Medical application of olopatadine hydrochloride in treating primary liver cancer disease

Abstract

The invention discloses a medical application of olopatadine hydrochloride in treatment of primary liver cancer diseases, and discloses a novel medical application of olopatadine hydrochloride, the olopatadine hydrochloride has the capabilities of remarkably inhibiting proliferation of primary liver cancer cells and promoting apoptosis of the primary liver cancer cells, and the prepared medicine for treating the primary liver cancer has an obvious medical effect and relatively high safety. The invention belongs to the technical field of biological medicines, and relates to a novel medicine for treating primary liver cancer, which has extremely high clinical application value and medicine popularization potential, provides a novel effective medicine for treating primary liver cancer, has better clinical transformation potential and belongs to the technical field of biological medicines.

Description

Technical Field

[0001] The present invention discloses the medical use of olopatadine hydrochloride in the treatment of primary liver cancer, which has obvious medical effects, provides a new medical use of olopatadine hydrochloride, and belongs to the field of biomedical technology. Background Art

[0002] Primary liver cancer is a malignant tumor originating from the liver. The pathogenesis of primary liver cancer is complex and involves the interaction of multiple pathogenic factors. Treatment methods include surgical resection, ablation therapy, transcatheter arterial chemoembolization (TACE), radiotherapy, systemic therapy, and liver transplantation, etc. However, these treatment methods have certain limitations. For example, surgical resection and ablation therapy are applicable to early-stage small liver cancers, but have limited effects on advanced or metastatic liver cancers. TACE may cause liver function damage. Radiotherapy is not applicable to patients with poor liver function and may cause complications such as radiation-induced liver disease. Although molecular targeted drugs and immune checkpoint inhibitors in systemic therapy provide new treatment options for patients with intermediate and advanced liver cancers, the problems of drug resistance and side effects still plague the clinical efficacy. Therefore, in order to improve the treatment effect, solve the drug resistance problem, reduce side effects, and achieve precise treatment, the research and development of new drugs is particularly important and urgent.

[0003] Olopatadine Hydrochloride is an antihistamine H1 receptor antagonist, with the molecular formula C 21 H 24 ClNO3, and the molecular weight is 373.87 g / mol. It belongs to heterocyclic drugs. Olopatadine hydrochloride was originally used to treat allergic diseases. As a mast cell stabilizer, it has a dual effect of antihistamine and stabilizing mast cells. It mainly reduces the release of histamine, decreases the production of allergic cytokines in the body, and alleviates the stimulation of the immune system. Currently, olopatadine hydrochloride is mostly applied to allergic conjunctivitis, allergic rhinitis, urticaria, and other skin diseases. Summary of the Invention

[0004] The present invention discloses the medical use of olopatadine hydrochloride in the treatment of primary liver cancer, which can be made into a drug for treating liver cancer and has good curative effects, providing a new biomedical use of olopatadine hydrochloride.

[0005] This invention relates to the effect of olopatadine hydrochloride on the proliferation of two human hepatocellular carcinoma cell lines, HCCLM3 and HuH7. The CCK8 cell proliferation assay was used to investigate the inhibitory effect of olopatadine hydrochloride on the proliferation of HCCLM3 and HuH7 cells at different concentrations (0, 5, 10, 20, 30, 40 μmol / L). The results showed that olopatadine hydrochloride significantly inhibited the proliferation of these two cell lines, and the inhibitory effect was statistically significant ( p <0.05). The calculated IC50 values were 17.58 μmol / L and 11.83 μmol / L respectively, indicating that olopatadine hydrochloride has significant inhibitory activity against these two hepatocellular carcinoma cell lines.

[0006] Furthermore, the results of Western blot experiments confirmed that olopatadine hydrochloride can promote apoptosis of hepatocellular carcinoma cells, and this effect was verified at the protein molecular level. In addition, animal experiments also confirmed the ability of olopatadine hydrochloride to inhibit the growth of hepatocellular carcinoma cells in vivo. This invention not only verified the anti-hepatocellular carcinoma potential of olopatadine hydrochloride in vitro and in vivo, but also considering its known safety and effectiveness, olopatadine hydrochloride is expected to become a new type of anti-cancer drug with potential for clinical translation. These findings provide a scientific basis for the development of new treatment strategies for hepatocellular carcinoma and may have a positive impact on the treatment options for hepatocellular carcinoma patients.

[0007] Two human primary hepatocellular carcinoma cell lines, HuH7 and HCCLM3, were treated with olopatadine hydrochloride for 24 h, and double-distilled water was used as the blank control; the experimental data showed that at a concentration of 5-10 μmol / L of olopatadine hydrochloride, olopatadine hydrochloride had a significant inhibitory effect on the proliferation of HuH7 and HCCLM3 cell lines after 24 h ( p <0.05); the inhibitory effect of olopatadine hydrochloride on the normal hepatocyte cell line THLE-2 was no more than 50%, with low liver toxicity and good safety; the results of Western blot experiments verified at the protein molecular level that olopatadine hydrochloride promotes apoptosis of hepatocellular carcinoma cells; the animal experiment level verified that olopatadine hydrochloride can inhibit the growth of hepatocellular carcinoma cells in vivo.

[0008] The new medical use of olopatadine hydrochloride is disclosed, which has the ability to significantly inhibit the proliferation of primary liver cancer cells and promote their apoptosis. Preparing a drug for treating primary liver cancer has obvious medical effects and high safety, has extremely high clinical application value and drug promotion potential, provides a new and effective drug for the treatment of primary liver cancer, and has good clinical transformation potential. Experimental data further confirm that olopatadine hydrochloride has no obvious toxic effect on normal liver cells, thus ensuring its safety in clinical application. At the same time, the reasonable pricing of olopatadine hydrochloride helps to reduce the economic burden of primary liver cancer patients and effectively reduce the cost of public medical insurance, which is of great significance for improving the accessibility and affordability of patient treatment. Description of the Drawings

[0009] Figure 1 It is for detecting the inhibitory effect of olopatadine hydrochloride on the proliferation of liver cancer cells by CCK8; Figure 2 It is for detecting the effect of olopatadine hydrochloride on the proliferation of normal liver cells by CCK8; Figure 3 It is for the effect of olopatadine hydrochloride on the expression of apoptotic proteins in HuH7 and HCCLM3 cells; Figure 4 It is that olopatadine hydrochloride can significantly inhibit the growth of liver cancer at the level of in vivo animal experiments. Detailed Embodiments

[0010] The present invention is further described by the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change that is easily achieved by those of ordinary skill in the art to the present invention will fall within the scope of the claims of the present invention.

[0011] Experimental Example 1

[0012] Cells: Human liver cancer cell lines HCCLM3 and HuH7 were purchased from ATCC, USA, and were preserved and passaged by this laboratory; THLE-2 cells were purchased from Wuhan Procell Life Science & Technology Co., Ltd. HCCLM3 and HuH7 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin and streptomycin, while THLE-2 cells were cultured in BEGM complete medium containing 10% FBS and 1% penicillin and streptomycin.

[0013] Drugs and reagents: Olopatadine Hydrochloride was purchased from MCE; the Cell Counting Kit-8 (CCK-8) was purchased from Wuhan proteintech. Western blot reagents: Cleaved-caspase 3 monoclonal antibody (rabbit anti-human, dilution ratio 1:2000, Wuhan proteintech); Bcl-2 monoclonal antibody (rabbit anti-human, dilution ratio 1:2000, Wuhan proteintech); Bax monoclonal antibody (rabbit anti-human, dilution ratio 1:2000, Wuhan proteintech); Anti-β-Tubulin monoclonal antibody (mouse anti-human, dilution ratio 1:5000, Beijing TransGen Biotech); ECL luminescent solution was purchased from Wuhan proteintech.

[0014] Grouping: Different concentrations of Olopatadine Hydrochloride were used in the experiment. First, it was dissolved in high-pressure double-distilled water to prepare a stock solution. Subsequently, Olopatadine Hydrochloride was diluted to final concentrations of 0, 5, 10, 20, 30, 40 μmol / L respectively for cell treatment. The control group cells were only treated with high-pressure double-distilled water.

[0015] Cell viability detection by CCK-8 method:

[0016] Cells were seeded in 96-well tissue culture plates. The cells were treated with different concentrations of Olopatadine Hydrochloride, with concentrations of 0, 5, 10, 20, 30, 40 μmol / L respectively, for 24 hours. After treatment, 90 μl of medium containing 10 μL of CCK-8 reagent was added to each well, and the incubation was continued under the conditions of 37 °C and 5% CO2. After incubation, the absorbance (A) at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the absorbance value was used to represent cell viability.

[0017] Western Blot experiment:

[0018] The HuH7 and HCCLM3 cell lines were treated with olopatadine hydrochloride at drug concentrations of 0, 5, and 10 μmol / L for 24 hours. After the treatment, the cells were homogenized in a protein lysis buffer, and then centrifuged at 12,000 g for 15 minutes at 4°C to separate and collect the proteins in the supernatant. The protein concentration was quantitatively analyzed using a BCA assay kit. The protein samples were electrophoretically separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (PVDF). After transfer, the membrane was blocked with 5% skim milk at room temperature for 2 hours to reduce non-specific protein binding. After blocking, the membrane was incubated with specific primary antibodies overnight at 4°C to specifically recognize the target proteins. After incubation, the membrane was washed with TBST buffer to remove unbound primary antibodies. The membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibodies and detected by chemiluminescence using an ECL luminol solution, and the protein bands were observed and recorded using a gel imaging system.

[0019] Experimental Example 2: Olopatadine hydrochloride has a significant inhibitory effect on the proliferation of hepatoma cell lines HuH7 and HCCLM3

[0020] This invention conducted experiments on the effects of olopatadine hydrochloride on the proliferation of two classic human hepatocellular carcinoma cell lines, HCCLM3 and HuH7; different concentrations of olopatadine hydrochloride (0, 5, 10, 20, 30, 40 μmol / L) were used, and high-pressure double-distilled water was used as the blank control group. Through the CCK8 cell proliferation assay, compared with the control group, olopatadine hydrochloride significantly inhibited the proliferation of hepatoma cells HuH7 and HCCLM3 ( p <0.05); the IC50 values of each cell line were 17.58 and 11.83 μmol / L, respectively. These findings suggest that olopatadine hydrochloride can be used as a potential anti-hepatocellular carcinoma drug, and its specific mechanism of action and clinical application value are worthy of further study. See Figure 1 as shown, the inhibitory effect of olopatadine hydrochloride on the proliferation of hepatoma cells was detected by CCK8.

[0021] Experimental Example 3: Olopatadine hydrochloride has high safety for hepatocyte cell lines

[0022] Experiments on olopatadine hydrochloride treatment were conducted on two human hepatocellular carcinoma cell lines, with double-distilled water as the blank control. The experimental data showed that at olopatadine hydrochloride concentrations of 5 - 10 μmol / L, it had a significant inhibitory effect on the proliferation of HuH7 and HCCLM3 cell lines ( p <0.05) (see Figure 1 ). In addition, the inhibitory effect of olopatadine hydrochloride on the normal hepatocyte cell line THLE-2 was no more than 50% ( Figure 2, CCK8 was used to detect the effect of olopatadine hydrochloride on the proliferation of normal liver cells), indicating its low liver toxicity. Therefore, olopatadine hydrochloride is a drug for the treatment of primary liver cancer with high safety.

[0023] Experimental Example 4, Olopatadine Hydrochloride Induces Apoptosis in Hepatoma Cells

[0024] The effects of olopatadine hydrochloride at different concentrations (including 0 μmol / L as the control group, and 5 and 10 μmol / L) on two hepatoma cell lines, HuH7 and HCCLM3 cells, were investigated. Western blotting was used to detect the expression levels of apoptosis-related proteins in the cells. The WB analysis results revealed that in the cells treated with olopatadine hydrochloride, the expression levels of the apoptotic protein Cleaved Caspase-3 and the pro-apoptotic protein Bax were significantly increased compared with the control group, while the expression of the anti-apoptotic protein Bcl-2 was decreased. These results showed that olopatadine hydrochloride could promote the apoptosis of HuH7 and HCCLM3 cells by activating apoptosis-related proteins (see Figure 3 , the effect of olopatadine hydrochloride on the expression of apoptosis proteins in HuH7 and HCCLM3 cells).

[0025] Experimental Example 5, Olopatadine Hydrochloride Can Inhibit Hepatoma Growth in Vivo

[0026] HuH7 cells were inoculated subcutaneously into the upper-middle part of the right groin of 5-week-old male BALB / c nude mice at a density of 1×10 7 cells / mouse, and a xenograft hepatoma mouse model was successfully constructed. When the tumor volume reached 100 mm 3 or more, the nude mice were randomly divided into groups of 5 each, namely (1) Model group: The nude mice were intragastrically administered an equal volume of blank solvent ddH2O every day; (2) Experimental group: The nude mice were intragastrically administered olopatadine hydrochloride at 20 mg / kg every day; The administration was continued for 3 weeks, and the body weight and tumor volume of the nude mice were weighed and recorded every week; After 3 weeks, the nude mice were sacrificed. After the experiment, 5 mice were randomly selected from each group. The results were similar to those of the in vitro experiment. Compared with the control mice, the tumor volume of the mice treated with 20 mg / kg by gavage was significantly reduced ( Figure 4 , in the in vivo experiment of animals, olopatadine could significantly inhibit the growth of hepatoma), consistent with the in vitro analysis, olopatadine hydrochloride showed strong anti-hepatoma activity in vivo.

[0027] Statistical method:

[0028] The experimental data are presented in the form of mean ± standard error, reflecting the aggregated results of three independent experiments. To evaluate the statistical differences between different treatment groups, Student's test and one-way analysis of variance (ANOVA) were employed in this invention. All statistical comparisons were implemented using GraphPad Prism 5 software. In this invention, the set significance level threshold was p < 0.05, that is, when the p value of the statistical test is lower than 0.05, the difference is considered statistically significant.

[0029] Conclusion:

[0030] (1) Significantly effective against primary liver cancer: Olopatadine hydrochloride has the ability to significantly inhibit the proliferation of primary liver cancer cells and promote their apoptosis, thus having extremely high clinical application value and potential for drug promotion.

[0031] (2) High safety characteristics: Olopatadine hydrochloride has been widely used in the treatment of allergic diseases, showing good tolerance and a low incidence of side effects. In addition, the experimental data further confirmed that olopatadine hydrochloride has no obvious toxic effect on normal liver cells, thus ensuring its safety in clinical applications.

[0032] (3) Economic considerations: The reasonable pricing of olopatadine hydrochloride helps to reduce the economic burden on patients with primary liver cancer and effectively reduce the cost of public medical insurance, which is of great significance for improving the accessibility and affordability of patient treatment.

Claims

1. Application of olopatadine hydrochloride in the preparation of drugs for treating primary liver cancer.

2. The drug containing the active ingredient of olopatadine hydrochloride as claimed in claim 1 can be in any dosage form listed in the pharmacopoeia.

Images