Use of Amuc_1100 in the preparation of drugs for alleviating adriamycin chemotherapy cardiotoxicity

Abstract

The present invention discloses the role of Amuc_1100 in the preparation of drugs that alleviate doxorubicin cardiotoxicity. In vivo experimental results show that Amuc_1100 can effectively alleviate the toxic effects of the chemotherapy drug doxorubicin on the heart. By improving the homeostasis of the body and cardiac immune cells, reducing cell apoptosis in myocardial tissue, alleviating elevated myocardial enzymes, and enhancing left ventricular diastolic capacity and cardiac ejection fraction, it reduces the toxic side effects of the chemotherapy drug doxorubicin. It also improves the intestinal barrier function damage caused by doxorubicin chemotherapy. The present invention overcomes the current limitations on the clinical use of Dox and the drawbacks of existing chemotherapy cardioprotective drugs, while also solving the problem of preventing and treating the cardiotoxicity and intestinal side effects induced by Dox chemotherapy.

Description

Technical Field

[0001] The present invention relates to a new use of a drug, and in particular to a use of Amuc_1100 in preparing a drug for alleviating the cardiotoxicity of adriamycin chemotherapy. Background Art

[0002] Doxorubicin (Dox) is a broad-spectrum, highly effective anthracycline anticancer drug currently in routine clinical use, widely used to treat leukemia, malignant lymphoma, breast cancer, ovarian cancer, and osteosarcoma. Although anthracyclines, represented by Dox, have increased the long-term survival rate of cancer patients, their dose-dependent cardiotoxic side effects, which may ultimately lead to irreversible myocardial damage and congestive heart failure, have greatly limited their clinical application.

[0003] Current cardioprotective strategies for preventing anthracycline toxicity primarily involve the use of cardioprotective agents or reducing the cumulative chemotherapy dose to mitigate the cardiotoxic effect. Dexrazoxane is the only approved anthracycline cardioprotective agent. Although dexrazoxane can prevent a decrease in resting left ventricular ejection fraction and reduce the incidence of heart failure when used in combination with anthracycline chemotherapy, it can exacerbate chemotherapy-induced bone marrow suppression, impairing antitumor efficacy. Furthermore, clinical studies have shown that the addition of dexrazoxane to the FAC regimen (fluorouracil, doxorubicin, and cyclophosphamide) may result in patients receiving a higher cumulative dose of doxorubicin (compared to the group without dexrazoxane) but does not eliminate anthracycline-induced cardiotoxicity. Furthermore, the use of large amounts of reactive oxygen species (ROS) scavengers and iron chelators has been ineffective in preventing Dox-induced cardiomyopathy in cancer patients and has significant clinical limitations. Given these limitations in current clinical medications, the development of novel agents and interventions to treat anthracycline chemotherapy cardiotoxicity is urgent. In clinical practice, Dox chemotherapy can irritate the gastric mucosa, leading to symptoms such as nausea, vomiting, abdominal distension, abdominal pain, or constipation. While probiotic supplementation can improve these symptoms and aid digestion in chemotherapy patients, it does not alleviate Dox-induced cardiotoxicity. Dox treatment can also cause changes in intestinal epithelial function, Paneth cells, and mucus, leading to damage to the intestinal barrier mucosa. The intestinal barrier maintains homeostasis in the intestinal lining and prevents the translocation and release of pathogens and toxins into extraluminal tissues and organs. Damage to the intestinal barrier leads to the absorption and migration of large numbers of bacteria and endotoxins into the bloodstream and lymphatic system, potentially causing systemic multi-organ dysfunction, including sepsis and adverse vascular and cardiac remodeling. Studies have shown that activated intestinal immune cells during intestinal barrier damage can migrate and contribute to distal organ inflammation. We speculate that, in addition to the direct effects of Dox on the heart through the circulation, the resulting damage to the intestinal barrier, and the resulting imbalance in immune cell homeostasis in the body and the heart, may be a key factor exacerbating Dox cardiotoxicity.

[0004] Based on the above viewpoints, the present invention uses the biomacromolecule Amuc1100 with intestinal barrier protection function to intervene in the early stage of Dox treatment, so as to maintain the integrity of the intestinal barrier during Dox chemotherapy, further improve the imbalance of homeostasis of the body and cardiac immune cells, and reduce the risk of cardiotoxicity of Dox chemotherapy.

[0005] Amuc_1100 is an outer membrane protein of the intestinal bacterium Akkermansia muciniphila. Studies have found that Amuc_1100 can improve the metabolism of obese and diabetic mice, improve colorectal inflammation and prevent inflammatory colorectal cancer. It can also improve intestinal barrier damage caused by immunotherapy.

[0006] Although Amuc_1100 shows potential biological functions in improving metabolic diseases, its role and protective mechanism in alleviating cardiotoxicity caused by tumor Dox chemotherapy have not been reported.

[0007] The present invention provides a potential drug and intervention method for reducing the cardiac toxicity of doxorubicin chemotherapy, while also improving the intestinal function damage caused by doxorubicin. At the same time, due to the convenience of the above-mentioned intervention method, the present invention can effectively reduce the chemotherapy pain, as well as the psychological and economic burden of cancer patients. Summary of the Invention

[0008] The present invention aims to overcome the shortcomings of existing drugs and technologies by providing a method for preparing a drug for alleviating doxorubicin chemotherapy-induced cardiotoxicity. This invention proposes, for the first time, the oral administration of Amuc_1100 to prevent and treat doxorubicin (Dox) chemotherapy-induced cardiotoxicity. This approach overcomes current limitations on Dox clinical use and the drawbacks of existing chemotherapy cardioprotective drugs, while also addressing the prevention and treatment of Dox-induced cardiotoxicity.

[0009] The object of the present invention is achieved through the following technical solutions:

[0010] The present invention relates to use of a membrane protein Amuc_1100 in preparing a drug for alleviating adriamycin chemotherapy cardiotoxicity.

[0011] The amino acid sequence of Amuc_1100 is:

[0012] IVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSKFIK VYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEAAAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED.

[0013] As one embodiment of the present invention, Amuc_1100 is used as follows: oral gavage once a day starting one week before Dox treatment, and oral gavage once every other day after Dox treatment.

[0014] As one embodiment of the present invention, the dosage of Amuc_1100 for mice is: 200 μL / mouse for gavage, 3 μg / day per mouse.

[0015] As one embodiment of the present invention, the dosage of Amuc_1100 for adults is: based on a body weight of 70 kg, the oral dosage is about 0.58 to 2.31 mg / day.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention can effectively prevent and treat the cardiotoxicity caused by doxorubicin (Dox) chemotherapy by oral gavage of Amuc_1100 one week before chemotherapy, including significantly improving left ventricular systolic and diastolic function, increasing ejection fraction, reducing circulating myocardial enzyme levels, and reducing cardiac fibrosis and apoptosis levels; at the same time, it also has the effect of protecting the intestinal barrier damage caused by chemotherapy.

[0018] 2. The mechanism by which Amuc_1100 alleviates Dox chemotherapy-induced cardiotoxicity is:

[0019] 2.1. Damage to the intestinal epithelial barrier can lead to the absorption and migration of large amounts of bacteria and endotoxins into the blood circulation and lymphatic system, potentially causing systemic multi-organ dysfunction, including sepsis, vascular and cardiac adverse remodeling; Amuc_1100 regulates the intestinal immune barrier and the structure and expression of tight junction proteins (TJ), promoting the recovery of intestinal barrier damage.

[0020] 2.2. Amuc_1100 can effectively restore the integrity of the intestinal barrier, and thus may effectively reduce endotoxemia or the activation and migration of intestinal mucosal immune cells caused by intestinal barrier damage. This is important for reducing the protective pathway related to Dox-induced endotoxins entering the circulation from the intestine and then affecting cardiac function.

[0021] 2.3. The present invention found that after the intervention of Amuc_1100, the spleen index of Dox mice decreased, the spleen IFN-γ (Th1) level decreased significantly, while the IL-4 (Th2) level did not change significantly. The heart ROS level decreased, and the flow cytometric analysis results showed that the proportion of cardiac T cells decreased significantly. Amuc_1100 improves the intestinal barrier function, reduces the output and accumulation of splenic immune cells through the circulation to organs such as the intestine and heart, maintains the homeostasis of the body and cardiac immune cells, reduces the excessive response of T cells to myocardial tissue, reduces ROS levels, and thus inhibits myocardial cell apoptosis. This may be an important mechanism by which Amuc_1100 alleviates Dox cardiotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0023] Figure 1 A-B. Ultrasound examination of the left ventricle of the mouse heart after Amuc_1100 intervention; C. Myocardial enzyme spectrum analysis results; D. Western blot analysis of the expression levels of intestinal tight junction proteins ZO-1, Occludin, and Claudin1; E. Pathological tissue section analysis of mouse heart (Masson and TUNEL) and intestine (H&E); F. ELISA analysis of plasma levels of LPS and Zonulin, markers of intestinal barrier damage;

[0024] Figure 2 A is the spleen index, IFN-γ and IL-4 levels in the spleen, and ROS levels in the heart; B is the flow cytometric analysis of cardiac immune cell composition. DETAILED DESCRIPTION

[0025] The present invention will be described in detail below with reference to the examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several adjustments and improvements without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0026] Example 1

[0027] 1. Experimental Animals and Grouping

[0028] SPF male C57BL / 6 mice, 6–8 weeks old, weighing approximately 18–20 g, were purchased from Beijing Sibeifu Laboratory Animal Co., Ltd. They were raised at the Experimental Animal Center of Zhengzhou University.

[0029] The experimental groups included: oral PBS group (PBS group), oral PBS + Dox injection group (Dox group), oral Amuc_1100 group (Amuc group), and oral Amuc_1100 + Dox injection group (Amuc+Dox group).

[0030] 2. Treatment methods

[0031] PBS group and Dox group: One week before the first Dox injection, both groups were gavaged with 200 μl of PBS every day. After the first Dox injection, the gavage was continued every other day until the end of the experiment.

[0032] Amuc group and Amuc+Dox group: 1 week before the first Dox injection, 3ug of Amuc was gavaged every day. After the first Dox injection, the gavage was continued every other day until the end of the experiment.

[0033] After three injections of Dox, the mice were observed for 3-4 weeks before subsequent experimental operations.

[0034] 3. Modeling method

[0035] Dox-induced chronic cardiotoxicity mouse model: Dox was injected into the tail vein of mice at a final concentration of 15 mg / kg, three times at 5 mg / kg each injection, with an interval of 7 days. After the model was established, the mice were closely observed for feeding, mental state, and activity. The mice were weighed every other day after model establishment.

[0036] 4. Small Animal Ultrasound

[0037] Before Dox injection and every other week after injection, the left ventricular function changes of mice in each group were monitored regularly. When obvious changes in cardiac function appeared, the mice were euthanized and the intestinal tract, blood and heart samples were collected.

[0038] 5. Pathological Examination of Intestinal Tissue and Heart Tissue

[0039] A 2-cm segment of the distal ileum was removed and rapidly flushed with cold saline to remove intestinal contents. The sample was then fixed in a tissue fixative for 24-48 hours. Following H&E staining, intestinal histomorphological changes were observed under a light microscope and pathological scores were calculated.

[0040] Mouse heart tissue was collected, cleared of blood, and fixed in tissue fixative for 24-48 hours. H&E, Masson's, and TUNEL staining were used to analyze changes in cardiac tissue structure, the degree of myocardial fibrosis, and the level of cardiomyocyte apoptosis.

[0041] 6. Serum myocardial enzyme spectrum and intestinal barrier integrity monitoring

[0042] ELISA was used to analyze the expression levels of myocardial enzyme spectrum (CKMB, cTnT, NTpro-BNP) in each group of mice to confirm the differences in cardiac function among the mice groups; the intestinal tissue morphology of each group of mice was analyzed to evaluate the improvement of intestinal barrier.

[0043] 7. Results

[0044] 7.1 Growth status:

[0045] PBS group: The mice ate and defecated normally, were active, gained weight during the experiment, and no deaths occurred;

[0046] Dox group: Within three days after the first two injections of Dox, mice showed varying degrees of decreased activity, slowed movement, curled up, dull fur, and significant weight loss. After three days, their weight gradually recovered, and their mobility slowly recovered. However, after the third and fourth injections of Dox, their activity decreased significantly, and their weight remained the same or decreased.

[0047] Amuc group: rats ate and defecated normally, were in good condition, gained weight during the experiment, and no deaths occurred;

[0048] Amuc+Dox group: The mice experienced a slight weight loss within 3 days after Dox injection, and then recovered and maintained their weight; there was no significant decrease in activity.

[0049] 7.2 Improvement of cardiac function ( Figure 1 、 2 ):

[0050] PBS group: The left ventricular function of mice was normal during the experimental period, the myocardial enzyme levels were normal, there was no obvious myocardial fibrosis, and no obvious apoptosis of myocardial cells;

[0051] Dox group: After three injections of Dox, with a cumulative dose of 15 mg, mice experienced a decline in left ventricular function, a significant increase in serum myocardial enzyme expression, aggravated myocardial fibrosis, and significant myocardial cell apoptosis.

[0052] Amuc group: The left ventricular function of mice was normal during the experimental period, the myocardial enzyme levels were normal, there was no obvious myocardial fibrosis, and no obvious apoptosis of myocardial cells;

[0053] Amuc+Dox group: After three injections of Dox, with a cumulative dose of 15 mg, the left ventricular function of the mice did not decline significantly, the expression of serum myocardial enzymes decreased significantly compared with the Dox group, there was no obvious myocardial fibrosis, and myocardial cell apoptosis was not significant;

[0054] 7.3 Improvement of intestinal barrier ( Figure 1 ):

[0055] PBS group: At the end of the model, the intestinal tissue morphology of the mice was normal, inflammatory cell infiltration in the ileum was not obvious, villi were stretched normally, and LPS and zonulin levels were normal;

[0056] Dox group: At the end of the model, the intestinal tissue of mice was brittle, inflammatory cell infiltration in the ileum was obvious, villi were broken, LPS and Zonulin levels were significantly increased, and the expressions of intestinal epithelial tight junction proteins ZO-1, Occludin, and Cludin-1 were significantly decreased;

[0057] Amuc group: At the model endpoint, the intestinal tissue morphology of the mice was normal, inflammatory cell infiltration in the ileum was not obvious, villi were stretched normally, and LPS and zonulin levels were normal;

[0058] Amuc+Dox group: At the end of the model, the intestinal tissue morphology of mice was normal, ileal inflammatory cell infiltration was not obvious, villi were stretched normally, LPS and Zonulin levels returned to normal and were significantly lower than those in the Dox group, and the expression of intestinal epithelial tight junction proteins ZO-1, Occludin, and Cludin-1 was significantly increased.

[0059] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. Use of a membrane protein Amuc_1100 in the preparation of a drug for alleviating adriamycin chemotherapy cardiotoxicity; the amino acid sequence of Amuc_1100 is: IVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSKFIK VYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEAAAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED.

2. The use according to claim 1, characterized in that The dosage of Amuc_1100 for adults is: based on a body weight of 70 kg, the oral dose is 0.58 to 2.31 mg / day.

Images