Multi-mode combined treatment system and application thereof

Abstract

The invention provides a multi-mode combined treatment system and application thereof, the multi-mode combined treatment system comprises a drug bag and a hyperbaric oxygen device, and the drug bag comprises chemotherapeutic drugs; effective experiments prove that the combined treatment of the chemotherapeutic drug and hyperbaric oxygen can enhance the treatment effect of the chemotherapeutic drug, the combined treatment tumor volume is reduced by 73.0%, and the DNA damage and apoptosis levels of tumor tissues are increased; according to the multi-mode combined treatment system and the application thereof, a safe new mode and a new method are provided for resisting chemotherapy drug resistance and enhancing the tumor treatment effect.

Description

Technical Field

[0001] The invention relates to a multi-mode combined treatment system and application thereof, and belongs to the technical field of cell biology. Background Art

[0002] In the field of tumor treatment, traditional chemotherapy has long been one of the main treatment methods. Mitoxantrone (MIT) is a common chemotherapy drug used in the clinical treatment of a variety of tumors including acute leukemia, prostate cancer, breast cancer, etc. Its mechanism of action is mainly to interfere with DNA synthesis and inhibit topoisomerase II. However, although neoadjuvant chemotherapy involving genotoxic drugs such as mitoxantrone can effectively kill rapidly dividing tumor cells to a certain extent, it also causes the stromal cells in the tumor microenvironment to enter a state of cellular senescence. This effect significantly changes the tumor microenvironment and promotes the malignant enhancement of the remaining tumor cells, which manifests as chemotherapy resistance or even recurrence, limiting the overall efficacy of chemotherapy.

[0003] In order to break through the efficacy limitation of chemotherapy, researchers have searched for various drugs that can reduce the senescence-associated secretory phenotype (SASP) of senescent cells in vivo and in vitro, or even kill senescent cells, and improve the tumor treatment effect by combining them with genotoxic chemotherapy drugs. The former includes rutin, and the latter includes proanthocyanidin C1 (PCC1). However, since it involves small molecule drugs that have not yet entered the clinical application stage, and the drug development pipeline for new drugs to enter the clinic is very complex and time-consuming, the clinical transformation of this type of method has obvious limiting factors. Therefore, providing an accessible tumor chemotherapy optimization program to overcome chemotherapy resistance and improve tumor treatment effects is an urgent problem to be solved by basic medicine and clinical medicine researchers in this field.

[0004] Hyperbaric oxygen (HBO) medicine originated in the 17th century and was originally used to treat hypoxia and ischemic diseases. In the past 60 years, it has experienced a rise and flourishing in modern medicine, and its indications have been continuously expanded. Modern medicine's experience in the application of hyperbaric oxygen has fully proved its safety and non-invasiveness. So far, there is no research evidence that the use of hyperbaric oxygen for tumors will stimulate tumor growth or promote recurrence.

[0005] In summary, in view of the efficacy limitations of chemotherapy drugs in the existing technology, it is urgently necessary to seek new and safe methods to overcome the efficacy limitations of chemotherapy drugs, further resist chemotherapy resistance and enhance tumor treatment effects. Summary of the invention

[0006] The present invention provides a multi-mode combined treatment system and its application. The combined treatment of chemotherapy drugs and hyperbaric oxygen can enhance the therapeutic effect of chemotherapy drugs, and provides a new mode and method for resisting chemotherapy resistance and enhancing the effect of tumor treatment.

[0007] To achieve this object, the present invention provides the following technical solutions:

[0008] A first aspect of the present invention provides an application of a multi-modal combined therapy system in the preparation of a device for treating tumors, wherein the multi-modal combined therapy system comprises a drug package and a hyperbaric oxygen device, wherein the drug package comprises a chemotherapeutic drug.

[0009] Preferably, the chemotherapy drug is mitoxantrone.

[0010] Preferably, the dosage of mitoxantrone is 0.1-0.3 mg / kg, and the administration frequency is once a week; further preferably, the dosage of mitoxantrone is 0.2 mg / kg.

[0011] Preferably, the pressure of the high-pressure oxygen device is set to 1.0-3.0 ATA, and the oxygen concentration is set to ≥92%; further preferably, the pressure of the high-pressure oxygen device is set to 2.0 ATA.

[0012] Preferably, the use time of the hyperbaric oxygen device is set to 90-180 minutes each time.

[0013] Preferably, the high-pressure oxygen device is used at a frequency of not less than 5 times per week, and the interval between adjacent high-pressure oxygen treatments is not less than 24 hours.

[0014] Preferably, hyperbaric oxygen therapy is performed more than 24 hours after administration of the chemotherapy drug.

[0015] Preferably, the tumor includes, but is not limited to, malignant lymphoma, breast cancer, acute leukemia, lung cancer, melanoma, soft tissue sarcoma, multiple myeloma, liver cancer, colorectal cancer, kidney cancer, prostate cancer, endometrial cancer, testicular tumors, ovarian cancer and head and neck cancer.

[0016] In the present invention, tumor applies to all types of tumors in principle.

[0017] Preferably, the tumor treatment includes reducing tumor volume and increasing the level of DNA damage and apoptosis in tumor tissue.

[0018] A second aspect of the present invention provides a multi-modal combined treatment system, comprising a drug package and a hyperbaric oxygen device, wherein the drug package comprises a chemotherapeutic drug.

[0019] Preferably, the conditions of the high pressure oxygen device are set at 1.0-2.0 ATA and the time is 90-180 min.

[0020] Preferably, the high-pressure oxygen device is used at a frequency of not less than 5 times per week, and the interval between adjacent high-pressure oxygen treatments is not less than 24 hours.

[0021] Preferably, the chemotherapy drug is mitoxantrone.

[0022] A third aspect of the present invention provides a device for enhancing the efficacy of chemotherapeutic drugs, wherein the device is a hyperbaric oxygen device.

[0023] Preferably, the pressure of the high-pressure oxygen device is set to 1.0-3.0 ATA, and the oxygen concentration is set to ≥92%.

[0024] Preferably, the use time of the hyperbaric oxygen device is set to 90-180 minutes each time.

[0025] Preferably, the high-pressure oxygen device is used at a frequency of not less than 5 times per week, and the interval between adjacent high-pressure oxygen treatments is not less than 24 hours.

[0026] Preferably, the chemotherapy drug is mitoxantrone.

[0027] Compared with the prior art, the beneficial effects and significant progress of the technical solution of the present invention are: the multi-modal combined treatment system provided by the present invention, the combined treatment of chemotherapy drugs and hyperbaric oxygen can enhance the therapeutic effect of chemotherapy drugs, the combined treatment reduced the tumor volume by 73.0%, and the DNA damage and apoptosis levels of tumor tissues were increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments of the present invention are briefly introduced below.

[0029] Figure 1 It is the experimental operation flow chart of Example 2;

[0030] Figure 2 is a general schematic diagram and volume statistical result diagram of the four groups of tumors in Example 2;

[0031] Figure 3 It is the tissue β-gal staining diagram and statistical diagram of Example 3;

[0032] Figure 4 It is the histochemical staining diagram and statistical diagram of Example 3;

[0033] Figure 5 4 groups of different types of cancer cell staining images in Example 4;

[0034] Figure 6 This is a statistical chart of 4 groups of different types of cancer cells in Example 4. DETAILED DESCRIPTION

[0035] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0036] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0037] In order to more fully understand the present invention, the professional terms in the present invention are explained below.

[0038] DNA damage is a phenomenon in which the DNA nucleotide sequence is permanently changed during replication, resulting in changes in genetic characteristics. The situations are divided into: substitution, deletion, insertion, and exon skipping.

[0039] Apoptosis refers to a type of programmed cell death that occurs during the development of body cells or under the influence of certain factors through the regulation of intracellular genes and their products. It usually manifests as the death of a single cell without accompanying inflammatory response.

[0040] A hyperbaric oxygen device is a device that can provide a specific high pressure and a specific high oxygen environment.

[0041] The dosage refers to the amount of medicine that produces a certain reaction in the body when administered, usually referring to the amount used to prevent and treat diseases.

[0042] Chemotherapy is a drug used to treat tumors. Chemotherapy drugs can kill tumor cells. These drugs can act on different stages of tumor cell growth and reproduction, inhibiting or killing tumor cells. Chemotherapy drug treatment is currently one of the main means of treating tumors.

[0043] Cell proliferation is an important life characteristic of organisms. Cells proliferate by dividing. Single-celled organisms produce new individuals by cell division. Multicellular organisms produce new cells by cell division to replenish aging or dead cells in the body.

[0044] Cell migration, also known as cell crawling, cell movement or cell movement, refers to the movement of cells after receiving migration signals or sensing the gradient of certain substances. Cell migration is an alternating process in time and space of the extension of pseudopodia of the cell head, the establishment of new adhesions, and the contraction of the cell body tail. Cell migration is one of the basic functions of normal cells, a physiological process of normal growth and development of the body, and a common form of movement of living cells. Cell migration is involved in embryonic development, angiogenesis, wound healing, immune response, inflammatory response, atherosclerosis, cancer metastasis and other processes.

[0045] Cell invasion refers to the ability of cells to migrate from one area to another through the extracellular matrix or basement membrane matrix. Tumor cell invasion is the invasion or occupation of malignant tumors from primary tumors or secondary tumors to adjacent host tissues.

[0046] Example 1 Multimodal combined treatment method

[0047] 1.1. Construction of transplanted tumor

[0048] 7-8 week old NOD-SCID male mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. and maintained according to the animal husbandry guidelines of Shanghai Institutes for Biological Sciences. 5 1×10 human primary prostate stromal cells (PSC27) and 1×10 6 The initial amount of tumorigenic cells of human prostate cancer cells (PC3) was resuspended in PBS solution and injected subcutaneously into the posterior flank of the back to construct mouse tumor xenografts.

[0049] 1.2. Animals were observed and randomly divided into groups 2 weeks after tumor implantation for preclinical treatment.

[0050] 1.3 Combination therapy

[0051] The dosing frequency included weekly administration of mitoxantrone and five times of hyperbaric oxygen therapy per week starting from the end of the second week. The interval between two hyperbaric oxygen treatments was ≥ 24 hours. Hyperbaric oxygen therapy was avoided on the day of mitoxantrone administration to avoid excessive stress on the mice.

[0052] Mitoxantrone was administered intraperitoneally once a week at a dosage of 0.2 mg / kg in a dosing system of 100 μL / pig. Mitoxantrone was purchased from MedChemExpress (HY-13502), dissolved in DMSO to prepare a 10 mM stock solution, stored in a -20 degree refrigerator, equilibrated to room temperature before use, and fully diluted with a sterile PBS solution to ensure full dissolution without precipitation.

[0053] The hyperbaric oxygen treatment method is: the hyperbaric oxygen device is an animal experiment chamber purchased from Shandong Weifang Huaxin Oxygen Company. The treatment plan includes: placing NOD-SCID mice in a closed ventilation cage and putting them into the chamber; using high-purity oxygen (Shanghai Chloromin Gas Co., Ltd., oxygen purity ≥99.99%) to continuously ventilate the animal experiment chamber until the oxygen concentration in the chamber is replaced to ≥92%, which takes 20-30 minutes; setting automatic parameters and starting the program: the pressure increase and pressure reduction time are 10 minutes respectively, and HBO treatment is performed for 90 minutes at a pressure of 2.0ATA, and the chamber is flushed with pure oxygen for 5-10 seconds every 150-300 seconds to ensure the stability of the oxygen content in the chamber.

[0054] 1.4. During the entire treatment process, if the tumor exceeds the maximum threshold (size ≥ 15 mm in any dimension), the tumor is not allowed to continue to grow and the mouse is immediately killed according to the relevant animal care procedures. Otherwise, 4-5 weeks after tumor inoculation, the mouse is killed in accordance with relevant ethical requirements and the tumor size is measured.

[0055] Example 2 Effects of different treatment methods on tumor volume

[0056] Experimental methods

[0057] 2.1. Construction of transplanted tumor

[0058] 7-8 week old NOD-SCID male mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. and maintained according to the animal husbandry guidelines of Shanghai Institutes for Biological Sciences. 5 1×10 human primary prostate stromal cells (PSC27) and 1×10 6 The initial amount of tumorigenic cells of human prostate cancer cells (PC3) was resuspended in PBS solution and injected subcutaneously into the posterior flank of the back to construct mouse tumor xenografts.

[0059] 2.2. Animals were observed and randomly divided into groups 2 weeks after tumor implantation for preclinical treatment.

[0060] In order to illustrate the superiority of combined therapy over single therapy, four groups were set up and given different treatment plans (n=8). Figure 1 Group 1: placebo and sham treatment; Group 2: placebo and hyperbaric oxygen treatment; Group 3: mitoxantrone and sham treatment; Group 4: mitoxantrone and hyperbaric oxygen treatment.

[0061] 2.3. Mitoxantrone and hyperbaric oxygen therapy were performed according to the method in step 2.2, where placebo refers to an equal amount of sterile PBS solution, and sham treatment refers to placing the animals in the same ventilation cage and in the animal experimental chamber for the same period of time, but without ventilation treatment.

[0062] 2.4. All mice were killed and dissected on the same day (fifth week after tumor implantation), and the tumors were removed and measured in three dimensions. The tumor volume (v) was measured and calculated based on the length (l), width (w) and height (h) of the tumor. The calculation formula was: v = (π / 6) × ((l+w+h) / 3). The tumors were then quickly frozen and fresh frozen sections and paraffin sections were prepared for subsequent staining and analysis.

[0063] Experimental Results

[0064] The gross diagram and volume statistics of the tumor are shown in Figure 2 As shown, the effect of single hyperbaric oxygen therapy on tumor volume did not produce statistical differences, and the tumor volume of the mitoxantrone monotherapy group was reduced by 42.7%, while the tumor volume of the combined treatment group was reduced by 73.0%, compared with 52.9% in the mitoxantrone monotherapy group. This shows that combined treatment has better efficacy than single mitoxantrone treatment.

[0065] Example 3 Study on the mechanism of multimodal combined therapy

[0066] Fresh frozen sections were prepared from the mouse tumors dissected in Example 2 and subjected to tissue β-gal staining analysis. β-Gal staining has been used for immunofluorescence analysis, senescence assays, and histopathological analysis of tissue samples.

[0067] The results are as follows Figure 3 As shown, CTRL is a placebo and sham treatment; HBO is a placebo and hyperbaric oxygen treatment; MIT is mitoxantrone and sham treatment; MIT+HBO is mitoxantrone and hyperbaric oxygen treatment. The results showed that single mitoxantrone treatment significantly increased the level of tumor cell senescence, and the combined treatment group significantly reduced the level of cell senescence compared with the single mitoxantrone treatment group.

[0068] The paraffin sections prepared from the mouse tumors dissected in Example 2 were subjected to histochemical staining analysis of γ-H2AX and Cleaved-Caspase 3, and γ-H2AX-positive cells were defined as DNA damaged cells, and Cleaved-Caspase 3-positive cells were defined as apoptotic cells.

[0069] The results are as follows Figure 4 As shown in the figure, CTRL is a placebo and sham treatment; HBO is a placebo and hyperbaric oxygen treatment; MIT is mitoxantrone and sham treatment; MIT+HBO is mitoxantrone and hyperbaric oxygen treatment. The results showed that mitoxantrone treatment increased the DNA damage and apoptosis levels of tumor tissues to a certain extent, which was significantly increased after the combined treatment of hyperbaric oxygen and mitoxantrone.

[0070] Example 4 Hyperbaric oxygen inhibits cellular senescence of stromal cells and can limit their cancer-promoting effects

[0071] To clarify the mechanism of action, an in vitro experiment was performed to evaluate whether the conditioned medium of stromal cells (PSC27) treated with hyperbaric oxygen could limit the malignant enhancement phenotype of cancer cells associated with cell senescence.

[0072] Four prostate cancer cell lines, PC3, DU145, M12, and LNCaP, were used, and the conditioned medium of PSC27 cells treated with Bleomycin (Bleomycin is a classic drug that can induce stromal cells into a state of cell senescence in vitro) and hyperbaric oxygen alone or in combination was used to treat the cancer cells. Among them, CTRL is the blank control group, HBO is treated with HBO, BLEO is treated with Bleomycin, and BLEO+HBO is treated with Bleomycin and HBO in combination.

[0073] The results are as follows Figure 5-6 As shown, Figure 5 These are 4 groups of different types of cancer cell staining images. Figure 6 The statistical graphs of four different types of cancer cells are shown in Figure 6a, including the number of epithelial cells in Figure 6a, the cell activity in Figure 6b, the cell migration distance in Figure 6c, the number of invading cells in each field of view in Figure 6d, and the number of migrating cells in each field of view in Figure 6e. The results showed that the malignant phenotypes of the four types of cancer cells, including proliferation, migration and invasion levels, were significantly downregulated, indicating that hyperbaric oxygen inhibited the cell senescence of stromal cells and could limit their cancer-promoting effects.

[0074] The applicant declares that, in the description of the above specification:

[0075] The descriptions of the terms "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without causing any contradiction.

[0076] Finally, it should be noted that:

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them.

[0078] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection required by the present invention.

Claims

1. Application of a multi-modal combined treatment system in the preparation of a device for treating tumors, characterized in that: The multi-mode combined treatment system comprises a drug package and a hyperbaric oxygen device, wherein the drug package comprises chemotherapy drugs.

2. The use according to claim 1, characterized in that The chemotherapy drug is mitoxantrone.

3. The use according to claim 1, characterized in that The dosage of mitoxantrone is 0.1-0.3 mg / kg, and the administration frequency is once a week.

4. The use according to claim 1, characterized in that The pressure of the high-pressure oxygen device is set to 1.0-3.0 ATA, and the oxygen concentration is set to ≥92%.

5. The use according to claim 1, characterized in that The use time of the hyperbaric oxygen device is set to 90-180 minutes each time.

6. The use according to claim 1, characterized in that The frequency of use of the hyperbaric oxygen device is no less than 5 times per week, and the interval between adjacent hyperbaric oxygen treatments is no less than 24 hours.

7. The use according to claim 1, characterized in that The tumors include malignant lymphoma, breast cancer, acute leukemia, lung cancer, melanoma, soft tissue sarcoma, multiple myeloma, liver cancer, colorectal cancer, kidney cancer, prostate cancer, endometrial cancer, testicular tumors, ovarian cancer and head and neck cancer.

8. The use according to claim 1, characterized in that The tumor treatment includes reducing tumor volume and increasing the level of DNA damage and apoptosis in tumor tissue.

9. A multi-modal combined treatment system, characterized in that: The invention comprises a medicine package and a hyperbaric oxygen device, wherein the medicine package comprises chemotherapy drugs.

10. The multi-modal combined treatment system according to claim 9, characterized in that: The conditions of the high pressure oxygen device are set at 1.0-3.0 ATA and the time is 90-180 min.

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