Pharmaceutical combination of ursolic acid and multi-target tyrosine kinase receptor inhibitor

Abstract

The invention relates to a pharmaceutical composition of ursolic acid and a multi-target tyrosine kinase receptor inhibitor, which has a synergistic effect in the treatment of cancer, especially liver cancer.

Description

Technical Field

[0001] The present invention provides a use of a combination of therapeutic agents in preparing a drug for treating cancer; in particular, it provides a drug combination that produces synergistic effects in the treatment of liver cancer. Background Art

[0002] Primary hepatocellular carcinoma (HCC) is a common malignant tumor worldwide, with the morbidity and mortality ranking sixth and third respectively. China is a high-incidence area for HCC. In recent years, the incidence of HCC has shown a clear upward trend, seriously endangering human health and life safety. At present, clinical treatment methods for HCC are very limited. For HCC that cannot be surgically removed, targeted drugs are usually used to directly act on the tumor to prolong the life of advanced patients, but the treatment effect is not ideal.

[0003] Clinical targeted therapy for liver cancer usually uses small molecule compounds that inhibit multi-target tyrosine kinase receptors, such as Sorafenib, Lenvatinib, and Regorafenib, to achieve anti-tumor effects by targeting vascular endothelial growth factor receptors (VEGFR), fibroblast growth factor receptors (FGFR), and platelet-derived growth factor receptors (PDGFR) in tumors. However, the effect of such drugs in treating liver cancer is limited. In clinical work, patients have poor responses to such drugs or tumor progression after treatment.

[0004] Ursolic acid is a pentacyclic triterpenoid drug with high lipid solubility and low solubility. Its solubility in aqueous solution is very low, which is also the main reason for its low bioavailability in vivo. Therefore, many studies have used liposomes, solid dispersions and other preparations to improve its solubility and thus improve its bioavailability. Because there are differences in the bioavailability of ursolic acid APIs, liposomes, solid dispersions and other preparations, it is difficult to reasonably anticipate the combined effect of ursolic acid special preparations and multi-target tyrosine kinase receptor inhibitors based on the test data of ursolic acid APIs at the cell level.

[0005] Currently, the treatment methods for liver cancer are single and have limited efficacy. There is an urgent need to find other treatment methods to improve the anti-tumor treatment effect. Summary of the invention

[0006] One object of the present invention is to provide a drug combination of ursolic acid and a multi-target tyrosine kinase receptor inhibitor, which produces a synergistic effect in treating cancer, especially in treating liver cancer.

[0007] In a first aspect, the present invention provides a drug combination comprising a first active drug and a second active drug,

[0008] The first active drug is ursolic acid or a pharmaceutically acceptable salt thereof;

[0009] The second active drug is a multi-target tyrosine kinase receptor inhibitor.

[0010] In some embodiments, the second active drug is preferably lenvatinib or a pharmaceutically acceptable salt thereof, regorafenib or a pharmaceutically acceptable salt thereof, sorafenib or a pharmaceutically acceptable salt thereof, or any combination thereof. In some embodiments, the pharmaceutically acceptable salt is a mesylate, a hydrochloride, or a toluenesulfonate. Specifically, lenvatinib mesylate, regorafenib hydrochloride, or sorafenib toluenesulfonate.

[0011] In some embodiments, the weight ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient is 2.5:50 to 50:2.5; preferably 2.5:30 to 30:5. In some embodiments, the weight ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient is any integer or decimal value in the range of 2.5:50 to 50:2.5, specifically selected from: 15:5, 15:10, 15:15, 15:20, 15:25, 15:30, 20:5, 20:10, 20:15, 20:20, 20:25, 20:30, 25:5, 25:10, 25:15, 25:25, 25:30, 25:30, 30:5, 30:10, 30:15, 30:30, 30:35, 30:30.

[0012] In some embodiments, the first active drug is a preparation comprising ursolic acid or a pharmaceutically acceptable salt thereof, phospholipids and a pH adjuster; the second active drug is an oral preparation, preferably a tablet or capsule.

[0013] In some embodiments, the weight ratio of the first active drug to the second active drug is as follows:

[0014] The weight ratio of ursolic acid or a pharmaceutically acceptable salt thereof to lenvatinib or a pharmaceutically acceptable salt thereof in the form of free base is 5:10-30:10, preferably 5:10, 10:10, 15:10, 30:10; more preferably, 5:10, 10:10, 15:10; or

[0015] The weight ratio of ursolic acid or a pharmaceutically acceptable salt thereof to regorafenib or a pharmaceutically acceptable salt thereof in the form of free base is 5:5-30:5, preferably 5:5, 10:5, 15:5, 20:5, 25:5, 30:5; or

[0016] Calculated in free base form, the weight ratio of ursolic acid or a pharmaceutically acceptable salt thereof to sorafenib or a pharmaceutically acceptable salt thereof is 2.5:30-30:30, preferably 2.5:30, 5:30, 10:30.

[0017] In some embodiments, the first active drug is a lipid microsphere or liposome injection, and the average particle size D of the lipid microsphere or liposome is 50 It is 100-300nm, preferably 150-250nm.

[0018] In some embodiments, the phospholipid in the first active drug is selected from lecithin, soybean lecithin, hydrogenated soybean lecithin, phosphatidylethanolamine, synthetic phosphatidylserine, phosphatidylinositol, sphingomyelin, egg phosphatidylcholine, dicetyl phospholipids, dimyristoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, pegylated distearoyl phosphatidylethanolamine, methoxypegylated distearoyl phosphatidylethanolamine, or a mixture of more than two of the above.

[0019] In some embodiments, the first active drug is a lyophilized liposome injection, and the lyophilized liposome injection further comprises a lyophilized scaffold agent, and the lyophilized scaffold agent is selected from mannitol, sucrose, lactose or a combination thereof.

[0020] In some embodiments, the pH adjuster in the first active drug is a buffer, which controls the pH value of the aqueous phase in the range of 6-7, preferably 6.3-6.9 during the preparation process.

[0021] In some embodiments, a buffer solvent is used as the aqueous phase during the preparation of the freeze-dried liposome injection of the first active drug, and the buffer controls the pH value of the aqueous phase within the range of 6-7, preferably 6.3-6.9.

[0022] In some embodiments, the weight ratio of ursolic acid or a pharmaceutically acceptable salt thereof to phospholipid in the freeze-dried liposome injection of the first active drug is 0.1-10:5-500, preferably any value in the range of 0.1-10:10-50, 1-10:5-50, 2-5:10-50, 2-5:20-50, 2-5:30-50. The specific ratio can be selected from 0.1:10, 0.5:10, 1:10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, 10:10.

[0023] In some embodiments, the weight ratio of ursolic acid or its pharmaceutically acceptable salt to phospholipids and lyophilized excipients in the lyophilized liposome injection of the first active drug is 0.1-10:10-50:50-300, preferably 2-5:20-50:150-300. Specific weight ratios may be selected from 2:20:50, 2:20:100, 2:20:150, 2:20:200, 2:20:300, 3:20:150, 3:20:200, 3:20:250, 3:20:300, 3:25:150, 3:25:200, 3:25:250, 3:25:300, 3:30:150, 3:30:200, 3:30:250, 3:30:300, 3:50:150, 3:50:200, 3:50:250, 3:50:300.

[0024] In some embodiments, other pharmaceutically acceptable excipients or additives, such as surfactants and antioxidants, may be further added to the first active drug liposome.

[0025] In some embodiments, the pharmaceutical combination of the present invention is in the form of a pharmaceutical composition, which includes a first active drug and a second active drug; wherein the first active drug and the second active drug can be separate compositions, or compositions that are packaged together but not in contact with each other. In some embodiments, the "comprising" and "including" in the present invention can be replaced by "consisting of" or "consisting of".

[0026] In a second aspect, the present invention provides a kit comprising any of the first active drug and the second active drug described above. The kit can be a separate drug package or a combined drug package.

[0027] In some embodiments, the kit further comprises instructions for administering the first active drug and the second active drug to a human patient. The instructions state that the first active drug and the second active drug can be administered sequentially or simultaneously.

[0028] In a third aspect, the present invention provides a drug combination comprising any of the first active drug and the second active drug, and its use in preparing a drug for treating cancer. The present invention also provides a drug combination comprising any of the first active drug and the second active drug, and its use in treating cancer.

[0029] In some embodiments, the cancer to be treated is liver cancer. The liver cancer is selected from primary liver cancer or secondary liver cancer.

[0030] In some embodiments, the first active drug and the second active drug are administered sequentially or simultaneously.

[0031] In some embodiments, the cancer is a cancer resistant to a multi-target tyrosine kinase receptor inhibitor. In some embodiments, the multi-target tyrosine kinase receptor of the present invention includes any two or more of vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) or platelet-derived growth factor receptor (PDGFR).

[0032] In some embodiments, the cancer resistant to multi-target tyrosine kinase receptor inhibitors is selected from cancer resistant to lenvatinib, regorafenib or sorafenib. In some embodiments, the resistance is primary resistance or acquired liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Tumor growth inhibition of ursolic acid liposomes combined with lenvatinib or regorafenib in a human hepatocellular carcinoma PDX tumor model.

[0034] Figure 2 The combination of ursolic acid liposomes and sorafenib inhibited tumor growth in the orthotopic inoculation tumor model of human hepatoma cell PLC / PRF / 5. DETAILED DESCRIPTION

[0035] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0036] Example 1 Preparation of ursolic acid liposomes

[0037] Take 2g of ursolic acid and add 40ml of anhydrous ethanol to stir and dissolve at room temperature, then add 20g of soybean lecithin, heat to 48-52℃ and stir for 8-15 minutes to form an organic phase; prepare an aqueous phase solution with 40ml of distilled water, 100g of mannitol, and a buffer prepared by phosphoric acid and sodium hydroxide (control the pH value of the aqueous phase within the range of 6.3-6.9); slowly add the organic phase to the aqueous phase at 50-55℃ and stir for 30-35 minutes, then filter with a 0.8μ microporous filter membrane, and fill the filtrate into vials after making up the volume with injection water and pre-freeze at -50 to -40℃ for 10 hours, then perform gradient temperature increase vacuum freeze drying at a temperature range of -50℃ to 30℃ for 36 to 52 hours to obtain an ursolic acid liposome preparation (3mg / bottle). The freeze-dried ursolic acid liposomes are reconstituted with physiological salt and measured by a laser particle size analyzer. The particle size D 50 It is 100-300nm.

[0038] Example 2 Combination of ursolic acid liposomes and lenvatinib or regorafenib

[0039] The PDX model was established in immunodeficient mice using tissues removed from liver cancer patients during surgery. This was used to test the combined anti-tumor efficacy of ursolic acid liposomes and lenvatinib or regorafenib in vivo, as well as to compare the efficacy of each drug alone.

[0040] The ursolic acid liposomes prepared in Example 1 (provided by Wuhan Liyuanheng Pharmaceutical Technology Co., Ltd.) and commercially available lenvatinib mesylate capsules and regorafenib tablets were prepared into a test solution according to Table 1.

[0041] Table 1 Preparation and storage of test substances

[0042]

[0043]

[0044] The human hepatocellular carcinoma PDX tumor model was used for testing. When the subcutaneous tumor of the tumor-bearing mouse grew to 500-1000mm 3 Afterwards, mice (species strain: Mus musculus, BALB / c nude) were euthanized, the tumors were removed, the necrotic parts were removed, and the tumors were cut into pieces of approximately 2×2×2 mm. 3 After the experimental animals were anesthetized, the tumor masses were inoculated into the liver parenchyma of the anesthetized animals with a trocar, and one mass was inoculated per mouse. The day of inoculation was recorded as D0, and 4 days after inoculation (D4), the animals were randomly divided into groups for administration according to the weight of the mice (the day was recorded as PG-D0), and a total of 11 groups were divided, with 8 animals in each group. The administration was carried out according to the scheme in Table 2.

[0045] Table 2. Dosage schedule

[0046]

[0047]

[0048] Note: *, the administration volume is 10μL / g according to the animal's body weight; when the animal's body weight decreases by more than 15%, the administration volume can be suspended or adjusted until the body weight decreases to 10%; ip: intraperitoneal injection; po: oral gavage; # , qd×21: administer the drug once a day, for 21 times.

[0049] On the 22nd day after group administration (i.e., 1 day after the last administration of PG-D21), the entire experiment was terminated. The test results of the tumor inhibition effect (tumor weight) of the test substance are shown in Tables 3 and Figure 1 .

[0050] Table 3 Antitumor effects of the test substances on human liver cancer PDX tumor model

[0051]

[0052]

[0053] The doses of the tested drugs, low-dose and high-dose ursolic acid liposomes, low-dose and high-dose lenvatinib, and low-dose and high-dose regorafenib, were 15 mg / kg, 30 mg / kg, 10 mg / kg, 30 mg / kg, 5 mg / kg, and 10 mg / kg, respectively. The administration frequency was once a day, for a total of 21 times. At the end of the experiment (PG-D21), the ursolic acid liposomes (15 mg / kg) group, the ursolic acid liposomes (30 mg / kg) group, the lenvatinib (10 mg / kg) group, the regorafenib (5 mg / kg) group, the lenvatinib (30 mg / kg) group, the regorafenib (10 mg / kg) group, the ursolic acid liposomes + lenvatinib (15+10 mg / kg) group, the ursolic acid liposomes + lenvatinib (30+10 mg / kg) group, the ursolic acid liposomes + regorafenib (15+5 mg / kg) group, The tumor weights of the ursolic acid liposome + regorafenib (30 + 5 mg / kg) group were 0.513 ± 0.061 g, 0.750 ± 0.132 g, 0.533 ± 0.071 g, 0.275 ± 0.056 g, 0.261 ± 0.049 g, 0.377 ± 0.059 g, 0.252 ± 0.063 g, 0.231 ± 0.039 g, 0.319 ± 0.081 g and 0.269 ± 0.057 g, respectively. The tumor weight inhibition rate (TGI TW ) were 29%, -4%, 26%, 62%, 64%, 48%, 65%, 68%, 56% and 63%, respectively. Compared with the control vehicle group (0.720 ± 0.112 g), the tumor weights of the regorafenib (5 mg / kg) group, regorafenib (10 mg / kg) group, lenvatinib (30 mg / kg) group, ursolic acid liposome + lenvatinib (15 + 10 mg / kg) group, ursolic acid liposome + lenvatinib (30 + 10 mg / kg) group, ursolic acid liposome + regorafenib (15 + 5 mg / kg) group and ursolic acid liposome + regorafenib (30 + 5 mg / kg) group were significantly reduced (p < 0.05).

[0054] At the end of the experiment (PG-D21), the TGI of the ursolic acid liposome (15 mg / kg) group, the ursolic acid liposome (30 mg / kg) group, the lenvatinib (10 mg / kg) group, the ursolic acid liposome + lenvatinib (15 + 10 mg / kg) group, and the ursolic acid liposome + lenvatinib (30 + 10 mg / kg) group TWThe tumor weights of the ursolic acid liposome + lenvatinib (15+10mg / kg) group and the ursolic acid liposome + lenvatinib (30+10mg / kg) group were significantly lower than those of the corresponding monotherapy groups (p<0.05). The combination therapy of ursolic acid liposome + lenvatinib showed a tumor inhibition advantage that was significantly superior to ursolic acid liposome and lenvatinib monotherapy.

[0055] At the end of the experiment (PG-D21), the TGI of the ursolic acid liposome (15 mg / kg) group, the ursolic acid liposome (30 mg / kg) group, the regorafenib (5 mg / kg) group, the ursolic acid liposome + regorafenib (15 + 5 mg / kg) group, and the ursolic acid liposome + regorafenib (30 + 5 mg / kg) group TW They were 29%, -4%, 62%, 56% and 63% respectively. There was no significant difference in tumor recombination among the ursolic acid liposome + regorafenib (15+5mg / kg) group, the ursolic acid liposome + regorafenib (30+5mg / kg) group and the regorafenib (5mg / kg) group (p>0.05), but they were significantly lower than the ursolic acid liposome (15mg / kg) group and the ursolic acid liposome (30mg / kg) group (p<0.05). The combination therapy of ursolic acid liposome + regorafenib produced comparable tumor inhibition effect as regorafenib monotherapy.

[0056] During the treatment period, tumor-bearing mice showed good tolerance to the test substances ursolic acid liposomes (15mg / kg and 30mg / kg), lenvatinib (10mg / kg and 30mg / kg), and regorafenib (5mg / kg and 10mg / kg). The weight of the mice was relatively stable, and there was no significant difference between the treatment groups (p>0.05). They ate and drank water normally, were in good general condition, had no obvious abnormalities, and did not stop the drug or die. Gross autopsy of the mice at the time of euthanasia showed no clear ascites.

[0057] Example 3 Combination of ursolic acid lipid and sorafenib

[0058] Human hepatoma cell PLC / PRF / 5 was orthotopically inoculated into the liver parenchyma of male Balb / cNude mice, with a total of 65 mice inoculated. On the third day after tumor cell inoculation, the mice were divided into groups for drug administration, with a total of 7 groups, 8 animals in each group, including the vehicle control group, the high-dose group of ursolic acid liposomes (2.5 mg / kg), the medium-dose group (5.0 mg / kg), the low-dose group (10.0 mg / kg), the sorafenib group (30 mg / kg), the medium-dose group of ursolic acid liposomes and sorafenib combined application group, and the high-dose group of ursolic acid liposomes and sorafenib combined application group. The mice were weighed twice a week, and the relationship between the changes in the weight of the mice and the time of administration was recorded. At the end of the experiment, the mice were euthanized, the livers of the tumor-bearing mice were weighed, the in situ tumors were removed and weighed, and the tumors removed from the control group and the test group were neatly placed for photography. The tumor weight ratio (T / C) and tumor growth inhibition rate (1-T / C) of the treatment group and the solvent control group were calculated and statistically analyzed. The test results are shown in Table 4 and Figure 2 .

[0059] At the end of the experiment, the weight of the liver in situ tumor was weighed and the tumor was photographed. The tumor weight of each treatment group was significantly reduced compared with the vehicle control group (p<0.05). The tumor inhibition rates of the 2.5mg / kg, 5.0mg / kg and 10.0mg / kg groups of ursolic acid liposomes, the 30.0mg / kg group of sorafenib, the 5.0mg / kg group of ursolic acid liposomes + 30.0mg / kg group of sorafenib, and the 10.0mg / kg group of ursolic acid liposomes + 30.0mg / kg group of sorafenib were 20%, 31%, 69%, 82%, 74% and 90%, respectively. The anti-tumor effect of the 10.0mg / kg group of ursolic acid liposomes was significantly better than that of the medium and low dose groups (p<0.05), that is, the anti-tumor effect of ursolic acid liposomes in the range of 2.5mg / kg to 10.0mg / kg showed a dose-response relationship. The combined effect of ursolic acid liposome 5.0 mg / kg + sorafenib 30.0 mg / kg group was not much different from that of sorafenib 30.0 mg / kg alone group. Ursolic acid liposome 10.0 mg / kg + sorafenib 30.0 mg / kg had better synergistic effect than ursolic acid liposome 5.0 mg / kg + sorafenib 30.0 mg / kg group or sorafenib 30.0 mg / kg group.

[0060] During the treatment period, the tumor-bearing mice showed good tolerance to ursolic acid liposomes and sorafenib. The weight of mice in each group was normal, with no abnormal symptoms and in good general condition. Starting from the 14th day of administration (PD-D13), mice in each group became ill and their weight gradually decreased, but the weight curve was relatively stable. No obvious abnormalities were observed in the mice, and no drug discontinuation or death occurred.

[0061] Table 4 Antitumor effect of the test substances on mouse liver cancer PLC / PRF / 5

[0062]

Claims

1. A drug combination, characterized in that comprising a first active drug and a second active drug, The first active drug is ursolic acid or a pharmaceutically acceptable salt thereof; The second active drug is a multi-target tyrosine kinase receptor inhibitor, preferably lenvatinib or a pharmaceutically acceptable salt thereof, regorafenib or a pharmaceutically acceptable salt thereof, sorafenib or a pharmaceutically acceptable salt thereof, or a combination thereof.

2. The pharmaceutical combination according to claim 1, wherein The weight ratio of the first active drug to the second active drug is 2.5:50 to 50:2.5; preferably 2.5:30 to 30:

5.

3. The pharmaceutical combination according to claim 1 or 2, characterized in that The first active drug is a preparation comprising ursolic acid or a pharmaceutically acceptable salt thereof, phospholipids, and a pH regulator; The second active drug is an oral preparation, preferably a tablet or capsule.

4. The pharmaceutical combination according to any one of claims 1 to 3, wherein The weight ratio of ursolic acid or a pharmaceutically acceptable salt thereof to lenvatinib or a pharmaceutically acceptable salt thereof in the form of free base is 5:10-30:10, preferably 5:10, 10:10, 15:10, 30:10; more preferably, 5:10, 10:10, 15:10; or The weight ratio of ursolic acid or a pharmaceutically acceptable salt thereof to regorafenib or a pharmaceutically acceptable salt thereof in the form of free base is 5:5-30:5, preferably 5:5, 10:5, 15:5, 20:5, 25:5, 30:5; or In free base form, the weight ratio of ursolic acid or a pharmaceutically acceptable salt thereof to sorafenib or a pharmaceutically acceptable salt thereof is 2.5:30-30:30, preferably 2.5:30, 5:30, 7.5:30, 10:30, 15:

30.

5. The pharmaceutical combination of claim 4, wherein the first active drug is lipid microsphere or liposome injection.

6. The pharmaceutical combination according to any one of claims 3 to 5, wherein The phospholipid is selected from lecithin, soybean lecithin, hydrogenated soybean lecithin, phosphatidylethanolamine, synthetic phosphatidylserine, phosphatidylinositol, sphingomyelin, egg phosphatidylcholine, dicetyl phospholipids, dimyristoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, polyethylene glycol distearoyl phosphatidylethanolamine, methoxy polyethylene glycol distearoyl phosphatidylethanolamine, or a mixture of two or more of the above.

7. The drug combination according to any one of claims 3 to 6, wherein the first active drug is a lyophilized liposome injection, and the lyophilized liposome injection further comprises a lyophilized scaffold agent, and the lyophilized scaffold agent is selected from mannitol, sucrose, lactose or a combination thereof.

8. The pharmaceutical combination according to any one of claims 3 to 7, wherein the pH regulator in the first active drug is a buffer solvent, and the buffer controls the pH value of the aqueous phase within the range of 6-7 during the preparation process.

9. The pharmaceutical combination of claim 7, wherein the weight ratio of ursolic acid or a pharmaceutically acceptable salt thereof to phospholipid in the freeze-dried liposome injection of the first active drug is 0.1-10:5-500.

10. A kit comprising the first active drug and the second active drug of any one of claims 1 to 9.

11. The kit of claim 10, further comprising instructions for administering the first active drug and the second active drug to a human patient.

12. Use of the drug combination according to any one of claims 1 to 9 in the preparation of a drug for treating cancer, or use of the drug combination according to any one of claims 1 to 9 in treating cancer.

13. The use according to claim 12, wherein the cancer is liver cancer; the liver cancer is selected from primary liver cancer or secondary liver cancer.

14. The use according to claim 11 or 12, wherein the first active drug and the second active drug are administered sequentially or simultaneously.

15. The use according to claims 12-14, wherein the cancer is a cancer resistant to multi-target tyrosine kinase receptor inhibitors.

16. The use of claim 15, wherein the multi-target tyrosine kinase receptor comprises any two or more of vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) or platelet-derived growth factor receptor (PDGFR).

17. The use according to claim 15 or 16, wherein the cancer resistant to multi-target tyrosine kinase receptor inhibitors is selected from cancer resistant to lenvatinib, regorafenib or sorafenib, and preferably the resistance is primary resistance or acquired resistance to liver cancer.

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