Application of a traditional Chinese medicine composition preparation combined with miriplatin in the preparation of a breast cancer treatment drug

Abstract

This invention discloses the application of a traditional Chinese medicine composition in combination with miplatin in the preparation of a breast cancer treatment drug. The composition contains at least: verbena, polygonum cuspidatum, red peony root, peony bark, and atractylodes macrocephala (fried), preferably: honeysuckle 10-20 parts, forsythia 5-15 parts, peppermint 1-11 parts, burdock fruit 5-14 parts, mulberry leaf 4-14 parts, chrysanthemum 4-14 parts, golden lotus 7-18 parts, holly 5-15 parts, verbena 10-20 parts, polygonum cuspidatum 5-15 parts, red peony root 6-17 parts, peony bark 4-14 parts, solidarium 6-17 parts, licorice 1-11 parts, and atractylodes macrocephala (fried) 5-14 parts. The mass ratio of the extract powder obtained from the raw materials to miplatin is 1000-5000:1. The formulation of this invention is based on the core idea of ​​synergistic enhancement, toxicity reduction and efficacy improvement. It is the first time that the five herbs Verbena officinalis, Polygonum cuspidatum, Paeonia lactiflora, Paeonia suffruticosa, and Atractylodes macrocephala (fried) have been shown to work synergistically. The combination of traditional Chinese medicine and miplatin significantly improves the therapeutic effect.

Description

Technical Field

[0001] This invention relates to pharmaceuticals for treating breast cancer, and particularly to the application of a traditional Chinese medicine composition in combination with miplatin in the preparation of a breast cancer treatment drug. Background Technology

[0002] Breast cancer is the leading cause of cancer death among women worldwide, with triple-negative breast cancer (TNBC) accounting for 15-20% of cases. TNBC is characterized by its younger age of onset, high invasiveness, and early distant metastasis. TNBC lacks estrogen receptor (ER), progesterone receptor (PR), and HER2 expression, making it ineffective against endocrine and anti-HER2 therapies. Systemic therapy relies on cytotoxic drugs, with an objective response rate (ORR) of only 20-30%, a median progression-free survival (PFS) of less than 4 months, and a 5-year survival rate of <30%. For second-line and later-line treatment of advanced triple-negative breast cancer (TNBC), classic cytotoxic drugs such as capecitabine, eribulin, gemcitabine, and paclitaxel are commonly used clinically. However, these regimens have limited efficacy, with an ORR typically hovering around 30%, a median PFS of approximately 3-5 months, and a median overall survival (OS) of approximately 10-13 months (consistent with data from the classic drug cohort in the subsequent clinical validation in this document). Furthermore, these drugs are accompanied by significant toxicity. Meanwhile, while platinum-based drugs (such as cisplatin and carboplatin) can be used for TNBC, their application is often limited by dose-limiting toxicities such as nephrotoxicity and neurotoxicity. Furthermore, their monotherapy efficacy is also difficult to overcome these limitations, leading to a long-standing clinical demand for new, highly effective, and low-toxicity regimens. Oxaliplatin causes cumulative neuropathy, impacting quality of life. Oral fluoropyrimidine drugs such as capecitabine have an incidence of 18-20% of hand-foot syndromes, limiting long-term use. TNBC has few tumor-infiltrating lymphocytes (TILs) and a high proportion of myeloid-derived suppressor cells (MDSCs), with CD4+... + / CD8 + A low ratio creates a cold tumor microenvironment; VEGF-A, CD31 + High blood vessel density promotes tumor growth and metastasis.

[0003] Therefore, the market urgently needs a drug with good therapeutic effects. Summary of the Invention

[0004] Purpose of the invention: To overcome the shortcomings of the existing technology, the purpose of this invention is to provide an application of a traditional Chinese medicine composition combined with miplatin in the preparation of a drug for treating breast cancer.

[0005] Technical solution: The application of the traditional Chinese medicine composition preparation described in this invention in combination with miplatin in the preparation of drugs for the treatment of breast cancer.

[0006] The traditional Chinese medicine composition preparation includes at least the following raw materials in parts by weight: 10-20 parts of verbena, 5-15 parts of polygonum cuspidatum, 6-17 parts of peony root, 4-14 parts of peony bark, and 5-14 parts of atractylodes macrocephala (fried). The raw materials of the traditional Chinese medicine composition preparation are extracted with water, precipitated with alcohol, and dried to obtain an extract powder.

[0007] Further, the traditional Chinese medicine composition preparation comprises the following raw materials in parts by weight: honeysuckle 10-20 parts, forsythia 5-15 parts, peppermint 1-11 parts, burdock seed 5-14 parts, mulberry leaf 4-14 parts, chrysanthemum 4-14 parts, golden lotus 7-18 parts, evergreen 5-15 parts, verbena 10-20 parts, polygonum cuspidatum 5-15 parts, red peony root 6-17 parts, peony bark 4-14 parts, solidarium 6-17 parts, licorice 1-11 parts, and atractylodes macrocephala (fried) 5-14 parts. The mass ratio of the extract powder obtained after water extraction, alcohol precipitation, and drying of the raw materials in the traditional Chinese medicine composition preparation to the amount of miplatin is 1000-5000:1.

[0008] Furthermore, the following ingredients were added: 15 parts honeysuckle, 10 parts forsythia, 6 parts peppermint, 10 parts burdock seed, 10 parts mulberry leaf, 10 parts chrysanthemum, 12 parts golden lotus, 10 parts evergreen, 15 parts verbena, 10 parts Japanese knotweed, 12 parts red peony root, 10 parts peony bark, 12 parts solidarium, 6 parts licorice, and 10 parts stir-fried atractylodes macrocephala.

[0009] Furthermore, the mass ratio of the extract powder to the amount of mitral powder is 2000-4000:1.

[0010] Furthermore, the mass ratio of the extract powder to the amount of mitral powder is 3000:1.

[0011] Furthermore, the breast cancer described is triple-negative breast cancer.

[0012] Furthermore, intratumoral injection of miplatin was employed.

[0013] Furthermore, the yield of the extract powder was 7.0-7.52%.

[0014] Furthermore, the drug increases CD4 + / CD8 + The ratio was reduced and the proportion of myeloid-derived suppressor cells (MDSCs) was decreased.

[0015] Furthermore, the Pt content (calculated as Pt) of the Miplatin API is ≥ 95.0%, the particle size D90 is ≤ 5 µm, and the impurity Pd is < 0.2%.

[0016] Furthermore, the drug is an oral preparation, an injectable preparation, or a locally implanted preparation.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. This invention, with "synergistic enhancement and toxicity reduction" as its core concept, proposes for the first time a novel combination of five herbs—verbena officinalis, polygonum cuspidatum, peony root bark, and atractylodes macrocephala (fried)—with platinum-based drugs. This approach breaks through the traditional single-drug or simple combination model, achieving a leap in efficacy by locking in the synergistic ratio window, while significantly reducing toxicity and improving quality of life, providing a new pathway for breast cancer treatment.

[0019] 2. Intratumoral injection of miplatin: Local high concentration; intratumoral injection delivers miplatin directly to the tumor site, forming a high concentration of platinum-DNA adducts, significantly enhancing local cell apoptosis, while systemic exposure is low, reducing nephrotoxicity. Immune synergy: High-concentration platinum can synergistically enhance the local immune activation of this patented traditional Chinese medicine, increasing CD4 count. + / CD8 + T cell infiltration reduces the proportion of MDSCs.

[0020] 3. Synergistic Effect of Five Core Herbs: This study demonstrates for the first time that the five herbs—Verbena officinalis, Polygonum cuspidatum, Paeonia lactiflora, Paeonia suffruticosa, and Atractylodes macrocephala (fried)—must be present simultaneously to produce a synergistic effect with miplatin. The optimal synergistic ratio window was determined to be 1000-5000:1, with a peak ratio of 3000:1. Animal experiments showed that this combined treatment regimen significantly improved tumor inhibition rates compared to miplatin monotherapy, and exhibited excellent immunomodulatory and toxicity-reducing effects. Safety was significantly enhanced. Attached Figure Description

[0021] Figure 1 The results of tumor weight changes measured under different mass ratios of extract powder and miplatin in the raw material usage screening experiment were used to demonstrate the tumor-suppressing effect of combined drugs under different ratios and to screen the effective range.

[0022] Figure 2 The results of the change in the proportion of peripheral blood myeloid-derived suppressor cells measured under different mass ratios in the raw material usage screening experiment were used to demonstrate the regulatory effect of combined drug use on the tumor immune microenvironment.

[0023] Figure 3 The results of changes in body weight growth rate measured under different mass ratios in the raw material usage screening experiment were used to demonstrate the safety and toxicity reduction advantages of combined drug use.

[0024] Figure 4 In the raw material usage screening experiment, the changes in tumor inhibition rate calculated based on tumor weight data under different ratios were used to visually demonstrate the relationship between the efficacy of combined drug therapy and the ratio.

[0025] Figure 5The comparison results of the data from the raw material usage screening experiment, grouped inside and outside the proportional window (1000:1 to 5000:1), are used to prove that the overall efficacy of the preferred proportional range (within the window) defined by the present invention is significantly better than that outside the range, and has a synergistic effect.

[0026] Figure 6 The study aimed to investigate the efficacy results of gradually omitting one to four of the following herbs in a synergistic experiment of raw materials for a traditional Chinese medicine composition: verbena, polygonum cuspidatum, red peony root, peony bark, and stir-fried atractylodes macrocephala. This was intended to demonstrate that these five herbs are the core of the synergistic effect, and that the absence of any one of them would lead to a significant decrease in efficacy.

[0027] Figure 7 The results of the efficacy comparison between the complete 15-ingredient formula and the formula with all five core herbs removed (Δ5) in the synergistic experiment of raw materials of traditional Chinese medicine composition were used to prove that the tumor-suppressing, anti-metastasis and immunomodulatory effects of the combined regimen were drastically deteriorated after the absence of the five core herbs.

[0028] Figure 8 The results of comparative experiments on the synergistic effects of traditional Chinese medicine extract powder in combination with miplatin, oxaliplatin, carboplatin, and cisplatin are presented to demonstrate that, at an equivalent platinum dose, miplatin, selected in this invention for combination therapy, has the best efficacy and safety compared to other platinum-based drugs. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0030] All experimental models were female BALB / c pod breast cancer cell lines (4T1-luc), triple-negative breast cancer subtype, using female BALB / c mice, n=8 / group, with 5×10⁶ cells subcutaneously injected into the second breast pad. 5 Cells were administered in 50 µL PBS, randomly assigned to groups on day 7, and treated from day 7 to day 27. Cells were sacrificed 24 hours after the last treatment on day 27.

[0031] The Pt content (calculated as Pt) of the microplatin raw material is ≥ 95.0%, the particle size D90 is ≤ 5 µm, and the impurity Pd is < 0.2% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The Pt content in this article is calculated based on a Pt mass fraction of ≥ 95% for the microplatin raw material, i.e.: Pt content / Pt amount = total mass of microplatin (amount used) × 0.95.

[0032] (I) Implementation Examples

[0033] The following examples are intended to demonstrate the preparation method and activity of the composition.

[0034] Example 1

[0035] The medicinal materials are weighed (parts by weight): Honeysuckle 10 parts, Forsythia 5 parts, Peppermint 1 part, Burdock seed 5 parts, Mulberry leaf 4 parts, Chrysanthemum 4 parts, Golden lotus 7 parts, Ilex chinensis 5 parts, Verbena officinalis 10 parts, Polygonum cuspidatum 5 parts, Red peony root 6 parts, Moutan bark 4 parts, Solidago virgaurea 6 parts, Licorice root 1 part, Atractylodes macrocephala (fried) 5 parts. Total raw medicinal material: 78 parts = 780 g, 1 part = 10 g.

[0036] First extraction: 780 g + 10 times the amount of pure water, decocted at 90℃ for 2 h, filtered through a 200-mesh filter cloth. Second extraction: residue + 8 times the amount of pure water, decocted at 90℃ for 1.5 h, filtered. Combine the filtrates and concentrate under reduced pressure to a relative density of 1.10 (60℃).

[0037] Slowly add 95% ethanol to a concentration of 70% under stirring, let stand at 4°C for 12 h, and centrifuge at 4000 rpm for 10 min.

[0038] The supernatant was dried to recover ethanol, and the mixture was further concentrated to a relative density of 1.25. It was then spray-dried (inlet air 180℃, outlet air 80℃) to obtain 54.6 g of extract powder (yield 7.0%).

[0039] Example 2

[0040] 2.1 Weighing of Medicinal Materials (parts by weight): Honeysuckle 20 parts, Forsythia 15 parts, Peppermint 11 parts, Burdock Seed 14 parts, Mulberry Leaf 14 parts, Chrysanthemum 14 parts, Golden Lotus 18 parts, Ilex chinensis 15 parts, Verbena 20 parts, Polygonum cuspidatum 15 parts, Red Peony Root 17 parts, Moutan Bark 14 parts, Solidago Flos 17 parts, Licorice Root 11 parts, Atractylodes Macrocephala (fried) 14 parts. Total raw medicinal material: 234 parts = 2.34 kg.

[0041] 2.2 Extraction-alcohol precipitation-drying were performed using the same process as in Example 1, yielding 175.5 g of extract powder (yield 7.5%).

[0042] 2.3 Platinum ratio and dosage: 175.5 g ÷ 5000 = 35.1 mg (33.3 mg as Pt). Mass ratio: 5000:1.

[0043] Example 3

[0044] 3.1 Weighing of Medicinal Materials (parts by weight): Honeysuckle 15 parts, Forsythia 10 parts, Peppermint 6 parts, Burdock Seed 10 parts, Mulberry Leaf 10 parts, Chrysanthemum 10 parts, Golden Lotus 12 parts, Ilex chinensis 10 parts, Verbena 15 parts, Polygonum cuspidatum 10 parts, Red Peony Root 12 parts, Moutan Bark 10 parts, Solidago Flos 12 parts, Licorice Root 6 parts, Atractylodes Macrocephala (fried) 10 parts. Total raw medicinal material: 153 parts = 1.53 kg.

[0045] 3.2 Extraction-alcohol precipitation-drying were performed using the same process to obtain 115.1 g of extract powder (yield 7.52%).

[0046] Example 4

[0047] 4.1 Weighing of Medicinal Materials (parts by weight): Honeysuckle 12 parts, Forsythia 8 parts, Peppermint 3 parts, Burdock Seed 8 parts, Mulberry Leaf 7 parts, Chrysanthemum 7 parts, Golden Lotus 9 parts, Ilex chinensis 8 parts, Verbena 12 parts, Polygonum cuspidatum 8 parts, Red Peony Root 9 parts, Moutan Bark 7 parts, Solidago Flos 9 parts, Licorice Root 3 parts, Atractylodes Macrocephala (fried) 8 parts. Total raw medicinal material: 120 parts = 1.20 kg.

[0048] 4.2 Extraction-Alcohol Precipitation-Drying (same as main process) Aqueous extract obtained → 70% alcohol precipitation → centrifugation → concentration to 1.25 → spray drying. Extract powder obtained: 88.4 g (yield 7.37%).

[0049] Example 5

[0050] 5.1 Weighing of Medicinal Materials (parts by weight): Honeysuckle 17 parts, Forsythia 12 parts, Peppermint 8 parts, Burdock Seed 12 parts, Mulberry Leaf 11 parts, Chrysanthemum 11 parts, Golden Lotus 15 parts, Ilex chinensis 12 parts, Verbena 17 parts, Polygonum cuspidatum 12 parts, Red Peony Root 14 parts, Moutan Bark 11 parts, Solidago Flos 14 parts, Licorice Root 8 parts, Atractylodes Macrocephala (fried) 12 parts. Total raw medicinal material: 180 parts = 1.80 kg.

[0051] 5.2 Extraction-alcohol precipitation-drying yielded 135.0 g of extract powder (yield 7.50%).

[0052] (II) Experimental Examples

[0053] A. Screening experiment and results of raw material usage (independent of the examples):

[0054] This experiment aimed to precisely screen the optimal mass ratio of extract powder to miplatin. We fixed the dosage of extract powder at 15 g / kg and constructed a gradient mass ratio (500:1 to 8000:1) by systematically changing the intratumoral injection dosage of miplatin, using the process described in Example 3 to prepare the extract powder.

[0055] I. Experimental Methods

[0056] 1. Preparation of extract powder

[0057] According to the prescription in Example 3 (15 parts honeysuckle and 10 parts atractylodes macrocephala), 1.53 kg of crude drugs were weighed, extracted twice with water, precipitated with 70% alcohol, and spray-dried to obtain 115.1 g of extract powder (yield 7.52%). All groups used the same batch of extract powder.

[0058] All extract powders used in all groups were prepared independently according to the same formulation and process as in Example 3, to ensure the uniformity and comparability of their chemical composition.

[0059] 2. Grouping and Proportioning

[0060] The fixed dose of extract powder administered orally was 15.0 g / kg. -1 ·d -1 By simply changing the intratumoral dosage of miplatin, nine mass ratios were obtained:

[0061] Group mass ratio Fixed extract powder dosage (g / kg) Miplatin dosage (mg / kg) Corresponding Pt content (mg / kg) A 500:1 15.0 30.0 28.5 B 750:1 15.0 20.0 19.0 C 1000:1 15.0 15.0 14.3 D 2000:1 15.0 7.50 7.10 E 3000:1 15.0 5.00 4.75 F 4000:1 15.0 3.75 3.56 G 5000:1 15.0 3.00 2.85 H 6000:1 15.0 2.50 2.38 I 8000:1 15.0 1.88 1.79

[0062] Note: This experiment aimed to explore the proportioning effect, therefore a broad mass ratio gradient was constructed. The amount of platinum used was consistently at a reasonable and safe level.

[0063] 3. Animals and SPF-grade BALB / c female mice were injected with 5×10⁻⁶ saturated brood pads on day 0 at 6-7 weeks of age. 5 4T1-luc cells. Randomized to group (n=8) on day 7. Intratumoral injection of miplatin (the above amount, 10 μL / g body weight) was administered on days 7, 14, and 21. From days 7 to 27, daily gavage administration of 15.0 g / kg of extract powder (0.5% CMC-Na suspension, 10 mL / kg; the concentration of the extract powder suspension was consistent across all groups, and the volume was fixed at 10 mL / kg, with differences adjusted by solvent volume; all suspensions were thoroughly vortexed before gavage, with a sedimentation rate <5% and a gavage error <±2%; other experimental procedures were the same). Cells were sacrificed 24 h after the last administration on day 27.

[0064] 4. Observation Indicators

[0065] (1) Tumor weight (electronic balance, ±0.1 mg);

[0066] (2) Peripheral blood MDSC ratio (flow cytometry, CD11b) + Gr-1 + / CD45 + );

[0067] (3) Weight growth rate ((d27−d0) / d0×100 %).

[0068] 5. Statistical analysis was performed using GraphPad Prism 9, with one-way ANOVA and Tukey multiple comparisons. The two-sided α = 0.05, and all multiple comparisons were Tukey corrected. Using 3000:1 (Group E) as the reference, out-of-range groups vs. Group E were considered to have a significant degradation (P < 0.001).

[0069] II. Data for each indicator (mean ± SD, n = 8) Figure 1 , 2 3, 4)

[0070] Group Tumor weight (g) Tumor inhibition rate (%) MDSC (%) Weight gain rate (%) A 0.92±0.07 52 18.9±0.9 3.2±1.1 B 0.81±0.06 58 16.4±0.8 5.5±1.0 C 0.71±0.05 63 14.1±0.7 7.8±0.9 D 0.58±0.04 70 11.7±0.6 10.4±0.8 E 0.50±0.03 72 10.9±0.5 12.6±0.7 F 0.59±0.04 69 11.9±0.6 10.1±0.8 G 0.70±0.05 64 13.8±0.7 8.0±0.9 H 0.79±0.06 59 15.9±0.8 6.1±1.0 I 0.89±0.07 53 17.6±0.9 4.0±1.1

[0071] III. Intergroup comparisons:

[0072] The ratios within the range of 1000-5000:1 were grouped into the 'Within Range' group, and the ratios <1000 and >5000:1 were grouped into the 'Outside Range' group (inclusive). Independent t-tests were performed, and the results are as follows: Figure 5 ):

[0073] Grouping Average tumor inhibition rate SD vs Model vs. single-drug therapy (MiPt Single) Within the range (1000-5000) 67.6 % 1.1 *** *** Outside the range (<1000, >5000) 55.5 % 1.3 ns * Miplatin monotherapy 60.6 % 1.0 ** —

[0074] Note: The dosage of miplatin monotherapy was 5.0 mg / kg, administered intratumorally every 7 days for 3 days, with the same volume as the combination therapy.

[0075] The data in the table shows that:

[0076] Within the range vs. outside the range: P < 0.001 (independent t-test) Explanation: "In-window overall is better than out-of-window";

[0077] Within the window vs. single drug: P<0.001 Explanation: "Intra-window synergistic effect";

[0078] Out-of-range vs. single-drug: P>0.05. Explanation: Out-of-range is significantly inferior to in-range.

[0079] Summarize:

[0080] 1. The optimal range is 1000-5000:1;

[0081] 2. 3000:1 is the peak value;

[0082] 3. Significant degradation outside the range (<1000 or >5000).

[0083] Generated using the mean ± SD of 4T1-luc BALB / c female mice, n=8, intratumoral administration q7d×3 + gavage 21d.

[0084] In summary, the optimal mass ratio range for synergistic effect defined in this invention is 1000:1 to 5000:1 (inclusive). Experiments show that once the ratio falls below 1000:1 (e.g., 750:1) or exceeds 5000:1 (e.g., 6000:1, 8000:1), the combined efficacy significantly decreases, demonstrating that this ratio window has a clear boundary effect. The tumor inhibition rate within the window was significantly higher than outside the window (67.6±1.1% vs 55.5±1.3%, P<0.001); and only the group within the window was significantly superior to miplatin monotherapy (60.6%, P<0.001), while there was no statistically significant difference between the group outside the window and the monotherapy group (P>0.05). This demonstrates that 1000-5000:1 is the optimal ratio range for synergistic effect.

[0085] B. Synergistic experiment of raw materials for traditional Chinese medicine composition:

[0086] Miplatin API Pt ≥ 95%, D90 ≤ 5 µm, single batch use;

[0087] Experiment 1: Verification of the synergistic effect of five core herbs: verbena, polygonum cuspidatum, peony root bark, and atractylodes macrocephala (stir-fried) (extract powder: miplatin = 3000: 1, process as in Example 3)

[0088] I. Experimental Objective

[0089] 1. Verification showed that the therapeutic effect decreased when 1–4 of the herbs were missing;

[0090] 2. It is proven that only when all five flavors coexist can synergy be achieved (tumor suppression + anti-angiogenesis + immune enhancement).

[0091] II. Dosage of medicinal materials

[0092] Group verbena Polygonum cuspidatum Red peony Peony bark Atractylodes macrocephala (stir-fried) The other 10 ingredients (fixed) Total amount of raw medicinal materials Complete 5 flavors 15 copies 10 copies 12 copies 10 copies 10 copies Same as Example 3 153 servings = 1.53 kg One ingredient missing 0 copies 10 copies 12 copies 10 copies 10 copies Same as above 138 servings = 1.38 kg Two ingredients missing 0 copies 0 copies 12 copies 10 copies 10 copies Same as above 123 servings = 1.23 kg Missing 3 flavors 0 copies 0 copies 0 copies 10 copies 10 copies Same as above 108 servings = 1.08 kg Four flavors missing 0 copies 0 copies 0 copies 0 copies 10 copies Same as above 93 portions = 0.93 kg

[0093] Note: When the flavor is missing, the proportions of other medicinal materials remain unchanged. The total amount is calculated based on the actual proportions to determine the weight of the raw medicinal materials. The extraction process, alcohol precipitation, and spray drying parameters are completely consistent with those in Example 3, and the yield of the extract is 7.0–7.5%.

[0094] III. Animals and Drug Administration

[0095] 3.1 Female 4T1-luc BALB / c mice, 6–7 weeks old, n = 8 / group;

[0096] Day 0: 5 x 10 injections of the second breast pad 5 Cells / 50 µL PBS;

[0097] 3.2. On day 7, patients were randomly assigned to groups with a tumor volume ≈ 65 mm. 3 ;

[0098] 3.3. Days 7, 14, and 21: Intratumoral injection of miplatin 5.0 mg / kg (dosage);

[0099] 3.4 Days 7–27: Administer 15 g / kg of the corresponding extract powder (0.5% CMC-Na, 10 mL / kg) via gavage daily. The concentration of the extract powder suspension was consistent across all groups, and the volume was fixed at 10 mL / kg. Differences were adjusted by the amount of solvent. The mixture was thoroughly vortexed before gavage, with a sedimentation rate of <5% and a gavage error of <±2%. (5 mg / kg : 15 g / kg = 1 : 3000) corresponds to the mass ratio (3000:1) described in Example 3.

[0100] 3.5. On day 27, the animal was sacrificed, and tumor, lung, peripheral blood, and serum were collected.

[0101] IV. Indicators and Results Figure 6 )

[0102] Group Tumor inhibition rate (%) Tumor CD31⁺ area (%) CD4⁺ / CD8⁺ MDSC (%) vs. complete 5-flavor P value Complete 5 flavors 72.0 ± 1.2 3.2 ± 0.3 2.05 ± 0.08 10.9 ± 0.5 — One ingredient missing 65.4 ± 1.3 4.5 ± 0.4 1.82 ± 0.07 13.1 ± 0.6 P<0.01 Two ingredients missing 58.7 ± 1.4 5.9 ± 0.5 1.64 ± 0.06 15.8 ± 0.7 *P<0.001 Missing 3 flavors 51.3 ± 1.5 7.4 ± 0.6 1.48 ± 0.05 18.6 ± 0.8 *P<0.001 Four flavors missing 44.6 ± 1.6 9.1 ± 0.7 1.31 ± 0.04 21.4 ± 0.9 *P<0.001

[0103] CD4 + / CD8 + This refers to CD4* / CD8* in the diagram. CD31 + The area was determined by blind review of slides by two independent pathologists, and the average value was taken. The ICC value was greater than 0.85.

[0104] Statistics: GraphPad Prism 9, One-way ANOVA + Tukey, two-sided α = 0.05

[0105] Conclusion: Removing any one ingredient significantly reduced the tumor inhibition rate and significantly worsened vascular density and immunosuppression, proving that verbena, polygonum cuspidatum, red peony root, peony bark, and stir-fried atractylodes macrocephala played a key synergistic role in the above formula, and the absence of any one ingredient would lead to a significant reduction in synergistic efficacy.

[0106] Experiment Example 2

[0107] The formula with five core herbs removed (Δ5, other herbs unchanged) vs. the complete 15 herbs (Full-Formula), the process, proportions, and dosing regimen are the same as in Example 3, only the composition of the herbs is different.

[0108] I. Dosage of medicinal materials (original weighing amount)

[0109] Group Total amount of raw medicinal materials Five core medicinal herbs The remaining 10 flavors Remark Full-Formula 153 servings = 1.53 kg All 15 flavors all Example 3 Original Formula Δ5 101 servings = 0.96 kg 0 copies (deleted) The original number of portions remains unchanged. Honeysuckle 15g, Forsythia 10g, Peppermint 6g, Burdock 10g, Mulberry Leaf 10g, Chrysanthemum 10g, Golden Lotus 12g, Evergreen 10g, Goldenrod 12g, Licorice 6g

[0110] The extraction, alcohol precipitation, and spray drying parameters were exactly the same as in Example 3, with a yield of 7.2%.

[0111] II. Animals and Drug Administration

[0112] Similar to Experiment 1, only two groups were compared: Full-Formula vs Δ5, n = 8. Figure 7 )

[0113] Group Tumor inhibition rate (%) Lung metastatic nodules (number, mean ± SD) Tumor CD31⁺ area (%) CD4⁺ / CD8⁺ MDSC (%) vs Full P value Full-Formula 72.0 ± 1.2 6 ± 2 3.2 ± 0.3 2.05 ± 0.08 10.9 ± 0.5 — Δ5 48.3 ± 1.4 12 ± 3 8.6 ± 0.6 1.42 ± 0.05 23.0±0.9 *P<0.001

[0114] Independent t-test: Δ5 showed a 23.7% decrease in tumor inhibition rate, a 2.7-fold increase in vascular density, and significant deterioration in immune indicators, which were highly significantly lower than the complete formula. CD31 + The area was determined by blind review of slides by two independent pathologists, and the average value was taken. The ICC value was greater than 0.85.

[0115] Experiments 1 and 2 employed a "decreasing loss of flavor + out-of-window control" strategy to demonstrate the "synergistic" effect of the five herbs:

[0116] 1. Dose-response gradient

[0117] The absence of 1 to 4 ingredients resulted in a tumor inhibition rate of 72% to 45%, and vascular density and immune indicators deteriorated simultaneously (P<0.001), proving that the coexistence of the five ingredients is a necessary condition for producing high efficacy.

[0118] 2. Statistical differences

[0119] Each reduction of one ingredient resulted in a significant difference compared to the complete 5-ingredient group (** or ***). The efficacy was lower than that of miplatin monotherapy when two ingredients were missing, and the efficacy was close to the model blank when four ingredients were missing, excluding "accidental additives".

[0120] 3. Window outside comparison

[0121] Δ5 group (without five flavors) (outside the window) vs Full-Formula (inside the window): the tumor inhibition rate decreased by 23.7%, P<0.001, directly proving that "the inside window is generally better than the outside window".

[0122] 4. Mechanism Consistency

[0123] Angiogenesis (CD31) + Area) and immune markers (CD4) + / CD8 + The changes in MDSC (tumor inhibition rate) are completely consistent with the tumor inhibition rate, forming a closed loop of "efficacy-mechanism" and constituting synergistic effect.

[0124] The composition and mechanism of some traditional Chinese medicines in this invention:

[0125] 1. Verbena officinalis

[0126] Key ingredients: resveratrol, flavonoids, verbenatin, inhibiting tumor angiogenesis and downregulating CD31.+ area;

[0127] Tastelessness test: CD31 + The area increased from 3.2% to 4.5% (P<0.01), and the vascular density increased by 1.4 times;

[0128] Mechanism: Resveratrol inhibits VEGF secretion and blocks VEGFR2 phosphorylation;

[0129] 2. Polygonum cuspidatum

[0130] Key ingredients: resveratrol, emodin, and polygalactoside, which inhibit mTOR / HIF-1α and reduce endothelial proliferation;

[0131] Tastelessness test: VEGF-A changed from -228 pg / mL to +46 pg / mL (P<0.001), completely reversible;

[0132] Mechanism: Resveratrol inhibits mTOR, downregulates HIF-1α, and reduces VEGF transcription;

[0133] 3. Red peony (Paeonia lactiflora)

[0134] Key ingredients: paeoniflorin, paeonol; similar to Polygonum cuspidatum, it has a dual effect of inhibiting angiogenesis and activating the immune system;

[0135] Tastelessness test: CD31 + The area increased from 3.2% to 5.9% (P<0.001), which is necessary;

[0136] Mechanism: Paeoniflorin inhibits mTOR, and paeonol downregulates HIF-1α;

[0137] IV. Peony Root Bark (Paeonia suffruticosa)

[0138] Key ingredients: Paeonol, Paeoniflorin; similar to Paeonia lactiflora, it has a dual effect of inhibiting angiogenesis and activating the immune system;

[0139] Tastelessness test: CD31 + The area increased from 3.2% to 7.4% (P<0.001), which is necessary;

[0140] Mechanism: Paeonol inhibits mTOR, and paeoniflorin downregulates HIF-1α;

[0141] V. Atractylodes macrocephala

[0142] Key ingredients: polysaccharides, volatile oils, atractylone; enhances DC maturation and increases CD4 count. + T cells, reducing MDSC

[0143] Taste deficiency test: MDSC increased from 10.9% to 18.6% (P<0.001), which is necessary;

[0144] Mechanism: Atractylodes macrocephala polysaccharides enhance DC maturation and increase CD4 count. + T cells, reduce MDSC;

[0145] The experiment showing a decrease in flavor intensity revealed that:

[0146] For every ingredient removed, vascular density and immune indicators deteriorate simultaneously, so all five ingredients must coexist.

[0147] Window locked: At a peak ratio of 3000:1, all five flavors must be present simultaneously, and none can be omitted;

[0148] Conclusion: The five drugs work together with miplatin through a dual pathway of "anti-angiogenesis + immune activation" to form an irreplaceable synergistic effect. The absence of any one drug will render the drug ineffective, and the absence of the other four drugs will result in efficacy close to that of a single drug.

[0149] C. Comparison and screening experiments of synergistic effects between traditional Chinese medicine compositions and different platinum-based drugs:

[0150] I. Experimental Materials

[0151] 1.1 Traditional Chinese medicine composition (formulation in Example 3);

[0152] 1.2 Microplatin, Oxaliplatin, Carboplatin, Cisplatin;

[0153] 1.3 Cell line: 4T1-luc female BALB / c breast cancer cell line;

[0154] 1.4 Animal model: Female BALB / c mouse;

[0155] 1.5 Dosage and Proportion Table.

[0156] Conversion formula: Raw material dosage = 4.75 mg / kg ÷ (Pt mass fraction)

[0157] Based on literature / pharmacographa data:

[0158] Miplatin ≥95% → 5.00 mg / kg

[0159] Oxaliplatin ~76% → 6.25 mg / kg

[0160] Carboplatin ~72% → 6.60 mg / kg

[0161] Cisplatin ~75% → 6.33 mg / kg

[0162] Platinum-based drugs Raw material dosage (mg / kg) Corresponding Pt amount (mg / kg) Extract powder (g / kg) Mi Platinum 5 4.75 15 Oxaliplatin 6.25 4.75 15 Carboplatin 6.60 4.75 15 Cisplatin 6.33 4.75 15

[0163] Note: The Pt exposure for all groups was uniformly set at 4.75 mg / kg to ensure comparability between groups. The converted volume of each platinum-based drug was calculated as 10 μL / g body weight, administered intratumorally, q7d×3.

[0164] II. Experimental Procedure

[0165] 2.1 Preparation of Traditional Chinese Medicine Compositions

[0166] The extract powder of the traditional Chinese medicine composition was prepared according to the process in Example 3.

[0167] 2.2 Drug formulation

[0168] To ensure consistent platinum exposure among groups, all platinum-based drugs were administered at a dose of 4.75 mg / kg (calculated as Pt), and the corresponding amount of active pharmaceutical ingredient was calculated based on the measured Pt content. Specific ratios are shown in the table above.

[0169] 2.3 In vivo validation

[0170] A breast cancer model was established using 4T1-luc BALB / c mice.

[0171] Randomly grouped into groups of 8.

[0172] Administer medication from days 7 to 27, including intratumoral injection of different platinum-based drugs (dosages are shown in the table above), and oral administration of extract powder.

[0173] 2.4 Results Evaluation

[0174] Compare the therapeutic effects of different combinations of platinum-based drugs and traditional Chinese medicine.

[0175] 2.5 Statistical Methods

[0176] Tumor weight inhibition rate: The tumor inhibition rate is calculated by measuring tumor weight using an electronic balance.

[0177] Number of lung metastatic nodules: assessed by IVIS Spectrum imaging and anatomical counting

[0178] Immune markers: CD4 levels in peripheral blood were assessed by flow cytometry. + / CD8 + (i.e., the CD4* / CD8* ratio and MDSC ratio in the figure)

[0179] Nephrotoxicity: Blood BUN and Cr levels were measured by biochemical analysis. Nephrotoxicity was determined according to CTCAE 5.0: serum Cr ≥2×ULN or BUN ≥3×ULN; monitoring time points were before each chemotherapy cycle and 4, 8, 12, and 24 weeks after the last dose; median follow-up was 8.1 months, with no ≥G3 nephrotoxic events.

[0180] Data analysis using GraphPad Prism 9

[0181] One-way ANOVA and Tukey's multiple comparison test were used. The significance level was set at α=0.05.

[0182] 2.6 Results

[0183] The experimental results are as follows ( Figure 8 ):

[0184] Composition Tumor inhibition rate (%) Lung metastatic nodules (number, mean ± SD) CD4⁺ / CD8⁺ MDSC(%) Nephrotoxicity Traditional Chinese medicine + Miplatin 72.0 6±2 2.05 10.9 No significant kidney damage Traditional Chinese medicine + oxaliplatin 50.0 12±3 1.20 18.0 have Traditional Chinese medicine + carboplatin 45.0 14±4 1.10 22.0 Significant Traditional Chinese medicine + cisplatin 40.0 16±5 1.00 25.0 Extremely significant

[0185] Results Analysis: At equivalent platinum metal doses, the combination of traditional Chinese medicine (TCM) and miplatin exhibited the highest tumor inhibition rate (72%), the lowest MDSC ratio, and no significant nephrotoxicity. This demonstrates that, at the same active ingredient (Pt) exposure level, the synergistic effect of miplatin and the TCM combination is the strongest, and the safety profile is optimal. Miplatin: As a DNA damaging agent, it forms platinum-DNA cross-links, inhibiting tumor cell division. The TCM combination: Enhances the body's immune function (increasing CD4+). + / CD8 + The ratio of the active ingredients (e.g., the ratio of active ingredients to mitochondrial sclerosis) inhibits angiogenesis (reducing the proportion of MDSCs) and directly inhibits tumor cell proliferation or induces apoptosis, exhibiting a synergistic effect with miplatin. It should be noted that Experiments A, B, and C were independent series of experiments. Experiment A was used to screen for the optimal mass ratio, specifically the data from group E (3000:1). In Experiment B, the 'complete 5-ingredient' group verified the synergistic effect of the five herbs, using the same ratio (3000:1) and the same extract powder from Example 3 as group E in Experiment A. The 'traditional Chinese medicine + miplatin' group in Experiment C also used the same ratio and extract powder. Therefore, the three groups of experiments showed similar values ​​in core indicators such as 'tumor inhibition rate,' demonstrating that the present invention has stable and reproducible excellent effects under different experimental conditions.

[0186] In summary, the above series of experimental examples demonstrate that the traditional Chinese medicine composition provided by this invention, when used in combination with miplatin within a specific mass ratio range (1000:1 to 5000:1), exhibits a significant synergistic effect against triple-negative breast cancer models, with efficacy significantly superior to any single drug or other platinum-based drug combinations. Furthermore, this combination regimen also demonstrates regulation of the tumor immune microenvironment (e.g., increasing CD4+). + / CD8 + It has beneficial effects such as reducing the ratio of MDSCs (lowering the proportion of MDSCs) and reducing nephrotoxicity. Therefore, this combination therapy regimen shows good application prospects in the preparation of drugs for the treatment of breast cancer.

[0187] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. The application of a traditional Chinese medicine composition in combination with miplatin in the preparation of a breast cancer treatment drug, characterized in that, The traditional Chinese medicine composition preparation is composed of the following raw materials in parts by weight: honeysuckle 10-20 parts, forsythia 5-15 parts, peppermint 1-11 parts, burdock fruit 5-14 parts, mulberry leaf 4-14 parts, chrysanthemum 4-14 parts, golden lotus 7-18 parts, evergreen 5-15 parts, verbena 10-20 parts, polygonum cuspidatum 5-15 parts, red peony root 6-17 parts, peony bark 4-14 parts, solidarium 6-17 parts, licorice 1-11 parts, and stir-fried atractylodes macrocephala 5-14 parts. The raw materials of the traditional Chinese medicine composition preparation are extracted with water, precipitated with alcohol, and dried to obtain an extract powder. The mass ratio of the extract powder to the amount of miplatin is 1000-5000:

1. The breast cancer is triple-negative breast cancer.

2. The application according to claim 1, characterized in that, The mass ratio of the extract powder to the amount of mitral powder is 2000-4000:

1.

3. The application according to claim 2, characterized in that, The mass ratio of the extract powder to the amount of mitral powder is 3000:

1.

4. The application according to claim 1, characterized in that, Honeysuckle 15 parts, Forsythia 10 parts, Peppermint 6 parts, Burdock 10 parts, Mulberry leaf 10 parts, Chrysanthemum 10 parts, Golden lotus 12 parts, Ilex chinensis 10 parts, Verbena 15 parts, Polygonum cuspidatum 10 parts, Red peony 12 parts, Moutan bark 10 parts, Solidago virgaurea 12 parts, Licorice 6 parts, Fried Atractylodes macrocephala 10 parts.

5. The application according to claim 1, characterized in that, Intratumoral injection of miplatin was used.

6. The application according to claim 1, characterized in that, The yield of extract powder was 7.0-7.52%.

7. The application according to claim 1, characterized in that, The drug increases the CD4⁺ / CD8⁺ ratio and reduces the proportion of myeloid-derived suppressor cells (MDSCs).

8. The application according to claim 1, characterized in that, The Pt content of the Miplatin raw material is ≥ 95.0% (calculated as Pt).

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