Application of doxorubicin and paclitaxel in preparation of medicine for treating tumors

Abstract

The invention relates to the technical field of medicine, and discloses an application of doxorubicin liposome and albumin paclitaxel in preparation of tumor treatment drugs, a combined medication scheme of the doxorubicin liposome and albumin paclitaxel is not recorded by any colorectal cancer related diagnosis and treatment guideline at present, and the doxorubicin liposome and albumin paclitaxel liposome is an over-indication medication scheme.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of doxorubicin and paclitaxel in the preparation of drugs for treating tumors. Background Technology

[0002] Colorectal cancer is a malignant tumor with a high incidence and mortality rate. According to the latest statistics from the U.S. National Cancer Institute, more than 150,000 new cases of colorectal cancer will be diagnosed in the United States in 2025, accounting for 7.5% of all new cancer cases, and more than 50,000 patients will die from colorectal cancer, accounting for a staggering 16% of all cancer-related deaths. These figures are second only to lung cancer, breast cancer, and prostate cancer. Statistics released by the National Cancer Center of China in 2024 also show that colorectal cancer is a serious threat to the health of middle-aged and elderly residents in my country. As of 2022, there were 517,100 new cases of colorectal cancer in my country, ranking second among malignant tumors, and 240,000 deaths, ranking fourth. Many cities, including Beijing and Shanghai, have included early screening for colorectal cancer among middle-aged and elderly residents in their basic livelihood projects. It is evident that the huge economic and medical burden that colorectal cancer imposes on individuals and society as a whole has become a major focus of attention.

[0003] Surgical removal of the cancerous site is currently the foundation of colorectal cancer treatment. In addition, chemotherapy drugs such as oxaliplatin, capecitabine, and fluorouracil, as well as their combination regimens (such as the FOLFIRI regimen of irinotecan hydrochloride, leucovorin, and fluorouracil, and the FOLFOX regimen of oxaliplatin, leucovorin, and fluorouracil), are the most commonly used cornerstone drugs in colorectal cancer treatment. In recent years, targeted drugs such as bevacizumab and cetuximab, as well as immune checkpoint inhibitors such as PD-1 monoclonal antibodies, have also been included in colorectal cancer diagnosis and treatment guidelines, reflecting the progress and diversity of treatment options.

[0004] However, for patients with advanced disease, resistance to conventional chemotherapy drugs such as fluorouracil and platinum-based drugs often occurs, which is a major treatment challenge in clinical practice. Summary of the Invention

[0005] The main objective of this invention is to solve the technical problem of drug resistance to conventional chemotherapy drugs such as fluorouracil and platinum-based drugs in advanced colorectal cancer in the prior art.

[0006] The technical solution adopted in this invention is: the application of doxorubicin and paclitaxel in the preparation of drugs for treating tumors, wherein the tumor is advanced colorectal cancer resistant to chemotherapy drugs.

[0007] Preferably, the doxorubicin is a doxorubicin liposome; Preferably, the paclitaxel is albumin-bound paclitaxel; Preferably, the concentration of doxorubicin liposomes is 543.0 ng / ml; Preferably, the concentration of albumin-bound paclitaxel is 12.295 ug / ml.

[0008] The present invention also relates to a pharmaceutical composition for treating tumors, characterized in that the pharmaceutical composition comprises doxorubicin and paclitaxel.

[0009] Furthermore, the drug composition can be administered orally, sublingually, topically, or by injection.

[0010] Furthermore, the local administration refers to a method of administration that does not involve absorption through the gastrointestinal tract.

[0011] Furthermore, the injection administration includes subcutaneous injection, intramuscular injection, intravenous injection, and intradermal injection.

[0012] Furthermore, the pharmaceutical composition also includes other pharmaceutically recognized excipients, including adjuvants, carriers, excipients, gliding agents, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH adjusters and / or buffers, solvents, surfactants, or emulsifiers.

[0013] In one specific embodiment, the therapeutic and / or preventive effective amount of the present invention refers to the amount of tetrahydromethylene and magnolol that can exert the desired therapeutic effect, for example, an amount that can effectively prevent, alleviate, or improve the condition, treat the disease, or prolong the survival of the subject. As will be understood by those skilled in the art, the exact amount required varies depending on the patient, age, the general condition of the subject, and the severity of the condition, to determine the compounds and / or the composition and analogues to be administered, and their amounts. The appropriate therapeutic and / or preventive effective amount, in any individual case, can be determined by one of ordinary skill in the art by referring to relevant texts and documents and / or by using routine experiments.

[0014] The present invention has the following beneficial effects: The present invention, after evaluation by PDO drug sensitivity testing, confirmed that, compared with single-drug administration, the dosing regimen of doxorubicin liposome and albumin-bound paclitaxel has a more significant inhibitory effect on PDO growth, and therefore has a significant synergistic effect.

[0015] The combination therapy regimen of this invention has not yet been included in any colorectal cancer-related diagnosis and treatment guidelines. It is an off-label therapy regimen. Since it has achieved significant tumor inhibition effect in the drug sensitivity test described above, it has broad clinical application prospects. Attached Figure Description

[0016] Figure 1The present invention describes the morphological characteristics of patient-derived organoids at days 3, 7, and 10 of growth. It also examines the expression of colorectal cancer marker molecules on PDO after HE and immunohistochemical staining, and compares the consistency of marker molecule expression between PDO and the source tissue.

[0017] Figure 2 The study investigated the growth inhibition of organoids in three PDO models after drug sensitivity testing and analyzed statistically.

[0018] Figure 3 The study investigated the growth inhibition of organoids in three PDO models after drug sensitivity testing and analyzed statistically.

[0019] Figure 4 The study investigated the growth inhibition of organoids in three PDO models after drug sensitivity testing and analyzed statistically. Detailed Implementation

[0020] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0021] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0022] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0023] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0024] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0025] The reagents used in the following embodiments of this application are from the following sources: Tissue preservation solution (Shanghai Wanheyuan Biotechnology Co., Ltd., product number: B2303-PS100); Tissue digestion fluid (Shanghai Wanheyuan Biotechnology Co., Ltd., product number: X2307-DB100); Red blood cell lysis buffer (Shanghai Wanheyuan Biotechnology Co., Ltd., product number: H2305-LJ100); Tissue-specific cleaning solution (Shanghai Wanheyuan Biotechnology Co., Ltd., Product No.: Q2303-QX100); Complete tumor organoid culture medium (Shanghai Wanheyuan Biotechnology Co., Ltd., catalog number: ) Digestion Termination Solution (Shanghai Wanheyuan Biotechnology Co., Ltd., Product No.: Z2308-ZZ100); Organoid matrix gel (Shanghai Wanheyuan Biotechnology Co., Ltd., Product No.: J2306-MM010); Albumin-bound paclitaxel (MCE, catalog number: HY-P99974); Doxorubicin liposomes (Targetmol, catalog number: T1020). The combination therapy used in this invention is doxorubicin liposomes and albumin-bound paclitaxel. Doxorubicin is an anthracycline antitumor antibiotic produced by Streptomyces boswellia var. gravida. The mechanism of action of this type of drug in inhibiting tumors is mainly as follows: ① It inserts between DNA base pairs, interfering with the physiological process of DNA transcription; ② By inhibiting DNA polymerase, it interferes with DNA synthesis; ③ By disrupting the tertiary structure of DNA, it affects the normal functioning of cells; ④ It generates oxygen free radicals, which damage the integrity of the cell membrane barrier, leading to cell lysis and death. The doxorubicin formulation used in this invention is doxorubicin liposomes, which has the advantages of a longer half-life and better efficacy compared with traditional formulations, while also reducing side effects such as cardiotoxicity and hair loss.

[0026] Paclitaxel is a class of highly effective, low-toxicity diterpenoid alkaloids with broad-spectrum anticancer activity. Its main mechanism of tumor inhibition is as follows: ① By binding tightly to tubulin, it causes cell cycle arrest, thereby inhibiting cell mitosis; ②Mediates apoptosis.

[0027] The paclitaxel formulation used in this invention is albumin-bound paclitaxel, which has higher solubility in water compared to traditional paclitaxel. It no longer relies on solvents such as anhydrous ethanol and polyoxyethylene castor oil that can cause severe allergic reactions, thus effectively expanding the scope of application of this drug.

[0028] The objects tested in this invention are patient-derived organoid models (PDOs), whose source tissues include surgically removed tumors, puncture samples, ascites samples, etc.

[0029] Human cancer samples are not the only source for constructing organoids. Tumor tissues from mice, rats, rabbits, and non-human primates can also be used for organoid construction and should also be protected by this invention.

[0030] In this invention, a PDO model was constructed and drug sensitivity testing was performed to evaluate the tumor-suppressing efficiency of combination drug regimens. PDO, derived directly from the human body, is a self-renewing, self-organizing, and differentiating three-dimensional in vitro culture structure. Compared to traditional cell culture, it can fully inherit the pathological characteristics, cell type composition, and genetic background of the source tissue and can simulate some of the tissue's physiological functions. Compared to animal experiments, the PDO model has better stability and relatively lower cost, thus it can be considered an excellent model for simulating tissue responses to exogenous stimuli in vitro. In application areas, the PDO model is highly suitable for drug development and personalized medicine due to its high compatibility with the source tissue, its ability to meet high-throughput experimental needs, and its ability to obtain experimental results quickly. Patients seeking treatment alternatives to standard therapies for various reasons can use PDO drug sensitivity testing as an experimental tool to evaluate the tumor-suppressing efficiency of different drug regimens, and use these experimental results to guide clinical medication.

[0031] This invention, through PDO susceptibility testing, confirmed that the combination of anthracyclines and paclitaxel can more significantly inhibit PDO growth, demonstrating efficacy superior to monotherapy. This combination therapy is not currently included in any guidelines and falls outside the scope of its indications; therefore, it holds great promise for future clinical applications and can serve as a foundation for preliminary research in clinical trials related to expanding drug indications.

[0032] The method for constructing the PDO model is described below: ① Sample collection: This should be performed in a sterile operating room or a relatively clean laminar flow ward. Disposable sterile consumables or high-temperature sterilized equipment should be used throughout the procedure. After successful sample extraction, the sample should be immediately transferred to a sample preservation solution and completely submerged. Then, under a low-temperature environment of 4°C (no temperatures below 0°C), it should be immediately transferred to a cell culture laboratory. The time interval from sample collection to the start of PDO construction should not exceed 48 hours.

[0033] ② Tissue digestion: Use sharp instruments such as blades, tweezers, and scissors to remove visible necrotic, calcified, and fatty tissues, and wash the tissue several times. After washing, mix the digestive solution thoroughly with the shredded tissue and incubate until more than 70% of the suspension consists of cell clumps of 40-50 µm. Stop digestion and count the cells in the digested suspension and observe the morphology of the cell clumps to assess the quality of digestion.

[0034] ③ Organoid inoculation: On ice, mix an appropriate amount of cell suspension and matrix gel thoroughly, inoculate onto a cell culture plate, let stand for a period of time until the matrix gel solidifies, then add organoid culture medium to completely submerge the gel, and place the culture plate in a carbon dioxide incubator for culture.

[0035] ④ Change medium, observe and passage: Observe the growth status of organoids under a microscope every 2-3 days and change to fresh culture medium. When the cell confluence in the droplet reaches 70%~80%, passage the organoids at a dilution of 1:3 or higher.

[0036] After PDO is constructed, it can be used for antimicrobial susceptibility testing after the third generation and when the growth status is stable. The testing procedure is described below: ① When PDO grows to the standard morphology, discard the original culture medium and replace it with a culture medium containing the drug to be tested. The concentration of doxorubicin liposomes is 543.0 ng / ml, and the concentration of albumin-bound paclitaxel is 12.295 ug / ml.

[0037] ②On day 3-4 of drug treatment, stop the experiment, discard the culture medium containing the drug, wash the organoids with PBS, and wait for testing.

[0038] After the drug sensitivity test is completed, the tumor inhibition efficiency can be detected using the live cell ATP content assay and fluorescence staining method. The procedure for the ATP content assay is as follows: Mix the working solution from Promega's CellTiter-Glo® 3D cell viability assay kit with PBS at a 1:1 ratio, then add the mixture to organoid plates from which the supernatant has been completely discarded. Incubate for approximately 30 minutes. After incubation, measure the absorbance of each well using a microplate reader and plot a statistical graph of the tumor growth inhibition rate. The inhibition rate is calculated using the following formula.

[0039] Survival rate = (A 孔 -A control )×100% The procedure for fluorescent staining is described below: The fluorescent dyes used in this invention are calcein-acetylacetylmethyl ester (Calcein-AM) and propidium iodide (PI). Calcein-AM can penetrate the cell membrane of living cells and is converted into calcein by intracellular esterases, emitting fluorescence (green) under excitation light at 494 nm, used for labeling living cells. PI can penetrate the remaining cell membrane of dead or apoptotic cells and bind to DNA, emitting fluorescence (red) under excitation light at 535 nm, used for labeling dead / apoptotic cells. The combined use of these two dyes allows for the separate labeling of dead and living cells in the same culture, achieving the purpose of assessing cell growth inhibition rates.

[0040] After PDO stimulation is complete, discard the original culture medium and replace it with fresh culture medium containing 0.2 µmol / L calcein-AM. Incubate for about 60 minutes. After incubation, discard the dye-containing culture medium and wash once. Replace with blank culture medium and photograph under an inverted fluorescence microscope. PDO with clear edges and a diameter ≥30 µm that emits green fluorescence is considered to be live PDO.

[0041] The growth inhibition rate of PDO can be calculated based on the survival rate.

[0042] PDO drug susceptibility test results can be used to guide patients' clinical medication use.

[0043] Figure 1 This study presents the growth characteristics of the same cultured PDO at different time points, as well as the results of HE and immunohistochemical staining and photographic observation of cultured PDO and its source tissue. The indicators detected were colorectal cancer pathological markers CK7, CK20, CDX2, SATB2, and CEA. The results show that PDO and its source tissue have a high degree of consistency in terms of tissue morphology and expression of typical pathological markers. Therefore, PDO can be regarded as an in vitro reproduction of pathological tissue.

[0044] Figure 2This represents the results of drug sensitivity testing on three independent PDO models (CO178, CO196, and CO180). Based on different treatment regimens, they were divided into a combination therapy group, a doxorubicin monotherapy group, a paclitaxel monotherapy group, and a blank control group. After the three PDO models finished treatment, ATP content assay and fluorescence staining were used for evaluation, and the tumor growth inhibition rate was statistically analyzed. The results showed that, compared with the blank control group, the other three drug-treated groups significantly inhibited the growth of tumor cells in PDO. Compared with the two monotherapy groups, the combination therapy group showed a more significant tumor inhibition effect, indicating a certain synergistic effect between the two drugs, and achieving a better tumor growth inhibition rate when combined. Furthermore, compared with the first-line treatment groups FOLFOX (oxaliplatin + fluorouracil) and FOLFIRI (irinotecan + fluorouracil) for colorectal cancer, the combination therapy group achieved more significant tumor suppression in the CO178 and CO196 PDO models, indicating that in these two models, the anthracycline combined with paclitaxel is superior to the traditional first-line treatment; however, the efficacy of the combination therapy group in CO180 was not as good as that of the traditional first-line treatment.

[0045] Figure 3 This represents the results of drug sensitivity testing for three independent PDO models (CO-CA-T2506-059, CO404, and CO210). Based on different treatment regimens, they were divided into a combination therapy group, a doxorubicin monotherapy group, a paclitaxel monotherapy group, and a blank control group. After the three PDO models finished treatment, ATP content assay and fluorescence staining were used for evaluation, and the tumor growth inhibition rate was statistically analyzed. The results showed that, compared with the blank control group, the other three drug-treated groups significantly inhibited the growth of tumor cells in PDO. Compared with the two monotherapy groups, the combination therapy group achieved more significant tumor inhibition in the CO-CA-T2506-059 and CO404 PDO models, while in CO210, the paclitaxel monotherapy group showed the most significant tumor inhibition effect. Furthermore, compared with the FOLFOX and FOLFIRI groups, the combination therapy group achieved more significant tumor suppression effects in CO-CA-T2506-059 and CO404, while in CO210, the tumor suppression effects of both the paclitaxel monotherapy group and the combination therapy group were better than the FOLFOX group, but not as good as the FOLFIRI group.

[0046] Figure 4This data represents the results of drug sensitivity testing in three independent PDO models (CO12, CO118, and CO131). Based on different treatment regimens, the models were divided into a combination therapy group, a doxorubicin monotherapy group, a paclitaxel monotherapy group, and a control group. After the treatment in each PDO model ended, ATP content assays and fluorescence staining were used for evaluation, and the tumor growth inhibition rate was statistically analyzed. The results showed that, compared with the control group, the other three treatment groups significantly inhibited tumor cell growth in PDO. Compared with the two monotherapy groups, the combination therapy group achieved more significant tumor inhibition in both CO12 and CO131 models. However, in CO118, the paclitaxel monotherapy group showed the most significant tumor inhibition. Furthermore, compared with the FOLFOX and FOLFIRI groups, the combination therapy group showed more significant tumor inhibition in CO131; in CO12, the two first-line treatment regimens showed more significant tumor inhibition; and in CO118, the paclitaxel monotherapy group showed better tumor inhibition than all other groups.

[0047] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. The use of doxorubicin and paclitaxel in the preparation of drugs for treating tumors, wherein the tumor is colorectal cancer resistant to chemotherapy drugs; The Doxorubicin is a Doxorubicin liposome; The paclitaxel mentioned is albumin-bound paclitaxel.

2. The application as described in claim 1, characterized in that, The colorectal cancer mentioned is advanced colorectal cancer.

3. The application as described in claim 1, characterized in that, The concentration of doxorubicin liposomes is 500-600 ng / ml.

4. The application as described in claim 3, characterized in that, The concentration of albumin-bound paclitaxel is 10-15 ug / ml.

5. A pharmaceutical composition for treating tumors, characterized in that, The pharmaceutical composition comprises doxorubicin liposomes and albumin-bound paclitaxel, and the tumor is colorectal cancer resistant to chemotherapy drugs.

6. The pharmaceutical composition according to claim 5, characterized in that, The colorectal cancer mentioned is advanced colorectal cancer.

7. The pharmaceutical composition according to claim 5, characterized in that, The concentration of doxorubicin liposomes is 500-600 ng / ml.

8. The pharmaceutical composition according to claim 7, characterized in that, The concentration of albumin-bound paclitaxel is 10-15 ug / ml.

9. The pharmaceutical composition according to claim 5, characterized in that, The drug composition can be administered orally, sublingually, topically, or by injection.

10. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition further includes other pharmaceutically recognized excipients, including adjuvants, carriers, excipients, gliding agents, sweeteners, diluents, preservatives, colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH adjusters, solvents, surfactants, or emulsifiers.

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