0.001), and have protection effect on the hyperuricemia mouse;

(2) has significant inhibitory effect on the release of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) generated in serum and synovium of rats with gouty joint swelling induced by sodium urate crystals (compared with a control group, optimally P < 0.0001);

(3) also has significant inhibition effect on kidney injury;

(4) has no obvious body weight inhibition toxicity.

Drawings

FIG. 1 is a graph showing the effect of scopolamine on uric acid excretion in hyperuricemia mice;

FIG. 2 shows the result of HE staining of the kidney of mice in the normal group;

FIG. 3 shows the result of HE staining of the kidney of a hyperuricemia mouse;

FIG. 4 shows the staining of HE in kidney after treatment with scopolamine in hyperuricemia mice;

FIG. 5 is the mRNA expression level of GLUT 9;

FIG. 6 is the mRNA expression level of OAT 1;

FIG. 7 is the protein expression levels of URAT1 and GLUT 9;

FIG. 8 is a graph showing the effect of MSU on serum IL-1 β levels in various groups of rats;

FIG. 9 is a graph showing the effect of MSU on synovial TNF- α levels in various groups of rats;

FIG. 10 is a graph of the effect of MSU on the change in joint swelling in various groups of rats;

FIG. 11 is a graph of the effect of scopolamine on the level of IL-1 β in a MSU-induced gouty inflammatory cell model;

FIG. 12 is a graph of the effect of scopolamine on the level of IL-1 β mRNA in a MSU-induced model of gouty inflammatory cells.

Detailed Description

The technical solution of the present invention is further explained below with reference to the examples and the accompanying drawings.

Scopolamine, Isofraxidin-7-O-beta-D-glucoside, and fabbicin were purchased from Kyoto pusi Biotech, Inc.

Example 1: effect of scopolamine on Potassium Oxonate and hypoxanthine induced hyperuricemia mice

1. Experimental methods

60 male Kunming mice with the weight of 18-22 g. One week after acclimation feeding, randomized into 6 groups: (1) normal group, (2) model control group, (3) scopolamine I group: 50mg/kg, (4) scopolamine group II: 100mg/kg, (5) scopolamine group III: 200mg/kg, (6) positive control group: allopurinol 10 mg/kg. Injecting 0.8% CMC-Na solution into abdominal cavity of normal group mouse, injecting 300mg/kg potassium oxonate and 300mg/kg hypoxanthine into abdominal cavity of other group mouse, injecting 0.8% CMC-Na solution into abdominal cavity of normal group and model group mouse after 1h, and injecting administration into gastric cavity of other group mouse for 7 d. Taking blood from orbit 1h after 7d administration, taking urine from bladder, and measuring the levels of uric acid, creatinine and urea nitrogen in mouse serum and urine by using a kit method; taking a part of kidney, embedding in paraffin, slicing, dewaxing to water, dyeing with hematoxylin, separating color with 1% acid alcohol, promoting blue with saturated lithium carbonate, dyeing with eosin, dehydrating with gradient ethanol step by step, making the dehydrated product transparent with xylene, performing microscopic examination with gum sealing, and taking representative results of each group as shown in fig. 2-4; the other part of kidney tests the expression of uric acid related transporters such as URAT1, GLUT9 and OAT1 by Western Blot and PCR experiment.

2. Results of the experiment

TABLE 1 Effect of intragastric administration of scopolamine on the serum markers of hyperuricemia in mice induced by Potassium Oxonate and hypoxanthine (x + -SD, n ═ 10)

P < 0.0001, P < 0.05 (compared to control)

TABLE 2 influence of intragastric administration of scopolamine on urinary markers of hyperuricemia in mice caused by Potassium Oxonate and hypoxanthine (x + -SD, n ═ 10)

P < 0.0001, P < 0.01, P < 0.05 (compared to control)

TABLE 3 Effect of intragastric administration of scopolamine on the weight (g) of mice with hyperuricemia induced by Potassium Oxonate and hypoxanthine (x + -SD, n ═ 10)

P < 0.0001, P < 0.001, P < 0.01 (compare with control)

As can be seen from Table 1, when 50, 100 and 200mg/kg of scopolamine is administrated by intragastric administration, the dosage dependence inhibits the increase of the serum uric acid of mice caused by potassium oxonate and hypoxanthine, and the 50, 100 and 200mg/kg dosage groups can effectively reduce the serum creatinine and urea nitrogen levels, and the action effect is superior to that of allopurinol. As can be seen from fig. 1 and table 2, scopolamine can also dose-dependently promote uric acid excretion in the urine of hyperuricemia mice, but has no significant effect on urinary creatinine and urea nitrogen. As can be seen from Table 3, the weight of mice is not obviously affected by 50, 100 and 200mg/kg intragastric administration of scopolamine, the weight of the positive allopurinol group is obviously different from that of the control group from day 3, the toxic and side effects of the positive allopurinol on organisms can be preliminarily judged, and the scopolamine has no obvious toxicity and has a protective effect on hyperuricemia mice.

As can be seen from fig. 2 to 4, the kidney cells of the mice in the normal group are compact and uniform, have no obvious inflammatory cell infiltration, and have no vacuolar degeneration, renal interstitial edema or renal interstitial fibrosis (the black scale bar is 100 μm); the control group of mice has moderate granular and vacuole-like degeneration of renal tubular epithelial cells, and erythrocyte in renal tubules is deposited to form erythrocyte casts; dilatation of renal tubule lumen, renal interstitial edema, renal interstitial fibrosis; the kidney of the scopolamine group mice of 200mg/kg has no obvious lesion, injury, renal interstitial edema or renal interstitial fibrosis, the vacuolation is reduced, and no obvious erythrocyte siltation exists in the renal tubules. The results show that the scopolamine can inhibit the kidney injury of the mice with hyperuricemia.

As can be seen from FIGS. 5-7, at the mRNA level, scopolamine can inhibit the expression of GLUT9 and promote the expression of OAT 1; at the protein level, scopolamine can inhibit the expression of URAT1 and GLUT 9. The above results further indicate that scopolamine plays a role in preventing and/or treating hyperuricemia by promoting uric acid excretion.

Example 2: effect of scopolamine on sodium urate Crystal (MSU) -induced gouty arthritis in rats

1. Experimental methods

Male SD rats 12, weighing 180-. One week after acclimation feeding, 4 groups were randomized: (1) normal group, (2) model control group, (3) positive control group: indomethacin 3mg/kg, (4) scopolamine group: 100 mg/kg. The rats in the normal group and the rats in the model control group are subjected to intragastric gavage administration for 3 days, 1h after the last day of administration except the rats in the normal group, 100 mu L of sterile MSU (500 mu g/ml) suspension is injected into the right ankle joint cavity of the rats in the other groups, and 100 mu L of sterile PBS is injected into the right ankle joint cavity of the rats in the normal group. The joint circumferences of rats 0, 3, 6 and 24 hours after injection are respectively measured before injection by adopting a line-tying method, and the joint swelling rate is represented by the ratio of the difference value between the joint circumferences before and after the injection to the circumference before the injection. Blood was collected from the canthus 24h after the joint cavity injection, and synovial membrane was collected from the joint for detection of relevant inflammatory factors in serum and synovial membrane.

2. Results of the experiment

Table 4 effect of scopolamine on the rate (%) of gouty joint swelling in rats induced by sodium urate crystals (MSU) (x ± SD, n ═ 3)

P < 0.01, P < 0.05 (compared to control)

As can be seen in FIG. 8, the gavage administration of scopolamine at 100mg/kg reduced serum IL-1 β levels; as can be seen in FIG. 9, gavage of 100mg/kg scopolamine has a tendency to reduce TNF- α levels in the synovium; as can be seen from Table 4 and FIG. 10, scopolamine 100mg/kg can significantly inhibit the gouty joint swelling of rats caused by MSU 24 hours after intragastric administration.

Example 3: effect of scopolamine on MSU-induced gouty inflammatory cell model

1. Experimental methods

Stimulating THP-1 cells (mononuclear macrophages) with 500nM PMA for 3h to induce adherent differentiation into macrophages, discarding old culture medium, adding MCC950(100 μ M) into positive control group, adding 50, 25 and 6.25 μ M drugs into scopolamine high-medium low-dose group, respectively, and adding equal volume of PBS into normal group and control group; after 2h of administration, MSU (250. mu.M) solution was added, and the drug was incubated with MSU for 24h, and then culture supernatant and cell-extracted RNA were taken to detect IL-1. beta. level.

2. Results of the experiment

As can be seen from FIGS. 11-12, scopolamine can inhibit IL-1 β levels in cell supernatants and dose-dependently inhibit IL-1 β mRNA expression levels.

Example 4: effect of scopolamine structural analogues on Potassium Oxonate and hypoxanthine induced hyperuricemia mice

1. Experimental methods

50 male Kunming mice with the weight of 18-22 g. Randomized into 5 groups: (1) normal group, (2) model control group, (3) fabbicin: 50mg/kg, (4) isofraxidin-7-O- β -D-glucoside: 50mg/kg, (5) positive control group: allopurinol 10 mg/kg. Injecting 0.8% CMC-Na solution into abdominal cavity of normal group mouse, injecting 300mg/kg potassium oxonate and 300mg/kg hypoxanthine into abdominal cavity of other group mouse, injecting 0.8% CMC-Na solution into abdominal cavity of normal group and model group mouse after 1h, and injecting other group mouse into abdominal cavity for administration, and continuously making model for administration for two weeks. Blood is taken from the orbit 1h after the last day of administration, urine is taken from the bladder, and the levels of uric acid, creatinine and urea nitrogen in the serum and urine of the mouse are measured according to a kit method.

2. Results of the experiment

TABLE 5 Effect of scopolamine structural analogs on Potassium Oxonate and hypoxanthine induced hyperuricemia mouse serum indices (x + -SD, n ═ 10)

P < 0.0001, P < 0.001, P < 0.01 (compare with control)

TABLE 6 influence of structural analogs of scopolamine on hyperuricemia induced by Potassium Oxonate and hypoxanthine mouse urine index (x + -SD, n ═ 10)

P < 0.0001, P < 0.001, P < 0.01, P < 0.05 (compared to control)

TABLE 7 Effect of scopolamine structural analogs on Potassium Oxonate and hypoxanthine induced hyperuricemia mouse body weight (g) (x + -SD, n ═ 10)

P < 0.0001, P < 0.001, P < 0.05 (compared to control)

As can be seen from tables 5-6, gavage administration of the farnesoid glycoside at 50mg/kg can inhibit the increase of serum uric acid of mice caused by potassium oxonate and hypoxanthine; 50mg/kg of isofraxidin-7-O-beta-D-glucoside can inhibit the blood creatinine level of a mouse with hyperuricemia after intragastric administration; the administration of the piperlongumine and isofraxidin-7-O-beta-D-glucoside 50mg/kg through intragastric administration can inhibit the blood urea nitrogen level of mice with hyperuricemia and promote the excretion of uric acid in urine; 50mg/kg of isofraxidin-7-O-beta-D-glucoside can promote the excretion of creatinine in urine of mice with hyperuricemia by intragastric administration.

As can be seen from Table 7, the scopolamine analogue has no obvious influence on the body weight of the mice, the body weight of the positive allopurinol group is obviously different from that of the mice in the control group from the 7 th day, the positive allopurinol is preliminarily judged to have toxic and side effects on the body, and the scopolamine analogue has no obvious toxicity and has a protective effect on the hyperuricemia mice.