Application of bifidobacterium animalis H22B905 in preparation of medicine combined with gefitinib
By combining Bifidobacterium animalis H22B905 with gefitinib, the side effects of gefitinib were alleviated, the liver was protected, and the production of short-chain fatty acids was promoted. This solved the side effect problem of gefitinib in the treatment of non-small cell lung cancer and improved the treatment effect.
Patent Information
- Application Number
- CN202511251049.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-03
- Publication Date
- 2025-10-31
AI Technical Summary
Gefitinib has side effects such as diarrhea, nausea, and rash when used to treat non-small cell lung cancer, and long-term use may lead to abnormal liver function, which limits its application.
By combining Bifidobacterium animalis H22B905 with gefitinib, a drug containing this probiotic was prepared, which reduced the survival rate of lung cancer cells, alleviated the side effects of gefitinib, and promoted the production of short-chain fatty acids.
It significantly reduced the hepatotoxicity of gefitinib, protected liver tissue structure, and improved the therapeutic effect by promoting the regulation of the intestinal microecology through the addition of probiotics.
Smart Images
Figure CN120860073A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology and relates to the application of Bifidobacterium animalis H22B905 in the preparation of drugs used in combination with gefitinib. Background Technology
[0002] Gefitinib is an oral epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor (a small molecule compound). As a targeted therapy for non-small cell lung cancer, it primarily works by inhibiting EGFR activity to slow cancer cell growth and promote apoptosis, and is particularly suitable for advanced patients with EGFR19 or EGFR21 gene positivity. It can effectively prolong survival and alleviate symptoms, and can be used alone or as adjuvant chemotherapy.
[0003] However, gefitinib is not suitable for all lung cancer patients and may cause a range of side effects, such as diarrhea, nausea, and rash. Long-term use may also lead to elevated transaminase and bilirubin levels, as well as kidney dysfunction, thus limiting its application. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention aims to provide the application of Bifidobacterium animalis H22B905 in the preparation of drugs used in combination with gefitinib, offering new ideas and methods to reduce patient side effects and improve treatment efficacy.
[0005] The technical solution adopted by this invention to solve the technical problem is as follows:
[0006] The application of Bifidobacterium animalis H22B905 in the preparation of drugs used in combination with gefitinib. Bifidobacterium animalis H22B905 was deposited on October 23, 2024 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 65329, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Its taxonomic name is Bifidobacterium animalis.
[0007] Furthermore, the combined use of Bifidobacterium animalis H22B905 and gefitinib significantly reduced the survival rate of lung cancer cells.
[0008] Furthermore, the survival rate of the lung cancer cells is inversely proportional to the proportion of Bifidobacterium animalis H22B905 metabolites.
[0009] Furthermore, the concentration (v / v) of the Bifidobacterium animalis H22B905 metabolite is 10% to 30%.
[0010] Furthermore, the drugs used in combination with gefitinib include those that alleviate the side effects of gefitinib.
[0011] Furthermore, the side effects include liver damage.
[0012] Furthermore, the animal Bifidobacterium H22B905 can effectively promote the production of short-chain fatty acids in the body.
[0013] Furthermore, the short-chain fatty acids include acetic acid, propionic acid, and butyric acid.
[0014] The beneficial effects of this invention are as follows:
[0015] This invention identified *Bifidobacterium animalis* H22B905 from six probiotic strains of *Bifidobacterium animalis* through in vitro screening experiments. This probiotic exhibits a low degradation rate of gefitinib, ensuring it does not interfere with the drug's primary therapeutic effect. Furthermore, a non-small cell lung cancer model was established using nude mice to further verify the efficacy of *Bifidobacterium animalis* H22B905 in alleviating the side effects of gefitinib. Experimental results showed that compared to the group using gefitinib alone, the experimental group using a combination of gefitinib and *Bifidobacterium animalis* H22B905 exhibited significantly reduced liver damage. Specifically, liver tissue structure was better protected, demonstrating the positive effect of *Bifidobacterium animalis* H22B905 in reducing drug-induced hepatotoxicity. In addition, the addition of probiotics significantly promoted the production of short-chain fatty acids, further confirming the positive regulatory effect of probiotics on the intestinal microecology. This invention not only provides a new solution for alleviating the side effects of gefitinib but also provides strong scientific support for using probiotics as an adjunct to gefitinib treatment. Attached Figure Description
[0016] Figure 1 The degradation rate of gefitinib after co-culturing with six strains of Bifidobacterium animalis for 0h, 6h, 12h, and 24h is shown.
[0017] Figure 2 This shows the survival rate of non-small cell lung cancer HCC827 cells under the action of 20 μM gefitinib and different concentrations of Bifidobacterium animalis H22B905 metabolites.
[0018] Figure 3 The tumor volume is shown in the model group (T) and the treatment group (G, G+H22B905) after 5 weeks of treatment.
[0019] Figure 4 Liver sections of nude mice from the control group (Con), model group (T), and treatment group (G, G+H22B905) are shown.
[0020] Figure 5The table shows the content of acetic acid, propionic acid, and butyric acid in the control group (Con), model group (T), and treatment group (G, G+H22B905). Detailed Implementation
[0021] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.
[0022] Example
[0023] I. Materials and Methods
[0024] 1.1 In vitro experiments
[0025] 1.1.1 Experimental Materials
[0026] Bifidobacterium animalis strains H22B905, H22B970, H22B971, H22B976, H22B921, and H22B890 were all provided by the Tropical Probiotic Lactic Acid Bacteria Resource Bank of Hainan University. Among them, Bifidobacterium animalis H22B905 was deposited on October 23, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 65329, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Its taxonomic name is *Bifidobacterium animalis*.
[0027] The experimental drug gefitinib (pharmaceutical grade II standard) was purchased from AstraZeneca UK Limited. HCC827 human non-small cell lung cancer cells were purchased from the cell resource platform built by Xiamen Yimo Biotechnology Co., Ltd.
[0028] 1.1.2 Screening of bacteria using high performance liquid chromatography (HPLC)
[0029] In vitro, different types of probiotics were mixed with 33 μM gefitinib stock solution at a ratio of TPY liquid medium: bacterial culture: gefitinib stock solution = 12 mL: 400 μL: 200 μL, and co-cultured for 0 h, 6 h, 12 h, and 24 h. The six strains were cultured under identical conditions in an anaerobic workstation at 37 °C. Each liter of TPY medium contained: 10.0 g hydrolyzed casein, 5.0 g plant peptone, 2.0 g yeast extract, 5.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 0.5 g magnesium chloride, 0.25 g zinc sulfate, 0.15 g calcium chloride, 0.1 mg ferric chloride, 0.5 g cysteine-hydrochloric acid, and 1.0 mL Tween-80, with a final pH of 6.3-6.7. Subsequently, the peak area of gefitinib was measured using a high-performance liquid chromatograph (HPLC) from Agilent Technologies (column: octadecylsilane-bonded silica gel column; column temperature: 30℃; mobile phase: ammonium acetate solution: acetonitrile = 65:35; flow rate: 1.0 mL / min; detection: 243 nm UV detection). Using gefitinib cultured alone in culture medium as a blank, the peak area of gefitinib cultured with probiotics was compared to the peak area of gefitinib to calculate the degradation rate of gefitinib by the probiotics.
[0030] The formula for calculating the degradation rate of gefitinib is as follows:
[0031] V = (AB) / A
[0032] V: Degradation rate of gefitinib by probiotics
[0033] A: Peak area of gefitinib
[0034] B: Peak area of gefitinib after culturing with synergistic probiotics.
[0035] 1.1.3 MTT assay for HCC827 cell viability
[0036] Before the experiment, the metabolites of Bifidobacterium animalis H22B905 were prepared: Bifidobacterium animalis H22B905 was cultured to the maximum growth phase and then mixed by shaking. It was centrifuged at 6000 r / min and 4℃ for 5 min. After centrifugation, the supernatant was taken in a clean workbench and filtered twice with a 0.22 μM filter membrane. Then it was aliquoted and frozen at -80℃ until use.
[0037] HCC827 cells were digested and collected using trypsin, and then mixed with 1 mL of DMEM medium to prepare a cell suspension. Cells were counted and the concentration was adjusted to approximately 2000 cells / mL. After counting, 96-well culture plates were prepared, and HCC827 cells were evenly distributed into each well. The culture plates were then incubated at 37°C for 48 hours, during which time the cells proliferated normally until they covered the bottom of the wells.
[0038] Next, a 20% concentration of Bifidobacterium animalis H22B905 metabolite was added to each well (i.e., the volume ratio of the added Bifidobacterium animalis H22B905 metabolite to the cell culture medium was 20%), and incubation continued for 24 hours. 24 hours after cell treatment, the culture medium was removed, and then 100 μL of MTT dye solution was added to each well. The culture plate was then returned to the incubator for 4–6 hours of incubation.
[0039] After incubation, add 150 μL of DMSO to each well and shake for 10 minutes to ensure complete dissolution of the crystals. Finally, use Tecan... The 200PRO microplate reader (Swiss-made) measures absorbance (OD) at a wavelength of 570 nm.
[0040] 1.2 In vivo experiments
[0041] 1.2.1 Cell Culture
[0042] The HCC827 cell line for non-small cell lung cancer was cultured in DMEM basal medium supplemented with 10% fetal bovine serum, 1% penicillin, 1% streptomycin, 1% sodium pyruvate, and 1% glutamine. Cells were placed in a fully humidified incubator maintained at 37°C with 5% CO2. All experiments were performed when the cells were in the logarithmic growth phase. The medium was changed every two days, and cells were passaged using 0.25% trypsin.
[0043] 1.2.2 Laboratory Animals
[0044] This study was conducted with the approval of the Animal Ethics Committee of Hainan University, and all experimental procedures involving animals strictly followed the relevant regulations in the "Guidelines for the Care and Use of Laboratory Animals" issued by Hainan University. The applicant purchased 24 four-week-old female SPF-grade BALB / c Nude nude mice from Beijing Vital River Laboratory Animal Technology Co., Ltd. These mice were housed in a standard laboratory environment with a 12-hour day / night light cycle, humidity maintained at 60%, and temperature controlled at 23°C to ensure optimal living conditions. In addition, the mice had free access to standard food and drinking water provided by Beijing Keao Xieli Feed Co., Ltd. to meet their daily nutritional needs. Before the formal experiments began, the mice were given one week to acclimatize to the new environment to minimize the impact of environmental factors on the experimental results. During the experiments, both the control group and the lung cancer model group continued to consume this maintenance diet.
[0045] 1.2.3 Ectopic modeling of non-small cell lung cancer
[0046] HCC827 cells were cultured to the logarithmic growth phase and used to establish an ectopic cell model in nude mice. Cells were washed with DPBS, digested, and counted. The cell concentration was adjusted to approximately 1 × 10⁶ cells / mL using PBS and matrix gel. 7 Cells were placed on ice. The cell suspension was rapidly injected near the left hind leg of nude mice, 200 μL per mouse. Tumor volume was calculated as (L×W×H) / 2, where L is the tumor length, W is the tumor width, and H is the height.
[0047] 1.2.4 In vivo therapy
[0048] When the tumor grows to 50-100 mm 3 At that time, the tumors were grouped according to their size. The experiment was divided into 4 groups (n=6): blank control group (Con); non-small cell lung cancer model group (T); gefitinib treatment group (G); and gefitinib combined with probiotic Bifidobacterium animalis H22B905 treatment group (G+H22B905).
[0049] Groups Con and T received 200 μL of 0.5% sodium carboxymethyl cellulose via gavage daily; Group G received 200 μL of 100 mg / kg gefitinib via gavage daily, with the gefitinib dissolved in sodium carboxymethyl cellulose; Group G+H22B905 received 100 mg / kg gefitinib + 10% sodium carboxymethyl cellulose via gavage daily. 8 CFU animal Bifidobacterium H22B905 (dissolved in sodium carboxymethyl cellulose, 200 μL). The patient was weighed weekly, and the tumor volume was measured weekly.
[0050] Five weeks after treatment, the impact of the presence of probiotics on the therapeutic effect of gefitinib was assessed.
[0051] 1.2.5 Sample Collection and Measurement
[0052] Fecal samples were collected from mice before and after treatment and immediately frozen at -80°C. After 5 weeks of treatment, blood, tissue samples, and colon contents were collected from nude mice under sterile conditions. Blood samples were centrifuged to separate serum, which was then stored together with colon contents at -80°C until subsequent use. The colon contents were used to analyze the content of short-chain fatty acids.
[0053] The mouse livers obtained after dissection were divided into two equal parts. One part was placed in a sterile centrifuge tube, flash-frozen in liquid nitrogen, and then cryopreserved at -80°C. The other part was rinsed with 0.85% physiological saline and fixed in paraformaldehyde solution for subsequent histological section analysis (Wuhan Saive Biotechnology Co., Ltd.). Simultaneously, the dissected small intestine was also fixed in paraformaldehyde for the preparation of intestinal sections (Wuhan Saive Biotechnology Co., Ltd.).
[0054] For the determination of short-chain fatty acids, gas chromatography-mass spectrometry (GC-MS) using Agilent Technologies was employed. During the analysis, an Agilent DB-WAX column (0.25 mm × 0.25 μm × 50 cm) was used, with the injection port temperature and gas interface temperature both set at 250 °C. The carrier gas flow rate was 1.5 mL / min, the split ratio was 3:1, and the injection volume was 1 μL per sample.
[0055] 1.3 Statistical Analysis
[0056] All statistical analyses were performed using Graphpad Prism 9.5 software. A significance threshold of 0.05 was set for the p-value; that is, a p-value less than 0.05 was considered statistically significant, and a p-value less than 0.01 was considered highly significant. Furthermore, all graph and chart creation was also performed using Graphpad Prism 9.5 software.
[0057] II. Results
[0058] 2.1 Identification of the probiotic Bifidobacterium animalis H22B905
[0059] Gefitinib was co-cultured with Bifidobacterium animalis for 0 h, 6 h, 12 h, and 24 h, and its degradation rate was determined by high performance liquid chromatography. The results are as follows: Figure 1 As shown, Bifidobacterium animalis H22B905 showed the least change and its degradation degree approached zero during the 6h, 12h and 24h of co-culture. The degradation changes of the other 5 probiotic strains were relatively large compared with H22B905. Therefore, we chose to use H22B905 for subsequent experiments.
[0060] 2.2 MTT Test
[0061] In investigating the effect of metabolite concentration on cell viability, we observed large-scale cell death when the metabolite concentration exceeded 30%. This phenomenon may be attributed to the increased osmotic pressure caused by high metabolite concentrations, leading to a significant decrease in cell viability. Based on this, in subsequent experiments, we decided to control the metabolite concentration (v / v, i.e., the ratio of metabolite volume to cell culture medium volume) within the range of 10% to 30%.
[0062] The MTT assay was used to detect the effect of different concentrations of Bifidobacterium animalis H22B905 metabolites, combined with 20 μM gefitinib, on the survival rate of non-small cell lung cancer HCC827 cells. The results are as follows: Figure 2As shown, the survival rate of lung cancer cells is inversely proportional to the proportion of metabolites from Bifidobacterium animalis H22B905; that is, as the concentration of metabolites gradually increases, the survival rate of lung cancer cells decreases. This result strongly suggests that Bifidobacterium animalis H22B905 has a significant effect on inhibiting non-small cell lung cancer HCC827.
[0063] 2.3 The synergistic intake of probiotic H22B905 did not affect the therapeutic effect and alleviated hepatitis in tumor-bearing nude mice.
[0064] Co-intake of gefitinib with Bifidobacterium animalis H22B905 was found to have no effect on tumor treatment in mice (see [link]). Figure 3 Furthermore, it can alleviate the liver side effects of gefitinib, as shown in the results of liver slices from nude mice (see...). Figure 4 In the G group, numerous hepatocytes showed hydropic degeneration, with a small number exhibiting ballooning degeneration, indicating swollen, balloon-like cells. In contrast, the hepatocytes in the G+H22B905 group were round and plump, with only minor vascular congestion. This suggests that concurrent use of the probiotic H22B905 during gefitinib treatment can reduce the degree of liver damage caused by gefitinib.
[0065] 2.4 Ingestion of probiotic H22B905 increased short-chain fatty acid content.
[0066] After a five-week probiotic gavage experiment on nude mice, we observed an increase in short-chain fatty acid levels in their intestines, particularly acetic acid, propionic acid, and butyric acid. Specifically, compared to group G, the G+H22B905 group showed significantly increased levels of propionic acid and butyric acid (p<0.05), while the acetic acid level showed a highly significant difference (p<0.01). Figure 5 As shown in the figure. This discovery means that in the treatment of lung cancer, using Bifidobacterium animalis H22B905 as an adjunct to gefitinib therapy can effectively promote the production of short-chain fatty acids in the body.
[0067] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.
Claims
1. Application of Bifidobacterium animalis H22B905 in the preparation of drugs used in combination with gefitinib. The Bifidobacterium animalis H22B905 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on October 23, 2024, with accession number GDMCC No: 65329, deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China. Taxonomic name: Bifidobacterium animalis.
2. The application according to claim 1, characterized in that, The combination of Bifidobacterium animalis H22B905 and gefitinib significantly reduced the survival rate of lung cancer cells.
3. The application according to claim 2, characterized in that, The survival rate of the lung cancer cells was inversely proportional to the proportion of Bifidobacterium animalis H22B905 metabolites.
4. The application according to claim 3, characterized in that, The concentration of the animal Bifidobacterium H22B905 metabolite is 10% to 30%.
5. The application according to claim 1, characterized in that, The drugs used in combination with gefitinib include those that alleviate the side effects of gefitinib.
6. The application according to claim 5, characterized in that, The side effects include liver damage.
7. The application according to claim 1, characterized in that, The animal bifidobacterium H22B905 can effectively promote the production of short-chain fatty acids in the body.
8. The application according to claim 7, characterized in that, The short-chain fatty acids include acetic acid, propionic acid, and butyric acid.