Combined application of scopolamine and hyoscyamine

Abstract

The present invention discloses the combined use of scopolamine and hyoscyamine in the preparation of a drug for preventing or / and treating hyperuricemia. The combined use has an excellent synergistic effect, can significantly reduce the levels of serum uric acid, creatinine, and urea nitrogen, reduce the reabsorption of uric acid, creatinine, and urea nitrogen in urine by the kidney, and promote the excretion of the three, has significant anti-hyperuricemia activity, and has a protective effect on the kidney; it can also significantly reduce the generation of inflammatory factors, effectively improve the symptoms of joint swelling in gouty arthritis, and has clinical application value and development prospects.

Description

Technical Field

[0001] The present invention relates to a combined application of hyoscyamyl alcohol and scopoletin, and in particular to a combined application of hyoscyamyl alcohol and scopoletin in a drug for preventing or / and treating hyperuricemia with a synergistic effect. Background Art

[0002] Hyperuricemia is the cause of gout, a metabolic disorder caused by purine metabolism disorder. The diagnostic standard is that the blood uric acid level exceeds 420μmol / L (7mg / dL) twice on different days. Since there is no uricase gene in the human body, uricase cannot be further broken down into allantoin and promote its excretion in the kidneys, so uric acid is the final oxidation product of purine metabolism. When the human body consumes too much high-purine food such as meat, beer and seafood, the uric acid generated by liver metabolism increases. The accumulation of high-concentration uric acid leads to a decrease in the renal tubular filtration capacity of uric acid, and the uric acid metabolic balance is broken. Most hyperuricemia is caused by a decrease in the renal uric acid excretion efficiency.

[0003] Hyperuricemia is not only a key risk factor for gout, but is also closely related to kidney disease, hypertension, diabetes, cardiovascular disease, etc. At present, the drugs used to treat hyperuricemia are mainly divided into three types according to their mechanism of action. The first is drugs that inhibit uric acid production, such as allopurinol and febuxostat; the second is drugs that promote uric acid excretion, such as probenecid and benzbromarone; the third is drugs that promote uric acid decomposition, such as rasburicase. However, these drugs all have varying degrees of toxic side effects, such as liver damage, sensitization, diarrhea, nausea, etc. Therefore, the development of drugs that can effectively and low-toxicly treat gout and hyperuricemia has clinical application prospects. Summary of the invention

[0004] Purpose of the invention: The present invention aims to provide a combined application of scopolamine and hyoscine with synergistic effects.

[0005] Technical solution: The hyoscyamine alcohol and scopolamine described in the present invention are used in combination to prepare a drug for preventing and / or treating hyperuricemia, further a drug for preventing and / or treating gout caused by hyperuricemia, further a drug for preventing and / or treating gouty arthritis caused by hyperuricemia, and still further a drug for preventing and / or treating gouty joint swelling caused by hyperuricemia.

[0006]

[0007] Siegesbeckia orientalis L., Siegesbeckia pubescens Makino or Siegesbeckia glabrescens Makino, all of which are plants of the Asteraceae family, are dried aerial parts, and the whole plant is used as medicine. Siegesbeckia orientalis L., Siegesbeckia pubescens Makino, and Siegesbeckia glabrescens Makino and other similar plants are widely distributed in Northeast my country, North China, East China, Central South, and Southwest China, mainly in the wild. The main functions of Siegesbeckia are to dispel rheumatism, benefit joints, and detoxify. It can treat rheumatic pain, weakness of tendons and bones, soreness of waist and knees, paralysis of limbs, hemiplegia, and urticaria (Pharmacopoeia of the People's Republic of China 2020 edition). Kirenol is the main active ingredient in Siegesbeckia orientalis, with CAS number 52659-56-0 and molecular formula C 20 H 34 O 4 .

[0008]

[0009] Scopolamine is a coumarin compound that is widely used in the treatment of rheumatoid arthritis. Studies have found that scopolamine can reduce joint swelling induced by Freund's adjuvant in rats and significantly reduce the expression of IL-6 and VEGF in the synovium, thereby exerting its activity in inhibiting inflammation. In addition, previous studies have found that scopolamine has anti-hyperuricemia and anti-gout activities, CAS No. 531-44-2, molecular formula C 16 H 18 O 9 .

[0010] At present, there are no reports on the combined use of scopolamine and hyoscine, and there is no research on their activity against hyperuricemia and gouty arthritis.

[0011] Specifically, the drug inhibits the generation of creatinine, urea nitrogen and uric acid, inhibits their reabsorption and promotes their excretion.

[0012] More specifically, the drug downregulates the expression of GLUT9 protein, upregulates the expression of OAT1 and OAT3 proteins, and inhibits the generation of inflammatory factors IL-1β and TNF-α.

[0013] The mechanism of the combined application of the present invention is as follows:

[0014] After research, it was found that scopolamine and hyoscyamine alcohol, when used alone at a dose of 100 mg / kg, showed significant reduction in hyperuricemia and anti-gouty arthritis activity, respectively. When used in combination, their mechanism of action is to promote uric acid excretion by downregulating the GLUT9 transporter and upregulating the expression of uric acid secretory proteins OAT1 and OAT3, and to produce anti-gouty arthritis effects by inhibiting the expression of inflammatory factors IL-1β and TNF-α induced by sodium urate crystals.

[0015] In a specific application, the single dose ratio of the nonanol to scopolamine is 1:1; wherein the single doses of the nonanol and scopolamine are 1 to 200 parts by weight, respectively, preferably 1 to 200 mg / kg, further preferably 50 to 100 mg / kg, further preferably 50 to 75 mg / kg, and further preferably 50 mg / kg and 75 mg / kg.

[0016] The combination can be administered orally or by injection, specifically by intraperitoneal, subcutaneous, intravenous or intramuscular injection; diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and other pharmaceutically acceptable carriers can also be added to prepare a certain form of pharmaceutical composition, such as powders, pills, capsules, tablets, microcapsules, soft capsules, films, suppositories, injections, ointments, powders, granules, aerosols and other preparations. The dosage ratio of scopolamine to scopolamine in the pharmaceutical composition is 1:1, preferably 1 to 200 parts by weight, respectively, and can be specifically converted to 1 to 200 mg / kg, 50 to 100 mg / kg, and 50 to 75 mg / kg.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0018] The combination of scopolamine and hyoscyamine has an excellent synergistic effect, which can significantly reduce serum creatinine, urea nitrogen, and uric acid levels, reduce the renal reabsorption of uric acid, creatinine, and urea nitrogen in urine, and promote the excretion of the three. It has significant anti-hyperuricemia activity and has a protective effect on the kidneys. It can also significantly reduce the production of inflammatory factors and effectively improve the symptoms of joint swelling in gouty arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 HE staining of kidneys of mice with high uric acid; A is the control group, B is the model group, C is the positive drug allopurinol group, D is the positive drug benzbromarone group, and E is the 50 mg / kg nonanol group;

[0020] Figure 2 The results of GLUT9, OAT1, and OAT3 protein expression in mouse kidneys;

[0021] Figure 3 The results are the effects of drugs on gouty joint swelling in rats induced by sodium urate crystals (MSU);

[0022] Figure 4 The results are as follows: the effect of sodium urate crystals (MSU) on the synovial IL-1β level of rats in each group;

[0023] Figure 5 The results are as follows: the effect of sodium urate crystals (MSU) on the TNF-α level in the synovium of rats in each group;

[0024] Figure 6 The results show the effect of drugs on IL-1β levels in the gouty inflammatory cell model induced by sodium urate crystals (MSU). DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with embodiments.

[0026] Example 1: Effect of the pharmaceutical composition on hyperuricemia mice induced by potassium oxonate and hypoxanthine

[0027] 1. Experimental methods

[0028] 80 male Kunming mice, weighing 18-22 g, 6 weeks old. After 1 week of adaptive feeding, they were randomly divided into 8 groups: (1) normal group, (2) model group, (3) hyoscyamine group: 100 mg / kg, (4) scopolamine group: 100 mg / kg, (5) hyoscyamine and scopolamine combination group I: 50 mg / kg, (6) hyoscyamine and scopolamine combination group II: 75 mg / kg, (7) positive control group: allopurinol 10 mg / kg, (8) positive control group: benzbromarone 10 mg / kg. The mice in the normal group were gavaged and intraperitoneally injected with 0.5% CMC-Na solution, and the mice in the other groups were intraperitoneally injected with 300 mg / kg potassium oxonate and gavaged with 300 mg / kg hypoxanthine. One hour later, the mice in the normal group and the model group were gavaged with 0.5% CMC-Na solution, and the mice in the other groups were gavaged for 7 consecutive days. One hour after the 7th day of administration, the eyeballs were removed to collect blood, and the bladder was collected for urine. The levels of uric acid, creatinine, and urea nitrogen in the serum and urine of the mice were determined according to the kit method.

[0029] 2. Experimental results

[0030] Table 1. Effects of the drug combination on serum indices of hyperuricemia in mice induced by potassium oxonate and hypoxanthine (x±SD, n=10)

[0031]

[0032] * p<0.05, ** p<0.01,*** p<0.001 (compared with the model group).

[0033] Table 2. Effects of the drug combination on urine indexes of mice with hyperuricemia induced by potassium oxonate and hypoxanthine (x±SD, n=10)

[0034]

[0035] * p<0.05, ** p<0.01, *** p<0.001 (compared with the model group).

[0036] As shown in Table 1, the combination of scopolamine and hyoscyamine can inhibit the increase of serum uric acid in mice caused by potassium oxonate and hypoxanthine, and can reduce serum creatinine levels. As shown in Table 2, the pharmaceutical composition can inhibit the reabsorption of creatinine and urea nitrogen by the kidneys and promote the excretion of creatinine and urea nitrogen. Figure 1 It can be seen that the positive drugs have strong renal toxicity. After oral administration, the drug composition group I can inhibit the vacuolar degeneration of the kidneys of hyperuricemia mice, reduce the accumulation of red blood cells in the renal tubules, and significantly protect the kidneys. Figure 2 The results showed that compared with the normal group, the expression level of glucose transporter GLUT9 in the model group mice kidney increased, and the expression levels of uric acid secretion proteins OAT1 and OAT3 decreased, indicating that hypoxanthine and potassium oxonate caused hyperuricemia in mice by increasing the reabsorption of uric acid and reducing uric acid excretion. Compared with the model group, the positive drug allopurinol group and benzbromarone group significantly downregulated the expression of GLUT9 protein and upregulated OAT1 and OAT3 proteins; consistent with this, the drug composition group I also played the same role, with a significant decrease in the expression level of GLUT9 protein and a significant increase in OAT1 and OAT3 proteins. In summary, the experimental results show that the drug composition group I may play a role in lowering uric acid by reducing the reabsorption of uric acid in the kidneys of hyperuricemia mice and promoting the excretion of uric acid.

[0037] Example 2: Effect of the pharmaceutical composition on gouty arthritis in rats induced by sodium urate crystals (MSU)

[0038] 1. Experimental methods

[0039] Twenty-one male SD rats weighing 180-220 g were randomly divided into 7 groups after adaptive feeding for 1 week: (1) normal group, (2) model control group, (3) positive control group: indomethacin 3 mg / kg, (4) hyoscyamine group: 100 mg / kg, (5) scopolamine group: 100 mg / kg, (6) hyoscyamine and scopolamine combination group I: 50 mg / kg, (7) hyoscyamine and scopolamine combination group II: 75 mg / kg. Rats in the normal group and the model control group were gavaged with normal saline, and rats in the other groups were gavaged for 5 consecutive days. One hour after administration on the third day, rats in the other groups were injected with 50 μL of sterile MSU (20 mg / ml) suspension into the right ankle joint cavity, except for the normal group, and 50 μL of sterile PBS was injected into the right ankle joint cavity of rats in the normal group. The wire-tying method was used to measure the joint circumference of rats before injection and at 0, 24 and 48 hours after injection. The ratio of the difference between the joint circumference and the circumference before administration was used to express the joint swelling rate.

[0040] 2. Experimental results

[0041] Table 3. Effects of the drug composition on gouty joint swelling induced by sodium urate crystals (MSU) in rats (x±SD, n=3)

[0042]

[0043]

[0044] * p<0.05, ** p<0.01, *** p<0.001 (compared with the model group).

[0045] like Figure 3 As shown in Table 3, compared with the normal group, the right ankle joints of the mice in the model group were significantly swollen 24-48 hours after injection of 20 mg / mL MSU; compared with the model group, the degree of joint swelling in the groups treated with the combination of scopolamine and indomethacin was significantly reduced 48 hours after injection of MSU.

[0046] like Figure 4-5 As shown in the results, compared with the normal group, the levels of TNF-α and IL-1β in the ankle synovium of the model group rats showed an increasing trend; compared with the model group, the levels of IL-1β and TNF-α in the synovium of gouty arthritis rats after administration of indomethacin, chinonanol group and pharmaceutical composition group I all showed a decreasing trend, and the effect of pharmaceutical composition group I was better than that of chinonanol group, but the levels of IL-1β and TNF-α in the synovium of the scopoletin group did not decrease. In summary, pharmaceutical composition group I has certain activity in anti-acute gouty arthritis, and may exert its effect by inhibiting the levels of synovial inflammatory factors IL-1β and TNF-α.

[0047] Example 3: Effect of the pharmaceutical composition on the MSU-induced gouty inflammatory cell model

[0048] 1. Experimental methods

[0049] THP-1 cells (mononuclear macrophages) were stimulated with 500 nM PMA for 3 h to induce adherent differentiation into macrophages. The old culture medium was discarded and the cells were divided into 6 groups: (1) normal group, (2) model control group, (3) positive control group: MCC950 (100 μM), (4) hyoscyamine group: 100 mg / kg, (5) scopoletin group: 100 mg / kg, (6) hyoscyamine and scopoletin combination group I: 50 mg / kg, respectively. MSU (250 μM) solution was added 2 h after administration. After incubation of drugs and MSU for 24 h, the culture supernatant and cells were collected to extract RNA to detect IL-1β levels.

[0050] 2. Experimental results

[0051] like Figure 6 As shown in the results, chinonanol, scopolamine and their combination have anti-inflammatory activity in THP-1 cells. Compared with the blank group, the IL-1β protein level in the cell supernatant of the model group was significantly increased, indicating that MSU stimulation of THP-1 macrophages can promote the production of inflammatory factor IL-1β; compared with the model group, the NLRP3 inflammasome inhibitor MCC950 can significantly reduce the IL-1β level in the cell supernatant; chinonanol, scopolamine and their combination can also reduce the IL-1β level after treatment, and the combination group has a better effect, indicating that the chinonanol and scopolamine combination group can effectively inhibit the production of inflammatory factors in the in vitro model.

Claims

1. Use of a drug combination of scopoletin and hyoscine in the preparation of a drug for preventing and / or treating hyperuricemia.

2. The use according to claim 1, characterized in that: The medicine is a medicine for preventing and / or treating gout caused by hyperuricemia.

3. The use according to claim 2, characterized in that: The medicine is a medicine for preventing and / or treating gouty arthritis caused by hyperuricemia.

4. The use according to claim 3, characterized in that: The medicine is a medicine for preventing and / or treating gouty joint swelling caused by hyperuricemia.

5. The use according to claim 1, characterized in that: The drug inhibits the production of creatinine, urea nitrogen, and uric acid.

6. The use according to claim 1, characterized in that: The drug inhibits the reabsorption of creatinine, urea nitrogen, and uric acid.

7. The use according to claim 6, characterized in that: The drug promotes the excretion of creatinine, urea nitrogen and uric acid.

8. The use according to claim 1, characterized in that: The drug down-regulates the expression of GLUT9 protein and up-regulates the expression of OAT1 and OAT3 proteins.

9. The use according to claim 1, characterized in that: The drug inhibits the generation of inflammatory factors IL-1β and TNF-α.

10. The use according to claim 1, characterized in that: The dosage ratio of the nonanol to scopoletin is 1:1.

Images