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

Abstract

A pharmaceutical combination of ursolic acid and a multi-target tyrosine kinase receptor inhibitor, which have a synergistic effect in the treatment of cancer, especially in the treatment of liver cancer.

Description

Drug combination of ursolic acid and multi-target tyrosine kinase receptor inhibitor 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 efficacies in the treatment of liver cancer. Background Art

[0002] Primary hepatocellular carcinoma (HCC) is a common malignant tumor worldwide, ranking sixth in morbidity and third in mortality, respectively. China is a high-incidence region for HCC. In recent years, the incidence of HCC has shown a significant upward trend, posing a serious threat to human health and life. Currently, clinical treatment options for HCC are very limited. For HCC that cannot be surgically removed, targeted drugs are typically used to directly target the tumor in an attempt to prolong the lives of advanced patients, but these treatments are often ineffective.

[0003] Clinically, targeted therapies for liver cancer typically use small molecule compounds that inhibit multiple tyrosine kinase receptors, such as sorafenib, lenvatinib, and regorafenib. These drugs 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, these drugs have limited efficacy in treating liver cancer, and in clinical practice, patients experience poor responses to these 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 the main reason for its low bioavailability in vivo. Therefore, many studies have focused on improving its solubility and thus its bioavailability through formulations such as liposomes and solid dispersions. Due to the differences in the bioavailability of ursolic acid APIs, liposomes, solid dispersions, and other formulations, it is difficult to reasonably predict the efficacy of the combination of specific ursolic acid formulations with multi-target tyrosine kinase receptor inhibitors based on cellular test data of ursolic acid APIs.

[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, particularly 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 tosylate, specifically lenvatinib mesylate, regorafenib hydrochloride, or sorafenib tosylate.

[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, a phospholipid, and a pH adjuster; and 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, calculated as a 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, calculated as free base, is 5:5-30:5, preferably 5:5, 10:5, 15:5, 20:5, 25:5, 30:5; or

[0016] Calculated in the form of free base, 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, or 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 lecithin, distearoyl phosphatidylethanolamine, pegylated distearoyl phosphatidylethanolamine, methoxypegylated distearoyl phosphatidylethanolamine, or a mixture of two or more of the above.

[0019] In some embodiments, the first active drug is a lyophilized liposome injection, which further comprises a lyophilized scaffold agent, wherein 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 buffered 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 within the range of 0.1-10:10-50, 1-10:5-50, 2-5:10-50, 2-5:20-50, and 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, and 10:10.

[0023] In some embodiments, the weight ratio of ursolic acid or a pharmaceutically acceptable salt thereof 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 can 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, and 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 comprising a first active drug and a second active drug; wherein the first active drug and the second active drug can be separate compositions, or co-packaged compositions that are not in contact with each other. In some embodiments, the phrases "comprising" and "including" in the present invention can be replaced with "consisting of" or "consisting of."

[0026] In a second aspect, the present invention provides a kit comprising any of the aforementioned first active drug and second active drug. 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 agent and the second active agent to a human patient, indicating that the first active agent and the second active agent can be administered sequentially or simultaneously.

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

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

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

[0031] In some embodiments, the cancer is resistant to multi-target tyrosine kinase receptor inhibitors. In some embodiments, the multi-target tyrosine kinase receptors of the present invention include 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 a multi-target tyrosine kinase receptor inhibitor 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 the combination of ursolic acid liposomes and lenvatinib or regorafenib in a human HCC PDX tumor model.

[0034] Figure 2: The combination of ursolic acid liposomes and sorafenib inhibits tumor growth in the orthotopic inoculation tumor model of human hepatocellular carcinoma cells 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 understand 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 specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, 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 and stir to 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; add 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) to form an aqueous phase solution; 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 the filtrate is diluted to volume with injection water and then dispensed into a vial and pre-frozen at -50 to -40℃ for 10 hours, then subjected to 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 re-dissolved with physiological salt and measured by a laser particle size analyzer. The particle size D 50 100-300nm.

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

[0039] A PDX model was established in immunodeficient mice using surgically resected liver cancer tissue 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] The human hepatocellular carcinoma PDX tumor model was used for testing. When the subcutaneous tumor of the tumor-bearing mouse grew to 500-1000 mm 3 Afterwards, mice (species strain: Mus musculus, BALB / cnude) 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 anesthetizing the experimental animals, the tumor pieces were inoculated into the liver parenchyma using a trocar. One piece was inoculated per mouse. The day of inoculation was designated as D0. Four days after inoculation (D4), the animals were randomly divided into groups based on body weight for dosing (this day was designated as PG-D0). A total of 11 groups were divided, with 8 animals per group. Dosing was performed according to the schedule in Table 2.

[0043] Table 2. Dosage regimen

[0044] Note: *, the administration volume is 10 μL / g based on the animal's body weight; if 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.

[0045] The entire experiment was terminated on day 22 after group administration (i.e., 1 day after the last administration, PG-D21). The test results of the tumor inhibition effect (tumor weight) of the test substances are shown in Table 3 and Figure 1.

[0046] Table 3 Antitumor effects of the test substances on human liver cancer PDX tumor models

[0047] The doses of the test drugs ursolic acid liposomes low and high doses, lenvatinib low and high doses, and regorafenib low and high doses were 15 mg / kg, 30 mg / kg, 10 mg / kg, 30 mg / kg, 5 mg / kg, and 10 mg / kg, respectively. The frequency of administration was once a day, for a total of 21 doses. 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 / kg) group and ursolic acid liposome + regorafenib (30 + 5 mg / kg) group had tumor weights of 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±0.112g), the tumor weights of the regorafenib (5mg / kg) group, the regorafenib (10mg / kg) group, the lenvatinib (30mg / kg) group, the ursolic acid liposome + lenvatinib (15+10mg / kg) group, the ursolic acid liposome + lenvatinib (30+10mg / kg) group, the ursolic acid liposome + regorafenib (15+5mg / kg) group and the ursolic acid liposome + regorafenib (30+5mg / kg) group were significantly reduced (p<0.05).

[0048] At the end of the experiment (PG-D21), the TGI of the ursolic acid liposome (15 mg / kg), ursolic acid liposome (30 mg / kg), lenvatinib (10 mg / kg), ursolic acid liposome + lenvatinib (15 + 10 mg / kg) and ursolic acid liposome + lenvatinib (30 + 10 mg / kg) groups were TWThe tumor weights of the ursolic acid liposome + lenvatinib (15+10 mg / kg) group and the ursolic acid liposome + lenvatinib (30+10 mg / kg) group were significantly lower than those of the corresponding monotherapy groups (p<0.05). The combined treatment of ursolic acid liposome + lenvatinib showed a tumor-suppressing advantage that was significantly superior to that of ursolic acid liposome and lenvatinib monotherapy.

[0049] At the end of the experiment (PG-D21), the TGI of the ursolic acid liposome (15 mg / kg), ursolic acid liposome (30 mg / kg), regorafenib (5 mg / kg), ursolic acid liposome + regorafenib (15 + 5 mg / kg), and ursolic acid liposome + regorafenib (30 + 5 mg / kg) groups were TW The rates were 29%, -4%, 62%, 56% and 63% respectively. There was no significant difference in the tumor recombinant rates 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 those in 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 inhibitory effects as regorafenib monotherapy.

[0050] During treatment, tumor-bearing mice showed good tolerance to the test substances ursolic acid liposomes (15 mg / kg and 30 mg / kg), lenvatinib (10 mg / kg and 30 mg / kg), and regorafenib (5 mg / kg and 10 mg / kg). Their body weight remained relatively stable, with no significant differences between treatment groups (p>0.05). They consumed food and water normally and were generally in good condition, with no significant abnormalities, and no drug discontinuation or death occurred. Gross autopsy of the mice at euthanasia revealed no clear ascites.

[0051] Example 3 Combination of ursolic acid lipid and sorafenib

[0052] Human hepatocellular carcinoma (PLC / PRF / 5) cells were orthotopically inoculated into the liver parenchyma of male Balb / c nude mice. A total of 65 mice were inoculated. On the third day after tumor cell inoculation, seven groups of eight animals were divided and dosed: a vehicle control group, a high-dose ursolic acid liposome group (2.5 mg / kg), a medium-dose group (5.0 mg / kg), a low-dose group (10.0 mg / kg), a sorafenib group (30 mg / kg), a combination of a medium-dose ursolic acid liposome and sorafenib group, and a combination of a high-dose ursolic acid liposome and sorafenib group. Mice were weighed twice weekly, and changes in body weight were recorded in relation to dosing schedule. At the end of the experiment, mice were euthanized, and the livers of tumor-bearing mice were weighed. The orthotopic tumors were excised and weighed, and the excised tumors from the control and test groups were neatly arranged and photographed. The tumor weight ratio (T / C) and tumor growth inhibition rate (1-T / C) between the treatment and vehicle control groups were calculated and statistically analyzed. The test results are shown in Table 4 and Figure 2.

[0053] At the end of the experiment, the liver orthotopic tumors were weighed and photographed. Tumor weights in each treatment group were significantly reduced compared to the vehicle control group (p < 0.05). Tumor inhibition rates were 20%, 31%, 69%, 82%, 74%, and 90% in the 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg ursolic acid liposome groups, the 30.0 mg / kg sorafenib group, the 5.0 mg / kg ursolic acid liposome + 30.0 mg / kg sorafenib group, and the 10.0 mg / kg ursolic acid liposome + 30.0 mg / kg sorafenib group, respectively. The antitumor effect of the 10.0 mg / kg ursolic acid liposome group was significantly superior to that of the medium and low dose groups (p < 0.05), indicating that the antitumor effect of ursolic acid liposomes within the 2.5 mg / kg to 10.0 mg / kg range exhibited a dose-response relationship. The combined effect of ursolic acid liposomes 5.0 mg / kg and sorafenib 30.0 mg / kg was not significantly different from that of sorafenib 30.0 mg / kg alone. Ursolic acid liposomes 10.0 mg / kg and sorafenib 30.0 mg / kg had a more synergistic effect than ursolic acid liposomes 5.0 mg / kg and sorafenib 30.0 mg / kg or sorafenib 30.0 mg / kg.

[0054] During treatment, tumor-bearing mice showed good tolerance to both ursolic acid liposomes and sorafenib. All groups maintained normal weight, showed no abnormalities, and were generally in good condition. Starting on the 14th day of dosing (PD-D13), mice in all groups developed symptoms and gradually lost weight, but the weight curve remained relatively stable. No mice showed any significant abnormalities, and no discontinuation of treatment or death occurred.

[0055] Table 4 Antitumor effects of the test substances on mouse liver cancer PLC / PRF / 5

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 lecithin, 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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