CN118717798A - A drug for improving the effect of tumor chemotherapy and application of hydrogen molecules in the preparation of a drug for improving the effect of tumor chemotherapy - Google Patents

Abstract

The present invention discloses a drug for improving the effect of tumor chemotherapy and the use of hydrogen molecules in the preparation of drugs for improving the effect of tumor chemotherapy. The drug includes hydrogen molecules or hydrogen molecule source materials. The present invention combines hydrogen molecules with different chemotherapy drugs to increase the sensitivity of tumors to drugs, alleviate tumor drug resistance, promote the function of tumor drugs to inhibit tumor growth, and enable many tumor drugs that have lost their function due to drug resistance to be reapplied to tumor treatment.

Description

A drug for improving the effect of tumor chemotherapy and the use of hydrogen molecules in the preparation of drugs for improving the effect of tumor chemotherapy

Technical Field

The present invention belongs to the field of medical technology, and specifically relates to a drug for improving the effect of tumor chemotherapy and the application of hydrogen molecules in the preparation of a drug for improving the effect of tumor chemotherapy.

Background Art

At present, the standard treatment for hematological tumors and solid tumors is surgical resection combined with radiation and chemotherapy. For most malignant tumors, drug resistance is very easy to develop during the treatment process, making it difficult for tumor drugs to continue to inhibit the proliferation of tumor cells, or tumor metastasis occurs, making most chemotherapy drugs have little effect during the treatment process, and no widely used combined drug therapy method for auxiliary promotion of chemotherapy drugs has been found. At the same time, in recent decades, with the discovery and research of tumor stem cells, due to their unlimited proliferation, self-renewal ability and multidirectional differentiation potential, they have become the cause of tumor occurrence, drug resistance and metastasis. There is currently no clear treatment for tumor stem cells. Therefore, in actual treatment, improving the sensitivity of tumor cells to drugs and developing new treatment methods for tumor stem cells can fundamentally make most tumor drugs that have already developed drug resistance have therapeutic effects again.

Summary of the invention

The purpose of the present invention is to solve the above technical problems and provide a drug for improving the effect of tumor chemotherapy and the use of hydrogen molecules in the preparation of drugs for improving the effect of tumor chemotherapy. By using chemotherapy drugs in combination with hydrogen molecules, the sensitivity of tumor cells to chemotherapy drugs can be increased, tumor proliferation can be inhibited, and most tumor chemotherapy drugs that have developed drug resistance can have anti-tumor function again, promote the differentiation of tumor stem cells, reduce the malignancy of tumors, and improve the therapeutic effect of chemotherapy drugs.

A drug for improving the effect of tumor chemotherapy, comprising hydrogen molecules or hydrogen molecule source materials.

Preferably, the product form of the drug is one or more of gas, aerosol, solution, emulsion, foam, gel, colloidal dispersion, liposome, droplet, microbubble, paste, suppository, chewing gum, granule, pill, powder, lozenge, capsule, microcapsule.

Preferably, the medicine further comprises oxygen and/or medically acceptable pharmaceutical excipients.

Application of hydrogen molecules in the preparation of drugs for improving the effect of tumor chemotherapy.

Preferably, the hydrogen molecules are used in the preparation of drugs for reducing tumor chemotherapy resistance.

Preferably, the method for applying hydrogen molecules is to administer the drug before the tumor chemotherapy drug is administered, or to continuously administer the drug before, during or after the tumor chemotherapy drug is administered.

Preferably, the administration of hydrogen molecules includes one or more of respiratory administration, oral administration, injection administration or local administration.

Preferably, the tumor includes one or more of blood tumors, glioblastoma, liver cancer, lung cancer, gastric cancer, kidney cancer, breast cancer, nasopharyngeal carcinoma and melanoma;

The tumor chemotherapy drugs include cytotoxic drugs temozolomide, fluorouracil and cyclophosphamide, metabolic drugs metformin and letrozole, kinase inhibitor drugs sorafenib and trametinib, new anti-tumor drug TRAIL, immunotherapy drugs PD-1, cisplatin, dacarbazine, paclitaxel, methotrexate, vincristine, or one or more of the monoclonal antibody drugs bevacizumab, nimotuzumab and ramucirumab.

Preferably, the hydrogen molecules are administered by breathing, and the volume concentration of hydrogen molecules in the breathing gas is greater than 5% and less than 99%.

Preferably, when the tumor is glioblastoma, and:

When the chemotherapy drug is temozolomide, the temozolomide chemotherapy dosage is 2-3 mg/kg;

When the chemotherapy drug is TRAIL, the chemotherapy dosage of TRAIL is 4 to 6 mg/kg;

When the chemotherapy drug is metformin, the metformin chemotherapy dosage is 70-80 mg/kg;

When the chemotherapy drug is bevacizumab, the chemotherapy dosage of bevacizumab is 4 to 6 mg/kg;

When the tumor is liver cancer, lung cancer, stomach cancer or kidney cancer, and:

When the chemotherapy drug is sorafenib, the chemotherapy dosage of sorafenib is 25-35 mg/kg;

When the chemotherapy drug is fluorouracil, the chemotherapy dosage of fluorouracil is 15 to 25 mg/kg;

When the chemotherapy drug is nimotuzumab or bevacizumab, the chemotherapy dosage of nimotuzumab is 0.4-0.6 mg/kg, and the chemotherapy dosage of bevacizumab is 2-3 mg/kg;

When the chemotherapy drug is PD-1, the PD-1 chemotherapy dosage is 8 to 12 mg/kg;

When the chemotherapy drug is cyclophosphamide, the cyclophosphamide chemotherapy dosage is 25-35 mg/kg;

When the chemotherapy drug is cisplatin, the chemotherapy dosage of cisplatin is 1.5 to 2.5 mg/kg;

When the tumor is breast cancer, and:

When the chemotherapy drug is letrozole, the chemotherapy dosage of letrozole is 4 to 6 mg/kg;

When the chemotherapy drug is fluorouracil, the chemotherapy dosage of fluorouracil is 15 to 25 mg/kg;

When the chemotherapy drug is bevacizumab, the chemotherapy dosage of bevacizumab is 2 to 3 mg/kg;

When the chemotherapy drug is PD-1, the PD-1 chemotherapy dosage is 1.5 to 2.5 mg/kg;

When the tumor is melanoma, and:

When the chemotherapy drug is temozolomide, the temozolomide chemotherapy dosage is 8 to 12 mg/kg;

When the chemotherapy drug is dacarbazine, the dacarbazine chemotherapy dosage is 65-75 mg/kg;

When the chemotherapy drug is PD-1, the PD-1 chemotherapy dosage is 1.5 to 2.5 mg/kg;

When the chemotherapy drug is cisplatin, the chemotherapy dosage of cisplatin is 3.5-4.5 mg/kg;

When the tumor is nasopharyngeal carcinoma, and:

When the chemotherapy drug is cisplatin, the chemotherapy dosage of cisplatin is 10 to 20 mg/kg;

When the chemotherapy drug is fluorouracil, the chemotherapy dosage of fluorouracil is 35-45 mg/kg;

When the chemotherapy drug is paclitaxel, the chemotherapy dosage of paclitaxel is 5 to 15 mg/kg;

When the tumor is leukemia, and:

When the chemotherapy drug is cyclophosphamide, the cyclophosphamide chemotherapy dosage is 195-205 mg/kg;

When the chemotherapy drug is methotrexate, the methotrexate chemotherapy dosage is 95-105 mg/kg;

When the chemotherapy drug is vincristine, the chemotherapy dosage of vincristine is 145-155 ug/kg.

At present, many chemotherapy drugs are prone to drug resistance during clinical treatment, resulting in the inability of the drugs to kill tumors. The present invention combines hydrogen molecules with different chemotherapy drugs, which increases the sensitivity of tumors to drugs, alleviates tumor resistance, promotes the function of tumor drugs and thus inhibits tumor growth. At the same time, it can enable many tumor drugs that have lost their function due to drug resistance to be re-applied to tumor treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the therapeutic effect of hydrogen combined with TMZ administration in Example 1 of the present invention in a mouse GBM subcutaneous inoculation model, specifically a graph showing the change in tumor volume during the 15-day administration process.

FIG. 2 is a graph showing the detection of apoptosis levels of tumor cells in a brain glioma model treated with hydrogen molecules combined with TMZ in Example 1 of the present invention (TUNEL staining).

FIG3 is a diagram showing the effect of hydrogen molecules combined with TRAIL in treating brain glioma in Example 1 of the present invention.

FIG. 4 is a diagram showing the effect of hydrogen molecules combined with different anti-tumor drugs in Example 1 of the present invention on treating brain glioma.

Figure 5 is a diagram showing the effect of hydrogen molecules combined with sorafenib in treating liver cancer in Example 2 of the present invention, wherein the left side shows tumor images of mice in different groups (the upper and lower rows of tissues are samples of the same group with the same treatment), and the right side shows the change in tumor volume during the 15-day drug administration process.

FIG6 is a diagram showing the effect of hydrogen molecules combined with different anti-tumor drugs on liver cancer in Example 2 of the present invention;

FIG7 is a diagram showing the effect of hydrogen molecules combined with different anti-tumor drugs in treating lung cancer according to Example 3 of the present invention;

FIG8 is a diagram showing the effect of hydrogen molecules combined with different anti-tumor drugs in treating gastric cancer according to Example 4 of the present invention;

FIG9 is a diagram showing the effect of hydrogen molecules combined with different anti-tumor drugs in treating renal cancer according to Example 5 of the present invention;

FIG10 is a diagram showing the effect of hydrogen molecules combined with different anti-tumor drugs in treating breast cancer according to Example 6 of the present invention;

FIG11 is a diagram showing the effect of hydrogen molecules combined with different anti-tumor drugs in treating melanoma according to Example 7 of the present invention;

FIG12 is a graph showing the inhibitory effect of hydrogen molecules combined with TMZ on brain glioma cells in Example 8 of the present invention;

FIG13 is the immunofluorescence staining result of cells treated with hydrogen molecules combined with TMZ in Example 8 of the present invention;

FIG. 14 shows the growth inhibitory effect of hydrogen molecules combined with metformin on brain glioma cells according to Example 8 of the present invention.

FIG15 is a diagram showing the effect of hydrogen molecules combined with different anti-tumor drugs on nasopharyngeal carcinoma in Example 9 of the present invention;

FIG16 is a diagram showing the effect of hydrogen molecules combined with different anti-tumor drugs in treating leukemia according to Example 10 of the present invention;

FIG17 is a diagram showing the therapeutic effects of different hydrogen administration methods on a glioblastoma animal model according to Example 11 of the present invention;

FIG18 is a hydrogen production curve of the hydrogen patch in the local hydrogen supply method of Example 11 of the present invention;

FIG19 is a diagram showing the therapeutic effects of different hydrogen concentrations on a glioblastoma animal model according to Example 12 of the present invention;

FIG20 is a diagram showing the therapeutic effects of different hydrogen administration times on liver cancer animal models according to Example 13 of the present invention;

FIG. 21 is a diagram showing the therapeutic effect of different hydrogen administration times on a leukemia animal model according to Example 14 of the present invention.

DETAILED DESCRIPTION

As used herein, the term "therapeutically effective amount" is an amount sufficient to affect a desired biological effect, such as a beneficial outcome, including a clinical outcome.

The present application provides a drug for improving the effect of tumor chemotherapy, including hydrogen molecules or hydrogen molecule source materials, wherein the hydrogen molecules or hydrogen molecule source materials can be hydrogen, gas containing hydrogen, hydrogen-rich water or beverages rich in hydrogen molecules, solid hydrogen storage materials that can generate hydrogen molecules when used, etc.

Many chemotherapy drugs are prone to drug resistance during clinical treatment, resulting in the inability of the drugs to kill tumors. After research, the applicant found that the use of hydrogen molecules in combination with chemotherapy drugs can increase the sensitivity of tumors to drugs, alleviate tumor resistance, promote the effectiveness of tumor drugs and thus inhibit tumor growth, promote the killing effect of chemotherapy drugs on tumors, and alleviate chemotherapy side effects.

Therefore, the hydrogen molecules of the present invention can be widely used as an auxiliary treatment method for patients with various blood tumors and solid tumors during chemotherapy treatment. When used in combination with chemotherapy drugs, it can increase the sensitivity of tumors to drugs, alleviate tumor resistance, enhance the therapeutic effect of chemotherapy drugs, promote the killing effect of chemotherapy drugs on tumors, and alleviate chemotherapy side effects. At the same time, it can enable many tumor drugs that have lost their function due to drug resistance to be re-applied to tumor treatment.

Preferably, the product form of the above-mentioned drug includes but is not limited to one or more of gas, aerosol, solution, emulsion, foam, gel, colloidal dispersion, liposome, droplet, microbubble, paste, suppository, chewing gum, granule, pill, powder, lozenge, capsule, microcapsule.

Preferably, the medicine may also include oxygen and/or medically acceptable pharmaceutical excipients, etc. Oxygen may be mixed with hydrogen for use.

The administration of hydrogen molecules includes, but is not limited to, one or more of respiratory administration, oral administration, injection administration or local administration.

The method of administering hydrogen molecules is to administer them before taking tumor chemotherapy drugs, or to continuously administer them before, during or after taking tumor chemotherapy drugs.

Tumors include, but are not limited to, one or more of blood tumors, glioblastoma, liver cancer, lung cancer, gastric cancer, kidney cancer, breast cancer, nasopharyngeal carcinoma, and melanoma;

Tumor chemotherapy drugs include, but are not limited to, cytotoxic drugs temozolomide, fluorouracil and cyclophosphamide, metabolic drugs metformin and letrozole, kinase inhibitor drugs sorafenib and trametinib, new anti-tumor drug TRAIL, immunotherapy drugs PD-1, cisplatin, dacarbazine, paclitaxel, methotrexate, vincristine, or one or more of the monoclonal antibody drugs bevacizumab, nimotuzumab and ramucirumab.

Furthermore, after research, it is preferred that hydrogen molecules be administered by breathing, with the volume concentration of hydrogen molecules in the breathed gas being greater than 5% and less than 99%.

For different types of tumors, different types and different doses of chemotherapy drugs, the effective amount of hydrogen will also change accordingly. After research, this application found that:

When the tumor is a glioblastoma and:

When the chemotherapy drug is temozolomide, the temozolomide chemotherapy dosage is 2-3 mg/kg;

When the chemotherapy drug is TRAIL, the TRAIL chemotherapy dosage is 4 to 6 mg/kg;

When the chemotherapy drug is metformin, the metformin chemotherapy dosage is 70-80 mg/kg;

When the chemotherapy drug is bevacizumab, the dosage of bevacizumab chemotherapy is 4 to 6 mg/kg;

Further, when the tumor is liver cancer, lung cancer, stomach cancer or kidney cancer, and:

When the chemotherapy drug is sorafenib, the chemotherapy dosage of sorafenib is 25-35 mg/kg;

When the chemotherapy drug is fluorouracil, the chemotherapy dosage of fluorouracil is 15-25 mg/kg;

When the chemotherapy drug is nimotuzumab or bevacizumab, the dosage of nimotuzumab chemotherapy is 0.4-0.6 mg/kg, and the dosage of bevacizumab chemotherapy is 2-3 mg/kg;

When the chemotherapy drug is PD-1, the dosage of PD-1 chemotherapy is 8 to 12 mg/kg;

When the chemotherapy drug is cyclophosphamide, the dosage of cyclophosphamide chemotherapy is 25-35 mg/kg;

When the chemotherapy drug is cisplatin, the chemotherapy dosage of cisplatin is 1.5 to 2.5 mg/kg.

Further, when the tumor is breast cancer, and:

When the chemotherapy drug is letrozole, the chemotherapy dosage of letrozole is 4 to 6 mg/kg;

When the chemotherapy drug is fluorouracil, the chemotherapy dosage of fluorouracil is 15-25 mg/kg;

When the chemotherapy drug is bevacizumab, the dosage of bevacizumab chemotherapy is 2 to 3 mg/kg;

When the chemotherapy drug is PD-1, the dosage of PD-1 chemotherapy is 1.5 to 2.5 mg/kg.

When the tumor is melanoma and:

When the chemotherapy drug is temozolomide, the temozolomide chemotherapy dosage is 8-12 mg/kg;

When the chemotherapy drug is dacarbazine, the dacarbazine chemotherapy dosage is 65-75 mg/kg;

When the chemotherapy drug is PD-1, the dosage of PD-1 chemotherapy is 1.5 to 2.5 mg/kg;

When the chemotherapy drug is cisplatin, the chemotherapy dosage of cisplatin is 3.5 to 4.5 mg/kg.

When the tumor is nasopharyngeal carcinoma and:

When the chemotherapy drug is cisplatin, the dosage of cisplatin chemotherapy is 10-20 mg/kg;

When the chemotherapy drug is fluorouracil, the chemotherapy dosage of fluorouracil is 35-45 mg/kg;

When the chemotherapy drug is paclitaxel, the paclitaxel chemotherapy dosage is 5 to 15 mg/kg;

When the tumor is leukemia and:

When the chemotherapy drug is cyclophosphamide, the cyclophosphamide chemotherapy dosage is 195-205 mg/kg;

When the drug is methotrexate, the methotrexate chemotherapy dosage is 95 to 105 mg/kg;

When the chemotherapy drug is vincristine, the chemotherapy dosage of vincristine is 145-155ug/kg.

The following is a further explanation of the new application of hydrogen molecules in the preparation of drugs for improving the effect of tumor chemotherapy in combination with specific experiments.

Example 1 Therapeutic effect of hydrogen molecules combined with different anti-tumor drugs in a glioblastoma animal model

Glioblastoma (GBM) is the most malignant brain tumor with an extremely low survival rate and high recurrence rate. The alkylating agent temozolomide (TMZ) is currently the first-line drug for the treatment of brain tumors. However, drug resistance is easily generated during treatment, especially in the clinical treatment of GBM.

TRAIL can selectively induce apoptosis of tumor cells without obvious side effects. Therefore, TRAIL has a good prospect in the treatment of tumors. In vitro, soluble TRAIL can rapidly induce apoptosis of tumor cells, but is insensitive to normal tissue cells.

With the continuous development of medicine, the changes in the spectrum of human diseases, the gradual clarification of disease mechanisms and the changes in medical concepts, people have a new understanding of the efficacy of many classic drugs. "New uses of old drugs" has become a hot topic in the current medical research community. The representative drug is metformin. Metformin is a semi-synthetic oral hypoglycemic drug of the biguanide class, mainly used to treat type 2 diabetes, especially obese people. In recent years, the newly discovered metformin has anti-tumor effects, and a large number of related studies have emerged. Metformin can inhibit the proliferation, migration and invasion of tumor cells and induce cell apoptosis. The genes and signal transduction pathways involved are diverse and cross-linked with each other. In addition, the tumor microenvironment is the internal environment in which tumor cells are produced and live, which includes not only the tumor cells themselves, but also various cells such as fibroblasts, immune and inflammatory cells, and glial cells around them, as well as the interstitial cells and microvessels in the nearby area, as well as cytokines and inflammatory factors. The tumor microenvironment has three major characteristics: hypoxia, chronic inflammation and immunosuppression, and plays an important role in the occurrence, development and drug resistance of tumors. Metformin can exert its anti-tumor effect by improving the microenvironment of hypoxia, chronic inflammation and immunosuppression.

In recent years, the research and application of anti-angiogenic drugs have provided more options for the treatment of glioblastoma. The pathological histological characteristics of glioblastoma are microvascular proliferation, and the tumor tissue expresses high levels of pro-angiogenic factors. In 1971, Folkman first proposed the hypothesis that inhibiting angiogenesis would be an effective anti-cancer treatment for glioblastoma. Bevacizumab is a recombinant humanized monoclonal antibody that targets the VEGF-A receptor. Bevacizumab can improve the patient's progression-free survival. In 2009, the US Food and Drug Administration approved it for the treatment of recurrent glioblastoma.

This application conducted a hydrogen molecule combined with the above-mentioned drug chemotherapy experiment, the details are as follows:

(1) Therapeutic effect of hydrogen molecules combined with temozolomide (TMZ) on mouse glioblastoma model

Eight-week-old male C57BL/6N mice weighing 18-22 g were selected for the experiment. Cells of mouse GL261 malignant glioblastoma grown under normal culture conditions were collected and inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the GBM model mice were randomly divided into a control group (CTRL), a hydrogen breathing group (H ₂ ), a single drug group (TMZ), a hydrogen breathing group after temozolomide administration (TMZ+H ₂ ), and a temozolomide administration group after hydrogen breathing (H ₂ +TMZ). The TMZ drug concentration was 2.5 mg/kg (2.5 mg TMZ per kg of mouse body weight), and the control group was given an equal weight of normal saline. All experimental mice were housed at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the H ₂ group, TMZ+H ₂ group and H ₂ +TMZ group breathed hydrogen for 2 hours every day. The breathing environment of the mice contained 66% hydrogen (volume concentration, and the remaining gas contained 33% oxygen). During the hydrogen breathing process, the mice could move freely in the breathing box.

The administration cycle was 15 days. During and after the administration, the mouse tumors were collected to observe the changes in tumor volume and the level of tumor cell apoptosis. The specific results are shown in Figures 1-2.

The results in Figure 1 show that, unlike the result that breathing hydrogen before the tumor is formed can inhibit tumor growth, breathing hydrogen alone can no longer significantly inhibit tumor growth after the tumor is formed. Drug resistance begins to appear after 6 to 9 days of TMZ use, and the tumor begins to grow. Although breathing hydrogen combined with drug administration has a certain effect of reducing tumor volume, the improvement effect is not significant. Breathing hydrogen and then using TMZ significantly reduced the volume of the generated tumor, and has a significant anti-resistance effect. Compared with using TMZ alone, the tumors formed subcutaneously in mice treated with hydrogen after breathing hydrogen are significantly reduced (p=0.0003), proving that the treatment effect of hydrogen molecule treatment combined with TMZ is the best.

As shown in Figure 2, the statistical results show that the combination with hydrogen molecules can significantly increase the killing effect of TMZ on tumor cells.

(2) The therapeutic effect of hydrogen molecules combined with trail in the treatment of glioblastoma animal models

A mouse GL261 tumor stem cell subcutaneous GBM tumor model was constructed according to the above method. After three weeks, the mouse subcutaneous tumor grew to an appropriate size and was randomly divided into three groups, namely, a control group, a single drug group, and a hydrogen molecule combined drug group. The TRAIL administration concentration of the single drug group and the hydrogen molecule combined drug group was 5 mg/kg. Before administration, the mice in the hydrogen molecule combined drug group breathed hydrogen in the respiratory environment for 2 hours (hydrogen volume concentration 66%). During and after the administration, the mouse tumors were taken to observe the changes in tumor volume. The specific results are shown in Figure 3.

As shown in Figure 3, after the tumor is formed, the use of TRAIL to treat the tumor has a certain effect, and when combined with hydrogen molecules, the effect of TRAIL in inducing tumor cell apoptosis is greatly enhanced, tumor proliferation is slowed down, and tumor volume is significantly inhibited.

(3) The therapeutic effect of hydrogen molecules combined with other anti-tumor drugs on glioblastoma animal models

Metformin and bevacizumab were tested with reference to the above method. The metformin concentration was 75 mg/kg and the bevacizumab concentration was 5 mg/kg. The results are shown in Figure 4. The combination of hydrogen molecules with metformin and bevacizumab can significantly or extremely significantly enhance the tumor inhibition effect of anti-tumor drugs. After 15 days of administration to the subcutaneous animal model of mouse glioblastoma, the tumor tissue volume was significantly lower than that of the single drug group.

Example 2 Therapeutic effect of hydrogen molecules combined with different anti-tumor drugs on liver cancer animal models

Liver cancer is a highly malignant tumor with a high incidence in my country and a low survival rate of patients. As a first-line drug for the treatment of liver cancer, sorafenib is a multi-target kinase inhibitor that can simultaneously inhibit multiple intracellular kinases that exist in tumor cells and are involved in tumor cell signal transduction, angiogenesis and apoptosis, and can also inhibit cell surface kinases.

Fluorouracil has been synthesized for more than 60 years and has a wide anti-tumor spectrum. It is still a basic therapeutic drug for many malignant tumors, including digestive system tumors. 5-fluorouracil (5-FU) is a cell cycle-specific drug that acts on the S phase of cells. Its action is time-dependent. Continuous intravenous infusion can improve the efficacy, and it has a synergistic effect when combined with calcium folinate. Nimotuzumab targets the molecule-EGFR. EGFR (Epidermal Growth Factor Receptor) is a glycoprotein receptor that can be expressed or overexpressed with normal epithelial or epithelial cancer tissue cells. Preclinical studies have shown that blocking EGFR can stop tumor growth. EGFR tyrosine kinase activity can be selectively inhibited by drugs or competitively blocked by monoclonal antibodies from the extracellular ligand binding site. Nimotuzumab is an IgG1 humanized monoclonal antibody (Mab) that specifically binds to the antigen epitope of the extracellular domain of EGFR, preventing its activation and blocking cells in the G1 phase. PD-1/PD-L1 immune checkpoint inhibitors (ICI) have shown good efficacy and safety in clinical trials of monotherapy or combination therapy for liver cancer, providing a favorable scientific basis for their clinical application. PD-1/PD-L1 ICIs for monotherapy of liver cancer include nivolumab, pembrolizumab, and camrelizumab, which are mainly used for second-line treatment of liver cancer. Although some liver cancer patients can have a lasting response to PD-1/PD-L1 ICI monotherapy, the overall number of patients who benefit is still small. PD-1/D-L1 ICI combination therapy shows better immune response and control.

This application conducted a hydrogen molecule combined with the above-mentioned drug chemotherapy experiment, the details are as follows:

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. HepG2 hepatocellular carcinoma cells grown under normal culture conditions were collected and inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group (CTRL), a single drug group (Sorafenib), and a group that breathed hydrogen and then administered Sorafenib (H ₂ +Sorafenib). They were administered continuously for 15 days after 15 days of tumor growth (Sorafenib). Sorafenib was administered by gavage at a drug concentration of 30 mg/kg. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the _{H 2} +Sorafenib group breathed hydrogen for 2 hours before administration every day. The mouse breathing environment contained 66% hydrogen. The mice could move freely in the breathing box during the hydrogen breathing process. During and after the administration, the mouse tumors were taken to observe the changes in tumor volume. The specific results are as follows:

As shown in Figure 5, after using sorafenib alone to treat tumor-bearing mice, the growth of subcutaneous tumors in mice was significantly inhibited and the proliferation rate was reduced. After hydrogen molecules were used in combination with sorafenib, the subcutaneous tumors in mice showed a decreasing trend. The results showed that the combination of hydrogen molecules significantly increased the effect of sorafenib in inhibiting tumor proliferation, inhibited tumor formation, and had a therapeutic effect on tumors.

Referring to the above method, experiments were conducted on fluorouracil (dosing concentration was 20 mg/Kg), nimotuzumab (dosing concentration was 0.5 mg/Kg), sorafenib (dosing concentration was 30 mg/Kg) and PD-1 (dosing concentration was 10 mg/Kg). The results are shown in Figure 6.

As shown in Figure 6, the use of fluorouracil, sorafenib and PD-1 alone inhibited tumor growth to a certain extent compared with the control group, but there was no significant change. After combined use with hydrogen molecules, significant changes occurred. Compared with the control group, the use of nimotuzumab alone can significantly inhibit tumor proliferation (p < 0.01), but after combined use with hydrogen molecules, the inhibitory effect is stronger than that of the control group (p < 0.001), proving that the combined use of hydrogen molecules can increase the effect of the above drugs in inhibiting tumor proliferation.

Example 3 The therapeutic effect of hydrogen molecules combined with different anti-tumor drugs on lung cancer animal models

Lung cancer is a malignant tumor that originates from the lung bronchial mucosa and glandular epithelium. It is one of the common tumors of the respiratory system. At present, modern medicine mainly treats lung cancer with radiotherapy, chemotherapy, surgery, molecular targeted therapy, etc. Although it has achieved certain therapeutic effects, it is often because it only targets the cancer lesions and rarely improves the patient's overall systemic condition. Nimotuzumab, sorafenib, and PD-1 are also often used in the treatment of lung cancer.

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. After A549 lung cancer cells grown under normal culture conditions were collected, they were inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group (CTRL), a single-drug group (Drug), and a hydrogen breathing group (H ₂ +Drug). The drugs were administered for 15 consecutive days after 15 days of tumor growth. The drug concentrations of the single-drug group (Drug) and the hydrogen breathing group were: 0.5 mg/kg for nimotuzumab, 30 mg/kg for sorafenib, and 10 mg/kg for PD-1. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the _H2 +Drug group breathed hydrogen for 2 hours every day before administration, and the breathing environment of the mice contained 66% hydrogen. The mice were able to move freely in the breathing box during the hydrogen breathing process. After the administration, the mouse tumors were taken and the changes in tumor volume were observed. The specific results are as follows:

As shown in Figure 7, compared with the control group, the use of Nimotuzumab and Sorafenib alone inhibited tumor growth to a certain extent, but there was no significant change. After combined use with hydrogen molecules, significant changes occurred. Compared with the control group, the use of PD-1 alone can significantly inhibit tumor proliferation (p < 0.05), but after combined use with hydrogen molecules, the inhibitory effect is stronger than that of the control group (p < 0.01), proving that the combined use of hydrogen molecules can increase the effect of the above drugs in inhibiting tumor proliferation.

Example 4 Therapeutic effect of hydrogen molecules combined with different anti-tumor drugs on gastric cancer animal models

Gastric cancer is one of the common malignant tumors. About 2/3 of gastric cancers are already in the advanced stage when they are first treated. Even after radical surgery, many patients will experience local recurrence and/or distant metastasis. Chemoradiotherapy is a commonly used treatment method, but gastric cancer patients who fail first-line and second-line chemotherapy regimens often find it difficult to benefit from chemotherapy, and radiotherapy is only a local treatment method. Molecular targeted therapy is an important treatment method for advanced gastric cancer. Molecular targeted therapy targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) can prolong the overall survival of advanced gastric cancer. Drugs targeting HGF/c-Met, PI3K, PARP, FGFR2b and other targets have been clinically tested. Fluorouracil, cisplatin, and PD-1 are also often used in the treatment of gastric cancer.

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. MGC-803 gastric cancer cells grown under normal culture conditions were collected and inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the mice were waited for the tumor to grow. After the tumor grew, the mice were randomly divided into a control group (CTRL), a single drug group (Drug), and a hydrogen breathing group (H ₂ +Drug). The drugs were administered for 15 consecutive days after 15 days of tumor growth. The drug concentrations of the single drug group (Drug) and the hydrogen breathing group were: 20 mg/kg of fluorouracil, 2 mg/kg of cisplatin, and 10 mg/kg of PD-1. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the _H2 +Drug group breathed hydrogen for 2 hours every day before administration, and the breathing environment of the mice contained 66% hydrogen. The mice were able to move freely in the breathing box during the hydrogen breathing process. After the administration, the mouse tumors were taken and the changes in tumor volume were observed. The specific results are as follows:

As shown in Figure 8, although fluorouracil alone inhibited tumor growth to a certain extent compared with the control group, there was no significant change. After combined use with hydrogen molecules, there was a very significant change (p < 0.01). Cisplatin alone can significantly inhibit tumor proliferation compared with the control group (p < 0.05), but after combined use with hydrogen molecules, the inhibitory effect is stronger than the control group (p < 0.01). PD-1 alone can significantly inhibit tumor proliferation compared with the control group (p < 0.05), but after combined use with hydrogen molecules, the inhibitory effect is stronger than the control group (p < 0.001). The above results prove that the combination of hydrogen molecules can increase the effect of the above drugs in inhibiting tumor proliferation.

Example 5 The therapeutic effect of hydrogen molecules combined with different anti-tumor drugs on renal cancer animal models

Renal cell carcinoma (Rcc), also known as renal cancer, is one of the common malignant tumors of the urinary system. Many renal cancers still have no obvious symptoms in the late stage, and only about 20% of patients have the classic three major symptoms (hematuria, abdominal mass, and pain). 50% of patients are accidentally discovered through imaging examinations during physical examinations. At the time of diagnosis, about 16% of renal cancer has already metastasized. Most patients can only use palliative treatment, and their prognosis is poor, with a 5-year survival rate of less than 10%. Therefore, actively looking for sensitive markers for renal cancer plays an important role in the diagnosis of early and micro-renal cancer, which has always been the focus of renal cancer research. Cyclophosphamide is an alkylating immunosuppressant with a strong and lasting effect. In the clinical treatment of kidney disease, it is one of the immunosuppressants with a longer use time, but the side effects are also more obvious. Bevacizumab, sorafenib, and PD-1 are also often used in the treatment of renal cancer.

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. GRC-1 renal cancer cells grown under normal culture conditions were collected and inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group (CTRL), a single-drug group (Drug), and a hydrogen breathing group (H ₂ +Drug). The drugs were administered for 15 consecutive days after 15 days of tumor growth. The drug concentrations of the single-drug group and the hydrogen breathing group were: cyclophosphamide 30 mg/kg, bevacizumab 2.5 mg/kg, sorafenib 30 mg/kg, and PD-1 10 mg/kg. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the _H2 +Drug group breathed hydrogen for 2 hours before daily administration, and the breathing environment of the mice contained 66% hydrogen. The mice were able to move freely in the breathing box during the hydrogen breathing process. After the administration, the mouse tumors were taken and the changes in tumor volume were observed. The specific results are as follows:

As shown in Figure 9, the use of cyclophosphamide, bevacizumab, sorafenib and PD-1 alone inhibited tumor growth to a certain extent compared with the control group, but there was no significant change (p>0.05). After combined use with hydrogen molecules, the cyclophosphamide, bevacizumab, and PD-1 combined group produced extremely significant changes (p<0.01), and the sorafenib combined group produced significant changes (p<0.05). The above results prove that the combination of hydrogen molecules can increase the effect of the above drugs in inhibiting the proliferation of renal cancer.

Example 6 The therapeutic effect of hydrogen molecules combined with different anti-tumor drugs on breast cancer animal models

Breast cancer is the most common malignant tumor in women, which seriously threatens women's health and life. According to the latest global cancer burden data in 2020 released by the World Health Organization's International Agency for Research on Cancer, the number of new cases of breast cancer in the world is growing rapidly, and it has replaced lung cancer as the world's number one cancer. The occurrence and development of breast cancer is a pathological process in which multiple factors participate, including abnormal activation of oncogenes, loss of tumor suppressor gene function, abnormal activation or inactivation of signaling pathways, etc. In addition to changes in the biological activities of breast cancer cells themselves, information exchange and material exchange between cancer cells and other cells of the body are also important factors involved in regulating the occurrence and development of breast cancer. Letrozole is a third-generation aromatase inhibitor and the most commonly used adjuvant endocrine therapy drug in clinical practice. Letrozole has a highly selective effect and can strongly inhibit estrogen levels, while having little effect on corticosteroids, aldosterone, and adrenocortical hormones. Therefore, compared with anti-estrogen drugs, letrozole has a stronger anti-tumor effect. Because its action is more targeted and has no potential toxicity to various systems and target organs in the body, it has good safety and is tolerated by most patients. Letrozole was approved for marketing in the United States in 2001 and is currently recommended as an adjuvant endocrine therapy drug in more than 20 countries around the world. Currently, China has used letrozole as the first-line and second-line drug of choice for postmenopausal patients with advanced breast cancer, as well as the first-line adjuvant endocrine therapy drug of choice for metastatic breast cancer. Fluorouracil, bevacizumab, and PD-1 are also frequently used in the treatment of breast cancer.

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. MCF-7 breast cancer cells grown under normal culture conditions were collected and inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group (CTRL), a single drug group (Drug), and a hydrogen breathing group (H ₂ +Drug). The drugs were administered for 15 consecutive days after 15 days of tumor growth. The drug concentrations of the single drug group and the hydrogen breathing group were: letrozole 5 mg/kg, fluorouracil 20 mg/kg, bevacizumab 2.5 mg/kg, and PD-1 2 mg/kg. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the _H2 +Drug group breathed hydrogen for 2 hours before daily administration, and the breathing environment of the mice contained 66% hydrogen. The mice were able to move freely in the breathing box during the hydrogen breathing process. After the administration, the mouse tumors were taken and the changes in tumor volume were observed. The specific results are as follows:

As shown in Figure 10, the use of fluorouracil, letrozole, bevacizumab, and PD-1 alone inhibited tumor growth to a certain extent compared with the control group, but there was no significant change (p>0.05). After combined use with hydrogen molecules, the fluorouracil combination group produced a very significant change (p<0.01), and the letrozole, bevacizumab and PD-1 combination group produced a significant change (p<0.05). The above results prove that the combination of hydrogen molecules can increase the effect of the above drugs in inhibiting breast cancer proliferation.

Example 7 The therapeutic effect of hydrogen molecules combined with different anti-tumor drugs on melanoma animal models

Malignant melanoma (MM) is a tumor that arises from melanocytes in the skin and other organs and is one of the most serious types of skin cancer. Although the 5-year relative survival rate for all MM is 92%, the 5-year relative survival rates for local lesions and distant metastases are only 65% and 25%, respectively. Up to 4.3% of melanoma patients will develop metastasis. Targeted therapy for melanoma has always been a research hotspot.

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. A375 melanoma cells grown under normal culture conditions were collected and inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group (CTRL), a single-drug group (Drug), and a hydrogen breathing group (H ₂ +Drug). The drugs were administered for 15 consecutive days after 15 days of tumor growth. The drug concentrations of the single-drug group and the hydrogen breathing group were: 4 mg/kg cisplatin, 70 mg/kg dacarbazine, 10 mg/kg temozolomide, and 2 mg/kg PD-1. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the _H2 +Drug group breathed hydrogen for 2 hours before daily administration, and the breathing environment of the mice contained 66% hydrogen. The mice were able to move freely in the breathing box during the hydrogen breathing process. After the administration, the mouse tumors were taken and the changes in tumor volume were observed. The specific results are as follows:

As shown in Figure 11, the use of cisplatin, dacarbazine, temozolomide and PD-1 alone has a significant (p < 0.05) or extremely significant (p < 0.01) effect on inhibiting the growth of melanoma compared with the control group. After combined with hydrogen molecules, the inhibitory effect is further enhanced (p < 0.001). The above results prove that the combination of hydrogen molecules can increase the effect of the above drugs in inhibiting the proliferation of melanoma.

Example 8 Effects of hydrogen molecules combined with different drugs in treating tumors at the cellular level

(1) Effect of hydrogen molecules combined with temozolomide in the treatment of glioblastoma

Mouse GL261 cells were cultured and randomly divided into a control group (CTRL), a hydrogen breathing group (H ₂ ), a single drug group (TMZ), and a hydrogen breathing followed by drug administration group (H ₂ + TMZ). The control group and the single drug group were cultured in a conventional CO ₂ incubator for 2 h, the hydrogen breathing group and the hydrogen breathing followed by drug administration group were cultured in a hydrogen-containing incubator (hydrogen concentration 66%) for 2 hours, and then taken out. The single drug group and the hydrogen breathing followed by drug administration group were treated with TMZ (the control group and the hydrogen breathing group were given the same concentration of DMSO), and the TMZ administration concentration was 2.5 mg/Kg for detection.

As shown in Figure 12, the results of the cell half-inhibitory concentration IC50 experiment show that compared with the cells treated with TMZ alone, the IC50 value of GL261 cells was significantly reduced after TMZ combined with hydrogen molecules. At the same time, the results of cell immunofluorescence staining (see Figure 13) showed that hydrogen molecules can significantly increase the degree of DNA damage to tumor cells caused by a certain concentration of TMZ.

Phosphorylated H2A.X is a marker of cell DNA damage. The positive expression of phosphorylated H2A.X in cell immunofluorescence represents the level of damage caused by TMZ to GL261 tumor cells. As shown in Figure 13, TMZ alone can cause certain damage to cells, but after combined with hydrogen molecules, the level of cell phosphorylated H2A.X increased significantly, indicating that hydrogen molecules increase the drug sensitivity of tumor cells to TMZ, promote TMZ to cause cell DNA damage, thereby inducing tumor cell death and producing a therapeutic effect on tumors.

(2) Effect of hydrogen molecule combined with metformin in the treatment of glioblastoma

Mouse GL261 cells were randomly divided into a single drug group (MET) and a drug administration group after hydrogen breathing ( _H2 + MET). The single drug group was cultured in a conventional _CO2 incubator for 2 hours, and the drug administration group after hydrogen breathing was placed in a hydrogen-containing incubator (hydrogen concentration 66%) for 2 hours. After that, the cells were taken out and treated with MET at a MET concentration of 75 mg/kg for detection.

As shown in FIG14 , after combined use with molecular hydrogen, the growth of glioblastoma cells was significantly inhibited compared with the use of metformin alone.

Example 9 The therapeutic effect of hydrogen molecules combined with different anti-tumor drugs on nasopharyngeal carcinoma animal models

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. CNE1 nasopharyngeal carcinoma cells grown under normal culture conditions were collected and inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group (CTRL), a single drug group (Drug), and a hydrogen breathing group (H ₂ +Drug). The drugs were administered for 15 consecutive days after 15 days of tumor growth. The drug concentrations of the single drug group and the hydrogen breathing group were: cisplatin 15 mg/kg, fluorouracil 40 mg/kg, and paclitaxel 10 mg/kg. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the _{H 2} +Drug group breathed hydrogen for 2 hours a day, and the breathing environment of the mice contained 66% hydrogen. The mice could move freely in the breathing box during the hydrogen breathing process. After the administration, the mouse tumors were taken and the changes in tumor volume were observed. The specific results are as follows:

As shown in Figure 15, compared with the control group, the use of cisplatin alone has a significant effect on inhibiting the growth of nasopharyngeal carcinoma tumors (p < 0.05), and the use of fluorouracil and paclitaxel alone has no significant effect on inhibiting the growth of nasopharyngeal carcinoma tumors. After combined use with hydrogen molecules, the inhibitory effect was significantly enhanced (p < 0.01, p < 0.001). The above results prove that the combination of hydrogen molecules can increase the effect of the above drugs in inhibiting the proliferation of nasopharyngeal carcinoma tumors.

Example 10 The therapeutic effect of hydrogen molecules combined with different anti-tumor drugs on leukemia animal models

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. HL-60 leukemia cells grown under normal culture conditions were collected and inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group (CTRL), a single drug group (Drug), and a hydrogen breathing group (H ₂ +Drug). The drugs were administered for 15 consecutive days after the tumor grew for 15 days. The drug concentrations were: cyclophosphamide 200 mg/kg, methotrexate 100 mg/kg, and vincristine 150 ug/kg. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the _{H 2} +Drug group breathed hydrogen for 2 hours a day before administration. The breathing environment of the mice contained 66% hydrogen. The mice could move freely in the breathing box during the hydrogen breathing process. After the drug was taken, the mouse tumors were taken and the changes in tumor volume were observed. The specific results are as follows:

As shown in Figure 16, the use of cyclophosphamide, methotrexate and vincristine alone had a significant effect on inhibiting the growth of leukemia tumors compared with the control group (p < 0.05). After combined use with hydrogen molecules, the inhibitory effect was significantly enhanced (p < 0.001). The above results prove that the combination of hydrogen molecules can increase the effect of the above drugs in inhibiting the proliferation of leukemia tumors.

Example 11 The therapeutic effects of different hydrogen administration methods on glioblastoma animal models

Eight-week-old male C57BL/6N mice weighing 18-22 g were selected for the experiment. After collecting GL261 glioblastoma cells grown under normal culture conditions, they were inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group, a TMZ group, a hydrogen breathing + TMZ group, a hydrogen saline injection + TMZ group, a local hydrogen supply + TMZ group, and a hydrogen water drinking + TMZ group. After 15 days of subcutaneous tumor growth in mice, the drug was continuously administered for 15 days, the mice were killed, and the tumor volume was measured. The specific results are shown in Figure 17. The concentration of temozolomide was 2.5 mg/kg. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The hydrogen-breathing mice breathed hydrogen for 2 hours a day, in which the mouse breathing environment contained 66% hydrogen, and the mice could move freely in the breathing box during the hydrogen breathing process. The hydrogen concentration in drinking hydrogen water and injecting saturated hydrogen saline reached saturation (specifically, the hydrogen concentration was 800 ppb). Local hydrogen supply was given to the subcutaneous tumor site by means of a hydrogen patch (containing hydrogen storage material, releasing hydrogen when used). The hydrogen patch used a liquid hydrogen electrode to measure the hydrogen release curve (soaked in saline before testing, the ratio of patch to saline was: 2 cm ² /10 mL), and the results are shown in Figure 18.

The results in Figure 17 show that all hydrogen administration methods combined with TMZ can reduce tumor volume and inhibit tumor growth compared with TMZ administration alone, but breathing hydrogen is the most effective compared with other administration methods, so breathing hydrogen is the preferred administration method.

Example 12 The therapeutic effect of different hydrogen concentrations on glioblastoma animal model

The mouse modeling method was the same as in Example 11.

After the tumors grew, the mice were randomly divided into a control group, a TMZ group, a group breathing 5% hydrogen + temozolomide, and a group breathing 66% hydrogen + temozolomide. After 15 days of subcutaneous tumor growth in mice, the drug was continuously administered for 15 days, the mice were killed and the tumor volume was measured. The specific results are shown in Figure 19. The concentration of temozolomide was: 2.5 mg/kg. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. Mice breathing hydrogen breathed hydrogen for 2 hours a day, in which the mouse breathing environment contained 5% or 66% hydrogen, and the mice could move freely in the breathing box during the hydrogen breathing process.

The results in Figure 19 show that low concentration (5%) of breath hydrogen combined with TMZ has no significant inhibitory effect on tumor growth compared to TMZ alone, thus proving that breath hydrogen administration should be administered within a certain concentration.

Example 13 The therapeutic effect of different hydrogen administration times on liver cancer animal models

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. After collecting HepG2 hepatocellular carcinoma cells grown under normal culture conditions, they were inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group, a hydrogen breathing group, a sorafenib group, a sorafenib administration + hydrogen breathing group, and a hydrogen breathing + sorafenib group. The mice were administered for 15 consecutive days after 15 days of tumor growth. Sorafenib was administered by gavage at a drug concentration of 30 mg/kg. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. The mice in the hydrogen breathing group breathed hydrogen for 2 hours a day, and the mouse breathing environment contained 66% hydrogen. The mice could move freely in the breathing box during the hydrogen breathing process. During and after the administration, the mouse tumors were taken to observe the changes in tumor volume. The specific results are shown in Figure 20.

The results in Figure 20 show that after the tumor is formed, breathing hydrogen alone can no longer significantly inhibit tumor growth. Compared with the administration of sorafenib alone, breathing hydrogen after administration also has no significant improvement effect, while breathing hydrogen and then using sorafenib significantly reduced the volume of the generated tumor, which has a significant anti-resistance effect, proving that the therapeutic effect of hydrogen molecule treatment combined with sorafenib is the best, so it is preferred to use the method of hydrogen molecule treatment before medication.

Example 14 Therapeutic effects of different hydrogen administration times on leukemia animal models

Eight-week-old male NOD SCID mice weighing 18-22 g were selected for the experiment. After collecting HL-60 leukemia cells grown under normal culture conditions, they were inoculated subcutaneously at a cell concentration of 1×10 ^6. After the model was established, the tumor was allowed to grow. After the tumor grew, the mice were randomly divided into a control group, a cyclophosphamide group, a cyclophosphamide administration + hydrogen breathing group, and a hydrogen breathing + cyclophosphamide group. The drugs were administered for 15 consecutive days after 15 days of tumor growth, and the cyclophosphamide concentration was 200 mg/kg. All experimental mice were kept at 22-25°C and given a 12-hour light-dark cycle and a normal diet. Mice in the hydrogen breathing group breathed hydrogen for 2 hours a day, and the mouse breathing environment contained 66% hydrogen. During the hydrogen breathing process, the mice could move freely in the breathing box. After the administration, the mouse tumors were taken and the changes in tumor volume were observed. The specific results are shown in Figure 21.

The results in Figure 21 show that after the tumor is formed, breathing hydrogen alone can no longer significantly inhibit tumor growth. Compared with the administration of cyclophosphamide alone, breathing hydrogen after administration also has no significant improvement effect, while breathing hydrogen and then using cyclophosphamide significantly reduced the volume of the generated tumor, which has a significant anti-resistance effect, proving that the treatment effect of hydrogen molecule treatment combined with cyclophosphamide is the best.

Based on the results of Example 1 and Example 14, it is preferred to administer the drug after treatment with hydrogen molecules.

Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (10)

1. A drug for improving the effect of tumor chemotherapy, characterized in that it comprises hydrogen molecules or hydrogen molecule source materials. 2. The drug according to claim 1, characterized in that The product form of the drug is one or more of gas, aerosol, solution, emulsion, foam, gel, colloidal dispersion, liposome, droplet, microbubble, paste, suppository, chewing gum, granule, pill, powder, lozenge, capsule, microcapsule. 3. The drug according to claim 1, characterized in that The medicine also includes oxygen and/or medically acceptable pharmaceutical excipients. 4. The application of hydrogen molecules in the preparation of drugs for improving the effect of tumor chemotherapy. 5. The use according to claim 4, characterized in that The hydrogen molecule is used in preparing a drug for alleviating tumor chemotherapy resistance. 6. The use according to claim 4, characterized in that The hydrogen molecule application method is to administer the drug before the tumor chemotherapy drug is administered, or to continuously administer the drug before, during or after the tumor chemotherapy drug is administered. 7. The use according to claim 4, characterized in that The hydrogen molecule administration method includes one or more of respiratory administration, oral administration, injection administration or local administration. 8. The use according to claim 4, characterized in that The tumor includes one or more of blood tumor, glioblastoma, liver cancer, lung cancer, gastric cancer, kidney cancer, breast cancer, nasopharyngeal carcinoma and melanoma; The tumor chemotherapy drugs include cytotoxic drugs temozolomide, fluorouracil and cyclophosphamide, metabolic drugs metformin and letrozole, kinase inhibitor drugs sorafenib and trametinib, new anti-tumor drug TRAIL, immunotherapy drugs PD-1, cisplatin, dacarbazine, paclitaxel, methotrexate, vincristine, or one or more of the monoclonal antibody drugs bevacizumab, nimotuzumab and ramucirumab. 9. The use according to claim 4, characterized in that The hydrogen molecules are administered by breathing, and the volume concentration of hydrogen molecules in the breathed gas is greater than 5% and less than 99%. 10. The use according to claim 9, characterized in that When the tumor is a glioblastoma of the brain, and: When the chemotherapy drug is temozolomide, the temozolomide chemotherapy dosage is 2-3 mg/kg; When the chemotherapy drug is TRAIL, the chemotherapy dosage of TRAIL is 4 to 6 mg/kg; When the chemotherapy drug is metformin, the metformin chemotherapy dosage is 70-80 mg/kg; When the chemotherapy drug is bevacizumab, the chemotherapy dosage of bevacizumab is 4 to 6 mg/kg; When the tumor is liver cancer, lung cancer, stomach cancer or kidney cancer, and: When the chemotherapy drug is sorafenib, the chemotherapy dosage of sorafenib is 25-35 mg/kg; When the chemotherapy drug is fluorouracil, the chemotherapy dosage of fluorouracil is 15 to 25 mg/kg; When the chemotherapy drug is nimotuzumab or bevacizumab, the chemotherapy dosage of nimotuzumab is 0.4-0.6 mg/kg, and the chemotherapy dosage of bevacizumab is 2-3 mg/kg; When the chemotherapy drug is PD-1, the PD-1 chemotherapy dosage is 8 to 12 mg/kg; When the chemotherapy drug is cyclophosphamide, the cyclophosphamide chemotherapy dosage is 25-35 mg/kg; When the chemotherapy drug is cisplatin, the chemotherapy dosage of cisplatin is 1.5 to 2.5 mg/kg; When the tumor is breast cancer, and: When the chemotherapy drug is letrozole, the chemotherapy dosage of letrozole is 4 to 6 mg/kg; When the chemotherapy drug is fluorouracil, the chemotherapy dosage of fluorouracil is 15 to 25 mg/kg; When the chemotherapy drug is bevacizumab, the chemotherapy dosage of bevacizumab is 2 to 3 mg/kg; When the chemotherapy drug is PD-1, the PD-1 chemotherapy dosage is 1.5 to 2.5 mg/kg; When the tumor is melanoma, and: When the chemotherapy drug is temozolomide, the temozolomide chemotherapy dosage is 8 to 12 mg/kg; When the chemotherapy drug is dacarbazine, the chemotherapy dosage of dacarbazine is 65-75 mg/kg; when the chemotherapy drug is PD-1, the chemotherapy dosage of PD-1 is 1.5-2.5 mg/kg; When the chemotherapy drug is cisplatin, the chemotherapy dosage of cisplatin is 3.5-4.5 mg/kg; When the tumor is nasopharyngeal carcinoma, and: When the chemotherapy drug is cisplatin, the chemotherapy dosage of cisplatin is 10 to 20 mg/kg; When the chemotherapy drug is fluorouracil, the chemotherapy dosage of fluorouracil is 35-45 mg/kg; when the chemotherapy drug is paclitaxel, the chemotherapy dosage of paclitaxel is 5-15 mg/kg; When the tumor is leukemia, and: When the chemotherapy drug is cyclophosphamide, the chemotherapy dosage of cyclophosphamide is 195-205 mg/kg; when the chemotherapy drug is methotrexate, the chemotherapy dosage of methotrexate is 95-105 mg/kg; when the chemotherapy drug is vincristine, the chemotherapy dosage of vincristine is 145-155 ug/kg.