Use of homoharringtonine in the preparation of drugs for treating small cell lung cancer

Abstract

The application relates to the technical field of medicines, and discloses an application of homoharringtonine in the preparation of a medicine for treating small cell lung cancer, and further provides a medicine for treating small cell lung cancer and containing homoharringtonine. The application discloses an action mechanism of homoharringtonine, an active ingredient of traditional Chinese medicine, against SCLC, and discloses that the traditional Chinese medicine ingredient HHT inhibits a malignant phenotype by targeting tumor-specific glycosylation enzyme MGAT5, provides an innovative perspective of glyco-biology for the research of traditional Chinese medicine against tumors, provides an important scientific basis for the subsequent development and application of homoharringtonine, and provides a new medicine treatment approach for treating small cell lung cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of homoharringtonine in the preparation of drugs for treating small cell lung cancer. Background Technology

[0002] Lung cancer is the leading cause of cancer death worldwide, primarily classified into two types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). SCLC, accounting for approximately 15% of all lung cancers, is further divided into limited-stage and extensive-stage. It is the most malignant lung tumor, characterized by extremely high proliferation rates, a high risk of early metastasis, and poor prognosis, with a five-year survival rate of less than 5%, earning it the title of "refractory tumor." Currently, there are no definitive early screening methods for SCLC. Surgery is only suitable for stage I-II patients, while locally advanced treatment mainly involves a combination of radiotherapy and chemotherapy. Clinical drug research on SCLC has progressed slowly. For the past two decades, the primary first-line treatment regimen has been the combination of etoposide (VP16) and cisplatin (DDP). Therefore, actively exploring new therapeutic targets and drugs is crucial for prolonging the survival of SCLC patients. Traditional Chinese medicine has advantages in anti-tumor effects, including multi-target action, immunomodulatory function, and low toxicity. These advantages can improve patient tolerance, reduce drug resistance, and enhance immune surveillance to exert anti-tumor effects.

[0003] Homoharringtonine (HHT) is an alkaloid extracted from the traditional Chinese medicine Cephalotaxus fortunei. It is a classic drug for treating hematological malignancies and also has significant effects against various solid tumors such as liver cancer, bladder cancer, and colorectal cancer. Its structural formula is as follows:

[0004] .

[0005] However, the antitumor activity and specific molecular mechanism of HHT in SCLC are still unclear. Summary of the Invention

[0006] The first objective of this invention is to provide the use of homoharringtonine in the preparation of a medicament for treating small cell lung cancer. The second objective of this invention is to provide a medicament for treating small cell lung cancer comprising homoharringtonine.

[0007] Specifically, the drug comprises homoharringtonine and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients refer to various conventional excipients added when preparing different dosage forms, such as diluents, binders, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, and adsorbents. The dosage forms of the drug include injections, inhalations, tablets, granules, or capsules.

[0008] Specifically, drugs prepared from homoharringtonine can be administered via common routes of administration such as injection (intramuscular, intravenous, intraperitoneal), subcutaneous, inhalation, and rectal administration. They can be administered to patients with small cell lung cancer in combination via injection or inhalation. The preferred form is an injectable formulation, particularly a preparation used in combination with chemotherapy drugs. When intended for injection, it can be formulated as an injectable solution, aqueous solution, or oil suspension.

[0009] Furthermore, various dosage forms of the drug of this invention can employ optimized drug delivery systems to enhance the bioavailability of HHT. Novel delivery systems such as nanoparticles, polymer encapsulation, and liposomes can significantly improve drug solubility and stability, thereby increasing in vivo absorption efficiency. This not only enhances efficacy but also reduces toxic side effects.

[0010] The beneficial effects of this invention are as follows: This invention reveals the mechanism of action of homoharringtonine, an active ingredient in traditional Chinese medicine, against small cell lung cancer (SCLC), and reveals that the traditional Chinese medicine ingredient HHT inhibits malignant phenotypes by targeting the tumor-specific glycosylation enzyme MGAT5. This provides an innovative perspective of glycobiology for the research of anti-tumor effects of traditional Chinese medicine, provides important scientific basis for the subsequent development and application of homoharringtonine, and provides a new drug route for the treatment of small cell lung cancer. Attached Figure Description

[0011] Figure 1 Cell viability was detected using the CCK-8 assay. Figure A shows the effect of different DDP concentrations on the viability of three SCLC cell lines: H446, H1688, and H196. Figure B shows the effect of different VP16 concentrations on the viability of these three SCLC cell lines.

[0012] Figure 24D-FastDIA quantitative proteomics method was used to analyze differentially expressed proteins in HHT-treated SCLC cells. Figure A shows the extraction of total protein from SCLC cells treated with DMSO and HHT, the obtaining of peptide structures through enzymatic digestion, and analysis using tandem liquid chromatography-mass spectrometry. Figure B shows the quantitative proteomics study of the control and treatment groups, identifying the number of significantly upregulated and downregulated differentially expressed proteins between them. Figure C shows a volcano plot of the comparison groups including T-test P values. The horizontal axis represents the Ratio value of differential expression change in the comparison groups after Log2 transformation; the vertical axis represents the T-test P value after -Log10 transformation. The top 5 differentially expressed proteins for both upregulation and downregulation are also labeled in the figure. In the volcano plot, red dots indicate significant upregulation, blue dots indicate significant downregulation, and gray dots indicate no significant difference.

[0013] Figure 3 Western blot analysis was performed to detect the expression of ITGB1, MGAT5, and GAPDH proteins in H446, H1688, and H196 cells after treatment with different concentrations of HHT for 48 hours. ±s,n=3).

[0014] Figure 4 To observe the inhibitory effect of HHT on the invasion and migration of zebrafish xenografts. Figure A shows bright-field images of zebrafish xenografted embryos treated with HHT, EP, and HHT+EP combined treatment at 1 dpi and 3 dpi, acquired using stereofluorescence microscopy. Figure B shows the average fluorescence intensity of zebrafish xenograft models established with CM-DiI-labeled H446 cells after 3 days of treatment in the control group, HHT group, EP group, and HHT+EP combined treatment group. ±s,n=6)(Scale: 500 um).

[0015] Figure 5 The inhibitory effect of HHT on subcutaneous xenograft tumors in nude mice. Figure A shows tumor-bearing nude mice in the following groups: saline control group, low-concentration HHT group, high-concentration HHT group, EP (DDP+VP16) positive control group, and EP (DDP+VP16) + HHT combination group. Figure B shows a comparison of tumor masses in different groups. Figure C shows the change in body weight of nude mice over time in different groups. Figure D shows the change in tumor volume over time in different groups. Figure E shows a comparison of tumor weight in different groups. Detailed Implementation

[0016] This invention discloses the application of homoharringtonine in the preparation of drugs for treating small cell lung cancer. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention.

[0017] This invention provides the use of homoharringtonine in the preparation of a medicament for treating small cell lung cancer. Corresponding to this use, this invention also provides a medicament for treating small cell lung cancer comprising homoharringtonine.

[0018] The drug also includes one or more inert, non-toxic, and pharmacologically suitable excipients. These excipients may be carriers, solvents, emulsifiers, dispersants, wetting agents, binders, stabilizers, colorants, fragrances, etc. The dosage form of the drug may be tablets, powders, granules, capsules, injections, etc.

[0019] The medicaments according to the invention can act systemically and / or locally, and for this purpose can be taken in a suitable manner, such as via oral, parenteral, pulmonary, or nasal routes, and the medicaments according to the invention can be taken in forms suitable for these routes of administration.

[0020] Suitable for oral administration are dosage forms that, according to the level of existing technology, act rapidly and / or release the drug of the invention in an improved manner, and include drugs of the invention in crystalline and / or amorphous and / or dissolved forms, such as tablets (uncoated or coated tablets, having, for example, a coating that resists gastric juices or delays dissolution or is insoluble, for drug release according to the invention), tablets that break rapidly in the mouth, or films, films / lyophilized products, capsules (e.g., hard or soft capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols, or solutions. Parenteral administration can avoid absorption steps (e.g., intravenous, intra-arterial, intracardiac, intraspinal, or lumbar, or intra-articular) or simultaneously include absorption (e.g., intramuscular, subcutaneous, intradermal, percutaneous, or intraperitoneal). Suitable dosage forms for parenteral administration are particularly for injection and infusion of formulations in the form of solutions, suspensions, emulsions, lyophilized products, or sterile powders. Nasal administration generally refers to the method of drug delivery through absorption of medication via the nasal mucosa to treat local or systemic diseases. Common dosage forms for this method include nasal drops or nasal sprays.

[0021] The pharmaceutical products according to the invention can be converted into the described oral form. This can be done in a manner known per se by mixing with inert, non-toxic, and pharmacologically suitable excipients. These excipients particularly include carriers (e.g., microcrystalline cellulose, lactose, mannitol, starch), solvents (e.g., liquid polyethylene glycol), emulsifiers and dispersants or wetting agents (e.g., sodium lauryl sulfate, polysorbate oleate, propylene glycol), binders (e.g., polyvinylpyrrolidone), synthetic and natural polymers (e.g., albumin), stabilizers (e.g., antioxidants, such as ascorbic acid), colorants (e.g., inorganic pigments, such as iron oxides), and masking flavors and odors.

[0022] To improve the bioavailability of homoharringtonine, various dosage forms of the drug in this invention can optimize the drug delivery system. Novel delivery systems such as nanoparticles, polymer encapsulation, and liposomes can significantly improve drug solubility and stability, thereby increasing in vivo absorption efficiency, enhancing efficacy, and reducing toxic side effects.

[0023] To verify the therapeutic effect of homoharringtonine, this invention also provides several experimental examples for illustration. Unless otherwise specified, the raw materials, reagents, consumables, and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0024] Experiment 1: CCK-8 assay to detect the effect of DDP or VP16 on the viability of three SCLC cell lines.

[0025] Human normal lung epithelial cells BEAS-2B, human LUSC cells NCI-H1703, LTEP-s, human LUAD cells A549, NCI-H1299, and human SCLC cells NCI-H446, NCI-H1688, and NCI-H196 in logarithmic growth phase were seeded in 96-well plates according to their morphology, size, and growth rate, with the number of cells in each well adjusted to 3.5-10 × 10⁻⁶. 3The number of cells / well varied, and the cells were seeded in 96-well plates (3 replicates / group). The concentration gradients were set as follows: HHT stock solution concentration 1 mM, 0, 10, 31.25, 62.5, 125, 250, 500 nM; cisplatin stock solution concentration 1 mg / mL, 0, 1.25, 2.5, 5, 10, 20, 40, 80 μM; etoposide stock solution concentration 20 mg / mL, 0, 2.5, 5, 10, 20, 40, 80, 100 μM. After administration and incubation for 48 h, the culture supernatant was removed, and CCK-8 working solution (100 μL / well) was added at 10% of the final volume. The plates were then incubated at 37°C in the dark for 2 h. The absorbance (OD value) at 450 nm was measured using an ELISA reader. Cell viability was calculated using the formula: Viability (%) = (OD of drug-treated group - Zero-adjustment OD) / (OD of control - Zero-adjustment OD) × 100%, and IC50 was calculated. 50 The results are shown in Table 1 and Figure 1 As shown.

[0026] Table 1. IC50 of HHT on normal human bronchial epithelial cells and lung cancer cells after 48 hours of action. 50

[0027]

[0028] The results showed that DDP and VP16, when used on NCI-H446, NCI-H1688, and NCI-H196 for 48 hours, resulted in IC... 50 The effective concentrations were 7.38±2.3, 10.47±1.7, and 7.78±1.2 μM; and 6.62±1.3, 11.3±2.9, and 31.78±3.5 μM. These results indicate that HHT exhibits higher sensitivity to SCLC cells compared to DDP and VP16, suggesting that HHT could be considered a more potent anti-SCLC drug.

[0029] Experimental Example 2: Study on the anti-SCLC effect of homoharringtonine inhibiting MGAT5 protein using 4D-FastDIA quantitative proteomics.

[0030] This study investigated how homoharringtonine-regulated proteins affect the invasion and migration of SCLC cells using 4D-FastDIA quantitative proteomics. The SCLC cell line NCI-H446 was used as the research subject. Total protein was extracted from HHT-treated and untreated SCLC cells. A combination of cutting-edge technologies, including enzyme digestion, liquid chromatography-mass spectrometry (LC-MS / MS), and bioinformatics analysis, was employed to perform quantitative proteomics analysis on the samples. Results are as follows: Figure 2As shown, the distribution of differentially expressed proteins between the control and treatment groups can be visually compared. 174 and 193 proteins were identified as significantly upregulated and downregulated, respectively, with log2 changes greater than or less than 1 (P < 0.05). A volcano plot was then created for the comparison group including T-test P values. The horizontal axis represents the log2 transformed value of the differential expression change ratio in the comparison group; the vertical axis represents the -Log10 transformed value of the statistical test T-test P value. The top 5 differentially expressed proteins (in descending order of absolute log2 ratio) for both upregulation and downregulation are also labeled in the figure. The significantly downregulated protein α-1,6-mannosylglycoprotein 6 beta-N-acetylglucosaminyltransferase A (MGAT5) attracted our attention (P < 0.0001). MGAT5 is a glycosyltransferase that participates in and promotes cancer metastasis. It can generate β1,6-branchs on the N-glycans of target proteins such as cell adhesion molecules and cell surface receptors, thereby affecting cell adhesion, invasion and migration.

[0031] Experimental Example 3: Effect of HHT on MGAT5 protein levels in SCLC cells.

[0032] H446, H1688, and H196 cells in logarithmic growth phase were divided into a control group and several HHT treatment groups. The control group received no HHT, while the treatment groups received HHT at gradient concentrations. All cells were incubated under the same conditions for 48 hours. Cells from each group were collected and washed 2-3 times with pre-chilled PBS to remove residual culture medium. Then, RIPA lysis buffer containing 1% PMSF was added, and the cells were lysed on ice for 20 minutes, followed by sonication in an ice-water mixture for 5 minutes to ensure complete lysis and release of intracellular proteins. After lysis, the cells were centrifuged at 4°C, and the supernatant was collected as the sample containing total protein. The protein concentration of each group was determined using the BCA method to ensure consistent loading. SDS-PAGE electrophoresis was then performed to separate proteins of different molecular weights, including MGAT5, in the gel. After electrophoresis, the proteins on the gel were transferred to an NC membrane. The transferred membrane was blocked with PBST buffer containing 5% skim milk powder to block non-specific binding sites. Prepare 5 mL of primary antibody dilution buffer at a PBST:Tween-20 ratio of 4:1. Prepare the primary antibody incubation solution to an appropriate concentration according to the antibody instructions. Based on the protein marker markings on the membrane and the molecular weight of the target protein, place the membrane into the corresponding primary antibody incubation bag and incubate overnight at 4 °C. The next day, wash the membrane with TBST to remove unbound primary antibody, then add chemiluminescent secondary antibody incubation solution prepared with PBST buffer and incubate at room temperature for 1 hour. Wash again to remove excess secondary antibody. Use ECL chemiluminescence to develop the antibody-bound proteins on the membrane and acquire protein bands using an imaging system. Use ImageJ software to semi-quantitatively analyze the developed protein bands using grayscale values, normalize the internal control bands, and perform statistical analysis. Compare the changes in grayscale values ​​of each target protein to reflect changes in protein expression abundance after different treatments. Repeat the experiment three times.

[0033] like Figure 3 The results showed that, compared with the control group, treatment with HHT at doses of 20, 31.25, 62.5, and 125 nM for 48 h resulted in dose-dependent inhibition of MGAT5 and downstream ITGB1 protein expression. These results indicate that HHT exerts its anti-SCLC effect by inhibiting the downregulation of MGAT5 and ITGB1 in a dose-dependent manner.

[0034] Experiment Example 4: Observing the inhibitory effect of HHT on the invasion and migration of xenografts in zebrafish.

[0035] A zebrafish xenograft model was established by microinjecting CM-DiI-labeled H446 cells into the yolk sac of 48-h transgenic zebrafish TG (FLI-1:EGFP) embryos. Two-day-pass (dpf) zebrafish embryos were randomly assigned to 6-well plates (6 embryos / well) for yolk sac microinjection. One day after injection (1 dpi), the appropriate dose (MTD) was administered: control group (equal volume of systemic fish water), HHT group (5 μM), EP group (DDP 25 μM + VP16 50 μM), and HHT+EP combination group (DDP 25 μM + VP16 50 μM + HHT 2.5 μM), 4 mL per well, for 3 consecutive days. All drugs were administered via immersion. The combination group received half the MTD.

[0036] like Figure 4 The results showed that, compared with the control group, the fluorescence intensity at the yolk sac of zebrafish in the HHT group was reduced, and no metastatic cells were observed in the tail or brain. The EP group and the EP+HHT combination group also showed the same inhibitory effect. This indicates that HHT also has the ability to significantly inhibit the proliferation, invasion, and migration of SCLC cells in vivo.

[0037] Experiment 5: Observing the inhibitory effect of HHT on subcutaneous xenograft tumors in nude mice.

[0038] A subcutaneous xenograft model of SCLC in nude mice was constructed using NCI-1688 cells to investigate the in vivo effects of homoharringtonine on SCLC. BALB / c nude mice, half male and half female, were used in the study. They were 4 weeks old with an average weight of 13-16 g. Following the principle of equal male and female populations, the mice were divided into four groups: a saline control group, a low-concentration HHT group, a high-concentration HHT group, an EP (DDP+VP16) positive control group, and an EP (DDP+VP16) + HHT combination group, with 6 mice in each group. Log-phase NCI-1688 cells were subcutaneously injected into the right axilla of the mice to induce tumor formation. The dosage and frequency of administration were as follows: low-dose HHT group 1 mg / kg, high-dose HHT group 2.5 mg / kg, EP positive control group 4 mg / kg, and EP + HHT combination group 4 mg / kg + 1 mg / kg, administered intraperitoneally twice a week. After reaching the experimental endpoint, the mice were anesthetized by overdose. Nude mice were killed one by one using the intoxication method. The mice were arranged according to group and tumor size, photographed and recorded, and then dissected to remove subcutaneous tumor tissue, heart, liver, spleen, lung, and kidney tissue.

[0039] like Figure 5The results showed that HHT (1.0 mg / kg) significantly inhibited tumor growth after 21 days of treatment (P<0.05), with effects comparable to the EP group: the average tumor volume in the low-dose HHT group was reduced by 41.9% (550.68 mm³ vs 947.98 mm³) compared to the control group, and the average tumor weight was reduced by 27.3% (0.88 g vs 1.21 g). Furthermore, HHT (1.0 mg / kg) demonstrated superior safety—the nude mice experienced only a 4.1% weight loss during treatment (compared to 10.9% in the EP group), indicating that HHT can effectively inhibit tumor cell proliferation even at low doses.

[0040] The above experimental examples confirmed that HHT has good anti-SCLC activity both in vivo and in vitro, and can inhibit SCLC proliferation, invasion and metastasis. This invention utilizes 4D-FastDIA technology to study the molecular mechanism by which HHT targets the tumor-specific glycosylation enzyme MGAT5, providing a new strategy for the treatment of small cell lung cancer. It is the first time that the traditional Chinese medicine component HHT has been revealed to inhibit abnormal N-glycosylation by targeting the 2.MGAT5-ITGB1 axis, thereby suppressing the malignant phenotype, providing an innovative perspective in glycobiology for the research of anti-tumor effects of traditional Chinese medicine.

Claims

1. Application of homoharringtonine in the preparation of drugs for treating small cell lung cancer.

2. The application according to claim 1, characterized in that: The drug contains homoharringtonine and pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that: The dosage form of the drug is injection, inhalation, tablet, granule or capsule.

4. A drug for treating small cell lung cancer, characterized in that, It contains homoharringtonine.

5. The drug according to claim 4, characterized in that: The drug also contains pharmaceutically acceptable excipients.

6. The drug according to claim 4, characterized in that: The dosage form of the drug is injection, inhalation, tablet, granule or capsule.

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