CN114259566B - Anti-tumor composition based on oxidation-reduction balance destruction and application - Google Patents

Abstract

The invention discloses an anti-tumor composition, which comprises the following components: composition A or/and composition B; composition A is an NADPH regeneration inhibitor, comprising two or three of a G6PD inhibitor, an ME1 inhibitor and an ALDH1A1 inhibitor; composition B is a GSH synthesis regeneration inhibitor comprising two or three of a CBS inhibitor, xTC inhibitor and a GR inhibitor; and the mass ratio of any two components in the composition is 1; the composition A and the composition B can be independently prepared into a medicinal preparation as effective components or can be combined to prepare an AB compound preparation; moreover, any two compositions can be combined with FBP which can promote the oxidation phosphorylation of mitochondria and inhibit the de novo synthesis of NADPH and GSH to prepare a compound preparation.

Description

Anti-tumor composition based on oxidation-reduction balance destruction and application

Technical Field

The invention relates to the technical field of pharmacy, in particular to an anti-tumor composition based on oxidation-reduction balance destruction and application thereof.

Background

Tumor cells have multiple malignant biological characteristics compared to normal cells, with metabolic reprogramming being a core characteristic; metabolic reprogramming not only directly supports tumor cell proliferation, survival and tumor growth, but is also critical to maintaining other malignant characteristics. Reprogramming of tumor cell metabolism is also an important cause of drug resistance. Tumor cells are in a chronic metabolic oxidative stress state compared to normal cells, and therefore tumor cells need to achieve adaptive survival and growth by reprogramming related metabolic pathways to improve antioxidant capacity. Cancer cells of advanced tumors often exhibit multiple genetic alterations and high oxidative stress, suggesting that these cells can be preferentially eliminated by drug-generated reactive oxygen species damage. However, up-regulation of antioxidant stress capacity in tumor cells can lead to drug resistance.

Reduced Glutathione (GSH) and reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) are the most important substances for resisting oxidative damage in cells. GSH maintains sufficient intracellular cysteine levels, detoxifies xenobiotics including various chemotherapeutic agents, and enhances the resistance of cancer cells to chemotherapy, and high levels of GSH promote tumor progression including tumor metastasis. NADPH can reduce GSSG and TRX-TRX into GSH and TRX with ROS scavenging ability, and recent studies have demonstrated that NADPH, as an antioxidant stress injury substance, can exert an anti-iron-death effect independently of GSH and TRX, thereby inducing radiotherapy antagonism. In addition to NADPH providing reducing equivalents for the scavenging of Reactive Oxygen Species (ROS) produced by the rapid proliferation of tumor cells, NADPH is an essential cofactor for anabolic reactions such as lipid and nucleic acid biosynthesis. Therefore, GSH and NADPH exhausted from tumor cells can inhibit tumor growth and induce oxidative stress damage, and has important significance for treating tumors.

At present, research has found the antitumor effect of FBP and published the application of FBP in preparing antitumor medicine. With the progress of research, it was found that FBP in glioma cells could block the flow of glucose and glutamine to GSH precursor amino acid and macromolecule biosynthesis, and at the same time could promote mitochondrial oxidative phosphorylation to increase ROS production, eventually leading to intracellular NADPH and GSH depletion and oxidative stress damage. However, for other cancer cells such as lung cancer, only a few cell lines reproduce the drug sensitivity of glioma cells to FBP; moreover, the inability of FBP to effectively reduce intracellular NADPH and GSH levels is a major cause of relative insensitivity of other cancer cells.

Therefore, the technical problem to be solved by those skilled in the art is how to provide a composition capable of effectively reducing NADPH level or/and GSH level in tumor cells and apply the composition to antitumor drugs.

Disclosure of Invention

In view of the above, the present invention provides an anti-tumor composition based on the disruption of redox balance, which can inhibit the proliferation of cancer cells, and has synergistic effects between the components, thereby improving the cytotoxicity of the composition.

An antitumor composition based on disruption of redox balance comprising: an NADPH regeneration inhibitor or/and a GSH inhibitor; the two inhibitors are independently prepared or combined into a medicament respectively; wherein the NADPH regeneration inhibitor comprises two or three of a G6P D inhibitor, an ME1 inhibitor and an ALDH1A1 inhibitor; the GSH inhibitor comprises any two or three of a CBS inhibitor, an xCT inhibitor, and a GR inhibitor; and the mass ratio of any two components in the composition is 1.

The technical effect achieved by the technical scheme is as follows: NADPH is used as an antioxidant stress injury substance, can provide reducing equivalent for scavenging Reactive Oxygen Species (ROS) generated by rapid proliferation of tumor cells, and promotes the proliferation of the tumor cells, so that the NADPH inhibitor can effectively inhibit the proliferation of the tumor cells; PPP-generated NADPH is critical to prevent ROS damage caused by mitochondrial respiration and ionizing radiation. The cancer cells synthesize and regenerate NADPH and GSH de novo through PPP and serine synthesis pathways, thus alleviating oxidative damage caused by excess ROS and avoiding apoptosis, so that the depletion of NADPH and GSH can enhance the sensitivity of colon cancer, ovarian cancer and lung cancer cells to ROS-mediated apoptosis. PPP-generated NADPH is critical to prevent ROS damage caused by mitochondrial respiration and ionizing radiation. Glucose-6-phosphate dehydrogenase (G6 PD) is the rate-limiting enzyme of PPP and is the first step in catalyzing the partial oxidation of Glucose-6-phosphate to NADPH and ribose-5-phosphate. G6PD is highly expressed in various tumor cells, and the G6PD of a colon cancer cell is knocked out, so that the sensitivity to the treatment of the oxaliplatin can be increased; NADP ⁺ Dependent malic acidThe enzyme family (MEs) is another important source of NADPH, which catalyzes the oxidative decarboxylation of malic acid to carbon dioxide and pyruvate, during which NADP is converted ⁺ Reduced to NADPH. Malic enzyme (ME 2) in mitochondria regenerating NADPH is considered as a potential anti-cancer target. Citrate in mitochondria is transported to the cytosol where it can provide a substrate for either ME1 or isocitrate dehydrogenase 1 (IDH 1), both of which can regenerate NADPH. ME1 promotes gastric cancer cell growth, lung metastasis and peritoneal spread, and interference with ME1 can slow down tumor growth and inhibit epithelial-mesenchymal transition. Knocking out ME1 in gastric cancer cells can greatly reduce NADPH synthesis, so that ROS accumulation is caused, and apoptosis of tumor cells is promoted under an oxidative stress condition; aldehyde Dehydrogenase 1 family member A (Aldehydedehydrogenase 1-A, ALDH 1A) catalyzes the oxidation of the retinol metabolite retinal to retinoic acid, while NADP is simultaneously present ⁺ Reduced to NADPH. Patients with ulcerative colitis, a risk factor for colon cancer, have elevated levels of ALDH1A1 protein compared to normal colon cells, and play an important role in the conversion of colitis to colon cancer. In many tumors, ALDH1A1 high expression is closely related to the natural and acquired resistance of chemotherapy and the EGFR inhibitor erlotinib, and many other treatments but different cancer cells may be dependent on G6PD, ME1 and ALDH1A1 to varying degrees and, when one is inhibited, the rest may act as a compensation; therefore, to effectively reduce the NADPH level in cancer cells, it is necessary to use two or three inhibitors simultaneously, any two or three inhibitors have a synergistic effect, the cytotoxicity of the composition is improved, and the activity and proliferation rate of cancer cells are reduced compared with the single use.

The CBS inhibitor has more prominent cytotoxicity on cancer with high expression of CBS and xCT; the added GR inhibitor can further improve the lethality to tumor cells, and the lethality improvement has broad spectrum and is more sensitive to GR low-expression cancer cells, which shows that the GR inhibitor has pharmacodynamic value for killing GR high-expression and low-expression cancer cells, and also provides scientific basis for the combined application of the GR inhibitor and other anti-cancer substances or medicaments with action mechanisms.

As a preferred embodiment of the present invention, the method further comprises: FBP, the ratio between any two components in the composition being 1.

As a preferred technical scheme, the G6PD inhibitor comprises dehydroepiandrosterone or 6-aminonicotinamide; the ME1 inhibitor comprises piperine; the ALDH1A1 inhibitor comprises disulfiram.

As a preferable technical scheme of the invention, the CBS inhibitor comprises benserazide hydrochloride; the xCT inhibitor comprises sorafenib or sulfasalazine; the GR inhibitor comprises dihydroartemisinin or amitriptyline hydrochloride.

The technical effect achieved by the technical scheme is as follows: DHEA or 6-AN, piperine and DSF are inhibitors of three enzymes of G6PD, ME1 and ALDH1A respectively, and inhibit 2 or 3 key enzymes of NADPH regeneration simultaneously, so that the compound has obvious anticancer activity not only on non-small cell lung cancer but also on other types of tumors including liver cancer, intestinal cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer and the like. Moreover, the combination therapy of inhibitors of these three enzymes has a high safety profile.

The application of any one of the compositions in preparing anti-tumor medicaments, wherein tumors comprise various solid tumors and blood tumors; the composition can be used for resisting tumors alone or in combination with other treatments including targeted drugs, chemotherapy and radiotherapy, thereby playing roles in strengthening the cancer inhibition effect and resisting drug resistance.

As a preferable technical scheme, the medicine is prepared from the composition and pharmaceutically acceptable excipients or carriers, and comprises a solid preparation and a liquid preparation, wherein the preparation forms include an oral preparation, an injection preparation, a suppository, a film agent, a controlled release double-layer tablet, a controlled release three-layer tablet, a controlled release nano preparation, a microcapsule preparation, a microsphere preparation and an enteric preparation.

As a preferable technical scheme of the invention, the administration route of the medicament is oral administration or injection administration or oral nasal spray administration or external skin application administration or anal administration.

In conclusion, the anti-tumor composition provided by the invention achieves the technical effects that:

1) The components of the composition are all clinically used medicines, and most of the components are highly safe non-tumor treatment medicines, so that the composition has the advantage of being safer than the existing treatment and medicines, and has a shorter research and development period than the anticancer medicines starting from the lead compound, so that the composition can provide safe, effective and relatively cheap high-quality treatment for patients in a shorter period.

2) The medicine prepared from the composition can be used for independently treating various solid tumors and blood tumors, and can be administered before or after the operation of the solid tumors. Because cancer cells in metastasis are easily damaged by oxidative stress, the pharmaceutical preparation of the invention can inhibit tumor growth and kill tumor cells, and has important value for preventing and treating tumor metastasis.

3) The medicine provided by the invention can greatly improve the treatment value of the targeted medicine. The targeted drug has the advantages of obvious early treatment effect and the greatest defects of acquired drug resistance and congenital drug resistance, and the combination of the pharmaceutical preparation and the targeted drug can avoid or at least greatly delay the acquired drug resistance and overcome the congenital drug resistance, thereby greatly improving the treatment value of the targeted drug.

4) The medicine provided by the invention can improve the anticancer efficacy of radiotherapy and chemotherapeutic drugs taking ROS as a treatment principle, but does not increase toxicity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the effect of ABC compound on GSH level in non-small cell lung cancer cell strains A549 and H1975;

FIG. 2 is a diagram showing the effect of ABC compound on the NADPH level of non-small cell lung cancer cell strains A549 and H1975;

FIG. 3 is a graph showing the toxicity of Piperine (Piperine) on NSCLC cells;

FIG. 4 is a graph showing the toxicity of Dehydroepiandrosterone (DHEA) to non-small cell lung cancer cells;

FIG. 5 is a graph showing the toxicity of aminonicotinamide (6-AN) on non-small cell lung cancer cells;

FIG. 6 is a graph showing the toxicity of Disulfiram (DSF) against non-small cell lung cancer cells;

FIG. 7 is a graph showing the toxicity of benserazide hydrochloride (BEN) to non-small cell lung cancer cells;

FIG. 8 is a graph of the toxicity of Sorafenib (SOR) on non-small cell lung cancer cells;

FIG. 9 is a graph showing the toxicity of sulfasalazine (SAS) on non-small cell lung cancer cells;

FIG. 10 is a graph showing the toxicity of Dihydroartemisinin (DHA) on NSCLC cells;

FIG. 11 is a graph showing the toxicity of amitriptyline hydrochloride (AMI) on non-small cell lung cancer cells;

FIG. 12 is a graph showing the effect of AB Fufang on LLC transplantable tumor; wherein A, B is a tumor inhibition result graph of different groups; c is a survival time chart of different groups; d is a graph of the weight change results of different groups;

FIG. 13 is a graph showing the effect of AB combination FBP on LLC transplantable tumors; wherein A, B is a size and volume map of tumors of different groups; c is a graph of the weight change results of different groups;

FIG. 14 is a graph showing the effect of ABC compound on A549 xenograft tumors; wherein A, B is a map of tumor volumes of different groups; c is a survival time chart of different groups; d is a graph of the weight change results of different tissues;

FIG. 15 is a graph showing the effect of ABC complex on H1975 xenograft tumors; wherein A, B, C, D, E is a map of tumor volumes of different groups; f is a graph of survival time of different groups; g is a graph of the results of weight changes of different groups.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1ABC Complex decreases levels of GSH in tumor cells

The method comprises the following steps: non-small cell lung cancer cell strains A549 and H1975 cultured for 24 hours are subjected to pharmaceutical intervention for 36 hours according to tables 1-1 and tables 1-2, and the levels of cell GSH and GSSG are determined by using Biyunsian GSH and GSSG detection kits.

TABLE 1-1A 549 pharmaceutical combination

TABLE 1-2 H1975 pharmaceutical combination modalities

* Remarks are as follows: a1= pipeine; a2= DHEA; a3= DSF; a = a1+ a2+ a3; b1= BEN; b2= SOR;

b3＝DHA；B＝b1+b2+b3；C＝FBP。

as a result: as shown in fig. 1;

wherein FIG. 1-A, FIG. 1-B, FIG. 1-C and FIG. 1-D show: the AB compound and FBP treated cells 36h can obviously reduce the total glutathione (GSSH + GSSG) and GSH reserve in A549 cells and increase the ratio of GSSG and GSSG to GSSH + GSSG, while the ABC compound (A + B + FBP) can almost completely exhaust the total glutathione and GSH reserve and further improve the ratio of GSSG to GSSH + GSSG, and the FBP component C plays a key role in improving the ratio, which is consistent with the action principle that the FBP can increase the generation of ROS by promoting the oxidative phosphorylation of mitochondria. Accordingly, FBP in combination with components a and a + B, respectively, both significantly potentiate their effects in depleting intracellular glutathione stores. The a-component also significantly reduces the total amount of glutathione (GSH + GSSG), while the addition of the B-component (a + B1 and B2 or/and B3), especially the full component (a + B), further reduces the glutathione antioxidant reserve.

FIG. 1E, FIG. 1-F, FIG. 1-G, and FIG. 1-H show that in H1975A complexes significantly reduced intracellular GSSG content, AC complexes significantly reduced the total amount of GSH and GSSG, GSH content and GSSG content; the B compound remarkably improves the total amount of GSH and GSSG, the GSH content and the GSSG content, which are not in line with expectations, and the addition of the component C can reverse the effect of the B compound; the AB compound and the ABC compound obviously reduce the total amount of GSH and GSSG, the GSH content and the GSSG content, and simultaneously improve the ratio of the GSSG to the GSH plus the GSSG, wherein the influence of the ABC compound is more obvious than that of the AB compound. The result shows that the ABC compound can obviously reduce the glutathione antioxidant reserve in the H1975 cell, and the FBP (component C) plays a main role and is basically consistent with the theory.

Taken together, FBP, in combination with AB from the perspective of inhibiting GSH synthesis and enhancing its consumption, almost depletes glutathione antioxidant stores in tumor cells.

Example 2ABC complexes reduce the levels of NADPH in tumor cells

The method comprises the following steps: subjecting non-small cell lung cancer cell strains A549 and H1975 cultured for 24 hr to pharmaceutical intervention for 36 hr according to attached tables 2-1 and 2-2, and treating with Biyunshi NADP ⁺ NADPH and NADP of cells measured by NADPH detection kit ⁺ And (4) horizontal.

TABLE 2-1 drug combination regimen

TABLE 2-2 H1975 drug combination regimen

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* Remarks are as follows: a1= Piperine; a2= DHEA; a3= DSF; a = a1+ a2+ a3; b1= BEN; b2= SOR;

b3＝DHA；B＝b1+b2+b3；C＝FBP。

as a result: as shown in fig. 2;

wherein FIG. 2-A, FIG. 2-B and FIG. 2-C show: FBP can greatly reduce NADPH and NADP in A549 cells compared with model Control (CON) ⁺ Total amount, NADPH and NADP ⁺ The amounts of (a) and (b) are also significantly reduced, respectively. Both the A and A + bn fractions can significantly reduce NADPH and NADP ⁺ In particular, these components significantly increase NADPH levels but decrease NADP ⁺ Probably because a decrease in these components prevents biosynthesis which supports cell proliferation and thus less oxidation of the corresponding NADPH to NADP ⁺ . ABC compound for reducing NADPH and NADP ⁺ The intensity of the total amount was substantially consistent with that of FBP, while the amount of NADPH was also significantly higher than that of the FBP group.

FIGS. 2-D, 2-E, and 2-F show that in H1975 cells, FBP, A Complex, AB Complex, and AC Complex, AB Complex, ABC Complex all significantly reduced the total amount of NADP + and NADPH, the level of NADP +, and that FBP played a major role, compared to model Control (CON). However, these combinations not only did not reduce but increased intracellular NADPH levels. However, since the total amount of NADP + and NADPH was at a lower level, NADPH was also at a lower level, indicating that ABC complex decreased the level of NADPH in H1975.

The research results show that the ABC compound can reduce the NADPH level of cells.

Example 3 validation that NADPH-regenerating enzymes ME1, G6PD and ALDH1A inhibitors can produce synergistic effects

Reagent: ME1 inhibitor Piperine (Piperine), G6PD inhibitor Dehydroepiandrosterone (DHEA) or 6-aminonicotinamide (6-AN), ALDH1A1 inhibitor Disulfiram (DSF);

the method comprises the following steps: respectively culturing non-small cell lung cancer cell strains NCI-H1703, PC-9, NCI-H1975, NCI-H460, A549, NCI-H2170, NCI-H1299, NCI-H1395 or LLC which are cultured for 24 hours in a culture medium containing (1) 12.5, 25, 50, 100, 200 and 400 mu M Piperone; (2) 25, 50, 100, 200, 400 μ M DHEA; (3) 2, 5, 10, 20, 50, 100, 200 μ M6-AN; (4) Culturing in the culture medium of 25, 50, 100, 200, 400 and 800nM DSF for 72 hours. Meanwhile, a drug-free treatment group (also called a control group, CON) is provided. Cell viability was determined using Sulforhodamine B (SRB) staining assay.

As shown in FIG. 3, FIG. 4, FIG. 5 and FIG. 6, the single action of Piperone, DHEA or 6-AN, DSF, NADPH-regenerating enzyme inhibitor can significantly inhibit the cell viability of multiple non-small cell lung cancer cell lines, but the sensitivity of different cells to the inhibitors is not completely the same. The above results indicate that NADPH regenerating enzymes ME1, G6PD and ALDH1A1 inhibitors can inhibit proliferation of non-small cell lung cancer cells.

Then, the non-small cell lung cancer cell strains NCI-H1703, PC-9, NCI-H1975, NCI-H460, A549, NCI-H2170, NCI-H1299, NCI-H1395 or LLC cultured for 24 hours were subjected to drug intervention for 72 hours according to the following tables 3-1 respectively. At the same time, a normal control group (CON) was set without any drug treatment. Cell viability was determined using Sulforhodamine B (SRB) staining assay.

TABLE 3-1

* Remarks are as follows: a1= pipeine; a2= DHEA; a3= DSF; a = a1+ a2+ a3; c = FBP.

Meanwhile, triple negative breast cancer cell lines MDA-MB-231, SUM-159, HCC-1806, cervical cancer cell line Hela, colon cancer cell lines SW620, HCT-15, HCT-116, glioma cell lines U251, U87MG, SHG44, gastric cancer cell lines SGC-7901, leukemia cell lines Jurkat, myeloma cell lines U266, FO, RPMI-8226, liver cancer cell lines Huh7, hepG2, SMMC-7721, bel-7402, melanoma cell lines B16-F10, pancreatic cancer cell lines BxPC-3 and PANC-1 which are cultured for 24 hours are respectively combined according to the drug combination mode of the following table 3-1, but the concentration of the inhibitor is set as follows: a 180. Mu.M, a 250. Mu.M; a 30.2. Mu.M (these concentrations are the IC of the 9 lung cancer cell lines tested, respectively) ₃₀ Mean), intervene for 72 hours. Meanwhile, a normal control group (CON) without any drug treatment is set. Cell viability was determined using Sulforhodamine B (SRB) staining assay. The results are shown in Table 3-2;

TABLE 3-2 cancer cell proliferation rate (%) for inhibition of NADPH-regenerating enzyme

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* Remarks are as follows: a1= pipeine; a2= DHEA; a3= DSF; a = a1+ a2+ a3.

As can be seen from Table 3-2, 16 of the 31 tumor cell lines included lung cancer cells PC-9, H1975, H460, H1395, LLC, SUM-159, HCC-1806, glioma SHG-44, colon cancer cell HCT-116, hepatoma cell Huh7, SMMC-7721, pancreatic cancer cell BxPC-3, leukemia cell Jurkat, myeloma cells U266, FO, RPMI-8226, which were highly sensitive to ME1 inhibitor (a 1) in combination with G6PD inhibitor (a 2); the lung cancer cell A549, the intestinal cancer cell SW620, the HCT-15, the liver cancer cell HepG2 and the melanoma cell B16-F10 are highly sensitive to the A1 combined ALDH1A1 inhibitor (a 3), and the three inhibitors are remarkably highly sensitive to most of tumor cells. Therefore, the simultaneous inhibition of the 3 NADPH-regenerating enzymes can generate more broad-spectrum, stable and reliable antitumor effects than the inhibition of 2 of the 3 NADPH-regenerating enzymes, so that the 3 NADPH-regenerating enzyme inhibitors form the effective compound A reasonably.

Example 4 validation of the synergistic Effect of CBS, xCT and GR inhibitors of GSH synthase

Reagent: CBS inhibitor benserazide hydrochloride (BEN), xCT inhibitor Sorafenib (SOR) or sulfasalazine (SAS), GR inhibitor Dihydroartemisinin (DHA) or amitriptyline hydrochloride (AMI).

The method comprises the following steps: culturing non-small cell lung cancer cell strain NCI-H1703, PC-9, NCI-H1975, NCI-H460, A549, NCI-H2170, NCI-H1299, NCI-H1395 or LLC in the presence of (1) 10, 20, 40, 80, 160 or 320 μ M BEN, respectively; (2) 3.125, 6.25, 12.5, 25, 50 μ M SOR; (3) 0.05, 0.1, 0.2, 0.4, 0.8, 1.6mM SAS; (4) 1, 2, 4, 8, 16, 32 μ M DHA; (5) 6.25, 12.5, 25, 50, 100. Mu.M AMI for 72 hours. Meanwhile, a drug-free treatment group (also called a control group, CON) is provided. Cell viability was determined using Sulforhodamine B (SRB) staining assay.

As shown in fig. 7, 8, 9, 10 and 11, the activity of BEN, SOR, SAS, DHA or AMI, GSH synthesis or regeneration inhibitors alone can significantly inhibit the cell viability of multiple non-small cell lung cancer cell lines, but the sensitivity of different cells to these inhibitors is not completely the same. The above results indicate that inhibitors of the GSH synthase CBS, xCT and the GSH regenerating enzyme GR can inhibit proliferation of non-small cell lung cancer cells.

Non-small cell lung carcinoma NCI-H1975, cultured for 24 hours, was then drug-intervened for 72 hours according to Table 4-1. At the same time, a normal control group (CON) was set without any drug treatment. Cell viability was determined using Sulforhodamine B (SRB) staining assay.

TABLE 4-1

* Remarks are as follows: b1= BEN; b2= SOR; b3= DHA; b = B1+ B2+ B3; c = FBP.

Meanwhile, lung cancer cells NCI-H1975, colon cancer cell strains SW620 and HCT-116, liver cancer cell strains SMMC-7721 and Bel-7402, pancreatic cancer cells PANC-1, myeloma cell strains FO and RPMI-8226 which are cultured for 24 hours are respectively combined according to the drugs in the formula of table x1, but the concentration of the inhibitor is set as follows: b 185. Mu.M, b 23.5. Mu.M; b32.5 μ M (these concentrations are the IC30 mean of the 9 lung cancer cell lines tested), and intervene for 72 hours. At the same time, a normal control group (CON) was set without any drug treatment. Cell viability was determined using Sulforhodamine B (SRB) staining assay. The results are shown in Table 4-2;

TABLE 4-2 proliferation rate (%) of cancer cells inhibiting GSH synthesis and regeneration enzyme

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* Remarking: b1= BEN; b2= SOR; b3= DHA; b = B1+ B2+ B3.

As can be seen from Table 4-2, among the 8 tumor cell lines, colon cancer cells SW620, liver cancer cells Bel-7402 and SMMC-7721, and myeloma cells FO were highly sensitive to xCT inhibitor (b 2) in combination with GR inhibitor (b 3); the lung cancer cell H1975, the intestinal cancer cell HCT-116 and the pancreatic cancer cell PANC-1 are insensitive to the mutual combination of the two or the three inhibitors in the group B; it is noted that when the three inhibitors are combined, the sensitivity of the tumor cells is reduced except for the intestinal cancer cell HCT-116 and the pancreatic cancer cell PANC-1. Therefore, it is reasonable to determine that the 3 GSH synthesis and regeneration enzyme inhibitors constitute effective compound B because of the effective anti-tumor effect of inhibiting the 3 GSH synthesis and regeneration enzymes, especially inhibiting 2 of them.

Example 5 verification that NADPH inhibitors and GSH inhibitors have synergistic effects

To verify the synergistic effect of the components, non-small cell lung cancer cell lines NCI-H1703, PC-9, NCI-H1975, NCI-H460, A549, NCI-H2170, NCI-H1299, NCI-H1395 or LLC cultured for 24 hours were subjected to pharmacological intervention for 72 hours according to Table 5-1, respectively. At the same time, a normal control group (CON) was set without any drug treatment. Cell viability was determined using Sulforhodamine B (SRB) staining assay.

TABLE 5-1

Remarking: a1= Piperine; a2= DHEA; a3= DSF; a = a1+ a2+ a3; b1= BEN; b2= SOR;

b3＝DHA；B＝b1+b2+b3；C＝FBP。

meanwhile, triple negative breast cancer cell lines MDA-MB-231, SUM-159, HCC-1806, cervical cancer cell line Hela, glioma cell lines U251, U87MG, SHG44, colon cancer cell lines SW620, HCT-15, HCT-116, gastric cancer cell lines SGC-7901, liver cancer cell lines Huh7, hepG2, bel-7402, SMMC-7721, leukemia cell lines Jurkat, myeloma cell lines U266, FO, RPMI-8226, pancreatic cancer cell lines BxPC-3, PANC-1 and melanoma cell lines B16-F10 which are cultured for 24 hours are respectively combined according to the drug combination mode of the following table 3, but the concentration of the inhibitor is set as follows: a 180. Mu.M, a 250. Mu.M; a 30.2. Mu.M, b 185. Mu.M; b23.5 mu M; b 32.5. Mu.M (these concentrations are IC's of the 9 lung cancer cell lines tested ₃₀ Mean), intervene for 72 hours. At the same time, a normal control group (CON) was set without any drug treatment. The cell viability was measured by Sulforhodamine B (SRB) staining assay, and the results are shown in Table 5-2;

TABLE 5-2 effects of the combination of inhibitor of B group with Compound A on cancer cell viability (%)

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* Remarks are as follows: a1= pipeine; a2= DHEA; a3= DSF; a = a1+ a2+ a3; b1= BEN; b2= SOR; b3= DHA; b = B1+ B2+ B3.

As can be seen from Table 5-2, in 31 tested tumor cells, the cytotoxicity of the A compound can be significantly improved by adding the components b1, b2 or b3 of the inhibitor of the group H1703, hela, U87MG, U251 and PANC-1,B which are insensitive to the A compound (the inhibition rate is less than 50%); the cell strains H1975, H1299, SW620, SGC-7901 and Bel-7402 which are relatively insensitive to the A compound (the inhibition rate is less than 60 percent) can further improve the toxicity of the A compound to cells by adding any 1 or more than 1B components; for 21 cells sensitive to the A compound (the inhibition rate is more than 60%), the inhibitor in the B group is added in total, wherein the cell viability of 6 cells (comprising H460, A549, H2170, LLC, MDA-M-231, hela, SHG-44, HCT-15, hepG2, bxPC-3, U266, FO and RPMI-8226) is further reduced remarkably, however, a few cells are insensitive to the addition of B, and even the addition of B weakens the cytotoxicity of the A compound, such as PC-9, H1395, SUM-159, HCC-1806, HCT-116, huh7 and B16-F10. In general, the group B inhibitor can enhance the efficacy of the compound A in reducing cell viability, so that the compound AB has a broader anticancer spectrum and a stronger efficacy than the compound A.

Example 6 verification of the synergistic Effect of FBP with A-formulation, B-formulation or with A + B-formulation

Firstly, the antitumor activity of FBP-optimized compound A is verified, the method is the same as that of example 3, and the result is shown in table 6-1; thirdly, verifying the antitumor activity of the FBP optimized B compound by the same method as that of example 4, and showing the result in table 6-2; finally, the anti-tumor activity of the AB compound is optimized by FBP, and the method is the same as the example 5; the results are shown in tables 6-3.

TABLE 6-1A cancer cell proliferation Rate (%) with combination of Fufang and FBP (C = FBP)

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As can be seen from Table 6-1, FBP significantly increased the sensitivity of tumor cells to partial and full components of the A complex, as when FBP was combined with a1, the viability of 10 of the 31 tested cells (H1975, H2170, SUM-159, hela, U87MG, jurkat, U266, FO, hepG2 and B16-F10) was reduced by more than 40% by the addition of FBP, of which 12 were reduced by 20-40%. However, in this 31 tumor cell line, pancreatic cancer cells BxPC-3 were less sensitive to FBP in combination with a1 than to a 1.

TABLE 6-2B cancer cell proliferation (%) with combination of FUFANGS and FBPs (C = FBP)

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As can be seen from tables 6-2, FBP significantly increased the sensitivity of tumor cells to partial and full components of the B complex, as when FBP was combined with B1, the viability of 2 of the tested 8 cell lines (H1975 and FO) was reduced by more than 40% by the addition of FBP, and the viability of 2 cell lines was reduced by 20-40%. The above results indicate that FBP (component C) can enhance the antitumor effect of the B compound.

TABLE 6-3 cancer cell proliferation (%) with combination of AB Complex and FBP (C = FBP)

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As can be seen from tables 6-3, FBP can also significantly increase the sensitivity of tumor cells to the partial and complete components of the AB complex, as when FBP is combined with A + b1, 16 of the 30 tested cells (H1703, PC-9, H2170, LLC, SUM-159, HCC-1806, U251, SW620, HCT-15, SGC-7901, huh7, hepG2, S MMC-7721, bel-7402, bxPC-3, PANC-1) were more than 40% reduced in viability by the addition of FBP, with 9 cells reduced in viability by 10% -40%; when the FBP was combined with the AB full fraction, the activity of 16 of the 30 tested cells (PC-9, A549, H2170, MBA-MB-231, SUM-159, SW620, HCT-15, HCT-116, SGC-7901, huh7, hepG2, SMMC-7721, bel-7402, bxPC-3, PANC-1, RPMI-8226) was reduced by more than 20% by the addition of the FBP, of which 10 cells were reduced by 10-20%. However, in the 28 tumor cells, the sensitivity of the triple negative breast cancer cells MDA-MD-231, myeloma cells FO and RPMI-8226 to FBP combined A + b1 is lower than that to A + b 1.

In conclusion, FBP can generally and obviously improve the toxicity of the component A and the component B in the AB compound to tumor cells, so that the ABC compound is further formed by adding FBP (component C) into the AB compound.

Further verifies that the ABC compound has in vivo antitumor activity

The process is as follows: establishing a subcutaneous transplantation tumor model:

(1) LLC model: inoculating mouse non-small cell lung cancer cell strain LLC (100 ten thousand/mouse) to right axilla of adult male C57BL/6 black mice according to a conventional method;

(2) A549 model and H1975 model: the human non-small cell lung cancer cell strain A549 (200 ten thousand/mouse) or H1975 (200 ten thousand/mouse) is inoculated to the right armpit of an adult male BALB/c Nude mouse according to a conventional method.

Grouping: (1) LLC model: on the day of tumor bearing, the animals in each group are evenly grouped according to the weight, the number of the animals in each group of the model I is 10, and the number of the animals in each group of the model II is 6, and the number of the animals in each group of the model II is 4; (2) a549 model and H1975 model: measuring the long diameter and the wide diameter of the tumor by using a vernier caliper every three days after tumor loading according to the formula: tumor volume (mm) ³ ) =1/2 x long diameter (mm) x wide diameter (mm) ² Calculating the tumor volume until the tumor volume grows to 50mm ³ On the left and right, the a549 transplantation tumor model was divided into 4 groups according to the tumor volume, and the H1975 transplantation tumor model was divided into 7 groups according to the tumor volume, with the number of animals in each group being 7.

And (3) drug treatment: in all transplantable tumor models, the drug treatment dose and administration mode were: paclitaxel (PTX, 12mg/kg, once every two days, i.p.), cisplatin (CDDP, 5mg/kg, once a week, drug withdrawal after two weeks of treatment, i.p.), ocitinib (OSI, 5mg/kg, once a day, i.g.), STG (20 mg/kg, twice a day, i.g., S = STG below), FBP (500 mg/kg, twice a day, i.g., F = FBP below), piprine (50 mg/kg, once a day, i.g., a1= pine below), DHEA (60 mg/kg, once a day, i.g., a2= DHEA below), DSF (60 mg/kg, once a day, i.g., a3= DSF below), BEN (300 mg/kg, once a day, i.g., b1= BEN below), SOR (20 mg/kg, DHA, i.g., SOR, 2= b.g., below), and 3 g.g., DHA = b.g., DHA).

(1) LLC model I: (1) blank control group CON (purified water, i.g.), (2) positive control group PTX, (3) group of a1+ a2+ a3 (A = a1+ a2+ a 3), (4) group of A + b1+ b2, (5) group of A + b3, (6) group of A + b1+ b2+ b3 (B = B1+ B2+ B3); LLC model II: (1) blank control group CON (purified water, i.g.), (2) group A + B, (3) group S + F (C = SF) (FBP was administered 30min after STG), (4) group C + A + B; (2) a549 transplantable tumor model: (1) blank control group CON (purified water, i.g.), (2) SF (C) group (FBP was administered 30min after STG), (3) group of A + b3, (4) group of C + A + b3 (FBP was administered 30min after STG); (3) H1975 transplant tumor model: (1) blank control group CON (purified water, i.g.), (2) positive control group OSI, (3) positive control group CDDP, (4) SF (C) group (FBP administered 30min after STG), (5) group A + B, (6) group C + A + B (FBP administered 30min after STG), (7) group OSI + C + A + B (FBP administered 30min after STG). The dose per group was 10. Mu.L/g.

Index measurement: LLC model records weight and tumor volume once every 3 days, A549, H1975 and MDA-MB-231 models record weight and tumor volume once every 5 days, and reduce weight by more than 20% or increase tumor volume to 2000mm ³ The animal death endpoint was noted above.

Effects on LLC transplants:

the results are shown in fig. 12, fig. 13, fig. 14 and fig. 15, wherein, as shown in fig. 12-a and fig. 12-B, the tumor volumes of the compound whole formula a + B group, the compound component a + B1+ B2 group and the compound component a + B3 group are obviously smaller than those of the Control (CON) group, and the tumor inhibition effect of the compound whole formula a + B group is the most significant. The growth inhibition rate of the AB compound tumor reaches 81.3 percent, the tumor inhibition rate of the compound component A + b1+ b2 is 67.3 percent, the tumor inhibition rate of the compound component A + b3 is 25.7 percent, the tumor inhibition rate of the component A is only 14.8 percent, and the inhibition rate of the positive drug PTX group is 51.6 percent (table 7-1). In the aspect of prolonging the survival life of animals, the survival time of animals is prolonged remarkably by only 15.10d on average for CON group, by only 15.33d on average for A group, by 13.75d on average for A + b3 group, by 19.10d on average for PTX group, by 24.44 d on average for A + b1+ b2 group, by 25.00d on average for A + b1+ b2 group and by A + B group (FIG. 12-C); FIG. 12-D shows that the weight average of each treated group of animals increased compared to CON, while the weight increase was the slowest in the PTX group. Research results prove that the AB compound formula can obviously inhibit the growth of LLC transplanted tumors and prolong the life of animals, has better drug effect strength than the compound component A and the positive control drug PTX, and shows high safety.

As shown in FIGS. 13-A and 13-B, the AB cohort (A + B), SF (C), ABC cohort (C + A + B) all significantly inhibited tumor growth compared to the Control (CON) model control. At the end of the experiment (day 19 of treatment), the tumor inhibition rate of the ABC compound reaches 79.8%, the tumor inhibition rate of the AB compound is 61.8%, and the tumor inhibition rate of the SF group is 55.2% (Table 7-2). In addition, compared with the AB compound and the component C treatment groups, the ABC compound treatment group has smaller tumor individual difference, which shows that the ABC compound has more stable and reliable antitumor effect (figure 13-B). No weight loss was seen in each treatment group (FIG. 13-C). The results show that the FBP and the AB compound can generate better antitumor effect than the FBP and the AB compound, and support the antitumor application of the ABC compound formed by combining the FBP and the AB compound.

Effect on a549 transplantable tumor:

as shown in FIGS. 14-A and 14-B, tumor growth rates were significantly slower in the A + B3, SF (C), and C-associated A + B3 groups compared to the model control group Control (CON). Tumor growth to 2000mm ³ Model group was used for 47 days, and the 3 treatment groups were used for 72 days, 72 days and 117 days, respectively, and drug treatment was delayed by 1.5 times, 1.5 times and 2.5 times, respectively. Correspondingly, the life span of the treatment group is also significantly prolonged compared with that of CON group (FIG. 14-C), the average survival of CON group is only 57.00d, the average survival of SF (C) group is 79.50d, the average survival of A + b3 group is 81.14d, the average survival of C + A + b3 group is 117.00d, and the average survival of C + A + b3 group is prolonged by 60 days compared with that of CON group. The weight change in each group is shown in FIG. 14-D, and no weight loss was observed in the drug-treated group compared to the CON group. The results show that the FBP and AB compound component Ab3 has obvious antitumor effect on A549 xenograft tumors, and the combination of the FBP and AB compound component Ab3 can greatly improve the antitumor effect, and the research result also supports the antitumor application prospect of the ABC compound.

Effects on H1975 transplants:

15-A, 15-B, 15-C, and 15-D, model Control (CON) edema in the model control group on day 22Tumor growth to 2000mm ³ The SF (C) group has no drug effect, and the tumor volumes of the CDDP group, the OSI group, the AB compound group, the ABC compound (SF + A + B) group and the SOI + ABC compound group are all remarkably smaller. The CDDP group, the AB compound group and the ABC compound group are used for 32 days, 32 days and 42 days respectively, and the tumor grows to 2000mm ³ The tumor volume of OSI group and SOI + ABC compound group has not reached 2000mm ³ . Wherein the OSI group started to develop resistance trend at day 22, the OSI + ABC complex group still did not show significant resistance trend at day 62, and the tumor was in slow growth or even arrested growth state (fig. 15-E). Accordingly, as shown in fig. 15-F, animals in all treatment groups died within 62 days except the OSI group and OSI + ABC complex group, while no animal death was seen in OSI + ABC complex group at 62 days, significantly extending the life span of the animals, further confirming the antitumor advantage of the ABC complex in combination with the targeted drug OSI. The weight growth of each group is shown in fig. 15-G, except the group with CDDP had serious toxicity, no weight loss was seen in other groups, which indicates the in vivo safety of the ABC compound and each component.

The results show that the ABC compound is superior to CDDP in the drug effect and safety in the H1975 xenograft tumor model, can obviously delay the occurrence of OSI drug resistance, and shows clinical application value and wide application prospect for overcoming the drug resistance of targeted drugs

TABLE 7-1 tumor inhibition rates for each group of LLC transplanted tumor models

TABLE 7-2 tumor inhibition rates in groups of LLC transplanted tumor models

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (4)

1. An anti-tumor composition based on disruption of redox balance, comprising: NADPH regeneration inhibitors, GSH inhibitors and FBPs; wherein the NADPH regeneration inhibitor is a composition comprising piperine, dehydroepiandrosterone, and disulfiram; the GSH inhibitor is a composition of benserazide hydrochloride, sorafenib and dihydroartemisinin; and the mass ratio of any two components is 1 to 0.01-1.

2. The use of the composition of claim 1 for the preparation of an anti-neoplastic agent, wherein the neoplasm includes various solid tumors and hematological tumors; the composition can be used for resisting tumors alone or in combination with other treatments including targeted drugs, chemotherapy and radiotherapy, thereby playing roles in strengthening the cancer inhibition effect and resisting drug resistance.

3. The use of the composition according to claim 1 in the preparation of an anti-tumor medicament, wherein the medicament is prepared from the composition in combination with pharmaceutically acceptable excipients or carriers to prepare pharmaceutical preparations, including solid preparations and liquid preparations, and the preparations include oral preparations, injection preparations, suppositories, films, controlled-release bilayer tablets, controlled-release trilayer tablets, controlled-release nano preparations, microcapsule preparations, microsphere preparations and enteric preparations.

4. The use of a composition according to claim 1 for the preparation of a medicament for the treatment of tumors, wherein said medicament is administered orally or by injection or by nasal spray or by external application to the skin or by anal administration.

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