WO2022110568A1

WO2022110568A1 - Use of vitamin c and disulfiram in preparing anti-tumor combination drug

Info

Publication number: WO2022110568A1
Application number: PCT/CN2021/079565
Authority: WO
Inventors: 李为民; 何杨; 吴琼; 黄日东; 陈海; 罗雨蕉; 夏贞强
Original assignee: 四川大学华西医院; 成都华西精准医学产业技术研究院有限公司
Priority date: 2020-11-25
Filing date: 2021-03-08
Publication date: 2022-06-02
Also published as: CN112336716A

Abstract

A use of vitamin C and disulfiram in preparing a combination drug, which belongs to the field of anti-tumor drugs. The combination drug can achieve a vitamin C and disulfiram anti-tumor synergistic effect, an inhibitory effect on tumor cells is superior to using vitamin C or disulfiram in isolation, and said drug has good application prospects.

Description

The use of vitamin C and disulfiram in the preparation of anti-tumor combination drugs

technical field

The invention belongs to the field of antitumor drugs.

Background technique

Tumor treatment methods include surgery, radiotherapy and chemotherapy, molecular targeted therapy, and immunotherapy. Simple surgery has a low cure rate; radiotherapy and chemotherapy with obvious curative effects have strong toxic and side effects; molecular targeted therapy and immunotherapy have good curative effects and low toxic and side effects, but there are also problems such as drug resistance, genomic instability and response rate. This is also the reason why the prognosis and long-term survival rate of lung cancer patients are still very low.

As an antioxidant at physiological concentration (μM level), vitamin C (VC) can protect cells from oxidative stress, and at the same time, it has the functions of enhancing human immunity and preventing scurvy. Clinical trials and case studies in recent years have shown that intravenous injection of high-dose (mM grade) VC can selectively inhibit the growth of various tumors such as lung cancer, and at the same time, it has no obvious toxicity to normal tissues. However, the anti-tumor effect of VC alone is limited, and it is prone to drug resistance.

Disrlfiram (DSF) is a disulfide derivative of N,N-diethyldithiocarbamate (DEDTC), the chemical formula is C ₁₀ H ₂₀ N ₂ S ₄ , and the structural formula is as follows.

DSF has been used to treat alcohol dependence since its discovery in 1930. In recent years, DSF has been shown to have certain anticancer activity in various cancer models such as breast cancer, prostate cancer, glioblastoma, lung cancer, cervical cancer, liver cancer, myeloma, melanoma, neuroblastoma, etc. It has natural targeting to tumor tissue, but its specific mechanism of action is not yet clear.

DSF can produce a variety of side effects, currently reported are: optic neuritis and other neuropathies, hepatitis, rash, headache and so on. Therefore, the dose of DSF must be strictly controlled when it is used.

There is no report on the combined use of VC and DSF to treat tumors.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to provide a combination drug and an antitumor drug composition with excellent antitumor effect.

The technical scheme of the present invention is as follows:

An anti-tumor combined drug, the combined drug is

disulfiram and vitamin C;

The molar ratio of disulfiram and vitamin C is 1:2.5～4000.

Further, the molar ratio of the disulfiram and vitamin C is 1:2.5-400.

Further, the molar ratio of disulfiram and vitamin C is 1:5-400; preferably, it is 1:20-400.

An antitumor pharmaceutical composition, the composition is composed of vitamin C and disulfiram;

The molar ratio of disulfiram and vitamin C is 1:2.5～4000.

Further, the molar ratio of the disulfiram and vitamin C is 1:2.5-400.

The use of vitamin C and disulfiram in the preparation of anti-tumor combined drugs, or the use of vitamin C and disulfiram as anti-tumor active ingredients in the preparation of anti-tumor drugs.

Further, the tumor is lung cancer, preferably, non-small cell lung cancer.

Use of the aforementioned antitumor drug composition in the preparation of an antitumor drug.

Further, the tumor is lung cancer, preferably, non-small cell lung cancer.

Experiments show that vitamin C and disulfiram have a synergistic effect in inhibiting tumor cell activity. The anti-tumor combination drug or the anti-tumor drug composition of the invention has significantly better anti-tumor effect than vitamin C or disulfiram, and has good application prospect.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1: Cell viability results of H1299 cells inhibited by VC or DSF alone.

Figure 2: Results of the combined use of VC and SDF to inhibit cell viability of H1299 cells.

Figure 3: Cell viability results of H1975 cells inhibited by VC or DSF alone.

Figure 4: Results of cell viability inhibition of H1975 cells by combined use of VC and SDF.

Detailed ways

Example 1 VC combined with DSF inhibits H1299 cells

1. Method

The H1299 cell line (a non-small cell lung cancer cell line) was cultured in vitro in a complete medium (RPMI1640+10% fetal bovine serum), and the cells were cultured in a 37°C, 5% CO ₂ incubator. When the cells were confluent to about 80%, the adherent cells were digested with 0.25% trypsin, and different cells were prepared into a single cell suspension with complete medium, and seeded in a 96-well plate at a density of 15,000 cells per well. , the volume of each well is 100 μL. Transfer the culture plate into a CO ₂ incubator and incubate for 36h at 37°C and 5% CO ₂ . The original culture medium was aspirated and discarded, and the cells were divided into normal control group and drug groups with different concentrations. The normal control group was replaced with fresh culture medium; the other groups were replaced with different concentrations of VC, DSF, VC+DSF, and the volume of each well was 100 μL. Then continue to cultivate for 24h.

Add 5 μL of CCK8 to each well according to the operation manual, continue to incubate for 1-2 h, and terminate the culture. A group without cells is a blank group. Select 450nm wavelength, adjust the absorbance value of each well on the enzyme-linked immunosorbent assay instrument, and record the results. Plot the time as the horizontal axis and the absorbance value on the vertical axis. Calculate the cell viability according to formula 1-1. Each experiment was repeated at least 3 times.

Cell viability = (A _{drug solution} -A _blank )/(A _{normal control} -A _blank ) × 100% (1-1)

2. Results

The results of using VC or DSF alone are shown in Figure 1. The cells were not inhibited when the VC concentration was 0.5-2 mM, and the cells were significantly inhibited when the VC concentration was 3-4 mM. However, the cells were not inhibited when DSF was 1-200μM.

The results of the combined use of VC and DSF are shown in Figure 2 and Table 1.

When the DSF concentration was 1 μM and the VC concentration was 4 mM (DSF:VC=1:4000), the cell activity was significantly inhibited.

When the DSF concentration was 10 μM, and the VC concentration was 2-4 mM (DSF:VC=1:200-400), the cell activity was significantly inhibited.

When the DSF concentration was 50 μM, the cell activity was significantly inhibited when the VC concentration was above 2-4 mM (DSF:VC=1:40-80).

When the DSF concentration was 100 μM, the cell activity was significantly inhibited when the VC concentration was above 2-4 mM (DSF:VC=1:20-40).

When the DSF concentration was 200 μM, the cell activity was significantly inhibited when the VC concentration was above 2-4 mM (DSF:VC=1:10-20).

When the concentration of DSF was 10-200 μM and the concentration of VC was 4 mM (DSF:VC=1:20-400), the cell viability was significantly restricted, and the activity was lower than 10%. It can be seen that DSF:VC=1:20～400 is the preferred molar ratio range for the combined use of DSF and VC to inhibit H1299 cells.

Table 1. The cell viability of H1299 cells treated with different concentrations of VC and/or disulfiram for 24 h, the results are expressed as mean ± standard deviation (%).

In order to further analyze the synergistic effect of DSF and VC, the inventors analyzed the synergy index (CI, CI<1, indicating the existence of synergistic effect) of DSF and VC at each concentration by using CompuSyn software widely used in the art. The results are shown in Table 2. When the DSF concentration is 10-200 μM and the VC concentration is 0.5-4 mM (DSF:VC=1:2.5-400), DSF and VC have obvious synergistic effects; DSF concentration is 1 mM and VC concentration is 0.5 At ~1 mM (DSF:VC=1:500-1000) or 4mM (DSF:VC=1:4000), DSF and VC also had obvious synergistic effects. It is worth noting that the synergy index of DSF concentration of 1 μM and VC concentration of 2 to 3 mM in Table 2 is higher than 1, because the DSF and/or VC concentration is too low in a single case, with the increase of DSF (including DSF concentration exceeding 200 μM) ), DSF can generally synergize with VC (including when the concentration of VC exceeds 4mM).

The above ratios are all molar ratios.

Table 2 Combination Index (CI value) of VC combined with DSF on H1299 cells for 24 hours calculated by CompuSyn software

The results of this example show that the combined use of VC and DSF can produce a significant anti-tumor synergistic effect, which is beneficial to the treatment of lung cancer.

Example 2 VC combined with DSF inhibits H1975 cells

1. Method

Taking H1975 cells (a non-small cell lung cancer cell line) as the test object, they were treated by the method of Example 1.

2. Results

The results of using VC or DSF alone are shown in Figure 3. The cells were not inhibited when the VC concentration was 0.5-3 mM, and the cells were significantly inhibited when the VC concentration was 4 mM. However, the cells were not inhibited when the DSF concentration was 1-100 μM, and the cells were significantly inhibited when the concentration was 200 μM.

Table 3. The cell viability of H1975 cells treated with different concentrations of VC and/or DSF for 24 h, the results are expressed as mean ± standard deviation (%).

The results of the combined use of VC and DSF are shown in Figure 4 and Table 3.

When DSF is 1 μM, the dosage of 3-4mM (DSF:VC=1:3000-4000) of VC can significantly inhibit H1975 cells.

When DSF is 10μM, the dosage of VC is 2～4mM (DSF:VC=1:200～400), which can significantly inhibit H1975 cells.

When DSF is 50μM, the dosage of VC is 0.5～4mM (DSF:VC=1:10～80), which can significantly inhibit H1975 cells.

When DSF is 100μM, the dosage of VC is 0.5～4mM (DSF:VC=1:5～40), which can significantly inhibit H1975 cells.

When DSF is 200μM, the dosage of VC is 0.5～4mM (DSF:VC=1:2.5～10), which can significantly inhibit H1975 cells.

When DSF is 200 μM and VC is 1-4 mM (DSF:VC=1:5-20), and when DSF is 10-200 μM and VC is 4 mM (DSF:VC=1:20-400), and DSF is 50 μM In addition, when VC was 3 mM (DSF:VC=1:60), the inhibitory effect was extremely significant, and the cell activity was 3% or less. The combined range shows that DSF:VC=1:5～400 is the optimal molar ratio range for the combined use of DSF and VC to inhibit H1975 cells.

In order to further analyze the synergistic effect of DSF and VC, the inventors analyzed the synergy index (CI, CI<1, indicating the existence of synergistic effect) of DSF and VC at each concentration by using CompuSyn software widely used in the art. The results are shown in Table 4. When DSF is 10-200 μM and VC concentration is 0.5-4 (DSF:VC=1:2.5-400), DSF and VC have obvious synergistic effect; DSF concentration is 1 mM and VC concentration is 1 mM. At 3～4mM (DSF:VC=1:3000～4000), DSF and VC also have obvious synergistic effect. It is worth noting that in Table 4, when the DSF concentration is 1 μM and the VC concentration is below 2 mM, the synergy index is higher than 1, which is because the DSF and/or VC concentration is too low. 200μM), DSF can generally synergize with VC (including when the VC concentration exceeds 4mM).

The above ratios are molar ratios.

Table 4 Combination Index (CI value) calculated by VC combined with DSF on H1975 cells for 24 hours using CompuSyn software

The results of this example show that the combined use of VC and DSF can produce a significant anti-tumor synergistic effect.

In summary, the use of VC combined with DSF can produce significant anti-tumor synergistic effects; the use of VC and DSF as active ingredients to prepare anti-tumor drugs, or the use of VC and DSF to prepare anti-tumor combination drugs, will have good anti-tumor effects. prospect.

Claims

An anti-tumor combined drug, characterized in that: the combined drug is

disulfiram and vitamin C;

The molar ratio of disulfiram and vitamin C is 1:2.5～4000.
The combined medicine of claim 1, wherein the molar ratio of the disulfiram and vitamin C is 1:2.5-400.
The combined medicine of claim 1, wherein the molar ratio of the disulfiram to vitamin C is 1:5-400; preferably, it is 1:20-400.
An antitumor pharmaceutical composition, characterized in that: the composition is composed of vitamin C and disulfiram;

The molar ratio of disulfiram and vitamin C is 1:2.5～4000.
The composition of claim 4, wherein the molar ratio of the disulfiram and vitamin C is 1:2.5-400.
The composition of claim 5, wherein the molar ratio of the disulfiram to vitamin C is 1:5-400; preferably, it is 1:20-400.
The use of vitamin C and disulfiram in the preparation of anti-tumor combined drugs, or the use of vitamin C and disulfiram as anti-tumor active ingredients in the preparation of anti-tumor drugs.
The use according to claim 7, wherein the tumor is lung cancer, preferably non-small cell lung cancer.
Use of the antitumor pharmaceutical composition according to any one of claims 4 to 6 in the preparation of an antitumor drug.
The use according to claim 9, wherein the tumor is lung cancer, preferably non-small cell lung cancer.