Application of mitochondrial extract in reversing drug resistance in solid tumors

Abstract

This invention relates to the application of mitochondrial extracts in reversing drug resistance in solid tumors. By combining mitochondrial extracts with chemotherapeutic drugs, the resistance of chemotherapeutic-resistant solid tumors to these drugs can be reversed. Compared to using mitochondrial extracts and chemotherapeutic drugs alone, this method effectively enhances drug efficacy, thereby overcoming chemotherapeutic resistance in solid tumors. This invention, through intervention with mitochondrial extracts in different chemotherapeutic-resistant solid tumor cell lines, has verified that the combined use of mitochondrial transplantation and chemotherapeutic drugs can reverse chemotherapeutic resistance, thereby improving the efficacy of chemotherapy and opening up the possibility of continued treatment for patients with advanced solid tumors who are resistant to chemotherapeutic drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the application of mitochondrial extract as a drug for the treatment of solid tumors, and in particular to the application of a mitochondrial extract in reversing the drug resistance of solid tumors to chemotherapy drugs. Background Technology

[0002] Conventional treatments for solid tumors include surgery, chemotherapy, targeted therapy, immunotherapy, and radiotherapy. Among these, chemotherapy plays a crucial role in the treatment of solid tumors.

[0003] Lung cancer is often classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC accounting for the vast majority of lung cancer types. Clinically, targeted therapy and immunotherapy for NSCLC have limited suitable patient populations, and chemotherapy is still necessary after failure. Currently, platinum-based doublet chemotherapy is still the first choice, using cisplatin as the base, combined with another chemotherapy drug, such as etoposide, paclitaxel, docetaxel, gemcitabine, or vinorelbine. For non-squamous cell carcinoma patients, pemetrexed combined with cisplatin chemotherapy can be chosen. A clinical trial involving patients with advanced NSCLC without EGFR, ALK, or ROS1 gene mutations (Gogishvili M, et al. Cemiplimab plus chemotherapy versus chemotherapy alone in non-small cell lung cancer: a randomized, controlled, double-blind phase 3 trial) is also mentioned. Nature Medicine (2022, 28(11): 2374-2380.) indicates that the progression-free survival (PFS) for patients receiving platinum-based doublet chemotherapy is 5 months.

[0004] Ovarian cancer is a common malignant tumor that seriously threatens women's lives and health, with the highest mortality rate among gynecological cancers. According to the CSCO guidelines, postoperative adjuvant chemotherapy is a key measure to reduce the risk of recurrence. For patients with advanced ovarian cancer, commonly used chemotherapy drugs include paclitaxel, docetaxel, carboplatin, cisplatin, liposomal doxorubicin, and ifosfamide, but only 10-30% of patients show long-term survival, and most patients experience recurrence within two years of surgery (Zhang, L. et al. Bevacizumab with dose-dense paclitaxel / carboplatin as first-line chemotherapy for advanced ovarian cancer). Eur J Pharmacol . 2018, 837: 64-71.).

[0005] Triple-negative breast cancer (TNC) refers to breast cancer where immunohistochemical staining of the cancer tissue shows negative results for estrogen receptor (ER), progesterone receptor (PR), and the proto-oncogene HER2. It is unresponsive to endocrine therapy and HER2-targeted therapy, and is considered a non-immune inflammatory tumor, often referred to as an "immune-cold tumor," thus responding poorly to immunotherapy. Treatment options for TNC are limited, historically relying primarily on chemotherapy. Commonly used chemotherapy drugs include epirubicin, cyclophosphamide, docetaxel, paclitaxel, albumin-bound taxanes, carboplatin, and cisplatin. A clinical trial on advanced TNC (Jiang, Z. et al. Toripalimab plus nab-paclitaxel in metastatic or recurrent triple-negative breast cancer: a randomized phase 3 trial) is also relevant. Nature Medicine (2024, 30(1): 249-256.) indicates that the progression-free survival (PFS) for patients receiving paclitaxel chemotherapy is 5.6 months.

[0006] The vast majority of cancer patients, including those with esophageal cancer (common chemotherapy drugs include cisplatin, 5-fluorouracil, vincristine sulfate, bleomycin hydrochloride, leucovorin, oxaliplatin, capecitabine, and irinotecan), liver cancer (common chemotherapy drugs include cisplatin, oxaliplatin, paclitaxel, 5-fluorouracil, gemcitabine, and capecitabine), bladder cancer (common chemotherapy drugs include gemcitabine, cisplatin, methotrexate, vincristine, and doxorubicin), gastric cancer (common chemotherapy drugs include cisplatin, fluorouracil, etoposide, leucovorin, doxorubicin, and epirubicin), pancreatic cancer (common chemotherapy drugs include paclitaxel, gemcitabine, oxaliplatin, irinotecan, leucovorin, and 5-fluorouracil), and osteosarcoma (common chemotherapy drugs include doxorubicin, methotrexate, cisplatin, ifosfamide, etoposide, and mesna), will develop chemotherapy drug resistance during chemotherapy, leading to treatment failure. Currently, there is no clinical treatment that can reverse chemotherapy resistance in solid tumors. When cancer patients experience a decrease in sensitivity to first-line chemotherapy drugs, leading to relapse or worsening of their condition, other chemotherapy regimens must be used again.

[0007] However, the variety of chemotherapy drugs available clinically is limited. Developing drugs to reverse drug resistance in solid tumors is a crucial research area in current cancer prevention and treatment. To reverse drug resistance in solid tumors, improve the efficacy of chemotherapy, reduce the risk of resistance, and prolong survival, there is an urgent need to find synergistic combinations of novel therapeutic drugs and existing chemotherapy drugs that can reverse and mitigate drug resistance.

[0008] In recent years, with the deepening of research, people have gradually realized that mitochondrial energy homeostasis plays an important role in the occurrence and development of various diseases. Mitochondrial dysfunction can lead to a variety of diseases, including developmental disorders, neuromuscular diseases, metabolic diseases, tumor progression, etc. (Mammucari, C. et al. Signaling pathways inmitochondrial dysfunction and aging). Mech Ageing Dev. 2010, 131(7-8): 536-543.). Mitochondria in malignant tumor cells often undergo changes in number and structure to adapt to the rapid growth of tumors in acidic and hypoxic environments. Simultaneously, irreversible damage to mitochondrial function disrupts the balance between oxidative phosphorylation and glycolysis for energy, leading to malignant transformation of the cell. This phenomenon of altered bioenergy in tumor cells is known as "metabolic reprogramming" (Jin, X. et al. Dissecting the alternation landscape of mitochondrial metabolism-related genes in lung adenocarcinoma and their latent mechanisms). Aging ( Albany NY ) . 2023, 15(12): 5482-5496.). Mitochondria in malignant tumor cells are dysfunctional. In 1924, German scientists discovered that tumor energy metabolism is mainly based on rapid glycolysis under aerobic conditions, which is the famous Wenberge effect.

[0009] Mounting evidence suggests that mitochondrial agents play a crucial role in cancer treatment. Once inside hypoxic and acidic tumor tissue, mitochondria generate large amounts of oxygen free radicals, inducing cell death. This environmentally responsive pharmacological property of mitochondria can be used to eliminate tumor cells and restore the function of damaged tissues.

[0010] However, no studies have yet shown that mitochondrial agents can reverse chemotherapy resistance in solid tumors. Summary of the Invention

[0011] The purpose of this invention is to provide an application of mitochondrial extract to reverse drug resistance in solid tumors, so as to provide a more effective treatment for solid tumors using mitochondrial extract.

[0012] This invention has found that, compared to using mitochondrial extract and chemotherapy drugs alone, the combined use of mitochondrial extract and chemotherapy drugs can effectively enhance the efficacy of the drugs.

[0013] Therefore, the present invention first addresses the problem of drug resistance that easily occurs when using chemotherapy drugs to treat solid tumors, and proposes the application of mitochondrial extract in the preparation of drugs that reverse drug resistance in solid tumors.

[0014] In particular, this invention proposes the use of mitochondrial extracts in the preparation of drugs that reverse resistance to various solid tumor chemotherapy drugs, including platinum-based chemotherapy drugs or paclitaxel.

[0015] Furthermore, the solid tumors described in this invention are selected from various clinically confirmed solid tumors such as lung cancer, esophageal cancer, liver cancer, gastric cancer, pancreatic cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, or osteosarcoma.

[0016] More specifically, the solid tumor is preferably non-small cell lung cancer, ovarian cancer, or triple-negative breast cancer.

[0017] The experiments of this invention have shown that when mitochondrial extract is used in combination with chemotherapy drugs, it can significantly improve the inhibition rate of chemotherapy-resistant solid tumor cells, reverse the resistance of chemotherapy-resistant solid tumor cells to chemotherapy drugs, and improve the therapeutic effect of chemotherapy drugs on solid tumors.

[0018] Secondly, this invention further proposes the application of mitochondrial extract combined with chemotherapy drugs in the preparation of drugs for treating solid tumors.

[0019] More specifically, the present invention provides the use of the mitochondrial extract in combination with platinum-based chemotherapy drugs or paclitaxel in the preparation of a medicament for treating solid tumors.

[0020] This invention primarily targets several types of chemotherapy-resistant solid tumors with high malignancy, including lung cancer, ovarian cancer, and triple-negative breast cancer. It has verified that mitochondrial preparations can significantly improve the efficacy of second-line chemotherapy drugs and reverse chemotherapy resistance in different types of chemotherapy-resistant solid tumors.

[0021] Therefore, the present invention also provides a combination drug for treating chemotherapy-resistant solid tumors, wherein the active pharmaceutical ingredients of the combination drug include mitochondrial extract and chemotherapy drugs, and the mitochondrial extract and chemotherapy drugs are each independent drug delivery units.

[0022] Furthermore, the combination drug of the present invention for treating chemotherapy-resistant solid tumors more specifically includes mitochondrial extract and platinum-based chemotherapy drugs or paclitaxel as its active pharmaceutical ingredients, and the mitochondrial extract and platinum-based chemotherapy drugs or paclitaxel are each independent drug delivery units.

[0023] The chemotherapy drugs in the combined medication regimen are still used at the conventional dosage, and the preferred dosage of the mitochondrial extract is 10... 8 ~1011 One mitochondria.

[0024] This invention, through intervention with mitochondrial extracts in different chemotherapy-resistant solid tumor cell lines, verifies that the combined use of mitochondrial preparations and chemotherapeutic drugs can reverse chemotherapy resistance, thereby improving the efficacy of chemotherapy. This invention aims to enhance the efficacy of second-line chemotherapy drugs, reverse chemotherapy resistance in advanced solid tumors, provide a pathway and possibility for continued treatment for these patients, and make a beneficial exploration to alleviate the psychological stress and socioeconomic burden on patients and their families. Attached Figure Description

[0025] Figure 1 The result is that mitochondrial transplantation improves the efficacy of chemotherapy in cisplatin-resistant cancer tissues.

[0026] Figure 2 This involves observing the survival period of mice in each group.

[0027] Figure 3 Mitochondrial transplantation improves the efficacy of cisplatin-based chemotherapy on cisplatin-resistant lung cancer cells A549 / DDP.

[0028] Figure 4 Mitochondrial transplantation improves the efficacy of cisplatin chemotherapy against cisplatin-resistant Skov3 / DDP ovarian cancer cells.

[0029] Figure 5 Mitochondrial transplantation improves the efficacy of paclitaxel chemotherapy on paclitaxel-resistant MDA-MB-231 / Taxol-Re triple-negative breast cancer cells. Implementation

[0030] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, so that those skilled in the art can better understand and utilize the present invention, and are not intended to limit the scope of protection of the present invention.

[0031] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0032] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art. Example

[0033] Example 1

[0034] This embodiment uses a nude mouse model of cisplatin-resistant human lung adenocarcinoma cells transplanted subcutaneously to verify the effect of mitochondrial extract on reversing cisplatin chemotherapy resistance in advanced non-small cell lung cancer.

[0035] Several 6-week-old male nude mice (BALB / c) were housed in an SPF-grade environment. After one week of acclimatization, subcutaneous tumors of cisplatin (DDP)-resistant human lung adenocarcinoma cells (A549 / DDP) were implanted on their right side.

[0036] When the tumor volume reaches 50-80mm 3 Twenty-four tumor-bearing nude mice with appropriate tumor volume were selected and randomly divided into four groups: control group, tail vein injection of mitochondrial suspension group, cisplatin group, and tail vein injection of mitochondrial suspension + cisplatin group (DDP+Mito group), with six mice in each group. The mice were administered PBS, mitochondrial suspension, cisplatin, and mitochondrial suspension combined with cisplatin, respectively, to evaluate the efficacy of cisplatin administration and mitochondrial suspension combined with cisplatin administration.

[0037] Control group tumor-bearing mice were treated with intraperitoneal injection for 3 weeks, twice a week, 200 μl PBS each time;

[0038] Mito group tumor-bearing mice were treated with mitochondrial tail vein injection for 3 weeks, twice a week, 1×10⁻⁶ each time. 7 One mitochondria;

[0039] DDP group tumor-bearing mice were treated with intraperitoneal injection of cisplatin for 3 weeks, twice a week, 5 mg / kg each time;

[0040] DDP+Mito group tumor-bearing mice were treated with a combination of mitochondrial tail vein injection and cisplatin intraperitoneal injection for 3 weeks. Specifically, cisplatin was administered twice a week at 5 mg / kg each time, and mitochondrial tract infection was administered twice a week at 1×10 mg / kg each time. 7 One mitochondria.

[0041] The experimental indicators included mouse tumor volume, mouse body weight, and mouse survival time.

[0042] Throughout the experiment, tumor size and mouse weight were precisely measured weekly and recorded without knowledge of the experimental details. All animal experiments were conducted in strict accordance with ethical and scientific standards.

[0043] Tumor volume changes during mouse experiments, such as Figure 1As shown in (A). Compared with the control group, after 21 days of intervention, the tumor growth rate of mice in the DDP group receiving cisplatin monotherapy and the DDP+Mito group receiving mitochondrial injection combined with cisplatin chemotherapy was significantly reduced, and the reduction in tumor growth rate was more significant in the DDP+Mito group than in the DDP group.

[0044] Figure 1 Figures (B) and (D) further provide information on tumor volume in mice 21 days after intervention, where (B) represents the final tumor volume and (D) is an image of the tumor. Compared to the Control group, the tumor inhibition rate in the DDP group was 40% (P < 0.05), and the tumor inhibition rate in the DDP+Mito group was 65% (P < 0.05). Tail vein injection of mitochondria combined with cisplatin treatment improved efficacy by 62% compared to the DDP group (P < 0.05), indicating that tail vein injection of mitochondria can significantly improve the chemotherapy efficacy of cisplatin against drug-resistant cancer tissues, and mitochondrial transplantation can reverse cisplatin resistance in lung cancer.

[0045] Further evaluation of the safety of cisplatin monotherapy versus tail vein injection of mitochondria combined with cisplatin chemotherapy was conducted, with mouse body weight monitored during the experiment. Figure 1 (C) shows that, compared with the control group, mice in the DDP group experienced weight loss after cisplatin monotherapy, while mice in the DDP+Mito group who underwent mitochondrial transplantation combined with cisplatin chemotherapy showed a recovery in weight after mitochondrial transplantation. There was no significant decrease in body weight among all mice injected via the tail vein compared to other groups, indicating that mitochondrial transplantation does not cause weight loss in mice and has a certain degree of safety.

[0046] The survival observation results of each group of mice during the experiment are as follows: Figure 2 As shown, the survival time of the Control group and the Mito group was 28 days, the survival time of the DDP group was 35 days, and the survival time of the DDP+Mito group was 42 days. This indicates that tail vein injection of mitochondria can significantly prolong the survival time of chemotherapy mice, with an average extension of 20% (P<0.01).

[0047] The above experimental results show that mitochondrial transplantation can not only safely and effectively reverse cisplatin resistance in non-small cell lung cancer, but also prolong the survival of tumor-bearing mice and improve their prognosis.

[0048] Example 2

[0049] This embodiment verifies the role of mitochondrial transplantation in reversing cisplatin-resistant human lung adenocarcinoma CCK8 cells through an experiment.

[0050] Human lung adenocarcinoma cells (A549 / DDP) in logarithmic growth phase were collected, digested with trypsin, and suspended into a cell suspension for counting. 5000–10000 cells per well were seeded into 96-well cell culture plates, and 100 μL of culture medium was added. The plates were then incubated in a cell culture incubator (37°C, 5% CO2). After cell attachment, the viability of cisplatin-resistant A549 / DDP lung cancer cells was assessed by cisplatin administration and mitochondrial combined cisplatin administration to evaluate the efficacy of cisplatin and mitochondrial combined cisplatin administration. The cisplatin concentration was 100 μM, and the mitochondrial intervention concentration was 1 × 10⁻⁶. 7 per mL.

[0051] Figure 3 The effects of different interventions on the proliferation inhibition of A549 / DDP cells are presented, where (A) represents the drug concentration (IC50) corresponding to a 50% cell inhibition rate. 50 (B) represents cell viability, and (C) represents the cell proliferation inhibition rate.

[0052] Experimental results showed that cisplatin had an IC50 effect on A549 / DDP cells for 24 hours. 50 At 107.7 μM, the cell viability after 24 hours of cisplatin-only intervention was 47.70 ± 4.03% (P < 0.01), and the inhibition rate of cell proliferation was 52.29 ± 4.03% (P < 0.01); while the cell viability after 24 hours of cisplatin combined with mitochondrial intervention was 35.42 ± 6.71% (P < 0.01), and the inhibition rate of cell proliferation was 64.58 ± 6.71% (P < 0.01).

[0053] By combining mitochondrial transplantation with cisplatin, the efficacy of cisplatin in cisplatin-resistant lung cancer cells A549 / DDP was significantly improved, with an efficacy increase of 12.31±7.72% (P<0.01), indicating that combined mitochondrial transplantation can reverse cisplatin resistance in lung cancer.

[0054] Example 3

[0055] This embodiment uses a CCK8 cell line experiment to verify the effect of mitochondrial transplantation on reversing cisplatin-resistant ovarian cancer.

[0056] Log-phase cisplatin-resistant ovarian cancer cells (Skov3 / DDP) were collected, digested with trypsin, and suspended into a cell suspension for counting. 5000–10000 cells per well were seeded into 96-well cell culture plates, and 100 μL of culture medium was added. The plates were then incubated at 37°C (5% CO2). After cell attachment, the viability of Skov3 / DDP ovarian cancer cells was assessed by cisplatin administration and mitochondrial combined cisplatin administration to evaluate the efficacy of cisplatin and mitochondrial combined cisplatin administration. The cisplatin concentration was 15 μM, and the mitochondrial intervention concentration was 1 × 10⁻⁶. 7 per mL.

[0057] Figure 4 The effects of different interventions on the proliferation inhibition of Skov3 / DDP cells are presented, where (A) represents the drug concentration (IC50) corresponding to a 50% cell inhibition rate. 50 (B) represents cell viability, and (C) represents the cell proliferation inhibition rate.

[0058] Experimental results showed that cisplatin had an IC50 effect on Skov3 / DDP cells for 48 hours. 50 =20.4 μM (P < 0.01), the cell viability after 48 hours of cisplatin-only intervention was 56.84 ± 5.04% (P < 0.01), and the inhibition rate of cell proliferation was 43.16 ± 5.04% (P < 0.01); while the cell viability after 48 hours of cisplatin combined with mitochondrial intervention was 39.65 ± 2.01% (P < 0.01), and the inhibition rate of cell proliferation was 60.35 ± 2.01% (P < 0.01).

[0059] By combining mitochondrial transplantation with cisplatin, the efficacy of cisplatin in cisplatin-resistant Skov3 / DDP ovarian cancer cells was significantly improved by 17.19±4.65% (P<0.01), indicating that combined mitochondrial transplantation can reverse cisplatin resistance in lung cancer.

[0060] Example 4

[0061] This embodiment uses a CCK8 assay to verify the effect of mitochondrial transplantation on reversing paclitaxel resistance in advanced triple-negative breast cancer.

[0062] Triple-negative breast cancer paclitaxel-resistant cells (MDA-MB-231 / Taxol-Re) in logarithmic growth phase were collected, digested with trypsin, and suspended into cell suspensions for counting. 5000–10000 cells per well were seeded into 96-well cell culture plates, and 100 μL of culture medium was added. The plates were then incubated in a cell culture incubator (37℃, 5% CO2). After cell attachment, the viability of MDA-MB-231 / Taxol-Re cells was assessed by administering paclitaxel and by combining mitochondrial and paclitaxel administration, thus evaluating the efficacy of these two methods. The paclitaxel concentration was 2 μM, and the mitochondrial intervention concentration was 1 × 10⁻⁶. 7 per mL.

[0063] Figure 5 The effects of different interventions on the proliferation inhibition of MDA-MB-231 / Taxol-Re cells are presented, where (A) represents the drug concentration (IC50) corresponding to a 50% cell inhibition rate. 50 (B) represents cell viability, and (C) represents the cell proliferation inhibition rate.

[0064] Experimental results showed that paclitaxel had an IC50 effect on MDA-MB-231 / Taxol-Re cells for 48 hours. 50 At 22.0 μM, the cell viability of paclitaxel alone after 48 hours of intervention was 90.32 ± 3.89%, and the inhibition rate of cell proliferation was 9.68 ± 3.89%; while the cell viability of paclitaxel combined with mitochondrial intervention after 48 hours was 76.85 ± 1.47% (P < 0.05), and the inhibition rate of cell proliferation was 23.15 ± 1.47% (P < 0.05).

[0065] By combining mitochondria, the efficacy of paclitaxel against drug-resistant cells can be significantly improved, with an increase of 13.46±2.40% (P<0.01), indicating that combined mitochondrial transplantation can reverse paclitaxel resistance in triple-negative breast cancer.

[0066] The above embodiments of the present invention do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of mitochondrial extract in the preparation of drugs that reverse chemotherapy resistance in solid tumors.

2. Application of mitochondrial extract in the preparation of drugs that reverse resistance to platinum-based chemotherapy drugs or paclitaxel in solid tumors.

3. The application according to claim 1 or 2, wherein the solid tumor includes non-small cell lung cancer, ovarian cancer, or triple-negative breast cancer.

4. Application of mitochondrial extract in combination with chemotherapy drugs in the preparation of drugs for treating chemotherapy-resistant solid tumors.

5. Application of mitochondrial extract in combination with platinum-based chemotherapy drugs or paclitaxel in the preparation of drugs for treating chemotherapy-resistant solid tumors.

6. The application according to claim 5, wherein the solid tumor includes non-small cell lung cancer, ovarian cancer, or triple-negative breast cancer.

7. A combination drug for treating chemotherapy-resistant solid tumors, wherein the active pharmaceutical ingredients include a mitochondrial extract and a chemotherapy drug, wherein the mitochondrial extract and the chemotherapy drug are each a separate administration unit.

8. A combination drug for treating chemotherapy-resistant solid tumors, the active pharmaceutical ingredient comprising a mitochondrial extract and a platinum-based chemotherapy drug or paclitaxel, wherein the mitochondrial extract and the platinum-based chemotherapy drug or paclitaxel are each a separate administration unit.

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