Composition of capecitabine and pentacyclic triterpenoid and application of composition in preparation of antitumor drugs

Abstract

The invention discloses a pharmaceutical composition of a pentacyclic triterpenoid and capecitabine and application of the pharmaceutical composition, and belongs to the technical field of biological medicines. By reducing the hydrolysis of capecitabine in gastrointestinal tracts and livers, the pharmaceutical composition not only slows down the early release of capecitabine drug bodies in gastrointestinal tracts and livers, increases the total bioavailability of the drug bodies and 5-fluorouracil, reduces the non-uniform high distribution of capecitabine in normal tissues such as gastrointestinal tissues, but also improves the bioavailability of capecitabine in gastrointestinal tracts and livers. Reducing toxicity in these tissues; meanwhile, the effectiveness of the medicine is improved by relatively increasing the medicine distribution of the targeted tumor part. The combination will be suitable for all tumor treatment, especially solid tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a composition of capecitabine and a pentacyclic triterpenoid compound and its application in the preparation of antitumor drugs. Background Technology

[0002] Capecitabine (CAP) is an oral antitumor prodrug that requires multiple enzymatic reactions, including carboxylesterase, to be converted into its active metabolite, 5-fluorouracil (5'-FU), to exert its antitumor effect. Its advantage lies in the fact that the final activation step depends on thymidine phosphorylase, which is highly expressed in tumor tissue, giving it a certain degree of tumor targeting. However, premature hydrolysis of CAP in the gastrointestinal tract and liver leads to the accumulation of 5'-FU in normal tissues, causing significant adverse reactions such as diarrhea, hand-foot syndrome, and bone marrow suppression, affecting patient tolerability and treatment continuity.

[0003] Clinically, symptomatic and supportive treatments are often used to alleviate the above-mentioned side effects, such as using vitamin B6 to prevent hand-foot syndrome and using antiemetics and antidiarrheals to control gastrointestinal reactions. However, these methods can only relieve symptoms and cannot fundamentally inhibit the toxic metabolic pathways of CAP. In severe cases, it is still necessary to reduce the dosage or discontinue the medication.

[0004] To improve efficacy, CAP is often used in combination with drugs such as oxaliplatin and bevacizumab. While this can increase the objective response rate, it also increases the cumulative toxicity, leading to a higher incidence of adverse reactions and greater treatment risks. Therefore, there is an urgent need for a combination therapy strategy that can regulate CAP metabolism at the pharmacokinetic level to achieve "reduced toxicity and enhanced efficacy". Summary of the Invention

[0005] The present invention aims to provide a pharmaceutical composition comprising a pentacyclic triterpenoid compound and capecitabine, which reduces the toxicity of CAP in normal tissues by regulating its in vivo metabolism, while enhancing its antitumor effect.

[0006] In a first aspect, to achieve the above objectives, the present invention adopts the following technical solution: a pharmaceutical composition comprising capecitabine and pentacyclic triterpenoid compounds.

[0007] Preferably, as an improvement, the pentacyclic triterpenoid compound is selected from at least one of oleanolic acid, ursolic acid, crataegolic acid, or asiatic acid.

[0008] Preferably, as an improvement, the mass ratio of capecitabine to pentacyclic triterpenoid is 1:0.1 to 1:10.

[0009] Preferably, as an improvement, the mass ratio of capecitabine to the pentacyclic triterpenoid is 2:1. This ratio range can significantly reduce toxicity while ensuring antitumor efficacy.

[0010] Preferably, as an improvement, the dosage form of the pharmaceutical composition is an oral formulation, including tablets, capsules, granules, or sustained-release formulations, containing pharmaceutically acceptable excipients.

[0011] Preferably, as an improvement, the pentacyclic triterpenoid compound is an immediate-release formulation, and capecitabine is a delayed-release formulation.

[0012] Preferably, as an improvement, the release delay of capecitabine is 3 hours after administration. Administering capecitabine (CAP) 3 hours after administration of the pentacyclic triterpenoid ensures that it exerts its inhibitory effect on metabolic enzymes before CAP absorption, achieving a synergistic pharmacokinetic effect.

[0013] Secondly, the present invention also provides the use of the pharmaceutical composition described above in the preparation of a medicament for treating colorectal cancer, breast cancer, gastric cancer, or other CAP-sensitive solid tumors.

[0014] Preferably, as an improvement, the drug is used to reduce the gastrointestinal toxicity caused by capecitabine and enhance its antitumor effect.

[0015] Thirdly, the present invention also provides a compound preparation comprising the pharmaceutical composition and packaged as a single dose unit.

[0016] Beneficial technical effects of the present invention: 1. Reduced toxicity: Pentacyclic triterpenoids inhibit the activity of carboxylesterases in the gastrointestinal tract and liver, thus delaying the early hydrolysis of CAP in the gastrointestinal tract and reducing its conversion to 5'-FU in non-target tissues. This reduces toxic side effects such as gastrointestinal mucosal damage, diarrhea, nausea and vomiting, and significantly reduces adverse gastrointestinal reactions.

[0017] 2. Enhanced anti-tumor effects: Improved bioavailability of CAP and the proportion of the original drug in systemic circulation, increased drug distribution and sustained release in tumor tissues, and enhanced anti-cancer activity.

[0018] 3. Natural ingredients and high safety: Pentacyclic triterpenoids are derived from plants, and a large number of safety studies have supported their clinical application.

[0019] 4. Facilitates formulation development: Pentacyclic triterpenoids can be combined with CAP to form fixed-dose compound tablets, capsules, sustained-release formulations and other oral preparations, improving the convenience of medication. Attached Figure Description

[0020] Figure 1 The inhibitory effects of oleanolic acid (A), crataegolic acid (B), ursolic acid (C), and asiatic acid (D) on CAP metabolism in Caco-2 cells; Figure 2The inhibitory effects of oleanolic acid (A), crataegolic acid (B), ursolic acid (C), and asiatic acid (D) on CAP metabolism in human intestinal microsomes; Figure 3 The IC50 values ​​of arugula acid (A), oleanolic acid (B), ursolic acid (C), and asiatic acid (D) on carboxylesterase inhibition 50 The determination; Figure 4 Concentrations of CAP (A) and 5'-FU (B) in various tissues of rats; Figure 5 Results of tumor inhibition in nude mice by the combination of oleanolic acid, ursolic acid, crataegolic acid and asiatic acid in CAP. Detailed Implementation

[0021] The following detailed description illustrates the specific implementation methods: The structural formulas of oleanolic acid (CAS: 508-02-1), ursolic acid (CAS: 77-52-1), crataegolic acid (CAS: 4373-41-5), asiatic acid (CAS: 464-92-6), and capecitabine (CAS: 464-92-6) in the following examples are shown in formulas (1), (2), (3), and (4), respectively: , , , , .

[0022] Example 1: Inhibitory effects of crataegolic acid, oleanolic acid, ursolic acid, and asiatic acid on CAP metabolism Caco-2 cells were fed at a rate of 2 × 10 5 Cells were seeded at a density of cells / ml in 6-well plates. One group was pretreated with 20 μM arugula acid, oleanolic acid, ursolic acid and asiatic acid for 1 hour, respectively, while the control group was not treated. Cells were then treated with 5, 10, 25 and 50 μM CAP, respectively. After culturing in a CO2 incubator for 24 hours, the cells were scraped off, sonicated and then an equal volume of ice-cold acetonitrile containing diphenhydramine (4 ng / mL) was added. The mixture was vortexed for 30 seconds and centrifuged at 18000g for 20 min. The supernatant was then analyzed by LC-MS / MS.

[0023] Add 10 μM each of hawthorn acid, oleanolic acid, ursolic acid and asiatic acid, CAP (500 μM) and human intestinal microsomes (0.2 mg / mL) to PBS (pH=7.4). Incubate the reaction solution at 37°C for 30 min, then add an equal volume of ice-cold acetonitrile to terminate the reaction. Vortex the reaction solution for 30 s, centrifuge at 18000 g for 20 min, and then take the supernatant for LC-MS / MS analysis.

[0024] In Caco-2 cell and intestinal microsomal metabolism experiments, the sum of the three metabolites of CAP was used to represent the inhibitory effect of four pentacyclic triterpenoids on CAP. Results are as follows: Figure 1 , Figure 2 As shown, ursolic acid and oleanolic acid can significantly inhibit CAP hydrolysis and reduce the production of total metabolites in Caco-2 cells and human intestinal microsomes.

[0025] Example 2: IC50 of the inhibitory effects of hawthorn acid, oleanolic acid, ursolic acid, and asiatic acid on carboxylesterase 50 Measurement Stock solutions of hawthorn acid, oleanolic acid, ursolic acid, and asiatic acid were prepared in a series of concentration gradients (2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0 mM). The stock solutions of hawthorn acid, oleanolic acid, ursolic acid, and asiatic acid, PBS (pH=7.4), CAP (500 μM), and HLMs (0.2 mg / mL) were added sequentially to the incubation system. After incubating the reaction solution at 37°C for 30 min, an equal volume of ice-cold acetonitrile containing diphenhydramine (4 ng / mL) was added to terminate the reaction. The reaction solution was vortexed for 30 s, centrifuged at 18000 g for 20 min, and the supernatant was analyzed by LC-MS / MS.

[0026] The residual activity of carboxylesterase at each inhibitor concentration was calculated according to formula (1), and the results are as follows: Figure 3 As shown, the horizontal axis represents the logarithm of CAP concentration, and the vertical axis represents residual activity. Crataegus acid, oleanolic acid, ursolic acid, and asiatic acid have strong inhibitory effects on CAP metabolism, IC50... 50 The concentrations were 0.33 μM, 0.25 μM, 0.06 μM, and 0.23 μM, respectively.

[0027] (1) Example 3: Effects of oleanolic acid on CAP hydrolysis in rats 1. Effects of oleanolic acid on pharmacokinetic parameters of CAP Twelve SPF-grade male rats, weighing 200±20g, were randomly divided into two groups of six each. The control group received CAP at 200mg / kg via gavage, while the experimental group received CAP at 200mg / kg via gavage three hours after administration of oleanolic acid at 100mg / kg. Blood samples of 0.2ml were collected from the orbital sinus of each rat at 0, 5, 10, 20, 30, 45 min, 1, 2, 3, 4, 6, 8, and 12 h. The blood samples were placed in heparin sodium anticoagulant tubes, centrifuged at 3000rpm and 4℃ for 20 min, and the supernatant plasma was separated and analyzed by LC-MS / MS.

[0028] The results are shown in Tables 1 and 2. Compared with the single-dose CAP group, the AUC of the original CAP drug in the plasma of rats in the combination therapy group was significantly higher. （0-12） Increased by 2.7 times; AUC of metabolite 5'-FU （0-12） The concentration increased by 2.3 times, and the half-life of 5'-FU in the combined administration group was prolonged by 1.2 times, with the plasma concentration remaining at a high level for a longer period; indicating that oleanolic acid inhibited the premature release of CAP in the intestine. Based on these pharmacokinetic results, the AUC of 5'-FU after combined administration of oleanolic acid and CAP was... （0-12） The dosage was increased from 37.69 to 86.22 μg / L*h, meaning that the therapeutic effect could still be achieved even with a reduced dosage of CAP, further broadening the therapeutic window of CAP.

[0029] Table 1 Pharmacokinetic parameters of CAP

[0030] Table 2 Pharmacokinetic parameters after combined administration

[0031] 2. Tissue distribution of CAP after combined drug administration Twelve SPF-grade rats (200±20g) were randomly divided into two groups of six each. The control group was administered CAP 200mg / kg by gavage, while the experimental group was first administered oleanolic acid 100mg / kg by gavage, followed by CAP 200mg / kg by gavage three hours later. Two hours after CAP administration, heart, liver, spleen, lung, kidney, stomach, and small intestine tissues were collected from both groups. PBS was added to each tissue at a 1:4 ratio, and the tissues were homogenized. A small amount of homogenate was centrifuged at 12000g for 20min, and the supernatant was collected. Three times the volume of ice-cold acetonitrile was added to the supernatant to remove protein, and the mixture was centrifuged at 18000g for 20min. The supernatant was dried under a nitrogen stream, and finally reconstituted with 10% acetonitrile containing diphenhydramine (4ng / ml). After centrifugation at 18000g for 20min, the supernatant was analyzed by LC-MS / MS.

[0032] The results are as follows Figure 4As shown, in the CAP monotherapy group, the concentration of the original drug in the stomach and intestines was significantly higher than in other tissues. However, after combining with oleanolic acid, the concentration of CAP increased significantly not only in the gastrointestinal tract but also in organs including the heart, lungs, liver, spleen, and kidneys. Furthermore, 5'-FU, the active metabolite of CAP, showed a slightly decreased concentration in the intestines, while its levels increased in other organs. Additionally, we calculated the ratio of drug concentration in tissues to plasma to reflect the distribution of CAP and 5'-FU in specific tissues. If the drug ratios in different tissues are similar, the distribution can generally be considered relatively uniform. The results are shown in Table 3. Compared to the CAP monotherapy group, the concentrations of CAP and 5'-FU in all tissues in the combination therapy group were significantly higher than in plasma, and the concentration differences between different tissues were small, indicating a relatively uniform distribution.

[0033] Table 3. Ratios of drug concentrations in tissues to plasma in various organs of rats.

[0034] Example 4: The ameliorative effects of oleanolic acid, ursolic acid, crataegolic acid and asiatic acid on gastrointestinal toxicity induced by CAP. SD rats (200±20g) were randomly divided into four groups: CAP group, oleanolic acid combined treatment group, ursolic acid combined treatment group, crataegolic acid combined treatment group, and asiatic acid combined treatment group, with six rats in each group. The CAP group received 200 mg / kg via gavage; in the combined treatment groups, oleanolic acid, ursolic acid, crataegolic acid, and asiatic acid were administered at 100 mg / kg via gavage, followed by 200 mg / kg of CAP three hours later; administration was once daily for 14 consecutive days. Diarrhea was observed during the treatment period, and the dietary intake of rats in each group was recorded. Diarrhea scores were calculated using the Hart diarrhea scoring method (see Table 4).

[0035] Table 4 Hart's Diarrhea Scoring Criteria

[0036] The rats' defecation was observed daily, and a total score for the day was obtained. A total score of ≥12 points was considered diarrhea, and the higher the score, the more severe the diarrhea.

[0037] The results showed that from day 3 onwards, rats in the CAP group experienced varying degrees of decreased appetite, with some rats showing slow or slight weight gain. Their stools were predominantly liquid, with occasional pasty stools. Compared to the CAP group, the combined drug group showed significantly improved mental state, faster recovery of appetite, slower weight gain, and stools that were mostly semi-solid or soft. Diarrhea scores during continuous drug administration are shown in Table 5.

[0038] Table 5. Statistics of Diarrhea Scores

[0039] Therefore, it can be concluded that when the four pentacyclic triterpenoids are used in combination with CAP, they can significantly reduce the gastrointestinal toxicity caused by CAP, as evidenced by a decrease in the incidence of diarrhea, a reduction in the severity of diarrhea, and an improvement in the overall condition of rats.

[0040] Example 5: The inhibitory effect of oleanolic acid, ursolic acid, crataegolic acid and asiatic acid in combination with CAP on tumors. CT26 tumor cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics and placed in a 37°C, 5% CO2 incubator to allow them to adhere and grow. When the tumor cells reached the logarithmic growth phase, they were digested with trypsin and counted. The skin on the back of nude mice was disinfected with alcohol swabs, and each mouse was subcutaneously injected with 1×10⁻⁶ mmol / L. 6 1 cell / 200μL PBS, until the tumor volume is approximately 100mm² 3 Mice were divided into 6 groups of 6 mice each. The solvent group was given CMC-Na by gavage, the CAP group was given CAP 200 mg / kg by gavage, and the combination drug group was given oleanolic acid, ursolic acid, crataegolic acid and asiatic acid 100 mg / kg respectively. Three hours later, CAP 200 mg / kg was given. The administration was repeated once a day for 14 days. Tumor volume changes were observed during the administration period.

[0041] The results are as follows Figure 5 As shown, compared with the blank solvent group, the tumor volume of both the CAP group and the combined drug group decreased significantly, and the tumor volume of the combined drug group decreased significantly compared with the CAP group, indicating that oleanolic acid, ursolic acid, crataegolic acid and asiatic acid combined with CAP can effectively inhibit tumor proliferation.

[0042] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises capecitabine and a pentacyclic triterpene compound.

2. The pharmaceutical composition of claim 1, wherein: The pentacyclic triterpene compound is at least one selected from the group consisting of oleanolic acid, ursolic acid, maslinic acid and asiatic acid.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that: The mass ratio of capecitabine to the pentacyclic triterpene compound is 1:0.1-1:

10.

4. The pharmaceutical composition of claim 3, wherein: The mass ratio of capecitabine to the pentacyclic triterpene compound is 2:

1.

5. The pharmaceutical composition of claim 1 or 2, wherein: The dosage form of the pharmaceutical composition is oral preparation.

6. The pharmaceutical composition of claim 5, wherein: The pentacyclic triterpene compound is immediate release preparation and capecitabine is delayed release preparation.

7. A pharmaceutical composition according to claim 6, wherein: The release delay time of capecitabine is 3 hours after administration.

8. Use of the pharmaceutical composition according to any one of claims 1, 2, 4, 6, 7 in the preparation of a medicament for treating colorectal cancer, breast cancer, gastric cancer or other solid tumors sensitive to CAP.

9. Use according to claim 8, characterized in that, The medicament is used for reducing gastrointestinal toxicity caused by capecitabine and enhancing its anti-tumor effect.

10. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition according to any one of claims 1, 2, 4, 6, 7 is contained and packaged as a single dosage unit.

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