CN105963304B - A kind of pharmaceutical composition and its application in preparation of anti-tumor drugs of melbine and ursolic acid and its derivative - Google Patents

Abstract

The present invention provides a pharmaceutical composition with synergistic anti-tumor metastasis or anti-tumor effect and its application in the preparation of anti-tumor drugs, the pharmaceutical composition is characterized in that it contains the compound shown in formula I and metformin I; wherein R in formula I is hydroxyl or acetoxy; wherein the compound shown in formula I is in a ratio of 10-20:500-20000 to metformin. The pharmaceutical composition, that is, the combined use of ursolic acid and its derivatives and metformin has a significant inhibitory effect on the proliferation of cancer cells.

Description

A kind of pharmaceutical composition of metformin, ursolic acid and its derivatives and its preparation Application in antineoplastic drugs

technical field

The invention relates to a composition of metformin, ursolic acid and its derivatives and its application in the preparation of antitumor drugs, belonging to the field of antitumor drug compositions.

Background technique

Cancer (malignant tumor) refers to a disease caused by a disorder in the mechanism regulating cell proliferation. Cancer is a serious threat to human health and is one of the most serious diseases in humans today. The risk of cancer is increasing year by year. In China, more than 1.6 million people are diagnosed with cancer and 1.2 million people die of cancer every year. Cancer has become one of the main chronic diseases of the health of Chinese residents, ranking first in the cause of death in cities and second in rural areas.

Drug combination refers to the simultaneous or sequential application of two or more drugs to achieve therapeutic purposes. Combination drugs often have in vivo or in vitro drug interactions. For certain drug combinations, combination therapy also allows for optimal combined dosages to minimize side effects; combination therapy of two compounds may reveal unexpected synergies and elicit effects not induced by a single compound.

Ursolic acid is 3β-hydroxy-ursolic-12-ene-28-acid (3β-hydroxy-urs-12-en-28-oic acid, referred to as UA), also known as ursolic acid, which belongs to a-amyresinol (a-amyrin) type pentacyclic triterpenes, the structure is shown in formula II, its relative molecular weight is 456.68, molecular formula is C ₃₀ H ₄₈ O ₃ , it is a natural active compound widely distributed in nature, mainly in the form of free or It exists in the form of glycosides, which are widely distributed in many natural plants such as loquat leaves, bearberries, hawthorns, and Hedyotis diffusa. It is also one of the main active ingredients of many traditional Chinese medicines, and has a wide range of pharmacological effects, such as anti-cancer, liver protection , anti-inflammation, anti-virus, anti-oxidation, etc. Among them, the anti-cancer activity is the most significant. It not only has resistance to various carcinogens, but also has inhibitory effects on various tumor cells both in vivo and in vitro. Because of its small side effects and low toxicity, it shows great potential for clinical application. In recent years, anti-tumor research on UA has been deepened at home and abroad, and it has been found that it has unique advantages and potential application prospects in tumor prevention, treatment, and prevention of late recurrence and metastasis. Ursolic acid and its derivatives have a broad anti-cancer spectrum. Patent N201510466210.4 disclosed for the first time that the ursolic acid derivative ASP-UA has a significant inhibitory effect on the adhesion, migration, and invasion of breast cancer cells. Patent N201410189256.1 disclosed for the first time that ursolic acid derivative US597 can effectively reduce the experimental lung metastasis of B16-F10 melanoma in mice, but currently anti-ursolic acid and its derivatives are combined with metformin for anti-cancer metastasis Not yet reported.

UA1, whose structure is shown in formula Ⅲ, this compound can significantly improve the solubility of UA, and its physical and chemical properties are more stable; in addition, compared with UA, it has a more significant inhibitory effect on the proliferation of different types of tumor cells, and has lower toxicity to normal cells , suggesting that it has the characteristics of safety, high efficiency and low toxicity, and it is expected to be applied in the early prevention and treatment of tumors.

Metformin (Met for short), with a relative molecular weight of 129.10 and a molecular formula of C ₄ H ₁₁ N ₅ , is considered to be one of insulin sensitizers, and the result is shown in formula IV. As a classic and effective drug for the treatment of type 2 diabetes, metformin has a history of clinical application for nearly 50 years. Metformin is a strong water-soluble drug, which is mainly absorbed in the small intestine after oral administration, with a bioavailability of about 50-60%. At present, metformin is recommended as the first choice drug in the diabetes treatment guidelines of various countries, and has no contraindications. Its mechanism of action is different from other types of oral anti-glycemic drugs. It can reduce the production of glycogen and reduce the absorption of sugar in the small intestine. And it can also increase the uptake and utilization of peripheral sugar, thereby improving the sensitivity of insulin. Its prestige in diabetes treatment is also increasing day by day, and it has become the core drug for the global fight against diabetes. Patent N201310069025.2 first discloses a new use of metformin, which proves that metformin can inhibit the proliferation of endometrial cancer cells. Patent N201080048060.0 also discloses the use of metformin in cancer treatment and prevention.

In the present invention, SCL-1, MCF-7, HepG2 and A549 cells are used as research objects, and ursolic acid and its derivatives are used in combination with metformin to anti-cancer in vitro on SCL-1, MCF-7, HepG2 and A549 cells. Activity test, the results show that the combination of ursolic acid and its derivatives and metformin has a significant inhibitory effect on the proliferation of cancer cells.

Contents of the invention

The purpose of the present invention is to provide a composition of metformin, ursolic acid and its derivatives and its application in the preparation of antitumor drugs, by combining metformin with strong water solubility and antitumor natural product bearberry with high efficiency and low toxicity It is expected to obtain relatively safe and reliable new candidate drugs for the prevention and treatment of cancer by conducting combined medication with acid and its derivatives, and investigating their synergistic effects.

A pharmaceutical composition with anti-tumor effect and its application in the preparation of anti-tumor drugs, characterized in that it comprises a compound as shown in formula I and metformin

I;

Wherein R in formula I is hydroxyl or acetoxy;

Wherein the substance ratio of the compound represented by formula I to metformin is 10-20:500-20000.

Description of drawings

Figure 1. The results of inhibition of SCL-1 cell proliferation after ursolic acid and metformin were used alone or in combination for 24 hours;

Figure 2. The results of inhibition of SCL-1 cell proliferation after ursolic acid and metformin were used alone or in combination for 48 hours;

Figure 3. Ursolic acid and metformin used alone and in combination for 72h inhibited the proliferation of SCL-1 cells;

Figure 4. Ursolic acid and metformin alone and in combination 24h inhibit the proliferation of MCF-7 cells;

Figure 5. Ursolic acid and metformin used alone and in combination for 48h inhibited the proliferation of MCF-7 cells;

Figure 6. Ursolic acid and metformin alone and in combination after 72h inhibit the proliferation of MCF-7 cells;

Fig. 7. ursolic acid and metformin are used alone and combined with the results of inhibition of HepG2 cell proliferation after 24h;

Figure 8. ursolic acid and metformin used alone and in combination for 48h inhibited the proliferation of HepG2 cells;

Figure 9. Ursolic acid and metformin alone and in combination with the results of inhibition of HepG2 cell proliferation after 72h;

Figure 10. Ursolic acid and metformin are used alone and combined with the results of inhibition of A549 cell proliferation after 24h;

Figure 11. Ursolic acid and metformin alone and in combination with the results of inhibition of A549 cell proliferation after 48h;

Figure 12. Ursolic acid and metformin alone and in combination after 72h inhibit the proliferation of A549 cells;

Figure 13. The results of inhibition of SCL-1 cell proliferation after UA1 and Met were used alone or in combination for 24 hours;

Figure 14. The results of inhibition of MCF-7 cell proliferation after UA1 and Met used alone or in combination for 24 hours;

Figure 15. The results of inhibition of HepG2 cell proliferation after UA1 and Met used alone or in combination for 24 hours;

Figure 16. The results of inhibition of A549 cell proliferation after UA1 and Met used alone or in combination for 24 hours.

Detailed ways

In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

Example 1

Synthesis of UA1

①In a 250 mL round bottom flask, add 20 mL of dichloromethane, 20 mL of pyridine, 1 g of ursolic acid, and 3.5 mL of acetic anhydride. Add a small amount of DMAP, magnetically stir overnight at room temperature;

② Add 100 ml of dichloromethane to the bottle ①, extract the organic layer obtained in ② twice with 100 mL 1 N HCl solution respectively, and collect the organic layer;

③ Same as step ②, extract the organic layer obtained in ② with saturated NaHCO ₃ solution twice, each time 100mL, and finally separate the organic layer;

④ Add anhydrous sodium sulfate to the organic layer obtained in ③, fully stir and dry (about 4 scoops), and filter;

⑤ Rotary evaporator evaporates the solvent to obtain the crude product;

⑥ Purified by column chromatography to obtain pure white UA1;

Example 2

Inhibitory effect of ursolic acid and metformin alone and in combination on SCL-1 cell proliferation: The proliferation inhibitory activity of ursolic acid and metformin in different combination ratios on SCL-1 cell lines was measured by standard MTT colorimetry.

The specific steps are: Digest the SCL-1 cells in the logarithmic phase with trypsin, count the cells, and prepare the cell concentration of 8×10 ⁴ /ml, and inoculate 100 μl of the prepared cell suspension into 96 Well plates were placed in a 37°C, 5% CO ₂ incubator for 24 h, the old medium was discarded, and the prepared medium containing different drug concentrations was added to continue culturing for 24, 48, and 72 h, and the containing Add 100 μL of diluted MTT solution (0.5 mg/ml MTT stock solution:serum-free phenol red-free medium = 1:9) to each well, continue incubation for 4 h, then terminate the culture; carefully aspirate and discard Add 100 μL DSMO to each well of the supernatant in a 96-well plate, shake for 10 min, measure the light absorption value (OD value) of each well on a microplate reader at a wavelength of 570 nm, and calculate the cell survival rate (%) = (medication The average OD value of the group/the average OD value of the blank control group) × 100%. GraphPad Prism software was used for data processing, and the results are shown in Figure 1-3 below.

As shown in Figure 1, after 24 hours of drug action, 10 μM ursolic acid has no obvious effect on inhibiting the proliferation of SCL-1 cells; the cell survival rate of metformin is about 80% under the condition of 20 mM; At 24 h, when Met≥5 mM, it can obviously inhibit the proliferation of SCL-1 cells, and at this time it can play a synergistic anti-tumor effect; as shown in Figure 2, after 48 h of drug action, ursolic acid Under the condition of 10 μM, it can significantly inhibit the proliferation of SCL-1 cells; Metformin can inhibit the proliferation of SCL-1 cells when it is ≥20 mM; when the two are combined for 48 hours, when Met≥1 mM It can significantly inhibit the proliferation of cells; as shown in Figure 3, after 72 h of drug action, ursolic acid can kill half of the cells under the condition of 10 μM; metformin can’t be obviously Metformin can significantly inhibit the growth of SCL-1 cells under the condition of 0.5 mM after the combined administration of the two for 72 hours, and with the increase of the concentration, the inhibition of cell proliferation is more obvious. In summary, it can be found that the combined administration of the two can significantly reduce the dosage of Met, and can synergistically inhibit the proliferation of SCL-1 cells in a concentration- and time-dependent manner.

Example 3

King’s modified formula was used to calculate the synergistic effect of the combined administration of ursolic acid and metformin on SCL-1 cells at different ratios for 24, 48, and 72 h, and the optimal drug ratio was screened out. The results are shown in Table 1. -3 shown. The inhibitory rate of SCL-1 cells was calculated as EA under the condition of UA=10 μM, and the inhibitory rates of different metformin concentrations on SCL-1 cells were calculated as EB1, EB2, EB3, etc. The inhibition rates of SCL-1 cells under different ratios are EC1, EC2, EC3, etc., and the combined drug index q value is calculated by the following formula, q=EC/(EB+EA-EB*EA), when the q value>1.15 It is a synergistic effect, 0.85<q<1.15 is an additive effect, and q<0.85 is an antagonistic effect. The final drug efficacy of the combination of the two drugs was further judged by the calculation of the combination drug index.

As shown in Table 1-3, the combined administration of ursolic acid and metformin acted on SCL-1 cells for 24, 48, and 72 hours at different ratios. It is an additive effect; when metformin is used at a high concentration, the combined effect of the two drugs is basically a synergistic effect, which can synergistically inhibit the proliferation of SCL-1 cells, and the greater the drug concentration, the more obvious the inhibition.

Example 4

The inhibitory effect of ursolic acid and metformin on the proliferation of MCF-7 cells when used alone or in combination is as in Example 2, and the results are shown in Figures 4-6.

As shown in Figure 4, after 24 h of drug action, ursolic acid has no obvious effect on inhibiting the proliferation of MCF-7 cells under the condition of 10 μM; only under the condition of metformin ≥ 20 mM can it effectively inhibit MCF-7 cell proliferation; the combined administration of the two can significantly inhibit the proliferation of MCF-7 cells when Met≥5 mM for 24 hours, and can play a synergistic anti-tumor effect at this time; as shown in Figure 5, when After 48 hours of drug action, ursolic acid can significantly inhibit the proliferation of cells under the condition of 10 μM; Metformin alone can inhibit the proliferation of MCF-7 cells under the condition of ≥10 mM, when ursolic acid and metformin combined Metformin ≥ 1 mM can effectively inhibit the proliferation of cells; as shown in Figure 6, after 72 hours of drug action, ursolic acid in the condition of 10 μM SCL-1 cell death reached half; both After 72 h of combined administration, the metformin concentration ≥ 0.5 mM can significantly inhibit cell proliferation. In summary, it can be found that the combined administration of the two can significantly reduce the dosage of Met, and the combination of the two can obviously play a coordinated anti-tumor effect. With the increase of the concentration of Met, the proliferation of MCF-7 cells can be inhibited. more obvious.

Example 5

Adopt Kim's revised formula to calculate the synergistic effect of 24, 48, 72 h on MCF-7 cells under different ratios of combined administration of ursolic acid and metformin, the method is the same as in Example 3, and the results are shown in Table 4-6. Show.

As shown in Table 5-6, the combined administration of ursolic acid and metformin acted on MCF-7 cells for 24, 48, and 72 h at different ratios. It is an additive effect; when metformin is used at a high concentration, the combined effect of the two drugs is basically a synergistic effect, which can synergistically inhibit the proliferation of MCF-7 cells, and the greater the drug concentration, the more obvious the inhibition.

Example 6

The inhibitory effect of ursolic acid and metformin on the proliferation of HepG2 cells when used alone or in combination is as in Example 2, and the results are shown in Figures 7-9.

As shown in Figure 7, after 24 h of drug action, the effect of ursolic acid on inhibiting the proliferation of HepG2 cells was not obvious under the condition of 10 μM; the proliferation of HepG2 cells could be effectively inhibited under the condition of metformin ≥ 20 mM; The combined administration of the two drugs can significantly inhibit the proliferation of HepG2 cells when Met ≥ 5 mM for 24 hours, and can play a synergistic anti-tumor effect at this time; as shown in Figure 8, after 48 hours of drug action, Ursolic acid can significantly inhibit the proliferation of cells under the condition of 10 μM; the combination of ursolic acid and metformin, when metformin is ≥1mM, inhibits the proliferation of HepG2 cells more significantly than metformin alone; as shown in Figure 9 It was shown that after 72 h of drug action, ursolic acid at 10 μM killed and injured HepG2 cells by half; after 72 h of combined administration of the two, the concentration of metformin ≥ 0.5 mM could significantly inhibit cell proliferation. In summary, it can be found that the combined administration of the two can significantly reduce the dosage of Met, and the combined administration of the two can obviously play a coordinated anti-tumor effect. With the increase of the Met concentration, the inhibition of the proliferation of HepG2 cells is more obvious .

Example 7

King's correction formula was used to calculate the synergistic effect of ursolic acid and metformin combined administration on HepG2 cells in different ratios for 24, 48, and 72 h. The method was the same as in Example 3, and the results were shown in Tables 7-9.

As shown in Table 7-9, the combined administration of ursolic acid and metformin at different ratios for 24, 48, and 72 hours on HepG2 cells is basically additive when metformin is used at low concentrations. Effect; when metformin is used at high concentrations, the combined effect of the two medicines is basically a synergistic effect, which can synergistically inhibit the proliferation of HepG2 cells. The greater the drug concentration, the more obvious the inhibition.

Example 8

Inhibitory effect of ursolic acid and metformin alone and in combination on A549 cell proliferation: The proliferation inhibitory activity of ursolic acid and metformin in different combination ratios on A549 cell line was measured by standard MTT colorimetry. The method is as in Example 2, and the results are shown in Figures 10-12.

As shown in Figure 10, after 24 hours of drug action, 10 μM ursolic acid has no obvious effect on inhibiting the proliferation of A549 cells; under the condition of 20 mM metformin, the cell survival rate is about 80%; When Met≥5 mM, it can obviously inhibit the proliferation of A549 cells, and it can play a synergistic anti-tumor effect; as shown in Figure 11, after 48 h of drug action, ursolic acid at 10 μM Metformin can significantly inhibit the proliferation of A549 cells under these conditions; Metformin can inhibit the proliferation of A549 cells when it is ≥20 mM; the combined administration of the two can significantly inhibit the proliferation of cells under the condition of Met≥1 mM for 48 hours ; As shown in Figure 12, after 72 hours of drug action, ursolic acid under the condition of 10 μM cell death reached half; metformin under the condition of low concentration ≤1 mM, still can not significantly inhibit the growth of SCL-1 cells Metformin can significantly inhibit cell proliferation under the condition of 0.5 mM after 72 hours of combined administration of the two, and with the increase of the concentration, the inhibition of cell proliferation is more obvious. In summary, it can be found that the combined administration of the two can significantly reduce the dosage of Met, and can synergistically inhibit the proliferation of A549 cells in a concentration- and time-dependent manner.

Example 9

King's modified formula was used to calculate the synergistic effect of the combined administration of ursolic acid and metformin under different ratios for 24, 48, and 72 h on A549 cells. The method was the same as in Example 3, and the results were shown in Tables 10-12.

As shown in Table 10-12, the combined administration of ursolic acid and metformin acted on A549 cells for 24, 48, and 72 hours at different ratios. Additive effect; when metformin is in a high concentration condition, the combined effect of the two medicines is basically a synergistic effect, which can synergistically inhibit the proliferation of A549 cells. The greater the drug concentration, the more obvious the inhibition.

Example 10

UA1 and metformin used alone or in combination inhibited the proliferation of SCL-1 cells. The method was the same as in Example 2, and the results are shown in FIG. 13 .

As shown in Figure 13, UA1 has no obvious inhibitory effect on the proliferation of SCL-1 cells under the condition of 10 μM; Metformin can inhibit the proliferation of SCL-1 cells only under the condition of ≥20 mM; the combined administration of the two 24 h, when Met ≥ 5mM, it can effectively inhibit the proliferation of SCL-1 cells, and with the increase of metformin concentration, the inhibition of cell proliferation is more obvious.

Example 11

King's modified formula was used to calculate the synergistic effect of the combined administration of UA1 and Met on SCL-1 cells under different ratios for 24 hours. The method was the same as in Example 3, and the results are shown in Table 13.

As shown in Table 13, the combined administration of UA1 and Met at different ratios for 24 h had an additive effect on SCL-1 cells when metformin was used at a low concentration; when metformin was used at a high Under the condition of concentration, the drug combination effect of the two drugs is basically a synergistic effect, which can synergistically inhibit the proliferation of SCL-1 cells, and the greater the drug concentration, the more obvious the inhibition.

Example 12

The inhibitory effect of UA1 and metformin on the proliferation of MCF-7 cells when used alone or in combination is the same as in Example 2, and the results are shown in FIG. 14 .

As shown in Figure 14, after 24 hours of drug action, the effect of UA1 on inhibiting the proliferation of MCF-7 cells under the condition of 10 μM is not obvious; Metformin can effectively inhibit the proliferation of MCF-7 cells under the condition of ≥20 mM ; The combined administration of the two can effectively inhibit the proliferation of MCF-7 cells when Met≥5 mM for 24 hours.

Example 13

King's revised formula was used to calculate the synergistic effect of the combined administration of UA1 and Met on MCF-7 cells for 24 h at different ratios, and the best drug ratio was screened out. The method was the same as in Example 3, and the results were shown in Table 14. shown.

As shown in Table 14, the combined administration of UA1 and Met at different ratios for 24 h had an additive effect on MCF-7 cells when metformin was used at low concentrations; when metformin was used at high Under the condition of the concentration, the combined effect of the two drugs is basically a synergistic effect, which can synergistically inhibit the proliferation of MCF-7 cells, and the greater the drug concentration, the more obvious the inhibition.

Example 14

The inhibitory effect of UA1 and metformin on the proliferation of HepG2 cells when used alone or in combination is the same as in Example 2, and the results are shown in FIG. 15 .

As shown in Figure 15, after 24 hours of drug action, the effect of UA1 on inhibiting the proliferation of HepG2 cells was not obvious under the condition of 0 μM; Metformin could significantly inhibit the proliferation of HepG2 cells under the condition of ≥20 mM; both Combined administration can significantly inhibit the proliferation of HepG2 cells when Met≥5 mM for 24 hours.

Example 15

King's correction formula was used to calculate the synergistic effect of the combined administration of UA1 and Met on HepG2 cells for 24 h at different ratios, and the best drug ratio was screened out. The method was the same as in Example 3, and the results were shown in Table 15. .

As shown in Table 15, the combined administration of UA1 and Met at different ratios for 24 h had an additive effect on HepG2 cells when metformin was used at a low concentration; when metformin was used at a high concentration Under the conditions, the combined effect of the two drugs is basically a synergistic effect, which can synergistically inhibit the proliferation of HepG2 cells, and the greater the drug concentration, the more obvious the inhibition.

Example 16

UA1 and metformin used alone or in combination inhibited the proliferation of A549 cells. The method was the same as in Example 2, and the results are shown in FIG. 16 .

As shown in Figure 16, UA1 has no obvious inhibitory effect on the proliferation of A549 cells under the condition of 10 μM; Metformin can only inhibit the proliferation of A549 cells under the condition of ≥20 mM; Met ≥ 5 mM can effectively inhibit the proliferation of SCL-1 cells, and with the increase of metformin concentration, the inhibition of cell proliferation is more obvious.

Example 17

King's correction formula was used to calculate the synergistic effect of the combined administration of UA1 and Met on A549 cells for 24 h at different ratios. The method was the same as in Example 3, and the results are shown in Table 16.

As shown in Table 16, the combined administration of UA1 and Met at different ratios for 24 h has an additive effect on A549 cells when metformin is used at a low concentration; when metformin is used at a high concentration Under the conditions, the combined drug effect of the two drugs is basically a synergistic effect, which can synergistically inhibit the proliferation of A549 cells, and the greater the drug concentration, the more obvious the inhibition.

Claims (2)

1. a kind of pharmaceutical composition with antitumor action, it is characterised in that double comprising compound and diformazan shown in formula I Guanidine

I ；

R wherein in Formulas I is hydroxyl or acetoxyl group；

Wherein the amount ratio of the substance of Formulas I compound represented and melbine is 10-20:500-20000.

2. pharmaceutical composition application in preparation of anti-tumor drugs as described in claim 1.