Synergistic anti-tumor pharmaceutical composition

Abstract

The invention provides application of a combination of astragaloside and lobetyolin in preparation of an anti-breast cancer drug with a synergistic interaction effect on cis-platinum. The pharmaceutical composition has the potential to be developed into a medicament for reducing toxicity and enhancing effect on cis-platinum.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to an anti-tumor pharmaceutical composition with synergistic effect on cisplatin. BACKGROUND

[0002] Breast cancer is one of the most common malignant tumors in women worldwide, with high incidence and mortality. Among them, triple-negative breast cancer accounts for about 15-20% of all breast cancers, with high recurrence rate and poor prognosis. It is the most invasive type of breast cancer, and chemotherapy is the key means of triple-negative breast cancer comprehensive treatment, especially for patients with advanced, recurrent or metastatic breast cancer (Canha-Borges A, et al. Cancer Treat Rev. 2025; 140:103026.). Cisplatin is a classic platinum-based broad-spectrum anti-tumor drug, which can interfere with DNA replication and transcription by cross-linking with tumor cell DNA, and then induce apoptosis. In the treatment of breast cancer, cisplatin is used for specific subtypes (such as triple-negative breast cancer) or as a salvage treatment regimen (Xu T, et al. Acta Pharm Sin B. 2025; 15(5):2703-2722.).

[0003] Cisplatin faces two major challenges in clinical application, which severely limits its efficacy and patient benefits: first, acquired drug resistance of tumor cells. After long-term or multiple use of cisplatin, tumor cells can develop resistance through various mechanisms (such as enhancing DNA damage repair, reducing drug accumulation, activating anti-apoptotic pathways, etc.), leading to treatment failure (Lv GY, J Hepatol. 2025; 83(4):917-930.). Second, dose-limiting toxic side effects. Cisplatin is significantly toxic to normal tissues, especially the kidneys, nervous system, and auditory system. These toxic side effects often force clinicians to reduce drug doses or interrupt treatment, thereby affecting the anti-tumor effect (Gupta S, et al. JAMA Oncol. 2025; 11(6):636-643.). Therefore, finding strategies to enhance the efficacy of cisplatin while reducing its toxic side effects or reversing drug resistance is a key problem that needs to be solved in the field of breast cancer treatment.

[0004] “Synergistic Effect” refers to the therapeutic effect of two drugs used in combination being greater than the sum of the therapeutic effects of the same doses of the two drugs used alone. According to the principle of Chou TC. Cancer Res. 2010, 70(2):440-6., the effect of two drugs used in combination can be judged by “Combination Index” (CI):

[0005]

[0006] wherein (Dx)1 and (Dx)2 are the doses of drug 1 and drug 2, respectively, required to produce an x% effect (e.g. IC 50 (D)1 and (D)2 are the doses of drug 1 and drug 2, respectively, required to produce an x% effect (e.g. IC

[0007] CI < 1 is synergistic; CI = 1 is additive; CI > 1 is antagonistic. SUMMARY

[0008] The present application provides the application of the combination of astragaloside and lognansapogenin in the preparation of anti-breast cancer drugs with synergistic effect on cisplatin.

[0009] To achieve the above object, the present application adopts the following technical solutions:

[0010] The present application provides the application of the combination of astragaloside and lognansapogenin in the preparation of anti-breast cancer drugs with synergistic effect on cisplatin.

[0011] In a preferred embodiment, the weight ratio of astragaloside to lognansapogenin in the combination is (2-16): 5.

[0012] In another preferred embodiment, the breast cancer is triple-negative breast cancer.

[0013] In another preferred embodiment, the combination can alleviate the kidney damage caused by cisplatin.

[0014] The details of each aspect of the present application will be described in the subsequent chapters. The features, objects and advantages of the present application will be more apparent through the following description and claims. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure 1 shows the effect of different drug regimens on the survival rate of triple-negative breast cancer cells 4T1 in Example 1 of the present application. (A) Survival rate of 4T1 cells treated with different concentrations of drugs; (B) Effect of AS-LB combination; (C) Combination index (CI).

[0016] Figure 2 Figure 2 shows the effect of different drug regimens on the tumor volume (A) and body weight (B) of triple-negative breast cancer-bearing mice and the combination index (CI) (C) in Example 1 of the present application. *, P < 0.05; **, P < 0.01.

[0017] Figure 3In Example 2 of this invention, the effects of different drug regimens on the levels of serum creatinine (CRE) and urinary kidney injury factor-1 (KIM) in triple-negative breast cancer-bearing mice were investigated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0018] Figure 4 This is a comparative HE staining pathological image of kidney tissue sections from mice with cisplatin-induced acute kidney injury in each group, as shown in Example 2 of this invention.

[0019] Figure 5 The figure shows the detection results of serum creatinine (Scr), blood urea nitrogen (BUN) and tumor necrosis factor-α (TNF-α) levels in mice with cisplatin-induced acute kidney injury in each group in Example 2 of the present invention. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, ns, no significant difference.

[0020] Figure 6 This is a comparative diagram of the level of apoptosis in renal tissue cells of mice with cisplatin-induced acute kidney injury detected by TUNEL staining in Example 2 of the present invention.

[0021] Figure 7 This is a comparative diagram showing the detection of macrophage infiltration (F4 / 80 labeling) and expression of cell proliferation protein (Ki67) in the renal tissue of mice with cisplatin-induced acute kidney injury in each group by immunohistochemical staining in Example 2 of the present invention. Detailed Implementation

[0022] This invention provides a traditional Chinese medicine active ingredient composition for synergistic enhancement of cisplatin's efficacy in treating triple-negative breast cancer. The composition consists of astragaloside A and codonopsis glycoside, which work synergistically to enhance the efficacy of cisplatin in treating triple-negative breast cancer while significantly reducing its nephrotoxic side effects. The weight ratio of astragaloside A to codonopsis glycoside in the composition is (2-16):5.

[0023] As used in this invention:

[0024]

[0025] Astragaloside A, molecular formula: C 41 H 68 O 14 Molecular weight: 784.97, CAS number: 84687-43-4.

[0026]

[0027] Codonopsis glycosides, molecular formula: C 20 H 28O8, molecular weight: 396.43, CAS number: 136085-37-5.

[0028] The inventors have confirmed through their research that, in the constructed triple-negative breast cancer mouse model, compared with single-drug therapy, the combination of the above-mentioned composition with cisplatin can synergistically inhibit tumor growth (combination index CI<1) and effectively maintain the animal's body weight.

[0029] In a cisplatin-induced acute kidney injury mouse model, the composition significantly improved renal tissue pathological damage, reduced renal tubular epithelial cell apoptosis and macrophage infiltration, restored serum urea nitrogen (BUN) and creatinine (Scr) levels, and reduced the expression of pro-inflammatory factors, indicating that it has a good nephroprotective effect.

[0030] Details of various aspects of the invention will be described in detail in the following sections. The features, objects, and advantages of the invention will become more apparent from the following description and the claims.

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0033] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0034] Example 1: Experiment on enhancing the effect of cisplatin against triple-negative breast cancer with a combination of active ingredients from traditional Chinese medicine

[0035] (1) In vitro triple-negative breast cancer 4T1 cells

[0036] The mouse triple-negative breast cancer cell line 4T1 was purchased from Shanghai Fuheng Biotechnology Co., Ltd. It was cultured in DMEM (high glucose) medium containing 10% serum and incubated at 37°C with 5% CO2. The cells were diluted to 5 × 10⁶ cells / mL. 4Cell suspension was prepared at 1 / mL. 200 μL of cell suspension was injected into each well of a 96-well plate, and the mixture was gently shaken after addition. The plates were then incubated until cells adhered. Culture media containing different concentrations of cisplatin were prepared using cisplatin stock solution: 5, 10, 20, 40, and 80 µmol / L; astragaloside A (AS) concentrations were 5, 10, 20, 40, 80, and 160 µmol / L; and codonopsis glycoside (LB) concentrations were 6.25, 12.5, 25, 50, and 100 µmol / L. After 12 h of drug treatment, 20 μL of MTT solution was added to each well of the 96-well plate, and the plates were incubated for 4 h. Crystals were retained in the wells, and the solution was carefully aspirated. 150 μL of DMSO was added to each well. The plates were shaken for 10 min, and the OD value of each well was measured using a microplate reader at 490 nm. Cell viability was calculated using the following formula:

[0037] Cell viability (%) = (OD value of experimental group - OD value of blank group / OD value of control group - OD value of blank group) × 100%.

[0038] Experimental results showed that cisplatin intervention for 12 h resulted in a half-maximal inhibitory concentration (IC50) in 4T1 cells. 50 ) were 20 µmol / L ( Figure 1 A). To further verify the synergistic effect of the AS-LB combination, we used a low-toxicity concentration of cisplatin (5 µmol / L, 89.2 ± 3.2%) in combination with AS at concentrations of 5, 10, and 20 µmol / L and LB at concentrations of 6.25, 12.5, and 25 µmol / L, respectively. The combination regimens were: AS 5 µmol / L + LB 6.25, 12.5, and 25 µmol / L + CIS 5 µmol / L; AS 10 µmol / L + LB 6.25, 12.5, and 25 µmol / L + CIS 5 µmol / L; AS 20 µmol / L + LB 6.25, 12.5, and 25 µmol / L + CIS 5 µmol / L (… Figure 1 B).

[0039] The results are shown in Table 1. For AS 10 µmol / L + LB 25 µmol / L + CIS (CI = 0.57 < 1), AS 20 µmol / L + LB 6.25 µmol / L + CIS (CI = 0.845 < 1), AS 20 µmol / L + LB 12.5 µmol / + CIS (CI = 0.75 < 1), and AS 20 µmol / L + LB 25 µmol / + CIS (CI = 0.67 < 1), these four concentration combinations significantly enhanced the sensitivity to cisplatin, and AS and LB produced a synergistic effect in enhancing the anti-breast cancer effect of cisplatin. At this time, the concentration ratios of AS to LB were 2:5, 16:5, 8:5, and 4:5 ( Figure 1 C).

[0040] Table 1

[0041]

[0042]

[0043] (2) In vivo triple-negative breast cancer tumor-bearing mouse model

[0044] Female SPF-grade BALB / c mice at 7 weeks of age were selected as experimental animals and were housed in the standardized environment of the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine (temperature 22 - 26 °C, relative humidity 40% - 70%, 12 h light / dark cycle). All experimental protocols for the experimental animals were approved by the Shanghai Traditional Chinese Medicine Research Ethics Committee (PZSHUTCM220919006), and the animal welfare guidelines were strictly followed. (Animal use license number: SYXK (Shanghai) 2020 - 0009)

[0045] Triple-negative breast cancer 4T1 cells in the logarithmic growth phase were resuspended to 5×10 6 cells / mL. The cell suspension was inoculated into the mammary fat pad of mice. After about one week, a mass was palpable, and the mice were randomly divided into groups of 8 each, including: normal group, model group, cisplatin (3 mg / kg) group, astragaloside (AS) (20, 40 mg / kg) + cisplatin group, lobetyolin (LB) (12.5, 50 mg / kg) + cisplatin group, and groups of combined use of astragaloside and lobetyolin at four different doses (dose ratios were 20 + 12.5, 20 + 50, 40 + 12.5, 40 + 50 mg / kg) + cisplatin group.

[0046] The model group was given an equal amount of normal saline; cisplatin was intraperitoneally injected (3 mg / kg, once every 3 days); astragaloside and lobetyolin were administered by gavage (once a day). The body weight and tumor volume were recorded every 3 days, and samples were taken after 21 days.

[0047] Experimental results showed that the combined use of four different doses of astragaloside A and codonopsis pilosula (dose ratios of 20+12.5, 20+50, 40+12.5, and 40+50 mg / kg) significantly enhanced the inhibitory effect of cisplatin on tumor growth and maintained the tumor growth in animals, with a combination index CI < 1 (Table 2), indicating that the combination of the two drugs has a synergistic anti-tumor effect. Figure 2 ).

[0048] Table 2

[0049]

[0050] Example 2: Effects of a combination of active ingredients from traditional Chinese medicine on cisplatin-induced kidney injury

[0051] (1) In vivo triple-negative breast cancer mouse model (same as above)

[0052] Serum and urine samples were collected from triple-negative breast cancer-bearing mice, and serum creatinine (CRE) and urinary kidney injury molecule-1 (KIM) were measured according to the kit instructions. The results showed that four different doses of astragaloside combined with codonopsis pilosula (dose ratios of 20+12.5, 20+50, 40+12.5, and 40+50 mg / kg) plus cisplatin significantly reduced CRE and KIM levels, alleviating cisplatin-induced renal function damage. Figure 3 ).

[0053] (2) Cisplatin-induced acute kidney injury mouse model

[0054] To further verify the effects of astragaloside A and codonopsis pilosula on cisplatin-induced kidney injury and to explore their related mechanisms, we used male SPF-grade BALB / c mice, with 8 mice in each group: normal control group, cisplatin model group (25 mg / kg), low-dose group of cisplatin + astragaloside A (20 mg / kg) + codonopsis pilosula (12.5 mg / kg) (ABL), high-dose group of cisplatin + astragaloside A (20 mg / kg) + codonopsis pilosula (50 mg / kg) (ABH), and positive control group (Shenqi Fuzheng Injection, 80 mL / kg).

[0055] Except for the normal control group, all other groups underwent a single intraperitoneal injection of cisplatin (25 mg / kg) to establish the model. The ABL and ABH groups received daily gavage administration starting 7 days before modeling, and continued administration after modeling until tissue collection. The positive control group received intraperitoneal injection. Serum and kidney tissue samples were collected on day 10.

[0056] Sample Collection: Animals were anesthetized, placed supine on a surgical frame, and the abdominal aorta was exposed. The distal end of the aorta was ligated, and the proximal end was clamped with an arterial clamp. A parallel needle was then inserted between the two ends, the clamp released, and blood was immediately collected. Whole blood was collected from the mouse's abdominal aorta into a micro-volume blood collection tube. After high-speed centrifugation, the supernatant serum was collected, aliquoted, and frozen at -80°C for subsequent testing. The abdominal and thoracic cavities were cut along the midline of the abdomen to expose both kidneys and the heart. 20 ml of pre-cooled saline was drawn up, and the needle tip was inserted into the left ventricle. Water was slowly injected until both kidneys turned pale. Both kidneys were then removed and placed in pre-cooled saline. The kidney tissue was trimmed and weighed. A long-axis incision was made along the coronal plane of the kidney, dividing it into two halves. One half of the kidney was immersed in 4% paraformaldehyde and fixed for 24 hours; the other half of the kidney tissue was preserved in liquid nitrogen for subsequent experiments.

[0057] Kidney histopathological observation

[0058] Kidney tissue collected 24 hours after fixation was routinely harvested, dehydrated, embedded in paraffin, prepared into 4 μm thick sections, stained with hematoxylin and eosin (HE), and observed and described after scanning with a panoramic scanner. Lesion sites were excised. Results are shown in […]. Figure 4 HE staining showed that the cisplatin model group exhibited significant vacuolation, swelling, and necrosis of renal tubular epithelial cells. The renal tissue structure in the ABL and ABH groups was essentially normal, with no obvious pathological changes observed in the glomeruli and tubules, indicating that this combination has a protective effect against cisplatin-induced kidney injury.

[0059] Blood biochemistry and inflammatory factor testing

[0060] The collected serum was used to measure creatinine (Scr), blood urea nitrogen (BUN), and TNF-α according to the kit instructions. The results showed that Scr and BUN levels were significantly reduced in both the ABL and ABH groups; the level of the pro-inflammatory factor TNF-α was also significantly reduced in the ABL group, indicating that this composition can improve renal function and inhibit the inflammatory response. Figure 5 ).

[0061] Apoptosis detection in kidney tissue of cisplatin-induced kidney injury model mice

[0062] The paraffin-embedded kidney tissue was dewaxed, and the prepared paraffin sections were processed. Proteinase K was added to the sections, and the sections were incubated at 37 °C for 20 minutes. Then, TUNEL reaction mixture was added, and the sections were incubated at 37 °C in the dark for 60 minutes. DAPI staining solution was added, and the sections were incubated at room temperature in the dark for 10 minutes. The sections were then observed and photographed using a fluorescence microscope.

[0063] TUNEL staining results showed that the number of apoptotic renal tubular epithelial cells in the ABL and ABH groups was significantly reduced compared with the model group, suggesting that the composition can inhibit cisplatin-induced apoptosis. Figure 6 ).

[0064] Immunohistochemical detection of kidney tissue in cisplatin-induced kidney injury model mice

[0065] Paraffin sections were dewaxed using a gradient of ethanol and then hydrated. The sections were soaked in sodium citrate buffer (pH 6.0) and then microwaved at 110 °C for 10 minutes. They were then incubated in 3% H₂O₂ deionized water at room temperature for 10 minutes. The sections were rinsed three times with PBS, each time for 3 minutes. After removing the PBS, the sections were blocked with 5% BSA at room temperature for 30 minutes. Primary antibody was added and incubated overnight at 4 °C. The sections were then rinsed three times with PBS, each time for 3 minutes. Secondary antibody was added and incubated at room temperature in the dark for 1 hour. The sections were then rinsed three times with PBS, each time for 3 minutes. 100 μL of diaminobenzidine chromogenic solution was added to the sections. The sections were rinsed with distilled water, dehydrated using a gradient of ethanol, cleared with xylene, dried, and mounted. The sections were then observed and photographed under a microscope.

[0066] Ki67 staining showed that the cell proliferation activity in the ABL and ABH groups was higher than that in the model group; F4 / 80 staining showed that the composition could reduce macrophage infiltration in renal tissue, indicating that it could alleviate the inflammatory microenvironment and relieve renal injury. Figure 7 ).

[0067] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. Application of the combination of astragaloside A and codonopsis glycoside in the preparation of an anti-breast cancer drug with synergistic effect against cisplatin.

2. The application as described in claim 1, characterized in that, The weight ratio of astragaloside A to codonopsis glycoside in the combination is (2~16):

5.

3. The application as described in claim 1, characterized in that, The breast cancer mentioned is triple-negative breast cancer.

4. The application as described in claim 1, characterized in that, The combination can alleviate cisplatin-induced kidney damage.

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