Use of digoxin and lactobacillus reuteri in combination for the treatment of ampullary cancer
By combining Lactobacillus reuteri with digetoxin, the therapeutic effect of ampullary carcinoma is enhanced, which solves the problems of lack of targeted drugs for ampullary carcinoma and the limited efficacy of digetoxin monotherapy, achieving significant anti-cancer effects and reducing toxic side effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LANZHOU UNIV
- Filing Date
- 2026-04-28
- Publication Date
- 2026-06-19
AI Technical Summary
Existing treatments for ampullary carcinoma lack targeted therapies, traditional drugs are costly to develop, and digetoxin monotherapy for ampullary carcinoma has limited efficacy and toxic side effects.
When Lactobacillus reuteri was used in combination with digetoxin, Lactobacillus reuteri acted as a sensitizer of digetoxin, enhancing its inhibitory effect on ampullary cancer cells. Furthermore, by regulating drug metabolism and immune response, it significantly inhibited cancer cell proliferation.
It significantly inhibits the proliferation of ampullary cancer cells, reduces the toxic side effects of digetoxin, and provides a new combination therapy regimen with good application prospects.
Smart Images
Figure CN122229897A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of medicine, specifically relating to the application of the combined use of digetoxin and Lactobacillus reuteri in the treatment of ampullary carcinoma. Background Technology
[0002] Ampullary carcinoma (AC) is a rare digestive system cancer originating in the ampulla of Vater. It accounts for approximately 0.2% of all gastrointestinal tumors and only 20% of periampullary carcinomas. The annual incidence of ampullary carcinoma is approximately 5.7 cases per million people, with a slightly higher incidence in men than women. The ampullary region, as a unique anatomical and functional area, forms the junction of the bile duct, pancreatic duct, and digestive tract, possessing special physiological significance. Currently, drug treatment for ampullary carcinoma typically employs chemotherapy regimens based on 5-fluorouracil and gemcitabine; there are currently no targeted therapies for this cancer. Traditional drug development processes are usually time-consuming and costly, while repurposing existing drugs for new indications can reduce drug development costs to some extent.
[0003] With the advancement of microbiome technologies leading to a deeper understanding of the interaction between microorganisms and tumors, the synergistic effects between microorganisms and anti-tumor drugs have become a new frontier in precision oncology research. Studies have shown that microorganisms can significantly influence or even enhance the efficacy of traditional chemotherapy drugs, novel targeted drugs, and immunotherapies through multiple pathways, including regulating drug metabolism and immune responses. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention has discovered that *Lactobacillus reuteri* can enhance the efficacy of digatexin against ampullary carcinoma and can be used as a sensitizer for digatexin in treating ampullary carcinoma. Combining the two can significantly inhibit the proliferation of ampullary cancer cells, providing a new drug delivery strategy for the prevention or treatment of ampullary carcinoma. Specifically, it includes the following:
[0005] In a first aspect, the present invention provides the use of Lactobacillus reuteri or its bacterial culture as a sensitizer for digetoxin in the treatment of ampullary carcinoma, wherein the Lactobacillus reuteri enhances the effect of digetoxin in the treatment of ampullary carcinoma.
[0006] Preferably, the Lactobacillus reuteri is a live bacterium.
[0007] In a second aspect, the present invention provides a pharmaceutical composition in which the active ingredients comprise digitoxane or a pharmaceutically acceptable salt thereof and Lactobacillus reuteri or a bacterial culture thereof.
[0008] Preferably, the Lactobacillus reuteri is a live bacterium.
[0009] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients.
[0010] Preferably, the pharmaceutical composition can be formulated into any dosage form such as tablets, sprays, granules, capsules, oral liquids, injections, or suspensions.
[0011] Thirdly, the present invention provides the use of the pharmaceutical composition described in the second aspect above in the preparation of a drug for the prevention of ampullary cancer.
[0012] Fourthly, the present invention provides the use of the pharmaceutical composition described in the second aspect above in the preparation of a medicament for treating ampullary cancer.
[0013] The beneficial effects of this invention are as follows: Firstly, this invention unexpectedly discovered that *Lactobacillus reuteri* can enhance the inhibitory function of digetuxin against ampullary cancer cells, and can be used as a sensitizer of digetuxin against ampullary cancer. Secondly, the combined use of *Lactobacillus reuteri* and digetuxin significantly inhibited the proliferation of ampullary cancer cells, and its effect was superior to 5-fluorouracil. Moreover, the combined use reduced the toxic side effects of digetuxin, providing a unique drug-bacterial combination therapy for the treatment of ampullary cancer, which has good application prospects. Attached Figure Description
[0014] Figure 1 Cell proliferation in Lactobacillus reuteri co-cultured with digetoxin.
[0015] Figure 2 Tumor volume growth curves and body weight change curves of subcutaneous xenograft tumor models in nude mice in each group.
[0016] Figure 3 The experimental endpoints were the weight of isolated tumors and serum calcium ion concentration in nude mice in each group.
[0017] Figure 4 Comparison of the weights of the heart, liver, spleen, lungs, kidneys, stomach and intestines of nude mice in each group.
[0018] Figure 5 HE staining of pathological sections of the heart, liver, spleen, lungs, kidneys, stomach and intestines of nude mice after drug administration in each group. Detailed Implementation
[0019] The following embodiments further describe and illustrate the present invention, but do not limit the scope of protection claimed by the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.
[0020] The Lactobacillus reuteri described in the following examples was obtained from the Guangdong Provincial Microbial Culture Collection Center (GDMCC1.614).
[0021] The aforementioned digetoxin is sourced from Shanghai Maclean Biotechnology Co., Ltd., product number D860643.
[0022] The ampullary cancer cell line DPC-X1 originated from the team led by Director Xu Hao at the Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine.
[0023] Example 1: Lactobacillus reuteri enhances the inhibitory effect of digetoxin on the proliferation of ampullary cancer cells.
[0024] 1. Experimental Methods
[0025] (1) Cell culture: Resuscitate and culture ampullary carcinoma cell line DPC-X1, take ampullary carcinoma cells in logarithmic growth phase, discard the culture medium and add 0.25% trypsin for digestion. After digestion, count the cell suspension using a hemocytometer, adjust the cell suspension concentration so that 1.5 × 10⁶ cells are seeded per well of a 96-well plate. 4 100 μL of liquid was added to each well to 100 cells. The appropriate concentration of cell suspension was mixed and transferred to a 96-well plate, and incubated for 24 h until the cells adhered.
[0026] (2) Drug treatment: After the culture was completed, the culture medium was discarded. Live Lactobacillus reuteri and inactivated Lactobacillus reuteri were used to set up a control group, a digetoxin administration group, a Lactobacillus reuteri inoculation group, and a digetoxin and Lactobacillus reuteri combined group, respectively. Culture medium, digetoxin at a concentration of 1 μM, and Lactobacillus reuteri at a concentration of 4 × 10⁻⁶ were added to each well. 7 CFU / mL culture medium, and simultaneously containing 1 μM digetoxin and 4 × 10 7 Culture medium containing CFU / mL *Lactobacillus reuteri*. After co-culturing for 4 h, the supernatant was discarded, and the sample was washed three times with PBS. Culture medium containing both antibiotics was added again, and after culturing for 2 h, the medium was discarded, and the sample was washed three times again with PBS. Complete culture medium was then added, and the sample was cultured for 48 h. In the digetoxin-treated group and the digetoxin and *Lactobacillus reuteri* combined group, the culture medium added after washing always contained 1 μM digetoxin.
[0027] (3) MTT assay: After treatment, add 10 μL of 5 mg / mL MTT solution to each well and continue incubation for 4 h. After incubation, remove the supernatant, retain the formazan crystals at the bottom of the plate, add 100 μL of DMSO to each well, shake at 120 rpm for 15 min to completely dissolve the crystals, and measure the absorbance at 490 nm. The absorbance is directly proportional to the number of viable cells. The drug inhibition rate is calculated based on the number of viable cells in the control group and the drug-treated group.
[0028] 2. Results Analysis
[0029] The MTT test results are as follows Figure 1As shown, the digatexin group exhibited a significant inhibitory effect on the proliferation of ampullary cancer cells. Furthermore, compared with the digatexin monotherapy group, the relative survival rate of ampullary cancer cells was further significantly reduced when digatexin was combined with *Lactobacillus reuteri* (P < 0.01), indicating that *Lactobacillus reuteri* has a sensitizing effect on digatexin, and the combined treatment significantly inhibited the proliferation of ampullary cancer cells. Inactivated *Lactobacillus reuteri*, however, failed to achieve this effect and even promoted tumor cell proliferation to some extent.
[0030] Example 2: In vivo antitumor activity of digetoxin, Lactobacillus reuteri, and their combinations
[0031] 1. Experimental Methods
[0032] (1) Nude mouse rearing: Nude mice are first reared for one week to adapt. During the rearing process, they can freely obtain sterilized feed and sterile drinking water. The bedding is changed every three days.
[0033] (2) Tumor modeling: After adaptive feeding, tumor modeling was performed. DPC-X1 cells in the logarithmic growth phase were digested with 0.25% trypsin. After digestion, the cells were pipetted with PBS solution to prepare a cell suspension. After counting, the cells were centrifuged and the supernatant was discarded. The suspension was then resuspended with PBS solution to a concentration of 5×10⁶ cells / mL. 7 The cell suspension was placed on ice at a concentration of cells / mL. Then, using a sterile syringe, 100 μL of the cell suspension was dispensed and injected at 5 × 10⁻⁶ ppm. 6 A concentration of 1 cell / mouse was administered subcutaneously to the axilla of the right forelimb of nude mice. Tumor formation was observed daily until the tumor volume reached approximately 50-100 mm. 3 Subcutaneous xenograft tumor.
[0034] (3) Animal drug administration: After tumor formation, tumor-bearing mice were randomly divided into a control group, a digatexin administration group, a Lactobacillus reuteri inoculation group, a combined drug administration group, and a 5-FU positive drug group, and drug administration experiments were conducted. The control group was administered 100 μL of PBS solution containing 2% DMSO by gavage every 2 days; the digatexin administration group was administered 100 μL of digatexin solution with a concentration of 1 mg / kg by gavage every 2 days; and the Lactobacillus reuteri inoculation group was administered 100 μL of PBS solution with a concentration of 1×10⁻⁶ mg / kg by gavage. 9 CFU / animal Lactobacillus reuteri suspension, once every 2 days; combined treatment group: 100 μL by gavage containing 1×10⁻⁶ CFU / animal 9 A mixture of CFU / mouse of *Lactobacillus reuteri* and 1 mg / kg of digetoxin was administered every 2 days; the 5-FU positive group received an intraperitoneal injection of 1 mg / kg of 5-FU solution every 2 days. A total of 7 administrations were administered over a total of 14 days. PBS solution containing 2% DMSO was used as the solvent in all administrations. Tumor volume and mouse weight were measured every 2 days. Tumor volume was calculated using the formula: V = 0.5 × L × W2 Where V, W, and L represent tumor volume, short diameter, and long diameter, respectively.
[0035] (4) Sample collection and testing: After drug administration, mice were euthanized, and blood was collected from the eyeballs, organs, and tumors. Whole blood samples were left to stand at room temperature for 4 h, then centrifuged at 3500 rpm for 10 min in a pre-cooled centrifuge at 4°C. Serum samples were then stored at -80°C. Serum calcium ion detection was performed by Wuhan Saiweier Biotechnology Co., Ltd., using the Changchun Huili Calcium (Ca) assay kit. The kit was operated strictly according to the instructions and the assay was performed using a fully automated biochemical analyzer. Organ and tumor samples were weighed and recorded, then stored in 4% paraformaldehyde fixative. After fixation, organ samples were embedded in paraffin and sectioned, stained with hematoxylin and eosin (HE), and then dewaxed twice in xylene for 10 min each time. Afterwards, the tissues were rehydrated for 5 minutes each with anhydrous ethanol solutions containing 100%, 95%, 85%, and 75%, followed by staining in hematoxylin solution for 5 minutes. After washing off the dye, the tissues were stained in eosin solution for 1 minute. After dehydration with gradient ethanol (75%, 85%, 95%, 100%), clearing with xylene, and mounting with neutral resin, the pathological changes of each group of organs were observed and photographed under an optical microscope.
[0036] 2. Results Analysis
[0037] Experimental results are as follows Figure 2 As shown, compared with the control group, the 5-FU group showed no significant difference. The antitumor activity of the digintuximab monotherapy group and the Lactobacillus reuteri monotherapy group was moderate. However, after combined administration of digintuximab and Lactobacillus reuteri, the tumor volume was significantly smaller than that of the digintuximab-administered group. The average tumor volume in the combined group was reduced by approximately 82.2% compared to the control group, approximately 68.2% compared to the digintuximab group, and approximately 76.7% compared to the positive control group. This indicates that the combination of digintuximab and Lactobacillus reuteri exhibits a good tumor-suppressing effect (P<0.0001). Simultaneously, the body weight of mice in each group remained stable during the experiment, with no significant differences observed. Furthermore, no obvious diarrhea, infection, or behavioral abnormalities were observed, indicating that this combined treatment regimen has good tolerability and low toxicity in nude mice.
[0038] Tumor terminal weight analysis results as follows Figure 3 As shown, the average tumor weight in the group receiving the combined administration of digetoxin and Lactobacillus reuteri was significantly lower than that in the control group, and the blood calcium concentration was significantly increased, demonstrating the possibility that Lactobacillus reuteri enhances the effect of digetoxin through calcium ions.
[0039] The weight results of organs such as heart, liver, spleen, lung, kidney, stomach and intestines in mice of each group are as follows: Figure 4 As shown, the weights of organs such as the heart, liver, spleen, lungs, kidneys, stomach, and intestines of mice in each group did not change significantly, indicating the low toxicity of digetoxin and Lactobacillus reuteri and their combination therapy.
[0040] HE staining results of major organ sections of mice as follows Figure 5 As shown, HE staining of major organ sections from mice revealed no significant pathological abnormalities in the heart, liver, spleen, lungs, kidneys, and stomach tissues across all groups. The tissue structures remained intact, without inflammatory infiltration, necrosis, or fibrosis. However, the intestinal tissues showed different characteristics among the different treatment groups. In the digetoxin group and the 5-fluorouracil positive drug group, varying degrees of sloughing and necrosis of the intestinal villi epithelium were observed, indicating some degree of intestinal damage. In contrast, the intestinal tissue structure in the digetoxin and Lactobacillus reuteri combined administration group remained largely intact, with neatly arranged villi and well-continuous epithelial cells, showing no significant difference from the control group. This indicates that the combined administration of Lactobacillus reuteri can effectively reduce the intestinal toxicity induced by digetoxin.
[0041] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.
Claims
1. The use of Lactobacillus reuteri or its bacterial culture as a sensitizer for digetoxin in the treatment of ampullary carcinoma, wherein the Lactobacillus reuteri enhances the effect of digetoxin in the treatment of ampullary carcinoma.
2. The application as described in claim 1, characterized in that, The Lactobacillus reuteri mentioned is a live bacterium.
3. A pharmaceutical composition, characterized in that, The active ingredients of the pharmaceutical composition include digitoxane or a pharmaceutically acceptable salt thereof and Lactobacillus reuteri or a bacterial culture thereof.
4. The pharmaceutical composition according to claim 3, characterized in that, The Lactobacillus reuteri mentioned is a live bacterium.
5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.
6. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition can be formulated into any dosage form, such as tablets, sprays, granules, capsules, oral liquids, injections, or suspensions.
7. Use of the pharmaceutical composition according to any one of claims 3-6 in the preparation of a medicament for the prevention of ampullary cancer.
8. Use of the pharmaceutical composition according to any one of claims 3-6 in the preparation of a medicament for treating ampullary cancer.